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WO2025032534A2 - Protéines modifiées - Google Patents

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Publication number
WO2025032534A2
WO2025032534A2 PCT/IB2024/057693 IB2024057693W WO2025032534A2 WO 2025032534 A2 WO2025032534 A2 WO 2025032534A2 IB 2024057693 W IB2024057693 W IB 2024057693W WO 2025032534 A2 WO2025032534 A2 WO 2025032534A2
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Prior art keywords
amino acid
modified
seq
protein
als3
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WO2025032534A9 (fr
WO2025032534A3 (fr
Inventor
Fabio SERVENTI
Sandra MARKOVIC-MULLER
Gerd Martin LIPOWSKY
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GlaxoSmithKline Biologicals SA
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GlaxoSmithKline Biologicals SA
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Publication of WO2025032534A3 publication Critical patent/WO2025032534A3/fr
Publication of WO2025032534A9 publication Critical patent/WO2025032534A9/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • 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
    • A61K39/0002—Fungal antigens, e.g. Trichophyton, Aspergillus, Candida
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61P31/10—Antimycotics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/40—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Candida
    • 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
    • C12N9/88—Lyases (4.)
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • 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
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/99—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
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/02—Aldehyde-lyases (4.1.2)
    • C12Y401/02013—Fructose-bisphosphate aldolase (4.1.2.13)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55583—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation

Definitions

  • the present invention relates to the field of modified proteins, immunogenic compositions and vaccines comprising the modified proteins, their manufacture and the use of such compositions in medicine. More particularly, it relates to a modified Als3 (Agglutinin-like sequence 3 of Candida albicans) protein.
  • the modified Als3 protein can be used as a carrier protein for other antigens, particularly saccharide antigens or other antigens lacking T cell epitopes.
  • Candida Cryptococcus, Aspergillus, and Pneumocystis are the most common fungal genera causing invasive human infections.
  • Candida species in particular, cause some of the most prevalent fungal infections and is the leading fungal pathogen worldwide.
  • Candida species are early colonizers acquired at or near human birth primarily by physical contact.
  • Candida infections can be superficial or invasive. Invasive candidiasis are difficult to treat. Globally, an estimated 700,000 persons a year suffer from invasive candidiasis, with an associated mortality that may exceed 50%. Furthermore, Candida can also cause mucocutaneous infections, such as vulvovaginal candidiasis which, while rarely lethal, are associated with significant morbidity.
  • Candida albicans is the most studied member of the genus and is the most common opportunistic pathogen and cause of invasive fungal infection in hospitalized human patients (Sobel, 2007; Pfuller , 2011).
  • C. albicans is a highly adaptable fungal species that is prevalent in nosocomial infections, and immunocompromised individuals are particularly at risk.
  • C. albicans has a large repertoire of virulence factors that allows its transition from commensal organism (yeast form) to pathogen (hyphal form). For example, C. albicans is a commensal in the vaginal epithelium. Certain environmental conditions trigger the morphogernesis of C.
  • VVC vulvovaginal candidiasis
  • albicans shows that the inner cell wall is composed mainly of ⁇ -glucans ( ⁇ -1,3 and ⁇ -1,6 linked polymers of glucose).
  • the outer cell wall is comprised of highly glycosylated cell wall proteins that are decorated with N- and O-linked terminal mannnans (branched polymer of mannose linked via ⁇ -1,2, ⁇ -1,3, ⁇ -1,4, ⁇ -1,6 and ⁇ -1,2 glycosidic bonds) (Ahmadipour et al., 2021, The Cell Surface, 7:100063). Consequently, these polysaccharide components of the fungal cell wall, ⁇ -glucans and mannans, could be interesting candidates for developing safe and efficacious subunit fungal vaccines.
  • N- and O-linked terminal mannnans branched polymer of mannose linked via ⁇ -1,2, ⁇ -1,3, ⁇ -1,4, ⁇ -1,6 and ⁇ -1,2 glycosidic bonds
  • polysaccharides are T-independent antigens that elicit antibody production via B lymphocytes without involvement of T-cells.
  • Polysaccharides may elicit a long-lasting T-cell-dependent immune response in humans if they are coupled to a protein carrier that contains T-cell epitopes.
  • conjugation of T-independent antigens to carrier proteins has been established as a way of enabling T-cell 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 boost stability of the response.
  • the present invention provides, for the first time, a new class of eukaryotic Candida glycoconjugate vaccines produced using the process of bioconjugation in a prokaryotic bacteria, namely Escherichia coli.
  • a glycoconjugate is a hybrid molecule composed of a carrier protein and multiple polysaccharide chains, wherein the polysaccharides are covalently linked to the carrier protein. This linkage of the antigenic polysaccharide to a carrier protein has brought significant advances in the field of vaccinology, eliciting a T-cell-dependent response characterized by the induction of immunological memory and improved immunogenicity.
  • the standard approach to production of glycoconjugate vaccines is a chemical conjugation process that necessitates long development timelines, as the process requires extensive optimization for each individual target antigen. Additionally, the complexity of the production process results in high costs for such products.
  • bioconjugation is an innovative technology that allows the production of glycoconjugate vaccines in a biological environment (e.g., E. coli) to preserve native immunogenic structures.
  • a biological environment e.g., E. coli
  • the E. coli glycan biosynthesis machinery is genetically modified to produce the target polysaccharide antigen and covalently link it to an asparagine residue in a consensus sequence on a carrier protein.
  • the glycoconjugate vaccine is produced entirely in E. coli in a single-step process, resulting in advantages for process reproducibility and robustness, while decreasing manufacturing cost.
  • glycoconjugate vaccines against prokaryotic organisms such as Gram negative bacteria (e.g.
  • the present invention provides modified Agglutinin-like sequence 3 (Als3) proteins from C. albicans comprising at least one consensus sequence for glycosylation (e.g.
  • D/E-X-N-Z-S/T for use in conjugation to an antigen (e.g. Candida polysaccharide).
  • an antigen e.g. Candida polysaccharide.
  • the present invention further provides a glycoconjugate comprising the modified Als3 carrier protein linked to the Candida polysaccharide antigen at one or more asparagine residues on the modified Als3 protein, as well as methods of producing the Candida glycoconjugate vaccine in a host cell (e.g., E. coli).
  • the present invention further provides methods of preventing and/or treating RVVC using the Candida glycoconjugate vaccine.
  • a modified Agglutinin-like sequence 3 (Als3) protein comprising amino acid residues 18-316 of amino acid residues 18-316 of SEQ ID NO: 1 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1, modified in that the amino acid sequence comprises one or more consensus sequences comprising an amino acid sequence of D/E-X-N-Z-S/T, wherein X and Z are independently any amino acid except proline.
  • modified Als3 protein of the invention wherein the modified Als3 protein further comprises at least one Fructose biphosphate aldolase-1 (Fba) peptide comprising an amino acid sequence of YGKDVKDLFDYAQE (SEQ ID NO: 3) or an amino acid sequence at least 70%, 80%, 85%, 90%, or 92% identical to SEQ ID NO: 3.
  • Fba Fructose biphosphate aldolase-1
  • SEQ ID NO: 3 amino acid sequence of YGKDVKDLFDYAQE
  • modified Als3 protein of the invention comprising an amino acid sequence of SEQ ID NO: 10.
  • modified Als3 protein of the invention comprising an amino acid sequence of SEQ ID NO: 11.
  • a conjugate comprising a modified Als3 protein of the invention and at least one saccharide antigen.
  • a modified Als3 protein of Candida albicans consisting of: (1) an amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11; and (2) at least one saccharide antigen of Candida, wherein the at least one saccharide antigen is a ⁇ -1,3 glucan polymer consisting of at least six consecutive ⁇ -1,3 linked glucose molecules, and wherein the at least one saccharide antigen is linked to at least one of three asparagine residues at positions 20, 92, and 324 of SEQ ID NO: 10 or positions 20, 92, and 337 of SEQ ID NO: 11.
  • a polynucleotide encoding a modified Als3 protein of the invention there is provided a vector comprising a polynucleotide encoding a modified Als3 protein of the invention.
  • a host cell comprising: (1) one or more polynucleotide sequences that encode one or more heterologous glycosyltransferases; (2) a polynucleotide sequence that encodes a heterologous oligosaccharyl transferase; (3) a polynucleotide sequence that encodes a modified Als3 protein of the invention; and, optionally, (4) a polynucleotide sequence that encodes a polymerase.
  • a method for producing a bioconjugate that comprises (or consists of) a modified Als3 protein linked to at least one saccharide antigen, the method comprising: (1) culturing a host cell of the invention under conditions suitable for the production of proteins; and (2) isolating the bioconjugate produced by said host cell, optionally isolating the bioconjugate from a periplasmic extract from the host cell.
  • an immunogenic composition comprising a modified Als3 protein of the invention, a conjugate of the invention, or a bioconjugate of the invention and optionally a pharmaceutically acceptable excipient and/or carrier.
  • a method of making the immunogenic composition of the invention comprising the step of mixing a modified Als3 protein of the invention, a conjugate of the invention, or a bioconjugate of the invention, with a pharmaceutically acceptable excipient or carrier.
  • a vaccine comprising an immunogenic composition of the invention and, optionally, a pharmaceutically acceptable excipient or carrier and optionally an adjuvant.
  • a Candida albicans vaccine comprising: (1) a modified Als3 protein of the invention; (2) at least one Candida albicans saccharide antigen linked to said modified Als3 protein; and, optionally, (3) a pharmaceutically acceptable carrier or adjuvant.
  • a method for treatment or prevention of Candida albicans infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention.
  • a method for immunizing a subject against Candida albicans infection comprising administering to the subject an immunoprotective dose of a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of any of the invention, an immunogenic composition of the invention, or a vaccine of the invention.
  • a method of inducing immune response to Candida albicans infection in a subject comprising administering to the subject a therapeutically or prophylactically effective amount of a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention.
  • a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention for use in treatment or prevention of a disease caused by Candida albicans infection.
  • a modified Als3 protein of the invention a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention for use in the manufacture of a medicament for the treatment or prevention of a disease caused by Candida albicans infection.
  • a method for increasing expression level of a modified Als3 protein of the invention comprising substituting the one or more consensus sequences for the amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1, wherein the modified Als3 protein exhibits an increased expression level relative to a control Als3 protein which does not comprise one or more consensus sequences substituted for amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a host cell comprising: i. a nucleotide sequence encoding one or more heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,3 glucan polymer; ii. A nucleotide sequence encoding a glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetyl glucosamine (GlcNac) molecule; iii. a nucleotide sequence encoding a heterologous oligosaccharyl transferase; and iv.
  • a method of producing a glycoconjugate comprising a modified carrier protein and a ⁇ -1,3 glucan wherein said method comprises culturing the host cell of the invention under conditions suitable for the production of proteins.
  • a glucan having the structure: wherein n is 2-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.
  • a saccharide which is a glucan having the structure: [ ⁇ 3)- ⁇ -D-Glcp-(1 ⁇ ] n ⁇ 3)- ⁇ -D-Glcp-(1 ⁇ 6)- ⁇ -D-Glcp-(1 ⁇ 6)- ⁇ -D-Glcp-(1 ⁇ 4)- ⁇ -D-Glcp- (1 ⁇ 4)- ⁇ -D-Glcp-(1 ⁇ 3)- x-D-GlcpNAc wherein n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.
  • a conjugate e.g.
  • a host cell comprising a nucleotide sequence comprising (i) a wzm gene comprising a nucleotide sequence of SEQ ID NO: 36, optionally comprising a nucleotide sequence at least 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 36; and (ii) a wzt gene comprising a nucleotide sequence of SEQ ID NO: 37, optionally comprising a nucleotide sequence at least 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 37.
  • a method of producing a ⁇ - 1,3 glucan polymer in a prokaryotic host cell comprising the steps of introducing and expressing in the host cell: i. a nucleotide sequence encoding a first glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetyl glucosamine (GlcNAc) molecule, wherein the first glycosyltransferase is WfaP from E. coli O56; ii.
  • nucleotide sequence encoding additional glycosyltransferases capable of synthesizing a fungal ⁇ -1, 3 glucan, wherein the additional glycosyltransferases comprise SleC, SleE, SleF, SleU and SleW from rhizobia, optionally from Agrobacterium, optionally from Agrobacterium sp. ZX09, and wherein the host cell produces more SleW than SleC, SleE, SleF or SleU; and iii.
  • a nucleotide sequence encoding a translocase capable of translocating the ⁇ -1, 3 glucan to periplasmic side of an inner membrane of the prokaryotic host cell wherein the translocase comprises Wzm-Wzt from Klebsiella sp., optionally from Klebsiella pneumoniae, wherein the ⁇ -1,3 glucan polymer is linked to a lipid carrier via the GlcNAc and wherein the ⁇ -1,3 glucan polymer comprises at least four ⁇ -1,3 linked glucose molecules.
  • a prokaryotic host cell of the invention that produces a ⁇ -1,3 glucan polymer; and b. further introducing and expressing in the host cell: i. a nucleotide sequence encoding a modified carrier protein comprising a glycosylation site comprising a consensus sequence D/E-X-N-Z-S/T, wherein X and Z are any amino acid except proline, and wherein the modified carrier protein further comprises an N-terminal bacterial signal sequence capable of transporting the modified carrier protein to the periplasmic side of the inner membrane of the prokaryotic host cell; and ii.
  • FIG. 1 shows the structure of Als3-NT protein from C. albicans of SEQ ID NO: 27 (comprising residues 18-316 of SEQ ID NO: 1). Spheres indicate the position of insertion of glycosites.
  • FIG. 2 shows the biosynthesis scheme for modified Als3-NT protein-glucan bioconjugate in E. coli.
  • FIG. 3 shows glycosylation tests with a series of modified Als3-NT proteins, each comprising a single glycosite.
  • FIG. 4 shows the expression levels and glycosylation levels of various modified Als3-NT proteins compared to the wild type Als3-NT protein.
  • FIG. 4A shows the relative expression level of modified Als3-NT proteins compared to the wild type Als3-NT protein.
  • FIG. 4B shows the glycosylation efficiency of modified Als3-NT proteins.
  • FIG. 5 shows SDS-PAGE and Western Blot analyses of purified modified Als3-NTprotein-glucan conjugates.
  • FIG. 5A SDS-PAGE analysis.
  • FIG. 5A SDS-PAGE analysis.
  • FIG. 5B Western Blot analysis with anti-Als3 antibody.
  • FIG. 5C Western Blot analysis with anti-Fba antibody.
  • FIG. 5D Western Blot analysis with anti- Glucan antibody.
  • FIG. 5E Western Blot analysis with anti-Dectin antibody.
  • FIG. 6 shows Surface Plasma Resonance (SPR) assays for testing binding of wt Als3-NT protein (“Als 318-316 wt;” left panel), engineered unglycosylated modified Als3-NT protein (“uAls 318-316 -3S;” middle panel), and glycosylated modified Als3-NT protein (“ ⁇ -glucan-Als 318-316 -3S;” right panel) to their natural ligand, fibronectin.
  • SPR Surface Plasma Resonance
  • FIG. 7 shows the preclinical testing of a modified Als3-NT protein-glucan bioconjugate (Als3-NT-3S- Fba_bgluc d+ ; Als3-3FG) in rabbit.
  • FIG. 7A shows the bioconjugate attributes.
  • FIG. 7B shows a 3D representation of the modified Als3-NT protein-glucan bioconjugate.
  • Fig. 7C shows a rabbit immunization scheme with a purified modified Als3-NT protein-glucan bioconjugate.
  • FIG.8 shows the immunogenicity of the modified Als3-NT protein-glucan bioconjugate in rabbit.
  • FIG. 8A shows that the modified Als3-NT protein-glucan bioconjugate is immunogenic.
  • FIG. 8A shows that the modified Als3-NT protein-glucan bioconjugate is immunogenic.
  • FIG. 8A shows that the Fba peptide (which is a part of the Als3-NT bioconjugate) is immunogenic.
  • FIG. 9 shows the immunogenicity of the modified Als3-NT protein-glucan bioconjugate in rabbit.
  • FIG. 10 shows the capacity of antibodies against modified Als3-NT protein-glucan bioconjugate to inhibit adhesion of C. albicans hyphae to plastic.
  • FIG. 11 shows the capacity of antibodies against modified Als3-NT protein-glucan bioconjugate to inhibit adhesion of C. albicans to vaginal epithelial cells.
  • FIG. 11A shows the results of adhesion quantification.
  • FIG. 11B shows a microscopy image of Candida adhered to epithelial cells.
  • FIG. 12 shows the capacity of antibodies against modified Als3-NT protein-glucan bioconjugate to bind to C. albicans hyphae using whole cell ELISA.
  • FIG. 13 shows a microscopy image of antibodies (against modified Als3-NT protein-glucan bioconjugate) bound to C. albicans cells.
  • FIG. 14 shows a microscopy image of antibodies (against modified Als3-NT protein-glucan bioconjugate) bound to C. auris VPCI479/P/13 cells.
  • FIG. 15 shows the capacity of antibodies against modified Als3-NT protein-glucan bioconjugate to inhibit biofilm formation of C. albicans hyphae on 96-well plates.
  • FIG. 13 shows a microscopy image of antibodies (against modified Als3-NT protein-glucan bioconjugate) bound to C. albicans cells.
  • FIG. 14 shows a microscopy image of antibodies (against modified Als3-NT protein-
  • Als3 protein or Als3 refers to a wild type Agglutinin-like sequence 3 protein comprising a wild type leader sequence (amino acid residues 1-17) at its N-terminus.
  • the Als3 protein is from Candida, optionally from Candida albicans.
  • the Als3 protein comprises the amino acid sequence of SEQ ID NO.: 1.
  • the term “Als3-NT protein or Als3-NT” refers to an N-terminal fragment of an Agglutinin-like sequence 3 (Als3) protein.
  • the Als3-NT protein is from Candida, optionally from Candida albicans.
  • the Als3-NT protein comprises amino acid residues 18-316 of SEQ ID NO.: 1.
  • the Als3-NT protein comprises amino acid residues 18-329 of SEQ ID NO.: 1.
  • the Als3-NT protein comprises amino acid residues 1-316 of SEQ ID NO.: 1.
  • the Als3-NT protein comprises amino acid residues 1-329 of SEQ ID NO.: 1.
  • the Als3-NT protein comprises an amino acid sequence selected from, but not limited to, the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41.
  • modified protein refers to a protein that is altered (in one or more way) as compared to wild type protein (e.g. a “modified Als3 protein” excludes a wild type Als3 protein).
  • a modified Als3 protein refers to an Als3 protein that comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acid residues of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1.
  • a modified Als3 protein refers to an Als3 protein that comprises a C-terminal or N-terminal deletion 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.
  • a modified Als3 protein refers to an Als3 protein that comprises additions/deletions/substitutions of one or more amino acids 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.
  • a modified Als3 protein refers to an Als3 protein that comprises one or more consensus sequences (e.g.
  • a modified Als3 protein refers to an Als3 protein that comprises one or more consensus sequences (e.g.
  • a modified Als3 protein refers to an Als3 protein that comprises one or more consensus sequences (e.g.
  • a modified Als3 protein refers to an Als3 protein that comprises one or more consensus sequences (e.g.
  • a modified Als3 protein refers to an Als3 protein that comprises one or more consensus sequences (e.g.
  • a modified Als3 protein of the invention is an isolated modified Als3 protein. In other embodiments, a modified Als3 protein of the invention is a recombinant modified Als3 protein.
  • a modified Als3 protein of the invention is an isolated recombinant modified Als3 protein.
  • modified Als3-NT protein or modified Als3-NT refers to a Als3-NT protein into which one or more glycosite sequences (e.g. D/E-X-N-Z-S/T) have been introduced.
  • the modified Als3-NT protein is from Candida, optionally from Candida albicans.
  • the modified Als3-NT protein comprises an amino acid sequence of SEQ ID NO: 10.
  • the modified Als3-NT protein comprises an amino acid sequence of SEQ ID NO: 11.
  • the modified Als3-NT protein comprises an amino acid sequence selected from, but not limited to, the amino acid sequences of Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7, Mut8, Mut9, Mut10, Mut11, Mut12, Mut13, Mut14, Mut15, Mut16, Mut17, and Mut18.
  • control Als3 protein means, without limitation,: (1) Als3 protein that does not comprise one or more consensus sequences inserted next to or substituted for one or more amino acids of SEQ ID NO: 1 (or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1) or added to the N- terminal and/or C-terminal end 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); (2) an Als3 protein that does not comprise one or more consensus sequences inserted next to or substituted for one or more amino acids of amino acid residues 18-316 of SEQ ID NO: 1 (or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO:
  • a control Als3 protein includes, without limitation, a wild type Als3 protein, a wild type Als3 protein of SEQ ID NO: 1, an Als3 protein comprising amino acid residues 1-316 of SEQ ID NO: 1, an Als3 protein comprising amino acid residues 18-316 of SEQ ID NO: 1, an Als3 protein comprising amino acid residues 1-329 of SEQ ID NO: 1, an Als3 protein comprising amino acid residues 18-329 of SEQ ID NO: 1, and a modified Als3 protein of the invention that does not comprise one or more consensus sequences substituted for amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • carrier protein refers to a protein which may be linked to an antigen (e.g. saccharide antigen, such as a fungal polysaccharide antigen) to create a conjugate (e.g. bioconjugate).
  • an antigen e.g. saccharide antigen, such as a fungal polysaccharide antigen
  • a carrier protein activates T-cell mediated immunity in relation to the antigen to which it is conjugated.
  • carrier protein refers to a protein that comprises one or more consensus sequences to which a saccharide antigen of the invention is linked.
  • a carrier protein is a modified Als3 protein of the invention.
  • 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 (gln, 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).
  • alanine ala, A
  • arginine arg, R
  • asparagine asparagine
  • aspartic acid aspartic acid
  • cysteine
  • 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 (gln, 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, they catalyze 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 sulfur-based.
  • GTFs, Gtfs activated nucleotide sugar
  • nucleophilic glycosyl acceptor molecule the nucleophile of which can be oxygen- carbon-, nitrogen-, or sulfur-based.
  • oligosaccharyl transferases refers to enzymes that catalyze a mechanistically unique and selective transfer of an oligo- or polysaccharide (glycosylation) to the asparagine (N) residue at the consensus sequence of nascent or folded proteins.
  • OSTs transfer of a 14-sugar oligosaccharide from dolichol to nascent protein.
  • OST is a type of glycosyltransferase. The reaction catalyzed by OST is the central step in the N-linked glycosylation pathway.
  • OST is a component of the translocon in the endoplasmic reticulum (ER) membrane.
  • 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.
  • LPS“ refers to large molecules comprising a lipid and a polysaccharide joined by a covalent bond.
  • capsule polysaccharide (CP) refers to a polysaccharide found on the bacterial cell wall. Examples include 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 a 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.
  • UPP undecaprenyl-diphosphate
  • reducing end refers to the reducing end of an oligosaccharide or polysaccharide is the monosaccharide with a free anomeric carbon that is not involved in a glycosidic bond and is thus capable of converting to the open-chain form.
  • conjugate refers to carrier protein covalently linked to an antigen.
  • bioconjugate refers to conjugate between a protein (e.g. a carrier protein) 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).
  • an antigen e.g. a saccharide antigen, such as a bacterial polysaccharide antigen
  • host cell machinery links the antigen to the protein (e.g. N-linked glycosylation).
  • the polysaccharide is linked to asparagine via N- acetylglucosamine.
  • immunogenic fragment refers to a portion of an antigen smaller than the whole, that is capable of eliciting a humoral and/or cellular immune response in a host animal, e.g. human, specific for that fragment.
  • Fragments of a protein can be produced using techniques known in the art, e.g. recombinantly, by proteolytic digestion, or by chemical synthesis.
  • Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide.
  • fragments comprise at least 10, 20, 30, 40 or 50 contiguous amino acids of the full length sequence.
  • Fragments may be readily modified by adding or removing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 amino acids from either or both of the N and C termini.
  • a fragment of a modified Als3 protein of the invention still comprises the recited modifications that are made to the Als3 protein.
  • 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 may produce polypeptides having functional and chemical characteristics similar to those of a parental polypeptide.
  • the term “deletion” refers to the removal of one or more amino acid residues from the protein sequence.
  • no more than about from 1 to 6 residues are deleted at any one site within the protein molecule.
  • the terms “insertion” or “addition” refers to the addition of one or more non-native amino acid residues in the protein sequence or, as the context requires, addition of one or more non-native nucleotides in the polynucleotide sequence.
  • no more than about from 1 to 10 residues, (e.g. 1 to 7 residues, 1 to 6 residues, or 1 to 4 residues) are inserted at any one site within the protein molecule.
  • the term “added next to” refers to the addition of one or more non-native amino acid residues in the protein sequence at a position adjacent to the referenced amino acid or amino acid region.
  • “added next to one or more amino acids between amino acid residues 33-37” means the addition at a position adjacent to any one of amino acid residues 33-37 (including adjacent to amino acid residues 33 or 37).
  • the term “glycosite” refers to an amino acid sequence recognized by a bacterial oligosaccharyl transferase, e.g. PglB of Campylobacter jejuni.
  • a glycosite refers to an amino acid sequence within a carrier protein (e.g.
  • a “consensus sequence” refers to a sequence have a specific structure and/or function. As used herein, the term “consensus sequence” is a sequence comprising a glycosite.
  • a consensus sequence of the invention includes, but is not limited to, a five amino acid consensus sequence D/E- X-N-Z-S/T, a seven amino acid consensus sequence K-D/E-X-N-Z-S/T-K, and an extended consensus sequence (e.g.
  • the term “introduced at” is used herein to reference the location and manner of inserting a consensus sequence into an amino acid sequence.
  • a consensus sequence (or glycosite) which is introduced at an N-terminal or C-terminal position of a protein may be added next to the amino acid sequence at the N-terminus or C-terminus, whereas a consensus sequence (or glycosite) which is introduced at a specific amino acid residue within the protein (e.g. amino acid residue 18 of SEQ ID NO: 1), may be substituted for that amino acid.
  • a numeric range e.g.
  • “33-37”) is inclusive of endpoints (i.e. includes the values 33 and 37).
  • “between amino acids 18 to 316 of SEQ ID NO: 1” refers to position in the amino acid sequence between amino acid 18 and amino acid 316 of SEQ ID NO: 1 including both amino acids 18 and 316.
  • the term “identical” or percent “identity” refers to nucleotide sequences or amino acid sequences that are the same or have a specified percentage of nucleotide residues or amino acid residues that are the same (e.g.
  • Identity between polypeptides may be calculated by various algorithms. In general, when calculating percentage identity the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. For example 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.
  • 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 refers to a protein, conjugate (e.g.
  • bioconjugate polynucleotide, or vector in a form not found in nature.
  • 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).
  • subject refers to an animal, in particular a mammal such as a primate (e.g. human).
  • the term “therapeutically or prophylactically effective amount,” in the context of administering a therapy e.g.
  • an immunogenic composition or a vaccine of the invention) to a subject refers to the amount of a therapy which has a prophylactic and/or therapeutic effect(s).
  • an “therapeutically or prophylactically 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 fungal infection or symptom associated therewith; (ii) reduce the duration of a fungal infection or symptom associated therewith; (iii) prevent the progression of a fungal infection or symptom associated therewith; (iv) cause regression of a fungal infection or symptom associated therewith; (v) prevent the development or onset of a fungal infection, or symptom associated therewith; (vi) prevent the recurrence of a fungal infection or symptom associated therewith; (vii) reduce organ failure associated with a fungal infection; (viii) reduce hospitalization of a subject having a fungal infection; (ix) reduce hospitalization length of
  • the term “immunoprotective dose,” in the context of administering a therapy (e.g. an immunogenic composition or a vaccine of the invention) to a subject refers to the amount of a therapy which has a prophylactic and/or therapeutic effect(s).
  • an “immunoprotective dose” 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 fungal infection or symptom associated therewith; (ii) reduce the duration of a fungal infection or symptom associated therewith; (iii) prevent the progression of a fungal infection or symptom associated therewith; (iv) cause regression of a fungal infection or symptom associated therewith; (v) prevent the development or onset of a fungal infection, or symptom associated therewith; (vi) prevent the recurrence of a fungal infection or symptom associated therewith; (vii) reduce organ failure associated with a fungal infection; (viii) reduce hospitalization of a subject having a fungal infection; (ix) reduce hospitalization length of a subject having a fungal infection; (x) increase the survival of a subject with a fungal infection; (xi) eliminate a fungal infection in a subject; and
  • conjugate vaccine refers to a vaccine created by covalently linking a polysaccharide antigen to a carrier protein. Conjugate vaccine elicit immune response against a pathogen (e.g. a fungus) and immunological memory.
  • glycoconjugate vaccine refers to a vaccine comprising a protein carrier linked to an antigenic or immunogenic oligosaccharide.
  • undecaprenyl or “und” refers to undecaprenol lipid composed of eleven prenol units.
  • Und-P refers to undecaprenyl phosphate, which is a universal lipid carrier (derived from Und) of glycan biosnyhetic intermediates for carbohydrate polymers.
  • Und-PP refers to undecaprenyl pyrophosphate, which is a phosdphorylated version of Und-P.
  • Periodic space or “periplasm” refers to the space between the inner cytoplasmic membrane and external outer membrane of a host cell (e.g. Gram-negative bacteria, e.g. E. coli). The terms “of” and “from” are used herein interchangeably.
  • a saccharide antigen “from” Candida means, without limitation, (1) a saccharide antigen obtained from Candida or (2) a saccharide antigen of Candida, i.e., a saccharide antigen comprising a structure similar to a saccharide antigen from Candida, but, e.g., produced recombinantly in a host cell (e.g. a bacterial cell).
  • sleC refers to a glycosyltransferase.
  • sleC is a glycosyltransferase of Agrobacterium sp. ZX09.
  • sleC is a wild type glycosyltransferase.
  • sleC is a non-naturally occurring (e.g., mutant and/or recombinant) glycosyltransferase.
  • sleE refers to a glycosyltransferase.
  • sleE is a glycosyltransferase of Agrobacterium sp. ZX09.
  • sleE is a wild type glycosyltransferase.
  • sleE is a non-naturally occurring (e.g., mutant and/or recombinant) glycosyltransferase.
  • sleF refers to a glycosyltransferase.
  • sleF is a glycosyltransferase of Agrobacterium sp. ZX09. In some aspects, sleF is a wild type glycosyltransferase. In other aspects, sleF is a non-naturally occurring (e.g., mutant and/or recombinant) glycosyltransferase.
  • sleU refers to a glycosyltransferase. In certain embodiments, sleU is a glycosyltransferase of Agrobacterium sp. ZX09. In some aspects, sleU is a wild type glycosyltransferase.
  • sleU is a non-naturally occurring (e.g., mutant and/or recombinant) glycosyltransferase.
  • sleW refers to a glycosyltransferase.
  • sleW is a glycosyltransferase of Agrobacterium sp. ZX09.
  • sleW is a wild type glycosyltransferase.
  • sleW is a non-naturally occurring (e.g., mutant and/or recombinant) glycosyltransferase.
  • WfaP refers to a glycosyltransferase capable of covalently bonding a glucose to GlcNac.
  • WfaP is a glycosyltransferase of E. coli 056.
  • WfaP is a wild type glycosyltransferase.
  • wfaP is a non-naturally occurring (e.g., mutant and/or recombinant) glycosyltransferase.
  • Wzm-Wzt refers to a translocase.
  • Wzm-Wzt is a translocase of Klebsiella pneumoniae.
  • Wzm-Wzt is a wild type translocase. In other aspects, Wzm- Wzt is a non-naturally occurring (e.g., mutant and/or recombinant) translocase.
  • PglB refers to a oligosaccharyl transferase. In certain embodiments, pglB is a oligosaccharyl transferase obtained from an organism including, but not limited to, Campylobacter jejuni, Campylobacter coli, or Sinorhizobium meliloti 1021. In certain embodiments, pglB is a oligosaccharyl transferase of Campylobacter coli.
  • pglB is a wild type oligosaccharyl transferase. In other aspects, PglB is a non-naturally occurring oligosaccharyl transferase. In specific embodiments, the pglB of the invention compirises an amino acid sequence of SEQ ID NO: 20. In additional asects, the pglB protein is an evolved pglB, i.e., an evolved oligosacharyl transferase. By “evolved” is meant a protein or nucleic acid that has undergone directed evolution. Directed evolution is a method used is protein engineering that mimics the process of natural selection to steer proteins or nucleic acids toward a user-defined goal.
  • the process of directed evolution consists of subjecting a gene to iterative rounds of mutagenesis (creating a library of variants), selection (expressing those variants and isolating members with the desired function) and amplification (generating a template for the next round). It can be performed in vivo (in living organisms), or in vitro (in cells or free in solution). Directed evolution is used both for protein engineering as an alternative to rationally designing modified proteins, as well as for experimental evolution studies of fundamental evolutionary principles in a controlled, laboratory environment.
  • the pglB contains one or more mutations which enhances the activity of pglB for the saccharide antigen of the invention.
  • the pglB of the invention is an evolved pglB that transfers a saccharide antigen of the invention to a modified Als3 protein of the invention more efficiently in comparison to a wild-type pglB (e.g., a wild type pglB obtained from Campylobacter jejuni).
  • Als3 Protein Agglutinin-like sequence 3 protein of Candida albicans also known as “Als3” is a C. albicans hypha-specific cell surface protein that is a multifunctional adhesin and invasin, which enables C.
  • Als3 is a member of the agglutininlike sequence (Als) family of proteins and is encoded by the ALS3 gene.
  • SP signal peptide
  • NT 300-amino-acid immunoglobulin- like domain
  • T 104-amino-acid threonine-rich domain that contains ⁇ -sheets
  • the N-terminal domain of Als3 protein (“Als3-NT”), which contains a peptide binding cavity, is required for its adhesive function (Lin J et al., 2014, J. Biol. Chem., 280(26):18401-18412). Antibodies obtained using Als3-NT domain for immunization block adherence of Candida to host endothelial and epithelial cells (Coleman, DA et al., 2009, J. Mol. Meth.).
  • the central domain of the Als3 protein is composed of a variable number of 36-amino-acid tandem repeats (“TR”) (Fig. 1A). These repeats are rich in serine and threonine, exposed on the cell surface, and required for adherence function.
  • the tandem repeats can directly mediate adherence to some substrates, such as polystyrene.
  • C terminus (“CT”) of Als3 protein is serine and threonine rich and predicted to be heavily glycosylated. It contains a glycosylphosphatidylinositol anchorage sequence that is cleaved when the protein is covalently linked to the cell wall. Functioning as an adhesin, Als3 mediates C. albicans invasion by attachment of the organism to epithelial cells, endothelial cells, and extracellular matrix proteins. It also plays an important role in biofilm formation on prosthetic surfaces, both alone and in mixed infection with Streptococcus gordonii.
  • Als3 is one of two known C. albicans invasins. It binds to host cell receptors such as E- cadherin and N-cadherin and thereby induces host cells to endocytose the organism. Als3 also binds to host cell ferritin and enables C. albicans to utilize this protein as a source of iron. Als3 is produced in C. albicans in a precursor from which a leader sequence of 17 amino acids (“signal peptide”) is removed during the maturation process (Liu Y and Filler SG, 2011, Eukaryot Cell, 10(2):168-173).
  • signal peptide a leader sequence of 17 amino acids
  • an Als3 protein useful in the invention can be produced by methods known in the art in view of the present disclosure, see for example (Gong J et al., 2019, Antimicrob Agents Chemother, 64(1):e01975-79).
  • a full-length wild-type Als3 protein of C. Albicans comprises the amino acid sequence of SEQ ID NO: 1: SEQ ID NO: 1: full-length wild-type Als3 protein sequence from C.
  • modified Als3 protein refers to a Als3 protein comprising an amino acid sequence (for example, having a amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1), which Als3 amino acid sequence has been modified by the addition, substitution or deletion of one or more amino acids (for example, by addition of a consensus sequence(s) selected from D/E-X-N-Z-S/T, K-D/E-X-N-Z-S/T and/ or an extended consensus sequence (e.g.
  • a modified Als3 protein may be an Als3 amino acid sequence of SEQ ID NO: 1 which has been modified in that the amino acid sequence comprises one or more consensus sequences selected from from D/E-X-N-Z-S/T, K-D/E-X-N-Z-S/T and/or an extended consensus sequence (e.g. J-U-B-D/E- X-N-Z-S/T-J-U-B).
  • X and Z are independently any amino acid except proline; preferably, X is Q (glutamine) and Z is A (alanine).
  • a modified Als3 protein of the invention may comprise further modifications (e.g., additions, substitutions, and/or deletions of one or more amino acid residues).
  • a modified Als3 protein of the invention comprises a C-terminal deletion of 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 amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises amino acid residues 1-316 of SEQ ID NO.: 1 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises amino acid residues 1-329 of SEQ ID NO.: 1 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises amino acid residues 18-329 of SEQ ID NO.: 1 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises amino acid residues 18-316 of SEQ ID NO.: 1 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • the modified Als3 protein of the invention is a non-naturally occurring Als3 protein (i.e. not native).
  • a modified Als3 protein of the invention may have an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. In some aspects, a modified Als3 protein of the invention may have an amino acid sequence at least 80% identical to SEQ ID NO: 1. In other aspects, a modified Als3 protein of the invention may have an amino acid sequence at least 85% identical to SEQ ID NO: 1. In yet other aspects, a modified Als3 protein of the invention may have an amino acid sequence at least 90% identical to SEQ ID NO: 1. In still other aspects, a modified Als3 protein of the invention may have an amino acid sequence at least 91% identical to SEQ ID NO: 1.
  • a modified Als3 protein of the invention may have an amino acid sequence at least 92% identical to SEQ ID NO: 1. In other aspects, a modified Als3 protein of the invention may have an amino acid sequence at least 93% identical to SEQ ID NO: 1. In still other aspects, a modified Als3 protein of the invention may have an amino acid sequence at least 94% identical to SEQ ID NO: 1. In yet other aspects, a modified Als3 protein of the invention may have an amino acid sequence at least 95% identical to SEQ ID NO: 1. In certain aspects, a modified Als3 protein of the invention may have an amino acid sequence at least 96% identical to SEQ ID NO: 1. In other aspects, a modified Als3 protein of the invention may have an amino acid sequence at least 97% identical to SEQ ID NO: 1.
  • a modified Als3 protein of the invention may have an amino acid sequence at least 98% identical to SEQ ID NO: 1. In yet other aspects, a modified Als3 protein of the invention may have an amino acid sequence at least 99% identical to SEQ ID NO: 1. In certain aspects, a modified Als3 protein of the invention comprises one or more consensus sequences. The terms “glycosite sequence”, “consensus glycosite sequence” and “consensus sequence” are used herein interchangeably. In certain aspects, a modified Als3 protein of the invention comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten consensus sequences.
  • a modified Als3 protein of the invention contains one, two, three, four, five, six, seven, eight, nine, or ten consensus sequences. In specific aspects, a modified Als3 protein of the invention contains at least three consensus sequences. In preferred aspects, a modified Als3 protein of the invention contains three consensus sequences. In certain embodiments, a modified Als3 protein of the invention comprises one or more consensus sequences, wherein all of the consensus sequences have identical amino acid sequences. In other embodiments, a modified Als3 protein of the invention comprises one or more consensus sequences, wherein all of the consensus sequences have different amino acid sequences.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein at least two of the consensus sequences have identical amino acid sequences.
  • the present invention provides a modified Als3 protein comprising amino acid residues 18-316 of SEQ ID NO: 1 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1, modified in that the amino acid sequence comprises one or more consensus sequences comprising an amino acid sequence of D/E-X-N-Z-S/T, wherein X and Z are independently any amino acid except proline.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have each been added next to or substituted for one or more amino acids, selected from specific amino acid residues within a modified Als3 protein of the invention (consensus sequence sites).
  • These one or more consensus sequence sites are independently selected from (1) one or more amino acids between amino acid residues 18- 23 (e.g. amino acid residue 18), (2) one or more amino acids between amino acid residues 311-316 (e.g. amino acid residue 316), (3) one or more amino acids between amino acid residues 28-42 (e.g. one or more amino acids between amino acid residues 33-37), (4) one or more amino acids between amino acid residues 75-87 (e.g.
  • one or more amino acids between amino acid residues 80-82 (5) one or more amino acids between amino acid residues 82-92 (e.g. amino acid residue 87), (6) one or more amino acids between amino acid residues 99-113 (e.g. one or more amino acids between amino acid residues 104-108), (7) one or more amino acids between amino acid residues 114-126 (e.g. one or more amino acids between amino acid residues 119-121), (8) one or more amino acids between amino acid residues 118-132 (e.g. one or more amino acids between amino acid residues 123-127), (9) one or more amino acids between amino acid residues 150-164 (e.g.
  • amino acid residue 220 amino acid residue 220
  • amino acid residues 231-242 e.g. one or more amino acids between amino acid residues 236-237
  • amino acid residues 265-275 e.g. amino acid residue 270
  • amino acid residues 271-281 e.g. amino acid residue 276
  • amino acid residues 281-292 e.g. one or more amino acids between amino acid residues 286-287
  • amino acid residues 294-305 e.g.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids selected from the group consisting of: one or more amino acids between amino acid residues 18-23, one or more amino acids between amino acid residues 28-42, one or more amino acids between amino acid residues 75-87, one or more amino acids between amino acid residues 82-92, one or more amino acids between amino acid residues 99-113, one or more amino acids between amino acid residues 114-126, one or more amino acids between amino acid residues 118-132, one or more amino acids between amino acid residues 150-164, one or more amino acids between amino acid residues 158-169, one or more amino acids between amino acid residues 163- 177, one or more amino acids between amino acid residues 170-184, one or more amino acids between amino acid residues 202-212, one or more amino acids between amino acid residues 215- 225, one or more amino acids between amino acid residues 231-242
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for amino acid residue 18 of amino acid residues 18-316 of SEQ ID NO: 1 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids between amino acid residues 33-37 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids between amino acid residues 80-82 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for amino acid residue 87 of amino acid residues 18-316 of SEQ ID NO: 1 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • substitution of the one or more consensus sequences for the amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1 results in an increase in expression level of the modified Als3 protein relative to a control Als3 protein.
  • the expression level of the modified Als3 protein is increased at least about 2-fold, at least about 3-fold, at least about 4- fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold relative to the control Als3 protein. In other aspects, the expression level of the modified Als3 protein is increased about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or about 10-fold relative to the control Als3 protein.
  • substitution of the one or more consensus sequences for the amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1 results in an increase in expression level of the modified Als3 protein of about 4-fold relative to a control Als3 protein.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids between amino acid residues 119-121 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids between amino acid residues 123-127 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids between amino acid residues 155-159 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids between amino acid residues 163-164 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids between amino acid residues 168-172 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids between amino acid residues 175-179 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for amino acid residue 207 of amino acid residues 18-316 of SEQ ID NO: 1 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for amino acid residue 220 of amino acid residues 18-316 of SEQ ID NO: 1 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids between amino acid residues 236-237 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for amino acid residue 270 of amino acid residues 18-316 of SEQ ID NO: 1 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for amino acid residue 276 of amino acid residues 18-316 of SEQ ID NO: 1 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids between amino acid residues 286-287 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids between amino acid residues 299-300 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been substituted for (i) the amino acids between amino acid residues 33-37 and (ii) the amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for (i) the amino acids between amino acid residues 33-37; (ii) the amino acids between amino acid residues 104-108; and (iii) amino acid residue 316 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for (i) the amino acids between amino acid residues 33-37; (ii) the amino acids between amino acid residues 104-108; and (iii) amino acid residue 329 of amino acid residues 18-329 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-329 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for (i) the amino acids between amino acid residues 33-37; (ii) the amino acids between amino acid residues 104-108; (iii) the amino acids between amino acid residues 163-164; (iv) amino acid residue 220; (v) the amino acids between amino acid residues 299-300; and (vi) amino acid residue 316 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for (i) the amino acids between amino acid residues 33-37; (ii) the amino acids between amino acid residues 104-108; (iii) the amino acids between amino acid residues 163-164; (iv) amino acid residue 220; (v) the amino acids between amino acid residues 299-300; and (vi) amino acid residue 329 of amino acid residues 18-329 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-329 of SEQ ID NO: 1.
  • the modified Als3 protein of the invention is from a fungi.
  • the fungi is Candida.
  • the modified Als3 protein of the invention is from Candida.
  • the Candida includes, but is not limited to, Candida albicans, Candida auris, Candida guilliermondi, Candida lusitaniaea and Candida tropicalis.
  • the modified Als3 protein of the invention is from Candida albicans.
  • at least one of the one or more consensus sequences comprises an amino acid sequence of K-D/E-X-N-Z-S/T, wherein X and Z are independently any amino acid except proline.
  • X is Q (glutamine).
  • Z is A (alanine).
  • the one or more consensus sequences include, but are not limited to, KDQNAT (SEQ ID NO: 5), KDQNAS (SEQ ID NO: 6) and DQNAT (SEQ ID NO: 7).
  • X is Q (glutamine)
  • Z is A (alanine)
  • the one or more consensus sequences are selected from the group consisting of KDQNAT (SEQ ID NO: 5), KDQNAS (SEQ ID NO: 6) and DQNAT (SEQ ID NO: 7).
  • a modified Als3 protein of the invention further comprises at least one Fructose biphosphate aldolase (Fba) peptide.
  • Fba peptide is a 14-mer peptide derived from the N-terminal portion of fructose-bisphosphate aldolase protein (Fba-1).
  • Fba-1 protein is a multifunctional C. albicans cell wall protein and an important enzyme of glycolytic pathway. It can facilitate fungal attachment to human cells or abiotic surfaces, and protects Candida cells from the host’s immune system (Elamin E, et al., 2021, J. Immunol. Res., 2021:1-19). In addition to this, it promotes the detoxification of reactive oxygen species generated during respiratory burst.
  • Fba1 is the most abundant and stable enzyme in Candida and is considered to be one of the main immunodominant proteins (Elamin E, et al., 2021, J. Immunol. Res., 2021:1-19).
  • the Fba peptide has been previously been used to generate a self-adjuvanting vaccine (Xin H et al., 2012, PLoS ONE, 7:e35106).
  • the at least one Fba peptide comprises (or consists of) an amino acid sequence of YGKDVKDLFDYAQE (SEQ ID NO: 3).
  • the at least one Fba peptide comprises (or consists of) an amino acid sequence at least 70%, 80%, 85%, 90%, or 92% identical to SEQ ID NO: 3.
  • a modified Als3 protein of the invention comprises at least one Fba peptide.
  • the at least one Fba peptide comprises an amino acid sequence of YGKDVKDLFDYAQE (SEQ ID NO: 3) or an amino acid sequence at least 70%, 80%, 85%, 90%, or 92% identical to SEQ ID NO: 3.
  • the at least one Fba peptide is linked to a modified Als3 protein of the invention.
  • the at least one Fba peptide is non- covalently linked to a modified Als3 protein of the invention. In other embodiments, the at least one Fba peptide is covalently linked to a modified Als3 protein of the invention. In additional embodiments, the Fba peptide is linked to a modified Als3 protein of the invention at a single amino acid residue. In other embodiments, the Fba peptide is linked to a modified Als3 protein of the invention at more than one amino acid residues. In additional embodiments, the Fba peptide is linked to a modified Als3 protein of the invention at one or more amino acid residues.
  • the one or more amino acid residues include, but are not limited to, amino acid residues 89, 163, 259, 199 and 316.
  • the Fba peptide is linked to a modified Als3 protein of the invention at amino acid residue 316.
  • the numbering of the amino acid residues as specified herein refers to the amino acid positions in SEQ ID NO: 1 (or where an amino acid sequence is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1 equivalent positions to that of SEQ ID NO: 1 if this sequence was lined up with an amino acid sequence of SEQ ID NO: 1 in order to maximise the sequence identity between the two sequences).
  • the Fba peptide is covalently linked to a modified Als3 protein of the invention at one or more amino acid residues selected from the group consisting of 89, 163, 259, 199 and 316 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent position(s) within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • the Fba peptide is covalently linked to a modified Als3 protein of the invention at amino acid residue 316 of amino acid residues 18-316 of SEQ ID NO: 1 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a modified Als3 protein of the invention comprises at least one additional consensus sequence.
  • the at least one additional consensus sequence has been added next to C-terminal amino acid residue of a modified Als3 protein of the invention.
  • a modified Als3 protein of the invention comprises at least one Fba peptide and the at least one additional consensus sequence has been added next to C-terminal amino acid residue of the at least one Fba peptide.
  • a modified Als3 protein of the invention comprises an Fba peptide comprising an amino acid sequence of YGKDVKDLFDYAQE (SEQ ID NO: 3) or an amino acid sequence at least 70%, 80%, 85%, 90%, or 92% identical to SEQ ID NO: 3, and the at least one consensus sequence has been added next to C-terminal amino acid residue of SEQ ID NO: 3 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3.
  • the modified Als3 protein comprises at least one additional consensus sequence comprising an amino acid sequence of J-U-B-D/E-X-N-Z-S/T-J-U-B, wherein X and Z are independently any amino acid except proline and J, U and B independently comprise 1 to 5 naturally occurring amino acid residues, wherein the at least one additional consensus sequence has been added next to C-terminal amino acid residue of SEQ ID NO: 3 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 3.
  • the at least one additional consensus sequence comprises an amino acid sequence of J-U-B-D/E-X-N- Z-S/T-J-U-B, wherein X and Z are independently any amino acid except proline and J, U and B independently comprise one or more naturally occurring amino acid residues.
  • X is Q (glutamine).
  • Z is A (alanine).
  • J comprises at least one Glycine (G) residue.
  • J comprises 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 Glycine (G) residues.
  • J comprises 1 to 5 Glycine (G) residues.
  • B comprises at least one Glycine (G) residue.
  • B comprises 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 Glycine (G) residues.
  • B comprises 1 to 5 Glycine (G) residues.
  • each of J and B comprises 1 to 5 Glycine (G) residues.
  • U comprises at least one Glycine (G) residue.
  • U comprises 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 Glycine (G) residues.
  • U comprises 1 to 5 serine (S) residues.
  • X is Q
  • Z is A
  • each of J and B comprises 1 to 5 Glycine (G) residues
  • U comprises 1 to 5 serine (S) residues.
  • the additional consensus sequence comprises (or consists of) an amino acid sequence of GSGGGDQNATGSGGG (SEQ ID NO: 9).
  • a modified Als3 protein of the invention comprises (or consists of) an amino acid sequence of SEQ ID NO: 10: SKTITGVFNSFNSLTWKDQNATYNYKGPGTPTWNAVLGWSLDGTSASPGDTFTLNMPCVFKFTTSQ TSVDLTAHGVKYATCQFQAGEEKDQNASTLTCTVSNTLTPSIKALGTVTLPLAFNVGGTGSSVDLEDSKCFTAG TNTVTFNDGGKKISINVDFERSNVDPKGYLTDSRVIPSLNKVSTLFVAPQCANGYTSGTMGFANTYGDVQIDCS NIHVGITKGLNDWNYPVSSESFSYTKTCSSNGIFITYKNVPAGYRPFVDAYISATDVNSYTLSYANEYTCAGGYW QRAPFTLRWTGYRYGKDVKDLFDYAQEGSGGGDQNATGSGGG
  • a modified Als3 protein of the invention comprises (or consists of) an amino acid sequence of SEQ ID NO: 10: SKTIT
  • the modified Als3 protein of the invention is N-glycosylated.
  • the present inventors unexpectedly found that, when expressed in a host cell, a modified Als3 protein of the invention comprising at least one glycosite between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 (“Mut4 variant”) showed a >2-fold increase in protein expression relative to control (e.g., wild-type) Als3-NT expression.
  • the present invention provides a method for increasing expression level of a modified Als3 protein of the invention.
  • the method for increasing expression level of a modified Als3 protein of the invention comprises substituting the one or more consensus sequences for the amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1, wherein the modified Als3 protein exhibits an increased expression level relative to a control Als3 protein.
  • the present invention provides a method for increasing expression level of a modified Als3 protein of the invention in a host cell, the method comprising substituting the one or more consensus sequences for the amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1, wherein the modified Als3 protein when expressed in a host cell exhibits an increased expression level relative to a control Als3 protein which does not comprise one or more consensus sequences substituted for amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • the one or more consensus sequences may have been added next to or substituted for any one (or more) of amino acid numbers 33, 34, 35, 36, and 37 in SEQ ID NO: 1.
  • the Als3 amino acid sequence is a variant and/or fragment of an amino acid sequence of SEQ ID NO: 1, such as an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, the reference to “between amino acids ...” refers to a position that would be equivalent to the defined position, if this sequence was lined up with an amino acid sequence of SEQ ID NO: 1 in order to maximise the sequence identity between the two sequences (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).
  • the sequence alignment tool used is Clustal Omega (www(.)ebi(.)ac(.)ac(.)uk).
  • the amino acid numbers referred to herein correspond to the amino acids in SEQ ID NO: 1 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: 1 by alignment.
  • a modified Als3 protein of the invention is an isolated modified Als3 protein.
  • a modified Als3 protein of the invention is a recombinant modified Als3 protein.
  • a modified Als3 protein of the invention is an isolated recombinant modified Als3 protein.
  • a modified Als3 protein of the invention comprises a D/E-X-N-Z-S/T, K-D/E-X-N-Z-S/T or J-U-B-D/E-X-N-Z-S/T-J-U-B consensus sequence, wherein X and Z are independently any amino acid except proline and J, U and B independently comprise 1 to 5 naturally occurring amino acid residues.
  • the classical 5 amino acid glycosylation consensus sequence (D/E-X- N-Z-S/T) may be extended by 1-5 other amino acid residues either side of the consensus sequence for more efficient glycosylation J-U-B-D/E-X-N-Z-S/T-J-U-B (e.g.
  • consensus sequences in the modified Als3 protein of the invention may comprise (or consist of) a D/E- X-N-Z-S/T consensus sequence.
  • the consensus sequence(s) may be selected from: D/E-X-N-Z-S/T, K-D/E-X-N-Z-S/T (SEQ ID NO: 4) or J-U-B-D/E-X-N-Z-S/T-J-U-B, wherein X is Q (glutamine) and Z is A (alanine).
  • the consensus sequence(s) may be selected from: D/E-X-N-Z-S/T and K-D/E-X-N-Z-S/T (SEQ ID NO: 4), wherein X is Q (glutamine) and Z is A (alanine).
  • the consensus sequence is D/E-X-N-Z- S/T, wherein X is Q (glutamine) and Z is A (alanine), e.g. D-Q-N-A-T (SEQ ID NO: 7) also referred to as “DQNAT” (SEQ ID NO: 7).
  • the consensus sequence is K-D/E-X-N-Z-S/T (SEQ ID NO: 4), wherein X is Q (glutamine) and Z is A (alanine), e.g. K-D-Q-N-A-T (SEQ ID NO: 5) also referred to as “KDQNAT” (SEQ ID NO: 5).
  • the consensus sequence is K-D/E-X-N-Z-S/T (SEQ ID NO: 4), wherein X is Q (glutamine) and Z is A (alanine), e.g. K-D-Q-N-A- S (SEQ ID NO: 6) also referred to as “KDQNAS” (SEQ ID NO: 6).
  • the consensus sequence(s) may be selected from: D/E-X-N-Z-S/T, K-D/E-X-N-Z-S/T (SEQ ID NO: 4) or J-U-B-D/E-X-N-Z-S/T-J-U-B, wherein X is Q (glutamine), Z is A (alanine), J, U and B are indepedently 1 to 5 amino acid residues independently selected from glycine and/or serine.
  • the consensus sequence is J-U-B-D/E-X-N-Z-S/T-J-U-B (SEQ ID NO: 8), wherein X is Q (glutamine), Z is A (alanine), each of J and B comprises 1 to 5 Glycine (G) residues and U comprises 1 to 5 serine (S) residues, e.g. G-S-G-G-G-D-Q-N-A-T-G-S-G-G-G (SEQ ID NO: 9) also referred to as “GSGGGDQNATGSGGG (SEQ ID NO: 9)”.
  • the modified Als3 protein of the invention comprises at least two D/E-X-N-Z-S/T or K-D/E-X-N-Z-S/T consensus sequences. In other embodiment, the modified Als3 protein of the invention comprises at least three D/E-X-N-Z-S/T or K-D/E-X-N-Z-S/T consensus sequences. In yet other embodiments, the modified Als3 protein of the invention comprises at least four D/E-X-N-Z-S/T or K-D/E-X-N-Z-S/T consensus sequences.
  • the modified Als3 protein of the invention comprises at least five D/E-X-N-Z-S/T or K-D/E-X-N-Z-S/T consensus sequences. In additional embodiments, the modified Als3 protein of the invention comprises at least six D/E-X-N-Z-S/T or K-D/E-X-N-Z-S/T consensus sequences. In further embodiments, the modified Als3 protein of the invention comprises at least seven D/E-X-N-Z-S/T or K-D/E-X-N-Z-S/T consensus sequences.
  • the modified Als3 protein of the invention contains three to seven D/E-X-N-X-S/T or K-D/E-X-N-Z-S/T consensus sequences. In yet other embodiments, the modified Als3 protein of the invention contains four to seven D/E-X-N-X-S/T or K-D/E-X-N-Z-S/T consensus sequences. In additional embodiments, the modified Als3 protein of the invention contains five to seven D/E-X-N-X-S/T or K-D/E-X-N-Z-S/T consensus sequences. Introduction of such glycosylation sites can be accomplished by, e.g. adding new amino acids to the primary structure of the protein (i.e.
  • the glycosylation sites are added, in full or in part), or by mutating existing amino acids in the protein in order to generate the glycosylation sites (i.e. amino acids are not added to the protein, but selected amino acids of the protein are mutated so as to form glycosylation sites).
  • the consensus sequence(s) are recombinantly introduced into the Als3 amino acid sequence of SEQ ID NO: 1 or a Als3 amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1.
  • a modified Als3 protein of the invention may further comprise a “peptide tag” or “tag”, i.e.
  • 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.
  • HIS histidine
  • FLAG- TAG FLAG- TAG
  • HA tags hexa-histidine tags.
  • the tags used herein are removable, e.g.
  • a modified Als3 protein of the invention may further comprise a peptide tag.
  • the peptide tag is located at the C-terminus of the amino acid sequence of a modified Als3 protein of the invention.
  • the peptide tag comprises six histidine residues at the C-terminus of the amino acid sequence of a modified Als3 protein of the invention.
  • the present invention provides a modified Als3 protein comprising a tag (e.g., a histidine tag).
  • the present invention provides a modified Als3 protein not comprising a tag (e.g., a histidine tag), e.g., a modified Als3 protein with the histidine tag removed.
  • the modified Als3 protein of the invention comprises (or consists of): (i) amino acid residues 18-316 of SEQ ID NO: 1 or an amino acid sequence which is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to amino acid residues 18-316 of SEQ ID NO: 1 and (ii) a peptide tag (e.g. six histidine residues at the C-terminus of the amino acid sequence).
  • a modified Als3 protein of the invention comprises (or consists of) amino acid residues 18-316 of SEQ ID NO: 1 or an amino acid sequence which is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to amino acid residues 18-316 of SEQ ID NO: 1, with the peptide tag (e.g. histidine tag) removed.
  • a modified Als3 protein of the invention comprises a signal sequence which is capable of directing the modified Als3 protein to the periplasm of a host cell (e.g. bacterium). Signal sequences, including periplasmic signal sequences, are usually removed during translocation of the protein into, for example, the periplasm by signal peptidases (i.e.
  • a mature protein is a protein from which at least the signal sequence has been removed).
  • the signal sequence is selected from, but is not limited to, E. coli flagellin (FlgI) [MIKFLSALILLLVTTAAQA (SEQ ID NO: 21)], E. coli outer membrane porin A (OmpA) [MKKTAIAIAVALAGFATVAQA (SEQ ID NO: 22)], E. coli maltose binding protein (MalE) [MKIKTGARILALSALTTMMFSASALA (SEQ ID NO: 23)], E.
  • E. coli outer membrane porin C [MKVKVLSLLVPALLVAGAANA] (SEQ ID NO: 24)]
  • Erwinia carotovorans pectate lyase PelB
  • MKYLLPTAAAGLLLLAAQPAMA SEQ ID NO: 63
  • heat labile E. coli enterotoxin LTIIb MSFKKIIKAFVIMAALVSVQAHA (SEQ ID NO: 64)]
  • Bacillus subtilis endoxylanase XynA [MFKFKKKFLVGLTAAFMSISMFSATASA (SEQ ID NO: 65)]
  • the signal sequence is from E. coli flagellin (FlgI) [MIKFLSALILLLVTTAAQA (SEQ ID NO: 21)].
  • the present invention provides a modified Als3 protein, wherein the amino acid sequence further comprises a signal sequence which is capable of directing the modified Als3 protein to the periplasm of a host cell (e.g. bacterium).
  • the signal sequence is FlgI.
  • the Flgl comprises an amino acid sequence of SEQ ID NO: 21.
  • the bacterial signal sequence is removed from the modified Als3 protein of the invention after the modified Als3 protein is transported to the periplasmic side of the inner membrane of a host cell of the invention.
  • the present invention provides a polynucleotide encoding a modified Als3 protein of the invention.
  • the present invention provides a polynucleotide encoding a modified Als3 protein of the invention, having a nucleotide sequence that encodes a polypeptide with an amino acid sequence that is at least 97%, 98%, 99% or 100% identical to SEQ ID NO: 10 or 11.
  • the present invention provides a nucleotide sequence according to SEQ ID NO: 69, or a nucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 69.
  • the present invention provides a nucleotide sequence according to SEQ ID NO: 70, or a nucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 70.
  • a nucleotide sequence of the invention comprises nucleotides encoding for amino acids corresponding to one (or more) consensus sequence(s) selected from: D/E-X-N-Z-S/T, K-D/E-X-N-Z-S/T-K, and J-U- B-D/E-X-N-Z-S/T-J-U-B.
  • a nucleotide sequence of the invention comprises nucleotides encoding for amino acids corresponding to one (or more) consensus sequence(s) selected from: KDQNAT (SEQ ID NO: 5), KDQNAS (SEQ ID NO: 6), DQNAT (SEQ ID NO: 7), and GSGGGDQNATGSGGG (SEQ ID NO: 9).
  • a nucleotide sequence of the invention comprises nucleotides encoding for a modified Als3 protein of the invention comprising one or more consensus sequences, wherein the one or more consensus sequences have been added next to or substituted for one or more amino acids selected from the group consisting of: amino acid residue 18, one or more amino acids between amino acid residues 33-37, one or more amino acids between amino acid residues 80-82, amino acid residue 87, one or more amino acids between amino acid residues 104-108, one or more amino acids between amino acid residues 119-121, one or more amino acids between amino acid residues 123-127, one or more amino acids between amino acid residues 155-159, one or more amino acids between amino acid residues 163-164, one or more amino acids between amino acid residues 168-172, one or more amino acids between amino acid residues 175- 179, amino acid residue 207, amino acid residue 220, one or more amino acids between amino acid residues 236-237, amino acid residue 270, amino acid residue 276,
  • a nucleotide sequence of the invention comprises nucleotides encoding for a modified Als3 protein of Candida albicans comprising one or more consensus sequences, wherein the one or more consensus sequences have been added next to one or more amino acids selected from the group consisting of: amino acid residue 16, amino acid residue 88, and amino acid residue 321 of SEQ ID NO: 10 and amino acid residue 16, amino acid residue 88, and amino acid residue 334 of SEQ ID NO: 11.
  • the present invention provides a vector comprising a polynucleotide encoding a modified Als3 protein of the invention. Conjugates In certain embodiments, the present invention provides a conjugate comprising a modified Als3 protein of the invention.
  • the conjugate of the invention may be a conjugate of a modified Als3 protein (e.g. chemical conjugate or bioconjugate).
  • the conjugate of the invention may be a conjugate of a modified Als3 protein and an antigen, e.g. a saccharide antigen (i.e. bioconjugate).
  • the present invention provides a conjugate comprising (or consisting of) a modified Als3 protein of the invention and at least one saccharide antigen.
  • the conjugate of the invention is a bioconjugate.
  • the present invention provides a conjugate comprising a saccharide of the invention linked to a modified carrier protein.
  • the saccharide of the invention is linked to an asparagine residue of the modified carrier protein.
  • the present invention provides a conjugate comprising a saccharide of the invention linked to an asparagine residue of a modified carrier protein.
  • the conjugate is a bioconjugate.
  • the modified carrier protein of the invention includes, but is not limited to, Als3, Sap2, detoxified Exotoxin A of P. aeruginosa (EPA), CRM197, Diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of S. aureus, clumping factor A of S. aureus, clumping factor B of S. aureus, E.
  • the modified carrier protein is a modified Als3 protein of the invention.
  • the modified Als3 protein of the invention is linked to the at least one saccharide antigen.
  • the modified Als3 protein of the invention is directly linked to the at least one saccharide antigen. In other aspects, the modified Als3 protein of the invention is linked to the at least one saccharide antigen through a linker. In certain embodiments, the modified Als3 protein of the invention is non-covalently linked to the at least one saccharide antigen. In some aspects, the modified Als3 protein of the invention is non-covalently linked to the at least one saccharide antigen via an avidin-strepatvidin interaction. In other embodiments, the modified Als3 protein of the invention is covalently linked to the at least one saccharide antigen through a chemical linkage obtainable using a chemical conjugation method (i.e. the conjugate is produced by chemical conjugation).
  • 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). Other methods are described in EP- 0-161-188, EP-208375 and EP-0-477508.
  • the conjugation method may alternatively rely on activation of the saccharide with 1-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester.
  • CDAP 1-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. et al. Infect. Immunity, 1983245256.
  • 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.
  • Amino group for instance via lysine.
  • this group is linked to carboxyl groups on saccharides directly or to a carboxyl group on a linker with carbodiimide chemistry e.g. with EDAC.
  • this group is linked to hydroxyl groups activated with CDAP or CNBr on saccharides directly or to such groups on a linker; to saccharides or linkers having an aldehyde group; to saccharides or linkers having a succinimide ester group.
  • C) Sulphydryl for instance via cysteine).
  • this group is linked to a bromo or chloro acetylated saccharide or linker with maleimide chemistry. In one embodiment this group is activated/modified with bis diazobenzidine.
  • D) Hydroxyl group for instance via tyrosine). In one embodiment this group is activated/modified with bis diazobenzidine.
  • E) Imidazolyl group for instance via histidine). In one embodiment this group is activated/modified with bis diazobenzidine.
  • a saccharide in general the following groups can be used for a coupling: OH, COOH or NH 2 .
  • Aldehyde groups can be generated after different treatments such as: periodate, acid hydrolysis, hydrogen peroxide, etc.
  • Conjugates can be purified by any method known in the art for purification of a protein, 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. ID0312709 (p. 1-18); see also the methods described in WO 2009/104074.
  • the amino acid residue on the modified Als3 protein of the invention to which the at least one saccharide antigen is linked includes, without limitation, Ala, Arg, Asp, Cys, Gly, Glu, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
  • the amino acid is: an amino acid comprising a terminal amine group, a lysine, an arginine, a glutaminic acid, an aspartic acid, a cysteine, a tyrosine, a histidine or a tryptophan.
  • the amino acid residue on the modified Als3 protein of the invention to which the at least one saccharide antigen is linked is not an asparagine residue and in this case, the conjugate is typically produced by chemical conjugation.
  • the at least one saccharide antigen is linked to an amino acid on a modified Als3 protein of the invention selected from asparagine, aspartic acid, glutamic acid, lysine, cysteine, tyrosine, histidine, arginine or tryptophan (e.g. asparagine) and in the case of asparagine, the conjugate may be a bioconjugate (for example an enzymatic conjugation using a oligosaccharyl transferase such as PglB).
  • the amino acid residue on a modified Als3 protein of the invention to which the at least one saccharide antigen is linked is an asparagine residue.
  • the amino acid residue on the modified Als3 protein to which the at least one saccharide antigen is linked is part of the consensus sequence, e.g. the asparagine in D/E-X-N-Z-S/T, K-D/E-X-N-Z-S/T-K or J-U-B-D/E-X-N-Z-S/T-J-U-B consensus sequence.
  • the conjugate of the invention is a conjugate of a recombinant modified Als3 protein (e.g. chemical conjugate or bioconjugate).
  • the conjugate of the invention is a conjugate of an isolated recombinant modified Als3 protein and a recombinant antigen, e.g.
  • the modified Als3 protein of the invention is linked to the at least one saccharide antigen at one or more amino acid residues on the modified Als3 protein.
  • the one or more residues include, without limitations, one or more asparagine residues, one or more aspartic acid residues, one or more glutamic acid residues, one or more lysine residues, one or more cysteine residues, one or more tyrosine residues, one or more histidine residues, one or more arginine residues, one or more tryptophan residues, one or more serine residues, and one or more threonine residues.
  • the modified Als3 protein of the invention is linked to the at least one saccharide antigen at one or more asparagine residues on the modified Als3 protein of the invention.
  • the at least one saccharide antigen is linked to at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten asparagine residues of a modified Als3 protein of the invention.
  • the at least one saccharide antigen is linked to one, two, three, four, five, six, seven, eight, nine, or ten asparagine residues of a modified Als3 protein of the invention.
  • the at least one saccharide antigen is linked to at least three asparagine residues of a modified Als3 protein of the invention.
  • the at least one saccharide antigen is linked to three asparagine residues of a modified Als3 protein of the invention.
  • the modified Als3 protein of the invention comprises (or consists of) an amino acid sequence of SEQ ID NO: 10, and the three asparagine residues include, but are not limited to, positions 20, 92, and 324 of SEQ ID NO: 10.
  • the modified Als3 protein of the invention comprises (or consists of) an amino acid sequence of SEQ ID NO: 11, the three asparagine residues include, but are not limited to, positions 20, 92, and 337 of SEQ ID NO: 11.
  • the at least one saccharide antigen is linked to at least one of three asparagine residues of a modified Als3 protein of the invention.
  • the modified Als3 protein of the invention comprises (or consists of) an amino acid sequence of SEQ ID NO: 10, and the three asparagine residues include, but are not limited to, positions 20, 92, and 324 of SEQ ID NO: 10.
  • the modified Als3 protein of the invention comprises (or consists of) an amino acid sequence of SEQ ID NO: 11, the three asparagine residues include, but are not limited to, positions 20, 92, and 337 of SEQ ID NO: 11.
  • the present invention provides a modified Als3 protein of Candida albicans comprising (or consisting of): (1) an amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11; and (2) at least one saccharide antigen of Candida, wherein the at least one saccharide antigen is linked to at least one of three asparagine residues at positions 20, 92, and 324 of SEQ ID NO: 10 or positions 20, 92, and 337 of SEQ ID NO: 11.
  • the present invention provides conjugates (e.g., bioconjugates) wherein a carrier protein (e.g., a modified Als3 protein of the invention) may be linked (e.g., covalently or non-covalently linked) to a number of different antigens.
  • a carrier protein e.g., a modified Als3 protein of the invention
  • the modified Als3 protein is linked to at least one antigen which is a saccharide antigen.
  • the antigen comprises at least one saccharide antigen.
  • the at least one saccharide antigen is a fungal polysaccharide, a yeast polysaccharide or a mammalian polysaccharide.
  • Polysaccharides comprise 2 or more monosaccharides, typically greater than 6, 8 or 10 monosaccharides.
  • the at least one saccharide antigen in a conjugate (e.g. bioconjugate) of the invention includes, but is not limited to, O antigens of E. coli, Salmonella sp. O antigens, Pseudomonas sp., Klebsiella sp. O antigens, Acinetobacter O antigens, Chlamydia trachomatis O antigens, Vibrio cholera O antigens, Listeria sp.
  • O antigens Legionella pneumophila serotypes 1 to 15 O antigens, Bordetella parapertussis O antigens, Burkholderia mallei and pseudomallei O antigens, Francisella tularensis O antigens, Campylobacter sp.
  • O antigens capsular polysaccharides of Clostridium difficile, Staphylococcus aureus type 5 and 8, Streptococcus pyrogenes, E.
  • coli Streptococcus agalacticae, Neisseria meningitidis, Candida sp., Candida albicans, Haemophilus influenza, Enterococcus faecalis capsular polysaccharides type I-V, and other surface polysaccharide structures, e.g. the Borrelia burgdorferi glycolipids, Neisseria meningitidis pilin O glycan and lipooligosaccharide (LOS), Haemophilus influenza LOS, Leishmania major lipophosphoglycan, tumor associated carbohydrate antigens, malaria glycosyl phosphatidylinositol, and mycobacterium tuberculosis arabinomannan.
  • LOS lipooligosaccharide
  • the at least one saccharide antigen is an O-antigen e.g. from a Gram- negative bacterium. In certain embodiments, the at least one saccharide antigen is an O-antigen from Salmonella species, Shigella species, Pseudomonas species or Klebsiella species. In other embodiments, the at least one saccharide antigen is an O-antigen from Shigella species, Pseudomonas species or Klebsiella species (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, or Klebsiella pneumoniae).
  • Shigella dysenteriae Shigella flexneri
  • Shigella sonnei Shigella sonnei
  • Pseudomonas aeruginosa or Klebsiella pneumoniae.
  • the at least one saccharide antigen is an O-antigen from Shigella dysenteriae, Shigella flexneri or Shigella sonnei.
  • the antigen may be 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. 8; Liu et al Structure and genetics of Shigella O antigens FEMS Microbiology Review, 2008. 32: p. 27).
  • the at least one saccharide 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 at least one saccharide antigen is an O-antigen from Klebsiella pneumoniae.
  • the at least one saccharide antigen is a capsular polysaccharide from Neisseria meningitidis serogroup A (MenA), N. meningitidis serogroup C (MenC), N.
  • the at least one saccharide antigen is a capsular polysaccharide from Streptococcus species or Staphylococcus species. (e.g. Streptococcus pneumoniae or Staphylcoccus aureus). In additional embodiments, the at least one saccharide antigen is a capsular polysaccharide from Staphylococcus aureus.
  • the at least one saccharide antigen may be a capsular polysaccharide from Staphylococcus aureus type 5 and 8.
  • the at least one saccharide antigen is a capsular polysaccharide from Streptococcus pneumoniae.
  • the at least one saccharide antigen is a fungal saccharide antigen.
  • the fungi is Candida species.
  • the at least one saccharide antigen is a saccharide antigen of Candida species.
  • the Candida species includes, but is not limited to, Candida albicans, Candida auris, Candida guilliermondi, Candida lusitaniaea and Candida tropicalis, Candida glabrata, Candida krusei, and Candida parapsilosis.
  • the Candida species is Candida albicans.
  • the at least one saccharide antigen is a saccharide antigen of Candida albicans.
  • the at least one saccharide antigen is a ⁇ -1,3 glucan polymer.
  • the at least one saccharide antigen is a ⁇ -1,3 glucan polymer of Candida albicans. ⁇ -1,3 glucans are widespread in nature.
  • Candida ⁇ -1,3 glucans consist of long linear polymers with occasional ⁇ -1,6 branchings. They confer strength and shape to the cell wall.
  • the ⁇ -1,3 glucan polymer of the invention is a naturally-occuring glucan.
  • the ⁇ -1,3 glucan polymer of the invention is a recombinantly produced glucan.
  • the at least one saccharide antigen comprises a ⁇ -1,3 glucan polymer.
  • the ⁇ -1,3 glucan polymer of the invention comprises at least four ⁇ -1,3 linked glucose molecules.
  • the ⁇ -1,3 glucan polymer comprises four to hundred, four to fifty, four to forty, four to thirty five, four to thirty, four to twenty five, four to twenty, four to ten, four to nine, four to eight, four to seven, four to six, four to five, four, five to hundred, five to fifty, five to forty, five to thirty five, five to thirty, five to twenty five, five to twenty, five to ten, five to nine, five to eight, five to seven, five to six, five, six to hundred, six to fifty, six to forty, six to thirty five, six to thirty, six to twenty five, six to twenty, six to ten, six to nine, six to eight, six to seven, six, seven to hundred, seven to fifty, seven to forty, seven to thirty five, seven to thirty, seven to twenty five, seven to twenty, seven to ten, seven to nine, seven to eight, or seven ⁇ -1,3 linked glucose molecules.
  • the ⁇ -1,3 glucan polymer comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 consecutive ⁇ -1,3 linked glucose molecules.
  • the ⁇ -1,3 glucan polymer comprises at least 11 ⁇ -1,3 linked glucose molecules.
  • the ⁇ -1,3 glucan polymer comprises at least 11 consecutive ⁇ -1,3 linked glucose molecules.
  • the present invention provides a glucan having the structure:
  • n 2-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.
  • the present invention provides a saccharide which is a glucan having the structure: [ ⁇ 3)- ⁇ -D-Glcp-(1 ⁇ ] n ⁇ 3)- ⁇ -D-Glcp-(1 ⁇ 6)- ⁇ -D-Glcp-(1 ⁇ 6)- ⁇ -D-Glcp-(1 ⁇ 4)- ⁇ -D-Glcp- (1 ⁇ 4)- ⁇ -D-Glcp-(1 ⁇ 3)- x-D-GlcpNAc wherein n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.
  • the at least one saccharide antigen comprises a glucan having the structure: wherein n is 2-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.
  • the at least one saccharide antigen comprises a glucan having the structure: [ ⁇ 3)- ⁇ -D-Glcp-(1 ⁇ ] n ⁇ 3)- ⁇ -D-Glcp-(1 ⁇ 6)- ⁇ -D-Glcp-(1 ⁇ 6)- ⁇ -D-Glcp-(1 ⁇ 4)- ⁇ -D-Glcp- (1 ⁇ 4)- ⁇ -D-Glcp-(1 ⁇ 3)- x-D-GlcpNAc wherein n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.
  • the present invention provides a modified Als3 protein of Candida albicans comprising (or consisting of): (1) an amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11; and (2) at least one saccharide antigen of Candida, wherein the at least one saccharide antigen is a ⁇ -1,3 glucan polymer comprising (or consisting of) at least six consecutive ⁇ -1,3 linked glucose molecules, and wherein the at least one saccharide antigen is linked to at least one of three asparagine residues at positions 20, 92, and 324 of SEQ ID NO: 10 or positions 20, 92, and 337 of SEQ ID NO: 11.
  • the at least one saccharide antigen is a ⁇ -1,2 mannan polymer.
  • the at least one saccharide antigen is a ⁇ -1,2 mannan polymer of Candida albicans.
  • Mannans are the outermost layers of the cell wall and they participate in Candida cell adherence and immune system evasion. Mannans are highly complex and branched structures which are linked to secreted proteins. ⁇ -1,2 mannans are a part of Candida’s mannans present at the non-reducing end. Structures comprising at least two consecutive ⁇ -1,2 mannoses are exclusive to the Candida genus and are conserved in several Candida species (e.g., including C. albicans, C. auris, C.
  • the ⁇ -1,2 mannan polymer of the invention is a naturally-occuring mannan. In other embodiments, the ⁇ -1,2 mannan polymer of the invention is a recombinantly produced mannan. In certain embodiments, the at least one saccharide antigen comprises a ⁇ -1,2 mannan polymer. In some aspects, the ⁇ -1,2 mannan polymer comprises at least two ⁇ -1,2 linked mannose molecules.
  • the ⁇ -1,2 mannan polymer comprises two to fifty, two to forty, two to thirty, two to twenty, two to ten, two to nine, two to eight, two to seven, two to six, two to five, two to four, two to three, two, three to fifty, three to forty, three to thirty, three to twenty, three to ten, three to nine, three to eight, three to seven, three to six, three to five, three to four, three, four to fifty, four to forty, four to thirty, four to twenty, four to ten, four to nine, four to eight, four to seven, four to six, four to five, four, five to fifty, five to forty, five to thirty, five to twenty, five to ten, five to nine, five to eight, five to seven, five to six, or five, six to forty, six to thirty, six to twenty, six to ten, six to nine, six to eight, six to seven, six, seven to fifty, seven to forty, seven to thirty, seven to twenty, seven to ten, seven to nine, six to forty, six
  • the ⁇ -1,2 mannan polymer comprises at least 2, at least 3, at least 4, or at least 5 consecutive ⁇ -1,2 linked mannose molecules.
  • Host cell In certain embodiments, the present invention provides host cells comprising a polynucleotide sequence that encodes a modified Als3 protein of the invention. In certain embodiments, the present invention provides host cells that can be used to produce the bioconjugates of the invention. Host cells of the invention include, without limitation, archea, prokaryotic host cells, and eukaryotic host cells. In certain embodiments, the host cell is a non-human host cell. In certain embodiments, the host cell is a eukaryotic host cell.
  • the eukarytoic host cell includes, but is not limited to, a yeast cell, an insect cell and a mammalian cell.
  • the host cell is a prokaryotic host cell.
  • the prokaryotic host cell is a bacterial cell.
  • the bacteria is a gram positive bacteria. In other aspects, the bacteria is a gram negative bacteria.
  • the bacteria includes, but is not limited to, an Escherichia species, a Shigella species, Klebsiella species, a Xhantomonas species, a Salmonella species, a Yersinia species, a Lactococcus species, a Lactobacillus species, a Pseudomonas species, a Corynebacterium species, a Streptomyces species, a Streptococcus species, a Staphylococcus species, a Bacillus species, and a Clostridium species.
  • the bacteria is an E. coli species. In specific aspects, the bacteria is E. coli.
  • exemplary host cells include, but are not limited to, an Escherichia species, a Shigella species, Klebsiella species, a Xhantomonas species, a Salmonella species, a Yersinia species, a Lactococcus species, a Lactobacillus species, a Pseudomonas species, a Corynebacterium species, a Streptomyces species, a Streptococcus species, a Staphylococcus species, a Bacillus species, and a Clostridium species.
  • the host cell is an E. coli species. In specific embodiments, the host cell is E. coli.
  • Host cells of the invention 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 antigen 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 the invention, 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 (rfb or wb), enterobacterial common antigen cluster (wec), 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 waaL gene, gtrA gene, gtrB gene, gtrS gene, or a gene or genes from the wec cluster or a gene, or a gene or genes from the colonic acid cluster (wc), or a gene or genes from the rfb gene cluster are deleted or functionally inactivated from the genome of a prokaryotic host cell of the invention.
  • one or more of the waaL gene, gtrA gene, gtrB gene, gtrS gene, or a gene or genes from the wec 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 the invention.
  • the host cell of the invention is E. coli, wherein the native enterobacterial common antigen cluster (ECA, wec) with the exception of wecA, the colanic acid cluster (wca), and the O16-antigen cluster have been deleted.
  • ECA enterobacterial common antigen cluster
  • wca the colanic acid cluster
  • O16-antigen cluster the native lipopolysaccharide O-antigen ligase waaL may be deleted from the host cell of the invention.
  • the native gtrA gene, gtrB gene and gtrS gene may be deleted from the host cell of the invention.
  • 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 Als3 protein of the invention, optionally within a plasmid.
  • the host cells of the invention also comprise one or more nucleotide sequences comprising polysaccharide synthesis genes.
  • host cells of the invention can produce a bioconjugate comprising an antigen, for example a saccharide antigen (e.g. a fungal, bacterial, yeast or mammalian polysaccharide antigen) which is linked to a modified Als3 protein of the invention.
  • a saccharide antigen e.g. a fungal, bacterial, yeast or mammalian polysaccharide antigen
  • one or more heterologous nucleotide sequences encode for the polysaccharide synthesis proteins to produce the fungal polysaccharide antigen, bacterial polysaccharide antigen, yeast polysaccharide antigen or mammalian polysaccharide antigen.
  • the present invention provides a host cell comprising: (1) one or more polynucleotide sequences that encode one or more heterologous glycosyltransferases; (2) a polynucleotide sequence that encodes a heterologous oligosaccharyl transferase; (3) a polynucleotide sequence that encodes a modified Als3 protein of the invention; and, optionally, (4) a polynucleotide sequence that encodes a polymerase.
  • the one or more heterologous glycosyltransferases includes, without limitation, exoL, exoM, exoO, exoU and exoW.
  • the exoL, exoM, exoO, exoU and exoW are from a rhizobia.
  • the rhizobia is Sinorhizobium.
  • the Sinorhizobium is Sinorhizobium meliloti 1021.
  • the one or more heterologous glycosyltransferases comprises exoL, exoM, exoO, exoU and exoW from a rhizobia, optionally from Sinorhizobium, optionally from Sinorhizobium meliloti 1021.
  • the one or more heterologous glycosyltransferases includes, without limitation, SleC, SleE, SleF, SleU and SleW.
  • the SleC, SleE, SleF, SleU and SleW are from a rhizobia.
  • the rhizobia is Agrobacterium.
  • the Agrobacterium is Agrobacterium sp. ZX09.
  • the one or more heterologous glycosyltransferases comprises SleC, SleE, SleF, SleU and SleW from a rhizobia, optionally from Agrobacterium, optionally from Agrobacterium sp. ZX09.
  • the present invention provides a host cell comprising: i. a nucleotide sequence encoding one or more heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,3 glucan polymer; ii. A nucleotide sequence encoding a glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetyl glucosamine (GlcNac) molecule; iii. a nucleotide sequence encoding a heterologous oligosaccharyl transferase; and iv.
  • a host cell comprising: i. a nucleotide sequence encoding one or more heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,3 glucan polymer; ii. A nucleotide sequence encoding a glycosyltransferase capable of covalently bonding
  • the present invention provides a host cell comprising: (1) one or more polynucleotide sequences that encode one or more heterologous glycosyltransferases; (2) a polynucleotide sequence that encodes a glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetyl glucosamine (GlcNac) molecule; (3) a polynucleotide sequence that encodes a heterologous oligosaccharyl transferase; (4) a polynucleotide sequence that encodes a modified Als3 protein of the invention; and, optionally, a polynucleotide sequence that encodes a polymerase.
  • a host cell comprising: (1) one or more polynucleotide sequences that encode one or more heterologous glycosyltransferases; (2) a polynucleotide sequence that encodes a glycosyltransferase capable of covalently bonding
  • the present invention provides a host cell comprising: i. a nucleotide sequence encoding one or more heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,3 glucan polymer; ii. A nucleotide sequence encoding a glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetyl glucosamine (GlcNac) molecule; iii. a nucleotide sequence encoding a heterologous oligosaccharyl transferase; and iv.
  • a host cell comprising: i. a nucleotide sequence encoding one or more heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,3 glucan polymer; ii. A nucleotide sequence encoding a glycosyltransferase capable of covalently bonding
  • the modified carrier protein of the invention includes, but is not limited to, Als3, Sap2, detoxified Exotoxin A of P. aeruginosa (EPA), CRM197, Diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of S. aureus, clumping factor A of S. aureus, clumping factor B of S. aureus, E.
  • the modified carrier protein is a modified Als3 protein of the invention.
  • the one or more heterologous glycosyltransferases capable of synthesizing a ⁇ -1,3 glucan polymer includes, without limitation, exoL, exoM, exoO, exoU and exoW.
  • the exoL, exoM, exoO, exoU and exoW are from a rhizobia.
  • the rhizobia is Sinorhizobium.
  • the Sinorhizobium is Sinorhizobium meliloti 1021.
  • the one or more heterologous glycosyltransferases comprises exoL, exoM, exoO, exoU and exoW from a rhizobia, optionally from Sinorhizobium, optionally from Sinorhizobium meliloti 1021.
  • the one or more heterologous glycosyltransferases includes, without limitation, SleC, SleE, SleF, SleU and SleW.
  • the SleC, SleE, SleF, SleU and SleW are from a rhizobia.
  • the rhizobia is Agrobacterium.
  • the Agrobacterium is Agrobacterium sp.
  • the one or more heterologous glycosyltransferases of i. comprises SleC, SleE, SleF, SleU and SleW from a rhizobia, optionally from Agrobacterium, optionally from Agrobacterium sp. ZX09.
  • the one or more heterologous glycosyltransferases capable of synthesizing a ⁇ -1,3 glucan polymer has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of exoL of Sinorhizobium meliloti 1021.
  • the one or more heterologous glycosyltransferases capable of synthesizing a ⁇ -1,3 glucan polymer has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleC of Agrobacterium sp. ZX09. In some aspects, the one or more heterologous glycosyltransferases capable of synthesizing a ⁇ -1,3 glucan polymer has an amino acid sequence identical to the amino acid sequence of SleC of Agrobacterium sp. ZX09.
  • the one or more heterologous glycosyltransferases capable of synthesizing a ⁇ -1,3 glucan polymer has an amino acid sequence identical to the amino acid sequence of exoL of Sinorhizobium meliloti 1021.
  • SleC of Agrobacterium sp. ZX09 comprises the amino acid sequence of SEQ ID NO: 12.
  • the one or more heterologous glycosyltransferases capable of synthesizing a ⁇ -1,3 glucan polymer has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 12.
  • the one or more heterologous glycosyltransferases capable of synthesizing a ⁇ -1,3 glucan polymer has an amino acid sequence identical to SEQ ID NO: 12.
  • the one or more heterologous glycosyltransferases capable of synthesizing a ⁇ -1,3 glucan polymer has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleC of Agrobacterium sp. ZX09 comprising SEQ ID NO: 12.
  • the one or more heterologous glycosyltransferases capable of synthesizing a ⁇ -1,3 glucan polymer has an amino acid sequence identical to the amino acid sequence of SleC of Agrobacterium sp. ZX09 comprising SEQ ID NO: 12: MIHILYLAHDLSDPAIRRRVLTLLAGGARVTLAGFRRGQNRLAEIEGVVPVVLGETADGQFLQRMAAVA KASLSLGKVLNGIPAPDVVLARNLEMLALAKRAMSIYSGRPALVYECLDIHRLLLHEGKPGQMLNAAQRYFARDA KLLVTSSPAFVEHYFKPVSGLDLPVLLQENKVLALDDTIAATPRPRAPAPGEPWKIGWFGALRCRKSLEILAEFAR RMEGRVEIILRGRPAYSEFADFDGFVAAAPHVHFHGPYKNPEDLAAIYNEVQFTWAIDFFEEGQNSSWLLPNRLY EGCLYGTLPIALAGTETARFIEK
  • ZX09 optionally a nucleotide sequence encoding sleC from Agrobacterium sp.
  • ZX09 comprising an amino acid sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12, optionally within a plasmid.
  • host cells of the present invention comprise a nucleotide sequence encoding sleC, optionally sleC from Agrobacterium sp.
  • ZX09 optionally a nucleotide sequence encoding sleC from Agrobacterium sp.
  • ZX09 comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 34: ATGATCCATATTCTCTACCTCGCGCATGATCTGTCAGACCCCGCCATTCGTCGGCGGGTGCTGACGC TGCTTGCAGGCGGGGCGCGGGTCACGCTGGCCGGTTTCCGGCGCGGACAGAACCGGCTGGCGGAGATCGAA GGCGTCGTACCTGTCGTGCTCGGGGAAACCGCCGACGGGCAATTTCTGCAGCGCATGGCGGCAGTGGCGAA AGCCAGCCTTTCGCTCGGCAAAGTCTTGAACGGAATTCCGGCACCCGACGTCGTTCTCGCCAGGAACCTCGA AATGCTGGCTGGCAAAGCGCCATGTCGATCTATTCCGGCCGCCCGGCGCTGGTTTACGAATGTCTCGA TATTCATCGCCTGCTGCTCCACGAAGGCAAGCCCGGACAGATGCTGAATGCCGCGCAGCGTTATTTC
  • the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleE of Agrobacterium sp. ZX09. In some aspects, the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of SleE of Agrobacterium sp. ZX09. In other aspects, the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of exoM of Sinorhizobium meliloti 1021. In certain embodiments, SleE of Agrobacterium sp. ZX09 comprises the amino acid sequence of SEQ ID NO: 13.
  • the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 13. In other aspects, the one or more heterologous glycosyltransferases has an amino acid sequence identical to SEQ ID NO: 13. In specific aspects, the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleE of Agrobacterium sp. ZX09 comprising SEQ ID NO: 13.
  • the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of SleE of Agrobacterium sp. ZX09 comprising SEQ ID NO: 13: MTDNTVTGTQYLKTVDIGICTYRRPALVATLLSLFELDVPEGVKVRLIVADNDEEPSAKASVDRLRETAP FEITYVHCPKSNISIARNACLSECKADYLAFIDDDETAPPHWLAALLEKADETGAETVLGPVTAVYRDNAPGWMK RGDFHSTVPVWVNGEIITGYTCNTLLRMEAPSVKGRRFALALGQSGGEDTHFFSHLHAAGGRIVFAEDAVLSEP VPENRASFLWLAKRRFRSGQTHGRVLAEKKPGARRVVQVVKAGSKVLYCALFAALSGFNAVRRNRYALRGALHM GSMSGAFGVREIRQYGAVEAT
  • host cells of the present invention comprise a nucleotide sequence encoding sle
  • ZX09 optionally a nucleotide sequence encoding sleE from Agrobacterium sp.
  • ZX09 comprising an amino acid sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 13, optionally within a plasmid.
  • host cells of the present invention comprise a nucleotide sequence encoding sleE, optionally sleE from Agrobacterium sp.
  • ZX09 optionally a nucleotide sequence encoding sleE from Agrobacterium sp.
  • ZX09 comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 33: ATGACCGACAACACCGTCACCGGCACGCAATATCTCAAGACCGTCGATATCGGCATCTGCACCTACA GACGCCCGGCTTGTCGCCACACTTCTGTCACTCTTCGAGCTGGATGTGCCTGAAGGCGTGAAAGTTAGGC TGATTGTCGCCGATAATGACGAGGAGCCCAGCGCAAAGGCAAGCGTCGATCGCCTGCGCGAAACCGCCCCCT TCGAGATCACCTATGTGCATTGCCCGAAATCTAATATTTCGATTGCCCGCAATGCCTGTCGGAATGCAA GGCGGACTATCTCGCCTTTATCGATGACGACGAAACGGCCGCCACACTGGCTGGCCGCGCTTCTGGAAAA GGCAGACGAGACCGGTGCAGAAACAGTTCTCGGCCCCGTCACTGCGGTTTACCGGGACAAC
  • the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleF of Agrobacterium sp. ZX09. In some aspects, the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of SleF of Agrobacterium sp. ZX09. In other aspects, the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of exoO of Sinorhizobium meliloti 1021. In certain embodiments, SleF of Agrobacterium sp. ZX09 comprises the amino acid sequence of SEQ ID NO: 14.
  • the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 14. In other aspects, the one or more heterologous glycosyltransferases has an amino acid sequence identical to EQ ID NO: 13. In specific aspects, the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleF of Agrobacterium sp. ZX09 comprising SEQ ID NO: 14.
  • the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of SleF of Agrobacterium sp. ZX09 comprising SEQ ID NO: 14: MENLTSPPDISFVIAAYNAADTIEAAVQSALDQQGVTLEVIVVDDRSADDTIPFVEAIAAIDPRVRLLALE ENRGPGGARNAGIEAATGRWIAVLDSDDVIRPERSACMMCRAEAANADIAVDNLDVVYTDGRPMETMFPEEFL EERPVLTLEDFISSNILFRSTFNFGYMKPMFRRDFLNNEALRFREDIRIGEDYILLASALAAGGLCVIEPKPGYIYNI REGSISRVLELHHVEAMMRADEEFLSHYTLLPAAMDAQQARARSLRLAHNFLTLVENIKRRSVLGALKTTIRDPA VLGHLRMPIAVRLRRLRDAVFAPAANTGVKRQIS
  • host cells of the present invention comprise a nucleotide sequence
  • ZX09 optionally a nucleotide sequence encoding sleF from Agrobacterium sp.
  • ZX09 comprising an amino acid sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 14, optionally within a plasmid.
  • host cells of the present invention comprise a nucleotide sequence encoding sleF, optionally sleF from Agrobacterium sp.
  • ZX09 optionally a nucleotide sequence encoding sleF from Agrobacterium sp.
  • ZX09 comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 32: ATGGAAAACCTCACCTCTCCGCCAGACATCAGCTTCGTTATCGCCGCCTATAATGCCGCCGATACGA TTGAGGCCGCGGTTCAAAGCGCTCGATCAGCAGGGCGTGACGCTGGAAGTAATCGTCGTCGACGACCGC TCCGCAGACGATACCATTCCCTTCGTTGAAGCGATCGCCGCAATCGATCCGCGCGTGCGGCTGCTTGCTC GAAGAAAACCGCGGTCCAGGCGGCGCCCGCAACGCCGGCATCGAGGCCGCGACGGGGCGCTGGATCGCCGT GCTCGACTCCGACGATGTCATCCGCCCGGAGCGCTCCGCCTGCATGATGTGCCGGGCGGAAGCCGCCAACG CTGACATCGCTGTCGATAATCTCGATGTCGTCTACACCGATGGCCGGCCGATGGAGACGATGTTTCGT
  • the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleU of Agrobacterium sp. ZX09. In some aspects, the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of SleU of Agrobacterium sp. ZX09. In other aspects, the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of exoU of Sinorhizobium meliloti 1021. In certain embodiments, SleU of Agrobacterium sp. ZX09 comprises the amino acid sequence of SEQ ID NO: 15.
  • the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 15. In other aspects, the one or more heterologous glycosyltransferases has an amino acid sequence identical to SEQ ID NO: 15. In specific aspects, the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleU of Agrobacterium sp. ZX09 comprising SEQ ID NO: 15.
  • the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of SleU of Agrobacterium sp. ZX09 comprising SEQ ID NO: 15: MVSGSQPICVIIAAKNASDTIDIAIRSALAEPEVGEVVVIDDGSTDTTSDVAHAADDGTGRLRVVRFDV NRGPSAARNHAISISSAPLISILDADDFFFHGRFAAMLADDDWDLVADNIAFIQQSVPGASSMQPARFEPQARFL SLTEFVEGNISRPGVERGETGFLKPVIRRAFLDKHALRYDEALRLGEDYELYVRALAAGARYKVIRHCGYGAIVRG NSLSGRHRTEDLRLLYEADRAILAGCRLSAEETAILREHEKHIRAKFELRHFLDTKKQKGVSGALSHALARLPALP AITRGIWSDKTARFRKAAPVRDVRYLLDGTPVS
  • host cells of the present invention comprise a nucleotide sequence encoding s
  • ZX09 optionally a nucleotide sequence encoding sleU from Agrobacterium sp.
  • ZX09 comprising an amino acid sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 15, optionally within a plasmid.
  • host cells of the present invention comprise a nucleotide sequence encoding sleU, optionally sleU from Agrobacterium sp.
  • ZX09 optionally a nucleotide sequence encoding sleU from Agrobacterium sp.
  • ZX09 comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 31: ATGGTGAGCGGTTCACAGCCCATTTGCGTCATCATCGCTGCAAAGAATGCATCTGATACCATCGATA TCGCCATTCGTTCCGCCCTTGCGGAGCCAGAGGTCGGTGAAGTCGTGGTGATCGACGATGGTTCCACCGATA CAACGAGCGACGTGGCGCATGCGGCGGATGACGGCACGGGCCGCCTGCGGGTGGTGCGATTCGACGTCAAC CGCGGGCCATCGGCTGCGCGCAATCATGCCATCTCGATTTCCTCCGCTCCCCTCATCAGCATTCTCGATGCG GACGATTTTTTCTTCCACGGCCGTTTCGCCGCCATGCTGGCCGATGACGACTGGGACCTCGTGGCCGATAAT ATCGCCTTCATTCAGCAATCCGTTCCAGGCCATGCTGCCTGCCTGCCAGCCAGGCGG
  • the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleW of Agrobacterium sp. ZX09. In some aspects, the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of SleW of Agrobacterium sp. ZX09. In other aspects, the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of exoW of Sinorhizobium meliloti 1021. In certain embodiments, SleW of Agrobacterium sp. ZX09 comprises the amino acid sequence of SEQ ID NO: 16.
  • the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 16. In other aspects, the one or more heterologous glycosyltransferases has an amino acid sequence identical to SEQ ID NO: 16. In specific aspects, the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleW of Agrobacterium sp. ZX09 comprising SEQ ID NO: 16.
  • the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of SleW of Agrobacterium sp. ZX09 comprising SEQ ID NO: 16: LINARGETMARFTVVIPYYQKQHGVLGRALASVFAQTYQDFDLVIVDDESPYPIDQELAELSQEQKDRI LVIKQANAGPGGARNTGLDNVPDGTDYVAFLDSDDIWTPDHLRNAAFALTTYGGECYWASMQASDEFYYHFAI SELEKNEGAARLSEKPLVIELPDLASVMLRNWSFLHLSCMVIGRPLFEKIRFDPALRLAAEDVLFFCDSILASKRTLL CDDAGAMRGMGVNIFHSIDNTSPEFLRQQFNTWVALDTLEGRFSRRPADVASIASYKNTARKQALWSQAGNLK RRKAPEFGLLLKWAMRDPALLRAAFELGAGKIVRSR
  • host cells of the present invention comprise a nucleotide
  • ZX09 optionally a nucleotide sequence encoding sleW from Agrobacterium sp.
  • ZX09 comprising an amino acid sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 16, optionally within a plasmid.
  • host cells of the present invention comprise a nucleotide sequence encoding sleW, optionally sleW from Agrobacterium sp.
  • ZX09 optionally a nucleotide sequence encoding sleW from Agrobacterium sp.
  • ZX09 comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 30: TTGATCAACGCAAGGGGCGAAACAATGGCAAGATTTACTGTCGTCATTCCCTACTATCAAAAGCAGC ACGGTGTCCTGGGACGTGCACTCGCATCGGTTTTTGCGCAGACTTACCAGGACTTCGATCTTGTCATCGTCG ATGACGAATCGCCATACCCGATCGATCAGGAACTTGCGGAACTTTCGCAGGAACAGAAAGACCGGATTCTTG TCATTAAGCAGGCCAATGCCGGCCCGGGCGGCGCCCGCAACACCGGTCTGGACAATGTGCCTGACGGCACC GACTACGTTGCCTTTCTTGATTCCGACGATATCTGGACGCCCGATCATCTGCGGAATGCGGCCTTTGCTC ACGACCTATGGCGGCGAGTGCTACTGGGCGTCCATGCAAGTGACGAATTTTATTATCATTTCGCCATT T
  • the host cell comprises one gene copy of SleW. In other aspects, the host cell comprises more than one gene copy of SleW. In certain embodiments, the host cell comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten gene copies of SleW. In specific embodiments, the host cell contains at least two gene copies of a SleW of Agrobacterium sp. ZX09. In additional embodiments, the host cell contains at least one copy of a SleW of Agrobacterium sp. ZX09 under the control of a strong promoter that enables higher expression of SleW compared to expression of SleC, SleE, SleF or SelU.
  • the glycosyltransferase capable of covalently bonding a glucose to GlcNac is WfaP.
  • WfaP is from a bacteria.
  • the bacteria is a gram positive bacteria. In other aspects, the bacteria is a gram negative bacteria.
  • the bacteria includes, but is not limited to, an Escherichia species, a Shigella species, Klebsiella species, a Xhantomonas species, a Salmonella species, a Yersinia species, a Lactococcus species, a Lactobacillus species, a Pseudomonas species, a Corynebacterium species, a Streptomyces species, a Streptococcus species, a Staphylococcus species, a Bacillus species, and a Clostridium species.
  • the bacteria is an Escherichia species.
  • the Escherichia species is E. coli 056.
  • the glycosyltransferase capable of covalently bonding a glucose to GlcNac is is WfaP from E. coli 056.
  • the glycosyltransferase capable of covalently bonding a glucose to GlcNac has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WfaP from E. coli 056.
  • the glycosyltransferase capable of covalently bonding a glucose to GlcNac has an amino acid sequence identical to the amino acid sequence WfaP from E. coli 056.
  • glycosyltransferase capable of covalently bonding a glucose to GlcNac comprises an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 17.
  • the glycosyltransferase capable of covalently bonding a glucose to GlcNac comprises an amino acid sequence identical to SEQ ID NO: 17.
  • glycosyltransferase capable of covalently bonding a glucose to GlcNac comprises an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WfaP from E.
  • the glycosyltransferase capable of covalently bonding a glucose to GlcNac comprises an amino acid sequence identical to the amino acid sequence of WfaP from E. coli 056 comprising SEQ ID NO: 17: MELVSIIIAAYNCKDTIYATVESALSQTYKNIEIIICDDSSTDDTWDIINKIKDSRIICIKNNYCKGAAGARNCALKI AKGRYIAFLDSDDYWVTTKISNQIHFMETEKVFFSYSNYYIEKDFVITGVFSSPPEINYGAMLKYCNIACSTVILDR TGVKNISFPYIDKEDYALWLNILSKGIKARNTNLVDTYYRVHAGSVSANKFKELIRQSNVLKSIGIKAHHRIICLFY YAINGLIKHCFSYRDKRNA
  • host cells of the present invention comprise a nucleotide sequence encoding WfaP, optionally Wfa
  • host cells of the present invention comprise a nucleotide sequence encoding WfaP, optionally WfaP from E. coli 056, optionally a nucleotide sequence encoding WfaP from E.
  • coli 056 comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 35: ATGGAACTGGTCTCTATCATCATTGCCGCCTACAACTGTAAGGACACGATCTATGCCACCGTCGAATCCGCCC TGAGCCAAACCTACAAGAACATCGAAATTATCATTTGCGATGACAGCTCTACCGATGACACGTGGGATATTAT TAACAAAATCAAGGACAGCCGTATCATTTGCATCAAGAACAACTACTGTAAGGGTGCGGCGGGCGCGTAA CTGTGCTCTGAAAATCGCGAAGGGCCGCTATATTGCCTTTCTGGATTCAGATGACTACTGGGTGACCACGAA AATCTCGAACCAGATTCATTTCATGGAAACCGAAAAGGTGTTTTTCAGCTACTCGAACTACTACATCGAAAAG GATTTCGTGATTACCGGCGTTTTCAGTTCCCCGCCGGAAATCAACTATGGTGCTATGCTGAAATACT
  • the process is accomplished by the enzymatic oligosaccharyl transferase 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 oligosaccharyl transferase complex
  • the machinery responsible of this reaction is encoded by a cluster called “pgl” (for protein glycosylation).
  • the C. jejuni glycosylation machinery is transferred to E. coli to allow for the glycosylation of recombinant proteins expressed by the host E. coli cells.
  • Previous studies have demonstrated how to generate E. coli strains that can perform N-glycosylation (see, e.g. Wacker et al. Science. 2002; 298 (5599):1790-3; Nita-Lazar et al. Glycobiology. 2005; 15(4):361-7; Feldman et al. Proc Natl Acad Sci U S A. 2005; 102(8):3016-21; Kowarik et al.
  • the host cells of the present invention comprise a nucleotide sequence encoding a heterologous oligosaccharyl transferase, optionally within a plasmid.
  • the heterologous oligosaccharyl transferase is a PglB.
  • the PglB is from Campylobacter.
  • the Campylobacter includes, but is not limited to, Campylobacter jejuni or Campylobacter coli.
  • the Campylobacter is Sinorhizobium meliloti 1021.
  • the PglB is optionally from Campylobacter, optionally from Campylobacter jejuni or Campylobacter coli comprising an amino acid sequence of SEQ ID NO: 20.
  • the PglB comprises an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of PglB of Campylobacter coli.
  • the PglB comprises an amino acid sequence identical to the amino acid sequence of PglB of Campylobacter coli.
  • PglB of Campylobacter coli comprises the amino acid sequence of SEQ ID NO: 20.
  • the PglB has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 20.
  • the PglB has an amino acid sequence identical to SEQ ID NO: 20.
  • the PglB has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of PglB of Campylobacter coli comprising SEQ ID NO: 20.
  • the PglB has an amino acid sequence identical to the amino acid sequence of PglB of Campylobacter coli comprising SEQ ID NO: 20: MLKKEYLKNPYLVLFAMIILAYVFSVFCRFYWVWWASEFNEYFFNNQLMIISNDGYAFAEGARDMIAGF HQPNDLSYYGSSLSALTYWLYKITPFSFESIILYMSTFLSSLVVIPTILLANEYKRPLMGFVAALLASIANSYYNRTM SGYYDTDMLVIVLPMFILFFMVRMILKKDFFSLIALPLFIGIYLWWYPSSYTLNVALIGLFLIYTLIFHRKEKIFYIAVI LSSLTLSNIAWFYQSAIIVILFALFALEQKRLNFMIIGILGSATLIFLILSGGVDPILYQLKFYIFRSDESANLTQGFM YFNVNQTIQEVENVDLSEFMRRISGSEIVFLFSLFGFVWLLRKHKSMIMALPILVLG
  • host cells of the present invention comprise a nucleotide sequence encoding PglB, optionally PglB from Campylobacter jejuni, optionally a nucleotide sequence encoding PglB from Campylobacter jejuni or Campylobacter coli comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 29, optionally within a plasmid: ATGCTGAAGAAAGAATATCTGAAAAATCCGTATCTGGTGCTGTTCGCAATGATTATCCTGGCGTATG TGTTTAGCGTGTTCTGTCGTTTCTACTGGGTGTGGTGGGCAAGTGAATTTAACGAATATTTCTTTAACAACC AGCTGATGATCATCTCCAATGATGGCTATGCCTTCGCAGAAGGTGCCCGTGACATGATTGCAGGCTTTCATC AGCCGAACGATCTGAGTTATTACGGTAGCTCTCTGTCCGCC
  • Polymerase 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 the invention (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 the invention may further comprise a nucleotide sequence encoding a heterologous wzy polymerase.
  • a host cell of the invention may also comprise a nucleotide sequence encoding a heterologous 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 periplam 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 the invention comprises a nucleotide sequence encoding a flippase (e.g.
  • the flippase is a capsular polysaccharide flippase (e.g. wzx) of Streptococcus pneumoniae.
  • a host cell of the invention may also comprise a nucleotide sequence encoding a translocase (e.g. wzm-wzt), e.g. a heterologous translocase.
  • a translocase e.g. wzm-wzt
  • the translocase is a Wzx/Wzy dependent transporter.
  • the translocase is a ATP-binding cassette (ABC) dependent transporter.
  • the translocase is a synthase dependent transporter.
  • the translocase is an ABC transporter.
  • Polysaccharides assembled by ABC transporters are fully polymerized by sequential glycosyl transfer at the cytoplasmic face of the inner membrane of a host cell.
  • the glycan can be assembled as an Undecaprenyl diphosphate (Und-PP)-linked intermediate (e.g. for most O-antigen polysaccharides).
  • Und-PP Undecaprenyl diphosphate
  • the acceptor is diacylglycerol phosphate.
  • the polysaccharide chain is elongated by addition of monomers to the nonreducing terminus of the lipid- linked intermediate and is completed in the cytoplasm, prior to export to the periplasmic space via the translocase (e.g.
  • a saccharide antigen of the invention is synthesized by a ABC transporter-dependent pathway.
  • a scaccharide antigen of the invention is completely synthesized on the cytosolic leaflet of the plasma membrane of a host cell (e.g. E. coli).
  • the saccharide antigen of the invention is built on a lipid acceptor (e.g.
  • Und-PP Undecaprenyl diphosphate
  • Und-PP serves as a lipid acceptor and is modified by the addition of an acetylated amino sugar phosphate (e.g., N-acetylglucosamine-1-P) to generate a biosynthesis primer.
  • a translocase e.g. wzm-wzt
  • the translocase is a bacterial translocase, e.g. a translocase of the polysaccharide biosynthetic pathway of interest.
  • the host cell of the invention comprises a nucleotide sequence encoding a translocase (e.g. wzm-wzt) of a polysaccharide biosynthetic pathway of a Klebsiella species, Streptococcus species, Shigella species, Escherichia species, Pseudomonas species, or Staphylococcus species. (e.g.
  • the heterologous translocase that is introduced into a host cell of the invention is an ABC transporter (e.g. wzm-wzt) of Klebsiella pneumoniae.
  • Other translocases that can be introduced into the host cells of the invention are for example from Campylobacter jejuni (e.g. pglK).
  • the present invention provides a host cell comprising a nucleotide sequence comprising (i) a wzm gene comprising a nucleotide sequence of SEQ ID NO: 36, optionally comprising a nucleotide sequence at least 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 36; and (ii) a wzt gene comprising a nucleotide sequence of SEQ ID NO: 37, optionally comprising a nucleotide sequence at least 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 37.
  • the wzm and wzt genes are from Klebsiella pneumoniae.
  • the present invention provides a wzm gene, optionally from Klebsiella pneumoniae, optionally comprising a nucleotide sequence of SEQ ID NO: 36, optionally comprising a nucleotide sequence at least 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 36: ATGAAGTACAATTTAGGGTATTTATTTGATTTACTTGTTGTCATAACAAATAAAGATCTAAAAGTGC GCTATAAGAGCAGCATGCTAGGCTATTTATGGTCAGTAGCAAATCCATTGCTTTTTGCCATGATTTATTT TATATTTAAGCTGGTAATGAGAGTACAAATTCCAAATTATACCGTTTTCCTCATTACCGGCTTGTTTCCGTGG CAATGGTTTGCCAGTTCGGCCACTAACTCATTATTTTCATTCATCGCTAACGCTCAAATTATCAAGAAGACAG TTTTTCCCCGGTCCGTGATTCCGCTAATGTAATGATGGAAGGGTTGCATTCTTTGTACC
  • the wzm gene encodes a Wzm protein comprising (or consisting of) an amino acid sequence of SEQ ID NO: 18: VISAMPKGTRRTSMKYNLGYLFDLLVVITNKDLKVRYKSSMLGYLWSVANPLLFAMIYYFIFKLVMRVQI PNYTVFLITGLFPWQWFASSATNSLFSFIANAQIIKKTVFPRSVIPLSNVMMEGLHFLCTIPVIVVFLFVYGMTPSL SWVWGIPLIAIGQVIFTFGVSIIFSTLNLFFRDLERFVSLGIMLMFYCTPILYASDMIPEKFSWIITYNPLASMILSW RDLFMNGTLNYEYISILYFTGIILTVVGLSIFNKLKYRFAEIL
  • the present invention provides a wzt gene, optionally from Klebsiella pneumoniae, optionally comprising a nucleotide sequence of SEQ ID NO: 37, optionally comprising a nucleotide sequence at least 80%
  • the wzt gene encodes a Wzt protein comprising (or consisting of) an amino acid sequence of SEQ ID NO: 19: MHPVINFSHVTKEYPLYHHIGSGIKDLIFHPKRAFQLLKGRKYLAIEDVSFTVGKGEAVALIGRNGAGKS TSLGLVAGVIKPTKGTVTTEGRVASMLELGGGFHPELTGRENIYLNATLLGLRRKEVQQRMERIIEFSELGEFIDE PIRVYSSGMLAKLGFSVISQVEPDILIIDEVLAVGDIAFQAKCIQTIRDFKKRGVTILFVSHNMSDVEKICDRVIWIE NHRLREVGSAERIIELYKQAMA
  • the present invention provides a method of producing a ⁇ -1,3 glucan polymer in a host cell of the invention, the method comprising the steps of introducing and expressing in the host cell: i.
  • nucleotide sequence encoding a first glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetyl glucosamine (GlcNAc) molecule; ii. a nucleotide sequence encoding additional glycosyltransferases capable of synthesizing a fungal ⁇ -1, 3 glucan, wherein the additional glycosyltransferases comprise SleC, SleE, SleF, SleU and SleW from rhizobia; and iii.
  • the present invention provides a method of producing a ⁇ -1,3 glucan polymer in a prokaryotic host cell, the method comprising the steps of introducing and expressing in the host cell: i.
  • the host cell produces more SleW than SleC, SleE, SleF or SleU; and iii. optionally, a nucleotide sequence encoding a translocase capable of translocating the ⁇ -1, 3 glucan to periplasmic side of an inner membrane of the prokaryotic host cell, wherein the translocase comprises Wzm-Wzt from Klebsiella sp., optionally from Klebsiella pneumoniae, wherein the ⁇ -1,3 glucan polymer is linked to a lipid carrier via the GlcNAc and wherein the ⁇ -1,3 glucan polymer comprises at least four ⁇ -1,3 linked glucose molecules.
  • the ⁇ -1,3 glucan polymer is a fungal ⁇ -1,3 glucan polymer.
  • the fungal ⁇ -1,3 glucan polymer is of Candida.
  • the fungal ⁇ -1,3 glucan polymer is of Candida albicans.
  • the ⁇ -1,3 glucan polymer has the structure: or 6-25. In other embodiments, the ⁇ -1,3 glucan polymer has the structure: or 6-25.
  • the ⁇ -1,3 glucan polymer has the structure: [ ⁇ 3)- ⁇ -D-Glcp-(1 ⁇ ] n ⁇ 3)- ⁇ -D-Glcp-(1 ⁇ 6)- ⁇ -D-Glcp-(1 ⁇ 6)- ⁇ -D-Glcp-(1 ⁇ 4)- ⁇ -D-Glcp- (1 ⁇ 4)- ⁇ -D-Glcp-(1 ⁇ 3)- x-D-GlcpNAc wherein n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.
  • the prokaryotic host cell is a bacterial cell.
  • the bacteria is a gram positive bacteria.
  • the bacteria is a gram negative bacteria.
  • the bacteria includes, but is not limited to, an Escherichia species, a Shigella species, Klebsiella species, a Xhantomonas species, a Salmonella species, a Yersinia species, a Lactococcus species, a Lactobacillus species, a Pseudomonas species, a Corynebacterium species, a Streptomyces species, a Streptococcus species, a Staphylococcus species, a Bacillus species, and a Clostridium species.
  • the bacteria is an E. coli species. In specific aspects, the bacteria is E. coli.
  • the prokaryotic host cell includes, but is not limited to, an Escherichia species, a Shigella species, Klebsiella species, a Xhantomonas species, a Salmonella species, a Yersinia species, a Lactococcus species, a Lactobacillus species, a Pseudomonas species, a Corynebacterium species, a Streptomyces species, a Streptococcus species, a Staphylococcus species, a Bacillus species, and a Clostridium species.
  • the prokaryotic host cell is an E. coli species. In specific embodiments, the prokaryotic host cell is E. coli.
  • the GlcNAc is linked to a lipid carrier by WecA from the host E. coli cell.
  • the lipid carrier is undecaprenyl.
  • Bioconjugates the present invention provides a bioconjugate comprising a modified Als3 protein of the invention linked to an antigen, as described herein.
  • the antigen is linked to an amino acid on the modified Als3 protein 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.
  • the present invention provides a method of producing a bioconjugate that comprises a modified Als3 protein of the invention linked to at least one saccharide antigen, the method comprising: (1) culturing a host cell of the invention under conditions suitable for the production of proteins; and (2) isolating the bioconjugate produced by a host cell of the invention.
  • the bioconjugate is isolated or purified from a whole cell extract of the host cell.
  • the bioconjugate is isolated or purified from a cytoplasmic extract from the host cell.
  • the bioconjugate is isolated or purified from a periplasmic extract from the host cell.
  • Methods of making Biocojugates are known in the art.
  • Bioconjugates of the invention 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 bioconjugates of the invention.
  • the aim is to increase glycosylation efficiency and recombinant protein yield per cell and while maintaining simplicity and reproducibility in the process.
  • Bioconjugates of the invention can be manufactured on a commercial scale by developing an optimized manufacturing method using typical E. coli production processes.
  • a bioconjugate of the invention comprises at least one saccharide antigen linked to a modified Als3 protein of the invention.
  • the bioconjugates of the invention 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.
  • the bioconjugates of the invention may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.
  • the present invention provides a method of producing a glycoconjugate comprising a modified carrier protein and at least one saccharide antigen.
  • the term “glycoconjugate” refers to a hybrid molecule composed of a carrier protein and multiple polysaccharide chains, wherein the polysaccharides are covalently linked to the carrier protein.
  • the modified carrier protein of the invention includes, but is not limited to, Als3, Sap2, detoxified Exotoxin A of P.
  • aeruginosa EPA
  • CRM197 Diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of S. aureus, clumping factor A of S. aureus, clumping factor B of S. aureus, E. coli FimH, E. coli FimHC, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli sat protein, the passenger domain of E. coli sat protein, C. jejuni AcrA, and a C.
  • CTB Cholera toxin B subunit
  • the modified carrier protein is a modified Als3 protein of the invention.
  • the at least one saccharide antigen comprises a ⁇ -1,3 glucan polymer.
  • the ⁇ -1,3 glucan polymer comprises at least four ⁇ -1,3 linked glucose molecules.
  • the ⁇ -1,3 glucan polymer comprises four to hundred, four to fifty, four to forty, four to thirty five, four to thirty, four to twenty five, four to twenty, four to ten, four to nine, four to eight, four to seven, four to six, four to five, four, five to hundred, five to fifty, five to forty, five to thirty five, five to thirty, five to twenty five, five to twenty, five to ten, five to nine, five to eight, five to seven, five to six, five, six to hundred, six to fifty, six to forty, six to thirty five, six to thirty, six to twenty five, six to twenty, six to ten, six to nine, six to eight, six to seven, six, seven to hundred, seven to fifty, seven to forty, seven to thirty five, seven to thirty, seven to twenty five, seven to twenty, seven to ten, seven to nine, seven to eight, or seven ⁇ -1,3 linked glucose molecules.
  • the ⁇ -1,3 glucan polymer comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 consecutive ⁇ -1,3 linked glucose molecules.
  • the ⁇ -1,3 glucan polymer comprises at least 11 ⁇ -1,3 linked glucose molecules.
  • the ⁇ -1,3 glucan polymer comprises at least 11 consecutive ⁇ -1,3 linked glucose molecules.
  • the present invention provides a method of producing a glycoconjugate comprising a modified carrier protein and a ⁇ -1,3 glucan.
  • the method of producing a glycoconjugate comprising a modified carrier protein and a ⁇ -1,3 glucan comprises culturing the host cell the invention under conditions suitable for the production of proteins.
  • the present invention provides a method of producing a bioconjugate in a host cell of the invention, the method comprising the steps of: i. obtaining a host cell of the invention that produces a ⁇ -1,3 glucan polymer; and ii. further introducing and expressing in the host cell: a.
  • nucleotide sequence encoding a modified carrier protein comprising at least one glycosylation site comprising a consensus sequence D/E-X-N-Z-S/T, wherein X and Z are any amino acid except proline, and wherein the modified carrier protein further comprises an N-terminal bacterial signal sequence capable of transporting the modified carrier protein to the periplasmic side of the inner membrane of the host cell; and b. a nucleotide sequence encoding an oligosaccharyl transferase capable of producing a bioconjugate by transferring the ⁇ -1,3 glucan polymer from a lipid carrier to the modified carrier protein.
  • the present invention provides a method of producing a bioconjugate in a prokaryotic host cell, the method comprising the steps of: a. obtaining a prokaryotic host cell of the invention that produces a ⁇ -1,3 glucan polymer; and b. further introducing and expressing in the host cell: i.
  • a nucleotide sequence encoding a modified carrier protein comprising a glycosylation site comprising a consensus sequence D/E-X-N-Z-S/T, wherein X and Z are any amino acid except proline, and wherein the modified carrier protein further comprises an N-terminal bacterial signal sequence capable of transporting the modified carrier protein to the periplasmic side of the inner membrane of the prokaryotic host cell; and ii.
  • the present invention provides a method of producing a bioconjugate in a prokaryotic host cell, the method comprising the steps of: c. obtaining a prokaryotic host cell of the invention that produces a ⁇ -1,3 glucan polymer; and d. further introducing and expressing in the host cell: iii.
  • nucleotide sequence encoding a modified carrier protein comprising a glycosylation site comprising a consensus sequence D/E-X-N-Z-S/T, wherein X and Z are any amino acid except proline (optionally a polynucleotide sequence of the invention), and wherein the modified carrier protein further comprises an N-terminal bacterial signal sequence capable of transporting the modified carrier protein to the periplasmic side of the inner membrane of the prokaryotic host cell; and a nucleotide sequence encoding an oligosaccharyl transferase capable of producing a bioconjugate by transferring the ⁇ -1,3 glucan polymer from a lipid carrier to the modified carrier protein, wherein the oligosaccharyl transferase is PglB from Campylobacter, optionally from Campylobacter jejuni or Campylobacter coli.
  • the modified carrier protein of the invention includes, but is not limited to, Als3, Sap2, detoxified Exotoxin A of P. aeruginosa (EPA), CRM197, Diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of S. aureus, clumping factor A of S. aureus, clumping factor B of S. aureus, E. coli FimH, E. coli FimHC, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, E.
  • CTB Cholera toxin B subunit
  • the modified carrier protein is a modified Als3 protein of the invention.
  • the bacterial signal sequence is selected from, without limitation, Flgl, MalE, OmpA, and OmpC.
  • the bacterial signal sequence is Flgl.
  • the Flgl comprises an amino acid sequence of SEQ ID NO: 21.
  • the bacterial signal sequence is removed from the modified carrier protein after the modified carrier protein is transported to the periplasmic side of the inner membrane of the prokaryotic host cell.
  • 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 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.
  • the free glycans are purified on carbon columns and subsequently labeled at the reducing end with the fluorophor 2-amino benzamide. See Bigge JC, Patel TP, Bruce JA, Goulding PN, Charles SM, Parekh RB, Anal Biochem 1995, 230(2):229-238.
  • the labeled polysaccharides are separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al.. See Royle L, Mattu TS, Hart E, Langridge JI, Merry AH, Murphy N, Harvey DJ, Dwek RA, Rudd PM, Anal Biochem 2002, 304(1):70-90.
  • the resulting fluorescence chromatogram indicates the polysaccharide length and number of repeating units.
  • Structural information can be gathered by collecting individual peaks and subsequently performing MS/MS analysis. Thereby the monosaccharide composition and sequence of the repeating unit can be confirmed and additionally in homogeneity of the polysaccharide composition can be identified.
  • high mass MS and size exclusion HPLC can be applied to measure the size of the complete bioconjugates. Yield may be measured as carbohydrate amount derived from a liter of bacterial production culture grown in a bioreactor under controlled and optimized conditions. After purification of bioconjugate, the carbohydrate yields can be directly measured by either the anthrone assay or ELISA using carbohydrate specific antisera.
  • Indirect measurements are possible by using the protein amount (measured by BCA, Lowry, or bardford assays) and the glycan length and structure to calculate a theoretical carbohydrate amount per gram of protein.
  • yield can also be measured by drying the glycoprotein preparation from a volatile buffer and using a balance to measure the weight.
  • Various methods can be used to analyze the conjugates of the invention including, for example, SDS-PAGE or capillary gel electrophoresis. Polymer length is defined by the number of repeat units that are linearly assembled. This means that the typical ladder like pattern is a consequence of different repeat unit numbers that compose the glycan. Thus, two bands next to each other in SDS PAGE (or other techniques that separate by size) differ by only a single repeat unit.
  • glycoproteins for glycan size the unglycosylated carrier protein and the bioconjugate with different polymer chain lengths separate according to their electrophoretic mobilities.
  • the first detectable repeat unit number (n1) and the average repeat unit number (n average ) present on a bioconjugate are measured. These parameters can be used to demonstrate batch to batch consistency or polysaccharide stability, for example.
  • Glycosylation site usage may be quantified by, for example, glycopeptide LC-MS/MS: conjugates are digested with protease(s), and the peptides are separated by a suitable chromatographic method (C18, Hydrophilic interaction HPLC HILIC, GlycoSepN columns, SE HPLC, AE HPLC), and the different peptides are identified using MS/MS. This method can be used with or without previous sugar chain shortening by chemical (smith degradation) or enzymatic methods. Quantification of glycopeptide peaks using UV detection at 215 to 280nm allows relative determination of glycosylation site usage.
  • site usage may be quantified by size exclusion HPLC: Higher glycosylation 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 Briliant Blue following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • the conjugates (e.g. bioconjugate), of the invention are particularly suited for inclusion in immunogenic compositions and vaccines.
  • the present invention provides an immunogenic composition comprising a conjugate or a bioconjugate of the invention.
  • the immunogenic composition additionally comprises a pharmaceutically acceptable excipient and/or carrier.
  • the present invention provides an immunogenic composition comprising a modified Als3 protein of the invention, a conjugate of the invention, or a bioconjugate of the invention.
  • the immunogenic composition additionally comprises a pharmaceutically acceptable excipient and/or carrier.
  • Immunogenic compositions comprise an immunologically effective amount of a modified Als3 protein of the invention, or conjugate (e.g. bioconjugate) of the invention, as well as any other components.
  • immunologically effective amount it is meant that the administration of that amount to an individual, either as a single dose or as part of a series is effective for treatment or prevention.
  • compositions and carriers are described, for example, in Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co. Easton, PA, 5th Edition (1975).
  • Pharmaceutically acceptable excipients can include a buffer, such as a phosphate buffer (e.g. sodium phosphate).
  • Pharmaceutically acceptable excipients can include a salt, for example sodium chloride.
  • Pharmaceutically acceptable excipients can include a solubilizing/stabilizing agent, for example, polysorbate (e.g. TWEEN 80).
  • compositions of the invention can include a preservative, for example 2-phenoxyethanol or thiomersal.
  • Pharmaceutically acceptable excipients can include a carrier such as water or saline.
  • the present invention provides a method of making the immunogenic composition of the invention comprising the step of mixing a modified Als3 protein of the invention, a conjugate of the invention, or a bioconjugate of the invention, with a pharmaceutically acceptable excipient or carrier.
  • an immunogenic composition of the invention is formulated as a vaccine for in vivo administration to a subject (e.g. human) wherein the individual components of the composition are formulated such that the immunogenicity of individual components is not impaired by other individual components of the composition (see above definition).
  • an immunogenic composition of the invention is formulated as a vaccine for in vivo administration to a subject (e.g. human), which confers an antibody titre superior to the criterion for seroprotection for each antigenic component for an acceptable percentage of human subjects.
  • a vaccine comprising an immunogenic composition of the invention.
  • the vaccine additionally comprises a pharmaceutically acceptable excipient or carrier.
  • the vaccine additionally comprises an adjuvant.
  • the present invention provides a vaccine comprising an immunogenic composition of the invention and, optionally, a pharmaceutically acceptable excipient or carrier.
  • the present invention provides a Candida albicans vaccine comprising: (1) a modified Als3 protein of the invention; (2) at least one Candida albicans saccharide antigen linked to said modified Als3 protein; and, optionally, (3) a pharmaceutically acceptable carrier or adjuvant.
  • adjuvant refers to a compound that when administered in conjunction with or as part of an immunogenic composition of vaccine of the invention augments, enhances and/or boosts the immune response to modified Als3 protein conjugate/bioconjugate, but when the compound is administered alone does not generate an immune response to the modified Als3 protein conjugate/bioconjugate.
  • 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 include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (see United Kingdom Patent GB2220211), MF59 (Novartis), AS01 (GlaxoSmithKline), and saponins, such as QS21 (see Kensil et al. in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540).
  • the adjuvant is Freund’s adjuvant (complete or incomplete).
  • 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)).
  • a suitable adjuvant is an adjuvant comprising an oil in water emulsion, wherein said oil in water emulsion comprises a metabolisable oil, a tocol and an emulsifier.
  • the metabolisable oil is squalene
  • the tocol is alpha-tocopherol
  • the emulsifying agent is polyoxyethylene sorbitan monooleate.
  • the adjuvant includes an oil-in-water emulsion.
  • the oil- in-water emulsion can include an oil phase that incorporates a metabolisable oil, and an additional oil phase component, such as a tocol.
  • the oil-in-water emulsion may also contain an aqueous component, such as a buffered saline solution (e.g., phosphate buffered saline).
  • the oil- in-water emulsion typically contains an emulsifier.
  • the metabolizable oil is squalene.
  • the tocol is alpha-tocopherol.
  • the emulsifier is a nonionic surfactant emulsifier (such as polyoxyethethylene sorbitan monooleate, TWEEN80TM).
  • the oil-in-water emulsion contains squalene and alpha tocopherol in a ratio which is equal or less than 1 (w/w).
  • the metabolisable oil in the oil-in-water emulsion may be present in an amount of 0.5-10mg.
  • the tocol in the oil-in-water emulsion may be present in an amount of 0.5 – 11 mg.
  • the emulsifying agent may be present in an amount of 0.4 – 4 mg.
  • the oil phase of the emulsion system has to comprise a metabolisable oil.
  • metabolisable oil is well known in the art. Metabolisable can be defined as ‘being capable of being transformed by metabolism’ (Dorland’s Illustrated Medical Dictionary, W.B. Sanders Company, 25th edition (1974)).
  • the oil may be any vegetable oil, fish oil, animal oil or synthetic oil, which is not toxic to the recipient and is capable of being transformed by metabolism. Nuts, seeds, and grains are common sources of vegetable oils.
  • Synthetic oils are also part of this invention and can include commercially available oils such as NEOBEE ⁇ (caprylic/capric triglycerides made using glycerol from vegetable oil sources and medium-chain fatty acids (MCTs) from coconut or palm kernel oils) and others.
  • a particularly suitable metabolisable oil is squalene.
  • Squalene (2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22- tetracosahexaene) is an unsaturated oil which is found in large quantities in shark-liver oil, and in lower quantities in olive oil, wheat germ oil, rice bran oil, and yeast, and is a particularly preferred oil for use in this invention.
  • Squalene is a metabolisable oil by virtue of the fact that it is an intermediate in the biosynthesis of cholesterol (Merck index, 10th Edition, entry no.8619).
  • Tocols are well known in the art and are described in EP0382271.
  • the tocol is alpha- tocopherol or a derivative thereof such as alpha-tocopherol succinate (also known as vitamin E succinate).
  • said tocol is suitably present in in an amount of 0.5-11 mg.
  • the oil in water emulsion further comprises an emulsifying agent.
  • the emulsifying agent may suitably be polyoxyethylene sorbitan monooleate.
  • the emulsifying agent may be Polysorbate® 80 (Polyoxyethylene (20) sorbitan monooleate) or Tween® 80.
  • said emulsifying agent is suitably present in the adjuvant composition in an amount of 0.4-4mg.
  • a method of making the immunogenic composition of the invention comprising the step of mixing the modified Als3 protein or the conjugate (e.g. bioconjugate) of the invention with a pharmaceutically acceptable excipient and/or carrier and an adjuvant.
  • Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M.F. & Newman M.J.) (1995) Plenum Press New York).
  • the immunogenic compositions of the invention can be included in a container, pack, or dispenser together with instructions for administration.
  • the immunogenic compositions or vaccines of the invention 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 the invention may be stored in solution or lyophilized. In certain embodiments, the solution is lyophilized in the presence of a sugar such as sucrose, trehalose or lactose. In additional embodiments, the vaccines of the invention are lyophilized and extemporaneously reconstituted prior to use.
  • Immunogenic compositions or vaccines of the invention may be used to protect or treat a subject (e.g. human), by means of administering said immunogenic composition or vaccine via systemic or mucosal route.
  • administrations may include injection via the intramuscular (IM), intraperitoneal, intradermal (ID) or subcutaneous (SC) routes; or via mucosal administration to the oral/alimentary, respiratory, genitourinary tracts.
  • the immunogenic composition or vaccine of the invention 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 ml.
  • the immunogenic composition or vaccine of the invention is administered by the intradermal administration.
  • Human skin comprises an outer "horny" cuticle, called the stratum corneum, 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.
  • the immunogenic composition or vaccine of the invention 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. It is desirable to use 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 the invention are spray devices.
  • Suitable commercially available nasal spray devices include ACCUSPRAYTM (Becton Dickinson).
  • the amount of conjugate (e.g. 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 a conjugate (e.g. a bioconjugate) will typically be in the range 1-100 ⁇ g, suitably 5-50 ⁇ g for the sachcharide dose, e.g., glucan dose.
  • the present invention also provides an immunogenic composition of the invention, or the vaccine of the invention, for use in medicine.
  • the use of an immunogenic composition of the invention in the manufacture of a medicament for the treatment or prevention of diseases caused by infection by C. albicans is also envisioned, as is a method of immunising a subject (e.g. a human) against disease caused by C. albicans, which method comprises administering to the subject an immunoprotective dose of an immunogenic composition of the invention.
  • the present invention provides a method of inducing immune response to a fungal infection in a subject (e.g.
  • the method comprising administering to the subject a therapeutically or prophylactically effective amount of a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention.
  • the modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention can be used to induce an immune response against fungi.
  • the fungi is Candida species.
  • the Candida species includes, but is not limited to, Candida albicans, Candida auris, Candida guilliermondi, Candida lusitaniaea and Candida tropicalis, Candida glabrata, Candida krusei, and Candida parapsilosis.
  • the Candida species is Candida albicans.
  • said subject has fungal infection at the time of administration.
  • said subject does not have a fungal infection at the time of administration.
  • the present invention provides a method of inducing immune response to Candida albicans infection in a subject (e.g.
  • the method comprising administering to the subject a therapeutically or prophylactically effective amount of a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention.
  • the present invention also provides methods of treating and/or preventing a fungal infection in a subject comprising administering to the subject a conjugate (e.g. bioconjugate) of the invention.
  • the conjugate e.g. bioconjugate
  • the conjugate may be in the form of an immunogenic composition or vaccine.
  • the present invention provides a method for treatment or prevention of fungal infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention.
  • the conjugate e.g. bioconjugate
  • the present invention provides a method for immunizing a subject against fungal infection, the method comprising administering to the subject an immunoprotective dose of a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of any of the invention, an immunogenic composition of the invention, or a vaccine of the invention.
  • the present invention provides a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention for use in the manufacture of a medicament for the treatment or prevention of a disease caused by fungal infection in a subject (e.g. human).
  • the fungi is Candida species.
  • the Candida species includes, but is not limited to, Candida albicans, Candida auris, Candida guilliermondi, Candida lusitaniaea and Candida tropicalis, Candida glabrata, Candida krusei, and Candida parapsilosis.
  • the Candida species is Candida albicans.
  • the present invention provides a method for treatment or prevention of Candida albicans infection in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention.
  • the present invention provides a method for immunizing a subject against Candida albicans infection, the method comprising administering to the subject an immunoprotective dose of a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of any of the invention, an immunogenic composition of the invention, or a vaccine of the invention.
  • the present invention provides a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention for use in the manufacture of a medicament for the treatment or prevention of a disease caused by Candida albicans infection in a subject (e.g. human).
  • the present invention provides a method of inhibiting adhesion of Candida albicans hyphae to vaginal epithelial cells in a subject (e.g. human), the method comprising administering to the subject a therapeutically or prophylactically effective amount of a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention.
  • the present invention provides a method of mediating neutrophile killing of Candida albicans hyphae in a subject (e.g.
  • the method comprising administering to the subject a therapeutically or prophylactically effective amount of a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention.
  • the present invention provides a method of inhibiting biofilm formation of Candida albicans, the method comprising: (i) administering to a subject (e.g. human) a therapeutically or prophylactically effective amount of a modified Als3 protein of the invention, a conjugate of the invention, a bioconjugate of the invention, an immunogenic composition of the invention, or a vaccine of the invention.
  • a modified Agglutinin-like sequence 3 (Als3) protein comprising amino acid residues 18-316 of SEQ ID NO: 1 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1, modified in that the amino acid sequence comprises one or more consensus sequences comprising an amino acid sequence of D/E-X-N-Z-S/T, wherein X and Z are independently any amino acid except proline.
  • amino acids between amino acid residues 168-172 one or more amino acids between amino acid residues 170-184 (e.g. one or more amino acids between amino acid residues 175-179), one or more amino acids between amino acid residues 202-212, one or more amino acids between amino acid residues 215-225, one or more amino acids between amino acid residues 231-242, one or more amino acids between amino acid residues 265-275, one or more amino acids between amino acid residues 271-281, one or more amino acids between amino acid residues 281-292, and one or more amino acids between amino acid residues 294-305 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • the modified Als3 protein of paragraph 20 wherein the expression level of the modified Als3 protein is increased at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold relative to the control Als3 protein.
  • the modified Als3 protein of paragraph 23 wherein the Candida is selected from the group consisting of Candida albicans, Candida auris, Candida guilliermondi, Candida lusitaniaea and Candida tropicalis.
  • Fba Fructose biphosphate aldolase-1
  • the modified Als3 protein of paragraph 32 wherein X is Q (glutamine), Z is A (alanine) and the one or more consensus sequences are selected from the group consisting of KDQNAT (SEQ ID NO: 5), KDQNAS (SEQ ID NO: 6) and DQNAT (SEQ ID NO: 7). 34.
  • a modified Als3 protein comprising an amino acid sequence of SEQ ID NO: 10.
  • a modified Als3 protein comprising an amino acid sequence of SEQ ID NO: 11.
  • O antigens Legionella pneumophila serotypes 1 to 15 O antigens, Bordetella parapertussis O antigens, Burkholderia mallei and pseudomallei O antigens, Francisella tularensis O antigens, Campylobacter sp.
  • O antigens capsular polysaccharides of Clostridium difficile, Staphylococcus aureus type 5 and 8, Streptococcus pyrogenes, E.
  • coli Streptococcus agalacticae, Neisseria meningitidis, Candida sp., Candida albicans, Haemophilus influenza, Enterococcus faecalis capsular polysaccharides type I-V, and other surface polysaccharide structures, e.g. the Borrelia burgdorferi glycolipids, Neisseria meningitidis pilin O glycan and lipooligosaccharide (LOS), Haemophilus influenza LOS, Leishmania major lipophosphoglycan, tumor associated carbohydrate antigens , malaria glycosyl phosphatidylinositol, and mycobacterium tuberculosis arabinomannan. 41.
  • LOS Lipooligosaccharide
  • ⁇ -1,3 glucan polymer comprises four to hundred, four to fifty, four to forty, four to thirty five, four to thirty, four to twenty five, four to twenty, four to ten, four to nine, four to eight, four to seven, four to six, four to five, four, five to hundred, five to fifty, five to forty, five to thirty five, five to thirty, five to twenty five, five to twenty, five to ten, five to nine, five to eight, five to seven, five to six, five, six to hundred, six to fifty, six to forty, six to thirty five, six to thirty, six to twenty five, six to twenty, six to ten, six to nine, six to eight, six to seven, six, seven to hundred, seven to fifty, seven to forty, seven to thirty five, seven to thirty, seven to twenty five, seven to twenty, seven to ten, seven to nine, seven to eight, or seven ⁇ -1,3 linked glucose molecules.
  • the ⁇ -1,2 mannan polymer comprises two to fifty, two to forty, two to thirty, two to twenty, two to ten, two to nine, two to eight, two to seven, two to six, two to five, two to four, two to three, two, three to fifty, three to forty, three to thirty, three to twenty, three to ten, three to nine, three to eight, three to seven, three to six, three to five, three to four, three, four to fifty, four to forty, four to thirty, four to twenty, four to ten, four to nine, four to eight, four to seven, four to six, four to five, four, five to fifty, five to forty, five to thirty, five to twenty, five to ten, five to nine, five to eight, five to seven, five to six, or five, six to forty, six to thirty, six to twenty, six to ten, six to nine, six to eight, six to seven, six, seven to fifty, seven to forty, seven to thirty, seven to twenty, six to ten, six to nine, six to eight, six
  • a modified Als3 protein of Candida albicans consisting of: (1) an amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11; and (2) at least one saccharide antigen of Candida, wherein the at least one saccharide antigen is a ⁇ -1,3 glucan polymer consisting of at least six consecutive ⁇ -1,3 linked glucose molecules, and wherein the at least one saccharide antigen is linked to at least one of three asparagine residues at positions 20, 92, and 324 of SEQ ID NO: 10 or positions 20, 92, and 337 of SEQ ID NO: 11.
  • a vector comprising the polynucleotide sequence of paragraph 57.
  • a host cell comprising: (1) one or more polynucleotide sequences that encode one or more heterologous glycosyltransferases; (2) a polynucleotide sequence that encodes a heterologous oligosaccharyl transferase; (3) a polynucleotide sequence that encodes a modified Als3 protein according to any of paragraphs 1 to 35; and, optionally, (4) a polynucleotide sequence that encodes a polymerase.
  • a method of producing a bioconjugate that comprises a modified Als3 protein linked to at least one saccharide antigen comprising: (1) culturing the host cell of any of paragraphs 59 and 60 under conditions suitable for the production of proteins; and (2) isolating the bioconjugate.
  • An immunogenic composition comprising the modified Als3 protein of any of paragraphs 1 to 37 and 56, the conjugate of any of paragraphs 38 to 54, or the bioconjugate of any of paragraphs 55 and 62. 64.
  • a method of making the immunogenic composition of paragraph 63 comprising the step of mixing the modified Als3 protein of any of paragraphs 1 to 37 and 56, the conjugate of any of paragraphs 38 to 54, or the bioconjugate any of paragraphs 55 and 62, with a pharmaceutically acceptable excipient or carrier.
  • a vaccine comprising the immunogenic composition of paragraph 63 and, optionally, a pharmaceutically acceptable excipient or carrier.
  • a Candida albicans vaccine comprising: (1) the modified Als3 protein of any of paragraphs 1 to 35; (2) at least one Candida albicans saccharide antigen linked to said modified Als3 protein; and, optionally, (3) a pharmaceutically acceptable carrier or adjuvant.
  • a method for treatment or prevention of Candida albicans infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the modified Als3 protein of any of paragraph 1 to 37 and 56, the conjugate of any of paragraphs 38 to 55, the bioconjugate of any of paragraphs 55 and 62, the immunogenic composition of paragraph 63, or the vaccine of any of paragraphs 65 and 66.
  • the method of paragraph 54 wherein the Candida albicans infection causes Recurrent Vulvovaginal Candidiasis (RVVC) in the subject.
  • RVVC Recurrent Vulvovaginal Candidiasis
  • a method for immunizing a subject against Candida albicans infection comprising administering to the subject an immunoprotective dose of the modified Als3 protein of any of paragraphs 1 to 37 and 56, the conjugate of any of paragraphs 38 to 55, the bioconjugate of any of paragraphs 55 and 62, the immunogenic composition of paragraph 63, or the vaccine of any of paragraphs 65 and 66.
  • a method for inducing immune response to Candida albicans infection in a subject comprising administering to the subject a therapeutically or prophylactically effective amount of the modified Als3 protein of any of paragraphs 1 to 37 and 56, the conjugate of any of paragraphs 38 to 55, the bioconjugate of any of paragraphs 55 and 62, the immunogenic composition of paragraph 63, or the vaccine of any of paragraphs 65 and 66.
  • the modified Als3 protein of any of paragraphs 1 to 37 and 56, the conjugate of any of paragraphs 38 to 55, the bioconjugate of any of paragraphs 55 and 62, the immunogenic composition of paragraph 63, or the vaccine of any of paragraphs 65 and 66 for use in treatment or prevention of a disease caused by Candida albicans infection.
  • a method for increasing expression level of the modified Als3 protein of any of paragraphs 1 to 37 comprising substituting the one or more consensus sequences for the amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1, wherein the modified Als3 protein exhibits an increased expression level relative to a control Als3 protein which does not comprise one or more consensus sequences substituted for amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a host cell comprising: i.
  • nucleotide sequence encoding one or more heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,3 glucan polymer; ii. A nucleotide sequence encoding a glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetyl glucosamine (GlcNac) molecule; iii. a nucleotide sequence encoding a heterologous oligosaccharyl transferase; and iv.
  • the one or more heterologous glycosyltransferases of i. comprises exoL, exoM, exoO, exoU and exoW from a rhizobia, optionally from Sinorhizobium, optionally from Sinorhizobium meliloti 1021.
  • the host cell of any one of paragraphs 77 to 87, wherein the glycosyltransferase capable of covalently bonding a glucose to GlcNac of ii. is WfaP from E. coli 056.
  • the host cell of any one of paragraphs 77 to 90, wherein the oligosaccharyl transferase of iii. is a PglB.
  • the modified carrier protein of iv. is selected from the group consisting of Als3, Sap2, detoxified Exotoxin A of P.
  • aeruginosa EPA
  • CRM197 Diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of S. aureus, clumping factor A of S. aureus, clumping factor B of S. aureus, E. coli FimH, E. coli FimHC, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli sat protein, the passenger domain of E. coli sat protein, C. jejuni AcrA, and a C.
  • CTB Cholera toxin B subunit
  • the host cell of paragraph 93 wherein the modified carrier protein is the modified Als3 protein of any one of paragraphs 1 to 35.
  • 95 The host cell of any one of paragraphs 77 to 94, wherein said host cell is an 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, or a Clostridium species.
  • 96 The host cell of paragraph 95, wherein said host cell is an E.
  • a bioconjugate produced by the method of paragraph 97. 99.
  • n 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35 or 6-25.
  • a saccharide which is a glucan having the structure: [ ⁇ 3)- ⁇ -D-Glcp-(1 ⁇ ] n ⁇ 3)- ⁇ -D-Glcp-(1 ⁇ 6)- ⁇ -D-Glcp-(1 ⁇ 6)- ⁇ -D-Glcp-(1 ⁇ 4)- ⁇ -D-Glcp- (1 ⁇ 4)- ⁇ -D-Glcp-(1 ⁇ 3)- x-D-GlcpNAc wherein n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.
  • aeruginosa EPA
  • CRM197 Diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of S. aureus, clumping factor A of S. aureus, clumping factor B of S. aureus, E. coli FimH, E. coli FimHC, E. coli heat labile enterotoxin, detoxified variants of E. coli heat labile enterotoxin, Cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, E. coli sat protein, the passenger domain of E. coli sat protein, C. jejuni AcrA, and a C.
  • CTB Cholera toxin B subunit
  • a method of producing a ⁇ -1,3 glucan polymer in a prokaryotic host cell comprising the steps of introducing and expressing in the host cell: i.
  • the host cell produces more SleW than SleC, SleE, SleF or SleU; and iii. optionally, a nucleotide sequence encoding a translocase capable of translocating the ⁇ -1, 3 glucan to periplasmic side of an inner membrane of the prokaryotic host cell, wherein the translocase comprises Wzm-Wzt from Klebsiella sp., optionally from Klebsiella pneumoniae, wherein the ⁇ -1,3 glucan polymer is linked to a lipid carrier via the GlcNAc and wherein the ⁇ -1,3 glucan polymer comprises at least four ⁇ -1,3 linked glucose molecules. 110.
  • a method of producing a bioconjugate in a prokaryotic host cell comprising the steps of: a. obtaining a prokaryotic host cell of any of paragraphs 109 to 114 that produces a ⁇ - 1,3 glucan polymer; and b. further introducing and expressing in the host cell: i.
  • a nucleotide sequence encoding a modified carrier protein comprising a glycosylation site comprising a consensus sequence D/E-X-N-Z-S/T, wherein X and Z are any amino acid except proline, and wherein the modified carrier protein further comprises an N-terminal bacterial signal sequence capable of transporting the modified carrier protein to the periplasmic side of the inner membrane of the prokaryotic host cell; and ii.
  • the bacterial signal sequence is selected from the group consisting of: Flgl, MalE, OmpA, and OmpC.
  • the method of paragraph 116, wherein the bacterial signal sequence is Flgl. 118.
  • a method for increasing expression level of the modified Als3 protein of any of paragraphs 1 to 37 in a host cell comprising substituting the one or more consensus sequences for the amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1, wherein the modified Als3 protein when expressed in a host cell exhibits an increased expression level relative to a control Als3 protein which does not comprise one or more consensus sequences substituted for amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.
  • a method of producing a bioconjugate in a prokaryotic host cell comprising the steps of: a. obtaining a prokaryotic host cell of any of paragraphs 109 to 114 that produces a ⁇ - 1,3 glucan polymer; and b. further introducing and expressing in the host cell: i.
  • a host cell comprising: (1) one or more polynucleotide sequences that encode one or more heterologous glycosyltransferases; (2) a polynucleotide sequence that encodes a glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetyl glucosamine (GlcNac) molecule; (3) a polynucleotide sequence that encodes a heterologous oligosaccharyl transferase; (4) a polynucleotide sequence that encodes a modified Als3 protein according to any of paragraphs 1 to 35; and, optionally, a polynucleotide sequence that encodes a polymerase. .
  • a host cell comprising: i. a nucleotide sequence encoding one or more heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,3 glucan polymer; ii. A nucleotide sequence encoding a glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetyl glucosamine (GlcNac) molecule; iii.
  • a method of inhibiting adhesion of Candida albicans hyphae to vaginal epithelial cells in a subject e.g.
  • a method of mediating neutrophile killing of Candida albicans hyphae in a subject comprising administering to the subject a therapeutically or prophylactically effective amount of the modified Als3 protein of any of paragraphs 1 to 37 and 56, the conjugate of any of paragraphs 38 to 55, the bioconjugate of any of paragraphs 55 and 62, the immunogenic composition of paragraph 63, or the vaccine of any of paragraphs 65 and 66.
  • a method of mediating neutrophile killing of Candida albicans hyphae in a subject e.g.
  • a method of inhibiting biofilm formation of Candida albicans comprising: (i) administering to a subject (e.g.
  • SEQ ID NO: 27 Als3-NT (amino acids 18-316 of SEQ ID NO: 1) sequence of Candida albicans: SKTITGVFNSFNSLTWSNAATYNYKGPGTPTWNAVLGWSLDGTSASPGDTFTLNMPCVFKFTTSQTSVDLTAH GVKYATCQFQAGEEFMTFSTLTCTVSNTLTPSIKALGTVTLPLAFNVGGTGSSVDLEDSKCFTAGTNTVTFNDGG KKISINVDFERSNVDPKGYLTDSRVIPSLNKVSTLFVAPQCANGYTSGTMGFANTYGDVQIDCSNIHVGITKGLN DWNYPVSSESFSYTKTCSSNGIFITYKNVPAGYRPFVDAYISATDVNSYTLSYANEYTCAGGYWQRAPFTLRWT GYR 20 positions in total were selected for insertion of the consensus sequence for glycosylation i.e., glycosite (e.g.
  • D/E-X-N-Z-S/T by site directed mutagenesis (Table 1 and FIG.1). These positions included the N- and C-termini of the Als3-NT domain as well as its solvent accessible loops and some beta-strands.
  • One or more (up to 5) amino acids were substituted by the glycosite sequence to create a “modified Als3-NT protein.” In some cases, the glycosite was inserted in between two amino acids of the Als3-NT protein without creating any sequence substitution.
  • Modified Als3-NT proteins comprising single glycosites were tested for glycosylation with Klebsiella pneumoniae O5 antigen. The best performing glycosites were combined to generate modified Als3-NT proteins comprising between two to six glycosites in total.
  • the six positions that were selected for combinations were: 33-37 (Mut1), 104-108 (Mut4), 163-164 (Mut8), 220 (Mut12), 299-300 (Mut18), and 316 (C-terminus), where the numbering corresponds to the residues of the native Candida albicans Als3 sequence (SEQ ID NO: 1).
  • Modified Als3-NT proteins comprising a single inserted glycosite were tested for in vivo glycosylation efficiency using Klebsiella pneumoniae O5 antigen. For the data set presented in this work, the used E.
  • coli W3110-derivative strain included the deletion of the LPS-O antigen ligase waaL and contained the cluster of genes for the biosynthesis of Klebsiella pneumoniae O5 glycan replacing the native O antigen cluster rfbO16.
  • the E. coli strain producing KpO5 glycan was transformed with a pEC415 plasmid carrying a modified Als3-NT protein and a plasmid expressing PglB.
  • 5 ml TB medium comprising 10 mM MgCl2 and appropriate antibiotics was inoculated with a streak of colonies from the transformation plate and grown at 37°C o/n.
  • Als3 arabinose
  • PglB 0.1 mM IPTG
  • the expression and glycosylation of modified Als3-NT proteins was continued at 25°C overnight.
  • the selection criteria for modified Als3-NT proteins with single glycosite included the total expression level and the level of produced glycoconjugate, the later indicating suitability of glycosite position for modification by PglB.
  • lysis buffer 30 mM Tris-HCl pH 8.5, 1 mM EDTA (Ethylenediaminetetraacetic acid), 20% sucrose
  • IMAC immobilized metal affinity chromatography
  • the IMAC enriched PPE was analysed by SDS-PAGE. “Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4", Nature, 227 (5259): 680–685. Bibcode:1970Natur.227..680L. doi:10.1038/227680a0. ISSN 0028-0836. PMID 5432063). Unglycosylated Als3-NT proteins and glycoconjugates (i.e. modified Als3-NT proteins linked to one or more polysaccharide chains) glycosylated at one or more positions were detected on the gel by Coomassie staining (Fazekas de St. Groth, S.; Webster, R.
  • coli K12 W3110-derivative strain was constructed by subsequent replacements of the targeted gene clusters with the gene of interest, if any, followed by an FRT sites-flanked selection marker via ⁇ -Red homologous recombination followed by FLP recombinase-catalysed marker removal as described (TE Kuhlman and EC Cox. Nucleic Acids Res. 2010 Apr; 38(6): e92). Five homologous recombination / marker removal steps were carried out, removing genomic sequences of i. O16 O- antigen cluster (rfb or wb, GenBank NC_007779 position 2’114’113 to 2’103’814), ii.
  • colanic acid cluster (wca, GenBank NC_007779 position 2’138’241 to 2’118’033), iii. ECA cluster retaining wecA (wec, GenBank NC_007779 position 3’666’604 to 3’656’725), iv. O16wzz2 or cld (GenBank NC_007779 position 2’099’458 to 2’100’438), v. gtrABS or yfdGHI (GenBank NC_007779 position 2’473’301 to 2’475’908), vi. araBA (GenBank NC_007779 position 66’835 to 70’048). Moreover, a codon-optimized version of C.
  • jejuni pglB (GenBank WP_002866139) was introduced, replacing the LPS-O antigen ligase waaL (GenBank NC_007779 position 3’842’208 to 3’843’467).
  • An expression plasmid comprising the gene for the selected modified Als3-NT protein, followed by the genes necessary for the ⁇ -1,3-glucan biosynthesis in a pEC415 backbone (Schulz et al. J Biol Chem. 1998 Aug; 281(5380):1197-200) was constructed in different steps of classical restriction cloning and Gibson assembly (Gibson, D.G., et al. (2009) Nat. Methods 6, 343-345) starting from synthetic DNA templates.
  • the final plasmid contains the following genes under arabinose-inducible promoter, from 5’ to 3’: als3-NT, sleW, sleU, sleF, sleE, sleC, wfaP, wzm, and wzt.
  • Modified Als3-NT protein is described above; sleW, sleU, sleF, sleE, sleC sequences are from Agrobacterium sp.
  • ZX09 succingoglucan-like exopolysaccharide biosynthesis gene cluster (GenBank KT780309; Xu et al. Appl Microbiol Biotechnol. (2017) 101:585–598); wfaP sequence is from E.
  • coli O56 (GenBank DQ220293); wzm and wzt are from K. pneumoniae (GenBank CP052562 position 1’695’622 to 1’697’129).
  • a second expression plasmid was constructed by cloning a codon-optimized version of C. jejuni pglB (GenBank WP_002866139) into the vector pEXT21 (Dykxhoorn et al.Gene. 1996. 177(1- 2):133-6) under IPTG-inducible promoter.
  • the conjugate-producing strain was obtained by transforming the engineered strain with the two plasmids.
  • An osmotic shock protocol was applied in which the cells are diluted in a 5-fold volume of H 2 O for 1 hour, realising the content of the periplasm in the supernatant.
  • Cells were separated from the supernatant by centrifugation, and cell debris were removed by filtration through 0.45 and 0.2 ⁇ m filters.
  • the purification of the biconjugate from the filtered supernatant consisted in 4 chromatography steps: i. anion exchange, ii. anion exchange, iii. hydrophobic interaction, iv. size exclusion.
  • the elution profiles were followed with absorbance at 280 nm and SDS- PAGE/Coomassie staining.
  • Example 1 SDS PAGE analysis of Modified Als3-NT protein-glycosite variants purified from PPE by IMAC SDS-PAGE analysis (FIG.
  • FIGS. 3 and 4 correspond to the unglycosylated modified Als3-NT protein (“u carrier”), and to KpO5-modified Als3-NT bioconjugates (“conjugate”) with one occupied glycosite.
  • u carrier unglycosylated modified Als3-NT protein
  • Conjugate KpO5-modified Als3-NT bioconjugates
  • FIGS. 3 and 4 19 out of 20 modified Als3-NT proteins with single glycosite are expressed to a comparable or higher level than wild type (wt) Als3-NT protein (SEQ ID NO: 27).
  • the Mut4 variant has a >2-fold increase in expression compared to wt Als3-NT expression.
  • FIG. 4A shows the relative expression level of the modified Als3-NT proteins comprising a single glycosite compared to expression level of wt Als3-NT.
  • FIG. 4A shows the relative expression level of the modified Als3-NT proteins comprising a single glycosite compared to expression level of wt Als3-NT.
  • FIG. 4B shows the glycosylation efficiency of the modified Als3-NT proteins comprising a single glycosite.
  • Glycosylation of modified Als3-NT protein with KpO5 at 17 of the 19 positions was confirmed to be equally good or superior compared to unglycosylated wt Als3-NT control (FIG. 3, lane 2).
  • Als3-NT variants with a glycosite D/E-X-N-Z-S/T introduced at positions 33-37 (Mut1), 104- 108 (Mut4), 163-164 (Mut8), 220 (Mut12), 299-300 (Mut18), or 316 (C-terminal glycotag) look superior compared to unglycosylated wt Als3-NT control.
  • modified Als3-NT proteins used in this analysis comprised a histine tag, however a skilled person will recognize that modified Als3-NT proteins with the histidine tag removed could also be used for the analysis.
  • the invention provides for modified Als3-NT proteins with the histidine tag removed.
  • Example 2 Western Blot analysis of purified modified Als3-NT protein-glucan conjugate Immunoblot analysis was carried out on periplasmic extract of E.coli strains producing KpO5 antigen polysaccharide and expressing PglB and modified Als3-NT proteins with 1 glycosite introduced at the positions shown in Table 1 above.
  • FIGS. 5A shows SDS-PAGE analysis of purified unglycosylated Als3-NT-Fba (SEQ ID NO: 10) (lane 1) and purified Als3-NT-glucan conjugate (lane 2).
  • M represents the protein standard.
  • FIGS. 5B-5D represent immunoblots probed with anti-Als3 antibody (FIG. 5B), anti-Fba antibody (FIG. 5C), and anti-glucan antibody (FIG. 5D), respectively.
  • FIG. 5E represents recognition of the glucan conjugated to a modified Als3-NT protein by Dectin-1 receptor. The bands in FIGS.
  • 5B-5E correspond to the unglycosylated wt Als3-NT protein (lane 1) and to a modified Als3- NT protein-glucan bioconjugate with three occupied glycosites (lane 2).
  • the SDS-PAGE shows that a modified Als3-NT protein-glucan conjugate could be purified to high purity and that increase in molecular weight compared to unglycosylated Als3-NT-Fba indicates significant glycosylation level with beta-glucan.
  • the four independent Western blot analyses prove antigen identity, since specific antibodies against Als3, Fba peptide and ⁇ -1,3-glucan have been used.
  • Example 3 Assays to show functionality of modified Als3-NT-SPR with fibronectin
  • the adhesive function of Als3 is within the N-terminal (NT) region that carries peptide binding cavity to which various peptidic ligands can bind (Lin, J et al, J. Biol. Chem. 2014; 2. Coleman, DA et al, J. Mol. Meth. 2009; 3.
  • Fibronectin was added to the mobile phase and tested for binding at 8 concentrations (from 450 nM to 3.5 nM). Interaction between modified Als3-NT proteins and Fibronectin was analysed by multi-cycle kinetics. 1:1 binding mode was applied for kinetics fitting. SPR sensograms and the measured parameters are shown in FIG. 6. Conclusion: As shown in FIG.
  • an engineered unglycosylated modified Als3-NT protein (“uAls 318-316 -3S;” middle panel) or a glycosylated modified Als3-NT protein (“ ⁇ -glucan-Als 318-316 -3S;” right panel) shows very similar binding to fibronectin as unmodified wt Als3-NT protein (“Als 318-316 wt;” left panel) with KD values of 264, 272 and 280 nM, respectively. Therefore, the structure and function of Als3 is preserved upon engineering and glycosylation.
  • FIG.7 shows the preclinical testing of modified Als3-NT protein-glucan bioconjugate (Als3-NT- 3S-Fba_bgluc d+ ; Als3-3FG) in rabbit.
  • FIG. 7A shows the bioconjugate attributes.
  • FIG. 7B shows a 3D representation of a modified Als3-NT protein-glucan bioconjugate. Structure of a modified Als3-NT protein is shown as cartoon. Spheres represent positions of the three introduced glycosites. The conjugated beta-glucan chain is schematically represented, the position and the sequence of the Fba peptide sequence is shown in red.
  • FIG. 7 shows the preclinical testing of modified Als3-NT protein-glucan bioconjugate (Als3-NT- 3S-Fba_bgluc d+ ; Als3-3FG) in rabbit.
  • FIG. 7A shows the bioconjugate attributes.
  • FIG. 7B shows a 3D representation of a modified Als3-NT protein-glucan bioconju
  • FIGS. 8A and 8B show the immunogenicity of modified Als3-NT protein-glucan bioconjugate in rabbit.
  • Modified Als3-NT protein-glucan bioconjugate Als3 18-316 -3S-Fba-bgluc d+ ; Control indicates buffer immunized animals, all groups tested with AS03. New Zealand White (NZW) rabbits immunized three times on a two-week interval.
  • FIG. 9 shows the immunogenicity of modified Als3-NT protein-glucan bioconjugate in rabbit.
  • Modified Als3-NT protein-glucan bioconjugate Als3 18-316 -3S-Fba-bgluc d+ ; Control indicates buffer immunized animals, all groups tested with AS03. NZW rabbits immunized three times on a two-week interval.
  • Coating ELISA ⁇ -glucan extract. ⁇ -glucan specific serum IgG concentration (arbitrary units, AU) in pre-, post-II, post-III (d0, d28, d42) rabbit sera by treatment group.
  • FIG. 10 shows the capacity of antibodies against modified Als3-NT protein-glucan bioconjugate to inhibit adhesion of C. albicans hyphae to plastic. Sera was mixed with C. albicans hyphae (ATCC90028), added to plastic wells and incubated for 4hs. After wash, viable cells were measured using CellTiter Glo ® .
  • Modified Als3-NT protein-glucan bioconjugate Als3 18-316 -3S-Fba-bgluc d+ ; Control indicates buffer immunized animals, all groups tested with AS03. NZW rabbits immunized three times on a two-week interval. Graph depicts Mean+SD. ****: p ⁇ 0.0001, one-way ANOVA. Conclusion: As shown in FIG. 10, sera against modified Als3-NT protein-glucan bioconjugate comprising ⁇ -glucan inhibits adhesion of C. albicans hyphae to plastic. Significant reduction of adhesion to plastic wells is shown pre/post-III. Example 7: Quatitative adhesion assay of C. albicans hyphae FIGS.
  • FIG.11A and 11B show the capacity of antibodies against modified Als3-NT protein-glucan bioconjugate to inhibit adhesion of C. albicans to vaginal epithelial cells.
  • FIG.11A adhesion quantification
  • FIG 11B microscopy image of Candida adhered to epithelial cells. Sera was mixed with C. albicans (SC5314), added to epithelial cells (A431) and incubated for 1.5hs. After wash, adhered cells were measured using Concavalin A – Alexa fluor 488.
  • Modified Als3-NT protein-glucan bioconjugate Als3 18-316 -3S-Fba-bgluc d+ ; Control indicates buffer immunized animals, all groups tested with AS03.
  • FIGS. 11A and 11B show the capacity of antibodies against modified Als3-NT protein-glucan bioconjugate to bind to C. albicans cells.
  • FIG. 12 shows antibody binding to C.
  • FIG.13 shows microscopy image of antibodies bound to C. albicans cells. Cells were observed with differential interference contrast microscopy (DIC) and fluorescent microscopy (secondary antibody Alexa 488). Sera was mixed with C. albicans (SC5314) and coloured using Concavalin A – Alexa fluor 488.
  • DIC differential interference contrast microscopy
  • Alexa 488 fluorescent microscopy
  • Sera was mixed with C. albicans (SC5314) and coloured using Concavalin A – Alexa fluor 488.
  • Modified Als3-NT protein-glucan bioconjugate Als3 18-316 -3S-Fba-bgluc d+ ; Control indicates buffer immunized animals, all groups tested with AS03.
  • NZW rabbits immunized three times on a two-week interval As shown in FIG. 12 and in FIG. 13, antibodies against modified Als3-NT protein-glucan bioconjugate are able to bind C. albicans cells coated on plate. Using laser confocal microscopy it is possible to corroborate the binding of the antibodies to the yeast and hyphal segments of the Candida cells.
  • Example 9 Antibody binding to C. auris
  • FIG.14 shows a microscopy image of antibodies bound to C. auris VPCI479/P/13 cells. Cells were observed with differential interference contrast microscopy (DIC) and fluorescent microscopy (secondary antibody Alexa 488). Sera was mixed with C.
  • DIC differential interference contrast microscopy
  • fluorescent microscopy secondary antibody Alexa 488
  • auris VPCI479/P/13 South Asian clade
  • Concavalin A – Alexa fluor 488 Modified Als3-NT protein-glucan bioconjugate: Als3 18- 316 -3S-Fba-bgluc d+ ; Control indicates buffer immunized animals, all groups tested with AS03. NZW rabbits immunized three times on a two-week interval.
  • FIG.14 antibodies against modified Als3-NT protein-glucan bioconjugate are able to bind C. auris cells coated on plate and in Fig 13. Using laser confocal microscopy it is possible to corroborate the binding of the antibodies to C. auris cells.
  • FIG.15 shows the capacity of antibodies against modified Als3-NT protein-glucan bioconjugate to inhibit biofilm formation of C. albicans hyphae on 96-well plates.
  • Sera was added with C. albicans hyphae (SC5314) to plastic wells and incubated for 24hs at 37°C. After wash, viable cells were measured using XTT.
  • Modified Als3-NT protein-glucan bioconjugate Als3 18-316 -3S-Fba-bgluc d+ ; Control indicates buffer immunized animals, all groups tested with AS03. NZW rabbits immunized three times on a two-week interval. Graph depicts Mean+SD.
  • FIG.15 shows the capacity of antibodies against modified Als3-NT protein-glucan bioconjugate to mediate neutrophile killing of C. albicans hyphae.
  • C. albicans hyphae (SC5314) were mixed with modified Als3-NT protein-glucan bioconjugate sera, added to neutrophiles and incubated.
  • SEQUENCE LISTINGS SEQ ID NO: 1 Full-length Wild type Als3 sequence from Candida albicans (with wild type Leader sequence) MLQQYTLLLIYLSVATAKTITGVFNSFNSLTWSNAATYNYKGPGTPTWNAVLGWSLDGTSASPGDTFTLNMPCV FKFTTSQTSVDLTAHGVKYATCQFQAGEEFMTFSTLTCTVSNTLTPSIKALGTVTLPLAFNVGGTGSSVDLEDSK CFTAGTNTVTFNDGGKKISINVDFERSNVDPKGYLTDSRVIPSLNKVSTLFVAPQCANGYTSGTMGFANTYGDV QIDCSNIHVGITKGLNDWNYPVSSESFSYTKTCSSNGIFITYKNVPAGYRPFVDAYISATDVNSYTLSYANEYTCA GGYWQRAPFTLRWTGYRNSDAGS
  • coli flagellin FLSALILLLVTTAAQA SEQ ID NO: 22
  • E. coli outer membrane porin A OmpA
  • MKKTAIAIAVALAGFATVAQA SEQ ID NO: 23
  • E. coli maltose binding protein MKIKTGARILALSALTTMMFSASALA SEQ ID NO: 24
  • FlgI flagellin
  • MIKFLSALILLLVTTAAQA MIKFLSALILLLVTTAAQA
  • OmpA outer membrane porin A
  • MKKTAIAIAVALAGFATVAQA SEQ ID NO: 23
  • MalE maltose binding protein
  • coli outer membrane porin A (OmpC) signal sequence
  • MKVKVLSLLVPALLVAGAANA SEQ ID NO: 25
  • Als3-NT (1-316) sequence from Candida albicans (with wild type leader sequence)

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Abstract

La présente invention se rapporte au domaine des protéines modifiées, des compositions immunogènes et des vaccins comprenant les protéines modifiées, leur fabrication et l'utilisation de telles compositions en médecine. Plus particulièrement, elle se rapporte à une protéine Als3 (séquence 3 de type agglutinine de Candida albicans) modifiée. La protéine Als3 modifiée peut être utilisée en tant que protéine-support pour d'autres antigènes, en particulier des antigènes de saccharide ou d'autres antigènes dépourvus d'épitopes de lymphocytes T.
PCT/IB2024/057693 2023-08-09 2024-08-08 Protéines modifiées Pending WO2025032534A2 (fr)

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Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4365170A (en) 1979-09-21 1982-12-21 Hitachi, Ltd. Semiconductor switch
EP0161188A2 (fr) 1984-05-10 1985-11-13 Merck & Co. Inc. Polysaccharides bactériens modifiés de manière covalente, conjugués covalents stables de tels polysaccharides avec des protéines immunogéniques à l'aide de ponts bivalents, et méthodes pour préparer de tels polysaccharides et conjugués et pour confirmer la covalence
EP0208375A2 (fr) 1985-07-05 1987-01-14 SCLAVO S.p.A. Conjugués glycoprotéiniques ayant une activité immunogène trivalente
US4673574A (en) 1981-08-31 1987-06-16 Anderson Porter W Immunogenic conjugates
GB2220211A (en) 1988-06-29 1990-01-04 Ribi Immunochem Research Inc Modified lipopolysaccharides
EP0382271A1 (fr) 1989-02-04 1990-08-16 Akzo Nobel N.V. Tocols comme adjuvants de vaccins
US5057540A (en) 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
EP0477508A1 (fr) 1990-09-28 1992-04-01 American Cyanamid Company Vaccins améliorés à base de conjugués d'oligosaccharides
WO1993015760A1 (fr) 1992-02-11 1993-08-19 U.S. Government, As Represented By The Secretary Of The Army Structure immunogene a double vecteur
WO1995008348A1 (fr) 1993-09-22 1995-03-30 Henry M. Jackson Foundation For The Advancement Of Military Medicine Procede permettant d'activer un glucide soluble a l'aide de nouveaux reactifs cyanylants pour produire des structures immunogenes
WO1996029094A1 (fr) 1995-03-22 1996-09-26 Andrew Lees Preparation de produits de recombinaison immunogenes au moyen d'hydrates de carbone solubles actives par l'intermediaire de reactifs organiques de cyanylation
WO2003074687A1 (fr) 2002-03-07 2003-09-12 Eidgenössische Technische Hochschule Zürich Systeme et procede de fabrication de proteines glycosylees de recombinaison dans un hote procaryotique
WO2006119987A2 (fr) 2005-05-11 2006-11-16 ETH Zürich Proteines n-glycosylees de recombinaison produites a partir de cellules procaryotes
WO2009104074A2 (fr) 2008-02-20 2009-08-27 Glycovaxyn Ag Bioconjugués faits à partir de protéines n-glycosylées recombinées issues de cellules procaryotes
WO2011006261A1 (fr) 2009-07-17 2011-01-20 Ocean Harvest Technology (Canada) Inc. Formule d’algues naturelle et durable qui remplace les additifs synthétiques dans l’alimentation des saumons
WO2011138361A1 (fr) 2010-05-06 2011-11-10 Glycovaxyn Vaccins de bioconjugué de bactéries gram positif capsulaire

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012163533A1 (fr) * 2011-06-01 2012-12-06 Eth Zurich Epitope de cellule t de candida albicans
CN103998056B (zh) * 2011-07-22 2017-09-12 诺瓦蒂格姆疗法有限公司 用于接种抗金黄色葡萄球菌疫苗的方法和组合物
GB201721582D0 (en) * 2017-12-21 2018-02-07 Glaxosmithkline Biologicals Sa S aureus antigens and immunogenic compositions
US20230293657A1 (en) * 2020-06-25 2023-09-21 Glaxosmithkline Biologicals Sa Vaccine

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4365170A (en) 1979-09-21 1982-12-21 Hitachi, Ltd. Semiconductor switch
US4673574A (en) 1981-08-31 1987-06-16 Anderson Porter W Immunogenic conjugates
EP0161188A2 (fr) 1984-05-10 1985-11-13 Merck & Co. Inc. Polysaccharides bactériens modifiés de manière covalente, conjugués covalents stables de tels polysaccharides avec des protéines immunogéniques à l'aide de ponts bivalents, et méthodes pour préparer de tels polysaccharides et conjugués et pour confirmer la covalence
EP0208375A2 (fr) 1985-07-05 1987-01-14 SCLAVO S.p.A. Conjugués glycoprotéiniques ayant une activité immunogène trivalente
US5057540A (en) 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
GB2220211A (en) 1988-06-29 1990-01-04 Ribi Immunochem Research Inc Modified lipopolysaccharides
EP0382271A1 (fr) 1989-02-04 1990-08-16 Akzo Nobel N.V. Tocols comme adjuvants de vaccins
EP0477508A1 (fr) 1990-09-28 1992-04-01 American Cyanamid Company Vaccins améliorés à base de conjugués d'oligosaccharides
WO1993015760A1 (fr) 1992-02-11 1993-08-19 U.S. Government, As Represented By The Secretary Of The Army Structure immunogene a double vecteur
WO1995008348A1 (fr) 1993-09-22 1995-03-30 Henry M. Jackson Foundation For The Advancement Of Military Medicine Procede permettant d'activer un glucide soluble a l'aide de nouveaux reactifs cyanylants pour produire des structures immunogenes
WO1996029094A1 (fr) 1995-03-22 1996-09-26 Andrew Lees Preparation de produits de recombinaison immunogenes au moyen d'hydrates de carbone solubles actives par l'intermediaire de reactifs organiques de cyanylation
WO2003074687A1 (fr) 2002-03-07 2003-09-12 Eidgenössische Technische Hochschule Zürich Systeme et procede de fabrication de proteines glycosylees de recombinaison dans un hote procaryotique
WO2006119987A2 (fr) 2005-05-11 2006-11-16 ETH Zürich Proteines n-glycosylees de recombinaison produites a partir de cellules procaryotes
WO2009104074A2 (fr) 2008-02-20 2009-08-27 Glycovaxyn Ag Bioconjugués faits à partir de protéines n-glycosylées recombinées issues de cellules procaryotes
WO2011006261A1 (fr) 2009-07-17 2011-01-20 Ocean Harvest Technology (Canada) Inc. Formule d’algues naturelle et durable qui remplace les additifs synthétiques dans l’alimentation des saumons
WO2011138361A1 (fr) 2010-05-06 2011-11-10 Glycovaxyn Vaccins de bioconjugué de bactéries gram positif capsulaire

Non-Patent Citations (42)

* Cited by examiner, † Cited by third party
Title
"Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4", NATURE, vol. 227, no. 5259, pages 680 - 685
"Remington's Pharmaceutical Sciences", 1975, MACK PUBLISHING CO. EASTON
BI Y. ET AL., NATURE, vol. 553, no. 7688, 2018, pages 361 - 365
BIGGE JCPATEL TPBRUCE JAGOULDING PNCHARLES SMPAREKH RB, ANAL BIOCHEM, vol. 230, no. 2, 1995, pages 229 - 238
CHU C ET AL., INFECT. IMMUNITY, 1983, pages 245 - 256
COLEMAN, DA ET AL., J. MOL. METH, 2009, pages 3
COLEMAN, DA ET AL., J. MOL. METH., 2009
CUTHBERTSON L. ET AL., MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS, vol. 74, no. 3, 2010, pages 341 - 362
DMITRIEV, B.A. ET AL., SOMATIC ANTIGENS OF SHIGELLA EUR J. BIOCHEM, vol. 98, 1979, pages 8
DUFRESNE ET AL., NATURE BIOTECHNOLOGY, vol. 20, 2002, pages 1269 - 71
DYLOCHOORN ET AL., GENE, vol. 177, no. 1-2, 1996, pages 133 - 6
ELAMIN E ET AL., J. IMMUNOL. RES., 2021, pages 1 - 19
ELAMIN E ET AL., J. IMMUNOL. RES.,, 2021, pages 1 - 19
FAZEKAS DE STGROTH, SWEBSTER, R. GDATYNER, A.: "Two new staining procedures for quantitative estimation of proteins on electrophoretic strips", BIOCHIMICA ET BIOPHYSICA ACTA, vol. 71, 1963, pages 377 - 391
FELDMAN ET AL., PNAS USA, vol. 102, 2005, pages 3016 - 3021
FELDMAN ET AL., PROC NATL ACAD SCI U S A., vol. 102, no. 8, 2005, pages 3016 - 21
GIBSON, D.G. ET AL., NAT. METHODS, vol. 6, 2009, pages 343 - 345
GONG J ET AL., ANTIMICROB AGENTS CHEMOTHER,, vol. 64, no. 1, 2019, pages e01975 - 79
IELASI ET AL., MBIO, vol. 7, no. 4, 2016, pages e00584 - 16
KENSIL ET AL.: "Vaccine Design: The Subunit and Adjuvant Approach", 1995, PLENUM PRESS
KOWARIK ET AL., EMBO J., vol. 25, no. 9, 2006, pages 1957 - 66
LIN, J ET AL., J. BIOL. CHEM., vol. 280, no. 26, 2014, pages 18401 - 18412
LIU ET AL., STRUCTURE AND GENETICS OF SHIGELLA O ANTIGENS FEMS MICROBIOLOGY REVIEW, vol. 32, 2008, pages 27
LIU YFILLER SG, CELL, vol. 10, no. 2, 2011, pages 168 - 173
LIUFILLER, EUKARYOTIC CELL, vol. 10, no. 2, 2011, pages 168 - 173
MORAD HO ET AL., FRONT MICROBIOL,, 2018
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
NITA-LAZAR ET AL., GLYCOBIOLOGY., vol. 15, no. 4, 2005, pages 361 - 7
PALMA ET AL., J BIOL CHEM., vol. 281, no. 9, 2006, pages 5771 - 9
PHAN QT ET AL., PLOS BIOL., vol. 5, no. 3, 2007, pages e64
RAYMOND ET AL., J BACTERIOL., vol. 184, no. 13, 2002, pages 3614 - 22
ROYLE LMATTU TSHART ELANGRIDGE JIMERRY AHMURPHY NHARVEY DJDWEK RA, RUDD PM, ANAL BIOCHEM, vol. 304, no. 1, 2002, pages 70 - 90
SARASWAT ET AL., BIOMED. RES. INT., 2013, pages 1 - 18
SCHMIDT, CS A ET AL., VACCINE, 2012
SCHULZ ET AL., J BIOL CHEM., vol. 281, no. 5380, August 1998 (1998-08-01), pages 1197 - 200
SMITHWATERMAN, J. MOL. BIOL., vol. 147, 1981, pages 195 - 197
STOUTE ET AL., N. ENGL. J. MED., vol. 336, 1997, pages 86 - 91
TE KUHLMANEC COX, NUCLEIC ACIDS RES., vol. 38, no. 6, April 2010 (2010-04-01), pages e92
WACKER ET AL., PROC NATL ACAD SCI U S A., vol. 103, no. 18, 2006, pages 7088 - 93
WACKER ET AL., SCIENCE, vol. 298, no. 5599, 2002, pages 1790 - 3
XIN H ET AL., PLOS ONE, vol. 7, 2012, pages e35106
XU ET AL., APPL MICROBIOL BIOTECHNOL., vol. 101, 2017, pages 585 - 598

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