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WO2025032535A2 - Protéines modifiées - Google Patents

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Publication number
WO2025032535A2
WO2025032535A2 PCT/IB2024/057694 IB2024057694W WO2025032535A2 WO 2025032535 A2 WO2025032535 A2 WO 2025032535A2 IB 2024057694 W IB2024057694 W IB 2024057694W WO 2025032535 A2 WO2025032535 A2 WO 2025032535A2
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amino acid
modified
protein
seq
sap2
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WO2025032535A3 (fr
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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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    • 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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/23—Aspartic endopeptidases (3.4.23)
    • C12Y304/23024—Candidapepsin (3.4.23.24)
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins

Definitions

  • MODIFIED PROTEINS SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically hereby incorporated by reference in its entirety. Said XML copy, created on August 7, 2023, is named 70385US01P_SL.xml and is 124,012 bytes in size. FIELD OF THE INVENTION
  • 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 Sap2 (Secreted Aspartyl Proteinase 2 of Candida albicans) protein.
  • the modified Sap2 protein can be used as a carrier protein for other antigens, particularly saccharide antigens or other antigens lacking T cell epitopes.
  • Other antigens particularly saccharide antigens or other antigens lacking T cell epitopes.
  • Fungal infections are also detrimental to the well-being of grazing livestock, with milk production in dairy cows, and body and coat condition adversely affected by fungal infections.
  • 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. These organisms are able to colonize the skin, as well as the gastrointestinal, and reproductive tracts of humans [9,10].
  • these organisms can become pathogenic and can cause a broad spectrum of human infections.
  • the incidence of human infections caused by Candida genus has increased significantly (Sobel, 2007; Pfuller , 2011).
  • Candida infections can be superficial or invasive. Invasive candidiasis are difficult to treat.
  • 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.
  • albicans has a large repertoire of virulence factors that allows its transition from commensal organism (yeast form) to pathogen (hyphal form).
  • C. albicans is a commensal in the vaginal epithelium.
  • Certain environmental conditions trigger the morphogernesis of C. albicans from the commensal yeast form to the pathogenic hyphal form.
  • the hyphal form starts entering the lumen and breaches mucosal barriers, which leads to the symptomatic infection, e.g., vulvovaginal candidiasis (VVC) in women.
  • VVC vulvovaginal candidiasis
  • RVVC Recurrent VVC or RVVC (defined as > 3 episodes/year) is more serious and refractory to therapy. RVVC results in reduced quality of life, a strong negative impact on work and social life, as well as increased associated healthcare costs.
  • IFIs invasive fungal infections
  • treatment options are limited and the anti-fungal drug discovery pipeline is under developed (Perfect, 2017).
  • antifungal therapies are often ineffective.
  • multidrug-resistant fungal infections e.g.
  • 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 Secreted Aspartyl Proteinase 2 (Sap2) 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 Sap2 carrier protein linked to the Candida polysaccharide antigen at one or more asparagine residues on the modified Sap2 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 Secreted Aspartyl Proteinases 2 (Sap2) protein comprising amino acid residues 19-398 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 19-398 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 Sap2 protein of the invention wherein the modified Sap2 protein further comprises a substitution at amino acid residue 274 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1, optionally wherein the protein comprises an Aspartic Acid (D) to Asparagine (N) substitution at amino acid residue 274 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1.
  • D Aspartic Acid
  • N Asparagine
  • a modified Sap2 protein of the invention wherein the modified Sap2 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
  • a modified Sap2 protein of the invention comprising an amino acid sequence of SEQ ID NO: 9.
  • a modified Sap2 protein of the invention comprising an amino acid sequence of SEQ ID NO: 10.
  • a conjugate comprising a modified Sap2 protein of the invention and at least one saccharide antigen.
  • a modified Sap2 protein of protein of Candida albicans comprising (or consisting of): (1) an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and (2) at least one saccharide antigen of Candida, wherein the at least one saccharide antigen is a ⁇ -1,2 mannan polymer consisting of at least five consecutive ⁇ -1,2 linked mannose molecules, and wherein the at least one saccharide antigen is linked to at least one of four asparagine residues at positions 45, 94, 215, and 415 of SEQ ID NO: 9 or at least one of four asparagine residues at positions 6, 55, 176, and 376 of SEQ ID NO: 10.
  • a polynucleotide encoding a modified Sap2 protein of the invention there is provided a vector comprising a polynucleotide encoding a modified Sap2 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 Sap2 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 Sap2 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 Sap2 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 Sap2 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 Sap2 protein of the invention; (2) at least one Candida albicans saccharide antigen linked to said modified Sap2 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 Sap2 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 e.g. human
  • the method comprising administering to the subject an immunoprotective dose of a modified Sap2 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 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 Sap2 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 Sap2 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 Sap2 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 manufacture of a medicament for treatment or prevention of a disease caused by Candida albicans infection.
  • a host cell comprising: i. a nucleotide sequence encoding one or more first heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,2 mannan polymer; ii.
  • a method of producing a glycoconjugate comprising a modified carrier protein and a ⁇ -1,2 mannan comprising culturing the host cell of the invention under conditions suitable for the production of proteins.
  • a method of producing a glycoconjugate comprising a modified carrier protein and a ⁇ -1,2 mannan comprising the steps of i) culturing a host cell of the invention under conditions suitable for the production of proteins, ii) harvesting the culture to produce a harvested culture, and iii) isolating the glycoconjugate from the culture.
  • a saccharide that is a ⁇ -1,2 mannan polymer comprising the structure: .
  • a saccharide comprising the structure: ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 3)-x-D-GlcpNAc.
  • a conjugate e.g. bioconjugate
  • a saccharide of the invention linked to an asparagine residue of a modified carrier protein DESCRIPTION OF DRAWINGS/FIGURES FIGS.
  • FIG. 1A and 1B show the structure of mature Sap2 protein from C. albicans of SEQ ID NO: 88 (comprising residues 57-398 of SEQ ID NO: 1).
  • FIG. 1A spheres indicate the positions for insertion of glycosites.
  • FIG. 1B spheres indicate the best positions for insertion of glycosites.
  • N glycosite inserted next to position 19;
  • C glycosite inserted next to position 398;
  • Mut3a glycosite substituted for positions 98-102;
  • Mut 4d glycosite substituted for positions 109-113;
  • Mut 8a glycosite inserted next to position 220 (all positions are relative to wild-type full-length Sap2 sequence of C. albicans having SEQ ID NO: 1).
  • FIG. 1A spheres indicate the positions for insertion of glycosites.
  • FIG. 1B spheres indicate the best positions for insertion of glycosites.
  • N glycosite inserted next to position 19
  • C glycosite inserted next to position 3
  • FIG. 2 shows production of a proSap2-Fba- ⁇ -1,2-mannan bioconjugate in E. coli.
  • FIG. 2A shows the a schematic of the structure of the C. Albicans mannan.
  • FIG. 2B shows the biosynthesis scheme for the modified Sap2-mannan bioconjugate in E. coli.
  • Enterobacterial O-antigen cluster refers to Citrobacter fruedii P079F I.
  • FIG. 3 shows glycosylation tests with a series of modified proSap2 proteins, each comprising a single glycosite.
  • FIGS. 3A and 3B show SDS-PAGE analysis of modified proSap2 proteins purified from PPE by IMAC.
  • Modified proSap2 ”C” variant (FIG. 3A, lane 3) shows the highest expression level and glycosylation. Some positions (e.g., Mut14a-14e (FIG. 3B, lanes27-31)) seem to destabilize the modified proSap2 protein and lead to reduced protein expression.
  • FIG. 4 shows glycosylation tests with a series of modified proSap2 proteins comprising combined glycosites. Lanes 1-4 show SDS-PAGE analysis of modified proSap2 proteins (not comprising Fba peptide sequence) purified from PPE by IMAC.
  • Lanes 5-8 show SDS-PAGE analysis of modified proSap2 proteins (comprising the Fba peptide sequence) purified from PPE by IMAC. As shown, modified proSap2 proteins with up to 4 glycosites were expressed and purified.
  • FIG. 5 shows SDS-PAGE gels followed by coomassie blue staining (FIG. 5A) or Western Blot analyses (FIGS. 5B-5E)of purified modified Sap2 proteins.
  • Lane M protein standard
  • Lane 1 purified unglycosylated modified proSap2 (MutN3C-8a-Fba-C).
  • FIG. 5A SDS-PAGE analysis followed by coomassie blue staining.
  • FIG. 5B anti-proSap2 immunoblot (against N-terminal “pro- peptide” region);
  • FIG. 5C anti-Sap2 immunoblot (against peptide in the middle of the protein sequence).
  • FIG. 5D anti-Fba immunoblot;
  • FIG. 5E anti-mannan immunoblot.
  • FIG. 6 shows structural determination via crystallography modified proSap2 protein produced in E.
  • FIG. 7 shows Circular dichroism (CD) spectroscopy characterization of wild-type proSap2 protein and modified proSap2- ⁇ -1,2-mannan bioconjugate.
  • FIG. 7A shows near-UV CD spectroscopy analysis.
  • FIG. 7B shows far-UV CD spectroscopy analysis.
  • Fig. 7C shows the secondary structure element content after analysis with “CDNN” software (Applied Photophysics Ltd) for deconvolution of CD spectroscopy.
  • CDNN Circular dichroism
  • FIG. 8 shows preclinical testing of a purified modified proSap2-Fba- ⁇ -1,2-mannan bioconjugate (“proSap2-4FM”) in rabbit.
  • FIG. 8A shows the modified proSap2-Fba- ⁇ -1,2-mannan bioconjugate attributes.
  • FIG. 8B shows a 3D representation of the modified proSap2 protein-mannan bioconjugate;
  • FIG. 8C shows the rabbit immunization scheme with the modified proSap2-Fba - ⁇ - 1,2-mannan bioconjugate.
  • FIG. 9 shows immunogenicity of the purified modifed proSap2-Fba- ⁇ -1,2-mannan bioconjugate (“proSap2-4FM”) in rabbits.
  • FIG. 10 shows a protease activity inhibition assay of full-length wild-type Sap2 protein of C. albicans tested with a strong inhibitor of its activity (a mono-specific Fab fragment).
  • FIG.10A the measured activities in absence of Sap2 (negative control), in absence of the Fab fragment (positive control), and in presence of the Fab fragment (Fab anti-Sap2) were expressed as percentage of the positive control activity as determined via absorbance at 280 nm.
  • the employed Fab fragment was able to completely block the protease activity of Sap2.
  • FIG. 10B titration curves are shown where sera from a bicomponent immunization (“Candi5V”, where proSap2-Fba- ⁇ -1,2-mannan bioconjugate and Als3- Fba- ⁇ -1,3-glucan bioconjugate are formulated together) or proSap2-Fba- ⁇ -1,2-mannan rabbits immunization ”proSap2-4FM”) are used.
  • a bicomponent immunization (“Candi5V”, where proSap2-Fba- ⁇ -1,2-mannan bioconjugate and Als3- Fba- ⁇ -1,3-glucan bioconjugate are formulated together
  • proSap2-Fba- ⁇ -1,2-mannan rabbits immunization proSap2-4FM
  • Sap2 protein or “Sap2” refers to a wild type Secreted Aspartyl Proteinase 2 protein comprising a wild type leader sequence (amino acid residues 1-18) and a pro- peptide sequence (amino acid residues 19-56) at its N-terminus.
  • the Sap2 protein is from Candida, optionally from Candida albicans. In specific embodiments, the Sap2 protein comprises the amino acid sequence of SEQ ID NO.: 1.
  • the term “proSap2 protein” or “proSap2” refers to a C-terminal fragment of a wild type Sap2 protein.
  • the proSap2 protein is from Candida, optionally from Candida albicans.
  • the proSap2 protein comprises amino acid residues 19- 398 of SEQ ID NO.: 1.
  • the proSap2 protein comprises an amino acid sequence of SEQ ID NO.: 17.
  • modified protein refers to a protein that is altered (in one or more way) as compared to wild type protein (e.g. a “modified Sap2 protein” or “modified Sap2” excludes a wild type Sap2 protein and a “modified proSap2 protein” or “modified proSap2” excludes a wild type proSap2 protein).
  • modified Sap2 protein refers to a Sap2 protein that comprises one or more consensus glycosite sequences of the invention (e.g.
  • a modified Sap2 protein refers to a Sap2 protein that comprises one or more consensus glycosite sequences (e.g.
  • a modified Sap2 protein refers to a Sap2 protein that comprises one or more consensus glycosite sequences (e.g.
  • a modified Sap2 protein refers to a Sap2 protein that comprises one or more consensus glycosite sequences (e.g.
  • amino acid sequence of D/E-X-N-Z-S/T comprising an amino acid sequence of D/E-X-N-Z-S/T, wherein X and Z are independently any amino acid except proline), wherein the one or more consensus sequences have been added next to or substituted for one or more amino acid residues within amino acid residues 57-398 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 57-398 of SEQ ID NO: 1.
  • a modified Sap2 protein refers to an Sap2 protein that comprises amino acid residues 19-398 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 19-398 of SEQ ID NO: 1, modified in that the amino acid sequence comprises one or more consensus sequences (e.g.
  • amino acid sequence of D/E-X-N-Z-S/T comprising an amino acid sequence of D/E-X-N-Z-S/T, wherein X and Z are independently any amino acid except proline), wherein the one or more consensus sequences have been added next to or substituted for one or more amino acid residues within amino acid residues 19-398 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 protein further comprises a substitution at amino acid residue 88 and/or 274 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 SEQ ID NO: 1.
  • a modified Sap2 protein further comprises a substitution at amino acid residue 88 and/or 274 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 protein further comprises a substitution at amino acid residue 88 and/or 274 of amino acid residues 57-398 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 57-398 of SEQ ID NO: 1).
  • substitution is a D274N substitution.
  • a modified Sap2 protein of the invention is a naturally occuring modified Sap2 protein.
  • a modified Sap2 protein of the invention is a recombinant modified Sap2 protein.
  • a modified Sap2 protein of the invention is an isolated recombinant modified Sap2 protein.
  • the modified Sap2 protein is from Candida, optionally from Candida albicans.
  • the modified Sap2 protein comprises an amino acid sequence of SEQ ID NO: 9.
  • the modified Sap2 protein comprises an amino acid sequence of SEQ ID NO: 10.
  • the modified Sap2 protein comprises an amino acid sequence selected from, but not limited to, the amino acid sequences of N, N3C, C, Mut1a, Mut 1b, Mut2a, Mut 2b, Mut3a, Mut 3b, Mut4a, Mut4b, Mut4c, Mut4d, Mut4e, Mut5a, Mut5b, Mut6a, Mut6b, Mut7, Mut8a, Mut8b, Mut9, Mut10, Mut11, Mut12, Mut13, Mut14a, Mut14b, Mut14c, Mut14d, Mut14e, Mut15, Mut16, Mut17, Mut 18, Mut 19, and Mut20.
  • control Sap2 protein means, without limitation,: (1) a Sap2 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 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) a Sap2 protein that does not comprise one or more consensus sequences inserted next to or substituted for one or more amino acids of a Sap2 protein that comprises amino acid residues 19-398 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 19-398 of SEQ ID NO: 1 (or
  • a control Sap2 protein includes, without limitation, a wild type Sap2 protein, a wild type Sap2 protein of SEQ ID NO: 1, a wild type Sap2 protein comprising amino acid residues 19-398 of SEQ ID NO: 1, or a wild type Sap2 protein comprising amino acid residues 57-398 of SEQ ID NO: 1
  • the term “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).
  • 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 Sap2 protein of the invention.
  • any amino acid except proline (pro, P) 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), try
  • 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 Lipopolysaccharide
  • CP capsule polysaccharide
  • examples include capsular polysaccharide from Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis and Staphylcoccus aureus.
  • the term “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.
  • the term “conjugate” refers to a protein (e.g. a carrier protein) covalently linked to an antigen.
  • the term “bioconjugate” refers to a conjugate between a protein (e.g. a carrier protein) and 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.
  • 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 Sap2 protein of the invention still comprises the recited modifications that are made to the Sap2 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.
  • amino acid modifications to the sequence of a polypeptide may produce polypeptides having functional and chemical characteristics similar to those of a parental polypeptide.
  • deletion refers to the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 1 to 6 residues (e.g. 1 to 4 residues) are deleted at any one site within the protein molecule.
  • insertion 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. Typically, 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.
  • 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 modified Sap2 protein of the invention or a modified proSap2 protein of the invention) to which an antigen saccharide (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. J-U-B-D/E-X-N-Z-S/T-J-U-B).
  • 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 19 of SEQ ID NO: 1), may be substituted for that amino acid.
  • a numeric range e.g. “19-24” is inclusive of endpoints (i.e. includes the values 19 and 24).
  • “between amino acids 19 to 398 of SEQ ID NO: 1” refers to position in the amino acid sequence between amino acid 19 and amino acid 398 of SEQ ID NO: 1 including both amino acids 19 and 398.
  • 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. 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity over a specified region), when compared and aligned for maximum correspondence using, for example, sequence comparison algorithms or by manual alignment and visual inspection. Identity between polypeptides may be calculated by various algorithms.
  • 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.
  • the Needleman Wunsch algorithm (Needleman and Wunsch 1970, J. Mol. Biol.48: 443-453) for global alignment
  • the Smith Waterman algorithm (Smith and Waterman 1981, J. Mol. Biol. 147: 195- 197) for local alignment may be used, e.g. using the default parameters (Smith Waterman uses BLOSUM 62 scoring matrix with a Gap opening penalty of 10 and a Gap extension penalty of 1).
  • a preferred algorithm is described by Dufresne et al.
  • GenePAST Gene Quest Life Sciences, Inc. Boston, MA
  • the GenePAST “percent identity” algorithm finds the best fit between the query sequence and the subject sequence, and expresses the alignment as an exact percentage. GenePAST makes no alignment scoring adjustments based on considerations of biological relevance between query and subject sequences. Identity between two sequences is calculated across the entire length of both sequences and is expressed as a percentage of the reference sequence (e.g. SEQ ID NO: 1 of the invention).
  • reference sequence e.g. SEQ ID NO: 1 of the invention.
  • the term “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. This includes, for example, a a protein, conjugate (e.g.
  • an isolated or purified protein is a protein essentially free from all other polypeptides with which the protein is innately associated (or innately in contact with).
  • the term “subject” refers to an animal, in particular a mammal such as a primate (e.g. human).
  • the term “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 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
  • 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 phosphorylated 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).
  • wbaB refers to a glycosyltransferase.
  • wbaB is a glycosyltransferase obtained from an organism including, but not limited to, Citrobacter freundii P079F I, Salmonella O6,7 (C1) Thompson, or Escherichia coli O17.
  • wbaB is a glycosyltransferase of Citrobacter freundii P079F I.
  • wbaB is a wild type glycosyltransferase.
  • wbaB is a non-naturally occurring (e.g., mutant and/or recombinant) glycosyltransferase.
  • wbaC refers to a glycosyltransferase.
  • wbaC is a glycosyltransferase obtained from an organism including, but not limited to, Citrobacter freundii P079F I, Salmonella O6,7 (C1) Thompson, or Escherichia coli O17.
  • wbaC is a glycosyltransferase of Citrobacter freundii P079F I.
  • wbaC is a wild type glycosyltransferase.
  • wbaC is a non-naturally occurring (e.g., mutant and/or recombinant) glycosyltransferase.
  • wbaD refers to a glycosyltransferase.
  • wbaD is a glycosyltransferase obtained from an organism including, but not limited to, Citrobacter freundii P079F I, Salmonella O6,7 (C 1 ) Thompson, or Escherichia coli O17.
  • wbaD is a glycosyltransferase of Citrobacter freundii P079F I.
  • wbaD is a wild type glycosyltransferase.
  • wbaD is a non-naturally occurring (e.g., mutant and/or recombinant) glycosyltransferase.
  • Bmt3 refers to a glycosyltransferase.
  • Bmt3 is a glycosyltransferase obtained from an organism including, but not limited to, Candida albicans.
  • Bmt3 is a glycosyltransferase of C. albicans.
  • Bmt3 is a wild type glycosyltransferase gene.
  • Bmt3 is a non-naturally occurring (e.g., mutant and/or recombinant) glycosyltransferase gene.
  • the Bmt3 gene is a codon-optimized version of the gene from C. albicans.
  • the Bmt3 protein is N-terminally fused with the leader peptide sequence from E. coli OmpC for its periplasmic export.
  • manB refers to a guanylyltransferase.
  • manB is a guanylyltransferase obtained from an organism including, but not limited to Escherichia coli.
  • manB is a guanylyltransferase of E. coli K12 W3110.
  • manB is a wild type guanylyltransferase.
  • manB is a non-naturally occurring (e.g., mutant and/or recombinant) guanylyltransferase.
  • manC refers to a guanylyltransferase.
  • manC is a guanylyltransferase obtained from an organism including, but not limited to Escherichia coli.
  • manC is a guanylyltransferase of E. coli K12 W3110.
  • manC is a wild type guanylyltransferase. In other aspects, manC is a non-naturally occurring (e.g., mutant and/or recombinant) guanylyltransferase.
  • 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.
  • the pglB gene is an evolved oligosaccharyl transferase gene.
  • the pglB protein is an evolved pglB, i.e., an evolved oligosacharyl transferase.
  • 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 evolved pglB of the invention compirises an amino acid sequence of SEQ ID NO: 16.
  • the pglB amino acid sequence in SEQ ID NO:16 contains one or more mutations which enhances the activity of pglB for the saccharide antigen of the invention.
  • the evolved pglB of the invention transfers a saccharide antigen of the invention to a modified Sap2 protein of the invention more efficiently in comparison to a wild-type pglB (e.g., a wild type pglB obtained from Campylobacter jejuni).
  • Wzx refers to a translocase.
  • wzx is a translocase obtained from an organism including, but not limited to, Citrobacter freundii P079F I, Salmonella O6,7 (C 1 ) Thompson, or Escherichia coli O17.
  • wzx is a translocase of Citrobacter freundii P079F I.
  • wzx is a wild type translocase.
  • wzx is a non-naturally occurring (e.g., mutant and/or recombinant) translocase.
  • Sap2 Protein Secreted Aspartyl Proteinase 2 protein of Candida albicans (also known as “Sap2”) is an extracellular virulence factor that is the predominant enzyme in vaginal secretion of Candida infected symptomatic women. Sap2 provides nutrition for the Candida cells, facilitates attachment to host tissue, facilitates fungal epithelial and endothelial penetration, contributes to Candida’s capability to evade immune responses, and is immunogenic during infection (Kumar R. et al., 2015, Infect Immun, 83(7):2614-2626). Sap2 functions as a hydrolytic enzyme, exhibiting broad substrate specificity, and is expressed abundantly in a culture of C.
  • the Secreted Aspartyl Proteinase (Sap) proteins of are encoded by a family of 10 SAP genes and have been the most comprehensively studied as key virulence determinants of C. albicans (Id.).
  • Sap2 is a member of the secreted aspartyl Sap family of proteins and is encoded by the SAP2 gene. All 10 SAP genes of C. albicans encode preproenzymes approximately 60 amino acids longer than the mature enzymes, which are processed when transported via the secretory pathway (Id.).
  • the mature enzymes contain sequence motifs typical for all aspartyl proteinases, including the two conserved aspartate residues of the active site and conserved cysteine residues implicated in the maintenance of the three-dimensional structure (Id.).
  • the SAP2 gene encodes a 398-amino acid long preproprotein that is processed to a 342- residue mature enzyme, a typical aspartic proteinase of pH optimum 3-4, displaying sensitivity to pepstatin A, a peptide-based inhibitor (Cutfield S., et al., 1995, Structure, 3:1261-1271).
  • the C. albicans Sap2 preproprotein is processed to a 380- residue “intermediate” form (“proSap2”; SEQ ID NO: 17) that lacks the leader sequence, and then further processed to the 342-residue “mature” form (“mSap2”; SEQ ID NO: 88) that lacks the leader sequence and the pro-peptide sequence (Naglik et al. 2003).
  • proSap2 intermediate form
  • mSap2 342-residue “mature” form
  • This domain in SAP2 comprises residues 197–215 and 223–305, and contains one disulphide bond (between 256 and 294) which ties together a double loop (243–255, 282–293) of random structure.
  • These C-terminal loops are a peripheral feature of aspartic proteinases, forming part of the wide entrance to the binding site.
  • Another loop, formed by the disulphide 47–59, is more well defined. It also flanks the binding site but is considerably closer.
  • the pattern of secondary structure elements in SAP2 in particular the organisation of the many ⁇ strands and the two major ⁇ -helical sections (140–146, 228–237), is similar to other known aspartic proteinases. However, some of the turns linking these elements are different in conformation.
  • a highly conserved feature of the aspartic proteinases is the “flap” region (a ⁇ hairpin loop) which interacts centrally with bound inhibitor/substrate, shielding the active site from bulk solvent.
  • the flap comprises residues Lys81–Gln91, with the tyrosine at position 84 being highly conserved, as found in other aspartic proteinases (equivalent to Tyr75 in pepsin) (Cutfield S., et al., 1995, Structure, 3:1261-1271).
  • the overall structure of Sap2 conforms to the classical aspartic proteinase fold typified by pepsin.
  • One of the most noticeable properties of Sap2 is the variety of proteins it can cleave. Sap2 is known to degrade many human proteins including molecules that protect mucosal surfaces such as mucin and secretory immunoglobulin A (IgA).
  • Sap2 can also degrade molecules of the extracellular matrix such as keratin, collagen and vimentin (Naglik JR, et al, 2003, Microbiol. Mol. Biol. Rev., 67:400-428).
  • a Sap2 protein useful in the invention can be produced by methods known in the art in view of the present disclosure, see for example Smolenski G, Sullivan PA, Cutfield SM, Cutfield JF., 1997, Microbiology, 143 ( Pt 2):349-356. As shown below, a full-length wild-type Sap2 protein of C.
  • Albicans comprises the amino acid sequence of SEQ ID NO: 1 (with wild-type leader sequence underlined and the pro-peptide italicized; the amino acid residue (D) at position 274 is double underlined; in certain aspects, substitution of this residue (e.g.
  • modified Sap2 protein refers to a Sap2 protein comprising an amino acid sequence (for example, comprising an 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, or comprsing an amino acid sequence of amino acid residues 19-398 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 amino acid residues 19-398 of SEQ ID NO: 1), which Sap2 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 consensus sequence(s)
  • a modified Sap2 protein may be a Sap2 protein having an 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.
  • X and Z are independently any amino acid except proline; preferably, X is Q (glutamine) and Z is A (alanine).
  • a modified Sap2 protein of the invention may comprise further modifications (e.g., additions, substitutions, and/or deletions of one or more amino acid residues).
  • a modified Sap2 protein of the invention comprises amino acid residues 19-398 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 19-398 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 Sap2 protein of the invention comprises amino acid residues 57-398 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 57-398 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.
  • the modified Sap2 protein of the invention is a non-naturally occurring Sap2 protein (i.e. not native).
  • a modified Sap2 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.
  • a modified Sap2 protein of the invention may have an amino acid sequence at least 80% identical to SEQ ID NO: 1.
  • a modified Sap2 protein of the invention may have an amino acid sequence at least 85% identical to SEQ ID NO: 1.
  • a modified Sap2 protein of the invention may have an amino acid sequence at least 90% identical to SEQ ID NO: 1.
  • a modified Sap2 protein of the invention may have an amino acid sequence at least 91% identical to SEQ ID NO: 1.
  • a modified Sap2 protein of the invention may have an amino acid sequence at least 92% identical to SEQ ID NO: 1. In other aspects, a modified Sap2 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 Sap2 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 Sap2 protein of the invention may have an amino acid sequence at least 95% identical to SEQ ID NO: 1. In certain aspects, a modified Sap2 protein of the invention may have an amino acid sequence at least 96% identical to SEQ ID NO: 1. In other aspects, a modified Sap2 protein of the invention may have an amino acid sequence at least 97% identical to SEQ ID NO: 1.
  • a modified Sap2 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 Sap2 protein of the invention may have an amino acid sequence at least 99% identical to SEQ ID NO: 1. In certain aspects, a modified Sap2 protein of the invention comprises one or more consensus glycosite sequences. The terms “glycosite sequence”, “consensus glycosite sequence” and “consensus sequence” are used herein interchangeably. In certain aspects, a modified Sap2 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 Sap2 protein of the invention contains one, two, three, four, five, six, seven, eight, nine, or ten consensus sequences. In specific aspects, a modified Sap2 protein of the invention contains at least three consensus sequences. In preferred aspects, a modified Sap2 protein of the invention contains three consensus sequences. In certain embodiments, a modified Sap2 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 Sap2 protein of the invention comprises one or more consensus sequences, wherein all of the consensus sequences have different amino acid sequences.
  • a modified Sap2 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 Sap2 protein comprising amino acid residues 19-398 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 19-398 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.
  • the modified Sap2 protein of the invention is an inactive protein.
  • the modified Sap2 protein of the invention comprises a substitution at one or more positions selected from the group consisting of amino acid residue 88 of SEQ ID NO: 1 and amino acid residue 274 of SEQ ID NO: 1.
  • amino acid residue 88 of SEQ ID NO: 1 amino acid residue 1 and amino acid residue 274 of SEQ ID NO: 1.
  • SEQ ID NO: 88 amino acid residue 32 and amino acid residue 218, respectively, of SEQ ID NO: 88 (i.e., a Candida albicans Sap2 protein lacking the wild type leader sequence and lacking the wild type propeptide sequence).
  • a substitution of one or both amino acid residues at positions 88 and 274 of SEQ ID NO: 1 renders the Sap2 protein inactive.
  • a substitution of residue 274 of SEQ ID NO: 1 renders the Sap2 protein inactive (e.g., eliminates the hydrolytic activity of Sap2). In certain aspects, elimination of the hydrolytic activity of Sap2 eliminates the virulence of the protein.
  • a modified Sap2 protein of the invention further comprises a substitution at amino acid residue 274 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 protein of the invention comprises an Aspartic Acid (D) to Asparagine (N) substitution at amino acid residue 274 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 19-398 of SEQ ID NO: 1.
  • the substitution renders the modified Sap2 protein inactive.
  • a modified Sap2 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 Sap2 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 19- 24 (e.g. amino acid residue 19), (2) one or more amino acids between amino acid residues 52-62 (e.g. amino acid residue 57), (3) one or more amino acids between amino acid residues 93-107 (e.g. one or more amino acids between amino acid residues 98-102), (4) one or more amino acids between amino acid residues 104-118 (e.g. one or more amino acids between amino acid residues 109-113), (5) one or more amino acids between amino acid residues 142-144 (e.g. amino acid residue 143), (6) one or more amino acids between amino acid residues 187-197 (e.g.
  • amino acid residue 192 (7) one or more amino acids between amino acid residues 215-225 (e.g. amino acid residue 220), (8) one or more amino acids between amino acid residues 243-253 (e.g. amino acid residue 248), (9) one or more amino acids between amino acid residues 255-265 (e.g. amino acid residue 260), (10) one or more amino acids between amino acid residues 263-273 (e.g. amino acid residue 268), (11) one or more amino acids between amino acid residues 320-330 (e.g. amino acid residue 325), (12) one or more amino acids between amino acid residues 339-349 (e.g. amino acid residue 344), (13) one or more amino acids between amino acid residues 358-359 (e.g.
  • a modified Sap2 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 19-24 (e.g. amino acid residue 19), one or more amino acids between amino acid residues 52-62 (e.g.
  • amino acid residue 57 ), one or more amino acids between amino acid residues 93-107 (e.g. one or more amino acids between amino acid residues 98-102), one or more amino acids between amino acid residues 104-118 (e.g. one or more amino acids between amino acid residues 109-113), one or more amino acids between amino acid residues 142-144 (e.g. amino acid residue 143), one or more amino acids between amino acid residues 187-197 (e.g. amino acid residue 192), one or more amino acids between amino acid residues 215-225 (e.g. amino acid residue 220), one or more amino acids between amino acid residues 243-253 (e.g. amino acid residue 248), one or more amino acids between amino acid residues 255-265 (e.g.
  • amino acid residue 260 amino acid residue 260
  • amino acid residues 263-273 amino acid residue 268
  • amino acid residues 320-330 amino acid residue 325)
  • amino acid residues 339-349 e.g. amino acid residue 344
  • amino acid residues 358-359 e.g. amino acid residue 358 or 359
  • amino acid residues 388-398 one or more amino acids between amino acid residues 388-398
  • a modified Sap2 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 52-62 (e.g. amino acid residue 57), one or more amino acids between amino acid residues 93-107 (e.g.
  • amino acids between amino acid residues 98- 102 one or more amino acids between amino acid residues 104-118 (e.g. one or more amino acids between amino acid residues 109-113), one or more amino acids between amino acid residues 142- 144 (e.g. amino acid residue 143), one or more amino acids between amino acid residues 187-197 (e.g. amino acid residue 192), one or more amino acids between amino acid residues 215-225 (e.g. amino acid residue 220), one or more amino acids between amino acid residues 243-253 (e.g. amino acid residue 248), one or more amino acids between amino acid residues 255-265 (e.g. amino acid residue 260), one or more amino acids between amino acid residues 263-273 (e.g.
  • amino acid residue 268) amino acid residues 320-330 (e.g. amino acid residue 325), one or more amino acids between amino acid residues 339-349 (e.g. amino acid residue 344), one or more amino acids between amino acid residues 358-359 (e.g. amino acid residue 358 or 359), and (14) one or more amino acids between amino acid residues 388-398 (e.g. amino acid residue 398) of amino acid residues 57-398 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 57- 398 of SEQ ID NO: 1.
  • a modified Sap2 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 19 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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 57 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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 109-113 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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 19 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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 57 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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 109-113 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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 19-398 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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 398 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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 57 of amino acid residues 57-398 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 57-398 of SEQ ID NO: 1.
  • a modified Sap2 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 109-113 of amino acid residues 57-398 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 57-398 of SEQ ID NO: 1.
  • a modified Sap2 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 57-398 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 57-398 of SEQ ID NO: 1.
  • a modified Sap2 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 398 of amino acid residues 57-398 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 57-398 of SEQ ID NO: 1.
  • a modified Sap2 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) amino acid residue 57; and (ii) amino acid residue 398 of amino acid residues 57- 398 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 57-398 of SEQ ID NO: 1.
  • a modified Sap2 protein of the invention comprises the amino acid sequence of SEQ ID NO: 81.
  • a modified Sap2 protein of the invention comprises the amino acid sequence of SEQ ID NO: 85 (which additionally includes the fba sequence).
  • a modified Sap2 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) amino acid residue 57; (ii) amino acid resaidue 220; and (iii) amino acid residue 398 of amino acid residues 57-398 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 57-398 of SEQ ID NO: 1.
  • a modified Sap2 protein of the invention comprises the amino acid sequence of SEQ ID NO: 82. In other embodiments, a modified Sap2 protein of the invention comprises the amino acid sequence of SEQ ID NO: 86 (which additionally includes the fba sequence).
  • a modified Sap2 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) amino acid residue 57; (ii) the amino acids between amino acid residues 98-102; (iii) amino acid residue 220; and (iv) amino acid residue 398 of amino acid residues 57-398 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 57-398 of SEQ ID NO: 1.
  • a modified Sap2 protein of the invention comprises the amino acid sequence of SEQ ID NO: 83.
  • a modified Sap2 protein of the invention comprises the amino acid sequence of SEQ ID NO: 87 (which additionally includes the fba sequence).
  • a modified Sap2 protein of the invention comprising the amino acid sequence of SEQ ID NO: 87 was selected as the optimal protein carrier because it led to all the introduced glycosites being at least partially glycosylated, resulting in only minimal unglycosylated protein left and maximizing the sugar/protein ratio (see Fig. 4; MutN3C-3a-8a-fba-C).
  • the present invention provides a method of increasing efficiency of glycosylation of a modified Sap2 protein of the invention, the method comprising expressing in a host cell of the invention a modified Sap2 protein comprising at least four consensus sequences, wherein the consensus sequences have been added next to or substituted for (i) amino acid residue 57; (ii) the amino acids between amino acid residues 98-102; (iii) amino acid residue 220; and (iv) amino acid residue 398 of amino acid residues 57-398 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 57-398 of SEQ ID NO: 1, wherein glycosylation efficiency of the modified Sap2 protein is increased relative to glycosylation efficiency of a control Sap2 protein not comprising the at least four consensus sequences.
  • the modified Sap2 protein comprises the amino acid sequence of SEQ ID NO: 83. In other embodiments, the modified Sap2 protein comprises the amino acid sequence of SEQ ID NO: 87. In other embodiments, the present invention provides a method of increasing efficiency of glycosylation of a modified Sap2 protein of the invention, the method comprising expressing in a host cell of the invention a modified Sap2 protein comprising at least four consensus sequences, wherein the consensus sequences have been added next to or substituted for (i) amino acid residue 19; (ii) the amino acids between amino acid residues 98-102; (iii) amino acid residue 220; and (iv) amino acid residue 398 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1, wherein glycosylation efficiency of the modified Sap2 protein is increased relative to glycos
  • a modified Sap2 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) amino acid residue 220 and (ii) amino acid residue 398 of amino acid residues 57- 398 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 57-398 of SEQ ID NO: 1.
  • a modified Sap2 protein of the invention comprises the amino acid sequence of SEQ ID NO: 84.
  • a modified Sap2 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) amino acid residue 57; (ii) the amino acids between amino acid residues 98-102; and (iii) amino acid residue 220 of amino acid residues 57-398 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 57-398 of SEQ ID NO: 1.
  • a modified Sap2 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) amino acid residue 57; (ii) the amino acids between amino acid residues 98-102; (iii) the amino acids between amino acid residues 109-113; (iv) amino acid residue 220; and (v) amino acid residue 398 of amino acid residues 57-398 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 57-398 of SEQ ID NO: 1.
  • a modified Sap2 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) amino acid residue 19; and (ii) amino acid residue 398 of amino acid residues 19- 398 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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) amino acid residue 19; (ii) amino acid resaidue 220; and (iii) amino acid residue 398 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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) amino acid residue 19; (ii) the amino acids between amino acid residues 98-102; (iii) amino acid residue 220; and (iv) amino acid residue 398 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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) amino acid residue 220 and (ii) amino acid residue 398 of amino acid residues 19- 398 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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) amino acid residue 19; (ii) the amino acids between amino acid residues 98-102; and (iii) amino acid residue 220 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1.
  • a modified Sap2 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) amino acid residue 19; (ii) the amino acids between amino acid residues 98-102; (iii) the amino acids between amino acid residues 109-113; (iv) amino acid residue 220; and (v) amino acid residue 398 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1.
  • the modified Sap2 protein of the invention is from a fungi.
  • the fungi is Candida.
  • the modified Sap2 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 Sap2 protein of the invention is from Candida albicans.
  • at least one of the one or more consensus sequences comprises an amino acid sequence of 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, DQNAT (SEQ ID NO: 4) and DQNVT (SEQ ID NO: 5).
  • X is Q (glutamine)
  • Z is A (alanine)
  • the one or more consensus sequences are selected from the group consisting of DQNAT (SEQ ID NO: 4) and DQNVT (SEQ ID NO: 5).
  • a modified Sap2 protein of the invention further comprises at least one Fructose biphosphate aldolase (Fba) peptide.
  • the 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. Proteomics analysis revealed that 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.
  • the modified Sap2 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 Sap2 protein of the invention.
  • the at least one Fba peptide is non- covalently linked to a modified Sap2 protein of the invention.
  • the at least one Fba peptide is covalently linked to a modified Sap2 protein of the invention.
  • the Fba peptide is linked to a modified Sap2 protein of the invention at a single amino acid residue.
  • the Fba peptide is linked to a modified Sap2 protein of the invention at more than one amino acid residues. In additional embodiments, a Fba peptide is linked to a modified Sap2 protein of the invention at one or more amino acid residues. In specific aspects, the Fba peptide is linked to a modified Sap2 protein of the invention at amino acid residue 398.
  • 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 Sap2 protein of the invention at amino acid residue 398 of amino acid residues 19-398 of SEQ ID NO: 1 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, modified Sap2 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.
  • a modified Sap2 protein of the invention comprises at least one additional consensus sequence. In some embodiments, the at least one additional consensus sequence has been added next to C-terminal amino acid residue of a modified Sap2 protein of the invention.
  • a modified Sap2 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 Sap2 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 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 modified Sap2 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: 7).
  • the additional consensus sequence comprises (or consists of) an amino acid sequence of GSGGGDQNATGSGGGHHHHHHHHHH (SEQ ID NO: 8).
  • a modified Sap2 protein of the invention comprises (or consists of) an amino acid sequence of SEQ ID NO: 9: STPTTTKRSAGFVALDFSVVKTPKAFPVTNGQEGKTSKRGGGDQNATGGGQAVPVTLHNEQVTYAAD ITVGSNNQKLNVIVDTGSSDLWVPDQNVTCQVTYSDQTADFCKQKGTYDPSGSSASQDLNTPFKIGYGDGSSS QGTLYKDTVGFGGVSIKNQVLADVDSTSIDQGILGVGYKTNEAGGSYDNVPVTLKKQGVIAKNAYSLYLNSPDQ NATGQIIFGGVDNAKYSGSLIALPVTSDRELRISLGSVEVSGKTINTDNVDVLVNSGTTITYLQQDLADQIIKAFN GKLTQDSNGNSFYEVDCNLSGDVVFNFSKNAKISVPASEFAASLQGDDGQPYDKCQLLFDVNDANILGDNFLRS AYIVYDLDDNEISL
  • a modified Sap2 protein of the invention comprises (or consists of) an amino acid sequence of SEQ ID NO: 10: GGGDQNATGGGQAVPVTLHNEQVTYAADITVGSNNQKLNVIVDTGSSDLWVPDQNVTCQVTYSDQ TADFCKQKGTYDPSGSSASQDLNTPFKIGYGDGSSSQGTLYKDTVGFGGVSIKNQVLADVDSTSIDQGILGVGY KTNEAGGSYDNVPVTLKKQGVIAKNAYSLYLNSPDQNATGQIIFGGVDNAKYSGSLIALPVTSDRELRISLGSVE VSGKTINTDNVDVLVNSGTTITYLQQDLADQIIKAFNGKLTQDSNGNSFYEVDCNLSGDVVFNFSKNAKISVPAS EFAASLQGDDGQPYDKCQLLFDVNDANILGDNFLRSAYIVYDLDDNEISLAQVKYTSASSISALTYGKDVKDLFD YAQ
  • a modified Sap2 protein of the invention is glycosylated.
  • the modified Sap2 protein of the invention is N-glycosylated. It will be understood by a person skilled in the art, that reference to “between amino acids ...” (for example “between amino acids 19-24”) is referring to the amino acid number counting consecutively from the N-terminus of the amino acid sequence, for example “between amino acids 19 to 24...of SEQ ID NO: 1” refers to position in the amino acid sequence between amino acid 19 and amino acid 24 of SEQ ID NO: 1 including both amino acids 19 and 24.
  • the one or more consensus sequences may have been added next to or substituted for any one (or more) of amino acid numbers 19, 20, 21, 22, 23, and 24 in SEQ ID NO: 1.
  • Sap2 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 the 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 Sap2 protein of the invention is an isolated modified Sap2 protein.
  • a modified Sap2 protein of the invention is a recombinant modified Sap2 protein.
  • a modified Sap2 protein of the invention is an isolated recombinant modified Sap2 protein.
  • a modified Sap2 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 Sap2 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: 18) 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: 18), 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: 4) also referred to as “DQNAT” (SEQ ID NO: 4).
  • the consensus sequence is K-D/E-X-N-Z-S/T (SEQ ID NO: 18), wherein X is Q (glutamine) and Z is A (alanine), e.g. K-D-Q-N-A-T (SEQ ID NO: 19) also referred to as “KDQNAT” (SEQ ID NO: 19).
  • the consensus sequence is K-D/E-X-N-Z-S/T (SEQ ID NO: 18), wherein X is Q (glutamine) and Z is A (alanine), e.g. K-D-Q-N- A-S (SEQ ID NO: 20) also referred to as “KDQNAS” (SEQ ID NO: 20).
  • 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: 18) 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: 6), 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: 7) also referred to as “GSGGGDQNATGSGGG” (SEQ ID NO: 7).
  • the modified Sap2 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 Sap2 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 Sap2 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 Sap2 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 Sap2 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 Sap2 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 Sap2 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 Sap2 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 Sap2 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 Sap2 amino acid sequence of SEQ ID NO: 1 or a Sap2 amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1.
  • a modified Sap2 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 e.g. hexa-histidine tags.
  • the tags used herein are removable, e.g.
  • a modified Sap2 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 Sap2 protein of the invention.
  • the peptide tag comprises six histidine residues at the C-terminus of the amino acid sequence of a modified Sap2 protein of the invention.
  • the present invention provides a modified Sap2 protein comprising a tag (e.g., a histidine tag).
  • a modified Sap2 protein of the invention comprises (or consists of): (i) amino acid residues 19-398 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 19-398 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 Sap2 protein of the invention comprises (or consists of) amino acid residues 19-398 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 19-398 of SEQ ID NO: 1, with the peptide tag (e.g. histidine tag) removed.
  • a modified Sap2 protein of the invention comprises a signal sequence which is capable of directing the modified Sap2 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: 38)], heat labile E. coli enterotoxin LTIIb [MSFKKIIKAFVIMAALVSVQAHA (SEQ ID NO: 39)], Bacillus subtilis endoxylanase XynA [MFKFKKKFLVGLTAAFMSISMFSATASA (SEQ ID NO: 40)], E.
  • the signal sequence is from E. coli flagellin (FlgI) [MIKFLSALILLLVTTAAQA (SEQ ID NO: 21)].
  • the present invention provides a modified Sap2 protein, wherein the amino acid sequence further comprises a signal sequence which is capable of directing the modified Sap2 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 Sap2 protein of the invention after the modified Sap2 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 Sap2 protein of the invention.
  • the present invention provides a polynucleotide encoding a modified Sap2 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: 9 or 10.
  • the present invention provides a nucleotide sequence according to SEQ ID NO: 35, or a nucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 35.
  • the present invention provides a nucleotide sequence according to SEQ ID NO: 36, or a nucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 36.
  • the present invention provides a nucleotide sequence according to SEQ ID NO: 37, or a nucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37.
  • 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: 19), KDQNAS (SEQ ID NO: 20), DQNAT (SEQ ID NO: 4), and GSGGGDQNATGSGGG (SEQ ID NO: 7).
  • a nucleotide sequence of the invention comprises nucleotides encoding for a modified Sap2 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 19, amino acid residue 57, one or more amino acids between amino acid residues 98-102, amino acid residue 87, one or more amino acids between amino acid residues 104-108, one or more amino acids between amino acid residues 109-113, amino acid residue 220, one or more amino acids between amino acid residues 142-144, amino acid residue 198, amino acid residue 248, amino acid residue 261, amino acid residue 268, amino acid residue 325, amino acid residue 344, one or more amino acids between amino acid residues 358-359, and amino acid residue 398 of amino acid residues 19-398 of SEQ ID NO: 1 or at equivalent positions within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
  • the present invention provides a vector comprising a polynucleotide encoding a modified Sap2 protein of the invention.
  • Conjugates In certain embodiments, the present invention provides a conjugate comprising a modified Sap2 protein of the invention.
  • the conjugate of the invention may be a conjugate of a modified Sap2 protein (e.g. chemical conjugate or bioconjugate).
  • the conjugate of the invention may be a conjugate of a modified Sap2 protein and an antigen, e.g. a saccharide antigen (i.e. bioconjugate).
  • the present invention provides a conjugate comprising (or consisting of) a modified Sap2 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, Sap2, Als3, 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 Sap2 protein of the invention.
  • the modified Sap2 protein of the invention is linked to the at least one saccharide antigen.
  • the modified Sap2 protein of the invention is directly linked to the at least one saccharide antigen.
  • the modified Sap2 protein of the invention is linked to the at least one saccharide antigen through a linker.
  • the modified Sap2 protein of the invention is non-covalently linked to the at least one saccharide antigen.
  • the modified Sap2 protein of the invention is non-covalently linked to the at least one saccharide antigen via an avidin-strepatvidin interaction.
  • the modified Sap2 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).
  • the chemical conjugation method may be selected from the group consisting of carbodiimide chemistry, reductive animation, cyanylation chemistry (for example CDAP chemistry), maleimide chemistry, hydrazide chemistry, ester chemistry, and N-hydroysuccinimide chemistry.
  • Conjugates can be prepared by direct reductive amination methods as described in, US200710184072 (Hausdorff) US 4365170 (Jennings) and US 4673574 (Anderson).
  • conjugation method may alternatively rely on activation of the saccharide with 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 NH2.
  • 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 Sap2 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 Sap2 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 Sap2 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 Sap2 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 Sap2 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 Sap2 protein (e.g. chemical conjugate or bioconjugate).
  • the conjugate of the invention is a conjugate of an isolated recombinant modified Sap2 protein and a recombinant antigen, e.g.
  • the modified Sap2 protein of the invention is linked to the at least one saccharide antigen at one or more amino acid residues on the modified Sap2 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 Sap2 protein of the invention is linked to the at least one saccharide antigen at one or more asparagine residues on the modified Sap2 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 Sap2 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 Sap2 protein of the invention.
  • the at least one saccharide antigen is linked to at least three asparagine residues of a modified Sap2 protein of the invention. In specific embodiments, the at least one saccharide antigen is linked to four asparagine residues of a modified Sap2 protein of the invention.
  • the modified Sap2 protein of the invention comprises (or consists of) an amino acid sequence of SEQ ID NO: 9, and the four asparagine residues include, but are not limited to, positions 45, 94, 215, and 415 of SEQ ID NO: 9.
  • the present invention provides a modified Sap2 protein of Candida albicans comprising (or consisting of): (1) an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and (2) at least one saccharide antigen of Candida, wherein the at least one saccharide antigen is a ⁇ -1,2 mannan polymer consisting of at least five consecutive ⁇ -1,2 linked mannose molecules, and wherein the at least one saccharide chain is linked to at least one of four asparagine residues at positions 45, 94, 215, and 415 of SEQ ID NO: 9 or at least one of four asparagine residues at positions 6, 55, 176, and 376 of SEQ ID NO: 10.
  • the present invention provides conjugates (e.g., bioconjugates) wherein a modified Sap2 protein of the invention may be linked to a number of different antigens.
  • the modified Sap2 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, Citrobacter freundii O antigen, Agrobacterium sp. exopolysaccharide, 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 ⁇ -1,2 mannan polymer.
  • the at least one saccharide antigen is a ⁇ -1,2 mannan polymer of Candida albicans.
  • Mannans form 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. guilliermondii, C. lusitaniae, C. tropicalis, and excluding C.
  • Candida species e.g., including C. albicans, C. auris, C. guilliermondii, C. lusitaniae, C. tropicalis, and excluding 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.
  • the at least one saccharide antigen comprises a ⁇ -1,2 mannan polymer.
  • 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 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
  • the ⁇ -1,2 mannan polymer comprises at least 2, at least 3, at least 4, or at least 5 ⁇ - 1,2 linked mannose molecules. In additional aspects, 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.
  • the present invention provides a saccharide that is a ⁇ -1,2 mannan polymer comprising the structure: In some embodiments, the present invention provides a saccharide having the structure: In cert e comprising the structure: ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 3)-x-D-GlcpNAc In additional embodiments, the present invention provides a saccharide comprising the structure: ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp- (1 ⁇ 3)-x-D-GlcpNAc In certain embodiments, the at least one saccharide antigen comprises a glucan having the structure: In other embodiments, the at least one saccharide antigen comprises a glucan having the
  • the at least one saccharide antigen is a ⁇ -1,3 glucan polymer of Candida albicans.
  • ⁇ -1,3 glucans are widespread in nature. They are present in most yeasts and widespread in fungi and plants.
  • 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 twenty five, six to twenty, six to ten, six to nine, six to eight, six to seven, six, seven to hundred, seven to fifty, six to forty, six to thirty five, six to twenty five, six to twenty, six to ten, six to
  • 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. In specific embodiments, the ⁇ -1,3 glucan polymer comprises at least 11 ⁇ -1,3 linked glucose molecules. In particular embodiments, the ⁇ -1,3 glucan polymer comprises at least 11 consecutive ⁇ -1,3 linked glucose molecules.
  • the present invention provides a modified Sap2 protein of Candida albicans comprising (or consisting of): (1) an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and (2) at least one saccharide antigen of Candida, wherein the at least one saccharide antigen is a ⁇ -1,2 mannan polymer comprising (or consisting of) at least five consecutive ⁇ -1,2 linked mannose molecules, and wherein the at least one saccharide antigen is linked to at least one of four asparagine residues at positions 45, 94, 215, and 415 of SEQ ID NO: 9 or at least one of four asparagine residues at positions 6, 55, 176, and 376 of SEQ ID NO: 10.
  • the present invention provides host cells comprising a polynucleotide sequence that encodes a modified Sap2 protein of the invention.
  • 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.
  • the host cell is a non-human host cell.
  • 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.
  • 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.
  • 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.
  • 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 when nucleotide sequences are deleted from the genome of the host cells of the invention, they are replaced by a desirable sequence, e.g. a sequence that is useful for glycoprotein production.
  • a desirable sequence e.g. a sequence that is useful for glycoprotein production.
  • Exemplary genes that can be deleted in host cells (and, in some cases, replaced with other desired nucleotide sequences) include genes of host cells involved in glycolipid biosynthesis, such as waaL (see, e.g. Feldman et al.
  • 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.
  • uppS Undecaprenyl pyrophosphate synthase
  • uppP Undecaprenyl diphosphatase
  • 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 Sap2 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 Sap2 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 Sap2 protein of the invention; and, optionally, (4) a polynucleotide sequence that encodes a polymerase.
  • 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 Sap2 protein of the invention (optionally a polynucleotide sequence of the invention); and, optionally, (4) a polynucleotide sequence that encodes a polymerase.
  • the one or more heterologous glycosyltransferases includes, without limitation, WbaD, WbaC, and WbaB.
  • the WbaD, WbaC, and WbaB are from Citrobacter.
  • the Citrobacter is Citrobacter freundii.
  • the Citrobacter freundii is Citrobacter freundii P079F I.
  • the one or more heterologous glycosyltransferases comprises WbaD, WbaC, and WbaB from Citrobacter, optionally from Citrobacter freundii, optionally Citrobacter freundii P079F I.
  • the one or more heterologous glycosyltransferase(s) has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaD of Citrobacter freundii P079F I comprising SEQ ID NO: 11. In other embodiments, the one of more heterologous glycosyltransferase(s) has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaC of Citrobacter freundii P079F I comprising SEQ ID NO: 12.
  • the one of more heterologous glycosyltransferase(s) has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaB of Citrobacter freundii P079F I comprising SEQ ID NO: 13.
  • the present invention provides a host cell comprising: i. a nucleotide sequence encoding one or more heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,2 mannan polymer; ii.
  • nucleotide sequence encoding a second heterologous glycosyltransferase which is eukaryotic and is capable of covalently bonding a mannose molecule to a ⁇ -1,2 mannan polymer to extend a ⁇ -1,2 mannan polymer chain; iii. a nucleotide sequence encoding a heterologous oligosaccharyl transferase; and iv. optionally, 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 independently any amino acid except proline.
  • the present invention provides a host cell comprising: i. a nucleotide sequence encoding one or more heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,2 mannan polymer; ii. a nucleotide sequence encoding a second heterologous glycosyltransferase which is eukaryotic and is capable of covalently bonding a mannose molecule to a ⁇ -1,2 mannan polymer to extend a ⁇ -1,2 mannan polymer chain; iii. a nucleotide sequence encoding a heterologous oligosaccharyl transferase; and iv.
  • the modified carrier protein of the invention includes, but is not limited to, Sap2, Als3, 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 Sap2 protein of the invention.
  • the one or more heterologous glycosyltransferases capable of synthesizing a ⁇ -1,2 mannan polymer includes, without limitation, WbaD, WbaC, and WbaB.
  • the WbaD, WbaC, and WbaB are from Citrobacter.
  • the Citrobacter Citrobacter freundii is Citrobacter freundii P079F I.
  • the one or more heterologous glycosyltransferases comprises WbaD, WbaC, and WbaB from Citrobacter, optionally from Citrobacter freundii, optionally Citrobacter freundii P079F I.
  • the one or more heterologous glycosyltransferases capable of synthesizing a ⁇ -1,2 mannan polymer has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaD of Citrobacter freundii P079F I.
  • the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of WbaD of Citrobacter freundii P079F I.
  • WbaD of Citrobacter freundii P079F I comprises the amino acid sequence of SEQ ID NO: 11.
  • the one or more heterologous glycosyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 11.
  • the one or more heterologous glycosyltransferases has an amino acid sequence identical to SEQ ID NO: 11: MSNNPKKKILVLTPRYPFPVIGGDKLRIYKICQELSKYYDLTLLTLIDDIQDLTIPHDEEVFKYVHKIYLPK IKSFFNVLLALPTSTPLQVAYYKSDNFKQKLNELLPAYDATLSHLIRVGHYAKDVNGVNFLEMTDAISLNYKRVRE IKTLKSFKSFIFSLEQKRLERYERSIASSFDLTTFISAVDKNFLYPEERTDVIVSGNGVDTNFLQFKNRHIKSQEPVV LIFIGNMLSLQNMDAVTFFAKKILPLLNEKGNFIFKVIGKISEKSRRILSAIPDVIVTGTVDNILETASDGHIGICSM RLGAGVQNKVLEYMALGMPCVTTTVGFEGIGARDGNDIVIADSPREYVTAIEKLVNDSNYFSSIAINARNFVVSQ YSWEM
  • host cells of the present invention comprise a nucleotide sequence encoding WbaD, optionally WbaD of Citrobacter freundii P079F I, optionally a nucleotide sequence encoding WbaD of Citrobacter freundii P079F I comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 25: ATGAGCAATAACCCCAAGAAAAAAATTCTTGTTCTAACGCCTCGCTATCCATTTCCTGTAATCGGTG GTGATAAACTGAGAATTTATAAAATATGTCAGGAACTTTCAAAATACTATGATCTTACATTATTAACTTTAATT GATGATATTCAGGATTTAACCATACCTCATGATGAGGAAGTTTTTAAATACGTACATAAGATATATCTCCCTA AAATAAAGTCTTTTTTTAATGTACTTTTGGCATTACCTACTTCTACGCCATTGC
  • the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of WbaC of Citrobacter freundii P079F I.
  • WbaC of Citrobacter freundii P079F I comprises the amino acid sequence of SEQ ID NO: 12.
  • the one or more heterologous glycosyltransferases 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 has an amino acid sequence identical to SEQ ID NO: 12: MRIVVNNFFYGVLKRGIPIYTSELVAKLREEGVEVKELRCPKFLYGLPTWIHNFLFIIYDQIITPLFGLFHK TKYNIYPYNSLSLIDLFTNKPIVIIHDFISLNKNKKNISACYVKFCILSSSNRIRNVILISNTTAKIANKLSLFSNARQI LLPNTFFSFKSLSDGVQKEDHGFLLLVSGMGKNKDIDAALELYFSIPIEYRIPLKILGCGGGRDLLKAKIHGRDEFN TIEILKQIPLEDVVKLYAHCKFVWAHSLAEGYGRALAEGKISGKNILCTRIPAFIEQNCSNVFYYNDTESFQEHYFN LIENTPVVDVCELKEHIKFSEELKKIYEQ
  • host cells of the present invention comprise a nucleotide sequence encoding WbaC, optionally
  • host cells of the present invention comprise a nucleotide sequence encoding WbaC, optionally WbaC of Citrobacter freundii P079F I, optionally a nucleotide sequence encoding WbaC of Citrobacter freundii P079F I comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 26: ATGAGAATTGTAGTAAATAATTTCTTTTATGGTGTGCTAAAACGTGGAATCCCTATTTATACTTCTG AGTTAGTCGCTAAATTAAGAGAGGAAGGAGTTGAGGTTAAAGAACTAAGATGTCCTAAGTTTCTTTACGGCT TACCTACTTGGATTCATAATTTTCTTTTTATTATTTACGATCAAATTATTACTCCTCTATTTGGTCTTTTTCAT AAAACAAAATATAACATTTATCCATACAACAGTTTATCCTTAATAGACTTGTTC
  • the one or more heterologous glycosyltransferases has an amino acid sequence identical to the amino acid sequence of WbaB of Citrobacter freundii P079F I.
  • WbaB of Citrobacter freundii P079F I 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.
  • the one or more heterologous glycosyltransferases has an amino acid sequence identical to SEQ ID NO: 13: MIVFFTGSYPPSKCGVGDYLYKLIANILPHHSNVKVIKNSLLEFIFYAISNRKAIRHVNIQYPTIGYASNYL SAFKPHFVTLIARFLGIRVSITLHEFTSLSSKARFFANLFKIANNIVATSEYEHENLVKFGFNKDRVIVIPIGSNIKES DVKDKTIDFINFGIISPGKGIEDFLYVIEKIRVDYETLKVVLAGYIPDNSEYADKIIAQAKQLNIEFKPNQTEDELSIL VGESKRAILPYSDGISERRGTALAAMINKCVVYSYAGNSSEAFNTICMLARDRDELYNKLIDALHTDSADDCFIAK AYEYALARHWDKVSEKYLRMFYENCSK
  • host cells of the present invention comprise a nucleotide sequence encoding WbaB, optionally Wb
  • host cells of the present invention comprise a nucleotide sequence encoding WbB, optionally WbaB of Citrobacter freundii P079F I, optionally a nucleotide sequence encoding WbaB of Citrobacter freundii P079F I comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 27: ATGATAGTTTTTTTTACAGGCTCATATCCTCCGAGTAAGTGTGGTGTTGGTGATTATTTGTATAAAT TAATAGCTAATATTTTACCACACCATTCAAATGTTAAAGTTATAAAAAACTCATTGCTTGAATTTATTTTTTAT GCCATTTCAAATAGGAAGGCCATAAGACATGTAAATATACAATATCCGACAATTGGCTATGCTAGTAATTATT TAAGTGCATTTAAACCTCATTTTGTGACACTTATAGCTAGATTTTTGGGGATTAGAGTT
  • the one of more heterologous glycosyltransferase(s) has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaC of Citrobacter freundii P079F I comprising SEQ ID NO: 12. In still other embodiments, the one of more heterologous glycosyltransferase(s) has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaB of Citrobacter freundii P079F I comprising SEQ ID NO: 13.
  • the host cell of the invention further comprises a heterologous translocase, wherein the heterologous translocase has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of Wzx of Citrobacter freundii P079F I comprising SEQ ID NO: 14
  • host cells of the invention comprise one or more polynucleotide sequences that encode one or more heterologous guanylyltransferases.
  • the one or more heterologous guanylyltransferases includes, without limitation, manB and manC.
  • the manB and manC are from E. coli.
  • the E. coli is E.
  • the one or more heterologous guanylyltransferases comprises manB and manC from E. coli, optionally from E. coli K12 W3110. In some aspects, the one or more heterologous guanylyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of manB of E. coli K12 W3110. In some aspects, the one or more heterologous guanylyltransferases has an amino acid sequence identical to the amino acid sequence of manB of E. coli K12 W3110. In certain embodiments, manB of E. coli K12 W3110 comprises the amino acid sequence of SEQ ID NO: 31.
  • the one or more heterologous guanylyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 31.
  • the one or more heterologous guanylyltransferases has an amino acid sequence identical to SEQ ID NO: 31: MKKLTCFKAYDIRGKLGEELNEDIAWRIGRAYGEFLKPKTIVLGGDVRLTSETLKLALAKGLQDAGVDVL DIGMSGTEEIYFATFHLGVDGGIEVTASHNPMDYNGMKLVREGARPISGDTGLRDVQRLAEANDFPPVDETKRG RYQQINLRDAYVDHLFGYINVKNLTPLKLVINSGNGAAGPVVDAIEARFKALGAPVELIKVHNTPDGNFPNGIPNP LLPECRDDTRNAVIKHGADMGIAFDGDFDRCFLFDEKGQFIEGYYIVGLLAEAFLEKNPGAKIIHDPRLSWNTVD V
  • host cells of the present invention comprise a nucleotide sequence encoding manB, optionally manB of E. coli K12 W3110, optionally a nucleotide sequence encoding manB of E.
  • coli K12 W3110 comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 33: ATGAAAAAATTAACCTGCTTTAAAGCCTATGATATTCGCGGGAAATTAGGCGAAGAACTGAATGAAG ATATCGCCTGGCGCATTGGTCGCGCCTATGGCGAATTTCTCAAACCGAAAACCATTGTGTTAGGCGGTGATG TCCGCCTCACCAGCGAAACCTTAAAACTGGCGCTGGCGAAAGGTTTACAGGATGCGGGCGTTGACGTGCTGG ATATTGGTATGTCCGGCACCGAAGAGATCTATTTCGCCACGTTCCATCTCGGCGTGGATGGCGGCATTGAAG TTACCGCCAGCCATAATCCGATGGATTATAACGGCATGAAGCTGGTTCGCGAGGGGGCTCGCCCGATCAGCG GAGATACCGGACTGCGCGACGTCCAGCGTCTGGCTGAAGCCAACGACTTTCCTCCCGTCGATG
  • the one or more heterologous guanylyltransferases has an amino acid sequence identical to the amino acid sequence of manC of E. coli K12 W3110.
  • manC of E. coli K12 W3110 comprises the amino acid sequence of SEQ ID NO: 32.
  • the one or more heterologous guanylyltransferases has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 32.
  • the one or more heterologous guanylyltransferases has an amino acid sequence identical to SEQ ID NO: 32: MAQSKLYPVVMAGGSGSRLWPLSRVLYPKQFLCLKGDLTMLQTTICRLNGVECESPVVICNEQHRFIVA EQLRQLNKLTENIILEPAGRNTAPAIALAALAAKRHSPESDPLMLVLAADHVIADEDAFRAAVRNAMPYAEAGKLV TFGIVPDLPETGYGYIRRGEVSAGEQDMVAFEVAQFVEKPNLETAQAYVASGEYYWNSGMFLFRAGRYLEELKK YRPDILDACEKAMSAVDPDLNFIRVDEEAFLACPEESVDYAVMERTADAVVVPMDAGWSDVGSWSSLWEISAH TAEGNVCHGDVINHKTENSYVYAESGLVTTVGVKDLVVVQTKDAVLIADRNAVQDVKKVVEQIKADGRHEHRV HREVYRPWGKYDSIDAGDRYQVK
  • host cells of the present invention comprise a nucleotide sequence encoding manC, optionally manC of E. coli K12 W3110, optionally a nucleotide sequence encoding manC of E.
  • coli K12 W3110 comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 34: ATGGCGCAGTCGAAACTCTATCCAGTTGTGATGGCAGGTGGCTCCGGTAGCCGCTTATGGCCGCTT TCCCGCGTACTTTATCCCAAGCAGTTTTTATGCCTGAAAGGCGATCTCACCATGCTGCAAACCACCATCTGCC GCCTGAACGGCGTGGAGTGCGAAAGCCCGGTGGTGATTTGCAATGAGCAGCACCGCTTTATTGTCGCGGAA CAGCTGCGTCAACTGAACAAACTTACCGAGAACATTATTCTCGAACCGGCAGGGCGAAACACGGCACCTGCC ATTGCTGGCGGCGCTGGCGGCAAAACGTCATAGCCCGGAGAGCGACCCGTTAATGCTGGTATTGGCGGC GGATCATGTGATTGCCGATGAAGACGCGTTCCGTGCCGCCGTGCGTAATGCCATGC
  • the Bmt3 is from Candida.
  • the Candida is C. albicans.
  • the glycosyltransferase capable of covalently bonding a mannose molecule to a ⁇ -1,2 mannan polymer to extend a ⁇ -1,2 mannan polymer chain is Bmt3 of C. albicans.
  • the glycosyltransferase capable of covalently bonding a mannose molecule to a ⁇ -1,2 mannan polymer to extend a ⁇ -1,2 mannan polymer chain has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of Bmt3 of C. albicans.
  • the glycosyltransferase capable of covalently bonding a mannose molecule to a ⁇ -1,2 mannan polymer to extend a ⁇ -1,2 mannan polymer chain has an amino acid sequence identical to the amino acid sequence Bmt3 of C. albicans.
  • periplasmic expression of Bmt3 of C. albicans in the host cell is required to extend a ⁇ -1,2 mannan polymer chain.
  • addition of the terminal mannose residue to the ⁇ -1,2 mannan polymer chain requires periplasmic expression of Bmt3 of C. albicans in the host cell.
  • addition of the terminal mannose residue to the ⁇ -1,2 mannan polymer chain further requires presence of GDP-mannose in the culture medium.
  • the GDP-mannose is added to the harvested culture.
  • the conditions suitable for the production of a glycoconjugate of the invention includes the addition of GDP-mannose to the culture medium.
  • the GDP-mannose is added to the harvested culture.
  • the Bmt3 of C. albicans comprises the amino acid sequence of SEQ ID NO: 15. In other embodiments, the Bmt3 of C.
  • albicans comprises an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 15: MKVKVLSLLVPALLVAGAANASDYTPIKVSGYTFKNQVATKNLQCDSIVYDQDLDLQVSQAVDLNKPEDLKFFRD KLNELRSLNNIYDLFFQDNEDEVEESILERKWYKFCGSAVWLDKYGVYFMVNRIAYSKKGTRNNPTISVLAGQVF DKNWIELTGKKFPFSGLEFPTILPHYIDEGKEAEKVILGAEDPRVILHEYTNENGIRIQEPLIAFNALSTEVDWKRA MHIYRPLHDPHRIIRLSIENYAPREKEKNWAPFIDGNNLNFVYNFPLRILRCNINNGDCQKVSGPDFNDKSHENA GKLRGGTNLVEIPSQSLPKHLRSRKYWFGIARSHITDCGCVGELYRPHLILISRNKKSDQYELNYVSDLIDFNVNP EP
  • albicans optionally a nucleotide sequence encoding Bmt3 of C. albicans 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 Bmt3, optionally Bmt3 of C. albicans, optionally a nucleotide sequence encoding Bmt3 of C.
  • albicans comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 29: ATGAAAGTGAAAGTGCTGAGCCTGCTGGTTCCGGCACTGCTGGTTGCAGGTGCCGCCAATGCTAGCGACTAC ACCCCGATTAAAGTGAGCGGTTATACCTTCAAAAACCAGGTTGCGACCAAGAACCTGCAATGCGATAGCATC GTGTACGACCAGGATCTGGACCTGCAGGTGTCTCAAGCGGTTGATCTGAACAAGCCGGAGGACCTGAAATTC TTTCGTGATAAGCTGAACGAACTGCGTAGCCTGAACAACATTTACGACCTGTTCTTTCAAGATAACGAGGAC GAAGTGGAGGAAAGCATCCTGGAGCGTAAGTGGTATAAATTCTGCGGTAGCGCGGTTTGGCTGGATAAATA CGGCGTGTATTTTATGGTTAACCGTATCGCGTATAGCAAGAAAGGTACCCGTAACA
  • 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, optionally an evolved PglB comprising an amino acid sequence of SEQ ID NO: 16.
  • the PglB is an evolved pglB comprising an amino acid sequence at least 70%, 75%, 80%, 85%, 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.
  • the PglB is an evolved PglB enzyme comprising the amino acid sequence of SEQ ID NO: 16.
  • the evolved PglB has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 16.
  • the evolved PglB has an amino acid sequence identical to SEQ ID NO: 16: MLKKEYLKNPYLVLFAMIILAYVFSVFCRFYWVWWASEWNEYFHNNQLMIISNDGYAFAEGARDMIAG FHQPNDLSYYGSSLSALTYWLYKITPFSFESIILYMSTFLSSLVVIPQILLANEYKRRLMGFVAALLASIANSYYNRT MSGYYDTDMLVIVLPMFILFFMVRMILKKDFFSLIALPLFIGIYLWWYPSSYTLNVALIGLFLIYTLIFHRKEKIFYIA VILSSLTLSNIAWFYQSAIIVILFALFALEQKRLNFMIIGILGSAWLIFLILSGGVDPILYPLKFYIFRSDESTNLTQGF MYFNVVQTIQEVENVDLSEFMRRISGSEIVFLFSLLGFVWLLRKHKSMIMALPILVLGFLALKGGLRFTIYSVPVMA LGFGFLLSEFKA
  • host cells of the present invention comprise a nucleotide sequence encoding an evolved PglB, optionally a nucleotide sequence encoding an evolved PglB comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 30: ATGCTGAAGAAGGAATATCTGAAGAACCCGTATCTGGTGCTGTTTGCGATGATTATCCTGGCGTAT GTTTAGTGTGTTTTGTCGTTTCTACTGGGTGTGGTGGGCCAGTGAATGGAACGAATATTTCCACAACAAC CAGCTGATGATCATCTCCAATGATGGCTATGCCTTCGCAGAAGGTGCCCGTGACATGATTGCAGGCTTTCAT CAGCCGAACGATCTGAGTTATTACGGTAGCTCTCTGTCCGCGCTGACCTATTGGCTGTACAAAATCACGCCG TTTAGTTTCGAATCCATTATCCTGTACATGAGTACCTTCCTGAGTTC
  • 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. Wzx), e.g. a heterologous translocase.
  • a translocase e.g. Wzx
  • 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. ABC transporter) (Cuthbertson L. et al., 2010, Microbiology and Molecular Biology Reviews, 74(3):341-362; Bi Y. et al., 2018, Nature, 553(7688):361-365)).
  • 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. Undecaprenyl diphosphate (Und-PP)) leading to the formation of a lipid-linked antigen saccharide.
  • 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.
  • 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.
  • the heterologous translocase that is introduced into a host cell of the invention is an ABC transporter (e.g.
  • the heterologous translocase includes, without limitation, Wzx from Citrobacter.
  • the Citrobacter is Citrobacter freundii.
  • the Citrobacter freundii is Citrobacter freundii P079F I.
  • the translocase is Wzx from Citrobacter, optionally from Citrobacter freundii, optionally Citrobacter freundii P079F.
  • the heterologous translocase has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of Wzx of Citrobacter freundii P079F I.
  • the heterologous translocase has an amino acid sequence identical to the amino acid sequence of Wzx of Citrobacter freundii P079F I.
  • Wzx of Citrobacter freundii P079F I comprises the amino acid sequence of SEQ ID NO: 14.
  • the heterologous translocase has an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 14.
  • the heterologous translocase has an amino acid sequence identical to SEQ ID NO: 14: MCTKFIKKIPSHFVVAGSAWGSRFISIFVQFYSIKILLNYLGTNGYALFSLIASFSAWFLLVDIGMSTNLQ NKISERKAYGKAYFDLVKRTGCFLIVALTLFVILLWIFGPYLSRILLVSFDFLSEKDKNNIFFISSLLFIGNGIGFFAYK IWYAEHKGWISNILPAISSICGLLFLILLRTEQFNISHLIIVCLLSFYGPAAFLGVLSFLTTFASSLHYKEKPLSHSIVQ DIIRPSMGFFSFSVMAALVLQVDYIIMSHTLTGKDIVIYNVLSKIFGLINFLYAALLQSVWPLCAEAKYKDDKSIYN QIKVKYIGFGAILVFAIS
  • host cells of the present invention comprise a nucleotide sequence encoding Wzx, optionally Wzx of Citrobacter freundii P079F I, optionally a nucleotide sequence encoding Wzx of Citrobacter freundii P079F I comprising a nucleotide sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 28: ATGTGCACTAAATTTATAAAAAAAATCCCTAGTCATTTTGTTGTAGCAGGTAGTGCGTGGGGGAGTC GATTTATATCTATATTTGTACAATTCTATAGTATAAAGATATTATTAAACTATTTGGGTACAAACGGGTATGC TCTTCTCATTAATTGCGAGTTTCTCTGCATGGTTTCTCTTGGTTGATATTGGTATGTCAACCAATTTGCAA AATAAAATATCGGAGCGTAAAGCCTATGGAAAGGCATATTTTGATTTGGTTAAAAGA
  • the wzx gene is from Citrobacter freundii P079F I.
  • the present invention provides a method of producing a ⁇ -1,2 mannan polymer in a host cell of the invention, the method comprising the steps of introducing and expressing in the host cell: i. a nucleotide sequence encoding one or more first heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,2 mannan polymer; ii.
  • the present invention provides a method of producing a ⁇ -1,2 mannan polymer in a prokaryotic host cell, the method comprising the steps of introducing and expressing in the host cell: iv. a nucleotide sequence encoding one or more first heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,2 mannan polymer, wherein the one of more heterologous glycosyltransferase(s) comprises WbaD, WbaC, and WbaB from Citrobacter, optionally from Citrobacter freundii, optionally from Citrobacter freundii P079F I; v.
  • 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 Wzx from Citrobacter, optionally from Citrobacter freundii P079F I, wherein the ⁇ -1,2 mannan polymer comprises at least five consecutive ⁇ -1,2 linked mannose molecules.
  • the ⁇ -1,2 mannan polymer is a fungal ⁇ -1,2 mannan polymer.
  • the fungal ⁇ -1,2 mannan polymer is from Candida.
  • the fungal ⁇ -1,2 mannan polymer is from Candida albicans.
  • the ⁇ -1,2 mannan polymer has the structure: In additional embodiments, the ⁇ - the structure: ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 3)-x-D- GlcpNAc.
  • the prokaryotic host cell is a bacterial cell. In some aspects, 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.
  • 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.
  • the present invention provides a bioconjugate comprising a modified Sap2 protein of the invention linked to an antigen, as described herein.
  • the antigen is linked to an amino acid on the modified Sap2 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 Sap2 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 Sap2 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.
  • 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 present invention provides a method of producing a glycoconjugate comprising a modified carrier protein and a ⁇ -1,2 mannan, wherein said method comprises the steps of i) culturing the host cell of the invention under conditions suitable for the production of proteins, ii) harvesting the culture to produce a harvested culture, and iii) isolating the glycoconjugate from the culture.
  • the conditions suitable for the production of the glycoconjugate includes the addition of GDP-mannose to the culture medium.
  • the GDP-mannose is added to the harvested culture.
  • the modified carrier protein of the invention includes, but is not limited to, Sap2, Als3, 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 Sap2 protein of the invention.
  • the at least one saccharide antigen comprises a ⁇ -1,2 mannan polymer.
  • 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.
  • the present invention provides a method of producing a glycoconjugate comprising a modified carrier protein and a ⁇ -1,2 mannan.
  • the method of producing a glycoconjugate comprising a modified carrier protein and a ⁇ -1,2 mannan 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: a.
  • a host cell of the invention that produces a ⁇ -1,2 mannan polymer; and b. further introducing and expressing in the host cell: i. 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 ii.
  • 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 ⁇ -1,2 mannan 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: a. obtaining a prokaryotic host cell of the invention that produces a ⁇ -1,2 mannan polymer; and b. further introducing and expressing in the host cell: i.
  • the modified carrier protein of the invention includes, but is not limited to, Sap2, Als3, detoxified Exotoxin A of P. aeruginosa (EPA), CRM197, Diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of S.
  • the modified carrier protein is a modified Sap2 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.
  • Analytical Methods Various methods can be used to analyze the structural compositions and sugar chain lengths of the bioconjugates of the invention and to determine glycosylation site usage. Hydrazinolysis can be used to analyze glycans.
  • polysaccharides are released from their protein carriers by incubation with hydrazine according to the manufacturer’s instructions (Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK).
  • the nucleophile hydrazine attacks the glycosidic bond between the polysaccharide and the 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.
  • 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 (n 1 ) 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 Sap2 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 Sap2 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 Sap2 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 Sap2 protein of the invention; (2) at least one Candida albicans saccharide antigen linked to said modified Sap2 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 Sap2 protein conjugate/bioconjugate, but when the compound is administered alone does not generate an immune response to the modified Sap2 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 Sap2 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 saccharide dose, e.g., mannan 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 Sap2 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 Sap2 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 Sap2 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 Sap2 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 Sap2 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 modified Sap2 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 Sap2 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 Sap2 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 modified Sap2 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 Sap2 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 Sap2 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 Sap2 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 Secreted Aspartyl Proteinase 2 (Sap2) protein comprising amino acid residues 19- 398 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 19-398 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. 2.
  • the modified Sap2 protein of paragraph 2 wherein the protein comprises an Aspartic Acid (D) to Asparagine (N) substitution at amino acid residue 274 of amino acid residues 19-398 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 19-398 of SEQ ID NO: 1. 4.
  • the modified Sap2 protein of any of paragraphs 1 to 10 wherein the modified Sap2 protein is from Candida. 12.
  • the modified Sap2 protein of paragraph 11 wherein the Candida is selected from the group consisting of Candida albicans, Candida auris, Candida guilliermondi, Candida lusitaniaea, Candida tropicalis, Candida glabrata, Candida krusei, and Candida parapsilosis. 13.
  • Fba Fructose biphosphate aldolase-1
  • the modified Sap2 protein of paragraph 14 wherein the Fba peptide is covalently linked to the modified Sap2 protein at amino acid residue 398 of amino acid residues 19-398 of SEQ ID NO: 1 or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, modified Sap2 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.
  • the modified Sap2 protein of any of paragraphs 17 to 19, wherein the additional consensus sequence comprises an amino acid sequence of GSGGGDQNATGSGGGHHHHHHHHHH (SEQ ID NO: 8).
  • the modified Sap2 protein of any of paragraphs 1 to 20 comprising an amino acid sequence of SEQ ID NO: 9.
  • a modified Sap2 protein comprising an amino acid sequence of SEQ ID NO: 10. 23.
  • Salmonella sp. O antigens 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.
  • the at least one saccharide antigen is a saccharide antigen of Candida albicans.
  • the at least one saccharide antigen comprises a ⁇ -1,2 mannan polymer.
  • the ⁇ -1,2 mannan polymer comprises at least two ⁇ - 1,2 linked mannose molecules. 34.
  • the conjugate of paragraph 25 having the structure: . 35.
  • the conjugate of any of paragraphs 32 to 34, wherein the at least one saccharide antigen comprises the structure: ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 3)-x-D- 36.
  • the conjugate of any of paragraphs 25 to 31, wherein the at least one saccharide antigen comprises a ⁇ -1,2 mannan polymer.
  • 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
  • ⁇ -1,2 mannan polymer comprises at least 2, at least 3, at least 4, or at least 5 ⁇ -1,3 linked glucose molecules, optionally at least five consecutive ⁇ -1,2 linked mannan molecules, optionally five consecutive ⁇ -1,2 linked mannan molecules. 40.
  • the modified Sap2 protein is linked to the at least one saccharide antigen at one or more amino acid residues on the modified Sap2 protein, wherein the one or more residues are selected from the group consisting of 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. 41.
  • a modified Sap2 protein of protein of Candida albicans comprising (or consisting of): (1) an amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and (2) at least one saccharide antigen of Candida, wherein the at least one saccharide antigen is a ⁇ -1,2 mannan polymer consisting of at least five consecutive ⁇ -1,2 linked mannose molecules, and wherein the at least one saccharide antigen is linked to at least one of four asparagine residues at positions 45, 94, 215, and 415 of SEQ ID NO: 9 or at least one of four asparagine residues at positions 6, 55, 176, and 376 of SEQ ID NO: 10. 44.
  • a vector comprising the polynucleotide sequence of paragraph 44.
  • 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 Sap2 protein according to any of paragraphs 1 to 22; and, optionally, (4) a polynucleotide sequence that encodes a polymerase. 47.
  • the host cell of paragraph 46 wherein the host cell is Escherichia coli.
  • 48. A method of producing a bioconjugate that comprises a modified Sap2 protein linked to at least one saccharide, the method comprising: (1) culturing the host cell of any of paragraphs 46 and 47 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.
  • 49. A bioconjugate produced by the method of paragraph 48, wherein said bioconjugate comprises at least one saccharide linked to a modified Sap2 protein of any of paragraphs 1 to 22. 50.
  • An immunogenic composition comprising the modified Sap2 protein of any of paragraphs 1 to 24, the conjugate of any of paragraphs 25 to 41, or the bioconjugate of any of paragraphs 42 and 49.
  • a method of making the immunogenic composition of paragraph 50 the method comprising the step of mixing the modified Sap2 protein of any of paragraphs 1 to 24, the conjugate of any of paragraphs 25 to 41, or the bioconjugate of any of paragraphs 42 and 49 with a pharmaceutically acceptable excipient or carrier.
  • a vaccine comprising the immunogenic composition of paragraph 50 and, optionally, a pharmaceutically acceptable excipient or carrier.
  • a Candida albicans vaccine comprising: (1) the modified Sap2 protein of any of paragraphs 1 to 22; (2) at least one Candida albicans saccharide linked to said modified Sap2 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 Sap2 protein of any of paragraphs 1 to 24 and 43, the conjugate of any of paragraphs 25 to 41, the bioconjugate of any of paragraphs 42 and 49, the immunogenic composition of paragraph 50, or the vaccine of any of paragraphs 52 and 53. 55.
  • a method for immunizing a subject against Candida albicans infection comprising administering to the subject an immunoprotective dose of the modified Sap2 protein of any of paragraphs 1 to 24 and 43, the conjugate of any of paragraphs 25 to 41, the bioconjugate of any of paragraphs 42 and 49, the immunogenic composition of paragraph 50, or the vaccine of any of paragraphs 52 and 53. 57.
  • 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 modified Sap2 protein of any of paragraphs 1 to 24 and 43, the conjugate of any of paragraphs 25 to 41, the bioconjugate of any of paragraphs 42 and 49, the immunogenic composition of paragraph 50, or the vaccine of any of paragraphs 52 and 53.
  • the modified Sap2 protein of any of paragraphs 1 to 24 and 43, the conjugate of any of paragraphs 25 to 41, the bioconjugate of any of paragraphs 42 and 49, the immunogenic composition of paragraph 50, or the vaccine of any of paragraphs 52 and 53 for use in manufacture of a medicament for treatment or prevention of a disease caused by Candida albicans infection.
  • the saccharide of paragraph 63 comprising the structure: ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 2)- ⁇ -D-Manp-(1 ⁇ 3)-x-D-GlcpNAc.
  • the saccharide of paragraph 64 which is linked to a lipid carrier.
  • a conjugate comprising the saccharide of any one of paragraphs 61-64 linked to an asparagine residue of a modified carrier protein.
  • a host cell comprising: i.
  • nucleotide sequence encoding one or more first heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,2 mannan polymer; ii. a nucleotide sequence encoding a second heterologous glycosyltransferase which is eukaryotic and is capable of covalently bonding a mannose molecule to a ⁇ -1,2 mannan polymer to extend a ⁇ -1,2 mannan polymer chain; iii. a nucleotide sequence encoding a heterologous oligosaccharyl transferase; and iv.
  • the host cell of any one of paragraphs 73 to 77, wherein the eukaryotic glycosyltransferase of capable of covalently bonding a mannose molecule to a ⁇ -1,2 mannan polymer to extend a ⁇ -1,2 mannan polymer chain ii. is Bmt3, optionally of C. albicans. 79.
  • the host cell of any one of paragraphs 73 to 79, wherein the oligosaccharyltransferase of iii. is a PglB 81.
  • the host cell of paragraph 80 wherein the PglB is optionally from Campylobacter, optionally from Campylobacter jejuni or Campylobacter coli , optionally an evolved PglB comprising an amino acid sequence of SEQ ID NO: 16.
  • the modified carrier protein of iv. is selected from the group consisting of Sap2, Als3, 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.
  • the host cell of any one of paragraphs 73 to 83 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.
  • the host cell of paragraph 84 wherein said host cell is an E. coli species.
  • a method of producing a glycoconjugate comprising a modified carrier protein and a ⁇ -1,2 mannan comprising the steps of i) culturing the host cell of any one of paragraphs 73 to 85 under conditions suitable for the production of proteins, ii) harvesting the culture to produce a harvested culture, and iii) isolating the glycoconjugate from the culture.
  • the conditions suitable for the production of the glycoconjugate includes the addition of GDP-mannose to the culture medium.
  • 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 73 to 85 that produces a ⁇ -1,2 mannan 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. 91.
  • the method of paragraph 90, wherein the bacterial signal sequence is Flgl. 92.
  • a method of inhibiting adhesion of Candida albicans hyphae to vaginal epithelial cells in a subject comprising administering to the subject a therapeutically or prophylactically effective amount of the modified Sap2 protein of any of paragraphs 1 to 24 and 43, the conjugate of any of paragraphs 25 to 41, the bioconjugate of any of paragraphs 42 and 49, the immunogenic composition of paragraph 50, or the vaccine of any of paragraphs 52 and 53.
  • 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 the modified Sap2 protein of any of paragraphs 1 to 24 and 43, the conjugate of any of paragraphs 25 to 41, the bioconjugate of any of paragraphs 42 and 49, the immunogenic composition of paragraph 50, or the vaccine of any of paragraphs 52 and 53. 98.
  • 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 Sap2 protein according to any of paragraphs 1 to 22 (optionally a polynucleotide sequence of paragraph 44); and, optionally, (4) a polynucleotide sequence that encodes a polymerase.
  • a host cell comprising: i.
  • nucleotide sequence encoding one or more first heterologous glycosyltransferase(s) capable of synthesizing a ⁇ -1,2 mannan polymer; ii. a nucleotide sequence encoding a second heterologous glycosyltransferase which is eukaryotic and is capable of covalently bonding a mannose molecule to a ⁇ -1,2 mannan polymer to extend a ⁇ -1,2 mannan polymer chain; iii.
  • a method of producing a bioconjugate in a prokaryotic host cell comprising the steps of: a. obtaining a prokaryotic host cell of of any of paragraphs 72 to 84, 98 and 99 that produces a ⁇ -1,2 mannan polymer; and b.
  • All publications mentioned herein are herein incprporated by reference in their entirety. It is to be understood that the term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separatelt as well as their combination.
  • SEQ ID NO: 17 Wild type intermediate Sap2 ( “proSap2”) sequence from Candida albicans: TPTTTKRSAGFVALDFSVVKTPKAFPVTNGQEGKTSKRQAVPVTLHNEQVTYAADITVGSNNQKLNVIVDTGSS DLWVPDVNVDCQVTYSDQTADFCKQKGTYDPSGSSASQDLNTPFKIGYGDGSSSQGTLYKDTVGFGGVSIKNQ VLADVDSTSIDQGILGVGYKTNEAGGSYDNVPVTLKKQGVIAKNAYSLYLNSPDAATGQIIFGGVDNAKYSGSLI ALPVTSDRELRISLGSVEVSGKTINTDNVDVLVDSGTTITYLQQDLADQIIKAFNGKLTQDSNGNSFYEVDCNLS GDVVFNFSKNAKISVPASEFAASLQGDDGQPYDKCQLLFDVNDANILGDNFLRSAYIVYDLDDNEISLAQVKYTS ASSISALT For engineering proS
  • glycosite D/E-X-N-Z-S/T
  • site directed mutagenesis 23 positions were selected for insertion of the consensus sequence for glycosylation i.e. glycosite (D/E-X-N-Z-S/T) by site directed mutagenesis.
  • glycosite variants for some of the positions slight variation of the substituted region or introduced consensus sequence was applied, yielding 37 mutants in total.
  • ProSap2 variants comprising single glycosites were tested for glycosylation with Klebsiella pneumoniae O5 antigen. The best performing glycosites were combined to generate variants comprising between two to six glycosites in total.
  • Modified proSap2 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, an E.
  • coli W3110-derivative strain was used, which 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 proSap2 protein and a plasmid expressing PglB.
  • 5 ml TB medium containing 10 mM MgCl 2 and appropriate antibiotics was inoculated with a streak of colonies from the transformation plate and grown at 37°C overnight.
  • the expression and glycosylation of modified proSap2 proteins was continued at 25°C overnight.
  • the selection criteria for modified proSap2 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:1970 Natur.227..680L. doi:10.1038/227680a0. ISSN 0028-0836. PMID 5432063). Unglycosylated proSap2 proteins and glycoconjugates (i.e. modified proSap2 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.
  • Example 1 SDS PAGE analysis of Modified proSap2 protein-single glycosite variants purified from PPE by IMAC SDS-PAGE analysis (FIG.
  • 3A and 3B correspond to the unglycosylated modified proSap2 protein (“uCarrier”), and to KpO5-modified proSap2 bioconjugates (“Conjugate”) with one occupied glycosite.
  • modified proSap2 proteins with single glycosite showed variability in their expression.
  • the expression was almost completely lost, while for other ones the expression was similar to the ex[ression of the wild type (wt) proSap2 protein (SEQ ID NO: 17).
  • the proSap2 “C” variant SEQ ID NO: 46
  • the proSap2 “C” variant (SEQ ID NO: 46) was also the one with the highest degree of glycosylation (FIG. 3A, lane 3).
  • Several other variants showed a high level of glycosylation, such as N (SEQ ID NO: 44), Mut3a (SEQ ID NO: 52), Mut4d (SEQ ID NO: 56), and Mut8a (SEQ ID NO: 63) (FIG. 3A, lanes 9, 13, FIG.3B lane 20, respectively) and they were selected for glycosite combinations.
  • the modified proSap2 proteins used in this analysis comprised a histine tag, however a skilled person will recognize that modified proSap2 proteins with the histidine tag removed could also be used for the same analysis.
  • the invention provides for modified Sap2 or modified proSap2 proteins with the histidine tag removed.
  • the Fba peptide was introduced to selected variants at the C-terminal of the protein, followed by the “C” glycosite.
  • SDS- PAGE analysis (FIG.4) was carried out on IMAC enriched periplasmic extract of E.coli strains producing KpO5 polysaccharide and expressing PglB and proSap2 variant proteins carrying the following combination of glycosites (Table 3): Table 3: Modified No. of Position of Residues in Includes SEQ Lane Protein engineered engineered proSap2 Fba ID (FIG.
  • modified proSap2 proteins with combined glycosites were all expressed without any significant loss in yield. All the introduced glycosites were at least partially glycosylated, with species with up to four glycosites occupied, resulting in only minimal unglycosylated protein left and maximizing the sugar / protein ratio. The addition of the Fba peptide did not influence the expression level or the glycosylation efficiency.
  • the modified proSap2 variant, MutN3C-3a-8a-fba-C (SEQ ID NO: 87) was selected as the optimal protein carrier.
  • the modified proSap2 proteins used in this analysis comprised a histine tag, however a skilled person will recognize that modified proSap2 proteins with the histidine tag removed could also be used for the same analysis.
  • the invention provides for modified Sap2 or modified proSap2 proteins with the histidine tag removed.
  • Example 3 Production of the proSap2-Fba- ⁇ -1,2-mannan conjugate Construction of the Modified proSap2-Fba- ⁇ -1,2-mannan-producing strain An E. 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).
  • O16wzz2 or cld (GenBank NC_007779 position 2’099’458 to 2’100’438), vi. gtrABS or yfdGHI (GenBank NC_007779 position 2’473’301 to 2’475’908), vii. araBA (GenBank NC_007779 position 66’835 to 70’048).
  • a first expression plasmid comprising the gene for the mannosyltransferase Bmt from Candida albicans (GenBank XP_717972.1), fused with a periplasmic signal peptide at the N-terminal and codon optimized for E. coli, followed by the E. coli genes manC and manB (GenBank: NC_007779 position 2’123’746 to 2’126’657) for the enhanced biosynthesis of GDP-mannose, in a pEC415 backbone (Schulz et al. J Biol Chem. 1998 Aug; 281(5380):1197-200) was constructed.
  • the conjugate-producing strain was obtained by transforming the engineered strain with the three plasmids.
  • the role of each gene in the production of the bioconjugate, as well as the structure of the glycan are schematized in FIG. 2.
  • Production and analysis of the Modified proSap2- ⁇ -1,2-mannan bioconjugate The conjugate-producing strain was grown in a fed-batch 10-L bioreactor in a buffered rich medium at 37°C and pH 7. When the OD 600nm of the culture showed a value of 20-25, the temperature was changed to 25°C, the bioconjugate production was induced with IPTG and arabinose, and a rich medium feed was started and stopped 24 hours after the induction.
  • GDP-Mannose was then addded to the fermenter vessel for a span of 40 minutes to a final concentration of 50 ⁇ M.
  • Cells were harvested by centrifugation 26 hours after induction and washed and resuspended in TBSE buffer.
  • An osmotic shock protocol was applied in which the cells were diluted in a 5-fold volume of H2O for 1 hour, releasing 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.
  • Tangential Flow Filtration (TFF) with a 10 kDa cutoff was applied to exchange to an acidic buffer (10mM Citric Acid 40mM NaCl pH4.0).
  • Example 4 Structural determination of proSap2 produced in E. coli via crystallography Detoxified proSap2 was periplasmically overexpressed in E. coli, purified and concentrated using Amicon concentrator (6 ml, 10 kDa CO) at 3000 rpm to a concentration of 10 mg/mL.
  • Crystallization plates were setup on using a TTP Labtech Mosquito LCP robot; the drop size was 200 nl protein + 200 nl precipitant solutions; employed crystallization screens were SG1, PACT, Morpheus and PGA (Molecular Dimensions), and tested temperatures were 20°C and 4°C.
  • the structure of proSap2 was determined by the molecular replacement method using the structure of mSap2 (mature Sap2, lacking the propeptide) from C. albicans as a search model (PDB ID:1EAG). The structure was refined to 1.7 ⁇ resolution.
  • Example 5 Circular dichroism (CD) spectroscopy characterization of wild-type proSap2 protein and modified proSap2- ⁇ -1,2-mannan bioconjugate
  • the same batch of detoxified wild-type proSap2 analysed by crystallography and a modified Sap2 protein of the invention (modified proSap2-Fba- ⁇ -1,2-mannan bioconjugate, where proSap2 is the MutN3C-8a-fba-C variant) were analysed by near and far UV CD spectroscopy in order to obtain information on the secondary and tertiary structure characteristics of the proteins and to compare them.
  • near-UV CD a Chirascan Q100 CD spectrometer (Applied Photophysics Ltd., UK) was used.
  • the near-UV CD spectra in the wavelength range of 240-350 nm were recorded as co-addition of ten consecutive scans at a scanning speed of 30 nm min-1 by applying the following settings for bandwidth: 1 nm and wavelength step size: 0.5 nm, and by using a 1-cm path length flow-through cuvette (Applied Photophysics Ltd., UK).
  • a Chirascan Q100 CD spectrometer (Applied Photophysics Ltd., UK) was used for far-UV CD spectroscopy measurements.
  • the far-UV CD spectra in the wavelength range of 190-260 nm were recorded as co-addition of ten consecutive scans at a scanning speed of 60 nm min-1 by applying the following settings for bandwidth: 1 nm and wavelength step size: 1 nm, and by using a 0.01-cm path length flow-through cuvette (Applied Photophysics Ltd., UK).
  • the formulation buffer was used as a blank.
  • the mean residue ellipticities were extrapolated mathematically and plotted.
  • the far-UV CD spectra were analyzed by using CDNN software (version 2.1) with 33 untrained neural networks and a database containing secondary structure data for the wavelength range of 178-260 nm from 29 soluble proteins (SP29 database, compiled by Johnson et al., 1981 and 1988) and four additional components, i.e., carbopeptidase A, polyglutamic acid, rubredoxin and trypsin.
  • the retrieved data are reported in FigS. 7A (near-UV CD), 7B (far-UV CD) and 7C (secondary structure element content after analysis with “CDNN” software (Applied Photophysics Ltd) for deconvolution of CD spectroscopy).
  • FIG. 8B shows a 3D representation of the modified proSap2-Fba- ⁇ -1,2-mannan bioconjugate.
  • Structure of a modified proSap2 protein is shown as cartoon.
  • Spheres represent positions of the four introduced glycosites.
  • the conjugated beta-mannan chain is schematically represented, the position and the sequence of the Fba peptide sequence YGKDVKDLFDYAQE (SEQ ID NO: 3) is also shown.
  • FIG. 8C shows the rabbit immunization scheme with the modified proSap2-Fba - ⁇ -1,2-mannan bioconjugate.
  • proSap2-4FM refers to a modified proSap2-Fba- ⁇ -1,2- mannan bioconjugate (modified in that it comprises (i) the inactivating D218N substitution, (ii) the pro-peptide sequence, (iii) 4 glycosites, (iv) the Fba sequence, and (v) a glycan)).
  • FIG. 9 shows immunogenicity of the purified modifed proSap2-Fba- ⁇ -1,2-mannan bioconjugate (“proSap2-4FM”)in rabbits.
  • FIG. 9A, 9B and 9C show immunogenicity against Sap2, Fba and ⁇ -mannan, respectively, of the modified proSap2-Fba- ⁇ -1,2-mannan bioconjugate compared to mSap2 or Sap2-Fba- ⁇ -1,2-mannan bioconjugate, (“Sap2-4FM unmodif “) in rabbit. ; Control indicates buffer immunized animals, all vaccine groups tested with AS03. New Zealand White (NZW) rabbits immunized three times on a two-week interval.
  • Coating ELISA FIG. 9A, modified proSap2 not engineered with His-Tag, FIG. 9B, Fba peptide, FIG. 9C, C.albicans mannan extract.
  • ProSap2, Fba or mannan-specific serum IgG concentration (arbitrary units, AU) in pre-, post-II, post-III (d0, d28, d42) rabbit sera by treatment group.
  • Lines indicate Geometric Mean Concentration (GMC) +/-95% confidence interval. ****: p ⁇ 0.0001, one-way ANOVA. Ref: 36_009, 36_012.
  • proSap2- FM refers to a modified proSap2-Fba- ⁇ -1,2-mannan bioconjugate (modified in that it comprises (i) the inactivating D218N substitution, (ii) the pro-peptide sequence, (iii) 4 glycosites, (iv) the Fba sequence, and (v) a glycan);
  • mSap2 refers to a modified mature Sap2 protein (modified in that it comprises the inactivating D218N substitution, but lacks (i) the pro-peptide sequence, (ii) the fba sequence and (iii) the glycosites;
  • “Sap2-4FM unmodif ” refers to a modified mature Sap2-Fba- ⁇ -1,2- mannan bioconjugate (modified in that it comprises: (i) the inactivating D218N substitution, (ii) 4 glycosites, (iii) the Fba sequence, and (iv) a glycan
  • the raised antibodies present in the sera have the same binding epitope of the neutralizing Fab, they should be able to displace them in a concentration-dependent manner.
  • the Fab inhibits Sap2 activity.
  • 5 ⁇ g/mL anti-Sap2 Fab are complexed with 5 ⁇ g/mL proSap2wt in presence of 10 mg/mL BSA at pH 7.5 for 1h at 30°C, shaking at 450 rpm.
  • Activation of proSap2 to mSap2 was obtained by pH shift to pH 4 by adding citric acid to 0.1 M, incubating at 30°C for 4 hours, shaking at 450 rpm.
  • Trichloroacetic acid was added to 5% (w / w), tubes were incubated for 30 min on ice and centrifuged 10 minutes at 16,000 g to precipitate all the proteins. Peptides resulting from Sap2 digestion of BSA should not precipitate with the rest of the proteins; measurement of the residual absorbance at 280 nm in the supernatant is therefore proportional to the Sap2 protease activity.
  • Figure 10 A the measured activities in absence of Sap2 (negative control), in absence of the Fab fragment (positive control), and in presence of the Fab fragment (Fab anti-Sap2) were expressed as percentage of the positive control activity as determined via absorbance at 280 nm. The employed Fab fragment was able to completely block the protease activity of Sap2.
  • TMB (3,3',5,5' tetramethylbenzidine) was added to the plates to detect the presence of the secondary antibody by reacting with the HRP and emitting at 450 nm and therefore to assess whether the Fab fragment binding to proSap2 was compromised by the presence of the sera.
  • Example 8 Quantitative adhesion assay of C. albicans hyphae
  • Fig. 11 shows the capacity of antibodies against proSap2-4FM bioconjugate to inhibit adhesion of C. albicans to vaginal epithelial cells. Sera was mixed with C. albicans (SC5314), added to epithelial cells (A431) and incubated for 1.5hs.
  • proSap2-FM refers to a modified proSap2-Fba- ⁇ -1,2-mannan bioconjugate (modified in that it comprises (i) the inactivating D218N substitution, (ii) the pro-peptide sequence, (iii) 4 glycosites, (iv) the Fba sequence, and (v) a glycan);
  • mSap2 refers to a modified mature Sap2 protein (modified in that it comprises the inactivating D218N substitution, but lacks (i) the pro-peptide sequence, (ii) the fba sequence and (iii) the glycosites; and
  • Sap2-4F refers to a modified proSap2- Fba protein (modified in that it comprises: (i) the inactivating D218N substitution, (ii) 4 glycosites, and (iii) the Fba sequence,
  • C. albicans hyphae (SC5314) were mixed with sera, added to neutrophiles and incubated. Then, neutrophiles were lysed and Candida CFU were counted after incubation in YPD agar.
  • proSap2-FM refers to a modified proSap2-Fba- ⁇ -1,2-mannan bioconjugate (modified in that it comprises (i) the inactivating D218N substitution, (ii) the pro-peptide sequence, (iii) 4 glycosites, (iv) the Fba sequence, and (v) a glycan);
  • mSap2 refers to a modified mature Sap2 protein (modified in that it comprises the inactivating D218N substitution, but lacks (i) the pro-peptide sequence, (ii) the fba sequence and (iii) the glycosites; and
  • Sap2-4F refers to a modified proSap2- Fba protein (modified in that it comprises: (i) the inactivating D218N substitution, (ii) 4 glycosites, and (iii) the Fba sequence, but lacks the pro-peptide sequence and is not glycosylated); “control” indicates buffer
  • Fig. 13 shows the capacity of antibodies against proSap2-4FM bioconjugate to bind to C. albicans using fluorescent microscopy.
  • 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.
  • proSap2-FM refers to a modified proSap2-Fba- ⁇ -1,2-mannan bioconjugate (modified in that it comprises (i) the inactivating D218N substitution, (ii) the pro-peptide sequence, (iii) 4 glycosites, (iv) the Fba sequence, and (v) a glycan);
  • Candi5V refers to a multicomponent vaccine comprising Sap2-4FM and Als3-Fba- ⁇ -1,3- glucan; “control” indicates buffer immunized animals, all vaccine groups tested with AS03. NZW rabbits immunized three times on a two-week interval.
  • 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

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Abstract

La présente invention concerne le 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, l'invention concerne une protéine Sap2 (aspartyl protéinase sécrétée 2 de Candida albicans ') modifiée. La protéine Sap2 modifiée peut être utilisée en tant que protéine porteuse pour d'autres antigènes, en particulier des antigènes saccharidiques ou d'autres antigènes dépourvus d'épitopes de lymphocytes T.
PCT/IB2024/057694 2023-08-09 2024-08-08 Protéines modifiées Pending WO2025032535A2 (fr)

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