Modified protein

Abstract

This invention relates to modified proteins, immunogenic compositions and vaccines comprising modified proteins, the manufacture of the same, and the use of such compositions in medicine. More specifically, it relates to modified Sap2 (secreted aspartyl proteinase 2 of Candida albicans) protein. Modified Sap2 protein can be used as a carrier protein for other antigens, in particular sugar antigens or other antigens lacking T cell epitopes.

Description

[Technical Field]

[0001] Sequence List This application includes a sequence listing submitted electronically in XML format, the full text of which is incorporated herein by reference. The XML copy was created on 7 August 2023, named 70385US01P_SL.xml, and has a size of 124,012 bytes.

[0002] Field of Invention This invention relates to modified proteins, immunogenic compositions and vaccines containing the modified proteins, their manufacture, and the medical use of the compositions. More specifically, this invention relates to a modified Sap2 (Candida albicans-derived secretory aspartate proteinase 2) protein. The modified Sap2 protein can be used as a carrier protein for other antigens, particularly glycosylated antigens or other antigens lacking T cell epitopes.

[0003] Background of the Invention The impact of fungal infections on humans is a significant public health issue, and despite its ever-increasing incidence exacerbated by the rising prevalence of immunocompromised individuals in populations, it has received far less attention than bacterial infections and treatments. Fungal infections are also detrimental to the health of pasture livestock, negatively affecting milk production in dairy cows, as well as the physical and skin condition of these animals. The most common fungal genera that cause invasive infections in humans are Candida, Cryptococcus, Aspergillus, and Pneumocystis. Candida, in particular, is the cause of some of the most common fungal infections and is a major fungal pathogen globally. Candida is an early colonizer acquired in humans primarily through physical contact at or around birth. This fungus has the ability to colonize human skin, as well as the digestive tract and reproductive organs.[9,10] However, under certain circumstances (e.g., immunosuppressed states), this fungus can become pathogenic and cause widespread human infections. Over the past few decades, the incidence of human infections caused by Candida has increased significantly (Sobel, 2007; Pfuller, 2011). Candidiasis can be superficial or invasive. Invasive candidiasis is difficult to treat. Globally, an estimated 700,000 people suffer from invasive candidiasis annually, with an associated mortality rate exceeding 50%. Furthermore, Candida can also cause mucocutaneous infections, such as vulvovaginal candidiasis, which are rarely fatal but carry a significant morbidity. The estimated economic burden of healthcare costs associated with candidiasis is $3 billion.

[0004] Candida albicans is the most studied species in its genus, the most common opportunistic pathogen in hospitalized humans, and a cause of invasive fungal infections (Sobel, 2007; Pfuller, 2011). C. albicans is a highly adaptable fungal species commonly found in hospital-acquired infections, and immunocompromised patients are at particularly high risk. C. albicans possesses a variety of pathogenic factors that enable its transition from symbiotic (yeast) form to pathogenic (hyphal) form. For example, C. albicans is a commensal fungus that lives symbiotically in the vaginal epithelium. Certain environmental conditions trigger the morphogenesis of C. albicans from the symbiotic yeast form to the pathogenic hyphal form. The hyphal form then initiates invasion into the lumen and breaks down the mucosal barrier, causing symptomatic infections, such as vulvovaginal candidiasis (VVC) in women. Globally, approximately 70-80% of all women will experience VVC at least once in their lifetime, with over 85% of these infections caused by C. albicans. Up to 10% of women who contract VVC will suffer from relapses. Recurrent VVC, or RVVC (defined as four or more outbreaks per year), is more severe and resistant to treatment. RVVC leads to a reduced quality of life, severe negative impacts on work and social life, and increased associated healthcare costs. It is estimated that the number of RVVC patients will increase to 158 million by 2030.

[0005] Despite the high mortality and morbidity rates of invasive fungal infections (IFIs), treatment options are limited, and the drug pipeline for antifungal agents remains underdeveloped (Perfect, 2017). Furthermore, antifungal therapies are often ineffective. In addition, the widespread use of antifungal agents in agriculture and clinical settings has led to the emergence of multidrug-resistant fungal infections (e.g., multidrug-resistant Candida auris and other non-albicans species), which pose a significant threat to food security and human health (Nguyen et al., 2021). Moreover, despite the significant global burden of human fungal infections, there are currently no approved vaccines available for the prevention and / or control of human fungal infections (Oliviera et al., 2021). The lack of clinically available fungal vaccines is attributed to the extremely high costs associated with antigen production, as well as toxicological concerns. Furthermore, fungal vaccines composed of whole fungal cells (attenuated live or dead fungi) exhibit high immunogenicity and are therefore most likely to elicit a protective response, but they carry a potential risk of infection. Therefore, there is a clear and unfulfilled need to identify novel targets and develop new types of antifungal agents, including vaccines against pathogenic fungi (e.g., C. albicans) that can be safely mass-produced.

[0006] Based on theoretical safety benefits, identifying protective antigens for use in subunit vaccines has been the focus of much research. A major difference between the fungal and animal kingdoms is the presence of a cell wall in almost all fungal cells. The complex fungal cell wall is essential for maintaining cell shape and integrity (Garcia-Rubio et al., 2020). Analysis of cell wall composition across various Candida species, including C. albicans, has shown that the inner cell wall is mainly composed of β-glucans (β-1,3 and β-1,6 linked glucose polymers), while the outer cell wall is composed of highly glycosylated cell wall proteins modified with N-linked and O-linked terminal mannans (branched polymers of mannose linked via α-1,2, α-1,3, α-1,4, α-1,6, and β-1,2 glycosidic bonds) (Ahmadipour et al., 2021, The Cell Surface, 7:100063). Therefore, β-glucan and mannan, polysaccharide components of the fungal cell wall, could be interesting candidates in the development of safe and effective subunit fungal vaccines. However, polysaccharides are T cell-independent antigens, inducing antibody production via B lymphocytes without the involvement of T cells. Polysaccharides, when conjugated to protein carriers containing T cell epitopes, can induce long-lasting T cell-dependent immune responses in humans. In fact, conjugating T cell-independent antigens to carrier proteins is an established method that allows T cell help to be added to immune responses to antigens that are normally T cell-independent. This method can enhance the immune response and improve its stability by enabling the formation of immunological memory. Good conjugate vaccines against pathogenic prokaryotes have been developed by conjugating bacterial capsular polysaccharides to carrier proteins, which have a known effect of converting T cell-independent glycosylated antigens into T cell-dependent antigens that can induce immunological memory responses.However, until now, it has not been possible to successfully develop conjugate vaccines against pathogenic eukaryotic fungi such as Candida by conjugating fungal cell wall polysaccharides to fungal carrier proteins. This invention provides, for the first time, a novel type of eukaryotic Candida glycoconjugate vaccine produced using a bioconjugation process in prokaryotic bacteria (specifically Escherichia coli). [Overview of the project]

[0007] Glycoconjugates are hybrid molecules consisting of a carrier protein and multiple polysaccharide chains, with the polysaccharides covalently linked to the carrier protein. This binding of antigenic polysaccharides to the carrier protein represents a breakthrough in the field of vaccinology, triggering a T-cell-dependent response characterized by the induction of immunological memory and enhanced immunogenicity. Standard manufacturing methods for glycoconjugate vaccines involve a chemical binding process requiring extensive optimization for each individual target antigen, thus necessitating long development cycles. Furthermore, the complexity of the manufacturing process makes these products high-cost. In contrast, bioconjugation is an innovative technique that allows for the preservation of the natural immunogenic structure by producing glycoconjugate vaccines within a biological environment (e.g., E. coli). Using recombinant DNA technology, the glycosylation mechanism of E. coli is genetically engineered to produce target polysaccharide antigens, which are then covalently linked to asparagine residues on the consensus sequence of the carrier protein. This allows glycoconjugate vaccines to be produced entirely within E. coli in a single-step process, resulting in advantages in process reproducibility and robustness while reducing manufacturing costs. Using this bioconjugation technology, several glycoconjugate vaccines against prokaryotes such as Gram-negative bacteria (e.g., Shigella) and Gram-positive bacteria (e.g., Staphylococcus aureus or Streptococcus pneumoniae) have been developed and validated in clinical trials. However, because Candida is a eukaryote, transferring the biochemical pathways necessary for the bioconjugation and production of Candida glycoproteins to E. coli has not been as easy (as in the case of previous bacterial lipopolysaccharides or capsular polysaccharides). Consequently, due to the high complexity of the Candida bioconjugation process, the production of Candida glycoconjugate vaccines in E. coli has not been successful to date.

[0008] The present invention provides a modified secretory aspartyl proteinase 2 (Sap2) protein derived from C. albicans, comprising at least one consensus sequence (e.g., D / EXNZS / T) for glycosylation for use in binding to an antigen (e.g., a Candida polysaccharide). Furthermore, the present invention provides a glycoconjugate comprising a modified Sap2 carrier protein, which is linked to a Candida polysaccharide antigen at one or more asparagine residues on the modified Sap2 protein, and a method for producing a Candida glycoconjugate vaccine in host cells (e.g., Escherichia coli). The present invention further provides a method for the prevention and / or treatment of RVVC using a Candida glycoconjugate vaccine.

[0009] Accordingly, in a particular embodiment of the present invention, a modified secretory aspartyl proteinase 2 (Sap2) protein is provided, comprising an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residues 19-398 of SEQ ID NO: 1, wherein the amino acid sequence is modified in that it comprises one or more consensus sequences comprising the amino acid sequence D / EXNZS / T, where X and Z are each independently any amino acid other than proline.

[0010] According to another aspect of the present invention, a modified Sap2 protein of the present invention is provided, 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 a corresponding position in an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residues 19-398 of SEQ ID NO: 1, wherein the protein may optionally comprise a substitution from aspartic acid (D) to asparagine (N) at amino acid residue 274 of amino acid residues 19-398 of SEQ ID NO: 1, or at a corresponding position in an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residue 19-398 of SEQ ID NO: 1.

[0011] According to another aspect of the present invention, a modified Sap2 protein of the present invention is provided, wherein the modified Sap2 protein comprises at least one fructose diphosphate aldolase-1 (Fba) peptide, wherein the Fba peptide comprises the amino acid sequence YGKDVKDLFDYAQE (SEQ ID NO: 3), or an amino acid sequence having at least 70%, 80%, 85%, 90%, or 92% identity with SEQ ID NO: 3.

[0012] According to another aspect of the present invention, a modified Sap2 protein of the present invention is provided, comprising the amino acid sequence of SEQ ID NO: 9.

[0013] According to another aspect of the present invention, a modified Sap2 protein of the present invention is provided, comprising the amino acid sequence of SEQ ID NO: 10.

[0014] According to another aspect of the present invention, a conjugate (e.g., a bioconjugate) is provided comprising the modified Sap2 protein of the present invention and at least one glycosylation antigen.

[0015] In another aspect of the present invention, a modified Sap2 protein derived from Candida albicans is provided, which comprises (or consists of) (1) the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and (2) at least one Candida glycan antigen, wherein the at least one glycan antigen is a β-1,2 mannan polymer consisting of at least five consecutive β-1,2 linked mannose molecules, and the at least one glycan antigen is bound to at least one of the four asparagine residues at positions 45, 94, 215, and 415 of SEQ ID NO: 9, or to at least one of the four asparagine residues at positions 6, 55, 176, and 376 of SEQ ID NO: 10.

[0016] According to another aspect of the present invention, polynucleotides encoding the modified Sap2 protein of the present invention are provided.

[0017] According to another aspect of the present invention, a vector comprising a polynucleotide encoding the modified Sap2 protein of the present invention is provided.

[0018] According to another aspect of the present invention, a host cell is provided comprising: (1) one or more polynucleotide sequences encoding one or more heterologous glycosyltransferases; (2) a polynucleotide sequence encoding a heterologous oligosaccharide transferase; (3) a polynucleotide sequence encoding the modified Sap2 protein of the present invention; and optionally (4) a polynucleotide sequence encoding a polymerase.

[0019] Another aspect of the present invention provides a method for producing a bioconjugate comprising (or consisting of) a modified Sap2 protein bound to at least one glycosylation antigen, the method comprising: (1) culturing host cells of the present invention under conditions suitable for protein production; and (2) isolating the bioconjugate produced by the host cells, although the bioconjugate may be isolated from a periplasmic extract obtained from the host cells.

[0020] According to another aspect of the present invention, there is provided an immunogenic composition comprising a modified Sap2 protein of the present invention, a conjugate of the present invention, or a bioconjugate of the present invention, optionally comprising pharmaceutically acceptable additives and / or excipients.

[0021] According to another aspect of the present invention, there is provided a method for preparing an immunogenic composition of the present invention, the method comprising mixing a modified Sap2 protein of the present invention, a conjugate of the present invention, or a bioconjugate of the present invention with a pharmaceutically acceptable additive or excipient.

[0022] According to another aspect of the present invention, there is provided a vaccine comprising an immunogenic composition of the present invention, optionally comprising pharmaceutically acceptable additives or excipients, and optionally an adjuvant.

[0023] According to another aspect of the present invention, there is provided a Candida albicans vaccine comprising (1) a modified Sap2 protein of the present invention; (2) at least one Candida albicans sugar chain antigen bound to the modified Sap2 protein; and optionally (3) a pharmaceutically acceptable excipient or adjuvant.

[0024] According to another aspect of the present invention, there is provided a method for treating or preventing Candida albicans infection in a subject needing treatment or prevention of Candida albicans infection, the method comprising administering to the subject a therapeutically effective amount of a modified Sap2 protein of the present invention, a conjugate of the present invention, a bioconjugate of the present invention, an immunogenic composition of the present invention, or a vaccine of the present invention.

[0025] According to another aspect of the present invention, there is provided a method of conferring immunity to Candida albicans infection in a subject (e.g., a human), the method comprising administering to the subject an immunoprotective dose of a modified Sap2 protein of the present invention, a conjugate of the present invention, a bioconjugate of the present invention, an immunogenic composition of the present invention, or a vaccine of the present invention.

[0026] According to another aspect of the present invention, there is provided a method of inducing an immune response to Candida albicans infection in a subject (e.g., a human), the method comprising administering to the subject a therapeutically or prophylactically effective amount of a modified Sap2 protein of the present invention, a conjugate of the present invention, a bioconjugate of the present invention, an immunogenic composition of the present invention, or a vaccine of the present invention.

[0027] According to another aspect of the present invention, there is provided a modified Sap2 protein of the present invention, a conjugate of the present invention, a bioconjugate of the present invention, an immunogenic composition of the present invention, or a vaccine of the present invention for use in the treatment or prevention of a disease caused by Candida albicans infection.

[0028] According to another aspect of the present invention, there is provided a modified Sap2 protein of the present invention, a conjugate of the present invention, a bioconjugate of the present invention, an immunogenic composition of the present invention, or a vaccine of the present invention for use in the manufacture of a medicament for the treatment or prevention of a disease caused by Candida albicans infection.

[0029] According to another aspect of the present invention, there is provided a host cell comprising: i. a nucleotide sequence encoding one or more first heterologous glycosyltransferases capable of synthesizing a β-1,2 mannan polymer; [[ID=二十]]ii. a nucleotide sequence encoding a second heterologous glycosyltransferase that is eukaryote-derived and has the ability to covalently attach a mannose molecule to a β-1,2 mannan polymer to extend the β-1,2 mannan polymer chain; [[ID=二十一]] iii. Nucleotide sequences encoding heterologous oligosaccharide transferases; and iv. A nucleotide sequence encoding a modified carrier protein having a glycosylation site, which may include the consensus sequence D / EXNZS / T, wherein X and Z are each independently any amino acid other than proline.

[0030] Another aspect of the present invention provides a method for producing a glycoconjugate containing a modified carrier protein and β-1,2-mannan, the method comprising culturing the host cells of the present invention under conditions suitable for protein production.

[0031] Another aspect of the present invention provides a method for producing a glycoconjugate containing a modified carrier protein and β-1,2-mannan, the method comprising: i) culturing host cells of the present invention under conditions suitable for protein production; ii) recovering the culture to produce a recovered culture; and iii) separating the glycoconjugate from the culture.

[0032] According to another aspect of the present invention, a sugar that is a β-1,2-mannan polymer having the following structure is provided: [ka] According to another aspect of the present invention, a sugar having the following structure is provided:

[0033] [ka]

[0034] According to another aspect of the present invention, a conjugate (e.g., a bioconjugate) is provided which contains the sugar of the present invention linked to an asparagine residue of a modified carrier protein. [Brief explanation of the drawing]

[0035] [Figure 1A] Figures 1A and 1B show the structure of the mature Sap2 protein from C. albicans of sequence number 88 (including residues 57-398 of sequence number 1). Figure 1A: Spherical marks indicate the insertion sites of glycosites. Figure 1B: Spherical marks indicate the optimal insertion sites of glycosites. N: Insert a glycosite next to position 19; C: Insert a glycosite next to position 398; Mut 3a: Replace positions 98-102 with a glycosite; Mut 4d: Replace positions 109-113 with a glycosite; and Mut 8a: Insert a glycosite next to position 220 (all positions are relative to the wild-type full-length Sap2 sequence of C. albicans with sequence number 1). [Figure 1B] Figures 1A and 1B show the structure of the mature Sap2 protein from C. albicans of sequence number 88 (including residues 57-398 of sequence number 1). Figure 1A: Spherical marks indicate the insertion sites of glycosites. Figure 1B: Spherical marks indicate the optimal insertion sites of glycosites. N: Insert a glycosite next to position 19; C: Insert a glycosite next to position 398; Mut 3a: Replace positions 98-102 with a glycosite; Mut 4d: Replace positions 109-113 with a glycosite; and Mut 8a: Insert a glycosite next to position 220 (all positions are relative to the wild-type full-length Sap2 sequence of C. albicans with sequence number 1). [Figure 2A] Figure 2 shows the production of proSap2-Fba-β-1,2-mannan bioconjugate in Escherichia coli (E. coli). Figure 2A shows a schematic diagram of the mannan structure in C. albicans. Figure 2B shows the biosynthesis scheme of the modified Sap2-mannan bioconjugate in E. coli. The Enterobacteria O antigen cluster refers to Citrobacter fruedii P079F I. [Figure 2B]Figure 2 shows the production of proSap2-Fba-β-1,2-mannan bioconjugate in Escherichia coli (E. coli). Figure 2A shows a schematic diagram of the mannan structure in C. albicans. Figure 2B shows the biosynthesis scheme of the modified Sap2-mannan bioconjugate in E. coli. The Enterobacteria O antigen cluster refers to Citrobacter fruedii P079F I. [Figure 3] Figure 3 shows glycosylation tests using a series of modified proSap2 proteins, each with a single glycosite. Figures 3A and 3B show SDS-PAGE analysis of modified proSap2 proteins purified from PPE by IMAC. Most of the modified proSap2 proteins showed good glycosylation by Kp05. The modified proSap2 "C" mutant (Figure 3A lane 3) showed the highest expression level and glycosylation. Some sites (e.g., Mut14a-14e (Figure 3B lanes 27-31)) appear to destabilize the modified proSap2 protein, resulting in decreased protein expression. [Figure 4] Figure 4 shows glycosylation studies using a series of modified proSap2 proteins containing multiple combined glycosites. Lanes 1-4 show SDS-PAGE analysis of modified proSap2 proteins (without the Fba peptide sequence) purified from PPE by IMAC. Lanes 5-8 show SDS-PAGE analysis of modified proSap2 proteins (with the Fba peptide sequence) purified from PPE by IMAC. As shown in the figure, modified proSap2 proteins with up to four glycosites were expressed and purified. [Figure 5-1]Figure 5 shows the SDS-PAGE gel (stained with Coomassie blue after electrophoresis) (Figure 5A) and Western blot analysis (Figures 5B-5E) of the purified modified Sap2 protein. Lane M: Protein standard; Lane 1: Purified non-glycosylated modified proSap2 (MutN3C-8a-Fba-C). Lane 2: Purified modified proSap2-Fba-β-1,2-mannan bioconjugate (glycosylated MutN3C-8a-Fba-C). Figure 5A: SDS-PAGE analysis (stained with Coomassie blue). Figure 5B: Anti-proSap2 immunoblot (antibody against the N-terminal "propeptide" region). Figure 5C: Anti-Sap2 immunoblot (antibody against the peptide in the middle of the protein sequence). Figure 5D: Anti-Fba immunoblot; Figure 5E: Anti-mannan immunoblot. [Figure 5-2] Figure 5 shows the SDS-PAGE gel (stained with Coomassie blue after electrophoresis) (Figure 5A) and Western blot analysis (Figures 5B-5E) of the purified modified Sap2 protein. Lane M: Protein standard; Lane 1: Purified non-glycosylated modified proSap2 (MutN3C-8a-Fba-C). Lane 2: Purified modified proSap2-Fba-β-1,2-mannan bioconjugate (glycosylated MutN3C-8a-Fba-C). Figure 5A: SDS-PAGE analysis (stained with Coomassie blue). Figure 5B: Anti-proSap2 immunoblot (antibody against the N-terminal "propeptide" region). Figure 5C: Anti-Sap2 immunoblot (antibody against the peptide in the middle of the protein sequence). Figure 5D: Anti-Fba immunoblot; Figure 5E: Anti-mannan immunoblot. [Figure 6] Figure 6 shows the structure determined by crystal structure analysis, overlaying the modified proSap2 protein produced in E. coli onto the published wild-type mature Sap2 (mSap2) protein from C. albicans. The spherical marks represent the residue range boundaries obtained from the AlphaFold model. [Figure 7-1]Figure 7 shows the circular dichroism (CD) spectroscopy characterization of wild-type proSap2 protein and modified proSap2-β-1,2-mannan bioconjugate. Figure 7A shows the near-ultraviolet CD spectroscopy analysis. Figure 7B shows the far-ultraviolet CD spectroscopy analysis. Figure 7C shows the secondary structure element content after deconvolution analysis using CD spectroscopy with "CDNN" software (Applied Photophysics Ltd). [Figure 7-2] Figure 7 shows the circular dichroism (CD) spectroscopy characterization of wild-type proSap2 protein and modified proSap2-β-1,2-mannan bioconjugate. Figure 7A shows the near-ultraviolet CD spectroscopy analysis. Figure 7B shows the far-ultraviolet CD spectroscopy analysis. Figure 7C shows the secondary structure element content after deconvolution analysis using CD spectroscopy with "CDNN" software (Applied Photophysics Ltd). [Figure 8A] Figure 8 shows the preclinical trials of the purified modified proSap2-Fba-β-1,2-mannan bioconjugate ("proSap2-4FM") in rabbits. Figure 8A shows the properties of the modified proSap2-Fba-β-1,2-mannan bioconjugate. Figure 8B shows a 3D representation of the modified proSap2 protein-mannan bioconjugate. Figure 8C shows the rabbit immunization scheme using the modified proSap2-Fba-β-1,2-mannan bioconjugate. [Figure 8B] Figure 8 shows the preclinical trials of the purified modified proSap2-Fba-β-1,2-mannan bioconjugate ("proSap2-4FM") in rabbits. Figure 8A shows the properties of the modified proSap2-Fba-β-1,2-mannan bioconjugate. Figure 8B shows a 3D representation of the modified proSap2 protein-mannan bioconjugate. Figure 8C shows the rabbit immunization scheme using the modified proSap2-Fba-β-1,2-mannan bioconjugate. [Figure 8C]Figure 8 shows the preclinical trials of the purified modified proSap2-Fba-β-1,2-mannan bioconjugate ("proSap2-4FM") in rabbits. Figure 8A shows the properties of the modified proSap2-Fba-β-1,2-mannan bioconjugate. Figure 8B shows a 3D representation of the modified proSap2 protein-mannan bioconjugate. Figure 8C shows the rabbit immunization scheme using the modified proSap2-Fba-β-1,2-mannan bioconjugate. [Figure 9-1] Figure 9 shows the immunogenicity of the purified modified proSap2-Fba-β-1,2-mannan bioconjugate ("proSap2-4FM") in rabbits. Figures 9A, 9B, and 9C show the immunogenicity of the modified proSap2-Fba-β-1,2-mannan bioconjugate ("proSap2-4FM") to Sap2, Fba, and β-mannan, respectively, compared to the modified mature Sap2 protein ("mSap2") or the modified Sap2-Fba-β-1,2-mannan bioconjugate ("Sap2-4FMunmodif"). [Figure 9-2] Figure 9 shows the immunogenicity of the purified modified proSap2-Fba-β-1,2-mannan bioconjugate ("proSap2-4FM") in rabbits. Figures 9A, 9B, and 9C show the immunogenicity of the modified proSap2-Fba-β-1,2-mannan bioconjugate ("proSap2-4FM") to Sap2, Fba, and β-mannan, respectively, compared to the modified mature Sap2 protein ("mSap2") or the modified Sap2-Fba-β-1,2-mannan bioconjugate ("Sap2-4FMunmodif"). [Figure 10]Figure 10 shows the inhibition test of the protease activity of full-length wild-type Sap2 protein derived from C. albicans. This test was performed using a substance (single-specific Fab fragment) that strongly inhibits its protease activity. Figure 10A: The measured activity in the absence of Sap2 (negative control), the absence of Fab fragment (positive control), and the presence of Fab fragment (Fab anti-Sap2) is shown as a percentage of the positive control activity by absorbance measurement at 280 nm. The Fab fragment used was able to completely inhibit the protease activity of Sap2. Figure 10B: Titration curves are shown using serum obtained from bicomponent immunization ("Candi5V": a combination of proSap2-Fba-β-1,2-mannan bioconjugate and Als3-Fba-β-1,3-glucan bioconjugate) or rabbit immunization with proSap2-Fba-β-1,2-mannan ("proSap2-4FM"). As a control, serum from mock-immunized rabbits was used. [Figure 11] Figure 11 shows that antibodies against the proSap2-4FM bioconjugate have the ability to inhibit the adhesion of C. albicans to vaginal epithelial cells. [Figure 12] Figure 12 shows the ability of antibodies against the proSap2-4FM bioconjugate to participate in the killing of C. albicans mycelial forms by neutrophils. [Figure 13] Figure 13 shows the ability of antibodies against the proSap2-4FM bioconjugate to bind to C. albicans, as observed by fluorescence microscopy.

[0036] Detailed description of the invention definition As used herein, the terms “Sap2 protein” or “Sap2” refer to the wild-type secreted aspartyl proteinase 2 protein having a wild-type leader sequence (amino acid residues 1-18) and a propeptide sequence (amino acid residues 19-56) at its N-terminus. In certain embodiments, the Sap2 protein is derived from the genus Candida, and possibly from Candida albicans. In certain embodiments, the Sap2 protein contains the amino acid sequence of Sequence ID No. 1.

[0037] As used herein, the terms “proSap2 protein” or “proSap2” refer to the C-terminal fragment of the wild-type Sap2 protein. In certain embodiments, the proSap2 protein is derived from the genus Candida, and possibly from Candida albicans. In certain embodiments, the proSap2 protein contains amino acid residues 19–398 of SEQ ID NO: 1. In some embodiments, the proSap2 protein contains the amino acid sequence of SEQ ID NO: 17.

[0038] As used herein, the term “modified” protein means a protein that has been altered (in one or more respects) compared to the wild-type protein (for example, “modified Sap2 protein” or “modified Sap2” excludes the wild-type Sap2 protein, and “modified proSap2 protein” or “modified proSap2” excludes the wild-type proSap2 protein). The term “modified Sap2 protein” means a Sap2 protein comprising one or more consensus glycosite sequences of the present invention (for example, a consensus sequence comprising the amino acid sequence D / EXNZS / T, where X and Z are each independently any amino acid other than proline). In certain embodiments, a modified Sap2 protein is a Sap2 protein comprising one or more consensus glycosite sequences (for example, comprising the amino acid sequence D / EXNZS / T, where X and Z are each independently any amino acid other than proline), wherein the one or more consensus sequences are added adjacent to or substituted for one or more amino acid residues of SEQ ID NO: 1, or equivalent positions within an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1. In other embodiments, a modified Sap2 protein is a Sap2 protein comprising one or more consensus glycosite sequences (for example, comprising the amino acid sequence D / EXNZS / T, where X and Z are each independently any amino acid other than proline), wherein one or more consensus sequences are added adjacent to or substituted for one or more amino acid residues in amino acid residues 19-398 of SEQ ID NO: 1, or at equivalent positions in an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residues 19-398 of SEQ ID NO: 1.In yet another embodiment, the modified Sap2 protein is a Sap2 protein comprising one or more consensus glycosite sequences (for example, comprising the amino acid sequence D / EXNZS / T, where X and Z are each independently any amino acid other than proline), wherein one or more consensus sequences are added adjacent 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 having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residues 57-398 of SEQ ID NO: 1. In certain embodiments, the modified Sap2 protein is a Sap2 protein comprising an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residues 19-398 of SEQ ID NO: 1, wherein the amino acid sequence comprises one or more consensus sequences (for example, comprising the amino acid sequence D / EXNZS / T, where X and Z are each independently any amino acid other than proline), and the one or more consensus sequences are added adjacent to or substituted for one or more amino acid residues within amino acid residues 19-398 of SEQ ID NO: 1, or equivalent positions within the amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residues 19-398 of SEQ ID NO: 1. In yet another embodiment, the modified Sap2 protein further includes substitutions at amino acid residues 88 and / or 274 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1.In another embodiment, the modified Sap2 protein further includes substitutions at amino acid residues 88 and / or 274 among amino acid residues 19-398 of SEQ ID NO: 1, or at equivalent positions in an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residues 19-398 of SEQ ID NO: 1. In yet another embodiment, the modified Sap2 protein further includes substitutions at amino acid residues 88 and / or 274 among amino acid residues 57-398 of SEQ ID NO: 1, or at equivalent positions in an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residue 57-398 of SEQ ID NO: 1. Those skilled in the art will know that these residues correspond to amino acid residues 32 and 218 of SEQ ID NO: 88 (i.e., Candida albicans Sap2 protein lacking the wild-type leader sequence and wild-type propeptide sequence), respectively. In certain embodiments, the substitution is a D274N substitution. In certain embodiments, the substitution causes the modified Sap2 protein to lose its biological activity. In certain embodiments, the modified Sap2 protein of the present invention is a naturally occurring modified Sap2 protein. In other embodiments, the modified Sap2 protein of the present invention is a recombinant modified Sap2 protein. In yet another embodiment, the modified Sap2 protein of the present invention is an isolated recombinant modified Sap2 protein. In certain embodiments, the modified Sap2 protein is derived from the Candida genus, and may also be derived from Candida albicans. In some embodiments, the modified Sap2 protein contains the amino acid sequence of SEQ ID NO: 9. In other embodiments, the modified Sap2 protein contains the amino acid sequence of SEQ ID NO: 10.In yet another embodiment, the modified Sap2 protein includes, but is not limited to, an amino acid sequence selected from the amino acid sequences of N, N3C, C, Mut1a, Mut1b, Mut2a, Mut2b, Mut3a, Mut3b, 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, Mut18, Mut19, and Mut20.

[0039] As used herein, the term “control Sap2 protein” means, but is not limited to, (1) a Sap2 protein that does not contain one or more consensus sequences inserted adjacent to one or more amino acids of SEQ ID NO: 1 (or an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1) or added to the N-terminus and / or C-terminus of SEQ ID NO: 1 (or an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1); (2) Sap2 proteins that do not contain one or more consensus sequences inserted adjacent to or substituted for one or more amino acids in the Sap2 protein containing amino acid residues 19-398 of SEQ ID NO: 1 (or amino acid sequences having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residues 19-398 of SEQ ID NO: 1), or that are attached to the N-terminus and / or C-terminus of the Sap2 protein containing amino acid residues 19-398 of SEQ ID NO: 1 (or amino acid sequences having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residues 19-398 of SEQ ID NO: 1); or (3) A Sap2 protein that does not contain one or more consensus sequences that are inserted adjacent to or substituted for one or more amino acids of the Sap2 protein containing amino acid residues 57-398 of SEQ ID NO: 1 (or an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residues 57-398 of SEQ ID NO: 1), or that are attached to the N-terminus and / or C-terminus of the Sap2 protein containing amino acid residues 57-398 of SEQ ID NO: 1 (or an amino acid sequence having at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity with amino acid residues 57-398 of SEQ ID NO: 1).Therefore, the control Sap2 protein includes wild-type Sap2 protein, wild-type Sap2 protein of SEQ ID NO: 1, wild-type Sap2 protein containing amino acid residues 19-398 of SEQ ID NO: 1, or wild-type Sap2 protein containing amino acid residues 57-398 of SEQ ID NO: 1.

[0040] As used herein, the term “carrier protein” refers to a protein that can bind to an antigen (for example, a glycan antigen such as a fungal polysaccharide antigen) to form a conjugate (for example, a bioconjugate). The carrier protein activates T cell-mediated immunity against the antigen to which it binds. As used herein, the term “carrier protein” refers to a protein containing one or more consensus sequences that link the glycan antigens of the present invention. In certain embodiments, the carrier protein is the modified Sap2 protein of the present invention.

[0041] As used herein, the term "any amino acid other than 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), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0042] As used herein, the term “naturally occurring amino acid residue” refers to amino acids that are naturally incorporated into polypeptides. Specifically, these are 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).

[0043] As used herein, the term “glycosyltransferase (GTF, Gtf)” refers to an enzyme that forms glycosidic bonds. Glycosyltransferases are enzymes that catalyze the formation of glycosidic bonds, thereby generating glycosides. For example, this enzyme catalyzes the transfer of a sugar chain portion from an activated sugar nucleotide (also known as a “glycosyl donor”) to a nucleophilic glycosyl acceptor molecule (whose nucleophilic group may be based on oxygen, carbon, nitrogen, or sulfur).

[0044] As used herein, the term “oligosaccharide transferase (OTase or OST)” refers to an enzyme that catalyzes the mechanistically specific and selective transfer of oligosaccharides or polysaccharides to asparagine (N) residues on the consensus sequence of a nascent or folded protein. OST transfers 14-saccharide oligosaccharides from dolichol to nascent proteins. OST is a type of glycosyltransferase. The reaction catalyzed by OST is a central step in the N-linked glycosylation pathway. OST is a component of the translocon located in the endoplasmic reticulum (ER) membrane. Lipid-bound core oligosaccharides are constructed in the ER membrane and transferred to selected asparagine residues of the nascent polypeptide chain by the oligosaccharide transferase complex.

[0045] As used herein, the term “O antigen (also known as O-specific polysaccharide or O side chain)” refers to a component of surface lipopolysaccharide (LPS) of Gram-negative bacteria. Examples include O antigens derived from Pseudomonas aeruginosa and Klebsiella pneumoniae.

[0046] As used herein, the term "lipopolysaccharide (LPS)" refers to a polymer containing covalently linked lipids and polysaccharides.

[0047] As used herein, the term “capsular polysaccharide (CP)” refers to polysaccharides present on the surface of bacterial cell walls. Examples include capsular polysaccharides derived from Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Staphylococcus aureus.

[0048] As used herein, the term “reducing end” refers to the reducing end of an oligosaccharide or polysaccharide, which is a monosaccharide having a free anomeric carbon that is not involved in glycosidic bonding and is therefore ring-opened and convertible into a chain.

[0049] As used herein, the term “conjugate” refers to a protein (e.g., a carrier protein) that is covalently bound to an antigen.

[0050] As used herein, the term "bioconjugate" refers to a conjugate between a protein (e.g., a carrier protein) and an antigen (e.g., a glycan antigen such as a bacterial polysaccharide antigen) prepared in the host cell environment, in which the host cell mechanism binds the antigen to the protein (e.g., N-linked glycosylation). Typically, in bioconjugates, the polysaccharide is bound to asparagine via N-acetylglucosamine.

[0051] As used herein, the term “immunogenic fragment” refers to a portion of an antigen smaller than the whole antigen that has the ability to evoke a humoral and / or cellular immune response specific to that fragment in a host animal, such as a human. Protein fragments can be prepared using methods known in the art, for example, by recombination, protease digestion, or chemosynthesis. Internal or terminal fragments of a polypeptide can be prepared by removing one or more nucleotides from one end (in the case of a terminal fragment) or both ends (in the case of an internal fragment) of the nucleic acid encoding the polypeptide. Typically, fragments contain at least 10, 20, 30, 40, or 50 consecutive amino acids from the full-length sequence. Fragments can be readily modified by removing or adding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 amino acids from either the N-terminus or the C-terminus, or both. In certain embodiments, the modified Sap2 protein fragments of the present invention still contain the described modifications made to the Sap2 protein.

[0052] As used herein, the term “conservative amino acid substitution” includes substitutions of native amino acid residues with non-native residues that have little to no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at the position in question, and do not result in a decrease in immunogenicity. For example, the above can be substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Conservative amino acid modifications (and corresponding coding base modifications) of polypeptide sequences can produce polypeptides having similar functional and chemical properties to the parent polypeptide.

[0053] As used herein, the term “deletion” refers to the removal of one or more amino acid residues from a protein sequence. Typically, the number of residues removed from any single site within a protein molecule does not exceed approximately 1 to 6 residues (for example, 1 to 4 residues).

[0054] As used herein, the terms “insertion” or “addition” (including other variations such as “inserted”) refer to the addition of one or more non-natural amino acid residues in a protein sequence, or, depending on the context, the addition of one or more non-natural nucleotides in a polynucleotide sequence. Typically, the number of residues inserted into any single site within a protein molecule does not exceed approximately 1 to 10 residues (e.g., 1 to 7 residues, 1 to 6 residues, or 1 to 4 residues).

[0055] As used herein, the expression "adjacently added" means the addition of one or more non-natural amino acid residues to a protein sequence at a position adjacent to a reference amino acid or amino acid region. For example, "adjacent to one or more amino acids between amino acid residues 19-24" means an addition at a position adjacent to any one of amino acid residues 19-24 (including positions adjacent to amino acid residues 19 or 24).

[0056] As used herein, the term “glycosyte” refers to an amino acid sequence recognized by a bacterial oligosaccharide transferase, for example, PglB of Campylobacter jejuni. Thus, a glycosite refers to an amino acid sequence within a carrier protein (for example, the modified Sap2 protein or the modified proSap2 protein of the present invention) to which an antigenic glycan (for example, a β-1,2-mannan polymer) is covalently or noncovalently bound.

[0057] A "consensus sequence" refers to a sequence having a specific structure and / or function. In this specification, the term "consensus sequence" means a sequence containing a glycosite. The consensus sequences of the present invention include, but are not limited to, the 5-amino acid consensus sequence D / EXNZS / T, the 7-amino acid consensus sequence KD / EXNZS / TK, and extended consensus sequences (e.g., JUBD / EXNZS / TJUB).

[0058] As used herein, the term “introduced at the position of ~” is used to refer to the location and method of inserting a consensus sequence into an amino acid sequence. A consensus sequence (or glycosite) introduced at the N-terminal or C-terminal position of a protein may be added adjacent to the N-terminal or C-terminal amino acid sequence, while a consensus sequence (or glycosite) introduced at a specific amino acid residue within a protein (e.g., amino acid residue 19 in SEQ ID NO: 1) may substitute for that amino acid residue.

[0059] Unless otherwise specified, a numerical range (e.g., "19-24") includes the endpoints (i.e., the values ​​of 19 and 24). For example, "between amino acids 19 to 398 of SEQ ID NO: 1" refers to the position in the amino acid sequence between amino acids 19 and 398 of SEQ ID NO: 1, which includes both amino acids 19 and 398.

[0060] The terms “identical” or “percent “identical” refer to the identicalness of nucleotide or amino acid sequences, or having a specific percentage of identical nucleotide or amino acid residues (e.g., 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity across a specific region), when the sequences are aligned and compared to maximize the match, for example, using a sequence comparison algorithm or by manually lining them up and visually examining them. Polypeptide identity can be calculated using various algorithms. Generally, when calculating percentage identity, the two sequences to be compared are aligned to maximize the correlation between them. This may involve inserting “gaps” into one or both sequences to increase the degree of alignment. For example, the Needleman-Wunsch algorithm (Needleman and Wunsch 1970, J. Mol. Biol. 48: 443-453) can be used for global alignment, or the Smith-Waterman algorithm (Smith and Waterman 1981, J. Mol. Biol. 147: 195-197) can be used for local alignment, for example, using default parameters (the Smith-Waterman method uses a BLOSUM 62 score matrix, with a gap start penalty of 10 and a gap extension penalty of 1). The preferred algorithm was described by Dufresne et al. in Nature Biotechnology 2002 (vol. 20, pp. 1269-71) and is used in the GenePAST software (Genome Quest Life Sciences, Inc. Boston, MA). GenePAST's "percent identity" algorithm detects the best match between the query sequence and the target sequence and expresses the alignment as an accurate percentage. GenePAST does not adjust the alignment score to account for the biological relevance between the query sequence and the target sequence.The identity between the two sequences is calculated over the entire length of both sequences and expressed as a percentage relative to the reference sequence (for example, sequence number 1 of the present invention).

[0061] In this specification, “recombinant” means artificial or synthetic. In certain embodiments, “recombinant protein” means a protein made using a recombinant nucleotide sequence (a nucleotide sequence introduced into a host cell). In certain embodiments, the nucleotide sequence encoding the “recombinant protein” is heterologous to the host cell.

[0062] In this specification, the terms “isolated” or “purified” refer to proteins, conjugates (e.g., bioconjugates), polynucleotides, or vectors in a form not found in nature. This includes, for example, proteins, conjugates (e.g., bioconjugates), polynucleotides, or vectors isolated (including crude extracts) from host cells or host organisms, or otherwise extracted from the natural environment. In certain embodiments, an isolated or purified protein refers to a protein that is essentially free from all other polypeptides that it would normally be associated with (or in contact with).

[0063] As used herein, the term “subject” refers to an animal, in particular a mammal such as a primate (e.g., human).

[0064] As used herein, the term “therapeutically or prophylactically effective amount” in the context of administering treatment to a subject (for example, administering the immunogenic composition or vaccine of the present invention) means an amount of therapeutic agent having a prophylactic and / or therapeutic effect. In a particular embodiment, “a therapeutically or prophylactically effective amount” means an amount of therapeutic agent sufficient to achieve one, two, three, four, or five or more of the following effects: (i) reducing or improving the severity of a fungal infection or related symptoms; (ii) shortening the duration of a fungal infection or related symptoms; (iii) preventing the progression of a fungal infection or related symptoms; (iv) causing regression of a fungal infection or related symptoms; (v) preventing the onset or development of a fungal infection or related symptoms; (vi) preventing the recurrence of a fungal infection or related symptoms; (vii) reducing organ failure associated with a fungal infection; (viii) reducing hospitalization of subjects with a fungal infection; (ix) shortening the length of hospitalization of subjects with a fungal infection; (x) extending the survival time of subjects with a fungal infection; (xi) eliminating a fungal infection in a subject; (xii) inhibiting or reducing fungal replication in a subject; and / or (xiii) To enhance or improve the preventive or therapeutic effects of other treatments.

[0065] As used herein, the term “immunoprotective dose” in the context of administering treatment to a subject (for example, administering the immunogenic composition or vaccine of the present invention) means the amount of therapeutic agent having a preventive and / or therapeutic effect. In a particular embodiment, “immunoprotective dose” means an amount of therapeutic agent sufficient to achieve one, two, three, four, or five or more of the following effects: (i) reducing or improving the severity of a fungal infection or related symptoms; (ii) shortening the duration of a fungal infection or related symptoms; (iii) preventing the progression of a fungal infection or related symptoms; (iv) causing regression of a fungal infection or related symptoms; (v) preventing the onset or development of a fungal infection or related symptoms; (vi) preventing the recurrence of a fungal infection or related symptoms; (vii) reducing organ failure associated with a fungal infection; (viii) reducing hospitalization of subjects with a fungal infection; (ix) shortening the length of hospitalization of subjects with a fungal infection; (x) extending the survival time of subjects with a fungal infection; (xi) eliminating a fungal infection in a subject; and / or (xii) inhibiting or reducing fungal replication in a subject.

[0066] The term “comprises” is not limiting in scope and simply means “includes.” Therefore, unless the context requires a different interpretation, the words “comprises” or “has,” and their inflections (such as “comprise” and “comprising,” or “have” and “having,” respectively), are understood to mean including the described compound, molecule, composition, or step, but not to exclude any other compound, molecule, composition, or step. In this specification, “comprising” and “having,” used as connecting phrases, are not limiting in scope, while “consisting of” is limiting (i.e., limited to what is described and not included beyond that). In certain embodiments, for readability, the word “is” may be used instead of “consists of” or “consisting of.” The abbreviation “eg” derives from the Latin “exempli gratia” and is used herein to indicate a non-limiting example. Therefore, the abbreviation "eg" is synonymous with the term "for example."

[0067] A "conjugate vaccine" is a vaccine created by covalently linking a polysaccharide antigen to a carrier protein. Conjugate vaccines induce an immune response and immunological memory against pathogens (for example, fungi). In infants and the elderly, binding polysaccharide antigens to proteins that induce T cell-dependent responses can induce a protective immune response against these antigens.

[0068] The term "glycoconjugate vaccine" refers to a vaccine that contains a protein carrier conjugated to an antigenic or immunogenic oligosaccharide.

[0069] As used herein, “undecaprenyl” or “und” refers to undecaprenol lipid, which consists of 11 prenol units. “Und-P” refers to undecaprenyl phosphate, which is a universal (Und-derived) lipid carrier for glycan biosynthesis intermediates of carbohydrate polymers. “Und-PP” refers to undecaprenyl pyrophosphate, which is a phosphorylated form of Und-P.

[0070] The term "periplasmic space" or "periplasm" refers to the space between the inner cell membrane and the outer membrane of a host cell (for example, Gram-negative bacteria such as E. coli).

[0071] In this specification, the terms “of” and “from” are used interchangeably. Therefore, Candida “derived” sugar antigens mean, but are not limited to, (1) sugar antigens obtained from Candida, or (2) sugar antigens of Candida, i.e., sugar antigens that contain a structure similar to Candida-derived sugar antigens, but are recombinantly produced in a host cell (such as a bacterial cell).

[0072] wbaB refers to a glycosyltransferase. In certain embodiments, wbaB is a glycosyltransferase obtained from organisms including, but not limited to, Citrobacter freundii P079F I, Salmonella O6,7 (C1) Thompson, or Escherichia coli O17. In certain embodiments, wbaB is the glycosyltransferase of Citrobacter freundii P079F I. In some embodiments, wbaB is a wild-type glycosyltransferase. In other embodiments, wbaB is a non-natural (e.g., mutant and / or recombinant) glycosyltransferase.

[0073] wbaC refers to a glycosyltransferase. In certain embodiments, wbaC is a glycosyltransferase obtained from organisms including, but not limited to, Citrobacter freundii P079F I, Salmonella O6,7 (C1) Thompson, or Escherichia coli O17. In certain embodiments, wbaC is the glycosyltransferase of Citrobacter freundii P079F I. In some embodiments, wbaC is a wild-type glycosyltransferase. In other embodiments, wbaC is a non-natural (e.g., mutant and / or recombinant) glycosyltransferase.

[0074] wbaD refers to a glycosyltransferase. In certain embodiments, wbaD is a glycosyltransferase obtained from organisms including, but not limited to, Citrobacter freundii P079F I, Salmonella O6,7 (C1) Thompson, or Escherichia coli O17. In certain embodiments, wbaD is the glycosyltransferase of Citrobacter freundii P079F I. In some embodiments, wbaD is a wild-type glycosyltransferase. In other embodiments, wbaD is a non-natural (e.g., mutant and / or recombinant) glycosyltransferase.

[0075] Bmt3 refers to a glycosyltransferase. In certain embodiments, Bmt3 is a glycosyltransferase obtained from an organism including, but not limited to, Candida albicans. In certain embodiments, Bmt3 is the glycosyltransferase of C. albicans. In one embodiment, Bmt3 is a wild-type glycosyltransferase gene. In other embodiments, Bmt3 is a non-natural (e.g., mutant and / or recombinant) glycosyltransferase gene. In certain embodiments, the Bmt3 gene is a codon-optimized version of the gene derived from C. albicans. In yet another embodiment, the Bmt3 protein is N-terminated with a leader peptide sequence derived from Escherichia coli OmpC for periplasmic transport.

[0076] manB refers to guanylyltransferase. In certain embodiments, manB is a guanylyltransferase obtained from organisms including, but not limited to, Escherichia coli. In certain embodiments, manB is the guanylyltransferase of Escherichia coli K12 W3110. In one embodiment, manB is wild-type guanylyltransferase. In other embodiments, manB is a non-natural (e.g., mutant and / or recombinant) guanylyltransferase.

[0077] manC refers to guanylyltransferase. In certain embodiments, manC is a guanylyltransferase obtained from an organism including, but not limited to, Escherichia coli. In certain embodiments, manC is the guanylyltransferase of Escherichia coli K12 W3110. In one embodiment, manC is wild-type guanylyltransferase. In other embodiments, manC is a non-natural (e.g., mutant and / or recombinant) guanylyltransferase.

[0078] PglB refers to an oligosaccharide transferase. In certain embodiments, pglB is an oligosaccharide transferase obtained from organisms including, but not limited to, Campylobacter jejuni, Campylobacter coli, or Sinorhizobium meliloti 1021. In certain embodiments, pglB is an oligosaccharide transferase derived from Campylobacter coli. In some embodiments, pglB is a wild-type oligosaccharide transferase. In other embodiments, PglB is a non-natural oligosaccharide transferase. In some embodiments, the pglB gene is an evolved oligosaccharide transferase gene. In yet another embodiment, the pglB protein is evolved pglB, i.e., an evolved oligosaccharide transferase. "Evolved" means a protein or nucleic acid that has undergone directed evolution. Directed evolution is a technique used in protein engineering that mimics the process of natural selection to guide a protein or nucleic acid toward a user-defined goal. The process of directed evolution consists of repeatedly subjecting a gene to a cycle of mutagenesis (creating a library of mutants), selection (expressing those mutants and isolating members with the desired function), and amplification (creating a template for the next round). This can be carried out in vivo (in a living organism) or in vitro (in cells or in solution). In addition to being used in protein engineering as an option for rationally designing modified proteins, directed evolution is also used for experimental evolutionary studies concerning the fundamental principles of evolution under controlled laboratory conditions. In certain embodiments, the evolved pglB of the present invention contains the amino acid sequence of SEQ ID NO: 16. Therefore, in certain embodiments, the pglB amino acid sequence of SEQ ID NO: 16 contains one or more mutations that enhance the activity of pglB toward the sugar antigen of the present invention.Therefore, in certain embodiments, the evolved pglB of the present invention transfers the sugar antigen of the present invention to the modified Sap2 protein of the present invention more efficiently than wild-type pglB (for example, wild-type pglB obtained from Campylobacter jejuni).

[0079] Wzx refers to a translocase. In certain embodiments, wzx is a translocase obtained from organisms including, but not limited to, Citrobacter freundii P079F I, Salmonella O6,7 (C1) Thompson, or Escherichia coli O17. In certain embodiments, wzx is the translocase of Citrobacter freundii P079F I. In some embodiments, wzx is a wild-type translocase. In other embodiments, wzx is a non-natural (e.g., mutant and / or recombinant) translocase.

[0080] Sap2 protein Candida albicans secretory aspartyl protein 2 (also known as "Sap2") is an extracellular pathogenic factor and the main enzyme in vaginal secretions of women exhibiting symptoms of Candida infection. Sap2 nourishes Candida cells, promotes adhesion to host tissues, facilitates fungal invasion of epithelium and endothelium, and contributes to Candida's ability to evade the immune response, thus exhibiting immunogenicity during infection (Kumar R. et al., 2015, Infect Immun, 83(7):2614-2626). Sap2 functions as a hydrolase, exhibits broad substrate specificity, and is expressed in large quantities during C. albicans culture (Naglik JR, et al, 2003, Microbiol. Mol. Biol. Rev., 67:400-428). The secreted aspartyl proteinase (Sap) protein is encoded by the SAP gene family, consisting of 10 genes, and has been the most extensively studied as a major pathogenicity determinant of C. albicans (ibid.). Sap2 is a member of the secreted aspartyl Sap protein family and is encoded by the SAP2 gene. All 10 SAP genes in C. albicans encode preproenzymes that are about 60 amino acids longer than the mature enzymes and are processed when transported via the secretory pathway (ibid.). The mature enzymes contain the typical sequence motif of all aspartyl proteinases, including two conserved aspartate residues in the active site and a conserved cysteine ​​residue involved in maintaining the three-dimensional structure (ibid.).

[0081] The SAP2 gene encodes a 398-amino acid preproprotein, which is processed into a 342-residue mature enzyme. This enzyme is a typical aspartate proteinase with an optimal pH of 3-4 and is sensitive to the peptide inhibitor pepstatin A (Cutfield S., et al., 1995, Structure, 3:1261-1271). The N-terminus of the Sap2 preproprotein contains an 18-amino acid signal peptide ("SP" or "leader sequence") followed by a 38-amino acid propeptide.

[0082] During maturation, the C. albicans Sap2 preproprotein is processed into an "intermediate" form of 380 residues lacking a leader sequence ("proSap2"; SEQ ID NO: 17), and then further processed into a "mature" form of 342 residues lacking both a leader sequence and a propeptide sequence ("mSap2"; SEQ ID NO: 88) (Naglik et al. 2003). Within the C-terminal domain, there is a structurally distinct subdomain of approximately 100 residues (Figures 1A and 1B), which has been shown to behave as a separate, robust structure in some other aspartate proteinases. This domain of SAP2 contains residues 197-215 and 223-305 and contains a single disulfide bond (between positions 256 and 294) that connects a randomly structured double loop (243-255, 282-293). These C-terminal loops are characteristic of the peripheral structure of aspartate proteinase, forming part of the large, open entrance to the binding site. Another loop, formed by disulfide bonds at 47–59, is more clearly defined. This is also adjacent to the binding site, but considerably closer. The pattern of secondary structural elements in SAP2, particularly the composition of numerous β-strands and two major α-helix moieties (140–146, 228–237), is similar to other known aspartate proteinases. However, some of the turns connecting these elements have different stereochemistry. A highly conserved feature of aspartate proteinase is the "flap" region (β-hairpin loop), which is central to the interaction with the bound inhibitor / substrate, shielding the active site from the bulk solvent. In SAP2, the flap consists of Lys81-Gln91 residues, and the tyrosine at position 84 is highly conserved, similar to other aspartate proteinases (corresponding to Tyr75 in pepsin) (Cutfield S. et al., 1995, Structure, 3:1261-1271). The overall structure of Sap2 is similar to the folding structure of classical aspartate proteinases, such as pepsin. One of the most notable characteristics of Sap2 is the diversity of proteins it can cleave.Sap2 is known to degrade many human proteins, including mucins and secretory immunoglobulin A (IgA), which are molecules that protect the mucosal surface. Sap2 can also degrade extracellular matrix molecules such as keratin, collagen, and vimentin (Naglik JR, et al, 2003, Microbiol. Mol. Biol. Rev., 67:400-428). Sap2 proteins useful for the present invention can be produced by methods known to those skilled in the art, taking into consideration this specification; see, for example, Smolenski G, Sullivan PA, Cutfield SM, Cutfield JF., 1997, Microbiology, 143 (Pt 2):349-356.

[0083] As shown below, the full-length wild-type Sap2 protein of C. albicans contains the amino acid sequence of SEQ ID NO: 1 (the wild-type leader sequence is underlined, and the propeptide is italicized; the amino acid residue at position 274 (D) is double-underlined; in certain embodiments, substitution of this residue (e.g., substitution with N) causes loss of biological activity of Sap2):

[0084] [ka] (Sequence ID 1).

[0085] In certain embodiments, the present invention provides a modified Sap2 protein. The term "modified Sap2 protein" includes an amino acid sequence in which the Sap2 amino acid sequence has been modified by the addition, substitution, or deletion of one or more amino acids (for example, by the addition of a consensus sequence selected from D / EXNZS / T, KD / EXNZS / T and / or an extended consensus sequence (e.g., JUBD / EXNZS / TJUB), and / or by the substitution of one or more amino acids by a consensus sequence selected from D / EXNZS / T, KD / EXNZS / T). For example, a Sap2 protein is defined as one that contains the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, or the amino acid sequence of amino acid residues 19-398 of SEQ ID NO: 1 or an amino acid sequence that is 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. For example, a modified Sap2 protein may be a Sap2 protein having the amino acid sequence of SEQ ID NO: 1, but modified in that the amino acid sequence contains one or more consensus sequences selected from D / EXNZS / T, KD / EXNZS / T and / or extended consensus sequences (e.g., JUBD / EXNZS / TJUB). As used herein, in the consensus sequence of the present invention, X and Z are independently any amino acid other than proline, preferably X is Q (glutamine) and Z is A (alanine). In certain embodiments, the modified Sap2 protein of the present invention may further include modifications (e.g., addition, substitution, and / or deletion of one or more amino acid residues).In certain embodiments, the modified Sap2 protein of the present invention has an amino acid sequence that is modified in that it includes one or more consensus sequences containing the amino acid sequence D / EXNZS / T (wherein X and Z are independently any amino acid other than proline), and contains an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1. In other embodiments, the modified Sap2 protein of the present invention comprises an amino acid sequence that is 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 it comprises one or more consensus sequences comprising the amino acid sequence D / EXNZS / T (wherein X and Z are independently any amino acid other than proline).

[0086] In some embodiments, the modified Sap2 protein of the present invention is a non-natural Sap2 protein (i.e., not natural). In certain embodiments, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least 80% identical to SEQ ID NO: 1. In other embodiments, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least 85% identical to SEQ ID NO: 1. In yet another embodiment, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least or 90% identical to SEQ ID NO: 1. In yet another embodiment, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least 91% identical to SEQ ID NO: 1. In yet another embodiment, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least 92% identical to SEQ ID NO: 1. In other embodiments, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least 93% identical to SEQ ID NO: 1. In yet another embodiment, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least 94% identical to SEQ ID NO: 1. In yet another embodiment, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. In a particular embodiment, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least 96% identical to SEQ ID NO: 1. In another embodiment, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least 97% identical to SEQ ID NO: 1. In yet another embodiment, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least 98% identical to SEQ ID NO: 1. In yet another embodiment, the modified Sap2 protein of the present invention may have an amino acid sequence that is at least 99% identical to SEQ ID NO: 1.

[0087] In certain embodiments, the modified Sap2 protein of the present invention contains one or more consensus glycosite sequences. The terms “glycosite sequence,” “consensus glycosite sequence,” and “consensus sequence” are used interchangeably herein. In certain embodiments, the modified Sap2 protein of the present invention contains 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. In other embodiments, the modified Sap2 protein of the present invention contains one, two, three, four, five, six, seven, eight, nine, or ten consensus sequences. In certain embodiments, the modified Sap2 protein of the present invention contains at least three consensus sequences. In preferred embodiments, the modified Sap2 protein of the present invention contains three consensus sequences. In certain embodiments, the modified Sap2 protein of the present invention includes one or more consensus sequences in which all consensus sequences have the same amino acid sequence. In other embodiments, the modified Sap2 protein of the present invention includes one or more consensus sequences in which all consensus sequences have different amino acid sequences. In yet another embodiment, the modified Sap2 protein of the present invention includes one or more consensus sequences in which at least two consensus sequences have the same amino acid sequence.

[0088] Thus, in certain embodiments, the present invention provides a modified Sap2 protein having an amino acid sequence that is modified in that its amino acid sequence includes one or more consensus sequences including the amino acid sequence D / EXNZS / T (wherein X and Z are independently any amino acid other than proline), and contains an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0089] In certain embodiments, the modified Sap2 protein of the present invention is an inactive protein. An “inactive” protein means that the protein lacks any biological activity. In certain embodiments, the modified Sap2 protein of the present invention includes substitutions at one or more positions selected from the group consisting of amino acid residue 88 and amino acid residue 274 of SEQ ID NO: 1. Those skilled in the art will know that these residues correspond to amino acid residues 32 and 218, respectively, of SEQ ID NO: 88 (i.e., the Candida albicans Sap2 protein lacking the wild-type leader sequence and the wild-type propeptide sequence). In certain embodiments, substitutions at one or both of the amino acid residues at positions 88 and 274 of SEQ ID NO: 1 inactivate the Sap2 protein. In certain embodiments, substitution at residue 274 of SEQ ID NO: 1 inactivates the Sap2 protein (e.g., eliminates the hydrolytic activity of Sap2). In certain embodiments, the removal of the hydrolytic activity of Sap2 eliminates the virulence of the protein.

[0090] Thus, in certain embodiments, the modified Sap2 protein of the present invention further includes a substitution at amino acid residue 274 of SEQ ID NO: 1, or at an equivalent position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1. In certain embodiments, the modified Sap2 protein of the present invention includes a substitution from aspartic acid (D) to asparagine (N) at amino acid residue 274 of SEQ ID NO: 1, or at an equivalent position within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1. In certain embodiments, the substitution inactivates the modified Sap2 protein.

[0091] In certain embodiments, the modified Sap2 protein of the present invention comprises one or more consensus sequences, each selected from specific amino acid residues (consensus sequence sites) within the modified Sap2 protein of the present invention, which are either adjacent to or substituted for one or more amino acids. These one or more consensus sequence sites are at equivalent positions within the amino acid sequence of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, specifically: (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), and (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., 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) are independently selected.

[0092] In other embodiments, the modified Sap2 protein of the present invention has one or more consensus sequences that are 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 equivalent positions within the same amino acid sequence, such as 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), and one or more amino acids between amino acid residues 187-197 (e.g., amino acid residue 192). (14) The sequence includes one or more consensus sequences that are adjacent to or substituted for one or more amino acids selected from the group consisting of (14) 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), one or more amino acids between 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).

[0093] In a further embodiment, the modified Sap2 protein of the present invention has one or more consensus sequences, one or more amino acids between amino acid residues 52-62 (e.g., amino acid residue 57) and one or more amino acids between amino acid residues 93-107, at equivalent positions within the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 57-398 of SEQ ID NO: 1. amino acids (e.g., 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), amino acids (14) It includes one or more consensus sequences that are adjacent to or substituted for one or more amino acids selected from the group consisting of 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), one or more amino acids between 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 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).

[0094] In certain embodiments, the modified Sap2 protein of the present invention comprises one or more consensus sequences which are added next to or replace amino acid residue 19 of SEQ ID NO: 1, or amino acid residues 19-398 of SEQ ID NO: 1 at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical.

[0095] In other embodiments, the modified Sap2 protein of the present invention comprises one or more consensus sequences which are added next to or replace amino acid residue 57 of SEQ ID NO: 1, or amino acid residues 19-398 of SEQ ID NO: 1 at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical.

[0096] In some embodiments, the modified Sap2 protein of the present invention comprises one or more consensus sequences which are added next to or replace one or more amino acids between amino acid residues 109-113 of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical amino acid residues 19-398 of SEQ ID NO: 1 at equivalent positions within an amino acid sequence.

[0097] In yet another embodiment, the modified Sap2 protein of the present invention comprises one or more consensus sequences which are added next to or replace amino acid residue 220 of SEQ ID NO: 1, or amino acid residues 19-398 of SEQ ID NO: 1 at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical within an equivalent position in an amino acid sequence.

[0098] In certain embodiments, the modified Sap2 protein of the present invention comprises one or more consensus sequences which are added next to or replace amino acid residue 19 of amino acid residues 19-398 of SEQ ID NO: 1, or equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0099] In other embodiments, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace amino acid residue 57 between amino acid residues 19-398 of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1 at an equivalent position within the amino acid sequence.

[0100] In some embodiments, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace one or more amino acids between amino acid residues 109-113 of amino acid residues 19-398 of SEQ ID NO: 1, or amino acid residues 19-398 of SEQ ID NO: 1 at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0101] In yet another embodiment, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace amino acid residue 220 between amino acid residues 19-398 of SEQ ID NO: 1, or at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0102] In yet another embodiment, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace amino acid residue 398 of amino acid residues 19-398 of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical in an equivalent position within an amino acid sequence to amino acid residues 19-398 of SEQ ID NO: 1.

[0103] In further embodiments, the modified Sap2 protein of the present invention comprises one or more consensus sequences which are added next to or replace amino acid residue 57 of amino acid residues 57-398 of SEQ ID NO: 1, or equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 57-398 of SEQ ID NO: 1.

[0104] In some embodiments, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace one or more amino acids between amino acid residues 109-113 of amino acid residues 57-398 of SEQ ID NO: 1, or amino acid residues 57-398 of SEQ ID NO: 1 at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 57-398 of SEQ ID NO: 1.

[0105] In yet another embodiment, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace amino acid residue 220 between amino acid residues 57-398 of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 57-398 of SEQ ID NO: 1 at an equivalent position within the amino acid sequence.

[0106] In yet another embodiment, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace amino acid residue 398 between amino acid residues 57-398 of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical in an amino acid sequence to amino acid residues 57-398 of SEQ ID NO: 1.

[0107] In certain embodiments, the modified Sap2 protein of the present invention includes one or more consensus sequences that are added next to or replace equivalent positions in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to (i) amino acid residue 57 and (ii) amino acid residue 398 of SEQ ID NO: 1 or amino acid residues 57 to 398 of SEQ ID NO: 1. In certain embodiments, the modified Sap2 protein of the present invention includes the amino acid sequence of SEQ ID NO: 81. In other embodiments, the modified Sap2 protein of the present invention includes the amino acid sequence of SEQ ID NO: 85 (fba sequence further included).

[0108] In certain embodiments, the modified Sap2 protein of the present invention includes one or more consensus sequences that are added next to or replace (i) amino acid residue 57, (ii) amino acid residue 220, and (iii) amino acid residue 398 of amino acid residues 57-398 of SEQ ID NO: 1, or amino acid residues 57-398 of SEQ ID NO: 1 at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical in position within the amino acid sequence. In certain embodiments, the modified Sap2 protein of the present invention includes the amino acid sequence of SEQ ID NO: 82. In other embodiments, the modified Sap2 protein of the present invention includes the amino acid sequence of SEQ ID NO: 86 (fba sequence further included).

[0109] In certain embodiments, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace amino acid residues 57-398 of SEQ ID NO: 1, (i) amino acid residue 57, (ii) amino acids between amino acid residues 98-102, (iii) amino acid residue 220, and (iv) amino acid residue 398, or amino acid residues 57-398 of SEQ ID NO: 1, at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical within an equivalent amino acid sequence. In certain embodiments, the modified Sap2 protein of the present invention includes the amino acid sequence of SEQ ID NO: 83. In other embodiments, the modified Sap2 protein of the present invention includes the amino acid sequence of SEQ ID NO: 87 (fba sequence further included). In certain embodiments, the modified Sap2 protein of the present invention, comprising the amino acid sequence of SEQ ID NO: 87, was selected as the optimal protein carrier because it results in at least partial glycosylation of all introduced glycosites, leaving only minimal unglycosylated proteins and maximizing the sugar / protein ratio (see Figure 4; MutN3C-3a-8a-fba-C).

[0110] Thus, in certain embodiments, the present invention provides a method for increasing the glycosylation efficiency of a modified Sap2 protein of the present invention, comprising the step of expressing a modified Sap2 protein containing at least four consensus sequences in a host cell of the present invention, wherein the consensus sequences are added to or substituted next to or at equivalent positions in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 57–398 of SEQ ID NO: 1, and the glycosylation efficiency of the modified Sap2 protein is increased compared to the glycosylation efficiency of a control Sap2 protein that does not contain at least four consensus sequences. In some embodiments, the modified Sap2 protein contains the amino acid sequence of SEQ ID NO: 83. In other embodiments, the modified Sap2 protein contains the amino acid sequence of SEQ ID NO: 87.

[0111] In other embodiments, the present invention provides a method for increasing the glycosylation efficiency of a modified Sap2 protein of the present invention, comprising the step of expressing a modified Sap2 protein containing at least four consensus sequences in a host cell of the present invention, wherein the consensus sequences are added to or substituted next to or at equivalent positions in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19–398 of SEQ ID NO: 1, and the glycosylation efficiency of the modified Sap2 protein is increased compared to the glycosylation efficiency of a control Sap2 protein that does not contain at least four consensus sequences.

[0112] In further embodiments, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace (i) amino acid residue 220 and (ii) amino acid residue 398 of amino acid residues 57-398 of SEQ ID NO: 1, or amino acid residues 57-398 of SEQ ID NO: 1 at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 57-398 of SEQ ID NO: 1. In certain embodiments, the modified Sap2 protein of the present invention includes the amino acid sequence of SEQ ID NO: 84.

[0113] In yet another embodiment, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace (i) amino acid residue 57, (ii) amino acids between amino acid residues 98 and 102, and (iii) amino acid residue 220, or amino acid residues 57 to 398 of SEQ ID NO: 1, or equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 57 to 398 of SEQ ID NO: 1.

[0114] In further embodiments, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace amino acid residues 57-398 of SEQ ID NO: 1, specifically (i) amino acid residue 57, (ii) amino acids between amino acid residues 98-102, (iii) amino acids between amino acid residues 109-113, (iv) amino acid residue 220, and (v) amino acid residue 398, or amino acid residues 57-398 of SEQ ID NO: 1 at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical.

[0115] In certain embodiments, the modified Sap2 protein of the present invention comprises one or more consensus sequences which are added next to or replace equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to (i) amino acid residue 19 and (ii) amino acid residue 398 of SEQ ID NO: 1 or amino acid residues 19 to 398 of SEQ ID NO: 1.

[0116] In other embodiments, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace (i) amino acid residue 19, (ii) amino acid residue 220, and (iii) amino acid residue 398 of SEQ ID NO: 1, or amino acid residues 19-398 of SEQ ID NO: 1, at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences.

[0117] In yet another embodiment, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace equivalent positions in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1, (i) amino acid residue 19, (ii) amino acids between amino acid residues 98-102, (iii) amino acid residue 220, and (iv) amino acid residue 398 or amino acid residue 19-398 of SEQ ID NO: 1.

[0118] In yet another embodiment, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace (i) amino acid residue 220 and (ii) amino acid residue 398 of amino acid residues 19-398 of SEQ ID NO: 1 or amino acid residues 19-398 of SEQ ID NO: 1 at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0119] In further embodiments, the modified Sap2 protein of the present invention comprises one or more consensus sequences which are added next to or replace (i) amino acid residue 19, (ii) amino acids between amino acid residues 98 and 102, and (iii) amino acid residue 220, or amino acid residues 19 to 398 of SEQ ID NO: 1, or equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19 to 398 of SEQ ID NO: 1.

[0120] In further embodiments, the modified Sap2 protein of the present invention includes one or more consensus sequences which are added next to or replace amino acid residues 19-398 of SEQ ID NO: 1, (i) amino acid residue 19, (ii) amino acids between amino acid residues 98-102, (iii) amino acids between amino acid residues 109-113, (iv) amino acid residue 220, and (v) amino acid residue 398, or amino acid residues 19-398 of SEQ ID NO: 1 at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0121] In certain embodiments, the modified Sap2 protein of the present invention is derived from a fungus. In certain embodiments, the fungus is Candida. Thus, in some embodiments, the modified Sap2 protein of the present invention is derived from Candida. In certain embodiments, Candida includes, but is not limited to, Candida albicans, Candida auris, Candida guilliermondi, Candida lusitaniaea, and Candida tropicalis. In certain embodiments, the modified Sap2 protein of the present invention is derived from Candida albicans. In certain embodiments, at least one of the one or more consensus sequences contains the amino acid sequence D / EXNZS / T (wherein X and Z are independently any amino acid other than proline). In certain embodiments, X is Q (glutamine). In other embodiments, Z is A (alanine). In some embodiments, one or more consensus sequences include, but are not limited to, DQNAT (sequence number 4) and DQNVT (sequence number 5). In certain embodiments, X is Q (glutamine), Z is A (alanine), and one or more consensus sequences are selected from the group consisting of DQNAT (sequence number 4) and DQNVT (sequence number 5).

[0122] In certain embodiments, the modified Sap2 protein of the present invention further comprises at least one fructose bisphosphate (biphosphate) aldolase (Fba) peptide. The Fba peptide is a 14-mer peptide (Fba-1) derived from the N-terminal portion of the fructose bisphosphate aldolase protein. The Fba-1 protein is a multifunctional C. albicans cell wall protein and a key enzyme in the glycolysis pathway. It facilitates fungal adhesion to human cells or non-living surfaces and protects Candida cells from the host immune system (Elamin E et al., 2021, J. Immunol. Res., 2021:1-19). In addition, it promotes the detoxification of reactive oxygen species generated during respiratory bursts. Proteomic analysis has shown that Fba1 is the most abundant and stable enzyme in Candida and is considered to be one of the major immunodominant proteins (Elamin E et al., 2021, J. Immunol. Res., 2021:1-19). Fba peptides have been previously used to generate autoadjuvant vaccines (Xin H et al., 2012, PLoS ONE, 7:e35106). In certain embodiments, at least one Fba peptide contains (or consists of) the amino acid sequence YGKDVKDLFDYAQE (SEQ ID NO: 3). In other embodiments, at least one Fba peptide contains (or consists of) an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 92% identical to SEQ ID NO: 3.

[0123] In certain embodiments, the modified Sap2 protein of the present invention further comprises at least one Fba peptide. In certain embodiments, the at least one Fba peptide contains the amino acid sequence of YGKDVKDLFDYAQE (SEQ ID NO: 3) or an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 92% identical to SEQ ID NO: 3. In certain embodiments, the at least one Fba peptide is ligated to the modified Sap2 protein of the present invention. In some embodiments, the at least one Fba peptide is non-covalently ligated to the modified Sap2 protein of the present invention. In other embodiments, the at least one Fba peptide is covalently ligated to the modified Sap2 protein of the present invention. In further embodiments, the Fba peptide is ligated to the modified Sap2 protein of the present invention at a single amino acid residue. In other embodiments, the Fba peptide is ligated to the modified Sap2 protein of the present invention at more than one amino acid residue. In further embodiments, the Fba peptide is ligated to the modified Sap2 protein of the present invention at one or more amino acid residues. In certain embodiments, the Fba peptide is ligated to the modified Sap2 protein of the present invention at amino acid residue 398. The amino acid residue numbering specified herein refers to the amino acid position in SEQ ID NO: 1 (or, if the amino acid sequence is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, the equivalent position in SEQ ID NO: 1 when this sequence is matched to the amino acid sequence of SEQ ID NO: 1 to maximize sequence identity between the two sequences). In certain embodiments, the Fba peptide is covalently ligated to the modified Sap2 protein of the present 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 that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0124] In certain embodiments, the modified Sap2 protein of the present invention comprises at least one further consensus sequence. In some embodiments, at least one further consensus sequence is added adjacent to the C-terminal amino acid residue of the modified Sap2 protein of the present invention. In other embodiments, the modified Sap2 protein of the present invention comprises at least one Fba peptide, and at least one further consensus sequence is added adjacent to the C-terminal amino acid residue of at least one Fba peptide. In further embodiments, the modified Sap2 protein of the present invention comprises the amino acid sequence of YGKDVKDLFDYAQE (SEQ ID NO: 3) or an Fba peptide comprising an amino acid sequence identical to SEQ ID NO: 3 by at least 70%, 80%, 85%, 90%, or 92%, and at least one further consensus sequence is added adjacent to the C-terminal amino acid residue of SEQ ID NO: 3, or to an equivalent position within an amino acid sequence identical to SEQ ID NO: 3 by at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%. In certain embodiments, the modified Sap2 protein includes at least one further consensus sequence comprising the amino acid sequence JUBD / EXNZS / TJUB (wherein X and Z are independently any amino acid other than proline, and J, U, and B independently comprise 1 to 5 naturally occurring amino acid residues), which is appended next to the C-terminal amino acid residue of SEQ ID NO: 3 or to an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In certain embodiments, the at least one further consensus sequence comprises the amino acid sequence JUBD / EXNZS / TJUB (wherein X and Z are independently any amino acid other than proline, and J, U, and B independently comprise 1 or more naturally occurring amino acid residues). In certain embodiments, X is Q (glutamine). In other embodiments, Z is A (alanine). In certain embodiments, J comprises at least one glycine (G) residue.In some embodiments, J contains 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 glycine (G) residues. In certain embodiments, J contains 1-5 glycine (G) residues. In other embodiments, B contains at least 1 glycine (G) residue. In certain embodiments, B contains 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 glycine (G) residues. In certain embodiments, B contains 1-5 glycine (G) residues. In further embodiments, J and B each contain 1-5 glycine (G) residues. In further embodiments, U contains at least 1 glycine (G) residue. In certain embodiments, U contains 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 glycine (G) residues. In certain embodiments, U contains 1-5 serine (S) residues. In certain embodiments, X is Q, Z is A, J and B each contain 1-5 glycine (G) residues, and U contains 1-5 serine (S) residues. In certain embodiments, a further consensus sequence contains (or consists of) the amino acid sequence GSGGGDQNATGSGGG (SEQ ID NO: 7). In other embodiments, a further consensus sequence contains (or consists of) the amino acid sequence GSGGGDQNATGSGGGHHHHHHHHHH (SEQ ID NO: 8).

[0125] In certain embodiments, the modified Sap2 protein of the present invention comprises (or consists of) the amino acid sequence of SEQ ID NO: 9: [ka]

[0126] Sequence ID 9 is an intermediate form of the modified Sap2 protein that lacks the native leader sequence, contains a propeptide sequence (underlined), four glycosites (bold), an inactivation substitution (double underlined), and further contains an Fba sequence (dashed underlined).

[0127] In other embodiments, the modified Sap2 protein of the present invention comprises (or consists of) the amino acid sequence of SEQ ID NO: 10: [ka]

[0128] Sequence ID 10 is a mature form of a modified Sap2 sequence that lacks a native leader or propeptide sequence, contains four glycosites (bold), contains an inactivating substitution (double underlined), and further contains an Fba sequence (dashed underlined).

[0129] In certain embodiments, the modified Sap2 protein of the present invention is glycosylated. In certain embodiments, the modified Sap2 protein of the present invention is N-glycosylated.

[0130] Those skilled in the art will understand that a reference to "between amino acids..." (for example, "between amino acids 19-24") refers to the numbers of amino acids counted consecutively from the N-terminus of an amino acid sequence, and for example, "between amino acids 19-24 of SEQ ID NO: 1" refers to the 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. Thus, in a particular embodiment, if "one or more consensus sequences are added next to or replace one or more amino acids between amino acid residues 19-24 of SEQ ID NO: 1", the one or more consensus sequences may be added next to or replace any one (or more) of amino acid numbers 19, 20, 21, 22, 23, and 24 in SEQ ID NO: 1. Those skilled in the art will understand that if the Sap2 amino acid sequence is a variant and / or fragment of the amino acid sequence of SEQ ID NO: 1, for example, an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, then the reference to "aminotherium..." will be understood to mean the position that is equivalent to the defined position when this sequence is aligned with the amino acid sequence of SEQ ID NO: 1 to maximize sequence identity between the two sequences (the sequence alignment tool is not limited to Clustal Omega (www.ebi.ac.ac.uk), MUSCLE (www.ebi.ac.uk), or T-coffee (www.tcoffee.org)). In one embodiment, the sequence alignment tool used is Clustal Omega (www.ebi.ac.ac.uk).

[0131] The amino acid numbers described herein correspond to those in SEQ ID NO: 1 and the amino acids described above, and those skilled in the art can determine, by alignment, equivalent amino acid positions in amino acid sequences that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. The addition or deletion of amino acids derived from variants and / or fragments of SEQ ID NO: 1 may result in differences in the actual amino acid positions of the consensus sequence in the mutated sequence, but by aligning the mutated sequence with the reference sequence, it is possible to identify amino acids at equivalent positions to the corresponding amino acids in the reference sequence and thus establish appropriate positions for addition or substitution of the consensus sequence.

[0132] In certain embodiments, the modified Sap2 protein of the present invention is an isolated modified Sap2 protein. In other embodiments, the modified Sap2 protein of the present invention is a recombinant modified Sap2 protein. In yet another embodiment, the modified Sap2 protein of the present invention is an isolated recombinant modified Sap2 protein.

[0133] Compass array In certain embodiments, the modified Sap2 protein of the present invention comprises a D / EXNZS / T, KD / EXNZS / T, or JUBD / EXNZS / TJUB consensus sequence (wherein X and Z are independently any amino acid other than proline, and J, U, and B independently comprise 1 to 5 naturally occurring amino acid residues). The classic five-amino acid glycosylation consensus sequence (D / EXNZS / T) can be extended to either side of the JUBD / EXNZS / TJUB consensus sequence by 1 to 5 other amino acid residues for more efficient glycosylation (e.g., GSGGGD / EXNZS / TGSGG (SEQ ID NO: 2)). The classic five-amino acid glycosylation consensus sequence (D / EXNZS / T) can be extended by lysine residues for more efficient glycosylation (e.g., KD / EXNZS / T). Thus, the consensus sequence in the modified Sap2 protein of the present invention may include (or be derived from) the D / EXNZS / T consensus sequence.

[0134] In the modified Sap2 protein of the present invention, the consensus sequence can be selected from D / EXNZS / T, KD / EXNZS / T (SEQ ID NO: 18), or JUBD / EXNZS / TJUB (wherein X is Q (glutamine) and Z is A (alanine)). In the modified Sap2 protein of the present invention, the consensus sequence can be selected from D / EXNZS / T and KD / EXNZS / T (SEQ ID NO: 18) (wherein X is Q (glutamine) and Z is A (alanine)). In some embodiments, the consensus sequence is D / EXNZS / T (wherein X is Q (glutamine) and Z is A (alanine)), for example, DQNAT (SEQ ID NO: 4), also known as "DQNAT" (SEQ ID NO: 4). In other embodiments, the consensus sequence is KD / EXNZS / T(sequence ID 18) (wherein X is Q (glutamine) and Z is A (alanine)), for example, KDQNAT(sequence ID 19), also known as "KDQNAT"(sequence ID 19). In yet another embodiment, the consensus sequence is KD / EXNZS / T(sequence ID 18) (wherein X is Q (glutamine) and Z is A (alanine)), for example, KDQNAS(sequence ID 20), also known as "KDQNAS"(sequence ID 20). In the modified Sap2 protein of the present invention, the consensus sequence can be selected from D / EXNZS / T, KD / EXNZS / T (SEQ ID NO: 18), or JUBD / EXNZS / TJUB (wherein X is Q (glutamine), Z is A (alanine), and J, U, and B are 1 to 5 amino acid residues independently selected from glycine and / or serine).In some embodiments, the consensus sequence is JUBD / EXNZS / TJUB (Sequence ID 6) (wherein X is Q (glutamine), Z is A (alanine), J and B each contain 1 to 5 glycine (G) residues, and U contains 1 to 5 serine (S) residues), for example, GSGGGDQNATGSGGG (Sequence ID 7), also known as "GSGGGDQNATGSGGG" (Sequence ID 7).

[0135] In certain embodiments, the modified Sap2 protein of the present invention contains at least two D / EXNZS / T or KD / EXNZS / T consensus sequences. In other embodiments, the modified Sap2 protein of the present invention contains at least three D / EXNZS / T or KD / EXNZS / T consensus sequences. In yet another embodiment, the modified Sap2 protein of the present invention contains at least four D / EXNZS / T or KD / EXNZS / T consensus sequences. In yet another embodiment, the modified Sap2 protein of the present invention contains at least five D / EXNZS / T or KD / EXNZS / T consensus sequences. In yet another embodiment, the modified Sap2 protein of the present invention contains at least six D / EXNZS / T or KD / EXNZS / T consensus sequences. In yet another embodiment, the modified Sap2 protein of the present invention contains at least seven D / EXNZS / T or KD / EXNZS / T consensus sequences. In other embodiments, the modified Sap2 protein of the present invention contains 3 to 7 D / EXNZS / T or KD / EXNZS / T consensus sequences. In yet another embodiment, the modified Sap2 protein of the present invention contains 4 to 7 D / EXNZS / T or KD / EXNZS / T consensus sequences. In yet another embodiment, the modified Sap2 protein of the present invention contains 5 to 7 D / EXNZS / T or KD / EXNZS / T consensus sequences.

[0136] The introduction of such glycosylation sites can be achieved, for example, by adding a new amino acid to the primary structure of the protein (i.e., by adding the glycosylation site entirely or partially), or by mutating an existing amino acid in the protein to generate a glycosylation site (i.e., by mutating a selected amino acid in the protein to form a glycosylation site, without adding an amino acid to the protein). In certain embodiments, the consensus sequence is recombinantly introduced into the Sap2 amino acid sequence of SEQ ID NO: 1 or into a Sap2 amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0137] In some embodiments, the modified Sap2 protein of the present invention may further include a "peptide tag" or "tag," i.e., a sequence of amino acids that enables the isolation and / or identification of the modified Sap2 protein. For example, tagging the modified Sap2 protein of the present invention may be useful in the purification of the protein, and therefore in the purification of a conjugate (e.g., bioconjugate) vaccine containing the tagged modified Sap2 protein. Exemplary tags that can be used herein include, but are not limited to, histidine (HIS) tags (e.g., hexahistidine tags, or 6XHis-Tag), FLAG-TAG, and HA tags. In certain embodiments, the tag is a hexahistidine tag. In certain embodiments, the tags used herein are removable when they are no longer needed, for example, when the modified Sap2 protein of the present invention has been purified, and can be removed, for example, by chemical agents or enzymatic means. Thus, the modified Sap2 protein of the present invention may further include a peptide tag. In certain embodiments, the peptide tag is located at the C-terminus of the amino acid sequence of the modified Sap2 protein of the present invention. In some embodiments, the peptide tag comprises six histidine residues at the C-terminus of the amino acid sequence of the modified Sap2 protein of the present invention. In certain embodiments, the present invention provides a modified Sap2 protein comprising a tag (e.g., a histidine tag). In other embodiments, the present invention provides a modified Sap2 protein without a tag (e.g., a histidine tag), e.g., a modified Sap2 protein from which the histidine tag has been removed. Thus, in certain embodiments, the modified Sap2 protein of the present invention comprises (i) an amino acid sequence that 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).In other embodiments, the modified Sap2 protein of the present invention comprises (or consists of) an amino acid sequence that 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.

[0138] In other embodiments, the modified Sap2 protein of the present invention includes a signal sequence that can direct the modified Sap2 protein to the periplasm of a host cell (e.g., bacteria). The signal sequence, including the periplasmic signal sequence, is typically removed by a signal peptidase, for example, during the translocation of the protein to the periplasm (i.e., the mature protein is a protein from which at least the signal sequence has been removed). The signal sequences are not limited to, but include: Escherichia coli (E. coli) flagellin (FlgI) [MIKFLSALILLLVTTAAQA (SEQ ID NO: 21)], Escherichia coli outer membrane porin A (OmpA) [MKKTAIAIAVALAGFATVAQA (SEQ ID NO: 22)], Escherichia coli maltose-binding protein (MalE) [MKIKTGARILALSALTTMMFSASALA (SEQ ID NO: 23)], Escherichia coli outer membrane porin C (OmpC) [MKVKVLSLLVPALLVAGAANA (SEQ ID NO: 24)], Erwinia carotovorans pectin lyase (PelB) [MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 38)], heat-unstable Escherichia coli enterotoxin LTIIb [MSFKKIIKAFVIMAALVSVQAHA (SEQ ID NO: 39)], and Bacillus subtilis. The subtilis endoxylanase XynA [MFKFKKKFLVGLTAAFMSISMFSATASA (SEQ ID NO: 40)], E. coli DsbA [MKKIWLALAGLVLAFSASA (SEQ ID NO: 41)], TolB [MKQALRVAFGFLILWASVLHA (SEQ ID NO: 42)], or SipA [MKMNKKVLLTSTMAASLLSVASVQAS (SEQ ID NO: 43)] is selected. In certain embodiments, the signal sequence is derived from E. coli flagellin (FlgI) [MIKFLSALILLLVTTAAQA (SEQ ID NO: 21)]. Thus, in certain embodiments, the present invention provides a modified Sap2 protein in which the amino acid sequence further comprises a signal sequence that can direct the modified Sap2 protein to the periplasm of a host cell (e.g., bacteria). In certain embodiments, the signal sequence is FlgI. In some embodiments, FlgI comprises the amino acid sequence of SEQ ID NO: 21.In certain embodiments, the bacterial signal sequence is removed from the modified Sap2 protein after it has been transported to the periplasmic side of the inner membrane of the host cell of the present invention.

[0139] In further embodiments, the present invention provides a polynucleotide encoding a modified Sap2 protein of the present invention. In certain embodiments, the present invention provides a polynucleotide encoding a modified Sap2 protein of the present invention having a nucleotide sequence encoding a polypeptide having an amino acid sequence that is at least 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 or 10. In other embodiments, the present invention provides the nucleotide sequence described in SEQ ID NO: 35, or a nucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 35. In further embodiments, the present invention provides the nucleotide sequence described in SEQ ID NO: 36, or a nucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 36. In yet another embodiment, the present invention provides the nucleotide sequence described in SEQ ID NO: 37, or a nucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37. In certain embodiments, the nucleotide sequence of the present invention comprises a nucleotide encoding an amino acid corresponding to one (or more) consensus sequences selected from D / EXNZS / T, KD / EXNZS / TK, and JUBD / EXNZS / TJUB. In other embodiments, the nucleotide sequence of the present invention comprises a nucleotide encoding an amino acid corresponding to one (or more) consensus sequences selected from KDQNAT (SEQ ID NO: 19), KDQNAS (SEQ ID NO: 20), DQNAT (SEQ ID NO: 4), and GSGGGDQNATGSGGG (SEQ ID NO: 7).In a particular embodiment, the nucleotide sequence of the present invention comprises one or more consensus sequences of amino acid residues 19-398 of SEQ ID NO: 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 The nucleotides comprising a nucleotide encoding the modified Sap2 protein of the present invention include one or more amino acids selected from the group consisting of base 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, or one or more consensus sequences that are added next to or replace an equivalent position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid residue of SEQ ID NO: 1.

[0140] In further embodiments, the present invention provides a vector comprising a polynucleotide encoding a modified Sap2 protein of the present invention.

[0141] Conjugate In certain embodiments, the present invention provides a conjugate comprising the modified Sap2 protein of the present invention. The conjugate of the present invention may be a conjugate of the modified Sap2 protein (e.g., a chemical conjugate or a bioconjugate). The conjugate of the present invention may be a conjugate of the modified Sap2 protein with an antigen, for example, a sugar antigen (i.e., a bioconjugate). In certain embodiments, the present invention provides a conjugate comprising (or consisting of) the modified Sap2 protein of the present invention and at least one sugar antigen. In certain embodiments, the conjugate of the present invention is a bioconjugate.

[0142] In some embodiments, the present invention provides a conjugate comprising the sugar of the present invention linked to a modified carrier protein. In certain embodiments, the sugar of the present invention is linked to an asparagine residue of the modified carrier protein. Thus, in certain embodiments, the present invention provides a conjugate comprising the sugar of the present invention linked to an asparagine residue of the modified carrier protein. In some embodiments, the conjugate is a bioconjugate. In certain embodiments, the modified carrier proteins of the present invention include, but are not limited to, Sap2, Als3, detoxified exotoxin A (EPA) of Pseudomonas aeruginosa, CRM197, diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus, clamping factor A of Staphylococcus aureus, clamping factor B of Staphylococcus aureus, FimH of Escherichia coli, FimHC of Escherichia coli, heat-unstable enterotoxin of Escherichia coli, detoxified variant of heat-unstable enterotoxin of Escherichia coli, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, sat protein of Escherichia coli, passenger domain of sat protein of Escherichia coli, Campylobacter jejuni AcrA, and Campylobacter jejuni natural glycoprotein. In certain embodiments, the modified carrier protein is the modified Sap2 protein of the present invention.

[0143] In some embodiments, the modified Sap2 protein of the present invention is ligated to at least one sugar antigen. In certain embodiments, the modified Sap2 protein of the present invention is directly ligated to at least one sugar antigen. In other embodiments, the modified Sap2 protein of the present invention is ligated to at least one sugar antigen via a linker. In certain embodiments, the modified Sap2 protein of the present invention is non-covalently ligated to at least one sugar antigen. In some embodiments, the modified Sap2 protein of the present invention is non-covalently ligated to at least one sugar antigen by avidin-streptavidin interaction. In other embodiments, the modified Sap2 protein of the present invention is covalently ligated to at least one sugar antigen via a chemical linkage obtained 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 amination, cyanylation chemistry (e.g., CDAP chemistry), maleimide chemistry, hydrazide chemistry, ester chemistry, and N-hydroxysuccinimide chemistry. The conjugates can be prepared by direct reductive amination as described in US200710184072 (Hausdorff), US 4365170 (Jennings), and US 4673574 (Anderson). Other methods are described in EP-0-161-188, EP-208375, and EP-0-477508. Alternatively, the conjugation method may rely on the activation of the sugar using 1-cyano-4-dimethylaminopyridinium tetrafluoroboric acid (CDAP) to form a cyanate ester. Such conjugates are described in PCT publications WO 93 / 15760 Uniformed Services University, WO 95 / 08348, and WO 96 / 29094. See also Chu C. et al., Infect. Immunity, 1983 245-256.

[0144] Generally, the following types of chemical groups on modified Sap2 proteins can be used for coupling / conjugation: A) Carboxyl group (e.g., via aspartic acid or glutamic acid). In one embodiment, this group is linked directly to an amino group on a sugar, or to an amino group on a linker using carbodiimide chemistry, for example, using EDAC.

[0145] B) Amino group (e.g., via lysine). In one embodiment, this group is linked directly to a carboxyl group on a sugar, or to a carboxyl group on a linker using carbodiimide chemistry, for example, EDAC. In another embodiment, this group is linked directly to a hydroxyl group activated with CDAP or CNBr on a sugar, or to such a group on a linker; to a sugar or linker having an aldehyde group; or to a sugar or linker having a succinimide ester group.

[0146] C) Sulfhydryl (e.g., via cysteine). In one embodiment, this group is linked to a bromo- or chloroacetylated sugar or linker using maleimide chemistry. In one embodiment, this group is activated / modified using bisdiazobenzidine.

[0147] D) A hydroxyl group (e.g., via tyrosine). In one embodiment, this group is activated / modified using bisdiazobenzidine.

[0148] E) Imidazolyl group (e.g., via histidine). In one embodiment, this group is activated / modified using bisdiazobenzidine.

[0149] F) Guanidyl group (e.g., via arginine).

[0150] G) Indolyl group (e.g., via tryptophan).

[0151] On sugars, the following groups can generally be used for coupling: OH, COOH, or NH2. Aldehyde groups can be generated after various treatments such as periodate production, acid hydrolysis, and hydrogen peroxide.

[0152] Conjugates can be purified by any method known in the industry for protein purification, for example, by chromatography (e.g., ion exchange, anion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. See, for example, Saraswat et al., 2013, Biomed. Res. Int. ID0312709 (p.1-18); also see the method described in WO 2009 / 104074. The actual conditions used to purify a particular conjugate will, in part (in the past), depend on the synthetic strategy (e.g., synthetic production vs. recombinant production) as well as factors such as the net charge, hydrophobicity, and / or hydrophilicity of the bioconjugate.

[0153] In some embodiments, the amino acid residues on the modified Sap2 protein of the present invention to which at least one sugar antigen is ligated include, but are not limited to, Ala, Arg, Asp, Cys, Gly, Glu, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. In further embodiments, the amino acids are amino acids containing a terminal amine group, lysine, arginine, glutamic acid, aspartic acid, cysteine, tyrosine, histidine, or tryptophan. In certain embodiments, the amino acid residues on the modified Sap2 protein of the present invention to which at least one sugar antigen is ligated are not asparagine residues, in which case the conjugate is typically produced by chemical conjugation. In other embodiments, at least one sugar antigen is ligated to an amino acid (e.g., asparagine) on the modified Sap2 protein of the present invention, selected from asparagine, aspartic acid, glutamic acid, lysine, cysteine, tyrosine, histidine, arginine, or tryptophan, and in the case of asparagine, the conjugate may be a bioconjugate (e.g., enzymatic conjugation using an oligosaccharide transferase such as PglB). In specific embodiments, the amino acid residue on the modified Sap2 protein of the present invention to which at least one sugar antigen is ligated is an asparagine residue. In specific embodiments, the amino acid residue on the modified Sap2 protein to which at least one sugar antigen is ligated is asparagine in a part of a consensus sequence, e.g., D / EXNZS / T, KD / EXNZS / TK, or JUBD / EXNZS / TJUB consensus sequence.

[0154] In certain embodiments, the conjugate of the present invention is a conjugate of a recombinant modified Sap2 protein (e.g., a chemical conjugate or a bioconjugate). In other embodiments, the conjugate of the present invention is a conjugate of an isolated recombinant modified Sap2 protein with a recombinant antigen, such as a recombinant glycoantigen (i.e., a bioconjugate).

[0155] In certain embodiments, the modified Sap2 protein of the present invention is linked to at least one sugar antigen at one or more amino acid residues on the modified Sap2 protein. In certain embodiments, the one or more residues include, but are not limited to, 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. In certain embodiments, the modified Sap2 protein of the present invention is linked to at least one sugar antigen at one or more asparagine residues on the modified Sap2 protein of the present invention. In certain embodiments, at least one sugar antigen is ligated 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 the modified Sap2 protein of the present invention. In some embodiments, at least one sugar antigen is ligated to one, two, three, four, five, six, seven, eight, nine, or ten asparagine residues of the modified Sap2 protein of the present invention. In certain embodiments, at least one sugar antigen is ligated to at least three asparagine residues of the modified Sap2 protein of the present invention. In certain embodiments, at least one sugar antigen is ligated to four asparagine residues of the modified Sap2 protein of the present invention. In certain embodiments, the modified Sap2 protein of the present invention comprises (or consists of) the 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.In certain embodiments, the present invention provides a modified Sap2 protein of Candida albicans comprising (or consisting of) (1) the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and (2) at least one sugar antigen of Candida, which is a β-1,2 mannan polymer consisting of at least five consecutive β-1,2 linked mannose molecules, and which is linked to at least one of the four asparagine residues at positions 45, 94, 215, and 415 of SEQ ID NO: 9 or at least one of the four asparagine residues at positions 6, 55, 176, and 376 of SEQ ID NO: 10.

[0156] antigen The present invention provides a conjugate (e.g., a bioconjugate) that can link the modified Sap2 protein of the present invention to several different antigens. In certain embodiments, the modified Sap2 protein is linked to at least one antigen, which is a sugar antigen. In certain embodiments, the antigen comprises at least one sugar antigen. In some embodiments, the at least one sugar antigen is a fungal polysaccharide, a yeast polysaccharide, or a mammalian polysaccharide. The polysaccharide comprises two or more monosaccharides, typically six, eight, or more than ten monosaccharides.

[0157] In certain embodiments, the at least one sugar antigen in the conjugate (e.g., bioconjugate) of the present invention may be, but is not limited to, the O antigen of Citrobacter freundii, extracellular polysaccharides of Agrobacterium species, or Escherichia coli (E. coli).O antigen of coli, O antigen of Salmonella species, O antigen of Pseudomonas species, O antigen of Klebsiella species, O antigen of Acinetobacter, O antigen of Chlamydia trachomatis, O antigen of Vibrio cholera, O antigen of Listeria species, O antigen of Legionella pneumophila serotypes 1-15, O antigen of Bordetella parapertussis, O antigen of Burkholderia mallei and pseudomalei, Francisella tularensis O antigen of *Campylobacter* species, O antigen of *Clostridium* difficile, *Staphylococcus aureus* types 5 and 8, *Streptococcus pyrogenes*, *Escherichia coli*, *Streptococcus agalacticae*, *Neisseria meningitidis*, *Candida* species, *Candida albicans*, *Haemophilus influenzae* capsular polysaccharides, *Enterococcus faecalis* capsular polysaccharides types I-V, and other surface polysaccharide structures, such as *Borrelia burgdorferi*. Examples include glycolipids from *Corbicula burgdorferi*, pyrine-O-glycans and lipo-oligosaccharides (LOS) from *Corbicula meningitidis*, LOS from *Haemophilus influenzae*, lipophosphoglycans from *Leishmania major*, tumor-associated glycosylation antigens, glycosylphosphatidylinositol from malaria, and arabinomannan from *Mycobacterium tuberculosis*.

[0158] In certain embodiments, at least one sugar antigen is, for example, an O antigen derived from Gram-negative bacteria. In certain embodiments, at least one sugar antigen is an O antigen derived from Salmonella, Shigella, Pseudomonas, or Klebsiella species. In other embodiments, at least one sugar antigen is an O antigen derived from Shigella, Pseudomonas, or Klebsiella species (e.g., Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas, or Klebsiella pneumoniae). In other embodiments, at least one sugar antigen is an O antigen derived from Shigella, Shigella flexneri, or Shigella sonnei. For example, the antigen may be an O antigen derived from S. dysenteriae type 1, S. sonnei, and S. flexneri type 6, as well as S. flexneri 2a and 3a0 (Dmitriev, BA 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). In another embodiment, at least one sugar antigen is an O antigen derived from Pseudomonas aeruginosa. For example, the antigen may be an O antigen derived from Pseudomonas aeruginosa serotypes 1-20 (Raymond et al., J Bacteriol. 2002 184(13):3614-22). In yet another embodiment, at least one sugar antigen is an O antigen derived from Klebsiella pneumoniae.

[0159] In certain embodiments, at least one sugar antigen is a capsular polysaccharide derived from meningococcal serogroup A (MenA), meningococcal serogroup C (MenC), meningococcal serogroup Y (MenY), meningococcal serogroup W (MenW), Haemophilus influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In other embodiments, at least one sugar antigen is a capsular polysaccharide derived from a Streptococcus species or a Staphylococcus species (e.g., Streptococcus pneumoniae or Staphylococcus aureus). In further embodiments, at least one sugar antigen is a capsular polysaccharide derived from Staphylococcus aureus. For example, at least one sugar antigen may be a capsular polysaccharide derived from Staphylococcus aureus types 5 and 8. In other embodiments, at least one sugar antigen is a capsular polysaccharide derived from Streptococcus pneumoniae.

[0160] In further embodiments, at least one sugar antigen is a fungal sugar antigen. In certain embodiments, the fungus is a Candida species. Thus, in certain embodiments, at least one sugar antigen is a Candida species sugar antigen. In some embodiments, the Candida species, but not limited to, candida albicans, candida auris, candida gilliermondii, candida lusitanie, and candida tropicalis, candida glabrata, candida krusei, and candida parapsilosis. In certain embodiments, the Candida species is candida albicans. Thus, in certain embodiments, at least one sugar antigen is a Candida albicans sugar antigen.

[0161] In certain embodiments, at least one sugar antigen is a β-1,2-mannan polymer. In certain aspects, at least one sugar antigen is a β-1,2-mannan polymer of Candida albicans. Mannans form the outermost layer of the cell wall, and they are involved in the adhesion of Candida cells and evasion of the immune system. Mannans are highly complex branched structures linked to secretory proteins. β-1,2-mannan is a part of Candida mannans located at the non-reducing end. Structures containing at least two consecutive β-1,2-mannose molecules are exclusive to the genus Candida and are conserved in several Candida species (e.g., Candida albicans, Candida auris, Candida gilliermondii, Candida lucitani, Candida tripicalis, but not Candida glabrata) (Gow NA &, Hube B; 2012; Curr Opin Microbiol; Miyakawa, Y et al., Infect. Immun 1992; Shibata N et al., Proc Jpn Acad, Ser B, 2012; Rudkin FM et al., Nat Commun 2018; Morad HO et al., Front Microbiol, 2018; Han et al., J. Infect. Dis, 1999; Xin et al., PNAS 2008). In certain embodiments, the β-1,2-mannan polymer of the present invention is a naturally occurring mannan. In other embodiments, the β-1,2-mannan polymer of the present invention is recombinantly produced mannan.

[0162] In certain embodiments, at least one sugar antigen comprises a β-1,2-mannan polymer. In some embodiments, the β-1,2-mannan polymer comprises at least two β-1,2-linked mannose molecules. In further embodiments, the β-1,2-mannan polymer comprises 2-50, 2-40, 2-30, 2-20, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 2, 3-50, 3-40, 3-30, 3-20, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 3, 4-50, 4-40, 4-30, 4-20, 4-10, 4-9 The polymer contains 1, 4-8, 4-7, 4-6, 4-5, 4, 5-50, 5-40, 5-30, 5-20, 5-10, 5-9, 5-8, 5-7, 5-6, or 5, 6-40, 6-30, 6-20, 6-10, 6-9, 6-8, 6-7, 6, 7-50, 7-40, 7-30, 7-20, 7-10, 7-9, 7-8, or 7 β-1,2 linked mannose molecules. In some embodiments, the β-1,2 mannan polymer contains at least 2, at least 3, at least 4, or at least 5 β-1,2 linked mannose molecules. In a further embodiment, the β-1,2-mannan polymer comprises at least two, at least three, at least four, or at least five consecutive β-1,2-linked mannose molecules.

[0163] In a particular embodiment, the present invention has a structure: [ka] This invention provides a sugar that is a β-1,2-mannan polymer containing the following:

[0164] In some embodiments, the present invention has a structure: [ka] We provide accreted sugar.

[0165] In a particular embodiment, the present invention has the following structure: [ka] It provides sugars containing [this ingredient].

[0166] In further embodiments, the present invention relates to a structure: [ka] It provides sugars containing [this ingredient].

[0167] In certain embodiments, at least one sugar antigen has the following structure: [ka] Contains glucans.

[0168] In other embodiments, at least one sugar antigen has the following structure: [ka] Contains glucans.

[0169] In yet another embodiment, at least one sugar antigen has the following structure: [ka] Contains glucans.

[0170] In other embodiments, at least one sugar antigen is a β-1,3-glucan polymer. Thus, in a particular embodiment, at least one sugar antigen is a Candida albicans β-1,3-glucan polymer. β-1,3-glucans are widely present in nature. They are present in many yeasts and are widely present in fungi and plants. Candida β-1,3-glucans consist of long linear polymers that are sometimes β-1,6-branched. They provide strength and shape to the cell wall. In a particular embodiment, the β-1,3-glucan polymer of the present invention is a naturally occurring glucan. In other embodiments, the β-1,3-glucan polymer of the present invention is a recombinantly produced glucan.

[0171] In certain embodiments, at least one sugar antigen comprises a β-1,3-glucan polymer. In some embodiments, the β-1,3-glucan polymer of the present invention comprises at least four β-1,3-linked glucose molecules. In further embodiments, the β-1,3-glucan polymer comprises 4-100, 4-50, 4-40, 4-35, 4-30, 4-25, 4-20, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 4, 5-100, 5-50, 5-40, 5-35, 5-30, 5-25, 5-20, 5-10, 5-9, 5-8, 5- The polymer contains 7, 5-6, 5, 6-100, 6-50, 6-40, 6-35, 6-30, 6-25, 6-20, 6-10, 6-9, 6-8, 6-7, 6, 7-100, 7-50, 7-40, 7-35, 7-30, 7-25, 7-20, 7-10, 7-9, 7-8, or 7 β-1,3-linked glucose molecules. In some embodiments, the β-1,3-glucan polymer contains 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 β-1,3-linked glucose molecules. In a particular embodiment, the β-1,3-glucan polymer contains at least 11 β-1,3-linked glucose molecules. In certain embodiments, the β-1,3-glucan polymer contains at least 11 consecutive β-1,3-linked glucose molecules.

[0172] Thus, in certain embodiments, the present invention provides a modified Sap2 protein of Candida albicans comprising (or comprising) at least one sugar antigen of Candida, wherein (1) the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and (2) at least one sugar antigen is a β-1,2 mannan polymer comprising (or comprising) at least five consecutive β-1,2 linked mannose molecules, and at least one sugar antigen is linked to at least one of the four asparagine residues at positions 45, 94, 215, and 415 of SEQ ID NO: 9 or at least one of the four asparagine residues at positions 6, 55, 176, and 376 of SEQ ID NO: 10.

[0173] host cell In certain embodiments, the present invention provides a host cell comprising a polynucleotide sequence encoding a modified Sap2 protein of the present invention. In certain embodiments, the present invention provides a host cell that can be used to produce the bioconjugate of the present invention. Host cells of the present invention include, but are not limited to, archaea, prokaryotic host cells, and eukaryotic host cells. In certain embodiments, the host cell is a non-human host cell. In certain embodiments, the host cell is a eukaryotic host cell. In some embodiments, eukaryotic host cells include, but are not limited to, yeast cells, insect cells, and mammalian cells. In further embodiments, the host cell is a prokaryotic host cell. In other embodiments, the prokaryotic host cell is a bacterial cell. In some embodiments, the bacteria are Gram-positive bacteria. In other embodiments, the bacteria are Gram-negative bacteria. In certain embodiments, the bacteria include, but are not limited to, species of Escherichia, Shigella, Klebsiella, Xhantomonas, Salmonella, Yersinia, Lactococcus, Lactobacillus, Pseudomonas, Corynebacterium, Streptomyces, Streptococcus, Staphylococcus, Bacillus, and Clostridium. In some embodiments, the bacteria are Escherichia coli species. In certain embodiments, the bacteria are Escherichia coli.Thus, exemplary host cells include, but are not limited to, Escherichia, Shigella, Klebsiella, Xhantomonas, Salmonella, Yersinia, Lactococcus, Lactobacillus, Pseudomonas, Corynebacterium, Streptomyces, Streptococcus, Staphylococcus, Bacillus, and Clostridium. In some embodiments, the host cell is Escherichia coli. In certain embodiments, the host cell is Escherichia coli.

[0174] The host cells of the present invention can be modified to delete or modify genes in the host cell's genetic background (genomics) that compete with or interfere with the synthesis of a target polysaccharide antigen (e.g., compete with or interfere with one or more heterologous polysaccharide synthesis genes recombinantly introduced into the host cell). These genes can be deleted or modified in the host cell background (genomics) in a manner that inactivates / defects them (i.e., the deleted / modified host cell nucleotide sequences do not encode a functional protein or do not encode a protein at all). In certain embodiments, if nucleotide sequences are deleted from the genome of the host cells of the present invention, they are replaced with desired sequences, e.g., sequences useful for glycoprotein production. Exemplary genes that may be deleted in host cells (and in some cases replaced by other desired nucleotide sequences) include host cell genes involved in glycolipid biosynthesis, such as waaL (see, e.g., Feldman et al., 2005, PNAS USA 102:3016-3021), O antigen clusters (rfb or wb), enterobacteria common antigen clusters (wec), lipid A core biosynthesis clusters (waa), galactose clusters (gal), arabinose clusters (ara), colonic acid clusters (wc), capsular polysaccharide clusters, 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, enterobacteria common antigen clusters, and prophage O antigen modification clusters such as the gtrABS cluster. In some embodiments, one or more of the following are deleted or functionally inactivated from the genome of the prokaryotic host cell of the present invention: the waaL gene, the gtrA gene, the gtrB gene, the gtrS gene, or a gene or multiple genes derived from the wec cluster or gene, or a gene or multiple genes derived from the colonic acid cluster (wc), or a gene or multiple genes derived from the rfb gene cluster.In other embodiments, one or more of the following genes are deleted or functionally inactivated from the genome of the prokaryotic host cell of the present invention: the waaL gene, the gtrA gene, the gtrB gene, the gtrS gene, or a gene or multiple genes derived from the wec cluster, or a gene or multiple genes derived from the rfb gene cluster. In further embodiments, the host cell of the present invention is Escherichia coli lacking the naturally occurring Enterobacteriaceae Common Antigen Cluster (ECA, wec), except for wecA, the cholanic acid cluster (wca), and the O16 antigen cluster. In further embodiments, the naturally occurring lipopolysaccharide O antigen ligase waaL can be deleted from the host cell of the present invention. In other embodiments, the naturally occurring gtrA gene, gtrB gene, and gtrS gene can be deleted from the host cell of the present invention.

[0175] The host cells of the present invention are engineered to contain heterologous nucleotide sequences. In certain embodiments, the host cells of the present invention are engineered to contain, optionally, a nucleotide sequence encoding the modified Sap2 protein of the present invention within a plasmid. In other embodiments, the host cells of the present invention also contain one or more nucleotide sequences containing polysaccharide synthesis genes. Thus, the host cells of the present invention can produce a bioconjugate containing an antigen linked to the modified Sap2 protein of the present invention, for example, a sugar antigen (e.g., a fungal, bacterial, yeast, or mammalian polysaccharide antigen). In certain embodiments, one or more heterologous nucleotide sequences encode a polysaccharide synthesis protein that produces a fungal polysaccharide antigen, a bacterial polysaccharide antigen, a yeast polysaccharide antigen, or a mammalian polysaccharide antigen.

[0176] Thus, in certain embodiments, the present invention provides a host cell comprising (1) one or more polynucleotide sequences encoding one or more heterologous glycosyltransferases; (2) a polynucleotide sequence encoding a heterologous oligosaccharide transferase; (3) a polynucleotide sequence encoding a modified Sap2 protein of the present invention; and optionally (4) a polynucleotide sequence encoding a polymerase.

[0177] In other embodiments, the present invention provides a host cell comprising (1) one or more polynucleotide sequences encoding one or more heterologous glycosyltransferases; (2) a polynucleotide sequence encoding a heterologous oligosaccharide transferase; (3) a polynucleotide sequence encoding a modified Sap2 protein of the present invention (optionally a polynucleotide sequence of the present invention); and optionally (4) a polynucleotide sequence encoding a polymerase.

[0178] In some embodiments, one or more heterologous glycosyltransferases include, but are not limited to, WbaD, WbaC, and WbaB. In certain embodiments, WbaD, WbaC, and WbaB are derived from citrobacter. In certain embodiments, citrobacter is citrobacter fraungi. In certain embodiments, citrobacter fraungi is citrobacter fraungi P079F I. In certain embodiments, one or more heterologous glycosyltransferases include WbaD, WbaC, and WbaB, which are derived from citrobacter fraungi, possibly citrobacter fraungi, and possibly citrobacter fraungi P079F I. In certain embodiments, one or more heteroglycosyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaD of Citrobacter freunge P079F I, including SEQ ID NO: 11. In other embodiments, one or more heteroglycosyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaC of Citrobacter freunge P079F I, including SEQ ID NO: 12. In yet another embodiment, one or more heteroglycosyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaB of Citrobacter freunge P079F I, including SEQ ID NO: 13.

[0179] In further embodiments, the present invention is i. A nucleotide sequence encoding one or more heterologous glycosyltransferases capable of synthesizing β-1,2-mannan polymers; ii. A nucleotide sequence encoding a second heterologous glycosyltransferase that is eukaryotic and capable of covalently bonding mannose molecules to β-1,2-mannan polymers, thereby extending the β-1,2-mannan polymer chain; iii. Nucleotide sequences encoding heterologous oligosaccharide transferases; and iv. A nucleotide sequence encoding a modified carrier protein that may include a glycosylation site containing the consensus sequence D / EXNZS / T (wherein X and Z are independently any amino acid other than proline). Provides host cells containing the following:

[0180] In yet another embodiment, the present invention is i. A nucleotide sequence encoding one or more heterologous glycosyltransferases capable of synthesizing β-1,2-mannan polymers; ii. A nucleotide sequence encoding a second heterologous glycosyltransferase that is eukaryotic and capable of covalently bonding mannose molecules to β-1,2-mannan polymers, thereby extending the β-1,2-mannan polymer chain; iii. Nucleotide sequences encoding heterologous oligosaccharide transferases; and iv. A nucleotide sequence encoding a modified carrier protein, optionally including a glycosylation site containing the consensus sequence D / EXNZS / T (wherein X and Z are independently any amino acid other than proline), optionally the polynucleotide sequence of the present invention. Provides host cells containing the following:

[0181] In certain embodiments, the modified carrier protein of the present invention may include, but is not limited to, Sap2, Als3, detoxified exotoxin A (EPA) of Pseudomonas aeruginosa, CRM197, diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus, clamping factor A of Staphylococcus aureus, clamping factor B of Staphylococcus aureus, FimH of Escherichia coli, FimHC of Escherichia coli, heat-unstable enterotoxin of Escherichia coli, detoxified variant of heat-unstable enterotoxin of Escherichia coli, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, sat protein of Escherichia coli, passenger domain of sat protein of Escherichia coli, Campylobacter jejuni AcrA, and Campylobacter jejuni natural glycoprotein. In certain embodiments, the modified carrier protein is the modified Sap2 protein of the present invention.

[0182] In some embodiments, one or more heterologous glycosyltransferases capable of synthesizing β-1,2-mannan polymers include, but are not limited to, WbaD, WbaC, and WbaB. In certain embodiments, WbaD, WbaC, and WbaB are derived from citrobacter. In certain embodiments, citrobacter is citrobacter fraeungi. In certain embodiments, citrobacter fraeungi is citrobacter fraeungi P079F I. In certain embodiments, the one or more heterologous glycosyltransferases include WbaD, WbaC, and WbaB, which are derived from citrobacter fraeungi, and optionally from citrobacter fraeungi, and optionally from citrobacter fraeungi P079F I.

[0183] In certain embodiments, one or more heterologous glycosyltransferases capable of synthesizing β-1,2-mannan polymers have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaD of Citrobacter freunge P079F I. In some embodiments, one or more heterologous glycosyltransferases have an amino acid sequence that is identical to the amino acid sequence of WbaD of Citrobacter freunge P079F I. In certain embodiments, WbaD of Citrobacter freunge P079F I contains the amino acid sequence of SEQ ID NO: 11. Thus, in certain embodiments, one or more heterologous glycosyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 11. In other embodiments, one or more heterologous glycosyltransferases have an amino acid sequence that is identical to SEQ ID NO: 11: [ka]

[0184] In certain embodiments, the host cell of the present invention comprises a nucleotide sequence encoding WbaD, which may be the WbaD of citrobacter freunge P079F I, and may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11, and may comprise this within a plasmid.

[0185] In further embodiments, the host cell of the present invention comprises a nucleotide sequence encoding WbaD, which in some cases is the WbaD of Citrobacter freunge P079F I, and in some cases comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Sequence ID No. 25: [ka] [ka]

[0186] In some embodiments, one or more heterologous glycosyltransferases capable of synthesizing β-1,2-mannan polymers have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaC of Citrobacter freunge P079F I. In some embodiments, one or more heterologous glycosyltransferases have an amino acid sequence that is identical to the amino acid sequence of WbaC of Citrobacter freunge P079F I. In a particular embodiment, WbaC of Citrobacter freunge P079F I contains the amino acid sequence of SEQ ID NO: 12. Thus, in a particular embodiment, one or more heterologous glycosyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 12. In other embodiments, one or more heterologous glycosyltransferases have an amino acid sequence that is identical to SEQ ID NO: 12: [ka]

[0187] In certain embodiments, the host cell of the present invention comprises a nucleotide sequence encoding WbaC, which may be the WbaC of citrobacter freunge P079F I, and may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12, and may comprise this within a plasmid.

[0188] In further embodiments, the host cell of the present invention comprises a nucleotide sequence encoding the WbaC of citrobacter freunge P079F I, which optionally comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the WbaC of citrobacter freunge P079F I, which optionally comprises a nucleotide sequence identical to SEQ ID NO: 26: [ka] [ka]

[0189] In other embodiments, one or more heterologous glycosyltransferases capable of synthesizing β-1,2-mannan polymers have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaB of Citrobacter freunge P079F I. In some embodiments, one or more heterologous glycosyltransferases have an amino acid sequence that is identical to the amino acid sequence of WbaB of Citrobacter freunge P079F I. In a particular embodiment, WbaB of Citrobacter freunge P079F I contains the amino acid sequence of SEQ ID NO: 13. Thus, in a particular embodiment, one or more heterologous glycosyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 13. In other embodiments, one or more heterologous glycosyltransferases have an amino acid sequence that is identical to SEQ ID NO: 13: [ka]

[0190] In certain embodiments, the host cell of the present invention comprises a nucleotide sequence encoding WbaB, which may be WbaB of citrobacter freunge P079F I, and which may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13, and may comprise this within a plasmid.

[0191] In further embodiments, the host cell of the present invention comprises WbaB, optionally WbaB of citrobacter freunge P079F I, and optionally a nucleotide sequence encoding WbaB of citrobacter freunge P079F I, which comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27: [ka] [ka]

[0192] In certain embodiments, one or more heteroglycosyltransferases capable of synthesizing β-1,2-mannan polymers have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaD of Citrobacter freunge P079F I, including SEQ ID NO: 11. In other embodiments, one or more heteroglycosyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaC of Citrobacter freunge P079F I, including SEQ ID NO: 12. In yet another embodiment, one or more heteroglycosyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaB of Citrobacter freunge P079F I, including SEQ ID NO: 13.

[0193] In other embodiments, the host cell of the present invention further comprises a heterologous translocase having an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of Wzx of citrobacter freunge P079F I, including SEQ ID NO: 14.

[0194] In further embodiments, the host cell of the present invention comprises one or more polynucleotide sequences encoding one or more heterologous guanylyltransferases. In certain embodiments, the one or more heterologous guanylyltransferases include, but are not limited to, manB and manC. In certain embodiments, manB and manC are derived from Escherichia coli. In certain embodiments, the Escherichia coli is Escherichia coli K12 W3110. In certain embodiments, the one or more heterologous guanylyltransferases include manB and manC derived from Escherichia coli, and optionally from Escherichia coli K12 W3110.

[0195] In some embodiments, one or more heterologous guanylyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of manB of E. coli K12 W3110. In some embodiments, one or more heterologous guanylyltransferases have an amino acid sequence that is identical to the amino acid sequence of manB of E. coli K12 W3110. In a particular embodiment, manB of E. coli K12 W3110 contains the amino acid sequence of SEQ ID NO: 31. Thus, in a particular embodiment, one or more heterologous guanylyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 31. In other embodiments, one or more heterologous guanylyltransferases have an amino acid sequence that is identical to SEQ ID NO: 31: [ka]

[0196] In certain embodiments, the host cell of the present invention comprises a nucleotide sequence encoding manB, which may be manB of E. coli K12 W3110, and which may comprise an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31, and may comprise this within a plasmid.

[0197] In further embodiments, the host cell of the present invention comprises a nucleotide sequence encoding manB, optionally a nucleotide sequence encoding manB of E. coli K12 W3110, optionally a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33: [ka]

[0198] In other embodiments, one or more heterologous guanylyltransferases have amino acid sequences that are at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of manC of E. coli K12 W3110. In some embodiments, one or more heterologous guanylyltransferases have amino acid sequences that are identical to the amino acid sequence of manC of E. coli K12 W3110. In a particular embodiment, manC of E. coli K12 W3110 contains the amino acid sequence of SEQ ID NO: 32. Thus, in a particular embodiment, one or more heterologous guanylyltransferases have amino acid sequences that are at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 32. In other embodiments, one or more heterologous guanylyltransferases have amino acid sequences that are identical to SEQ ID NO: 32: [ka]

[0199] In certain embodiments, the host cell of the present invention comprises a nucleotide sequence encoding manB, which is possibly manC of E. coli K12 W3110, and which comprises an amino acid sequence that is possibly at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32, and which is possibly contained within a plasmid.

[0200] In further embodiments, the host cell of the present invention comprises a nucleotide sequence encoding manC, optionally a nucleotide sequence encoding manC of E. coli K12 W3110, optionally a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34: [ka] [ka]

[0201] In certain embodiments, the eukaryotic glycosyltransferase capable of covalently bonding a mannose molecule to a β-1,2-mannan polymer to extend the β-1,2-mannan polymer chain is Bmt3. In certain embodiments, Bmt3 is derived from Candida. In some embodiments, Candida is Candida albicans. In certain embodiments, the glycosyltransferase capable of covalently bonding a mannose molecule to a β-1,2-mannan polymer to extend the β-1,2-mannan polymer chain is Bmt3 from Candida albicans. In certain embodiments, the glycosyltransferase capable of covalently bonding a mannose molecule to a β-1,2-mannan polymer to extend the β-1,2-mannan polymer chain has an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of Bmt3 from Candida albicans. In other embodiments, a glycosyltransferase capable of covalently bonding a mannose molecule to a β-1,2-mannan polymer and extending the β-1,2-mannan polymer chain has an amino acid sequence identical to that of Candida albicans Bmt3. In certain embodiments, periplasmic expression of Candida albicans Bmt3 in host cells is required for the extension of the β-1,2-mannan polymer chain. Thus, in certain embodiments, the addition of a terminal mannose residue to the β-1,2-mannan polymer chain requires periplasmic expression of Candida albicans Bmt3 in host cells. In other embodiments, the addition of a terminal mannose residue to the β-1,2-mannan polymer chain further requires the presence of GDP-mannose in the culture medium. In certain embodiments, GDP-mannose is added to the harvested culture. Thus, in certain embodiments, preferred conditions for the production of the sugar conjugate of the present invention include the addition of GDP-mannose to the culture medium. In certain embodiments, GDP-mannose is added to the harvested culture.

[0202] In certain embodiments, Bmt3 of Candida albicans contains the amino acid sequence of SEQ ID NO: 15. In other embodiments, Bmt3 of Candida albicans contains an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 15. [ka] [ka]

[0203] In certain embodiments, the host cell of the present invention comprises a nucleotide sequence encoding Bmt3, which may be Bmt3 of Candida albicans, and which may comprise a nucleotide sequence encoding Bmt3 of Candida albicans comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15, and which may comprise this within a plasmid.

[0204] In other embodiments, the host cells of the present invention include a nucleotide sequence encoding Bmt3, optionally a nucleotide sequence encoding Bmt3 of Candida albicans, optionally a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29: [ka]

[0205] Oligosaccharide transferase N-binding protein glycosylation (the addition of a carbohydrate molecule to an asparagine residue in the polypeptide chain of a target protein) is the most common type of post-translational modification occurring in the endoplasmic reticulum of eukaryotes. This process is accomplished by the enzymatic oligosaccharide transferase complex (OST), which is responsible for the transfer of pre-assembled oligosaccharides from a lipid carrier (dolichol phosphate) in the endoplasmic reticulum to an asparagine residue in the nascent protein within the conserved sequence Asn-X-Ser / Thr (where X is any amino acid other than proline).

[0206] The foodborne pathogen Campylobacter jejuni has also been shown to be able to N-glycosylate its proteins due to the fact that it possesses its own glycosylation mechanism (Wacker et al., Science. 2002; 298(5599):1790-3). The mechanism responsible for this reaction is encoded by a cluster called "pgl" (for protein glycosylation). In certain embodiments, the glycosylation mechanism of Campylobacter jejuni is introduced into E. coli to enable glycosylation of recombinant proteins expressed by host E. coli cells. Previous studies have demonstrated methods for generating E. coli strains capable of N-glycosylation (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 US A. 2005; 102(8):3016-21; Kowarik et al., EMBO J. 2006; 25(9):1957-66; Wacker et al., Proc Natl Acad Sci US A. 2006; 103(18):7088-93; International Patent Application Publications WO2003 / 074687, WO2006 / 119987, WO See issues 2009 / 104074, and WO / 2011 / 06261 and WO2011 / 138361.

[0207] Thus, in certain embodiments, the host cell of the present invention comprises a nucleotide sequence encoding a heterologous oligosaccharide transferase, which may be contained within a plasmid. In some embodiments, the heterologous oligosaccharide transferase is PglB. In certain embodiments, PglB is derived from Campylobacter. In certain embodiments, Campylobacter may include, but is not limited to, Campylobacter jejuni or Campylobacter coli. In some embodiments, Campylobacter is Sinorhizobium meliloti 1021. In certain embodiments, PglB may be derived from Campylobacter, may be derived from Campylobacter jejuni or Campylobacter coli, and may be an evolved PglB comprising the amino acid sequence of Sequence ID No. 16. In certain embodiments, PglB is an evolved pglB containing an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of Campylobacter coli PglB. In other embodiments, PglB contains an amino acid sequence identical to the amino acid sequence of Campylobacter coli PglB. In certain embodiments, PglB is an evolved PglB enzyme containing the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the evolved PglB has an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 16. In other embodiments, the evolved PglB has an amino acid sequence identical to SEQ ID NO: 16. [ka] [ka]

[0208] In certain embodiments, the host cell of the present invention comprises a nucleotide sequence encoding evolved PglB, which optionally comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16, and optionally comprises this within a plasmid.

[0209] In other embodiments, the host cell of the present invention includes a nucleotide sequence encoding evolved PglB, which optionally comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30: [ka] [ka]

[0210] In certain embodiments, the nucleotide sequence encoding PglB is codon-optimized.

[0211] polymerase The host cells of the present invention may also contain a nucleotide sequence encoding a polymerase (e.g., wzy). In certain embodiments, the polymerase (e.g., wzy) is introduced into the host cells of the present invention (i.e., the polymerase is heterologous to the host cells). In some embodiments, the polymerase is a bacterial polymerase. In other embodiments, the polymerase is a capsular polysaccharide polymerase (e.g., wzy) or an O antigen polymerase (e.g., wzy). In certain embodiments, the polymerase is an O antigen polysaccharide polymerase (e.g., wzy) derived from, for example, Shigella, Pseudomonas, or Escherichia species (e.g., Shigella shigaensis, Shigella flexinelli, Shigella group D, Pseudomonas aeruginosa, or Escherichia coli). In other embodiments, the polymerase is, for example, a capsular polysaccharide polymerase (e.g., wzy) derived from meningococcal serogroup A (MenA), meningococcal serogroup C (MenC), meningococcal serogroup Y (MenY), meningococcal serogroup W (MenW), Haemophilus influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In yet another embodiment, the polymerase is a capsular polysaccharide polymerase (e.g., wzy) from Streptococcus pneumoniae. In some embodiments, the wzy polymerase can be incorporated into the host cell as part of an rfb cluster or capsular polysaccharide cluster (e.g., by insertion into the genome or expression by plasmid). Thus, in certain embodiments, the host cell of the present invention may further contain a nucleotide sequence encoding a heterologous wzy polymerase.

[0212] Flippa The host cells of the present invention may also contain a heterologous flippase (e.g., wzx), for example, a nucleotide sequence encoding a heterologous flippase. The flippase translocates wild-type repeat units and / or their corresponding engineered (hybrid) repeat units from the cytoplasm of the host cell (e.g., Escherichia coli) to the periplasm. In certain embodiments, the flippase is a bacterial flippase, for example, a flippase of the polysaccharide biosynthesis pathway of interest. In some embodiments, the host cells of the present invention contain a nucleotide sequence encoding a flippase (e.g., the wzx gene) of the polysaccharide biosynthesis pathway of a Streptococcus species, Sigella species, Escherichia species, Pseudomonas species, or Staphylococcus species (e.g., Streptococcus pneumoniae, Shigella shiga, Shigella flexinelli, Shigella group D, Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus). In other embodiments, the flippase is a capsular polysaccharide flippase of Streptococcus pneumoniae (e.g., wzx). Other flippases that can be introduced into the host cells of the present invention include, for example, those derived from Campylobacter jejuni (e.g., pglK) or Wzx from Citrobacter (e.g., Citrobacter freungi P079F I).

[0213] Translocase The host cell of the present invention may also contain a translocase (e.g., Wzx), for example, a nucleotide sequence encoding a heterologous translocase. In certain embodiments, the translocase is a Wzx / Wzy-dependent transporter. In other embodiments, the translocase is an ATP-binding cassette (ABC)-dependent transporter. In yet another embodiment, the translocase is a synthase-dependent transporter. In certain embodiments, the translocase is an ABC transporter. The polysaccharide assembled by the ABC transporter is completely polymerized by sequential glycosyl transfer on the cytoplasmic surface of the inner membrane of the host cell. The glycan can be assembled as an undecaprenyl diphosphate (Und-PP)-bound intermediate (e.g., for many O-antigen polysaccharides). Alternatively, for certain types of polysaccharides (e.g., capsular polysaccharides), the acceptor is diacylglycerol phosphate. Polysaccharide chains are elongated by the addition of monomers to the non-reducing end of lipid-binding intermediates and are completed in the cytoplasm before being exported to the periplasmic space via translocases (e.g., ABC transporters) (Cuthbertson L. et al., 2010, Microbiology and Molecular Biology Reviews, 74(3):341-362; Bi Y. et al., 2018, Nature, 553(7688):361-365).

[0214] In certain embodiments, the sugar antigen of the present invention is synthesized by an ABC transporter-dependent pathway. Thus, in certain embodiments, the sugar antigen of the present invention is entirely synthesized on the cytoplasmic leaflet of the cell membrane of a host cell (e.g., Escherichia coli). In certain embodiments, the sugar antigen of the present invention is constructed on a lipid acceptor (e.g., undecaprenyl diphosphate (Und-PP)) resulting in the formation of a lipid-linked antigen sugar. In certain embodiments, Und-PP is modified by the addition of an acetylated aminoglycophosphate (e.g., N-acetylglucosamine-1-P) to act as a lipid acceptor and generate a biosynthetic primer. In certain embodiments, a translocase (e.g., Wzx) translocates the Und-PP-linked sugar antigen intermediate to the periplasmic side of the membrane, where it forms a substrate for glycosylation of a carrier protein (e.g., a modified Sap2 protein of the present invention). In certain embodiments, the translocase is a bacterial translocase, e.g., a translocase of the polysaccharide biosynthesis pathway of interest. Thus, in some embodiments, the host cells of the present invention contain a nucleotide sequence encoding a translocase (e.g., wzm-wzt) of the polysaccharide biosynthesis pathway of a Klebsiella, Streptococcus, Sigella, Escherichia, Pseudomonas, or Staphylococcus species (e.g., Klebsiella pneumoniae, Streptococcus pneumoniae, Shigella shiga, Shigella flexinelli, Shigella group D, Escherichia coli, Pseudomonas aeruginosa, or Staphylococcus aureus). In certain embodiments, the heterologous translocase introduced into the host cells of the present invention is the ABC transporter of Klebsiella pneumoniae (e.g., wzm-wzt). Other translocases that can be introduced into the host cells of the present invention include, for example, those derived from Campylobacter jejuni (e.g., pglK) or Wzx from Citrobacter (e.g., Citrobacter freungi P079F I).

[0215] In some embodiments, the heterologous translocase may include, but is not limited to, Wzx derived from Citrobacter. In certain embodiments, the Citrobacter is Citrobacter fraeungi. In certain embodiments, Citrobacter fraeungi is Citrobacter fraeungi P079F I. In certain embodiments, the translocase is Wzx derived from Citrobacter, possibly Citrobacter fraeungi, and possibly Citrobacter fraeungi P079F. In certain embodiments, the heterologous translocase has an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of Wzx of Citrobacter fraeungi P079F I. In some embodiments, the heterologous translocase has an amino acid sequence identical to the amino acid sequence of Wzx of Citrobacter fraeungi P079F I. In certain embodiments, Wzx of Citrobacter freunge P079F I contains the amino acid sequence of SEQ ID NO: 14. Thus, in certain embodiments, the heterologous translocase has an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 14. In other embodiments, the heterologous translocase has an amino acid sequence identical to SEQ ID NO: 14: [ka]

[0216] In certain embodiments, the host cell of the present invention comprises a nucleotide sequence encoding Wzx, which may be citrobacter freunge P079F I, which may comprise a nucleotide sequence encoding Wzx of citrobacter freunge P079F I having an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14, which may comprise this within a plasmid.

[0217] In a further embodiment, the host cell of the invention comprises Wzx, optionally a nucleotide sequence encoding Wzx of Citrobacter freundii P079F I, optionally a nucleotide sequence encoding Wzx of Citrobacter freundii P079F I comprising a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 28: [Chemical formula] [Chemical formula]

[0218] Thus, in certain embodiments, the invention provides a host cell comprising (i) a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 28 and optionally a nucleotide sequence comprising a wzx gene that is at least 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 28. In certain aspects, the wzx gene is derived from Citrobacter freundii P079F I.

[0219] In a further embodiment, the invention provides a method for producing a β-1,2 mannan polymer in the host cell of the invention, comprising: i. a nucleotide sequence encoding one or more first heterologous glycosyltransferases capable of synthesizing a β-1,2 mannan polymer; ii. a nucleotide sequence encoding a second heterologous glycosyltransferase that is eukaryotic and capable of covalently binding a mannose molecule to a β-1,2 mannan polymer to extend a β-1,2 mannan polymer chain, optionally Bmt3 of Candida albicans; and iii. optionally a nucleotide sequence encoding a translocase capable of translocating β-1,2 mannan to the periplasmic side of the inner membrane of the host cell and introducing and expressing the sequences into the host cell.

[0220] In certain embodiments, the present invention relates to a method for producing a β-1,2-mannan polymer in a prokaryotic host cell, iv. A nucleotide sequence encoding one or more first heterologous glycosyltransferases capable of synthesizing β-1,2-mannan polymers, comprising WbaD, WbaC, and WbaB derived from Citrobacter, possibly Citrobacter fraeungi, and possibly Citrobacter fraeungi P079F I; v. A nucleotide sequence encoding a second heteroglycosyltransferase, which is eukaryotic and capable of covalently bonding mannose molecules to β-1,2-mannan polymers and extending β-1,2-mannan polymer chains, and in some cases Bmt3 of Candida albicans; and vi. A translocase that can, in some cases, translocate β-1,3-glucan to the periplasmic side of the inner membrane of prokaryotic host cells, and which encodes a translocase containing Wzx derived from Citrobacter, and in some cases from Citrobacter fraeungi P079F I. The step includes introducing and expressing it in host cells, The present invention provides a method in which a β-1,2-mannan polymer contains at least five consecutive β-1,2-linked mannose molecules.

[0221] In certain embodiments, the β-1,2-mannan polymer is a fungal β-1,2-mannan polymer. In some embodiments, the fungal β-1,2-mannan polymer is derived from Candida. In certain embodiments, the fungal β-1,2-mannan polymer is derived from Candida albicans. In certain embodiments, the β-1,2-mannan polymer has the following structure: [ka] It holds.

[0222] In further embodiments, the β-1,2-mannan polymer has the following structure: [ka] It has.

[0223] In some embodiments, the prokaryotic host cell is a bacterial cell. In some embodiments, the bacteria are Gram-positive bacteria. In other embodiments, the bacteria are Gram-negative bacteria. In certain embodiments, the bacteria include, but are not limited to, Escherichia species, Sigella species, Klebsiella species, Xanthomonas species, Salmonella species, Yersinia species, Lactococcus species, Lactobacillus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Staphylococcus species, Bacillus species, and Clostridium species. In some embodiments, the bacteria are Escherichia coli species. In certain embodiments, the bacteria are Escherichia coli. In certain embodiments, the prokaryotic host cell may include, but is not limited to, Escherichia, Sigella, Klebsiella, Xanthomonas, Salmonella, Yersinia, Lactococcus, Lactobacillus, Pseudomonas, Corynebacterium, Streptomyces, Streptococcus, Staphylococcus, Bacillus, and Clostridium. In some embodiments, the prokaryotic host cell is Escherichia coli. In certain embodiments, the prokaryotic host cell is Escherichia coli. In certain embodiments, GlcNAc is linked to the lipid carrier by WecA derived from the host Escherichia coli cell. In certain embodiments, the lipid carrier is undecaprenyl.

[0224] Bioconjugate In certain embodiments, the present invention provides a bioconjugate comprising a modified Sap2 protein of the present invention, linked to an antigen described herein. In certain embodiments, the antigen is linked to an amino acid (e.g., asparagine) on a modified Sap2 protein selected from asparagine, aspartic acid, glutamic acid, lysine, cysteine, tyrosine, histidine, arginine, or tryptophan. The bioconjugate described herein has advantages over antigen-carrier protein chemical conjugates in that it requires fewer chemicals in production and is more consistent with respect to the final product produced.

[0225] In certain embodiments, the present invention provides a method for producing a bioconjugate comprising a modified Sap2 protein of the present invention, linked to at least one sugar antigen, the method comprising: (1) culturing host cells of the present invention under conditions suitable for protein production; and (2) isolating the bioconjugate produced by the host cells of the present invention. In some embodiments, the bioconjugate is isolated or purified from a whole cell extract of the host cells. In other embodiments, the bioconjugate is isolated or purified from a cytoplasmic extract derived from the host cells. In certain embodiments, the bioconjugate is isolated or purified from a periplasmic extract of the host cells.

[0226] Methods for preparing bioconjugates are well known in the industry. For example, the bioconjugate of the present invention can be prepared using a shaking flask process, for example, in an LB shaking flask. In certain embodiments, the bioconjugate of the present invention can be produced using a fed-batch process for the production of recombinant glycosylated proteins in bacteria. The objective is to increase the glycosylation efficiency and recombinant protein yield per cell while maintaining simplicity and reproducibility in the process. The bioconjugate of the present invention can be produced on a commercial scale by developing an optimized production method using a typical E. coli production process. Various types of supply strategies, such as batch, chemostat, and fed-batch, can be used.

[0227] Thus, in certain embodiments, the present invention provides a bioconjugate produced by the method of the present invention. In certain embodiments, the bioconjugate of the present invention comprises at least one sugar antigen linked to the modified Sap2 protein of the present invention.

[0228] The bioconjugates of the present invention can be purified, for example, by chromatography (e.g., ion exchange, anion exchange, affinity, and sizing column chromatography), centrifugation, fractional lysis, or by any other standard technique for protein purification. See, for example, Saraswat et al., 2013, Biomed. Res. Int. ID#312709 (p. 1-18); also see the method described in WO 2009 / 104074. Furthermore, the bioconjugates of the present invention can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0229] In a further embodiment, the present invention provides a method for producing a glycoconjugate comprising a modified carrier protein and at least one sugar antigen. As used herein, the term "glycoconjugate" refers to a hybrid molecule composed of a carrier protein and multiple polysaccharide chains, in which the polysaccharide is covalently linked to the carrier protein.

[0230] Thus, in certain embodiments, the present invention provides a method for producing a glycoconjugate comprising a modified carrier protein and β-1,2 mannan, comprising: i) culturing the host cell of the present invention under conditions suitable for protein production; ii) harvesting the culture to produce the harvested culture; and iii) isolating the glycoconjugate from the culture. In certain aspects, the conditions suitable for the production of the glycoconjugate include the addition of GDP-mannose to the culture medium. In certain aspects, GDP-mannose is added to the harvested culture.

[0231] In certain aspects, the modified carrier proteins of the present invention include, but are not limited to, Sap2, Als3, detoxified Pseudomonas aeruginosa exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus, clumping factor A of Staphylococcus aureus, clumping factor B of Staphylococcus aureus, Escherichia coli FimH, Escherichia coli FimHC, Escherichia coli heat-labile enterotoxin, detoxified variant of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, Escherichia coli sat protein, passenger domain of Escherichia coli sat protein, Campylobacter jejuni AcrA, and Campylobacter jejuni native sugar protein. In certain aspects, the modified carrier protein is the modified Sap2 protein of the present invention.

[0232] In certain embodiments, at least one sugar antigen comprises a β-1,2-mannan polymer. In some embodiments, the β-1,2-mannan polymer comprises at least two β-1,2-linked mannose molecules. In further embodiments, the β-1,2-mannan polymer comprises 2-50, 2-40, 2-30, 2-20, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 2, 3-50, 3-40, 3-30, 3-20, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 3, 4-50, 4-40, 4-30, 4-20, 4-10, 4- The polymer contains 9, 4-8, 4-7, 4-6, 4-5, 4, 5-50, 5-40, 5-30, 5-20, 5-10, 5-9, 5-8, 5-7, 5-6, or 5, 6-40, 6-30, 6-20, 6-10, 6-9, 6-8, 6-7, 6, 7-50, 7-40, 7-30, 7-20, 7-10, 7-9, 7-8, or 7 1,2-linked mannose molecules. In some embodiments, the β-1,2-mannan polymer contains at least 2, at least 3, at least 4, or at least 5 consecutive β-1,2-linked mannose molecules.

[0233] In certain embodiments, the present invention provides a method for producing a glycoconjugate containing a modified carrier protein and β-1,2-mannan. In some embodiments, the method for producing a glycoconjugate containing a modified carrier protein and β-1,2-mannan includes the step of culturing host cells of the present invention under conditions suitable for protein production.

[0234] In further embodiments, the present invention relates to a method for producing a bioconjugate in a host cell, a. The step of obtaining host cells of the present invention that produce β-1,2-mannan polymer; and A modified carrier protein comprising at least one glycosylation site containing the bi consensus sequence D / EXNZS / T (wherein X and Z are any amino acids other than proline), further comprising an N-terminal bacterial signal sequence capable of transporting the modified carrier protein to the periplasmic side of the inner membrane of a host cell, and a nucleotide sequence encoding the modified carrier protein; and ii. Nucleotide sequences encoding oligosaccharide transferases that can produce bioconjugates by transferring β-1,2-mannan polymers from lipid carriers to modified carrier proteins. The present invention provides a method that includes the step of further introducing and expressing the agent in host cells.

[0235] In certain embodiments, the present invention relates to a method for producing a bioconjugate in a prokaryotic host cell, a. The step of obtaining prokaryotic host cells of the present invention that produce β-1,2-mannan polymer; and A modified carrier protein comprising a glycosylation site containing the consensus sequence D / EXNZS / T (wherein X and Z are any amino acids other than proline), further comprising a nucleotide sequence encoding the modified carrier protein, the modified carrier protein comprising an N-terminal bacterial signal sequence capable of transporting the modified carrier protein to the periplasmic side of the inner membrane of a prokaryotic host cell; and ii. An oligosaccharide transferase capable of producing a bioconjugate by transferring a β-1,2-mannan polymer from a lipid carrier to a modified carrier protein, wherein the nucleotide sequence encoding the oligosaccharide transferase is PglB derived from Campylobacter, and in some cases from Campylobacter jejuni or Campylobacter coli. The present invention provides a method that includes the step of further introducing and expressing the substance in host cells.

[0236] In another embodiment, the present invention relates to a method for producing a bioconjugate in a prokaryotic host cell, a. The step of obtaining prokaryotic host cells of the present invention that produce β-1,2-mannan polymer; and A modified carrier protein comprising a glycosylation site containing the consensus sequence D / EXNZS / T (wherein X and Z are any amino acids other than proline) (optionally the polynucleotide sequence of the present invention), further comprising an N-terminal bacterial signal sequence capable of transporting the modified carrier protein to the periplasmic side of the inner membrane of a prokaryotic host cell, and a nucleotide sequence encoding the modified carrier protein; and ii. An oligosaccharide transferase capable of producing a bioconjugate by transferring a β-1,2-mannan polymer from a lipid carrier to a modified carrier protein, wherein the nucleotide sequence encoding the oligosaccharide transferase is PglB derived from Campylobacter, and in some cases from Campylobacter jejuni or Campylobacter coli. The present invention provides a method that includes the step of further introducing and expressing the substance in host cells.

[0237] In certain embodiments, the modified carrier protein of the present invention may include, but is not limited to, Sap2, Als3, detoxified exotoxin A (EPA) of Pseudomonas aeruginosa, CRM197, diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus, clamping factor A of Staphylococcus aureus, clamping factor B of Staphylococcus aureus, FimH of Escherichia coli, FimHC of Escherichia coli, heat-unstable enterotoxin of Escherichia coli, detoxified variant of heat-unstable enterotoxin of Escherichia coli, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, sat protein of Escherichia coli, passenger domain of sat protein of Escherichia coli, Campylobacter jejuni AcrA, and Campylobacter jejuni natural glycoprotein. In certain embodiments, the modified carrier protein is the modified Sap2 protein of the present invention.

[0238] In certain embodiments, the bacterial signal sequence is selected from, but is not limited to, FlgI, MalE, OmpA, and OmpC. In certain embodiments, the bacterial signal sequence is FlgI. In certain embodiments, FlgI comprises the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the bacterial signal sequence is removed from the modified carrier protein after the modified carrier protein has been transported to the periplasmic side of the inner membrane of the prokaryotic host cell.

[0239] Analysis method By using various methods, the structural composition and sugar chain length of the bioconjugate of the present invention can be analyzed to determine the use of glycosylation sites.

[0240] Glycans can be analyzed using hydrazinolysis. First, polysaccharides are released from their protein carrier by incubation with hydrazine according to the manufacturer's instructions (Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK). The nucleophilic hydrazine attacks the glycosidic bond between the polysaccharide and the carrier protein, causing the bound glycan to be released. The N-acetyl group is lost during this process and needs to be reconstituted by re-N-acetylation. The free glycan is purified on a carbon column and subsequently labeled at the reducing end with fluorophore 2-aminobenzamide. See Bigge JC, Patel TP, Bruce JA, Goulding PN, Charles SM, Parekh RB, Anal Biochem 1995, 230(2):229-238. The labeled polysaccharide is separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al. See Royle L, Mattu TS, Hart E, Langridge JI, Merry AH, Murphy N, Harvey DJ, Dwek RA, Rudd PM, Anal Biochem 2002, 304(1):70-90. The resulting fluorescence chromatogram shows the length of the polysaccharide and the number of repeating units. Structural information can be obtained by collecting individual peaks and performing MS / MS analysis. This allows for confirmation of the monosaccharide composition and sequence of the repeating units, and further identification of the homogeneity of the polysaccharide composition. Alternatively, high-mass MS and size exclusion HPLC can be applied to measure the size of the complete bioconjugate.

[0241] Yield can be measured as the amount of carbohydrates derived from 1 liter of bacterial production culture grown in a bioreactor under controlled and optimized conditions. After purification of the bioconjugate, carbohydrate yield can be measured directly by an anthron assay or ELISA using carbohydrate-specific antiserum. Indirect measurement is possible by using protein weight (measured by BCA, Lowry, or Bradford assay) as well as glycan length and structure to calculate the theoretical value of carbohydrates per gram of protein. Furthermore, yield can also be measured by drying the glycoprotein preparation from a volatile buffer and using a balance to measure its weight.

[0242] For example, the conjugates of the present invention can be analyzed using various methods, including SDS-PAGE or capillary gel electrophoresis. The polymer length is defined by the number of linearly assembled repeating units. This means that a typical ladder-like pattern is the result of different numbers of repeating units constituting the glycan. Thus, two adjacent bands in SDS-PAGE (or other techniques for size separation) differ by only a single repeating unit. These individual differences are utilized when analyzing glycoproteins for glycan size: non-glycosylated carrier proteins and bioconjugates with different polymer chain lengths are separated according to their electrophoretic mobility. The first detectable number of repeating units (n1) and the average number of repeating units (n1) present on the bioconjugate are determined. average These parameters are measured. These parameters can be used to demonstrate, for example, batch-to-batch consistency or polysaccharide stability.

[0243] For example, glycosylation site utilization can be quantified by LC-MS / MS of glycopeptides: the conjugate is digested with a protease, peptides are separated by a suitable chromatography method (C18, hydrophilic interaction HPLC, HILIC, GlycoSepN column, SE HPLC, AE HPLC), and various peptides are identified using MS / MS. This method can be used with or without prior shortening of the glycosylation chain by chemical (Smith degradation) or enzymatic methods. Quantification of glycopeptide peaks using UV detection at 215-280 nm allows for relative determination of glycosylation site utilization. In other embodiments, site utilization can be quantified by size exclusion HPLC: higher glycosylation site utilization is reflected by a faster elution time from the SE HPLC column. In yet another embodiment, site utilization can be quantified by quantitative density measurement of purified bioconjugates stained with Coomassie brilliant blue after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDA-PAGE).

[0244] Immunogenic compositions and vaccines The conjugate of the present invention (e.g., bioconjugate) is particularly suitable for inclusion in immunogenic compositions and vaccines.

[0245] Thus, in certain embodiments, the present invention provides an immunogenic composition comprising the conjugate or bioconjugate of the present invention. In certain embodiments, the immunogenic composition further comprises pharmaceutically acceptable excipients and / or carriers. In certain embodiments, the present invention provides an immunogenic composition comprising the modified Sap2 protein of the present invention, the conjugate of the present invention, or the bioconjugate of the present invention. In other embodiments, the immunogenic composition further comprises pharmaceutically acceptable excipients and / or carriers.

[0246] The immunogenic composition comprises an immunologically effective amount of the modified Sap2 protein of the present invention, or the conjugate of the present invention (e.g., a bioconjugate), and any other optional components. “Immunologically effective amount” means that the amount administered to an individual, either as a single dose or as part of a series of doses, is effective for treatment or prevention. This amount varies depending on the health and physical condition of the individual to be treated, age, desired level of protection, vaccine formulation, and other relevant factors.

[0247] Pharmaceutically acceptable excipients and carriers are described, for example, in Remington's Pharmaceutical Sciences, EW Martin, Mack Publishing Co., Easton, PA, 5th Edition (1975). Pharmaceutically acceptable excipients may include buffers such as phosphate buffer (e.g., sodium phosphate). Pharmaceutically acceptable excipients may include salts, such as sodium chloride. Pharmaceutically acceptable excipients may include solubilizers / stabilizers, such as polysorbates (e.g., TWEEN80®). Pharmaceutically acceptable excipients may include preservatives, such as 2-phenoxyethanol or thimerosal. Pharmaceutically acceptable excipients may include carriers such as water or saline solution.

[0248] In further embodiments, the present invention provides a method for preparing an immunogenic composition of the present invention, comprising the step of mixing the modified Sap2 protein of the present invention, the conjugate of the present invention, or the bioconjugate of the present invention with a pharmaceutically acceptable excipient or carrier.

[0249] Preferably, the immunogenic compositions of the present invention are formulated as vaccines for in vivo administration to a subject (e.g., human), such that the individual components of the composition are formulated such that the immunogenicity of the individual components is not impaired by the other individual components of the composition (see definition above). Thus, in some embodiments, the combination does not produce (significantly) harmful effects on the sugar antigen compared to its administration alone (with respect to protective efficacy). Preferably, the immunogenic compositions of the present invention are formulated as vaccines for in vivo administration to a subject (e.g., human), such that they result in antibody titers superior to the criteria for serum protection for each antigen component for an acceptable percentage of human subjects.

[0250] Thus, in further embodiments, the present invention provides a vaccine comprising the immunogenic composition of the present invention. In certain embodiments, the vaccine further comprises a pharmaceutically acceptable excipient or carrier. In other embodiments, the vaccine further comprises an adjuvant. In certain embodiments, the present invention provides a vaccine comprising the immunogenic composition of the present invention, optionally a pharmaceutically acceptable excipient or carrier.

[0251] In certain embodiments, the present invention provides a Candida albicans vaccine comprising (1) a modified Sap2 protein of the present invention; (2) at least one Candida albicans glycoantigen linked to the modified Sap2 protein; and optionally (3) a pharmaceutically acceptable carrier or adjuvant.

[0252] The term "adjuvant" refers to a compound that, when administered together with or as part of the immunogenic composition of the vaccine of the present invention, increases, enhances, and / or boosts the immune response to the modified Sap2 protein conjugate / bioconjugate, but does not generate an immune response to the modified Sap2 protein conjugate / bioconjugate when administered alone. Adjuvants can enhance the immune response through several mechanisms, including, for example, lymphocyte recruitment, B and / or T cell stimulation, and macrophage stimulation. Specific examples of adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3-deoxyacylated monophosphoryl lipid A (MPL) (UK Patent GB2220211), MF59 (Novartis), AS01 (GlaxoSmithKline), and saponins such as QS21 (see Kensil et al., Vaccine Design: The Subunit and Adjuvant Approach (Powell & Newman (eds.), Plenum Press, NY, 1995); U.S. Patent No. 5,057,540). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants include oil-in-water emulsions (such as squalene or peanut oil) combined with immunostimulants, such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)). Preferred adjuvants are those comprising an oil-in-water emulsion containing a metabolizable oil, tocopherol, and an emulsifier. Preferably, the metabolizable oil is squalene, the tocopherol is alpha-tocopherol, and the emulsifier is polyoxyethylene sorbitan monooleate.

[0253] In some embodiments, the adjuvant comprises an oil-in-water emulsion. For example, the oil-in-water emulsion may contain an oil phase comprising a metabolizable oil and further oil phase components such as tocol. The oil-in-water emulsion may also contain an aqueous component such as a buffered saline solution (e.g., phosphate-buffered saline). Furthermore, the oil-in-water emulsion typically contains an emulsifier. In some embodiments, the metabolizable oil is squalene. In certain embodiments, tocol is alpha-tocopherol. In some embodiments, the emulsifier is a nonionic surfactant emulsifier (e.g., polyoxyethylene sorbitan monooleate, TWEEN80®). In exemplary embodiments, the oil-in-water emulsion contains squalene and alpha-tocopherol in a ratio equal to or less than 1 (w / w).

[0254] In certain embodiments, the metabolic oil in the oil-in-water emulsion may be present in an amount of 0.5 to 10 mg. In some embodiments, the tocol in the oil-in-water emulsion may be present in an amount of 0.5 to 11 mg. In some embodiments, the emulsifier may be present in an amount of 0.4 to 4 mg.

[0255] For an oil-in-water composition to be suitable for administration to humans, the oil phase of the emulsion must contain a metabolizable oil. The meaning of the term metabolizable oil is well known in the art. Metabolizable can be defined as "can be converted by metabolism" (Dorland's Illustrated Medical Dictionary, WB Sanders Company, 25th edition (1974)). The oil may be any vegetable oil, fish oil, animal oil, or synthetic oil that is not toxic to the recipient and can be converted by metabolism. Nuts, seeds, and cereals are common sources of vegetable oils. Synthetic oils are also part of the present invention and may include commercially available oils such as NEOBEE® (caprylic / capric triglyceride made using glycerol derived from a vegetable oil source and medium-chain fatty acids (MCTs) derived from coconut or palm kernel oil) and others. A particularly preferred metabolizable oil is squalene. Squalene (2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene) is an unsaturated oil found in large quantities in shark liver oil and in small quantities in olive oil, wheat germ oil, rice bran oil, and yeast, and is a particularly preferred oil for use in the present invention. Squalene is a metabolizable oil due to the fact that it is an intermediate in the biosynthesis of cholesterol (Merck index, 10th edition, registration number 8619).

[0256] Tocol is well known in the industry and is described in EP0382271. Preferably, tocol is alpha-tocopherol or a derivative thereof, such as alpha-tocopherol succinate (also known as vitamin E succinate). In certain embodiments, the tocol is preferably present in an amount of 0.5 to 11 mg.

[0257] The oil-in-water emulsion further comprises an emulsifier. The emulsifier may preferably be polyoxyethylene sorbitan monooleate. In certain embodiments, the emulsifier may be polysorbate® 80 (polyoxyethylene (20) sorbitan monooleate) or Tween® 80. In certain embodiments, the emulsifier is preferably present in the adjuvant composition in an amount of 0.4 to 4 mg.

[0258] Furthermore, a method for preparing an immunogenic composition of the present invention is also provided, comprising the step of mixing the modified Sap2 protein or conjugate (e.g., bioconjugate) of the present invention with a pharmaceutically acceptable excipient and / or carrier and adjuvant. Vaccine preparations are generally described in Vaccine Design ("The subunit and adjuvant approach" (Powell MF & Newman MJ (eds.)) (1995) Plenum Press New York).

[0259] The immunogenic composition of the present invention may be contained in a container, pack, or dispenser, along with instructions for administration. The immunogenic composition or vaccine of the present invention may be stored before use, for example, by freezing the composition (e.g., at about -20°C or about -70°C); storing under refrigerated conditions (e.g., about 4°C); or storing at room temperature. The immunogenic composition or vaccine of the present 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 further embodiments, the vaccine of the present invention is lyophilized and reconstituted improvisationally before use.

[0260] Dosage and dosage An immunogenic composition or vaccine of the present invention can be used to protect or treat a subject (e.g., a human) by administering the immunogenic composition or vaccine systemically or via a mucosal route. These administrations may include injection via intramuscular (IM), intraperitoneal, intradermal (ID), or subcutaneous (SC) routes; or via mucosal administration to the oral / gastrointestinal tract, respiratory tract, or genitourinary tract.

[0261] In certain embodiments, the immunogenic composition or vaccine of the present invention is administered by an intramuscular delivery route. Intramuscular administration may be to the thigh or upper arm. The injection is typically by a needle (e.g., a subcutaneous injection needle), but needleless injection may be used as an alternative. A typical intramuscular dose is 0.5 ml.

[0262] In another embodiment, the immunogenic composition or vaccine of the present invention is administered by intradermal administration. Human skin includes an outer "hard" cuticle called the stratum corneum that covers the epidermis. Below this epidermis is a layer called the dermis, which then covers the subcutaneous tissue. The conventional technique of intradermal injection, the "Manto procedure," involves disinfecting the skin, extending one hand, and inserting a narrow-gauge needle (26-31 gauge) with the chamfered tip facing upward at an angle of 10-15°. Once the chamfered tip of the needle is inserted, the barrel of the needle is lowered and advanced further while applying slight pressure to raise it under the skin. The needle is then slowly withdrawn after a blister or bump has formed on the skin surface by very slowly injecting the liquid.

[0263] In a further embodiment, the immunogenic composition or vaccine of the present invention is administered by intranasal administration. Typically, the immunogenic composition or vaccine is administered locally to the nasopharynx region, for example, without inhalation into the lungs. It is desirable to use an intranasal delivery device that delivers the immunogenic composition or vaccine formulation to the nasopharynx region without allowing it to enter the lungs, or substantially without it entering them. A suitable device for intranasal administration of the vaccine according to the present invention is a spray device. A suitable commercially available nasal spray device is ACCUSPRAY® (Becton Dickinson).

[0264] The amount of conjugate (e.g., bioconjugate) in each immunogenic composition or vaccine dose is selected as an amount that induces an immunoprotective response without significant and adverse side effects in a typical vaccine. Such an amount will vary depending on the specific immunogen used and how it is delivered. The conjugate (e.g., bioconjugate) content is typically in the range of 1 to 100 μg, preferably 5 to 50 μg, with respect to the sugar dose, e.g., the mannan dose.

[0265] Preventive and therapeutic use The present invention also provides immunogenic compositions or vaccines of the present invention for use in pharmaceuticals. The use of immunogenic compositions of the present invention in the manufacture of pharmaceuticals for the treatment or prevention of diseases caused by Candida albicans infection is also intended, such as a method for immunizing a subject (e.g., a human) with a disease caused by Candida albicans, comprising the step of administering to the subject an immunoprotective dose of the immunogenic composition of the present invention.

[0266] In certain embodiments, the present invention provides a method for inducing an immune response to a fungal infection in a subject (e.g., a human), comprising the step of administering to the subject a therapeutic or prophylactic effective amount of the modified Sap2 protein of the present invention, the conjugate of the present invention, the bioconjugate of the present invention, the immunogenic composition of the present invention, or the vaccine of the present invention. In certain embodiments, an immune response to a fungus can be induced using the modified Sap2 protein of the present invention, the conjugate of the present invention, the bioconjugate of the present invention, the immunogenic composition of the present invention, or the vaccine of the present invention. In certain embodiments, the fungus is a Candida species. In some embodiments, Candida species include, but are not limited to, Candida albicans, Candida auris, Candida gilliermondi, Candida lucitanie and Candida tropicalis, Candida glabrata, Candida crusei and Candida parapsis. In certain embodiments, the Candida species is Candida albicans. In certain embodiments, the subject has a fungal infection at the time of administration. In other embodiments, the subject does not have a fungal infection at the time of administration.

[0267] In certain embodiments, the present invention provides a method for inducing an immune response to Candida albicans infection in a subject (e.g., a human), comprising the step of administering to the subject a therapeutic or prophylactic effective amount of the modified Sap2 protein of the present invention, the conjugate of the present invention, the bioconjugate of the present invention, the immunogenic composition of the present invention, or the vaccine of the present invention.

[0268] The present invention also provides a method for treating and / or preventing a fungal infection in a subject, comprising the step of administering to the subject a conjugate (e.g., a bioconjugate) of the present invention. The conjugate (e.g., a bioconjugate) may be in the form of an immunogenic composition or a vaccine. Thus, in certain embodiments, the present invention provides a method for treating or preventing a fungal infection in a subject requiring treatment or prevention of a fungal infection, comprising the step of administering to the subject a therapeutically effective dose of the modified Sap2 protein of the present invention, the conjugate of the present invention, the bioconjugate of the present invention, the immunogenic composition of the present invention, or the vaccine of the present invention. The conjugate (e.g., a bioconjugate) may be in the form of an immunogenic composition or a vaccine. In other embodiments, the present invention provides a method for immunizing a subject against a fungal infection, comprising the step of administering to the subject an immunoprotective dose of the modified Sap2 protein of the present invention, the conjugate of the present invention, the bioconjugate of the present invention, the immunogenic composition of the present invention, or the vaccine of the present invention. In further embodiments, the present invention provides modified Sap2 proteins, conjugates, bioconjugates, immunogenic compositions, or vaccines of the present invention for use in the manufacture of pharmaceuticals for the treatment or prevention of diseases caused by fungal infections in subjects (e.g., humans). In certain embodiments, the fungus is a Candida species. In some embodiments, Candida species include, but are not limited to, Candida albicans, Candida auris, Candida gilliermondi, Candida lucitani, and Candida tropicalis, Candida glabrata, Candida crusei, and Candida parapsis. In certain embodiments, the Candida species is Candida albicans.

[0269] Thus, in certain embodiments, the present invention provides a method for treating or preventing Candida albicans infection in a subject requiring treatment or prevention of Candida albicans infection, the method comprising the step of administering to the subject a therapeutically effective amount of the modified Sap2 protein of the present invention, the conjugate of the present invention, the bioconjugate of the present invention, the immunogenic composition of the present invention, or the vaccine of the present invention.

[0270] In other specific embodiments, the present invention provides a method for immunizing a subject against Candida albicans infection, comprising the step of administering to the subject an immunoprotective dose of the modified Sap2 protein of the present invention, the conjugate of the present invention, the bioconjugate of the present invention, the immunogenic composition of the present invention, or the vaccine of the present invention.

[0271] In certain embodiments, the present invention provides modified Sap2 proteins, conjugates, bioconjugates, immunogenic compositions, or vaccines for use in the manufacture of pharmaceuticals for the treatment or prevention of diseases caused by Candida albicans infection in subjects (e.g., humans).

[0272] In other embodiments, the present invention provides a method for inhibiting the attachment of Candida albicans hyphae to vaginal epithelial cells in a subject (e.g., a human), comprising the step of administering to the subject a therapeutic or prophylactic effective amount of the modified Sap2 protein of the present invention, the conjugate of the present invention, the bioconjugate of the present invention, the immunogenic composition of the present invention, or the vaccine of the present invention.

[0273] In further embodiments, the present invention provides a method for mediating neutrophil killing of Candida albicans hyphae in a subject (e.g., a human), comprising the step of administering to the subject a therapeutic or prophylactic effective amount of the modified Sap2 protein of the present invention, the conjugate of the present invention, the bioconjugate of the present invention, the immunogenic composition of the present invention, or the vaccine of the present invention.

[0274] Embodiments of the present invention are further described in the following numbered paragraphs: 1. A modified secreted aspartyl proteinase 2 (Sap2) protein that contains an amino acid sequence identical to amino acid residues 19-398 of SEQ ID NO: 1, or an amino acid sequence identical to amino acid residues 19-398 of SEQ ID NO: 1 by at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%, and is modified in that the amino acid sequence contains one or more consensus sequences that include the amino acid sequence D / EXNZS / T (wherein X and Z are independently any amino acid other than proline).

[0275] 2. A modified Sap2 protein as described in paragraph 1, further comprising a substitution at amino acid residue 274 between amino acid residues 19-398 of SEQ ID NO: 1, or at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0276] 3. A modified Sap2 protein as described in paragraph 2, comprising a substitution from aspartic acid (D) to asparagine (N) at amino acid residue 274 between amino acid residues 19-398 of SEQ ID NO: 1, or at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0277] 4. A modified Sap2 protein as described in either paragraph 2 or 3, wherein the substitution inactivates the Sap2 protein.

[0278] 5.1 or more consensus sequences include one or more amino acids between amino acid residues 19-24 of amino acid residues 19-398 of SEQ ID NO: 1, one or more amino acids between amino acid residues 52-62, one or more amino acids between amino acid residues 93-107, one or more amino acids between amino acid residues 104-118, one or more amino acids between amino acid residues 142-144, one or more amino acids between amino acid residues 187-197, one or more amino acids between amino acid residues 215-225, one or more amino acids between amino acid residues 243-253, and one or more amino acids between amino acid residues 255-265. A modified Sap2 protein as described in any of paragraphs 1 to 4, wherein one or more amino acids selected from the group consisting of one or more amino acids between amino acid residues 263 to 273, one or more amino acids between amino acid residues 320 to 330, one or more amino acids between amino acid residues 339 to 349, and one or more amino acids between amino acid residues 358 to 359, or is added next to or replaces an equivalent position in an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19 to 398 of SEQ ID NO: 1.

[0279] 6. A modified Sap2 protein as described in any of paragraphs 1 to 5, wherein one or more consensus sequences are added next to or replace amino acid residue 19 of amino acid residues 19 to 398 of SEQ ID NO: 1 at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19 to 398 of SEQ ID NO: 1.

[0280] 7. A modified Sap2 protein as described in any of paragraphs 1-6, wherein one or more consensus sequences are added next to or replace amino acid residue 57 between amino acid residues 19-398 of SEQ ID NO: 1 or amino acid residues 19-398 of SEQ ID NO: 1 at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0281] 8. A modified Sap2 protein as described in any of paragraphs 1-7, wherein one or more consensus sequences are added next to or replace one or more amino acids between amino acid residues 98-102 of amino acid residues 19-398 of SEQ ID NO: 1, or at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0282] 9. A modified Sap2 protein as described in any of paragraphs 1-8, wherein one or more consensus sequences are added next to or replace one or more amino acids between amino acid residues 109-113 of amino acid residues 19-398 of SEQ ID NO: 1, or at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0283] 10. A modified Sap2 protein as described in any of paragraphs 1-9, wherein one or more consensus sequences are added next to or replace amino acid residues 220 between amino acid residues 19-398 of SEQ ID NO: 1, or at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0284] 11. A modified Sap2 protein derived from Candida, as described in any of paragraphs 1-10.

[0285] 12. A modified Sap2 protein as described in paragraph 11, wherein the Candida is selected from the group consisting of Candida albicans, Candida auris, Candida gilliermondi, Candida lusitanie, Candida tropicalis, Candida glabrata, Candida crusei, and Candida parapsis.

[0286] 13. A modified Sap2 protein derived from Candida albicans, as described in either paragraph 11 or 12.

[0287] 14. A modified Sap2 protein as described in any of paragraphs 1 to 13, further comprising at least one fructose diphosphate aldolase-1 (Fba) peptide having an amino acid sequence identical to YGKDVKDLFDYAQE (SEQ ID NO: 3) or an amino acid sequence identical to SEQ ID NO: 3 by at least 70%, 80%, 85%, 90%, or 92%.

[0288] 15. The modified Sap2 protein described in 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 that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1.

[0289] 16. A modified Sap2 protein described in any of paragraphs 1 to 15, wherein one or more consensus sequences are selected from the group consisting of DQNAT (SEQ ID NO: 4) and DQNVT (SEQ ID NO: 5).

[0290] 17. A modified Sap2 protein according to any of paragraphs 14-16, wherein the modified Sap2 protein comprises at least one further consensus sequence comprising the amino acid sequence JUBD / EXNZS / TJUB (wherein X and Z are independently any amino acid other than proline, and J, U and B independently comprise 1 to 5 naturally occurring amino acid residues), and at least one consensus sequence is appended next to the C-terminal amino acid residue of SEQ ID NO: 3 or at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3.

[0291] 18. A modified Sap2 protein as described in paragraph 17, wherein X is Q, Z is A, J and B each contain 1 to 5 glycine (G) residues, and U contains 1 to 5 serine (S) residues.

[0292] 19. A modified Sap2 protein as described in either paragraph 17 or 18, wherein the additional consensus sequence includes the amino acid sequence GSGGGDQNATGSGGG (SEQ ID NO: 7).

[0293] 20. A modified Sap2 protein described in any of paragraphs 17-19, wherein the additional consensus sequence includes the amino acid sequence GSGGGDQNATGSGGGHHHHHHHHHH (Sequence ID 8).

[0294] 21. A modified Sap2 protein described in any of paragraphs 1-20, containing the amino acid sequence of Sequence ID No. 9.

[0295] 22. A modified Sap2 protein containing the amino acid sequence of Sequence ID No. 10.

[0296] 23. A modified Sap2 protein described in any of paragraphs 1 to 22, which is glycosylated.

[0297] 24. N-glycosylated modified Sap2 protein as described in any of paragraphs 1-23.

[0298] 25. A conjugate comprising a modified Sap2 protein described in any of paragraphs 1 to 22 and at least one sugar antigen.

[0299] 26. The conjugate according to paragraph 25, wherein a modified Sap2 protein is covalently linked to at least one sugar antigen.

[0300] 27. At least one sugar antigen is present in the O antigen of Citrobacter frewngii, extracellular polysaccharide of Agrobacterium species, O antigen of Escherichia coli, O antigen of Salmonella species, O antigen of Pseudomonas species, O antigen of Klebsiella species, O antigen of Acinetobacter, O antigen of Chlamydia trachomatis, O antigen of Vibrio cholerae, O antigen of Listeria species, O antigen of Legionella pneumophila serotypes 1-15, O antigen of Bordetella parapertussis, O antigen of Burgholderia malei and Pseudomalei, O antigen of Francisella tularensis, O antigen of Campylobacter species, capsular polysaccharide of Clostridium difficile, Staphylococcus aureus types 5 and 8, and Streptococcus piogeoge. Conjugates as described in either paragraphs 25 and 26, selected from the group consisting of Ness, Escherichia coli, Streptococcus agalactia, Neisseria meningitidis, Candida species, Candida albicans, Haemophilus influenzae, Enterococcus faecalis, capsular polysaccharides I-V, and other surface polysaccharide structures, such as glycolipids of Borrelia burgdorferi, pyrine O-glycan and lipooligosaccharide (LOS) of Neisseria meningitidis, LOS of Haemophilus influenzae, lipophosphoglycan of Leishmania major, tumor-associated glycosylation antigens, glycosylphosphatidylinositol of malaria, and arabinomannan of Mycobacterium tubercosis.

[0301] 28. A conjugate according to either paragraph 25 or 26, wherein at least one sugar antigen is a fungal sugar antigen.

[0302] 29. A conjugate according to any one of paragraphs 25, 26, and 28, wherein at least one sugar antigen is a sugar antigen of a Candida species.

[0303] 30. The conjugate described in paragraph 29, in which the Candida species is selected from the group consisting of Candida albicans, Candida auris, Candida gilliermondii, Candida lusitanie, Candida tropicalis, Candida glabrata, Candida crusei, and Candida parapsis.

[0304] 31. A conjugate described in any of paragraphs 25-30, wherein at least one sugar antigen is a sugar antigen of Candida albicans.

[0305] 32. A conjugate according to any one of paragraphs 25 to 31, wherein at least one sugar antigen comprises a β-1,2-mannan polymer.

[0306] 33. The conjugate according to paragraph 32, wherein the β-1,2-mannan polymer contains at least two β-1,2-linked mannose molecules.

[0307] 34. Structure: [ka] The conjugate described in paragraph 25, having the following characteristics.

[0308] 35. At least one sugar antigen, structure: [ka] The conjugates described in any of paragraphs 32-34, including the one mentioned above.

[0309] 36. A conjugate according to any one of paragraphs 25 to 31, wherein at least one sugar antigen comprises a β-1,2-mannan polymer.

[0310] 37. The conjugate according to paragraph 36, wherein the β-1,2-mannan polymer contains at least two β-1,2-linked mannose molecules.

[0311] 38. β-1,2 mannan polymers are present in quantities of 2-50, 2-40, 2-30, 2-20, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 2, 3-50, 3-40, 3-30, 3-20, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 3, 4-50, 4-40, 4-30, 4-20, 4-10, 4-9, 4-8, 4-7, 4-6, and 4 The conjugate described in either paragraph 36 or 37, comprising 5, 4, 5-50, 5-40, 5-30, 5-20, 5-10, 5-9, 5-8, 5-7, 5-6, or 5, 6-40, 6-30, 6-20, 6-10, 6-9, 6-8, 6-7, 6, 7-50, 7-40, 7-30, 7-20, 7-10, 7-9, 7-8, or 7 β-1,2-linked mannose molecules.

[0312] 39. The conjugate according to any one of paragraphs 36 to 38, wherein the β-1,2 mannan polymer comprises at least two, at least three, at least four, or at least five β-1,3-linked glucose molecules, optionally at least five consecutive β-1,2-linked mannan molecules, and optionally five consecutive β-1,2-linked mannan molecules.

[0313] 40. A conjugate as described in any of paragraphs 25-29, wherein a modified Sap2 protein is linked to at least one sugar antigen at one or more amino acid residues on the modified Sap2 protein, and 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.

[0314] 41. A conjugate as described in any of paragraphs 25-40, wherein a modified Sap2 protein is linked to at least one sugar antigen at one or more asparagine residues on the modified Sap2 protein.

[0315] 42. A bioconjugate, as described in any of paragraphs 25-41.

[0316] 43. (1) The amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and (2) A modified Sap2 protein of Candida albicans comprising (or consisting of) at least one sugar antigen of Candida, which is a β-1,2 mannan polymer consisting of at least five consecutive β-1,2 linked mannose molecules, and which is linked to at least one of the four asparagine residues at positions 45, 94, 215, and 415 of SEQ ID NO: 9 or at least one of the four asparagine residues at positions 6, 55, 176, and 376 of SEQ ID NO: 10.

[0317] 44. A polynucleotide sequence encoding a modified Sap2 protein as described in any of paragraphs 1-22.

[0318] 45. A vector containing the polynucleotide sequence described in paragraph 44.

[0319] 46. ​​A host cell comprising (1) one or more polynucleotide sequences encoding one or more heterologous glycosyltransferases; (2) a polynucleotide sequence encoding a heterologous oligosaccharide transferase; (3) a polynucleotide sequence encoding a modified Sap2 protein as described in any of paragraphs 1 to 22; and optionally (4) a polynucleotide sequence encoding a polymerase.

[0320] 47. The host cell described in paragraph 46, in which the host cell is E. coli.

[0321] 48. A method for producing a bioconjugate comprising a modified Sap2 protein linked to at least one sugar, comprising: (1) culturing host cells described in either paragraph 46 or 47 under conditions suitable for protein production; and (2) isolating the bioconjugate produced by the host cells, optionally isolating the bioconjugate from a periplasmic extract derived from the host cells.

[0322] 49. A bioconjugate produced by the method described in paragraph 48, comprising at least one sugar linked to a modified Sap2 protein as described in any of paragraphs 1 to 22.

[0323] 50. An immunogenic composition comprising a modified Sap2 protein described in any of paragraphs 1 to 24, a conjugate described in any of paragraphs 25 to 41, or a bioconjugate described in any of paragraphs 42 and 49.

[0324] 51. A method for preparing the immunogenic composition described in paragraph 50, comprising the step of mixing a modified Sap2 protein described in any of paragraphs 1 to 24, a conjugate described in any of paragraphs 25 to 41, or a bioconjugate described in any of paragraphs 42 and 49 with a pharmaceutically acceptable excipient or carrier.

[0325] 52. A vaccine comprising the immunogenic composition described in paragraph 50 and optionally a pharmaceutically acceptable excipient or carrier.

[0326] 53. A Candida albicans vaccine comprising (1) a modified Sap2 protein as described in any of paragraphs 1 to 22; (2) at least one Candida albicans sugar linked to the modified Sap2 protein; and optionally (3) a pharmaceutically acceptable carrier or adjuvant.

[0327] 54. A method for treating or preventing Candida albicans infection in a subject requiring treatment or prevention of Candida albicans infection, comprising the step of administering to the subject a therapeutically effective amount of a modified Sap2 protein described in any of paragraphs 1 to 24 and 43, a conjugate described in any of paragraphs 25 to 41, a bioconjugate described in any of paragraphs 42 and 49, an immunogenic composition described in paragraph 50, or a vaccine described in any of paragraphs 52 and 53.

[0328] 55. The method described in paragraph 54, which shows that Candida albicans infection causes recurrent vulvovaginal candidiasis (RVVC) in a subject.

[0329] 56. A method for immunizing a subject against Candida albicans infection, comprising the step of administering to the subject an immunoprotective dose of a modified Sap2 protein described in any of paragraphs 1-24 and 43, a conjugate described in any of paragraphs 25-41, a bioconjugate described in any of paragraphs 42 and 49, an immunogenic composition described in paragraph 50, or a vaccine described in any of paragraphs 52 and 53.

[0330] 57. A method for inducing an immune response to Candida albicans infection in a subject, comprising the step of administering to the subject a therapeutic or prophylactic dose of a modified Sap2 protein described in any of paragraphs 1-24 and 43, a conjugate described in any of paragraphs 25-41, a bioconjugate described in any of paragraphs 42 and 49, an immunogenic composition described in paragraph 50, or a vaccine described in any of paragraphs 52 and 53.

[0331] 58. A method described in any of paragraphs 54-57, wherein the subject is a human.

[0332] 59. A modified Sap2 protein as described in any of paragraphs 1-24 and 43, a conjugate as described in any of paragraphs 25-41, a bioconjugate as described in any of paragraphs 42 and 49, an immunogenic composition as described in paragraph 50, or a vaccine as described in any of paragraphs 52 and 53, for use in the treatment or prevention of diseases caused by Candida albicans infection.

[0333] 60. A modified Sap2 protein as described in any of paragraphs 1-24 and 43, a conjugate as described in any of paragraphs 25-41, a bioconjugate as described in any of paragraphs 42 and 49, an immunogenic composition as described in paragraph 50, or a vaccine as described in any of paragraphs 52 and 53, for use in the manufacture of a pharmaceutical for the treatment or prevention of a disease caused by Candida albicans infection.

[0334] 61. Structure: [ka] A sugar that is a β-1,2-mannan polymer containing this polymer.

[0335] 62. Structure: [ka] The sugar described in paragraph 61, having the properties of the sugar.

[0336] 63. Structure: [ka] Sugars containing sugars.

[0337] 64. Structure: [ka] The sugars listed in paragraph 63, including the sugars mentioned above.

[0338] 65. Sugars described in paragraph 64, linked to a lipid carrier.

[0339] 66. The sugar described in paragraph 65, wherein the lipid carrier is undecaprenyl.

[0340] 67. A conjugate containing the sugar described in any one of paragraphs 61-64, linked to an asparagine residue of a modified carrier protein.

[0341] 68. The conjugate described in paragraph 67, wherein the asparagine residue is located within the consensus sequence D / EXNZS / T (wherein X and Z are independently any amino acid other than proline).

[0342] 69. A conjugate as described in either paragraph 67 or 68, wherein the modified carrier protein is selected from the group consisting of the modified Sap2 protein described in any one of paragraphs 1 to 24, Sap2, Als3, detoxified exotoxin A (EPA) of Pseudomonas aeruginosa, CRM197, diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus, clamping factor A of Staphylococcus aureus, clamping factor B of Staphylococcus aureus, FimH of Escherichia coli, FimHC of Escherichia coli, heat-unstable enterotoxin of Escherichia coli, detoxified variant of heat-unstable enterotoxin of Escherichia coli, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, sat protein of Escherichia coli, passenger domain of sat protein of Escherichia coli, Campylobacter jejuni AcrA, and Campylobacter jejuni natural glycoprotein.

[0343] 70. A conjugate described in any one of paragraphs 67-69, wherein the carrier protein is a modified Sap2 protein described in any one of paragraphs 1-24.

[0344] 71. A conjugate described in any one of paragraphs 67-70, wherein the conjugate is a bioconjugate.

[0345] 72.i. A nucleotide sequence encoding one or more first heterologous glycosyltransferases capable of synthesizing β-1,2-mannan polymers; ii. A nucleotide sequence encoding a second heterologous glycosyltransferase that is eukaryotic and capable of covalently bonding mannose molecules to β-1,2-mannan polymers, thereby extending the β-1,2-mannan polymer chain; iii. Nucleotide sequences encoding heterologous oligosaccharide transferases; and iv. A nucleotide sequence encoding a modified carrier protein that may include a glycosylation site containing the consensus sequence D / EXNZS / T (wherein X and Z are independently any amino acid other than proline). Host cells containing these cells.

[0346] Host cells as described in paragraph 72, comprising WbaD, WbaC, and WbaB, wherein one or more heterologous glycosyltransferases of 73.i. are derived from Citrobacter, possibly Citrobacter fraeungi, and possibly Citrobacter fraeungi P079F I.

[0347] The host cell described in paragraph 73, wherein one or more heterologous glycosyltransferases of 74.i. have an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaD of Citrobacter freunge P079F I, including SEQ ID NO: 11.

[0348] A host cell as described in either paragraph 73 or 74, wherein one or more heterologous glycosyltransferases of 75.i. have an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaC of Citrobacter freunge P079F I, including SEQ ID NO: 12.

[0349] A host cell as described in any one of paragraphs 73-75, wherein one or more heterologous glycosyltransferases of 76.i. have an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WbaB of Citrobacter freunge P079F I, including SEQ ID NO: 13.

[0350] 77. A host cell according to any one of paragraphs 73-76, further comprising a heterologous translocase having an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of Wzx of Citrobacter freunge P079F I, including SEQ ID NO: 14.

[0351] 78. Host cells described in any one of paragraphs 73-77, in which Bmt3, and possibly Candida albicans, is a eukaryotic glycosyltransferase capable of covalently bonding mannose molecules to a β-1,2-mannan polymer and extending the β-1,2-mannan polymer chain ii.

[0352] 79. Host cells as described in paragraph 78, wherein Bmt3 has an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to sequence number 15.

[0353] Host cells described in any one of paragraphs 73-79, wherein the oligosaccharide transferase of 80.iii. is PglB.

[0354] 81. Host cells as described in paragraph 80, in which case PglB is derived from Campylobacter, in which case from Campylobacter jejuni or Campylobacter coli, in which case it is an evolved PglB containing the amino acid sequence of SEQ ID NO: 16.

[0355] Host cells as described in any one of paragraphs 73-81, wherein the modified carrier protein of 82.iv. is selected from the group consisting of Sap2, Als3, detoxified exotoxin A (EPA) of Pseudomonas aeruginosa, CRM197, diphtheria toxoid, tetanus toxoid, detoxified hemolysin A of Staphylococcus aureus, clamping factor A of Staphylococcus aureus, clamping factor B of Staphylococcus aureus, FimH of Escherichia coli, FimHC of Escherichia coli, heat-unstable enterotoxin of Escherichia coli, detoxified variant of heat-unstable enterotoxin of Escherichia coli, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, sat protein of Escherichia coli, passenger domain of sat protein of Escherichia coli, Campylobacter jejuni AcrA, and Campylobacter jejuni natural glycoprotein.

[0356] 83. The host cell described in paragraph 82, wherein the modified carrier protein is the modified Sap2 protein described in any one of paragraphs 1 to 24.

[0357] 84. Host cells described in any one of paragraphs 73-83, which are of the species Escherichia, Sigella, Klebsiella, Xanthomonas, Salmonella, Yersinia, Lactococcus, Lactobacillus, Pseudomonas, Corynebacterium, Streptomyces, Streptococcus, Staphylococcus, Bacillus, or Clostridium.

[0358] 85. The host cell described in paragraph 84, which is *E. coli*.

[0359] 86. A method for producing a modified carrier protein and a glycoconjugate containing β-1,2-mannan, comprising the steps of i) culturing one host cell from any one of paragraphs 73 to 85 under conditions suitable for protein production, ii) harvesting the culture to produce a harvested culture, and iii) isolating the glycoconjugate from the culture.

[0360] 87. Conditions suitable for the production of sugar conjugates are those described in paragraph 86, which include the addition of GDP-mannose to the culture medium.

[0361] 88. The method according to paragraph 86, wherein GDP-mannose is added to the harvested culture.

[0362] 89. A method for producing a bioconjugate in a prokaryotic host cell, a. The step of obtaining a prokaryotic host cell described in any of paragraphs 73-85 that produces a β-1,2-mannan polymer; and A nucleotide sequence encoding a modified carrier protein comprising a glycosylation site including the bi consensus sequence D / EXNZS / T (wherein X and Z are any amino acids other than proline), 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 a prokaryotic host cell; and ii. An oligosaccharide transferase capable of producing a bioconjugate by transferring a β-1,2-mannan polymer from a lipid carrier to a modified carrier protein, wherein the nucleotide sequence encoding the oligosaccharide transferase is PglB derived from Campylobacter, and in some cases from Campylobacter jejuni or Campylobacter coli. A method comprising the step of further introducing and expressing the substance in host cells.

[0363] 90. The method according to paragraph 89, wherein the bacterial signal sequence is selected from the group consisting of FlgI, MalE, OmpA, and OmpC.

[0364] 91. The method according to paragraph 90, wherein the bacterial signal sequence is FlgI.

[0365] 92. The method according to paragraph 91, wherein FlgI contains the amino acid sequence of SEQ ID NO: 21.

[0366] 93. The method according to any of paragraphs 90-92, wherein the bacterial signal sequence is removed from the modified carrier protein after the modified carrier protein has been transported to the periplasmic side of the inner membrane of a prokaryotic host cell.

[0367] 94. The method according to any of paragraphs 90 to 93, wherein the modified carrier protein is the modified Sap2 protein described in any of paragraphs 1 to 24.

[0368] 95. A modified Sap2 protein according to any of paragraphs 14-16, wherein the modified Sap2 protein comprises at least one further consensus sequence comprising the amino acid sequence JUBD / EXNZS / TJUB (wherein X and Z are independently any amino acid other than proline, and J, U and B independently comprise 1 to 5 naturally occurring amino acid residues), and the at least one further consensus sequence is added adjacent to the C-terminal amino acid residue of SEQ ID NO: 3 or at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3.

[0369] 96. A method for inhibiting the attachment of Candida albicans hyphae to vaginal epithelial cells in a subject (e.g., a human), comprising the step of administering to the subject a therapeutic or prophylactic effective amount of a modified Sap2 protein described in any of paragraphs 1 to 24 and 43, a conjugate described in any of paragraphs 25 to 41, a bioconjugate described in any of paragraphs 42 and 49, an immunogenic composition described in paragraph 50, or a vaccine described in any of paragraphs 52 and 53.

[0370] 97. A method for mediating neutrophil killing by Candida albicans hyphae in a subject (e.g., a human), comprising the step of administering to the subject a therapeutic or prophylactic effective dose of a modified Sap2 protein described in any of paragraphs 1-24 and 43, a conjugate described in any of paragraphs 25-41, a bioconjugate described in any of paragraphs 42 and 49, an immunogenic composition described in paragraph 50, or a vaccine described in any of paragraphs 52 and 53.

[0371] 98. A host cell comprising (1) one or more polynucleotide sequences encoding one or more heterologous glycosyltransferases; (2) a polynucleotide sequence encoding a heterologous oligosaccharide transferase; (3) a polynucleotide sequence encoding a modified Sap2 protein as described in any of paragraphs 1 to 22 (or optionally the polynucleotide sequence described in paragraph 44); and optionally (4) a polynucleotide sequence encoding a polymerase.

[0372] 99.i. A nucleotide sequence encoding one or more first heterologous glycosyltransferases capable of synthesizing β-1,2-mannan polymers; ii. A nucleotide sequence encoding a second heterologous glycosyltransferase that is eukaryotic and capable of covalently bonding mannose molecules to β-1,2-mannan polymers, thereby extending the β-1,2-mannan polymer chain; iii. Nucleotide sequences encoding heterologous oligosaccharide transferases; and In some cases, a nucleotide sequence encoding a modified carrier protein containing a glycosylation site including the consensus sequence D / EXNZS / T (wherein X and Z are independently any amino acid other than proline), and in some cases, the polynucleotide sequence described in paragraph 44. Host cells containing these cells.

[0373] 100. A method for producing a bioconjugate in a prokaryotic host cell, a. The step of obtaining a prokaryotic host cell that produces a β-1,2 mannan polymer as described in any of paragraphs 72-84, 98, and 99; and A modified carrier protein comprising a glycosylation site containing the consensus sequence D / EXNZS / T (wherein X and Z are any amino acids other than proline) (the polynucleotide sequence optionally described in paragraph 44), further comprising an N-terminal bacterial signal sequence capable of transporting the modified carrier protein to the periplasmic side of the inner membrane of a prokaryotic host cell, and a nucleotide sequence encoding the modified carrier protein; and ii. An oligosaccharide transferase capable of producing a bioconjugate by transferring a β-1,2-mannan polymer from a lipid carrier to a modified carrier protein, wherein the nucleotide sequence encoding the oligosaccharide transferase is PglB derived from Campylobacter, and optionally PglB derived from Campylobacter jejuni or Campylobacter coli. A method comprising the step of further introducing and expressing in host cells.

[0374] All publications mentioned herein are incorporated herein by reference in their entirety. The term “or” as used herein means options that may be combined; that is, the term “or” should be understood to include each listed option individually, as well as any combination thereof. Where used herein, unless the context otherwise indicates, singular references such as “a,” “an,” and “the” include the plural, and plural references include the singular.

[0375] To better understand the present invention, the following examples are provided. These examples are illustrative and should not be construed as limiting the scope of the invention in any way. (Examples)

[0376] material and method Modification of proSap2 for glycosylation by antigenic glycans To predict suitable insertion sites for glycosites, the crystal structure of the proSap2 protein of the present invention, having the amino acid sequence of SEQ ID NO: 17, was analyzed using various software. Sequence ID 17 - Wild-type intermediate Sap2 ("proSap2") sequence from Candida albicans: [ka]

[0377] For the modification of proSap2 for glycosylation, a fragment corresponding to the proenzyme (residues 19-398) was used, which contained a mutation in D218N (the second catalytic aspartic acid, numbered similarly to the mature Sap2 protein (mSap2; SEQ ID NO: 88)). Site-directed mutagenesis selected a total of 23 locations for inserting the consensus sequence for glycosylation, i.e., the glycosite (D / EXNZS / T). In designing glycosite mutants, slight mutations were added to the substituted region or introduced consensus sequence at some locations, resulting in a total of 37 mutants. ProSap2 mutants with a single glycosite were subjected to glycosylation testing using the Klebsiella pneumoniae O5 antigen. By combining the most effective glycosites, mutants containing a total of 2 to 6 glycosites were created. The six positions selected for the combination are position 19 (N-terminal proSap2), position 57 (N-terminal mSap2 = postpropeptide), positions 98-102 (Mut3a), positions 109-113 (Mut4d), position 220 (Mut8a), and position 398 (C-terminus). This numbering corresponds to the residues in the natural Candida albicans Sap2 sequence (SEQ ID NO: 1).

[0378] Glycosylation testing using modified proSap2 containing one or more glycosites. The in vivo glycosylation efficiency of a modified proSap2 protein containing a single inserted glycosite was tested using the Klebsiella pneumoniae O5 antigen. The dataset presented in this experiment used a strain derived from E. coli W3110, which contained a deletion of the LPS-O antigen ligase waaL and contained a gene cluster for Klebsiella pneumoniae O5 glycan biosynthesis in place of the native O antigen cluster rfbO16. The E. coli strain producing KpO5 glycans was transformed with the pEC415 plasmid containing the modified proSap2 protein and a plasmid expressing PglB. For pre-culture preparation, streaked colonies obtained from transformation plates were inoculated into 5 ml of TB medium containing 10 mM MgCl2 and a suitable antibiotic and incubated overnight at 37°C. This pre-culture was then inoculated into 50 ml of additive-containing TB medium in a shaking flask, and the starting concentration was OD 600 The value was set to = 0.1. The culture medium was prepared at 37°C with shaking at 200 rpm. 600 After culturing until the ratio reached 0.8-1, induction was performed by adding 0.001% arabinose (proSap2) and 0.1 mM IPTG (PglB). Expression and glycosylation of the modified proSap2 protein were continued overnight at 25°C.

[0379] Selection criteria for modified proSap2 proteins with a single glycosite included total expression level and the level of generated glycoconjugate, the latter indicating that the glycosite location is suitable for modification by PglB.

[0380] Preparation of periplasm extract OD 600The total volume of cells from the overnight culture, corresponding to 60 (measured by spectrophotometer), was recovered by centrifugation. The cell pellet was resuspended in 1.5 ml of lysis buffer (30 mM Tris-HCl pH 8.5, 1 mM EDTA (ethylenediaminetetraacetic acid), 20% sucrose), and lysozyme was added to a final concentration of 1 mg / ml. The suspension was incubated at 4°C for 25 minutes with gentle shaking, and then centrifuged at 16,000 rcf for 10 minutes. After centrifugation, the supernatant corresponding to the periplasm extract (PPE) was transferred to a new tube.

[0381] Concentration of periplasm extract by immobilized metal affinity chromatography (IMAC) To concentrate the periplasm extract containing the modified proSap2 protein and enable more direct reading by SDS-PAGE, the histidine-labeled modified proSap2 protein was purified using a one-step purification method with Ni-NTA (nickel nitrilotriacetate) agarose. 1 ml of PPE was mixed with 200 μl of pre-equilibriumized Ni-NTA slurry and incubated with gentle shaking for 30 minutes. The resin was then washed, and the bound protein was eluted with elution buffer (30 mM Tris pH 8.0, 500 mM imidazole, 50 mM NaCl). PPE concentrated by IMAC was analyzed 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). Non-glycosylated proSap2 proteins and glycoconjugates glycosylated at one or more sites (i.e., modified proSap2 proteins linked to one or more polysaccharides) were detected on the gel by Coomassi staining (Fazekas de St. Groth, S.; Webster, RG; Datyner, A. (1963). "Two new staining procedures for quantitative estimation of proteins on electrophoretic strips". Biochimica et Biophysica Acta. 71: 377-391. doi:10.1016 / 0006-3002(63)91092-8. PMID 18421828).

[0382] These experimental results are explained in Figure 3-13 and the following examples. [Example 1]

[0383] SDS-PAGE analysis of modified proSap2 protein-single glycosite variants purified from PPE by IMAC SDS-PAGE analysis (Figure 3) was performed on IMAC-enriched periplasm extracts of E. coli strains producing KpO5 polysaccharide and expressing PglB and modified proSap2 proteins. This modified proSap2 protein has a glycosite D / EXNZS / T introduced at a specific position in Sequence ID No. 17, the sequence of which is shown in Tables 1 and 2 below:

[0384] [Table 1]

[0385] [Table 2]

[0386] The protein bands in Figures 3A and 3B correspond to the non-glycosylated modified proSap2 protein ("uCarrier") and the KpO5 modified proSap2 bioconjugate with one glycosite occupied ("Conjugate").

[0387] As shown in Figures 3A and 3B, modified proSap2 proteins with a single glycosite exhibited variability in expression levels. In certain constructs (e.g., Mut14), expression was almost completely lost, while in other constructs, expression was similar to that of the wild-type (wt) proSap2 protein (SEQ ID NO: 17). Surprisingly, the proSap2 "C" mutant (SEQ ID NO: 46) showed higher expression levels than the wild type.

[0388] The proSap2 "C" mutant (SEQ ID NO: 46) also exhibited the highest degree of glycosylation (Figure 3A, lane 3). Several other mutants, including N (SEQ ID NO: 44), Mut3a (SEQ ID NO: 52), Mut4d (SEQ ID NO: 56), and Mut8a (SEQ ID NO: 63), also showed high levels of glycosylation (Figure 3A lanes 9, 13, Figure 3B lane 20), and these were selected for their glycosite combinations. The modified proSap2 proteins used in this analysis had histidine tags, but it will be understood by those skilled in the art that modified proSap2 proteins without histidine tags can also be used in the same analysis. Therefore, in certain embodiments, the present invention provides modified Sap2 or modified proSap2 proteins with histidine tags removed. [Example 2]

[0389] Analysis of glycosylation of proSap2 possessing combined glycosites and Fba peptide. By combining the glycosites exhibiting the best performance, mutants containing a total of 2 to 6 glycosites were created. The six positions selected for the combinations were position 19 (N-term proSap2), position 57 (N-term mSap2 = post-propeptide), positions 98-102 (Mut3a), positions 109-113 (Mut4d), position 220 (Mut8a), and position 398 (C-terminus), with this numbering corresponding to residues in the natural Candida albicans Sap2 sequence (SEQ ID NO: 1). Furthermore, in certain embodiments, the Fba peptide was introduced into the selected mutants at the C-terminal position of the protein, followed by a "C" glycosite. SDS-PAGE analysis (Figure 4) was performed on IMAC-enriched periplasm extracts of E. coli strains producing KpO5 polysaccharide and expressing proSap2 mutant proteins possessing PglB and the following glycosite combinations (Table 3).

[0390] [Table 3]

[0391] As shown in Figure 4, all modified proSap2 proteins with combined glycosites were expressed without a significant decrease in yield. All introduced glycosites were at least partially glycosylated, with some occupying up to four glycosites, resulting in minimal unglycosylated protein and a maximized sugar / protein ratio. The addition of the Fba peptide had no effect on expression levels or glycosylation efficiency. The modified proSap2 mutant MutN3C-3a-8a-fba-C (SEQ ID NO: 87) was selected as the optimal protein carrier. The modified proSap2 protein used in this analysis had a histidine tag, but those skilled in the art will understand that a modified proSap2 protein without the histidine tag can also be used in the same analysis. Therefore, in certain embodiments, the present invention provides a modified Sap2 or modified proSap2 protein without the histidine tag. [Example 3]

[0392] Production of proSap2-Fba-β-1,2-mannan conjugate Construction of a modified proSap2-Fba-β-1,2-mannan-producing strain The strain derived from *Escherichia coli* K12 W3110 was constructed by λ-Red homologous recombination, in which the target gene cluster was replaced with a selection marker flanked by the target gene (if any) and the subsequent FRT region, followed by removal of the marker by catalytic action of FLP recombinase (TE Kuhlman and EC Cox. Nucleic Acids Res. 2010 Apr; 38(6): e92). Seven homologous recombination / marker removal steps were performed to remove the following genomic sequences: i. LPS-O antigen ligase waaL (GenBank NC_007779 position 3'842'208~3'843'467), ii. O16 O antigen cluster (rfb or wb, GenBank NC_007779 position 2'114'113~2'103'814), iii. Coranic acid cluster (wca, GenBank NC_007779 position 2'138'241~2'118'033), iv. ECA cluster holding wecA (wec, GenBank NC_007779 position 3'666'604~3'656'725), v. O16wzz2 or cld (GenBank NC_007779 Position 2'099'458~2'100'438), vi. gtrABS or yfdGHI (GenBank NC_007779 Position 2'473'301~2'475'908), vii. araBA (GenBank NC_007779 Position 66'835~70'048). Furthermore, two copies of the wzx, wbaB, wbaC, and wbaD genes (GenBank QFTZ01000001.1 387'158~393'257) derived from Citrobacter freundii P079F I have been introduced into the O16 O antigen cluster locus (rfb or wb, GenBank NC_007779 position 2'114'113~2'103'814), replacing the yeaS gene (GenBank NC_007779 position 1'881'835~1'882'473).

[0393] To increase GDP-mannose biosynthesis, a first expression plasmid was constructed using pEC415 (Schulz et al. J Biol Chem. 1998 Aug; 281(5380):1197-200) as the plasmid backbone. This plasmid contains the Candida albicans mannosyltransferase Bmt gene (GenBank XP_717972.1), fused at the N-terminus with a periplasmic signal peptide and codon-optimized for E. coli, followed by the E. coli genes manC and manB (GenBank: NC_007779 position 2'123'746~2'126'657). A second expression plasmid was constructed, promoter-regulated with arabinose, encoding a selected proSap2-Fba variant within a plasmid backbone possessing trimethoprim resistance and a BBR ori (medium copy). A third expression plasmid was constructed within the pEXT21 plasmid skeleton (Dykxhoorn DM et al. Gene 1996;177(1-2):133-6.), encoding the pglB sequence obtained by directed evolution. All plasmids were constructed from synthetic DNA templates using one- or multi-step classical restriction enzyme cloning and Gibson assembly (Gibson, DG, et al. (2009) Nat. Methods 6, 343-345).

[0394] By transforming recombinant strains with three plasmids, we obtained strains that produce conjugates. The roles of each gene and the glycan structure in bioconjugate generation are schematically shown in Figure 2.

[0395] Production and analysis of modified proSap2-β-1,2-mannan bioconjugates The conjugate-producing strain was cultured in a 10 L fed-batch bioreactor in buffered nutrient-rich medium at 37°C and pH 7. The OD of the culture was also analyzed. 600nmWhen the temperature reached a value of 20-25, the temperature was changed to 25°C, and bioconjugate formation was induced with IPTG and arabinose. Addition of nutrient-rich medium was started and stopped 24 hours after induction. Subsequently, GDP-mannose was added to the culture vessel for 40 minutes until a final concentration of 50 μM was reached. 26 hours after induction, cells were collected by centrifugation, washed with TBSE buffer, and resuspended. An osmotic shock protocol was applied, and the cells were diluted with 5 times the volume of H2O for 1 hour to release the contents of the periplasm into the supernatant. Cells were separated from the supernatant by centrifugation and filtration was performed through 0.45 μm and 0.2 μm filters to remove cell debris. Tangential flow filtration (TFF) with a 10 kDa cutoff was applied and replaced with acidic buffer (10 mM citrate, 40 mM NaCl, pH 4.0). Cation exchange chromatography was used to almost completely separate the bioconjugate from impurities. The target fraction was pooled and diluted in pH 7 buffer, after which the bioconjugate was further purified completely by passing it through a hydrophobic interaction column. The bioconjugate-containing fraction was pooled and concentrated to the target volume by replacing the buffer with 10 mM NaPO4, 150 mM NaCl, pH 6.5 using TFF. As shown in Figure 5, the purified bioconjugate was analyzed on an SDS-PAGE gel and then analyzed by i. Coomassie blue staining (Figure 5A), ii. anti-proSap2 immunoblotting (against the N-terminal "propeptide" region; Figure 5B), iii. anti-Sap2 immunoblotting (against the peptide in the middle of the protein sequence; Figure 5C), iv. anti-Fba immunoblotting (Figure 5D), and iv. anti-mannan immunoblotting (Figure 5E). [Example 4]

[0396] Structural determination of proSap2 produced in E. coli by crystallography Detoxified proSap2 was overexpressed in the periplasm of E. coli, purified, and concentrated to a concentration of 10 mg / mL at 3000 rpm using an Amicon concentrator (6 ml, 10 kDa CO2). Crystallization plates were set up for use with the TTP Labtech Mosquito LCP robot; the drop volume was 200 nl of protein + 200 nl of precipitant solution; the crystallization screening agents used were SG1, PACT, Morpheus, and PGA (Molecular Dimensions), and the test temperatures were 20°C and 4°C. Since crystal formation was possible under several conditions, diffraction tests were performed. Data were collected at the X06DA beamline in Swiss Light Source.

[0397] The crystals were analyzed by diffraction at high resolution. The structure of proSap2 was determined by molecular substitution, using the structure of mSap2 (mature Sap2 lacking the propeptide) from C. albicans (PDB ID: 1EAG) as a search model.

[0398] The structure was refined to a resolution of 1.7 Å. High-quality electron density maps allowed for the construction of 362 of the 380 residues of proSap2; however, 18 residues of the propeptide could not be constructed, suggesting a flexible structure in this region. The remaining portion of the propeptide was modeled using an available AlphaFold model of proSap2. The resulting structure superimposed on the mSap2 structure derived from C. albicans, as shown in Figure 6.

[0399] Conclusion: proSap2, produced by E. coli, folds similarly to Candida-derived proteins, resulting in a remarkably similar structure. The propeptides present in proSap2 used in bioconjugation only slightly cover the active site, allowing potential antibodies to access it. [Example 5]

[0400] Circular dichroism (CD) spectroscopy characteristics of wild-type proSap2 protein and modified proSap2-β-1,2-mannan bioconjugate To obtain information on the characteristics of the secondary and tertiary structures of the protein and to compare them, detoxified wild-type proSap2 from the same batch analyzed by crystallography, and the modified Sap2 protein of the present invention (modified proSap2-Fba-β-1,2-mannan bioconjugate, where proSap2 is the MutN3C-8a-fba-C mutant) were analyzed by near-ultraviolet and far-ultraviolet CD spectroscopy.

[0401] Near-ultraviolet CD measurements were performed using a Chirascan Q100 CD spectrophotometer (Applied Photophysics Ltd., UK). Near-ultraviolet CD spectra in the wavelength range of 240–350 nm were obtained at a scan speed of 30 nm min. -1 Ten consecutive scans were summed up using the following settings: bandwidth 1 nm and wavelength step size 0.5 nm, recorded using a 1 cm path length flow-through cuvette (Applied Photophysics Ltd., UK). For far-ultraviolet CD measurements, a Chirascan Q100 CD spectrophotometer (Applied Photophysics Ltd., UK) was used. Far-ultraviolet CD spectra in the wavelength range 190–260 nm were recorded at a scan rate of 60 nm min. -1Ten consecutive scans were summed up using the following settings: bandwidth of 1 nm and wavelength step size of 1 nm, recorded using a flow-through cuvette with a 0.01 cm path length (Applied Photophysics Ltd., UK). The preparation buffer was used as a blank. The average residue ellipticity was mathematically extrapolated and plotted. To estimate the content of secondary structure elements, far-ultraviolet CD spectra were analyzed using CDNN software (version 2.1) with a database containing secondary structure data in the 178-260 nm wavelength range obtained from 33 untrained neural networks and 29 soluble proteins (SP29 database, constructed by Johnson et al. in 1981 and 1988) and four additional components (carbopeptidase A, polyglutamic acid, rubredoxin, and trypsin). The obtained data are shown in Figures S.7A (near-ultraviolet CD), 7B (far-ultraviolet CD), and 7C (content of secondary structural elements after deconvolution analysis by CD spectroscopy using "CDNN" software (Applied Photophysics Ltd)).

[0402] Conclusion: The analysis results show that both proSap2 (used for crystal structure analysis) and the proSap2-Fba-β-1,2-mannan bioconjugate have extremely similar tertiary structures. Therefore, neither the addition of glycosites nor their glycosylation impairs the tertiary structure of proSap2. [Example 6]

[0403] Immunogenicity of modified proSap2 protein-mannan bioconjugate Figure 8 shows the preclinical trials of the modified proSap2-Fba-β-1,2-mannan bioconjugate ("proSap2-4FM") in rabbits. Figure 8A shows the properties of the modified proSap2-Fba-β-1,2-mannan bioconjugate. Figure 8B is a 3D view of the modified proSap2-Fba-β-1,2-mannan bioconjugate. The structure of the modified proSap2 protein is shown schematicly. Spherical marks indicate the locations of the four introduced glycosites. The bound β-mannan chain is schematically represented, and the location and sequence of the Fba peptide sequence YGKDVKDLFDYAQE (SEQ ID NO: 3) are also shown. Figure 8C shows the rabbit immunization scheme using the modified proSap2-Fba-β-1,2-mannan bioconjugate. The term "proSap2-4FM" used herein refers to a modified proSap2-Fba-β-1,2-mannan bioconjugate (modified in that it includes (i) an inactivated D218N substitution, (ii) a propeptide sequence, (iii) four glycosites, (iv) an Fba sequence, and (v) a glycan).

[0404] Figure 9 shows the immunogenicity of purified modified proSap2-Fba-β-1,2-mannan bioconjugate ("proSap2-4FM") in rabbits. Figures 9A, 9B, and 9C show the immunogenicity of the modified proSap2-Fba-β-1,2-mannan bioconjugate to Sap2, Fba, and β-mannan, respectively, in rabbits, compared to mSap2 or Sap2-Fba-β-1,2-mannan bioconjugate ("Sap2-4FM"). unmodifShown in comparison to the immunogenicity of []. The control group represents animals immunized with buffer, and all vaccine groups were tested with AS03. New Zealand White (NZW) rabbits were immunized three times at two-week intervals. Coating ELISA: Figure 9A shows modified proSap2 without incorporation of the His-Tag, Figure 9B shows the Fba peptide, and Figure 9C shows the mannan extract of C. albicans. ProSap2-specific, Fba-specific, or mannan-specific IgG serum concentrations (arbitrary units, AU) in rabbit sera pre- (before immunization), post-II (after two immunizations), post-III (after three immunizations) (d0, d28, d42) by treatment group. Lines indicate geometric mean concentration (GMC) ± 95% confidence interval. ****: p<0.0001, one-way ANOVA. Ref: 36_009, 36_012. The "proSap2-FM" used here refers to a modified proSap2-Fba-β-1,2-mannan bioconjugate (modified in that it contains (i) inactivated D218N substitution, (ii) propeptide sequence, (iii) four glycosites, (iv) Fba sequence, and (v) sugar chain); "mSap2" refers to a modified mature form of the Sap2 protein (modified in that it contains the inactivated D218N substitution but lacks (i) propeptide sequence, (ii) Fba sequence, and (iii) four glycosites); and "Sap2-4FM unmodif " refers to a modified mature form of Sap2-Fba-β-1,2-mannan bioconjugate (modified in that it contains (i) inactivated D218N substitution, (ii) four glycosites, (iii) Fba sequence, and (iv) sugar chain but does not contain the propeptide sequence).

[0405] Conclusion: 1) The modified proSap2-Fba-β-1,2-mannan bioconjugate (proSap2-FM), Sap2-Fba-β-1,2-mannan bioconjugate (Sap2-4FM unmodif ), and mSap2 show immunogenicity against Sap2. A significant increase in titer was observed at pre / post-II (before / after two immunizations) and pre / post-III (before / after three immunizations) (Figure 9A).

[0406] 2) Fba peptide (modified proSap2-Fba-β-1,2-mannan bioconjugate and part of the modified Sap2 protein glucan bioconjugate) is immunogenic to Fba. In Sap2-4FM, a significant increase in titer was observed in pre / post-II (before immunization / after 2 immunizations) and pre / post-III (before immunization / after 3 immunizations). Sap2-4FM unmodif However, a significant increase in titer was observed only in the pre / post-III (before immunization / after 3 immunizations) (Figure 9B).

[0407] 3) The modified Sap2-Fba protein-β-1,2-mannan bioconjugate is immunogenic to β-mannan. Significant increases in titer were observed in pre / post-II (before immunization / after 2 immunizations) and pre / post-III (before immunization / after 3 immunizations) (Figure 9C). [Example 7]

[0408] Protease activity inhibition assay To evaluate whether antibodies produced in rabbits can bind to the active site of Sap2 and consequently inhibit its activity, we designed an assay in which a potent inhibitor of Sap2 activity (a single-specific Fab fragment) was mixed with Sap2, and the resulting serum was titrated in a solution. If the produced antibody present in the serum has the same binding epitope as the neutralizing Fab, it should be able to displace it in a concentration-dependent manner.

[0409] First, we demonstrated that Fab inhibits Sap2 activity. To this end, 5 μg / mL anti-Sap2 Fab was mixed with 5 μg / mL proSap2 wt in the presence of 10 mg / mL BSA at pH 7.5 and 30°C for 1 hour with shaking at 450 rpm to form a complex. Activation of proSap2 to mSap2 (cleavage of the propeptide) was achieved by adding citrate to 0.1 M to change the pH to 4 and incubating at 30°C for 4 hours with shaking at 450 rpm. Trichloroacetic acid (TCA) was added to 5% (w / w), the tube was incubated on ice for 30 minutes, and then the total protein was precipitated by centrifugation at 16,000 g for 10 minutes. The peptide produced by Sap2 digestion of BSA should not precipitate with the remaining protein; therefore, the 280 nm absorbance measurement remaining in the supernatant is proportional to the Sap2 protease activity. In Figure 10A, the measured activity was determined by absorbance at 280 nm in the absence of Sap2 (negative control), the absence of Fab fragment (positive control), and the presence of Fab fragment (Fab anti-Sap2), and is expressed as a percentage relative to the positive control activity. The Fab fragment used was able to completely inhibit the protease activity of Sap2.

[0410] Next, we tested the ability of rabbit-produced antiserum to replace Fab fragments. For this purpose, we developed an ELISA setup in which microtiter plates were coated overnight at 4°C with 1 μg / μL proSap2 in PBS pH 7.4 and blocked with Tween®-containing PBS buffer (PBST). Serum obtained from rabbit immunoassays, diluted in PBST, was added to the plates, dose-set, and incubated at 20°C for 1 hour with shaking at 450 RPM. Fab fragments containing His6 tags were pre-incubated for 1 hour with anti-His-HRP (horseradish peroxidase) secondary antibody in PBST at room temperature with shaking at 450 RPM. Subsequently, the Fab-secondary antibody complexes were added to the wells and the plates were washed. We evaluated whether the binding of Fab fragments to proSap2 was impaired in the presence of serum by adding TMB (3,3',5,5'-tetramethylbenzidine) to the plates and detecting the presence of the secondary antibody by reaction with HRP and color development at 450 nm. Figure 10B shows titration curves using serum obtained from two-component immunization ("Candi5V", using a combination of proSap2-Fba-β-1,2-mannan bioconjugate and Als3-Fba-β-1,3-glucan bioconjugate) or proSap2-Fba-β-1,2-mannan rabbit immunization ("proSap2-4FM"). Serum from mock-immunized (pseudoimmunized) rabbits was used as a control.

[0411] Conclusion: As shown in Figures 10A and 10B, the Fab used had the ability to completely inhibit Sap2 protease activity and was therefore demonstrated to bind to the Sap2 active site. Serum containing antibodies produced with proSap2-Fba-β-1,2-mannan (as a single-component immunization or as one component of a two-component immunization) had the ability to displace Fab in a concentration-dependent manner and was demonstrated to bind to the Sap2 active site. [Example 8]

[0412] Quantitative adhesion assay of C. albicans mycelium Figure 11 shows the ability of antibodies against the proSap2-4FM bioconjugate to inhibit the adhesion of C. albicans to vaginal epithelial cells. Serum was mixed with C. albicans (SC5314) and added to epithelial cells (A431), and incubated for 1.5 hours. After washing, the adhered cells were measured using concanavalin A-Alexa fluor 488. As used here, “proSap2-FM” refers to the modified proSap2-Fba-β-1,2-mannan bioconjugate (modified in that it contains (i) an inactivated D218N substitution, (ii) a propeptide sequence, (iii) four glycosites, (iv) an Fba sequence, and (v) a glycan); “mSap2” refers to the modified mature Sap2 protein (containing an inactivated D218N substitution, but with the addition of (i) a propeptide sequence, (ii) an Fba sequence, and (iii) Modified in that it lacks four glycosites; and “Sap2-4F” refers to the modified proSap2-Fba protein ((i) inactivated D218N substitution, (ii) four glycosites, and (iii) Fba sequence, but modified in that it lacks a propeptide sequence and is not glycosylated); “Control” refers to animals immunized with buffer, and preimmune controls were prepared by pooling preimmune serum from all animals. All vaccine groups were tested with AS03 adjuvant. NZW rabbits were immunized three times at 2-week intervals. Graphs show mean ± standard deviation. ****: p<0.0001, one-way ANOVA.

[0413] Conclusion: As shown in Figure 11, serum against the proSap2-4FM bioconjugate inhibits the adhesion of C. albicans hyphae to vaginal epithelial cells. After three immunizations (post-III), a significant reduction in adhesion to epithelial cells was observed compared to control serum. Serum against Sap2-4F and mSap2 did not inhibit adhesion to epithelial cells, suggesting that their effect is likely mediated by β-1,2-mannan antibodies. [Example 9]

[0414] Neutrophil bactericidal ability test Figure 12 shows the ability of antibodies against the proSap2-4FM bioconjugate to mediate the killing of C. albicans mycelial form by neutrophils. C. albicans mycelial form (SC5314) was mixed with serum and added to neutrophils for incubation. The neutrophils were then lysed and cultured on YPD agar, after which the CFU of Candida was counted. Herein, "proSap2-FM" refers to the modified proSap2-Fba-β-1,2-mannan bioconjugate (modified in that it contains (i) an inactivated D218N substitution, (ii) a propeptide sequence, (iii) four glycosites, (iv) an Fba sequence, and (v) a glycan); "mSap2" refers to the modified mature Sap2 protein (containing an inactivated D218N substitution, but also containing (i) a propeptide sequence, (ii) an Fba sequence, and (iii) Modified in that it lacks four glycosites; and “Sap2-4F” refers to the modified proSap2-Fba protein ((i) inactivated D218N substitution, (ii) four glycosites, and (iii) Fba sequence, but modified in that it lacks a propeptide sequence and is not glycosylated); “Control” refers to animals immunized with buffer, and preimmune controls were prepared by pooling preimmune serum from all animals. All vaccine groups were tested with AS03 adjuvant. NZW rabbits were immunized three times at 2-week intervals. Graphs show mean ± standard deviation. ***: p<0.001; **: p<0.01, PBS control, one-way ANOVA.

[0415] Conclusion: As shown in Figure 12, serum against proSap2-4FM bioconjugate and proSap2-F mediated bactericidal activity by C. albicans mycelial neutrophils. After three immunizations (post-III), a significant increase in bactericidal activity (reduction in CFU) was observed compared to control serum. Serum against mSap2 did not inhibit adhesion to epithelial cells, suggesting that its effect is likely mediated by β-1,2-mannan and Fba antibodies. [Example 10]

[0416] Binding of antibodies to C. albicans mycelium Figure 13 shows the ability of antibodies against the proSap2-4FM bioconjugate to bind to C. albicans using fluorescence microscopy. Cells were observed by differential interference contrast microscopy (DIC) and fluorescence microscopy (secondary antibody Alexa 488). Serum was mixed with C. albicans (SC5314) and stained with concanavalin A-Alexa fluor 488. Herein, “proSap2-FM” refers to the modified proSap2-Fba-β-1,2-mannan bioconjugate (modified in that it contains (i) an inactivated D218N substitution, (ii) a propeptide sequence, (iii) four glycosites, (iv) an Fba sequence, and (v) a glycan); “Candi5V” refers to a multicomponent vaccine containing Sap2-4FM and Als3-Fba-β-1,3-glucan; “control group” refers to animals immunized with buffer, and all vaccine groups were tested using AS03. The NZW rabbits were immunized three times at two-week intervals.

[0417] Conclusion: As shown in Figure 13, it is possible to confirm the binding of Sap2-4FM antibodies to the yeast and hyphal segments of Candida cells by using a laser confocal microscope.

[0418] Sequence ID 1: Full-length sequence of the wild-type Sap2 protein of Candida albicans (contains the wild-type leader sequence (italicized) and propeptide sequence (underlined); amino acid residue 274(D) is double-underlined). [ka]

[0419] Sequence ID 2: Consensus Sequence (Artificial Sequence) GSGGGD / EXNZS / TGSGG Sequence ID 3 Fba sequence YGKDVKDLFDYAQE Sequence ID 4: Consensus Sequence (Artificial Sequence) DQNAT Sequence ID 5: Consensus Sequence (Artificial Sequence) DQNVT Sequence ID 6: Consensus Sequence (Artificial Sequence) JUBD / EXNZS / TJUB

[0420] In the sequence, X is Q (glutamine), Z is A (alanine), J and B each contain 1 to 5 glycine (G) residues, and U contains 1 to 5 serine (S) residues. Sequence ID 7: Consensus Sequence (Artificial Sequence) GSGGGDQNATGSGGG Sequence ID 8: Consensus Sequence (Artificial Sequence) GSGGGDQNATGSGGGHHHHHHHHHH

[0421] Sequence ID 9: Modified Sap2 sequence (an intermediate Sap2-Fba fusion protein that lacks the native leader sequence and contains a propeptide sequence (underlined), four glycosites (bold), an inactivation substitution (double underlined), and an Fba sequence (dashed underlined)) (artificial sequence) [ka]

[0422] Sequence ID No. 10: Modified Sap2 sequence (a mature Sap2-Fba fusion protein that lacks a native leader sequence or propeptide sequence, has four glycosites (bold), an inactivating substitution (double underline), and also contains an Fba sequence (dashed underline)) (artificial sequence) [ka]

[0423] Sequence ID 11: Citrobacter freundii P079F I WbaD sequence [ka]

[0424] Sequence ID 12: Citrobacter freundii P079F I WbaC sequence [ka]

[0425] Sequence ID 13: Citrobacter freundii P079F I WbaB sequence [ka]

[0426] Sequence ID 14: Citrobacter freundii P079F I Wzx sequence [ka]

[0427] Sequence ID 15: Candida albicans Bmt3 sequence [ka]

[0428] Sequence ID 16: Campylobacter PglB (evolved form) sequence [ka]

[0429] Sequence ID 17: Candida albicans wild-type Sap2 sequence (intermediate Sap2 or "proSap2") (contains wild-type propeptide sequence (underlined) but lacks wild-type leader sequence; amino acid residue (D) at position 256 is double-underlined, which corresponds to position 274 in Sequence ID 1) (amino acid residues 19-398 of Sequence ID 1) [ka]

[0430] Sequence ID 18: Consensus Sequence (Artificial Sequence) KD / EXNZS / TK In the sequence, X is Q (glutamine) and Z is A (alanine). Sequence ID 19: Consensus Sequence (Artificial Sequence) KDQNAT Sequence ID 20: Consensus Sequence (Artificial Sequence) KDQNAS Sequence ID No. 21: Flagellin (FlgI) signaling sequence of Escherichia coli. MIKFLSALILLLVTTAAQA Sequence ID No. 22: Escherichia coli outer membrane porin A (OmpA) signal sequence MKKTAIAIAVALAGFATVAQA Sequence ID No. 23: E. coli maltose-binding protein (MalE) signal sequence MKIKTGARILALSALTTMMFSASALA Sequence ID No. 24: Escherichia coli outer membrane porin A (OmpC) signal sequence MKVKVLSLLVPALLVAGAANA

[0431] Sequence ID 25: Nucleotide sequence of WbaD of Citrobacter freundie's P079F I [ka] [ka]

[0432] Sequence ID 26: Nucleotide sequence of WbaC of Citrobacter freundie's P079F I [ka]

[0433] Sequence ID 27: Nucleotide sequence of WbaB of Citrobacter freundie's P079F I [ka]

[0434] Sequence ID 28: Nucleotide sequence of Wzx of Citrobacter freundiei P079F I [ka]

[0435] Sequence ID 29: Nucleotide sequence of Bmt3 in C. albicans [ka] [ka]

[0436] Sequence ID 30: Nucleotide sequence of Campylobacter PglB (evolved form) [ka] [ka]

[0437] Sequence ID 31: E. coli K12 W3110 manB sequence [ka]

[0438] Sequence ID 32: E. coli K12 W3110 manC sequence [ka]

[0439] Sequence ID 33: Nucleotide sequence of manB in E. coli K12 W3110 [ka] [ka]

[0440] Sequence ID 34: Nucleotide sequence of manC in E. coli K12 W3110 [ka]

[0441] Sequence ID 35: Nucleotide sequence of wild-type Sap2 in C. albicans [ka] [ka]

[0442] Sequence ID 36: Nucleotide sequence of modified Sap2 (an intermediate Sap2-Fba fusion protein lacking a native leader sequence) (including propeptide sequence; containing four glycosites and inactivation substitutions) [ka]

[0443] Sequence ID 37: Nucleotide sequence of a mature Sap2-Fba fusion protein lacking a native leader or propeptide sequence, containing four glycosites and including an inactivating substitution. [ka]

[0444] Sequence ID 38: Pectin acid lyase (PelB) sequence of the soft rot bacterium (Erwinia carotovora) ( MKYLLPTAAAGLLLLAAQPAMA Sequence ID No. 39: Escherichia coli heat-labile enterotoxin LTIIb sequence MSFKKIIKAFVIMAALVSVQAHA Sequence ID No. 40: Bacillus subtilis endoxylanase XynA sequence MFKFKKKFLVGLTAAFMSISMFSATASA Sequence ID 41: Escherichia coli DsbA sequence MKKIWLALAGLVLAFSASA Sequence ID 42: TolB sequence MKQALRVAFGFLILWASVLHA Sequence ID 43, SipA sequence MKMNKKVLLTSTMAASLLSVASVQAS

[0445] Sequence ID 44 proSap2 N sequence [ka]

[0446] Sequence ID 45 proSap2 N3C sequence [ka]

[0447] Sequence ID 46 proSap2 C sequence [ka]

[0448] Sequence ID 47: proSap2 Mut1a sequence [ka]

[0449] Sequence ID 48: proSap2 Mut1b sequence [ka]

[0450] Sequence ID 49: proSap2 Mut2a sequence [ka]

[0451] Sequence ID 50: proSap2 Mut2b sequence [ka]

[0452] Sequence ID 51: proSap2 Mut2c sequence [ka]

[0453] Sequence ID 52: proSap2 Mut3a sequence [ka]

[0454] Sequence ID 53: proSap2 Mut3b sequence [ka]

[0455] Sequence ID 54: proSap2 Mut4a sequence [ka]

[0456] Sequence ID 55 proSap2 Mut4c sequence [ka]

[0457] Sequence ID 56: proSap2 Mut4d sequence [ka] [ka]

[0458] Sequence ID 57: proSap2 Mut4e sequence [ka]

[0459] Sequence ID 58: proSap2 Mut5a sequence [ka]

[0460] Sequence ID 59: proSap2 Mut5b sequence [ka]

[0461] Sequence ID 60: proSap2 Mut6a sequence [ka] [ka]

[0462] Sequence ID 61: proSap2 Mut6b sequence [ka]

[0463] Sequence ID 62: proSap2 Mut7 sequence [ka]

[0464] Sequence ID 63: proSap2 Mut8a sequence [ka]

[0465] Sequence ID 64: proSap2 Mut8b sequence [ka]

[0466] Sequence ID 65: proSap2 Mut9 sequence [ka]

[0467] Sequence ID 66: proSap2 Mut10 sequence [ka]

[0468] Sequence ID 67: proSap2 Mut11 sequence [ka]

[0469] Sequence ID 68: proSap2 Mut12 sequence [ka]

[0470] Sequence ID 69: proSap2 Mut13 sequence [ka] [ka]

[0471] Sequence ID 70: proSap2 Mut14a sequence [ka]

[0472] Sequence ID 71: proSap2 Mut14b sequence [ka]

[0473] Sequence ID 72 proSap2 Mut14c sequence [ka]

[0474] Sequence ID 73: proSap2 Mut14d sequence [ka] [ka]

[0475] Sequence ID 74: proSap2 Mut14e sequence [ka]

[0476] Sequence ID 75 proSap2 Mut15 sequence [ka]

[0477] Sequence ID 76: proSap2 Mut16 sequence [ka]

[0478] Sequence ID 77 proSap2 Mut17 sequence [ka]

[0479] Sequence ID 78 proSap2 Mut18 sequence [ka]

[0480] Sequence ID 79: proSap2 Mut19 sequence [ka]

[0481] Sequence ID 80 proSap2 Mut20 sequence [ka]

[0482] Sequence ID 81: proSap2 MutN3C-C sequence [ka]

[0483] Sequence ID 82: proSap2 MutN3C-8a-C sequence [ka]

[0484] Sequence ID 83: proSap2 MutN3C-3a-8a-C sequence [ka]

[0485] Sequence ID 84 proSap2 MutN8a-fba-C sequence [ka]

[0486] Sequence ID 85 proSap2 MutN3C-fba-C sequence [ka]

[0487] Sequence ID 86 proSap2 MutN3C-8a-fba-C sequence [ka]

[0488] Sequence ID 87 proSap2 MutN3C-3a-8a-fba-C sequence [ka]

[0489] Sequence ID 88: Wild-type mature Sap2 sequence of Candida albicans (lacks wild-type leader sequence and wild-type propeptide sequence; amino acid residue (D) at position 218 is double-underlined; position 218 corresponds to position 274 of Sequence ID 1) (amino acid residues 57-398 of Sequence ID 1) [ka]

[0490] Sequence ID 89: Modified Sap2 sequence (histidine-labeled intermediate Sap2-Fba fusion protein, which lacks the native leader sequence and contains a propeptide sequence (underlined); contains four glycosites (bold); contains an inactivating substitution (double underlined); and further contains an Fba sequence (dashed underlined)) (artificial sequence) [ka] [ka]

[0491] Sequence ID 90: Modified Sap2 sequence (histidine-labeled mature Sap2-Fba fusion protein, which lacks the native leader or propeptide sequence, contains four glycosites (bold), contains inactivation substitutions (double underlined), and further contains an Fba sequence (dashed underlined)) (artificial sequence) [ka] Consensus sequence (artificial sequence) JUBD / EXNZS / TJUB Consensus sequence (artificial sequence) KD / EXNZS / TK

Claims

1. A modified secretory aspartyl proteinase 2 (Sap2) protein having an amino acid sequence identical to amino acid residues 19-398 of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of amino acid residues 19-398 of SEQ ID NO: 1, wherein the amino acid sequence is modified to include one or more consensus sequences containing the amino acid sequence D / EXNZS / T (wherein X and Z are independently any amino acid other than proline).

2. The modified Sap2 protein according to claim 1, wherein the 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 that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO: 1, and optionally the protein comprises a substitution from aspartic acid (D) to asparagine (N) 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 that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 19-398 of SEQ ID NO:

1.

3. A modified Sap2 protein according to any one of claims 1 and 2, wherein the modified Sap2 protein is derived from Candida albicans, and further comprises at least one fructose diphosphate aldolase-1 (Fba) peptide having an amino acid sequence identical to YGKDVKDLFDYAQE (SEQ ID NO: 3) or SEQ ID NO: 3 by at least 70%, 80%, 85%, 90%, or 92%.

4. A modified Sap2 protein according to any one of claims 1 to 3, wherein one or more consensus sequences are selected from the group consisting of DQNAT (SEQ ID NO: 4) and DQNVT (SEQ ID NO: 5), and the modified Sap2 protein includes at least one further consensus sequence comprising the amino acid sequence JUBD / EXNZS / TJUB (wherein X and Z are independently any amino acid other than proline, and J, U and B independently comprise 1 to 5 naturally occurring amino acid residues), which is added adjacent to the C-terminal amino acid residue of SEQ ID NO: 3 or at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

3.

5. A modified Sap2 protein containing the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO:

10.

6. A conjugate comprising a modified Sap2 protein according to any one of claims 1 to 5 and at least one sugar antigen, which is optionally a bioconjugate.

7. A modified Candida albicans Sap2 protein comprising (1) the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and (2) at least one glycan derived from Candida, which is a β-1,2 mannan polymer consisting of at least five consecutive β-1,2 linked mannose molecules, and is linked to at least one of the four asparagine residues at positions 45, 94, 215, and 415 of SEQ ID NO: 9 or at least one of the four asparagine residues at positions 6, 55, 176, and 376 of SEQ ID NO:

10.

8. A polynucleotide sequence encoding the modified Sap2 protein according to any one of claims 1 to 5.

9. A vector comprising the polynucleotide sequence described in claim 8.

10. A host cell comprising (1) one or more polynucleotide sequences encoding one or more heterologous glycosyltransferases; (2) a polynucleotide sequence encoding a heterologous oligosaccharide transferase; (3) a polynucleotide sequence encoding a modified Sap2 protein according to any one of claims 1 to 5; and optionally (4) a polynucleotide sequence encoding a polymerase.

11. An immunogenic composition comprising a modified Sap2 protein according to any one of claims 1 to 5, a conjugate according to claim 6, or a bioconjugate according to claim 6.

12. A Candida albicans vaccine comprising (1) a modified Sap2 protein according to any one of claims 1 to 5; (2) at least one Candida albicans sugar linked to the modified Sap2 protein; and optionally (3) a pharmaceutically acceptable carrier or adjuvant.

13. A method for treating or preventing Candida albicans infection in a subject requiring treatment or prevention of Candida albicans infection, comprising the step of administering to the subject a therapeutically effective amount of a modified Sap2 protein according to any one of claims 1 to 5 and 7, a conjugate according to claim 6, a bioconjugate according to any one of paragraphs 42 and 6, an immunogenic composition according to claim 11, or a vaccine according to claim 12.

14. A method for immunizing a subject against Candida albicans infection, comprising the step of administering to the subject an immunoprotective dose of a modified Sap2 protein according to any one of claims 1 to 5 and 7, a conjugate according to claim 6, a bioconjugate according to claim 6, an immunogenic composition according to claim 11, or a vaccine according to claim 12.

15. A method for inducing an immune response to Candida albicans infection in a subject, comprising the step of administering to the subject a therapeutic or prophylactic dose of a modified Sap2 protein according to any one of claims 1 to 5 and 7, a conjugate according to claim 6, a bioconjugate according to claim 6, an immunogenic composition according to claim 11, or a vaccine according to claim 12.

16. A modified Sap2 protein according to any one of claims 1 to 5 and 7, a conjugate according to claim 6, a bioconjugate according to claim 6, an immunogenic composition according to claim 11, or a vaccine according to claim 12, for use in the treatment or prevention of diseases caused by Candida albicans infection.

17. structure: 【Chemistry 1】 A sugar that is a β-1,2-mannan polymer containing this polymer.

18. structure: 【Chemistry 2】 Sugars containing sugars.

19. i. A nucleotide sequence encoding one or more first heterologous glycosyltransferases capable of synthesizing β-1,2-mannan polymers; ii. A nucleotide sequence encoding a second heterologous glycosyltransferase that is eukaryotic and capable of covalently bonding mannose molecules to β-1,2-mannan polymers, thereby extending the β-1,2-mannan polymer chain; iii. Nucleotide sequences encoding heterologous oligosaccharide transferases; and iv. A nucleotide sequence encoding a modified carrier protein that may include a glycosylation site containing the consensus sequence D / EXNZS / T (wherein X and Z are independently any amino acid other than proline). Host cells containing these cells.

20. A method for producing a modified carrier protein and a glycoconjugate containing β-1,2 mannan, comprising the steps of: i) culturing the host cell described in claim 19 under conditions suitable for protein production; ii) harvesting the culture to produce the harvested culture; and iii) isolating the glycoconjugate from the culture.

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