Modified protein

Abstract

The present invention relates to modified proteins, immunogenic compositions and vaccines containing modified proteins, their manufacture, and the use of such compositions in pharmaceuticals. More specifically, the present invention relates to the modified Als3 (aglutinin-like sequence 3 of Candida albicans) protein. The modified Als3 protein can be used as a carrier protein for other antigens, particularly glycogen 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, the filename is 70384US01P_SL.xml, and the size is 94,459 bytes.

[0002] Field of Invention The present invention relates to modified proteins, immunogenic compositions and vaccines containing the modified proteins, their manufacture, and the medical use of the compositions. More specifically, the present invention relates to the modified Als3 (aglutinin-like sequence 3 of Candida albicans) protein. The modified Als3 protein can be used as a carrier protein for other antigens, particularly glycosylated antigens lacking T cell epitopes or other antigens. [Background technology]

[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., immunosuppression), 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 bacterium 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. When polysaccharides are conjugated to protein carriers containing T cell epitopes, they 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 eukaryotes 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] [Problems that the invention aims to solve]

[0007] Glycoconjugates are hybrid molecules consisting of a carrier protein and multiple polysaccharide chains, with the polysaccharides covalently bound 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 periods. Furthermore, the complexity of the manufacturing process makes these products high-cost. In contrast, bioconjugation is an innovative technology that allows for the preservation of the natural immunogenic structure by producing glycoconjugate vaccines in 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 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. [Means for solving the problem]

[0008] The present invention provides a modified aglutinin-like sequence 3 (Als3) 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 Als3 carrier protein, which is linked to a Candida polysaccharide antigen at one or more asparagine residues on the modified Als3 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 aglutinin-like sequence 3 (Als3) 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 18-316 of Sequence 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] A further aspect of the present invention provides a modified Als3 protein comprising at least one fructose diphosphate aldolase-1 (Fba) peptide having 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.

[0011] A further aspect of the present invention provides a modified Als3 protein comprising the amino acid sequence of SEQ ID NO: 10.

[0012] According to a further aspect of the present invention, there is provided a modified Als3 protein of the present invention comprising the amino acid sequence of SEQ ID NO: 11.

[0013] According to a further aspect of the present invention, there is provided a conjugate (e.g., a bioconjugate) comprising a modified Als3 protein of the present invention and at least one sugar antigen.

[0014] According to a further aspect of the present invention, there is provided a modified Als3 protein of Candida albicans comprising (1) the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11 and (2) at least one sugar antigen of the genus Candida, wherein the at least one sugar antigen is a β-1,3 glucan polymer consisting of at least 6 consecutive β-1,3-linked glucose molecules, and the at least one sugar antigen is linked to at least one of the 3 asparagine residues at positions 20, 92 and 324 of SEQ ID NO: 10 or positions 20, 92 and 337 of SEQ ID NO: 11.

[0015] According to a further aspect of the present invention, there is provided a polynucleotide encoding a modified Als3 protein of the present invention.

[0016] According to a further aspect of the present invention, there is provided a vector comprising a polynucleotide encoding a modified Als3 protein of the present invention.

[0017] According to a further aspect of the present invention, there is provided a host cell comprising: (l) one or more polynucleotide sequences encoding one or more heterologous glycosyltransferases; (2) a polynucleotide sequence encoding a heterologous oligosaccharyltransferase; (3) a polynucleotide sequence encoding a modified Als3 protein of the present invention; and optionally (4) a polynucleotide sequence encoding a polymerase.

[0018] According to a further aspect of the present invention, there is provided a method for producing a bioconjugate comprising (or consisting of) a modified Als3 protein conjugated to at least one carbohydrate antigen, the method comprising: (1) culturing a host cell of the present invention under conditions suitable for protein production; and (2) isolating the bioconjugate produced by the host cell, optionally isolating the bioconjugate from a periplasmic extract obtained from the host cell.

[0019] According to a further aspect of the present invention, there is provided an immunogenic composition comprising a modified Als3 protein of the present invention, a conjugate of the present invention, or a bioconjugate of the present invention, and optionally a pharmaceutically acceptable excipient and / or carrier.

[0020] According to a further 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 Als3 protein of the present invention, a conjugate of the present invention, or a bioconjugate of the present invention with a pharmaceutically acceptable excipient or carrier.

[0021] According to a further aspect of the present invention, there is provided a vaccine comprising an immunogenic composition of the present invention, optionally a pharmaceutically acceptable excipient or carrier, and optionally an adjuvant.

[0022] According to a further aspect of the present invention, there is provided a Candida albicans vaccine comprising (1) a modified Als3 protein of the present invention; (2) at least one Candida albicans carbohydrate antigen conjugated to the modified Als3 protein; and optionally (3) a pharmaceutically acceptable carrier or adjuvant.

[0023] A further aspect of 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 administering to the subject a therapeutically effective amount of the modified Als3 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.

[0024] A further aspect of the present invention provides a method for conferring immunity to Candida albicans infection to a subject, the method comprising administering to the subject an immunoprotective dose of the modified Als3 protein of the present invention, the conjugate of the present invention, any of the bioconjugates of the present invention, the immunogenic composition of the present invention, or the vaccine of the present invention.

[0025] A further aspect of the present invention provides a method for 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 the modified Als3 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.

[0026] Further aspects of the present invention provide a modified Als3 protein, a conjugate, a bioconjugate, an immunogenic composition, or a vaccine for use in the treatment or prevention of diseases caused by Candida albicans infection.

[0027] A further aspect of the present invention provides a modified Als3 protein, a conjugate, a bioconjugate, an immunogenic composition, or a vaccine for use in the manufacture of pharmaceuticals for the treatment or prevention of diseases caused by Candida albicans infection.

[0028] A further aspect of the present invention provides a method for improving the expression level of the modified Als3 protein of the present invention, comprising substituting one or more consensus sequences for an amino acid between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1, or for 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 18-316 of SEQ ID NO: 1, wherein the modified Als3 protein exhibits an improved expression level compared to a control Als3 protein that does not contain one or more consensus sequences that substituted an amino acid between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1, or for 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 18-316 of SEQ ID NO: 1.

[0029] According to a further aspect of the present invention, a host cell, i. A nucleotide sequence encoding one or more heterologous glycosyltransferases capable of synthesizing β-1,3-glucan polymers. ii. A nucleotide sequence encoding a glycosyltransferase that can covalently bond a glucose molecule to an N-acetylglucosamine (GlcNac) molecule. iii. Nucleotide sequences encoding heterologous oligosaccharide transferases, and iv. A nucleotide sequence encoding a modified carrier protein which 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 [the specified substance] are provided.

[0030] A further aspect of the present invention provides a method for producing a glycoconjugate containing a modified carrier protein and β-1,3-glucan, comprising culturing host cells of the present invention under conditions suitable for protein production.

[0031] According to a further aspect of the present invention, the structure: [ka] A glucan having, Glucans are provided such that n is 2-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.

[0032] According to a further aspect of the present invention, the structure: [ka] A sugar that is a glucan having, Sugars are provided in which n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.

[0033] A further aspect of the present invention provides a conjugate (e.g., a bioconjugate) containing the sugar of the present invention linked to an asparagine residue of a modified carrier protein.

[0034] A host cell is provided comprising a nucleotide sequence comprising (i) a wzm gene comprising the nucleotide sequence of SEQ ID NO: 36, and optionally comprising a nucleotide sequence identical to at least 80%, 90%, 95%, 98%, or 99% of SEQ ID NO: 36, and (ii) a wzt gene comprising the nucleotide sequence of SEQ ID NO: 37, and optionally comprising a nucleotide sequence identical to at least 80%, 90%, 95%, 98%, or 99% of SEQ ID NO: 37.

[0035] According to a further aspect of the present invention, a method for producing a β-1,3-glucan polymer in a prokaryotic host cell, wherein in the host cell, i. A nucleotide sequence encoding a first glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetylglucosamine (GlcNAc) molecule, wherein the first glycosyltransferase is WfaP derived from E. coli O56. ii. A nucleotide sequence encoding a further glycosyltransferase capable of synthesizing fungal β-1,3-glucan, wherein the further glycosyltransferase comprises SleC, SleE, SleF, SleU, and SleW derived from rhizobia, possibly from the genus Agrobacterium, and possibly from the species Agrobacterium ZX09, wherein the host cell produces more SleW than SleC, SleE, SleF, or SleU, and iii. A nucleotide sequence encoding a translocase that can, in some cases, transfer β-1,3-glucan to the periplasmic side of the inner membrane of a prokaryotic host cell, wherein the translocase contains Wzm-Wzt derived from a species of the genus Klebsiella, and in some cases from Klebsiella pneumoniae. Steps to introduce and express A method is provided in which a β-1,3-glucan polymer is linked to a lipid carrier via GlcNAc, and the β-1,3-glucan polymer contains at least four β-1,3-linked glucose molecules.

[0036] According to a further aspect of the present invention, a method for generating a bioconjugate in a prokaryotic host cell, a. The step of obtaining a prokaryotic host cell of the present invention that produces a β-1,3-glucan polymer, and b. Furthermore, in host cells, i. A nucleotide sequence encoding a modified carrier protein comprising a glycosylation site including the consensus sequence D / EXNZS / T, wherein X and Z are any amino acids other than proline, and 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. A nucleotide sequence encoding an oligosaccharide transferase capable of generating a bioconjugate by transferring a β-1,3-glucan polymer from a lipid carrier to a modified carrier protein, wherein the oligosaccharide transferase is PglB derived from the genus Campylobacter, and in some cases from Campylobacter jejuni or Campylobacter coli. Steps to introduce and express A method including this is provided. [Brief explanation of the drawing]

[0037] [Figure 1] This diagram shows the structure of the Als3-NT protein from C. albicans (sequence number 27), including residues 18-316 of sequence number 1. The spheres indicate the insertion site of the glycosite. [Figure 2] This figure shows the biosynthesis scheme of the modified Als3-NT protein-glucan bioconjugate in E. coli. [Figure 3] This figure shows glycosylation tests using a series of modified Als3-NT proteins, each containing a single glycosite. [Figure 4] This figure shows the expression levels and glycosylation levels of various modified Als3-NT proteins compared to wild-type Als3-NT protein. Figure 4A shows the relative expression levels of modified Als3-NT proteins compared to wild-type Als3-NT protein. Figure 4B shows the glycosylation efficiency of modified Als3-NT proteins. [Figure 5]This figure shows SDS-PAGE and Western blot analyses of purified modified Als3-NT protein-glucan conjugates. Figure 5A: SDS-PAGE analysis. Figure 5B: Western blot analysis using anti-Als3 antibody. Figure 5C: Western blot analysis using anti-Fba antibody. Figure 5D: Western blot analysis using anti-glucan antibody. Figure 5E: Western blot analysis using anti-dectin antibody. [Figure 6] This figure shows surface plasma resonance (SPR) assays testing the binding of wt Als3-NT protein ("Als318-316wt", left panel), manipulated non-glycosylation modified Als3-NT protein ("uAls318-316-3S", center panel), and glycosylation modified Als3-NT protein ("β-glucan-Als318-316-3S", right panel) to their innate ligand, fibronectin. [Figure 7A] This figure shows the results of preclinical trials of modified Als3-NT protein-glucan bioconjugates (Als3-NT-3S-Fba_bglucd+, Als3-3FG) in rabbits. Figure 7A shows the properties of the bioconjugate. Figure 7B shows a 3D representation of the modified Als3-NT protein-glucan bioconjugate. Figure 7C shows the rabbit immunization scheme using the purified modified Als3-NT protein-glucan bioconjugate. [Figure 7B] This figure shows the results of preclinical trials of modified Als3-NT protein-glucan bioconjugates (Als3-NT-3S-Fba_bglucd+, Als3-3FG) in rabbits. Figure 7A shows the properties of the bioconjugate. Figure 7B shows a 3D representation of the modified Als3-NT protein-glucan bioconjugate. Figure 7C shows the rabbit immunization scheme using the purified modified Als3-NT protein-glucan bioconjugate. [Figure 7C]This figure shows the results of preclinical trials of modified Als3-NT protein-glucan bioconjugates (Als3-NT-3S-Fba_bglucd+, Als3-3FG) in rabbits. Figure 7A shows the properties of the bioconjugate. Figure 7B shows a 3D representation of the modified Als3-NT protein-glucan bioconjugate. Figure 7C shows the rabbit immunization scheme using the purified modified Als3-NT protein-glucan bioconjugate. [Figure 8] This figure shows the immunogenicity of the modified Als3-NT protein-glucan bioconjugate in rabbits. Figure 8A shows that the modified Als3-NT protein-glucan bioconjugate is immunogenic. Figure 8B shows that the Fba peptide (a component of the Als3-NT bioconjugate) is immunogenic. [Figure 9] This figure shows the immunogenicity of the modified Als3-NT protein-glucan bioconjugate in rabbits. [Figure 10] This figure shows the ability of an antibody against a modified Als3-NT protein-glucan bioconjugate to inhibit the attachment of C. albicans mycelium to plastics. [Figure 11] This figure shows the ability of an antibody against a modified Als3-NT protein-glucan bioconjugate to inhibit the adhesion of C. albicans to vaginal epithelial cells. Figure 11A shows the results of adhesion quantification. Figure 11B shows microscopic images of Candida species attached to epithelial cells. [Figure 12] This figure shows the ability of antibodies against a modified Als3-NT protein-glucan bioconjugate that binds to C. albicans mycelium, using whole-cell ELISA. [Figure 13] This figure shows a microscopic image of an antibody (against a modified Als3-NT protein-glucan bioconjugate) bound to C. albicans cells. [Figure 14] This figure shows a microscopic image of an antibody (against a modified Als3-NT protein-glucan bioconjugate) bound to C. auris VPCI479 / P / 13 cells. [Figure 15] This figure shows the ability of an antibody against a modified Als3-NT protein-glucan bioconjugate to inhibit biofilm formation of C. albicans mycelium on a 96-well plate. [Figure 16] This figure shows the ability of an antibody against a modified Als3-NT protein-glucan bioconjugate to mediate neutrophil killing in C. albicans mycelium. [Modes for carrying out the invention]

[0038] definition As used herein, the term “Als3 protein or Als3” means a wild-type agglutinin-like sequence 3 protein having a wild-type leader sequence (amino acid residues 1-17) at its N-terminus. In certain embodiments, the Als3 protein is derived from the Candida genus, and possibly from Candida albicans. In specific embodiments, the Als3 protein contains the amino acid sequence of Sequence ID No. 1.

[0039] As used herein, the term “Als3-NT protein or Als3-NT” means the N-terminal fragment of the aglutinin-like sequence 3 (Als3) protein. In certain embodiments, the Als3-NT protein is derived from the Candida genus, and possibly from Candida albicans. In some embodiments, the Als3-NT protein contains amino acid residues 18-316 of SEQ ID NO: 1. In other embodiments, the Als3-NT protein contains amino acid residues 18-329 of SEQ ID NO: 1. In yet another embodiment, the Als3-NT protein contains amino acid residues 1-316 of SEQ ID NO: 1. In yet another embodiment, the Als3-NT protein contains amino acid residues 1-329 of SEQ ID NO: 1. In yet another embodiment, the Als3-NT protein contains amino acid sequences selected from, but not limited to, the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and SEQ ID NO: 41.

[0040] As used herein, the term “modified” protein means a protein that has been modified (in one or more ways) compared to the wild-type protein (for example, “modified Als3 protein” excludes the wild-type Als3 protein). In certain embodiments, a modified Als3 protein means an Als3 protein containing one or more consensus sequences, where one or more consensus sequences are added to or substituted for one or more amino acid residues 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 SEQ ID NO: 1. In other embodiments, a modified Als3 protein means an Als3 protein containing a deletion at the C-terminus or N-terminus of SEQ ID NO: 1, or 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 yet another embodiment, a modified Als3 protein means an Als3 protein comprising the addition / deletion / substitution of one or more amino acids in 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 a particular embodiment, a modified Als3 protein means an Als3 protein comprising one or more consensus sequences (for example, comprising the amino acid sequence D / EXNZS / T, where X and Z are independently any amino acid other than proline), wherein one or more consensus sequences are added or substituted next to or in the same position as one or more amino acid residues of SEQ ID NO: 1, or in 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: 1.In other embodiments, a modified Als3 protein is an Als3 protein comprising one or more consensus sequences (for example, the amino acid sequence D / EXNZS / T, where X and Z are independently any amino acid other than proline), wherein one or more consensus sequences are added to or substituted for one or more amino acid residues within amino acid residues 1-329 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 1-329 of SEQ ID NO: 1. In yet another embodiment, a modified Als3 protein is an Als3 protein that includes one or more consensus sequences (for example, the amino acid sequence D / EXNZS / T, where X and Z are independently any amino acid other than proline), and the one or more consensus sequences are added to or substituted for one or more amino acid residues within amino acid residues 18-329 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 18-329 of SEQ ID NO: 1. In further embodiments, a modified Als3 protein is an Als3 protein comprising one or more consensus sequences (for example, the amino acid sequence D / EXNZS / T, where X and Z are independently any amino acid other than proline), wherein one or more consensus sequences are added to or substituted for one or more amino acid residues within amino acid residues 1-316 of SEQ ID NO: 1, or 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 1-316 of SEQ ID NO: 1.In specific embodiments, a modified Als3 protein is an Als3 protein containing one or more consensus sequences (for example, the amino acid sequence D / EXNZS / T, where X and Z are independently any amino acid other than proline), where one or more consensus sequences are added to or substituted for one or more amino acid residues within amino acid residues 18-316 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 18-316 of SEQ ID NO: 1. In certain embodiments, the modified Als3 protein of the present invention is an isolated modified Als3 protein. In other embodiments, the modified Als3 protein of the present invention is a recombinant modified Als3 protein. In yet another embodiment, the modified Als3 protein of the present invention is an isolated recombinant modified Als3 protein.

[0041] As used herein, the term “modified Als3-NT protein” or “modified Als3-NT” means an Als3-NT protein into which one or more glycosite sequences (e.g., D / EXNZS / T) have been introduced. In certain embodiments, the modified Als3-NT protein is derived from the genus Candida, and possibly from Candida albicans. In some embodiments, the modified Als3-NT protein contains the amino acid sequence of SEQ ID NO: 10. In other embodiments, the modified Als3-NT protein contains the amino acid sequence of SEQ ID NO: 11. In further embodiments, the modified Als3-NT protein contains, but is not limited to, an amino acid sequence selected from the amino acid sequences of Mut1, Mut2, Mut3, Mut4, Mut5, Mut6, Mut7, Mut8, Mut9, Mut10, Mut11, Mut12, Mut13, Mut14, Mut15, Mut16, Mut17 and Mut18.

[0042] As used herein, the term “control Als3 protein” means, but is not limited to: (1) an insertion or substitution of one or more amino acids adjacent to (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) one or more amino acids of SEQ ID NO: 1, or an N-terminus and / or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to one of SEQ ID NO: 1. (2) Als3 protein that does not contain one or more consensus sequences attached to the C-terminus, (3) an Als3 protein that is inserted next to or replaces one or more amino acids among amino acid residues 18-316 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 SEQ ID NO: 1), or amino acid residues 18-316 of SEQ ID NO: 1 (or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%). Als3 protein that does not contain one or more consensus sequences added to the N-terminus and / or C-terminus (of an amino acid sequence that is % or 99% identical), (3) an Als3 protein that is inserted next to or replaces one or more amino acids among amino acid residues 1-329 of SEQ ID NO: 1 (or at 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 1-329 of SEQ ID NO: 1 (or amino acid residues 1-3 of SEQ ID NO: 1) Als3 protein that does not contain one or more consensus sequences attached to the N-terminus and / or C-terminus (of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to 29), or (4) inserted or substituted next to one or more amino acids among amino acid residues 18-329 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 18-329 of SEQ ID NO: 1,Alternatively, an Als3 protein that does not contain one or more consensus sequences attached to the N-terminus and / or C-terminus of amino acid residues 18-329 of SEQ ID NO: 1 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-329 of SEQ ID NO: 1). Therefore, the control Als3 protein includes, but is not limited to, the wild-type Als3 protein, the wild-type Als3 protein of SEQ ID NO: 1, the Als3 protein containing amino acid residues 1-316 of SEQ ID NO: 1, the Als3 protein containing amino acid residues 18-316 of SEQ ID NO: 1, the Als3 protein containing amino acid residues 1-329 of SEQ ID NO: 1, the Als3 protein containing amino acid residues 18-329 of SEQ ID NO: 1, and the modified Als3 protein of the present invention which does not contain one or more consensus sequences in which amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 are substituted at equivalent positions within the same amino acid sequence as amino acid residues 18-316 of SEQ ID NO: 1 by at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%.

[0043] As used herein, the term "carrier protein" refers to a protein that can bind to an antigen (e.g., a glycan antigen such as a fungal polysaccharide antigen) to form a conjugate (e.g., 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 antigen of the present invention. In certain embodiments, the carrier protein is the modified Als3 protein of the present invention.

[0044] 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).

[0045] As used herein, the term “natural amino acid residue” refers to amino acids that are naturally incorporated into polypeptides. In particular, 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).

[0046] 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 forming glycosides. For example, these enzymes catalyze the transfer of a sugar chain portion from an activated nucleotide sugar (also known as a “glycosyl donor”) to a nucleophilic glycosyl acceptor molecule (whose nucleophile may be an oxygen, carbon, nitrogen, or sulfur-based nucleophile).

[0047] As used herein, the term “oligosaccharide transferase (OTase or OST)” refers to an enzyme that catalyzes the mechanistically specific and selective transfer (glycosylation) 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.

[0048] 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.

[0049] As used herein, the terms “lipopolysaccharide” or “LPS” refer to large molecules containing covalently linked lipids and polysaccharides.

[0050] 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.

[0051] As used herein, the term "wzy" refers to a polysaccharide polymerase gene that encodes an enzyme that catalyzes polysaccharide polymerization. The encoded enzyme transfers oligosaccharide units to non-reducing ends to form glycosidic bonds.

[0052] As used herein, the term "waaL" refers to the O antigen ligase gene that encodes a membrane-binding enzyme. The encoded enzyme transfers the undecaprenyl diphosphate (UPP)-bound O antigen to the lipid A core oligosaccharide to form a lipopolysaccharide.

[0053] 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 does not participate in glycosidic bonding and is therefore convertible to an open-chain form.

[0054] As used herein, the term "conjugate" refers to a carrier protein covalently bound to an antigen.

[0055] 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 background of a host cell, 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.

[0056] 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 Als3 protein fragments of the present invention still contain the described modifications made to the Als3 protein.

[0057] 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 substitutions may be 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 may produce polypeptides having similar functional and chemical properties to the parent polypeptide.

[0058] 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 (e.g., 1 to 4 residues).

[0059] 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).

[0060] As used herein, the expression "adjacently added" means adding 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, "adding adjacent to one or more amino acids between amino acid residues 33-37" means adding at a position adjacent to any one of amino acid residues 33-37 (including positions adjacent to amino acid residues 33 or 37).

[0061] As used herein, the term "glycosyte" refers to an amino acid sequence recognized by a bacterial oligosaccharide transferase, such as PglB of Campylobacter jejuni. Therefore, a glycosite is an amino acid sequence within a carrier protein (for example, the modified Als3 protein or the modified Als3-NT protein of the present invention) to which an antigenic sugar chain (for example, a β-1,3-glucan polymer) is covalently or noncovalently bound.

[0062] 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).

[0063] 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 18 in SEQ ID NO: 1) may substitute for that amino acid residue.

[0064] Unless otherwise specified, a numerical range (e.g., "33-37") includes the endpoints (i.e., the values ​​of 33 and 37). For example, "between amino acids 18 to 316 of SEQ ID NO: 1" refers to the amino acid sequence positions between amino acids 18 and 316 of SEQ ID NO: 1, which include both amino acids 18 and 316.

[0065] 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 include 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 (e.g., sequence number 1 of the present invention).

[0066] 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.

[0067] 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).

[0068] As used herein, the term “subject” refers to animals, particularly mammals such as primates (e.g., humans).

[0069] As used herein, the term “therapeutic or prophylactic effective dose” in the context of administering a therapeutic agent (e.g., the immunogenic composition or vaccine of the present invention) to a subject means an amount of therapeutic agent having a prophylactic and / or therapeutic effect. In a particular embodiment, “therapeutic or prophylactic effective 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 hospitalizations in subjects with a fungal infection; (ix) shortening the length of hospitalization in 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 effect(s) of other treatments.

[0070] As used herein, the term “immunoprotective dose” in the context of administering a therapeutic agent (e.g., the immunogenic composition or vaccine of the present invention) to a subject 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 hospitalizations in subjects with a fungal infection; (ix) shortening the length of hospitalizations in 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.

[0071] 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."

[0072] 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 (e.g., fungi). In infants and the elderly, conjugating polysaccharide antigens with proteins that induce T cell-dependent responses can induce a protective immune response against these antigens.

[0073] The term "glycoconjugate vaccine" refers to a vaccine that contains a protein carrier conjugated to an antigenic or immunogenic oligosaccharide.

[0074] As used herein, "undecaprenyl" or "und" refers to an undecaprenol lipid composed 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.

[0075] 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).

[0076] 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).

[0077] sleC refers to glycosyltransferase. In certain embodiments, sleC is the glycosyltransferase of Agrobacterium species ZX09. In some embodiments, sleC is wild-type glycosyltransferase. In other embodiments, sleC is a glycosyltransferase that does not exist in nature (e.g., mutant and / or recombinant).

[0078] sleE refers to glycosyltransferase. In certain embodiments, sleE is the glycosyltransferase of Agrobacterium species ZX09. In some embodiments, sleE is wild-type glycosyltransferase. In other embodiments, sleE is a glycosyltransferase that does not exist in nature (e.g., mutant and / or recombinant).

[0079] sleF refers to glycosyltransferase. In certain embodiments, sleF is the glycosyltransferase of Agrobacterium species ZX09. In some embodiments, sleF is a wild-type glycosyltransferase. In other embodiments, sleF is a glycosyltransferase that does not exist in nature (e.g., a mutant and / or recombinant).

[0080] sleU refers to glycosyltransferase. In certain embodiments, sleU is the glycosyltransferase of Agrobacterium species ZX09. In some embodiments, sleU is wild-type glycosyltransferase. In other embodiments, sleU is a glycosyltransferase that does not exist in nature (e.g., mutant and / or recombinant).

[0081] sleW refers to glycosyltransferase. In certain embodiments, sleW is the glycosyltransferase of Agrobacterium species ZX09. In some embodiments, sleW is wild-type glycosyltransferase. In other embodiments, sleW is a glycosyltransferase that does not exist in nature (e.g., mutant and / or recombinant).

[0082] WfaP refers to a glycosyltransferase capable of covalently bonding glucose to GlcNac. In certain embodiments, WfaP is the glycosyltransferase from E. coli O56. In some embodiments, WfaP is a wild-type glycosyltransferase. In other embodiments, WfaP is a glycosyltransferase that does not exist in nature (e.g., from mutants and / or recombinants).

[0083] Wzm-Wzt refers to a translocase. In certain embodiments, Wzm-Wzt is a translocase of Klebsiella pneumoniae. In some embodiments, Wzm-Wzt is a wild-type translocase. In other embodiments, Wzm-Wzt is a translocase that does not exist in nature (e.g., from mutants and / or recombinants).

[0084] 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 the Campylobacter coli oligosaccharide transferase. In some embodiments, pglB is a wild-type oligosaccharide transferase. In other embodiments, PglB is an oligosaccharide transferase that does not exist in nature. In specific embodiments, the pglB of the present invention contains the amino acid sequence of Sequence ID No. 20. In further embodiments, the pglB protein is an evolved pglB, i.e., an evolved oligosaccharide transferase. "Evolved" means a protein or nucleic acid that has undergone directed evolution. Directed evolution is a method used in protein engineering that mimics the process of natural selection to guide proteins or nucleic acids to meet user-defined goals. The process of directed evolution consists of supplying genes to iterative rounds of mutagenesis (creating a library of mutants), selection (expressing these mutants and isolating members with desired functions), and amplification (generating templates for the next round). The process of directed evolution can be carried out in vivo (in a living organism) or in vitro (in a cell or free in solution). Directed evolution is used in both protein engineering as an option for rationally designing modified proteins, and in experimental evolutionary studies of fundamental evolutionary principles in a controlled laboratory environment. Thus, in certain embodiments, pglB contains one or more mutations that enhance the activity of pglB against the sugar antigen of the present invention. In certain embodiments, the pglB of the present invention is an evolved pglB that more efficiently transfers the sugar antigen of the present invention to the modified Als3 protein of the present invention compared to wild-type pglB (e.g., wild-type pglB obtained from Campylobacter jejuni).

[0085] Als3 protein Candida albicans agglutinin-like sequence 3 protein ("Als3") is a multifunctional adhesin and invasin, a C. albicans hyphae-specific cell surface protein that enables C. albicans to adhere to biotic and abiotic surfaces, invade host cells, and acquire iron (Liu and Filler, 2011, Eukaryotic Cell, 10(2):168-173; Phan QT et al., 2007, PLOS biol., 5(3):e64). Als3 is a member of the agglutinin-like sequence (Als) family of proteins and is encoded by the ALS3 gene. The N-terminus of the Als3 protein contains a signal peptide ("SP") followed by a 300-amino acid immunoglobulin-like domain ("NT") and a 104-amino acid threonine-rich domain ("T") containing a β-sheet (Figure 1A). The N-terminal domain of the Als3 protein ("Als3-NT") contains a peptide bond cavity, which is necessary for its adhesion function (Lin J et al., 2014, J. Biol. Chem., 280(26):18401~18412). Antibodies obtained using the Als3-NT domain for immunization block the adhesion of Candida to host endothelial and epithelial cells (Coleman, DA et al., 2009, J. Mol. Meth.). The central domain of the Als3 protein consists of a variable number of 36 amino acid tandem repeats ("TR") (Figure 1A). These repeats are rich in serine and threonine, exposed on the cell surface, and necessary for adhesion. Because the tandem repeats are hydrophobic, they can directly mediate adhesion to some substrates, such as polystyrene. The C-terminus ("CT") of the Als3 protein is predicted to be rich in serine and threonine and highly glycosylated. This C-terminus contains a glycosylphosphatidylinositol anchor sequence, which is cleaved when the protein covalently attaches to the cell wall.

[0086] Als3 functions as an adhesin, mediating the entry of C. albicans by binding to epithelial cells, endothelial cells, and extracellular matrix proteins. Als3 also plays a crucial role in biofilm formation on prosthetic surfaces, both alone and in mixed infections with Streptococcus gordonii. Als3 is one of two known invasins of C. albicans. Als3 binds to host cell receptors, such as E-cadherin and N-cadherin, thereby inducing host cells to endocytose the organism. Als3 also binds to ferritin in host cells, allowing C. albicans to utilize this protein as an iron source.

[0087] Als3 is produced as a precursor within C. albicans, from which a 17-amino acid leader sequence ("signal peptide") is removed during the maturation process (Liu Y and Filler SG, 2011, Eukaryot Cell, 10(2):168-173). The Als3 protein useful in the present invention can be generated by methods known in the art in consideration of this disclosure, see, for example (Gong J et al., 2019, Antimicrob Agents Chemother, 64(1):e01975-79).

[0088] In a particular aspect of the present invention, the full-length wild-type Als3 protein of C. albicans contains the amino acid sequence of SEQ ID NO: 1:

[0089] Sequence ID 1: Full-length wild-type Als3 protein sequence from C. albicans (with underlined wild-type leader sequence) [ka] [ka]

[0090] In certain embodiments, the present invention provides a modified Als3 protein. The term "modified Als3 protein" means an Als3 protein comprising a certain amino acid sequence (for example, the amino acid sequence of SEQ ID NO: 1, or having 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), wherein the Als3 amino acid sequence is modified by the addition, substitution, or deletion of one or more amino acids (for example, by the addition of a consensus sequence(s) selected from D / EXNZS / T, KD / EXNZS / T and / or an extended consensus sequence(s) (e.g., JUBD / EXNZS / TJUB), and / or by the substitution of one or more amino acids by a consensus sequence(s) selected from D / EXNZS / T, KD / EXNZS / T). For example, a modified Als3 protein may have the Als3 amino acid sequence of SEQ ID NO: 1, but is modified in that the amino acid sequence includes one or more consensus sequences selected from D / EXNZS / T, KD / EXNZS / T, and / or an extended consensus sequence (e.g., JUBD / EXNZS / TJUB). As used herein, in the consensus sequences 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 Als3 protein of the present invention may include further modifications (e.g., addition, substitution, and / or deletion of one or more amino acid residues). In specific embodiments, the modified Als3 protein of the present invention includes a C-terminal deletion of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1. Therefore, in certain embodiments, the modified Als3 protein of the present invention contains an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-316 of SEQ ID NO: 1.In other embodiments, the modified Als3 protein of the present invention contains an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-329 of SEQ ID NO: 1, or amino acid residues 18-316 of SEQ ID NO: 1. In further embodiments, the modified Als3 protein of the present invention contains an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-329 of SEQ ID NO: 1, or amino acid residues 18-316 of SEQ ID NO: 1. In preferred embodiments, the modified Als3 protein of the present invention contains an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1, or amino acid residues 18-316 of SEQ ID NO: 1.

[0091] In some embodiments, the modified Als3 protein of the present invention is an Als3 protein that does not exist in nature (i.e., non-native). In certain embodiments, the modified Als3 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 Als3 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 Als3 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 Als3 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 Als3 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 Als3 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 Als3 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 Als3 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 Als3 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 Als3 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 Als3 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 Als3 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 Als3 protein of the present invention may have an amino acid sequence that is at least 99% identical to SEQ ID NO: 1.

[0092] In certain embodiments, the modified Als3 protein of the present invention contains one or more consensus sequences. The terms “glycocytic sequence,” “consensus glycocytic sequence,” and “consensus sequence” are used interchangeably herein. In certain embodiments, the modified Als3 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 Als3 protein of the present invention contains one, two, three, four, five, six, seven, eight, nine, or ten consensus sequences. In certain embodiments, the modified Als3 protein of the present invention contains at least three consensus sequences. In preferred embodiments, the modified Als3 protein of the present invention contains three consensus sequences. In certain embodiments, the modified Als3 protein of the present invention contains one or more consensus sequences, all of which have the same amino acid sequence. In other embodiments, the modified Als3 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 Als3 protein of the present invention includes one or more consensus sequences in which at least two consensus sequences have the same amino acid sequence.

[0093] Accordingly, in certain embodiments, the present invention provides a modified Als3 protein having an amino acid sequence that is modified in that the 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 has an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0094] In certain embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, each of which is adjacent to or substitutes for one or more amino acids selected from specific amino acid residues (consensus sequence sites) within the modified Als3 protein of the present invention. One or more of these consensus sequence sites independently correspond to: (1) one or more amino acids between amino acid residues 18-23 (e.g., amino acid residue 18), (2) one or more amino acids between amino acid residues 311-316 (e.g., amino acid residue 316), (3) one or more amino acids between amino acid residues 28-42 (e.g., one or more amino acids between amino acid residues 33-37), (4) one or more amino acids between amino acid residues 75-87 (e.g., one or more amino acids between amino acid residues 80-82), (5) one or more amino acids between amino acid residues 82-92 (e.g., amino acid residue 87), (6) one or more amino acids between amino acid residues 99-113 (e.g., one or more amino acids between amino acid residues 104-108), (7) one or more amino acids between amino acid residues 114-126 (e.g., one or more amino acids between amino acid residues 119-121), (8) one or more amino acids between amino acid residues 118-132 (e.g., (1) One or more amino acids between amino acid residues 123-127, (9) One or more amino acids between amino acid residues 150-164 (for example, one or more amino acids between amino acid residues 155-159), (10) One or more amino acids between amino acid residues 158-169 (for example, one or more amino acids between amino acid residues 163-164), (11) One or more amino acids between amino acid residues 163-177 (for example, one or more amino acids between amino acid residues 168-172), ( 12) One or more amino acids between amino acid residues 170-184 (for example, one or more amino acids between amino acid residues 175-179), (13) One or more amino acids between amino acid residues 202-212 (for example, amino acid residue 207), (14) One or more amino acids between amino acid residues 215-225 (for example, amino acid residue 220), (15) One or more amino acids between amino acid residues 231-242 (for example, one or more amino acids between amino acid residues 236-237),(16) One or more amino acids between amino acid residues 265-275 (e.g., amino acid residue 270), (17) One or more amino acids between amino acid residues 271-281 (e.g., amino acid residue 276), (18) One or more amino acids between amino acid residues 281-292 (e.g., one or more amino acids between amino acid residues 286-287), and (19) One or more amino acids between amino acid residues 294-305 (e.g., one or more amino acids between amino acid residues 299-300), or selected from equivalent positions (multiple positions are possible) within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0095] In certain embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, the one or more consensus sequences comprising one or more amino acids between amino acid residues 18-23, one or more amino acids between amino acid residues 28-42, one or more amino acids between amino acid residues 75-87, one or more amino acids between amino acid residues 82-92, one or more amino acids between amino acid residues 99-113, one or more amino acids between amino acid residues 114-126, one or more amino acids between amino acid residues 118-132, one or more amino acids between amino acid residues 150-164, one or more amino acids between amino acid residues 158-169, one or more amino acids between amino acid residues 163-177, and amino acids It is added next to or substituted for one or more amino acids selected from the group consisting of one or more amino acids between residues 170-184, one or more amino acids between amino acid residues 202-212, one or more amino acids between amino acid residues 215-225, one or more amino acids between amino acid residues 231-242, one or more amino acids between amino acid residues 265-275, one or more amino acids between amino acid residues 271-281, one or more amino acids between amino acid residues 281-292, and one or more amino acids between amino acid residues 294-305, or at 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 18-316 of SEQ ID NO: 1.

[0096] In certain embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for amino acid residue 18 of the amino acid residues 18-316 of SEQ ID NO: 1, or 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 18-316 of SEQ ID NO: 1.

[0097] In other embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for one or more amino acids between amino acid residues 33-37 of amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0098] In some embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for one or more amino acids between amino acid residues 80-82 of amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0099] In yet another embodiment, the modified Als3 protein of the present invention comprises one or more consensus sequences, one or more of which are added to or substituted for amino acid residue 87 among amino acid residues 18-316 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 18-316 of SEQ ID NO: 1.

[0100] In certain embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for one or more amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0101] In certain embodiments, substitution of an amino acid between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1, or one or more consensus sequences 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 18-316 of SEQ ID NO: 1, results in an increased expression level of the modified Als3 protein compared to the control Als3 protein. In some embodiments, the expression level of the modified Als3 protein is increased by at least approximately 2, at least approximately 3, at least approximately 4, at least approximately 5, at least approximately 6, at least approximately 7, at least approximately 8, at least approximately 9, or at least approximately 10 times compared to the control Als3 protein. In other embodiments, the expression level of the modified Als3 protein is increased by approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 7, approximately 8, approximately 9, or approximately 10 times compared to the control Als3 protein. In specific embodiments, substitution of an amino acid between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1, or one or more consensus sequences 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 18-316 of SEQ ID NO: 1, results in an approximately four-fold increase in the expression level of the modified Als3 protein compared to the control Als3 protein.

[0102] In further embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for one or more amino acids between amino acid residues 119-121 of amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0103] In other embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for one or more amino acids between amino acid residues 123-127 of amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0104] In yet another embodiment, the modified Als3 protein of the present invention comprises one or more consensus sequences, one or more of which are added to or substituted for one or more amino acids between amino acid residues 155-159 of amino acid residues 18-316 of SEQ ID NO: 1, or at 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 18-316 of SEQ ID NO: 1.

[0105] In yet another embodiment, the modified Als3 protein of the present invention comprises one or more consensus sequences, one or more of which are added to or substituted for one or more amino acids between amino acid residues 163-164 of amino acid residues 18-316 of SEQ ID NO: 1, or at 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 18-316 of SEQ ID NO: 1.

[0106] In yet another embodiment, the modified Als3 protein of the present invention comprises one or more consensus sequences, one or more of which are added to or substituted for one or more amino acids between amino acid residues 168-172 of amino acid residues 18-316 of SEQ ID NO: 1, or at 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 18-316 of SEQ ID NO: 1.

[0107] In a further embodiment, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for one or more amino acids between amino acid residues 175-179 of amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0108] In other embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for amino acid residue 207 among amino acid residues 18-316 of SEQ ID NO: 1, or 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 18-316 of SEQ ID NO: 1.

[0109] In yet another embodiment, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for amino acid residue 220 among amino acid residues 18-316 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 18-316 of SEQ ID NO: 1.

[0110] In specific embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for one or more amino acids between amino acid residues 236-237 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0111] In certain embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, one or more of which are added to or substituted for amino acid residue 270 among amino acid residues 18-316 of SEQ ID NO: 1, or 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 18-316 of SEQ ID NO: 1.

[0112] In yet another embodiment, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for amino acid residue 276 among amino acid residues 18-316 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 18-316 of SEQ ID NO: 1.

[0113] In yet another embodiment, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for one or more amino acids between amino acid residues 286-287 of amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0114] In further embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, which are added to or substituted for one or more amino acids between amino acid residues 299-300 of amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0115] In certain embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, one or more of which substitute (i) amino acids between amino acid residues 33-37 and (ii) amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0116] In other embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, one or more of which are added to or substituted for (i) amino acids between amino acid residues 33-37, (ii) amino acids between amino acid residues 104-108, and (iii) amino acid residue 316 of the amino acid residues 18-316 of SEQ ID NO: 1, or 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 18-316 of SEQ ID NO: 1.

[0117] In yet another embodiment, the modified Als3 protein of the present invention comprises one or more consensus sequences, one or more of which are added to or substituted for (i) amino acids between amino acid residues 33-37, (ii) amino acids between amino acid residues 104-108, and (iii) amino acid residue 329 of SEQ ID NO: 1, or 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 18-329 of SEQ ID NO: 1.

[0118] In further embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, one or more of which are added to or substituted for (i) amino acids between amino acid residues 33-37, (ii) amino acids between amino acid residues 104-108, (iii) amino acids between amino acid residues 163-164, (iv) amino acid residue 220, (v) amino acids between amino acid residues 299-300, and (vi) amino acid residue 316 of amino acid residue 1, or 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 18-316 of SEQ ID NO: 1.

[0119] In further embodiments, the modified Als3 protein of the present invention comprises one or more consensus sequences, one or more of which are added to or substituted for (i) amino acids between amino acid residues 33-37, (ii) amino acids between amino acid residues 104-108, (iii) amino acids between amino acid residues 163-164, (iv) amino acid residue 220, (v) amino acids between amino acid residues 299-300, and (vi) amino acid residue 329 of amino acid residue 1, or 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 18-329 of SEQ ID NO: 1.

[0120] In certain embodiments, the modified Als3 protein of the present invention is derived from a fungus. In certain embodiments, the fungus is of the genus Candida. Therefore, in some embodiments, the modified Als3 protein of the present invention is derived from the genus Candida. In certain embodiments, the genus Candida includes, but is not limited to, Candida albicans, Candida auris, Candida guilliermondi, Candida lusitaniaea, and Candida tropicalis. In specific embodiments, the modified Als3 protein of the present invention is derived from Candida albicans. In certain embodiments, at least one of one or more consensus sequences contains the amino acid sequence KD / EXNZS / T, where 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, KDQNAT (SEQ ID NO: 5), KDQNAS (SEQ ID NO: 6), and DQNAT (SEQ ID NO: 7). In specific embodiments, X is Q (glutamine), Z is A (alanine), and one or more consensus sequences are selected from the group consisting of KDQNAT (SEQ ID NO: 5), KDQNAS (SEQ ID NO: 6), and DQNAT (SEQ ID NO: 7).

[0121] In certain embodiments, the modified Als3 protein of the present invention further comprises at least one fructose diphosphate aldolase (Fba) peptide. The Fba peptide is a 14-mer peptide (Fba-1) derived from the N-terminal portion of the fructose diphosphate aldolase protein. The Fba-1 protein is a multifunctional cell wall protein of C. albicans and is an important enzyme in the glycolysis pathway. The Fba-1 protein can promote the binding of fungi to human cells or abiotic surfaces and protect Candida cells from the host immune system (Elamin E, et al., 2021, J. Immunol. Res., 2021: pp. 1-19). In addition, the Fba-1 protein promotes the detoxification of reactive oxygen species generated during respiratory bursts. Proteomic analysis has revealed that Fba1 is the most abundant and stable enzyme in the Candida genus and is considered to be one of the main immunodominant proteins (Elamin E, et al., 2021, J. Immunol. Res., 2021: pp. 1-19). Fba peptides have traditionally been used to produce autologous adjuvant 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.

[0122] In specific embodiments, the modified Als3 protein of the present invention comprises at least one Fba peptide. In certain embodiments, the at least one Fba peptide comprises the amino acid sequence 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 Als3 protein of the present invention. In some embodiments, the at least one Fba peptide is non-covalently ligated to the modified Als3 protein of the present invention. In other embodiments, the at least one Fba peptide is covalently ligated to the modified Als3 protein of the present invention. In further embodiments, the Fba peptide is ligated to the modified Als3 protein of the present invention by a single amino acid residue. In other embodiments, the Fba peptide is ligated to the modified Als3 protein of the present invention by two or more amino acid residues. In further embodiments, the Fba peptide is ligated to the modified Als3 protein of the present invention by one or more amino acid residues. In certain embodiments, one or more amino acid residues include, but are not limited to, amino acid residues 89, 163, 259, 199, and 316. In specific embodiments, the Fba peptide is linked to the modified Als3 protein of the present invention by amino acid residue 316. The numbering of amino acid residues as 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, it refers to the position equivalent to the numbering in SEQ ID NO: 1 when the sequence is aligned with 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 linked to the modified Als3 protein of the present invention at one or more amino acid residues selected from the group consisting of 89, 163, 259, 199, and 316 among amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions (multiple) within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.In specific embodiments, the Fba peptide is covalently linked to the modified Als3 protein of the present invention at amino acid residue 316 of amino acid residues 18-316 of SEQ ID NO: 1, or at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0123] In certain embodiments, the modified Als3 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 Als3 protein of the present invention. In other embodiments, the modified Als3 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 Als3 protein of the present invention comprises the amino acid sequence YGKDVKDLFDYAQE (SEQ ID NO: 3), or an Fba peptide having an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 92% identical to SEQ ID NO: 3, and at least one 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. In specific embodiments, the modified Als3 protein includes at least one further consensus sequence comprising the amino acid sequence JUBD / EXNZS / TJUB, where 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 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. In certain embodiments, the at least one further consensus sequence comprises the amino acid sequence JUBD / EXNZS / TJUB, where X and Z are independently any amino acid other than proline, and J, U, and B independently comprise one 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 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 glycine (G) residues.In specific embodiments, J contains 1 to 5 glycine (G) residues. In other embodiments, B contains at least 1 glycine (G) residue. In certain embodiments, B contains 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 glycine (G) residues. In specific embodiments, B contains 1 to 5 glycine (G) residues. In further embodiments, J and B each contain 1 to 5 glycine (G) residues. In further embodiments, U contains at least 1 glycine (G) residue. In certain embodiments, U contains 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 glycine (G) residues. In specific embodiments, U contains 1 to 5 serine (S) residues. In some embodiments, 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. In specific embodiments, a further consensus sequence includes (or consists of) the amino acid sequence GSGGGDQNATGSGGG (SEQ ID NO: 9).

[0124] In a particular embodiment, the modified Als3 protein of the present invention comprises (or consists of) the amino acid sequence of SEQ ID NO: 10: [ka]

[0125] In other embodiments, the modified Als3 protein of the present invention comprises (or consists of) the amino acid sequence of SEQ ID NO: 11: [ka]

[0126] In certain embodiments, the modified Als3 protein of the present invention is glycosylated. In certain embodiments, the modified Als3 protein of the present invention is N-glycosylated.

[0127] The inventors unexpectedly discovered that, when expressed in host cells, the modified Als3 protein of the present invention ("Mut4 mutant") containing at least one glycosite between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 shows a >2-fold improvement in protein expression compared to control (e.g., wild-type) Als3-NT expression. In further embodiments, the present invention provides a method for improving the expression level of the modified Als3 protein of the present invention. In certain embodiments, the method for improving the expression level of the modified Als3 protein of the present invention comprises substituting an amino acid between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 with one or more consensus sequences 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 18-316 of SEQ ID NO: 1, wherein the modified Als3 protein exhibits an improved expression level compared to the control Als3 protein.

[0128] In a particular embodiment, the present invention provides a method for improving the expression level of the modified Als3 protein of the present invention in host cells, comprising substituting an amino acid between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 with one or more consensus sequences 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 18-316 of SEQ ID NO: 1, wherein the modified Als3 protein, when expressed in host cells, exhibits an improved expression level compared to a control Als3 protein that does not contain one or more consensus sequences, which are substituted between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0129] Those skilled in the art will understand that a reference to "aminamino acids..." (for example, "aminamino acids 33-37") refers to the amino acid numbers counted consecutively from the N-terminus of an amino acid sequence, and for example, "aminamino acids 33-37... of SEQ ID NO: 1" refers to the position in the amino acid sequence between amino acid 33 and amino acid 37 of SEQ ID NO: 1, including both amino acids 33 and 37. Therefore, in certain embodiments, when "one or more consensus sequences are added next to or substituted for one or more amino acids between amino acid residues 33-37 of SEQ ID NO: 1", the one or more consensus sequences may be added next to or substituted for any one (or more) of amino acid numbers 33, 34, 35, 36, and 37 in SEQ ID NO: 1. Those skilled in the art will understand that if the Als3 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).

[0130] 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.

[0131] In certain embodiments, the modified Als3 protein of the present invention is an isolated modified Als3 protein. In other embodiments, the modified Als3 protein of the present invention is a recombinant modified Als3 protein. In yet another embodiment, the modified Als3 protein of the present invention is an isolated recombinant modified Als3 protein.

[0132] Compass array In certain embodiments, the modified Als3 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). Therefore, the consensus sequence in the modified Als3 protein of the present invention may (or may consist of) the D / EXNZS / T consensus sequence.

[0133] In the modified Als3 protein of the present invention, the consensus sequence can be selected from D / EXNZS / T, KD / EXNZS / T (SEQ ID NO: 4), or JUBD / EXNZS / TJUB (wherein X is Q (glutamine) and Z is A (alanine)). In the modified Als3 protein of the present invention, the consensus sequence(s) can be selected from D / EXNZS / T and KD / EXNZS / T (SEQ ID NO: 4) (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: 7), also known as "DQNAT" (SEQ ID NO: 7). In other embodiments, the consensus sequence is KD / EXNZS / T(sequence number 4) (wherein X is Q (glutamine) and Z is A (alanine)), for example, KDQNAT(sequence number 5), also known as "KDQNAT"(sequence number 5). In yet another embodiment, the consensus sequence is KD / EXNZS / T(sequence number 4) (wherein X is Q (glutamine) and Z is A (alanine)), for example, KDQNAS(sequence number 6), also known as "KDQNAS"(sequence number 6). In the modified Als3 protein of the present invention, the consensus sequence(s) can be selected from D / EXNZS / T, KD / EXNZS / T (SEQ ID NO: 4), 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 8) (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 9), also known as "GSGGGDQNATGSGGG" (Sequence ID 9).

[0134] In certain embodiments, the modified Als3 protein of the present invention contains at least two D / EXNZS / T or KD / EXNZS / T consensus sequences. In other embodiments, the modified Als3 protein of the present invention contains at least three D / EXNZS / T or KD / EXNZS / T consensus sequences. In yet another embodiment, the modified Als3 protein of the present invention contains at least four D / EXNZS / T or KD / EXNZS / T consensus sequences. In yet another embodiment, the modified Als3 protein of the present invention contains at least five D / EXNZS / T or KD / EXNZS / T consensus sequences. In yet another embodiment, the modified Als3 protein of the present invention contains at least six D / EXNZS / T or KD / EXNZS / T consensus sequences. In yet another embodiment, the modified Als3 protein of the present invention contains at least seven D / EXNZS / T or KD / EXNZS / T consensus sequences. In other embodiments, the modified Als3 protein of the present invention contains 3 to 7 D / EXNZS / T or KD / EXNZS / T consensus sequences. In yet another embodiment, the modified Als3 protein of the present invention contains 4 to 7 D / EXNZS / T or KD / EXNZS / T consensus sequences. In yet another embodiment, the modified Als3 protein of the present invention contains 5 to 7 D / EXNZS / T or KD / EXNZS / T consensus sequences.

[0135] 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 Als3 amino acid sequence of SEQ ID NO: 1 or into an Als3 amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.

[0136] In some embodiments, the modified Als3 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 Als3 protein. For example, tagging the modified Als3 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 Als3 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 Als3 protein of the present invention has been purified, and can be removed, for example, by chemical agents or enzymatic means. Thus, the modified Als3 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 Als3 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 Als3 protein of the present invention. In certain embodiments, the present invention provides a modified Als3 protein comprising a tag (e.g., a histidine tag). In other embodiments, the present invention provides a modified Als3 protein that does not comprise a tag (e.g., a histidine tag), e.g., a modified Als3 protein from which the histidine tag has been removed. Thus, in certain embodiments, the modified Als3 protein of the present invention comprises (i) an amino acid sequence identical to amino acid residues 18-316 of SEQ ID NO: 1 or at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% of amino acid residues 18-316 of SEQ ID NO: 1 and (ii) a peptide tag (e.g., six histidine residues at the C-terminus of the amino acid sequence) (or comprises (or comprises) such a tag).In other embodiments, the modified Als3 protein of the present invention contains (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 18-316 of SEQ ID NO: 1, with the peptide tag (e.g., histidine tag) removed.

[0137] In other embodiments, the modified Als3 protein of the present invention includes a signal sequence that can direct the modified Als3 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: 63)], heat-unstable Escherichia coli enterotoxin LTIIb [MSFKKIIKAFVIMAALVSVQAHA (SEQ ID NO: 64)], and Bacillus subtilis. The subtilis is selected from endoxylanase XynA [MFKFKKKFLVGLTAAFMSISMFSATASA (SEQ ID NO: 65)], E. coli DsbA [MKKIWLALAGLVLAFSASA (SEQ ID NO: 66)], TolB [MKQALRVAFGFLILWASVLHA (SEQ ID NO: 67)], or SipA [MKMNKKVLLTSTMAASLLSVASVQAS (SEQ ID NO: 68)]. 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 Als3 protein in which the amino acid sequence further comprises a signal sequence that can direct the modified Als3 protein towards 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 Als3 protein after it has been transported to the periplasmic side of the inner membrane of the host cell of the present invention.

[0138] In further embodiments, the present invention provides polynucleotides encoding the modified Als3 protein of the present invention. In certain embodiments, the present invention provides polynucleotides encoding the modified Als3 protein of the present invention having a nucleotide sequence encoding a polypeptide having an amino acid sequence at least 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 or 11. In some embodiments, the present invention provides a nucleotide sequence according to SEQ ID NO: 69, or a nucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 69. In further embodiments, the present invention provides a nucleotide sequence according to SEQ ID NO: 70, or a nucleotide sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 70. In one particular embodiment, the nucleotide sequence of the present invention includes 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 another embodiment, the nucleotide sequence of the present invention includes a nucleotide encoding an amino acid corresponding to one or more consensus sequences selected from KDQNAT (SEQ ID NO: 5), KDQNAS (SEQ ID NO: 6), DQNAT (SEQ ID NO: 7), and GSGGGDQNATGSGGG (SEQ ID NO: 9).In certain embodiments, the nucleotide sequence of the present invention comprises a nucleotide encoding the modified Als3 protein of the present invention, which comprises one or more consensus sequences, the one or more consensus sequences comprising one or more amino acids from amino acid residues 18-316 of SEQ ID NO: 1, amino acid residue 18, amino acid residues 33-37, amino acid residues 80-82, amino acid residue 87, amino acid residues 104-108, amino acid residues 119-121, amino acid residues 123-127, amino acid residues 155-159, amino acid residues 163-164, and amino acid It is added next to or substituted for one or more amino acids selected from the group consisting of one or more amino acids between residues 168-172, one or more amino acids between amino acid residues 175-179, amino acid residue 207, amino acid residue 220, one or more amino acids between amino acid residues 236-237, amino acid residue 270, amino acid residue 276, one or more amino acids between amino acid residues 286-287, one or more amino acids between amino acid residues 299-300, and amino acid residue 316, or at 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 18-316 of SEQ ID NO: 1. In specific embodiments, the nucleotide sequence of the present invention comprises a nucleotide encoding a modified Candida albicans Als3 protein containing one or more consensus sequences, wherein the one or more consensus sequences are added next to one or more amino acids selected from the group consisting of amino acid residues 16, 88, and 321 of SEQ ID NO: 10, and amino acid residues 16, 88, and 334 of SEQ ID NO: 11.

[0139] In further embodiments, the present invention provides a vector comprising a polynucleotide encoding the modified Als3 protein of the present invention.

[0140] Conjugate In certain embodiments, the present invention provides a conjugate comprising the modified Als3 protein of the present invention. The conjugate of the present invention may be a conjugate of the modified Als3 protein (e.g., a chemical conjugate or a bioconjugate). The conjugate of the present invention may be a conjugate of the modified Als3 protein with an antigen, such as a sugar antigen (i.e., a bioconjugate). In specific embodiments, the present invention provides a conjugate comprising (or consisting of) the modified Als3 protein of the present invention and at least one sugar antigen. In certain embodiments, the conjugate of the present invention is a bioconjugate.

[0141] 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 specific embodiments, the present invention provides a conjugate comprising the sugar of the present invention linked to an asparagine residue of a 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, Als3, Sap2, Pseudomonas aeruginosa detoxification exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, Staphylococcus aureus detoxification hemolysin A, Staphylococcus aureus clamping factor A, Staphylococcus aureus clamping factor B, Escherichia coli FimH, Escherichia coli FimHC, Escherichia coli thermolabile enterotoxin, detoxified variant of Escherichia coli thermolabile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, Escherichia coli sat protein, Escherichia coli sat protein passenger domain, C. jejuni AcrA, and C. jejuni natural glycoprotein. In specific embodiments, the modified carrier protein is the modified Als3 protein of the present invention.

[0142] In some embodiments, the modified Als3 protein of the present invention is ligated to at least one sugar antigen. In certain embodiments, the modified Als3 protein of the present invention is directly ligated to at least one sugar antigen. In other embodiments, the modified Als3 protein of the present invention is ligated to at least one sugar antigen via a linker. In certain embodiments, the modified Als3 protein of the present invention is non-covalently ligated to at least one sugar antigen. In some embodiments, the modified Als3 protein of the present invention is non-covalently ligated to at least one sugar antigen by avidin-streptavidin interaction. In other embodiments, the modified Als3 protein of the present invention is covalently ligated to at least one sugar antigen via a chemical conjugation 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.

[0143] Generally, the following types of chemical groups on modified Als3 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. 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. 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. D) A hydroxyl group (e.g., via tyrosine). In one embodiment, this group is activated / modified using bisdiazobenzidine. E) Imidazolyl group (e.g., via histidine). In one embodiment, this group is activated / modified using bisdiazobenzidine. F) Guanidyl group (e.g., via arginine). G) Indolyl group (e.g., via tryptophan).

[0144] 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.

[0145] Conjugates can be purified by any method known in the art 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 depend in part (in the past) 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.

[0146] In some embodiments, the amino acid residues on the modified Als3 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 acid is an amino acid 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 Als3 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 Als3 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 certain embodiments, the amino acid residue on the modified Als3 protein of the present invention to which at least one sugar antigen is ligated is an asparagine residue. In certain embodiments, the amino acid residue on the modified Als3 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.

[0147] In certain embodiments, the conjugate of the present invention is a conjugate of recombinant modified Als3 protein (e.g., a chemical conjugate or a bioconjugate). In other embodiments, the conjugate of the present invention is a conjugate of isolated recombinant modified Als3 protein with a recombinant antigen, for example, a recombinant glycoantigen (i.e., a bioconjugate).

[0148] In certain embodiments, the modified Als3 protein of the present invention is linked to at least one sugar antigen by one or more amino acid residues on the modified Als3 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 specific embodiments, the modified Als3 protein of the present invention is linked to at least one sugar antigen by one or more asparagine residues on the modified Als3 protein. In certain embodiments, the at least one sugar antigen is linked to at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten asparagine residues on the modified Als3 protein. 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 Als3 protein of the present invention. In a particular embodiment, at least one sugar antigen is ligated to at least three asparagine residues of the modified Als3 protein of the present invention. In a specific embodiment, at least one sugar antigen is ligated to three asparagine residues of the modified Als3 protein of the present invention. In a particular embodiment, the modified Als3 protein of the present invention includes (or consists of) the amino acid sequence of SEQ ID NO: 10, and the three asparagine residues include, but are not limited to, positions 20, 92, and 324 of SEQ ID NO: 10. In other embodiments, the modified Als3 protein of the present invention includes (or consists of) the amino acid sequence of SEQ ID NO: 11, and the three asparagine residues include, but are not limited to, positions 20, 92, and 337 of SEQ ID NO: 11. In further embodiments, at least one sugar antigen is ligated to at least one of the three asparagine residues of the modified Als3 protein of the present invention.In certain embodiments, the modified Als3 protein of the present invention comprises (or consists of) the amino acid sequence of SEQ ID NO: 10, and the three asparagine residues include, but are not limited to, positions 20, 92, and 324 of SEQ ID NO: 10. In other embodiments, the modified Als3 protein of the present invention comprises (or consists of) the amino acid sequence of SEQ ID NO: 11, and the three asparagine residues include, but are not limited to, positions 20, 92, and 337 of SEQ ID NO: 11. In certain embodiments, the present invention provides: a modified Als3 protein of Candida albicans comprising (or consisting of) (1) the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, and (2) at least one sugar antigen of the genus Candida, wherein at least one sugar antigen is ligated to at least one of three asparagine residues at positions 20, 92, and 324 of SEQ ID NO: 10 or positions 20, 92, and 337 of SEQ ID NO: 11.

[0149] antigen The present invention provides a conjugate (e.g., a bioconjugate) in which a carrier protein (e.g., the modified Als3 protein of the present invention) can be linked to several different antigens (e.g., covalently or non-covalently). In certain embodiments, the modified Als3 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.

[0150] 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, 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*.

[0151] 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.

[0152] 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.

[0153] In further embodiments, at least one sugar antigen is a fungal sugar antigen. In certain embodiments, the fungus is a Candida species. Therefore, 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. Therefore, in certain embodiments, at least one sugar antigen is a Candida albicans sugar antigen.

[0154] In certain embodiments, at least one sugar antigen is a β-1,3-glucan polymer. Thus, in certain embodiments, 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 certain embodiments, 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.

[0155] 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.

[0156] In some embodiments, the present invention has a structure:

[0157] [ka] It has, The present invention provides glucans in which n is 2-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.

[0158] In further embodiments, the present invention relates to a structure: [ka] A sugar that is a glucan having, The present invention provides sugars in which n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.

[0159] In a particular embodiment, at least one sugar antigen has the following structure:

[0160] [ka] It has, Contains glucans where n is 2-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.

[0161] In other embodiments, at least one sugar antigen has the following structure: [ka] It has, Contains glucans where n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.

[0162] Accordingly, in certain embodiments, the present invention provides a modified Als3 protein of Candida albicans comprising (or comprising) (1) the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, and (2) at least one sugar antigen of the genus Candida, wherein the at least one sugar antigen is a β-1,3-glucan polymer comprising (or comprising) at least six consecutive β-1,3-linked glucose molecules, and the at least one sugar antigen is linked to at least one of three asparagine residues at positions 20, 92 and 324 of SEQ ID NO: 10 or positions 20, 92 and 337 of SEQ ID NO: 11.

[0163] In other embodiments, at least one sugar antigen is a β-1,2-mannan polymer. In certain embodiments, at least one sugar antigen is a β-1,2-mannan polymer of Candida albicans. Mannan is the outermost layer of the cell wall and is involved in cell adhesion and immune system evasion in Candida species. Mannan is a highly complex and branched structure 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-mannoses are exclusive to the Candida genus and are conserved in several Candida species (e.g., C. albicans, C. auris, C. gilliermondi, C. lucitanie, and C. tropicalis, but excluding C. glabrata) (Morad HO et al., Front Microbiol, 2018). 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.

[0164] 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, and 4-10. , 4-9, 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 are included. In some embodiments, the β-1,2-mannan polymer includes at least 2, at least 3, at least 4 or at least 5 consecutive β-1,2-linked mannose molecules.

[0165] host cell In certain embodiments, the present invention provides a host cell comprising a polynucleotide sequence encoding the modified Als3 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.Therefore, 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.

[0166] 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.

[0167] 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 Als3 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 Als3 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.

[0168] Accordingly, 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 the modified Als3 protein of the present invention; and optionally (4) a polynucleotide sequence encoding a polymerase.

[0169] In some embodiments, one or more heterologous glycosyltransferases include, but are not limited to, exoL, exoM, exoO, exoU, and exoW. In certain embodiments, exoL, exoM, exoO, exoU, and exoW are derived from rhizobia. In certain embodiments, the rhizobia are of the genus Cinorhizobium. In specific embodiments, the genus Cinorhizobium is Cinorhizobium meliloti 1021. In certain embodiments, one or more heterologous glycosyltransferases include exoL, exoM, exoO, exoU, and exoW derived from rhizobia, possibly from the genus Cinorhizobium, and possibly from Cinorhizobium meliloti 1021. In other embodiments, one or more heterologous glycosyltransferases include, but are not limited to, SleC, SleE, SleF, SleU, and SleW. In certain embodiments, SleC, SleE, SleF, SleU, and SleW are derived from rhizobia. In certain embodiments, the rhizobia are of the genus Agrobacterium. In specific embodiments, the genus Agrobacterium is the species ZX09 of the genus Agrobacterium. In certain embodiments, one or more heterologous glycosyltransferases include SleC, SleE, SleF, SleU, and SleW derived from rhizobia, optionally from the genus Agrobacterium, and optionally from the species ZX09 of the genus Agrobacterium.

[0170] In further embodiments, the present invention relates to a host cell, i. A nucleotide sequence encoding one or more heterologous glycosyltransferases capable of synthesizing β-1,3-glucan polymers. ii. A nucleotide sequence encoding a glycosyltransferase that can covalently bond a glucose molecule to an N-acetylglucosamine (GlcNac) molecule. iii. Nucleotide sequences encoding heterologous oligosaccharide transferases, and iv. 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. Provides host cells containing the following:

[0171] In further 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 glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetylglucosamine (GlcNac) molecule; (3) a polynucleotide sequence encoding a heterologous oligosaccharide transferase; (4) a polynucleotide sequence encoding a modified A1s3 protein of the present invention; and optionally, a polynucleotide sequence encoding a polymerase.

[0172] In further embodiments, the present invention relates to a host cell, i. A nucleotide sequence encoding one or more heterologous glycosyltransferases capable of synthesizing β-1,3-glucan polymers. ii. A nucleotide sequence encoding a glycosyltransferase that can covalently bond a glucose molecule to an N-acetylglucosamine (GlcNac) molecule. iii. Nucleotide sequences encoding heterologous oligosaccharide transferases, and iv. A nucleotide sequence encoding a modified carrier protein comprising a glycosylation site including the consensus sequence D / EXNZS / T, wherein X and Z are independently any amino acid other than proline, and optionally the polynucleotide sequence of the present invention. Provides host cells containing the following:

[0173] In certain embodiments, the modified carrier proteins of the present invention include, but are not limited to, Als3, Sap2, Pseudomonas aeruginosa detoxification exotoxin A (EPA), CRM197, diphtheria toxoid, tetanus toxoid, Staphylococcus aureus detoxification hemolysin A, Staphylococcus aureus clamping factor A, Staphylococcus aureus clamping factor B, Escherichia coli FimH, Escherichia coli FimHC, Escherichia coli thermolabile enterotoxin, detoxified variant of Escherichia coli thermolabile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin, Escherichia coli sat protein, Escherichia coli sat protein passenger domain, C. jejuni AcrA, and C. jejuni natural glycoprotein. In specific embodiments, the modified carrier protein is the modified Als3 protein of the present invention.

[0174] In some embodiments, one or more heterologous glycosyltransferases capable of synthesizing β-1,3-glucan polymers include, but are not limited to, exoL, exoM, exoO, exoU, and exoW. In certain embodiments, exoL, exoM, exoO, exoU, and exoW are derived from rhizobia. In certain embodiments, the rhizobia are of the genus Sinorhizobium. In specific embodiments, the genus Sinorhizobium is Sinorhizobium meliloti 1021. In certain embodiments, one or more heterologous glycosyltransferases include exoL, exoM, exoO, exoU, and exoW derived from rhizobia, optionally from the genus Sinorhizobium, and optionally from Sinorhizobium meliloti 1021. In other embodiments, one or more heterologous glycosyltransferases include, but are not limited to, SleC, SleE, SleF, SleU, and SleW. In certain embodiments, SleC, SleE, SleF, SleU, and SleW are derived from rhizobia. In certain embodiments, the rhizobia are of the genus Agrobacterium. In specific embodiments, the genus Agrobacterium is the species ZX09 of the genus Agrobacterium. In certain embodiments, one or more heterologous glycosyltransferases of i. include SleC, SleE, SleF, SleU, and SleW derived from rhizobia, optionally from the genus Agrobacterium, and optionally from the species ZX09 of the genus Agrobacterium.

[0175] In certain embodiments, one or more heteroglycosyltransferases capable of synthesizing β-1,3-glucan polymers have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of exoL of Sinorhizobium melilotii 1021. In other embodiments, one or more heteroglycosyltransferases capable of synthesizing β-1,3-glucan polymers have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleC of Agrobacterium species ZX09. In some embodiments, one or more heteroglycosyltransferases capable of synthesizing β-1,3-glucan polymers have an amino acid sequence identical to the amino acid sequence of SleC of Agrobacterium species ZX09. In some embodiments, one or more heteroglycosyltransferases capable of synthesizing β-1,3-glucan polymers have the same amino acid sequence as exoL of Sinorhizobium meliloti 1021. In a particular embodiment, SleC of Agrobacterium species ZX09 contains the amino acid sequence of SEQ ID NO: 12. Therefore, in a particular embodiment, one or more heteroglycosyltransferases capable of synthesizing β-1,3-glucan polymers have the same amino acid sequence as SEQ ID NO: 12 by at least 80%, 90%, 95%, 98%, or 99%. In other embodiments, one or more heteroglycosyltransferases capable of synthesizing β-1,3-glucan polymers have the same amino acid sequence as SEQ ID NO: 12. In a specific embodiment, one or more heterologous glycosyltransferases capable of synthesizing β-1,3-glucan polymers have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleC of Agrobacterium species ZX09, which includes SEQ ID NO: 12. In a further embodiment, one or more heterologous glycosyltransferases capable of synthesizing β-1,3-glucan polymers have an amino acid sequence identical to the amino acid sequence of SleC of Agrobacterium species ZX09, which includes SEQ ID NO: 12: [ka]

[0176] In certain embodiments, the host cell of the present invention optionally contains within the plasmid a nucleotide sequence encoding sleC from Agrobacterium species ZX09, optionally containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 12.

[0177] In other embodiments, the host cell of the present invention comprises a nucleotide sequence encoding sleC, optionally derived from Agrobacterium species ZX09, optionally comprising a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34: [ka]

[0178] In certain embodiments, one or more heteroglycosyltransferases capable of synthesizing β-1,3-glucan polymers have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of exoM of Sinorhizobium meliloti 1021. 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 SleE of Agrobacterium species ZX09. In some embodiments, one or more heteroglycosyltransferases have an amino acid sequence identical to the amino acid sequence of SleE of Agrobacterium species ZX09. In other embodiments, one or more heteroglycosyltransferases have an amino acid sequence identical to the amino acid sequence of exoM of Sinorhizobium meliloti 1021. In a particular embodiment, the SleE of Agrobacterium species ZX09 contains the amino acid sequence of SEQ ID NO: 13. Therefore, in a particular embodiment, one or more heteroglycosyltransferases 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 heteroglycosyltransferases have an amino acid sequence identical to SEQ ID NO: 13. In a specific 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 Agrobacterium species ZX09 containing SEQ ID NO: 13. In a further embodiment, one or more heteroglycosyltransferases have an amino acid sequence identical to the amino acid sequence of Agrobacterium species ZX09 containing SEQ ID NO: 13: [ka]

[0179] In certain embodiments, the host cell of the present invention optionally contains within the plasmid a nucleotide sequence encoding sleE from Agrobacterium species ZX09, optionally containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13.

[0180] In other embodiments, the host cell of the present invention comprises a nucleotide sequence encoding sleE, optionally a nucleotide sequence encoding sleE from Agrobacterium species ZX09, optionally a nucleotide sequence encoding sleE from Agrobacterium species ZX09 that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33: [ka]

[0181] In certain embodiments, one or more heteroglycosyltransferases capable of synthesizing β-1,3-glucan polymers have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of exoO of Sinorhizobium meliloti 1021. 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 SleF of Agrobacterium species ZX09. In some embodiments, one or more heteroglycosyltransferases have an amino acid sequence identical to the amino acid sequence of SleF of Agrobacterium species ZX09. In other embodiments, one or more heteroglycosyltransferases have an amino acid sequence identical to the amino acid sequence of exoO of Sinorhizobium meliloti 1021. In one particular embodiment, SleF of Agrobacterium species ZX09 contains the amino acid sequence of SEQ ID NO: 14. Therefore, in one particular embodiment, one or more heteroglycosyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 14. In other embodiments, one or more heteroglycosyltransferases have an amino acid sequence identical to SEQ ID NO: 13. In a specific 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 SleF of Agrobacterium species ZX09 containing SEQ ID NO: 14. In a further embodiment, one or more heteroglycosyltransferases have an amino acid sequence identical to the amino acid sequence of SleF of Agrobacterium species ZX09 containing SEQ ID NO: 14: [ka]

[0182] In certain embodiments, the host cell of the present invention optionally contains within the plasmid a nucleotide sequence encoding sleF from Agrobacterium species ZX09, optionally containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14.

[0183] In other embodiments, the host cell of the present invention comprises a nucleotide sequence encoding sleF, optionally a nucleotide sequence encoding sleF from Agrobacterium species ZX09, optionally a nucleotide sequence encoding sleF from Agrobacterium species ZX09 that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32: [ka] [ka]

[0184] In certain embodiments, one or more heteroglycosyltransferases capable of synthesizing β-1,3-glucan polymers have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of exoU of Sinorhizobium meliloti 1021. 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 SleU of Agrobacterium species ZX09. In some embodiments, one or more heteroglycosyltransferases have an amino acid sequence identical to the amino acid sequence of SleU of Agrobacterium species ZX09. In other embodiments, one or more heteroglycosyltransferases have an amino acid sequence identical to the amino acid sequence of exoU of Sinorhizobium meliloti 1021. In a particular embodiment, the SleU of Agrobacterium species ZX09 contains the amino acid sequence of SEQ ID NO: 15. Therefore, in a particular embodiment, one or more heteroglycosyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 15. In other embodiments, one or more heteroglycosyltransferases have an amino acid sequence identical to SEQ ID NO: 15. In a specific 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 Agrobacterium species ZX09 containing SEQ ID NO: 15. In a further embodiment, one or more heteroglycosyltransferases have an amino acid sequence identical to the amino acid sequence of Agrobacterium species ZX09 containing SEQ ID NO: 15: [ka]

[0185] In certain embodiments, the host cell of the present invention optionally contains within a plasmid a nucleotide sequence encoding sleU from Agrobacterium species ZX09, optionally containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15.

[0186] In other embodiments, the host cell of the present invention comprises a nucleotide sequence encoding sleU, optionally a nucleotide sequence encoding sleU from Agrobacterium species ZX09, optionally a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31: [ka]

[0187] In one particular embodiment, one or more heterologous glycosyltransferases capable of synthesizing β-1,3-glucan polymers have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the exoW amino acid sequence of Sinorhizobium meliloti 1021. In other embodiments, one or more heterologous glycosyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the SleW amino acid sequence of Agrobacterium species ZX09. In some embodiments, one or more heterologous glycosyltransferases have an amino acid sequence identical to the SleW amino acid sequence of Agrobacterium species ZX09. In other embodiments, one or more heterologous glycosyltransferases have an amino acid sequence identical to the exoW amino acid sequence of Sinorhizobium meliloti 1021. In a particular embodiment, the SleW of Agrobacterium species ZX09 contains the amino acid sequence of SEQ ID NO: 16. Therefore, in a particular embodiment, one or more heteroglycosyltransferases have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 16. In other embodiments, one or more heteroglycosyltransferases have an amino acid sequence identical to SEQ ID NO: 16. In a specific 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 Agrobacterium species ZX09 containing SEQ ID NO: 16. In a further embodiment, one or more heteroglycosyltransferases have an amino acid sequence identical to the amino acid sequence of Agrobacterium species ZX09 containing SEQ ID NO: 16: [ka]

[0188] In certain embodiments, the host cell of the present invention optionally contains within a plasmid a nucleotide sequence encoding sleW from Agrobacterium species ZX09, optionally containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16.

[0189] In other embodiments, the host cell of the present invention comprises a nucleotide sequence encoding sleW, optionally from Agrobacterium species ZX09, optionally containing a nucleotide sequence identical to sequence number 30 by at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%: [ka]

[0190] In certain embodiments, the host cell of the present invention produces more SleW than SleC, SleE, SleF, or SleU. In certain embodiments, the host cell contains one gene copy of SleW. In other embodiments, the host cell contains two or more gene copies of SleW. In certain embodiments, the host cell 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 gene copies of SleW. In specific embodiments, the host cell contains at least two gene copies of SleW from Agrobacterium species ZX09. In further embodiments, the host cell contains at least one copy of SleW from Agrobacterium species ZX09 under the control of a potent promoter that enables higher expression of SleW compared to the expression of SleC, SleE, SleF, or SleU.

[0191] In certain embodiments, the glycosyltransferase capable of covalently bonding glucose to GlcNac is WfaP. In certain embodiments, WfaP is of bacterial origin. 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 the genera Escherichia, Shigella, Klebsiella, Xanthomonas, Salmonella, Yersinia, Lactococcus, Lactobacillus, Pseudomonas, Corynebacterium, Streptomyces, Streptococcus, Staphylococcus, Bacillus, and Clostridium. In some embodiments, the bacteria are species of the genus Escherichia. In certain embodiments, the species of Escherichia is Escherichia coli O56. In a specific embodiment, the glycosyltransferase capable of covalently bonding glucose to GlcNac is WfaP derived from E. coli O56. In a particular embodiment, the glycosyltransferase capable of covalently bonding glucose to GlcNac has an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WfaP derived from E. coli O56. In another embodiment, the glycosyltransferase capable of covalently bonding glucose to GlcNac has an amino acid sequence identical to the amino acid sequence of WfaP derived from E. coli O56. In a particular embodiment, WfaP derived from E. coli O56 contains the amino acid sequence of SEQ ID NO: 17. Therefore, in a particular embodiment, the glycosyltransferase capable of covalently bonding glucose to GlcNac contains an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 17. In another embodiment, the glycosyltransferase capable of covalently bonding glucose to GlcNac contains the amino acid sequence identical to SEQ ID NO: 17. In a specific embodiment, a glycosyltransferase capable of covalently bonding glucose to GlcNac contains an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of WfaP derived from E. coli O56, including SEQ ID NO: 17.In a further embodiment, a glycosyltransferase capable of covalently bonding glucose to GlcNac includes an amino acid sequence identical to that of WfaP derived from E. coli O56, as included in SEQ ID NO: 17. [ka]

[0192] In certain embodiments, the host cell of the present invention optionally contains within a plasmid WfaP, optionally a nucleotide sequence encoding WfaP derived from E. coli O56, optionally a nucleotide sequence encoding WfaP derived from E. coli O56 that optionally has an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17.

[0193] In other embodiments, the host cell of the present invention comprises a nucleotide sequence encoding WfaP, optionally a nucleotide sequence encoding WfaP derived from E. coli O56, optionally a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35: [ka] [ka]

[0194] 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).

[0195] 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.

[0196] Accordingly, in certain embodiments, the host cell of the present invention optionally contains a nucleotide sequence encoding a heterologous oligosaccharide transferase within the 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, and may be derived from Campylobacter jejuni or Campylobacter coli, and contains the amino acid sequence of Sequence ID No. 20. In certain embodiments, PglB contains an amino acid sequence that is at least 80%, 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, Campylobacter coli PglB contains the amino acid sequence of SEQ ID NO: 20. In certain embodiments, PglB has an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 20. In other embodiments, PglB has an amino acid sequence identical to SEQ ID NO: 20. In certain embodiments, PglB has an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of Campylobacter coli PglB containing SEQ ID NO: 20. In a further embodiment, PglB has the same amino acid sequence as PglB of Campylobacter coli and includes SEQ ID NO: 20. [ka]

[0197] In certain embodiments, the host cells of the present invention optionally contain within a plasmid PglB, which may include a nucleotide sequence encoding PglB derived from Campylobacter jejuni, which may optionally include a nucleotide sequence encoding PglB derived from Campylobacter jejuni or Campylobacter coli that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 20.

[0198] In other embodiments, the host cells of the present invention optionally contain PglB, a nucleotide sequence encoding PglB from Campylobacter jejuni, or a nucleotide sequence encoding PglB from Campylobacter jejuni or Campylobacter coli, which optionally contains a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29, optionally within a plasmid: [ka] [ka]

[0199] In certain embodiments, the nucleotide sequence encoding PglB is codon-optimized.

[0200] 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 include a nucleotide sequence encoding a heterologous wzy polymerase.

[0201] 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).

[0202] Translocase The host cells of the present invention may also contain a translocase (e.g., wzm-wzt), 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)-binding 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).

[0203] 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., E. 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., wzm-wzt) 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 Als3 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. Therefore, 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 are, for example, derived from Campylobacter jejuni (e.g., pglK).

[0204] Accordingly, in certain embodiments, the present invention provides a host cell comprising a nucleotide sequence comprising (i) a wzm gene comprising the nucleotide sequence of SEQ ID NO: 36, and optionally comprising a nucleotide sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 36, and (ii) a wzt gene comprising the nucleotide sequence of SEQ ID NO: 37, and optionally comprising a nucleotide sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 37. In certain embodiments, the wzm and wzt genes are derived from Klebsiella pneumoniae.

[0205] Therefore, in certain embodiments, the present invention provides a wzm gene which may include a nucleotide sequence derived from Klebsiella pneumoniae, may include a nucleotide sequence of SEQ ID NO: 36, and may include a nucleotide sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 36. [ka]

[0206] In certain embodiments, the wzm gene encodes a Wzm protein that contains (or consists of) an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to that of SEQ ID NO: 18. In specific embodiments, the wzm gene encodes a Wzm protein that contains (or consists of) the amino acid sequence of SEQ ID NO: 18: [ka]

[0207] Therefore, in a further embodiment, the present invention provides a wzt gene which optionally contains a nucleotide sequence derived from Klebsiella pneumoniae, optionally containing the nucleotide sequence of SEQ ID NO: 37, optionally containing a nucleotide sequence which is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 37 [ka]

[0208] In one embodiment, the wzt gene encodes a Wzt protein comprising (or consisting of) an amino acid sequence that is at least 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 19. In a specific embodiment, the wzt gene encodes a Wzt protein comprising (or consisting of) the amino acid sequence of SEQ ID NO: 19:

Chemical formula

[0209] In a further embodiment, the present invention provides a method for producing a β-1,3 glucan polymer in a host cell of the present invention, comprising, in the host cell, i. a nucleotide sequence encoding a first glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetylglucosamine (GlcNAc) molecule, ii. a nucleotide sequence encoding a further glycosyltransferase capable of synthesizing fungal β-1,3 glucan, wherein the further glycosyltransferase comprises SleC, SleE, SleF, SleU and SleW derived from Rhizobium, and iii. optionally, a nucleotide sequence encoding a translocase capable of translocating β-1,3 glucan to the periplasmic side of the inner membrane of the host cell introducing and expressing, and providing a method in which the β-1,3 glucan polymer is linked to a lipid carrier via GlcNAc.

[0210] In a specific embodiment, the present invention provides a method for producing a β-1,3 glucan polymer in a prokaryotic host cell, comprising, in the host cell, i. a nucleotide sequence encoding a first glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetylglucosamine (GlcNAc) molecule, wherein the first glycosyltransferase is WfaP derived from Escherichia coli O56,​ ii. A nucleotide sequence encoding a further glycosyltransferase capable of synthesizing fungal β-1,3-glucan, wherein the further glycosyltransferase comprises SleC, SleE, SleF, SleU, and SleW derived from rhizobia, possibly from the genus Agrobacterium, and possibly from the species Agrobacterium ZX09, wherein the host cell produces more SleW than SleC, SleE, SleF, or SleU, and iii. A nucleotide sequence encoding a translocase that can, in some cases, transfer β-1,3-glucan to the periplasmic side of the inner membrane of a prokaryotic host cell, wherein the translocase contains Wzm-Wzt derived from a species of the genus Klebsiella, and in some cases from Klebsiella pneumoniae. Steps to introduce and express The present invention provides a method comprising a β-1,3-glucan polymer linked to a lipid carrier via GlcNAc, wherein the β-1,3-glucan polymer contains at least four β-1,3-linked glucose molecules.

[0211] In certain embodiments, the β-1,3-glucan polymer is a fungal β-1,3-glucan polymer. In some embodiments, the fungal β-1,3-glucan polymer is of the genus Candida. In specific embodiments, the fungal β-1,3-glucan polymer is of Candida albicans. In certain embodiments, the β-1,3-glucan polymer has the structure:

[0212] [ka] It has, n is 2-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.

[0213] In other embodiments, the β-1,3-glucan polymer has the following structure:

[0214] [ka] It has, n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.

[0215] In further embodiments, the β-1,3-glucan polymer has the following structure: [ka] It has, n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.

[0216] 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.

[0217] Bioconjugate In certain embodiments, the present invention provides a bioconjugate comprising a modified Als3 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 Als3 protein selected from asparagine, aspartic acid, glutamic acid, lysine, cysteine, tyrosine, histidine, arginine, or tryptophan. The bioconjugate described herein has advantageous properties on antigen-carrier protein chemical conjugates in that it requires fewer chemicals in production and is more consistent with respect to the final product produced.

[0218] In certain embodiments, the present invention provides a method for producing a bioconjugate comprising a modified Als3 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.

[0219] Methods for preparing bioconjugates are known in the art. 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.

[0220] Accordingly, in certain embodiments, the present invention provides a biconjugate 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 Als3 protein of the present invention.

[0221] The bioconjugate of the present invention can be purified, for example, by chromatography (e.g., ion exchange chromatography, anion exchange chromatography, affinity chromatography, and sizing column chromatography), centrifugation, fractionation 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 bioconjugate of the present invention can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0222] In a further embodiment, the 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 a plurality of polysaccharide chains, wherein the polysaccharide is covalently linked to the carrier protein.

[0223] In certain embodiments, the modified carrier proteins of the invention include, but are not limited to, Als3, 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 variants of Escherichia coli heat-labile enterotoxin, cholera toxin B subunit (CTB), cholera toxin, detoxified variants of cholera toxin, Escherichia coli sat protein, the passenger domain of Escherichia coli sat protein, Campylobacter jejuni AcrA, and Campylobacter jejuni native sugar protein. In certain embodiments, the modified carrier protein is the modified Als3 protein of the invention.

[0224] In certain embodiments, at least one sugar antigen comprises a β-1,3-glucan polymer. In some embodiments, the β-1,3-glucan polymer 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, The β-1,3-linked glucose molecules are included in 5-7, 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 units. In some embodiments, the β-1,3-glucan polymer includes 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 specific embodiments, the β-1,3-glucan polymer includes 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.

[0225] In certain embodiments, the present invention provides a method for producing a glycoconjugate containing a modified carrier protein and β-1,3-glucan. In some embodiments, the method for producing a glycoconjugate containing a modified carrier protein and β-1,3-glucan includes the step of culturing host cells of the present invention under conditions suitable for protein production.

[0226] In further embodiments, the present invention relates to a method for generating a bioconjugate in a host cell of the present invention, i. The step of obtaining host cells of the present invention that produce β-1,3-glucan polymer; and ii Furthermore, in host cells, a. A nucleotide sequence encoding a modified carrier protein comprising at least one glycosylation site including the consensus sequence D / EXNZS / T, wherein X and Z are any amino acids other than proline, and the modified carrier protein further comprises an N-terminal bacterial signal sequence that can transport the modified carrier protein to the periplasmic side of the inner membrane of a host cell, and b. Nucleotide sequences encoding oligosaccharide transferases that can generate bioconjugates by transferring β-1,3-glucan polymers from lipid carriers to modified carrier proteins. Steps to introduce and express This provides a method that includes [something].

[0227] In a particular embodiment, the present invention relates to a method for generating a bioconjugate in a prokaryotic host cell, a. The step of obtaining a prokaryotic host cell of the present invention that produces a β-1,3-glucan polymer, and b. Furthermore, in host cells, i. A nucleotide sequence encoding a modified carrier protein comprising a glycosylation site including the consensus sequence D / EXNZS / T, wherein X and Z are any amino acids other than proline, and 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. A nucleotide sequence encoding an oligosaccharide transferase capable of generating a bioconjugate by transferring a β-1,3-glucan polymer from a lipid carrier to a modified carrier protein, wherein the oligosaccharide transferase is PglB derived from the genus Campylobacter, and possibly from Campylobacter jejuni or Campylobacter coli. Steps to introduce and express This provides a method that includes [something].

[0228] In a further embodiment, the present invention relates to a method for generating a bioconjugate in a prokaryotic host cell, c. The step of obtaining prokaryotic host cells of the present invention that produce β-1,3-glucan polymers; and d. Furthermore, in host cells, iii. A nucleotide sequence encoding a modified carrier protein comprising a glycosylation site containing the consensus sequence D / EXNZS / , wherein X and Z are any amino acids other than proline and T (optionally, the polynucleotide sequence of the present invention), and further comprising an N-terminal bacterial signal sequence that enables the modified carrier protein to transport to the periplasmic side of the inner membrane of a prokaryotic host cell, and A nucleotide sequence encoding an oligosaccharide transferase capable of generating a bioconjugate by transferring a β-1,3-glucan polymer from a lipid carrier to a modified carrier protein, wherein the oligosaccharide transferase is PglB derived from the genus Campylobacter, and optionally from Campylobacter jejuni or Campylobacter coli. Steps to introduce and express This provides a method that includes [something].

[0229] In certain embodiments, the modified carrier protein of the present invention may include, but is not limited to, Als3, Sap2, 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 Als3 protein of the present invention.

[0230] 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 includes 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] 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 separation by size) 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.

[0235] 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).

[0236] Immunogenic compositions and vaccines The conjugate of the present invention (e.g., bioconjugate) is particularly suitable for inclusion in immunogenic compositions and vaccines.

[0237] Accordingly, 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 Als3 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.

[0238] The immunogenic composition comprises an immunologically effective amount of the modified Als3 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.

[0239] 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.

[0240] 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 Als3 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.

[0241] 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.

[0242] Accordingly, 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.

[0243] In certain embodiments, the present invention provides a Candida albicans vaccine comprising (1) a modified Als3 protein of the present invention; (2) at least one Candida albicans glycoantigen linked to the modified Als3 protein; and optionally (3) a pharmaceutically acceptable carrier or adjuvant.

[0244] 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 Als3 protein conjugate / bioconjugate, but does not generate an immune response to the modified Als3 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 may 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.

[0245] 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).

[0246] 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.

[0247] 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).

[0248] Tocol is well known in the art 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.

[0249] 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.

[0250] Furthermore, a method for preparing an immunogenic composition of the present invention is also provided, comprising the step of mixing the modified Als3 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).

[0251] 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.

[0252] 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.

[0253] 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.

[0254] 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, which 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.

[0255] 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).

[0256] 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 glucan dose.

[0257] 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.

[0258] 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 Als3 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 Als3 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.

[0259] 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 Als3 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.

[0260] 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 of the present invention (e.g., a bioconjugate). 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 Als3 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 Als3 protein of the present invention, the conjugate of the present invention, any of the bioconjugates 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 Als3 proteins, conjugates, bioconjugates, immunogenic compositions, or vaccines 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.

[0261] 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 Als3 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.

[0262] 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 Als3 protein of the present invention, the conjugate of the present invention, any bioconjugate of the present invention, the immunogenic composition of the present invention, or the vaccine of the present invention.

[0263] In certain embodiments, the present invention provides modified Als3 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 Candida albicans infection in subjects (e.g., humans).

[0264] 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 Als3 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.

[0265] 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 Als3 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.

[0266] In further embodiments, the present invention provides a method for inhibiting the formation of a Candida albicans biofilm, comprising (i) administering a therapeutically effective or prophylactically effective amount of the modified Als3 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 to a subject (e.g., a human).

[0267] Embodiments of the present invention will be further described in the following numbered paragraphs: 1. A modified aglutinin-like sequence 3 (Als3) protein having an amino acid sequence identical to amino acid residues 18-316 of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of amino acid residues 18-316 of SEQ ID NO: 1, wherein the amino acid sequence is modified in that it includes one or more consensus sequences containing the amino acid sequence D / EXNZS / T, and X and Z are independently any amino acid other than proline.

[0268] 2. One or more consensus sequences are among the amino acid residues 18-316 of SEQ ID NO: one or more amino acids between amino acid residues 18-23, one or more amino acids between amino acid residues 28-42, one or more amino acids between amino acid residues 75-87, one or more amino acids between amino acid residues 82-92, one or more amino acids between amino acid residues 99-113, one or more amino acids between amino acid residues 114-126, one or more amino acids between amino acid residues 118-132, one or more amino acids between amino acid residues 150-164, one or more amino acids between amino acid residues 158-169, one or more amino acids between amino acid residues 163-177 (for example, one or more amino acids between amino acid residues 168-172), and one or more amino acids between amino acid residues 170-184 (for example, amino acids The modified Als3 protein of paragraph 1, wherein one or more amino acids selected from the group consisting of one or more amino acids between amino acid residues 175-179, one or more amino acids between amino acid residues 202-212, one or more amino acids between amino acid residues 215-225, one or more amino acids between amino acid residues 231-242, one or more amino acids between amino acid residues 265-275, one or more amino acids between amino acid residues 271-281, one or more amino acids between amino acid residues 281-292, and one or more amino acids between amino acid residues 294-305 are added next to or substituted with, or at 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 18-316 of SEQ ID NO: 1.

[0269] 3. A modified Als3 protein according to either paragraphs 1 and 2, wherein one or more consensus sequences are added to or substituted for amino acid residue 18 of the amino acid residues 18-316 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 18-316 of SEQ ID NO: 1.

[0270] 4. One or more consensus sequences are added to or substituted next to one or more amino acids between amino acid residues 33-37 of amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 3.

[0271] 5. One or more consensus sequences are added to or substituted next to or on one or more amino acids between amino acid residues 80-82 of amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 4.

[0272] 6. One or more consensus sequences are added to or substituted for amino acid residue 87 among amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 5.

[0273] 7. One or more consensus sequences are added to or substituted next to or on one or more amino acids between amino acid residues 104-108 of amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 6.

[0274] 8. One or more consensus sequences are added to or substituted next to or on one or more amino acids between amino acid residues 119-121 of amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 7.

[0275] 9. One or more consensus sequences are added to or substituted next to one or more amino acids between amino acid residues 123-127 of amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 8.

[0276] 10. One or more consensus sequences are added to or substituted next to or on one or more amino acids between amino acid residues 155-159 of amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 9.

[0277] 11. One or more consensus sequences are added to or substituted next to or on one or more amino acids between amino acid residues 163-164 of amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 10.

[0278] 12. One or more consensus sequences are added to or substituted for amino acid residue 207 among amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 11.

[0279] 13. One or more consensus sequences are added to or substituted for amino acid residue 220 among amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 12.

[0280] 14. One or more consensus sequences are added to or substituted next to one or more amino acids between amino acid residues 236-237 of amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 13.

[0281] 15. One or more consensus sequences are added to or substituted for amino acid residue 270 of SEQ ID NO: 1, or 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 14.

[0282] 16. A modified Als3 protein according to any of paragraphs 1 to 15, wherein one or more consensus sequences are added to or substituted for amino acid residue 276 among amino acid residues 18-316 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 18-316 of SEQ ID NO: 1.

[0283] 17. One or more consensus sequences are added to or substituted next to one or more amino acids between amino acid residues 286-287 of amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 16.

[0284] 18. One or more consensus sequences are added to or substituted next to one or more amino acids between amino acid residues 299-300 of amino acid residues 18-316 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 18-316 of SEQ ID NO: 1, according to any of paragraphs 1 to 17.

[0285] 19. One or more of the consensus sequences are substituted with (i) amino acids between amino acid residues 33-37 and (ii) amino acids between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1, according to either paragraphs 1 and 2.

[0286] 20. A modified Als3 protein according to any of paragraphs 1 to 19, wherein substitution of an amino acid between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1, or one or more consensus sequences 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 18-316 of SEQ ID NO: 1, results in an increased expression level of the modified Als3 protein compared to the control Als3 protein.

[0287] 21. The modified Als3 protein of paragraph 20, wherein the expression level of the modified Als3 protein is increased by at least approximately 2-fold, at least approximately 3-fold, at least approximately 4-fold, at least approximately 5-fold, at least approximately 6-fold, at least approximately 7-fold, at least approximately 8-fold, at least approximately 9-fold, or at least approximately 10-fold compared to the control Als3 protein.

[0288] 22. A modified Als3 protein according to any of paragraphs 20 and 21, wherein the expression level of the modified Als3 protein is increased by approximately 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to the control Als3 protein.

[0289] 23. A modified Als3 protein from any of paragraphs 1 through 22, belonging to the genus Candida.

[0290] 24. A modified Als3 protein of paragraph 23, wherein the genus Candida is selected from the group consisting of Candida albicans, Candida auris, Candida gilliermondii, Candida lucitaniae, and Candida tropicalis.

[0291] 25. A modified Als3 protein of either paragraph 23 or 24, belonging to Candida albicans.

[0292] 26. A modified Als3 protein according to any of paragraphs 1 to 25, further comprising the amino acid sequence of YGKDVKDLFDYAQE (SEQ ID NO: 3), or at least one fructose diphosphate aldolase-1 (Fba) peptide having an amino acid sequence that is at least 70%, 80%, 85%, 90%, or 92% identical to SEQ ID NO: 3.

[0293] 27. The modified Als3 protein of paragraph 26, wherein the Fba peptide is covalently linked to the modified Als3 protein at one or more amino acid residues selected from the group consisting of 89, 163, 259, 199, and 316 among amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions (may be multiple) within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0294] 28. A modified Als3 protein according to either paragraph 26 or 27, wherein the Fba peptide is covalently linked to the modified Als3 protein at amino acid residue 316 of amino acid residues 18-316 of SEQ ID NO: 1, or at an equivalent position within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0295] 29. A modified Als3 protein according to any of paragraphs 26 to 28, wherein the modified Als3 protein comprises at least one further consensus sequence containing the amino acid sequence JUBD / EXNZS / TJUB, where 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 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 amino acid residues 18 to 316 of SEQ ID NO: 3.

[0296] 30. A modified Als3 protein from paragraph 29, 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.

[0297] 31. A modified Als3 protein according to either paragraph 29 or 30, wherein the further consensus sequence includes the amino acid sequence GSGGGDQNATGSGGG (SEQ ID NO: 9).

[0298] 32. A modified Als3 protein according to any of paragraphs 1 to 31, wherein at least one of the one or more consensus sequences contains the amino acid sequence KD / EXNZS / T, where X and Z are independently any amino acid other than proline.

[0299] 33. A modified Als3 protein according to paragraph 32, wherein X is Q (glutamine), Z is A (alanine), and one or more consensus sequences are selected from the group consisting of KDQNAT (SEQ ID NO: 5), KDQNAS (SEQ ID NO: 6), and DQNAT (SEQ ID NO: 7).

[0300] 34. Modified Als3 protein containing the amino acid sequence of SEQ ID NO: 10.

[0301] 35. Modified Als3 protein containing the amino acid sequence of SEQ ID NO: 11.

[0302] 36. A glycosylated, modified Als3 protein according to any of paragraphs 1 through 35.

[0303] 37. An N-glycosylated, modified Als3 protein of any of the types described in paragraphs 1 through 36.

[0304] 38. A conjugate comprising any modified Als3 protein from paragraphs 1 to 35 and at least one sugar antigen.

[0305] 39. The conjugate of paragraph 38, wherein the modified Als3 protein is linked to at least one sugar antigen.

[0306] 40. At least one sugar antigen is present in the following antigens: Escherichia coli O antigen, Salmonella species O antigen, Pseudomonas species O antigen, Klebsiella species O antigen, Acinetobacter O antigen, Chlamydia trachomatis O antigen, Vibrio cholerae O antigen, Listeria species O antigen, Legionella pneumophila serotypes 1-15. Conjugates selected from the group consisting of O antigen, Bordetella parapertussis O antigen, Burgholderia malayi and Pseudomalayi O antigen, Tularemia O antigen, Campylobacter species O antigen, Clostridium difficile capsular polysaccharide, Staphylococcus aureus types 5 and 8, Streptococcus pyogenes, Escherichia coli, Group B Streptococcus, Neisseria meningitidis, Candida species, Candida albicans, Haemophilus influenzae, Enterococcus faecalis capsular polysaccharide types I-V, and other surface polysaccharide structures, such as Borrelia burgdorferi glycolipid, Neisseria meningitidis pyrin O glycan and lipooligosaccharide (LOS), Haemophilus influenzae LOS, Leushmania major lipophosphoglycan, tumor-associated carbohydrate antigen, Malaria glycosylphosphatidylinositol, and Mycobacterium tuberculosis arabinomannan, as specified in paragraphs 38 and 39.

[0307] 41. A conjugate of any of paragraphs 38 and 39, wherein at least one sugar antigen is a fungal sugar antigen.

[0308] 42. A conjugate of any of paragraphs 38 to 41, wherein at least one sugar antigen is a sugar antigen of a Candida species.

[0309] 43. The conjugate of paragraph 42, in which species of the genus Candida are selected from the group consisting of Candida albicans, Candida auris, Candida gilliermondii, Candida lusitanie, and Candida tropicalis, Candida glabrata, Candida crusei, and Candida parapsis.

[0310] 44. A conjugate of any of paragraphs 38 to 43, in which at least one sugar antigen is a sugar antigen of Candida albicans.

[0311] 45. A conjugate according to any of paragraphs 38 to 44, wherein at least one sugar antigen contains a β-1,3-glucan polymer.

[0312] 46. ​​The conjugate of paragraph 45, wherein the β-1,3-glucan polymer contains at least four β-1,3-linked glucose molecules.

[0313] 47. β-1,3 glucan polymers: 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, 5-7, 5-6, 5, 6-1 A conjugate according to any of paragraphs 45 and 46, comprising 00, 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.

[0314] 48. A conjugate according to any of paragraphs 45 to 47, wherein the β-1,3-glucan polymer comprises at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or at least 11 consecutive β-1,3-linked glucose molecules.

[0315] 49. A conjugate according to any of paragraphs 38 to 44, wherein at least one sugar antigen comprises a β-1,2-mannan polymer.

[0316] 50. The conjugate of paragraph 48, wherein the β-1,2-mannan polymer contains at least two β-1,2-linked mannose molecules.

[0317] 51. β-1,2 mannan polymers: 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, A conjugate according to any of paragraphs 49 and 50, comprising 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.

[0318] 52. A conjugate according to any of paragraphs 49 to 51, wherein 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.

[0319] 53. A conjugate according to any of paragraphs 38 to 52, wherein the modified Als3 protein is linked to at least one sugar antigen by one or more amino acid residues on the modified Als3 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.

[0320] 54. A conjugate according to any of paragraphs 38 to 53, wherein the modified Als3 protein is linked to at least one sugar antigen by one or more asparagine residues on the modified Als3 protein.

[0321] 55. A bioconjugate, which is any of the conjugates described in paragraphs 38 through 54.

[0322] 56. A modified Als3 protein of Candida albicans comprising (1) the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11, and (2) at least one sugar antigen of the genus Candida, wherein the at least one sugar antigen is a β-1,3-glucan polymer consisting of at least six consecutive β-1,3-linked glucose molecules, and the at least one sugar antigen is linked to at least one of three asparagine residues at positions 20, 92 and 324 of SEQ ID NO: 10 or positions 20, 92 and 337 of SEQ ID NO: 11.

[0323] 57. A polynucleotide sequence encoding one of the modified Als3 proteins described in paragraphs 1 through 35.

[0324] 58. A vector containing the polynucleotide sequence from paragraph 57.

[0325] 59. 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 A1s3 protein according to any of paragraphs 1 to 35, and optionally (4) a polynucleotide sequence encoding a polymerase.

[0326] 60. The host cell of paragraph 59, which is *E. coli*.

[0327] 61. A method for generating a bioconjugate comprising a modified Als3 protein linked to at least one sugar antigen, comprising (1) culturing host cells of any of paragraphs 59 and 60 under conditions suitable for protein generation, and (2) isolating the bioconjugate.

[0328] 62. A bioconjugate produced by the method of paragraph 61, comprising at least one sugar linked to a modified Als3 protein of any of paragraphs 1 to 35.

[0329] 63. An immunogenic composition comprising a modified Als3 protein from any of paragraphs 1 to 37 and 56, a conjugate from any of paragraphs 38 to 54, or a bioconjugate from any of paragraphs 55 and 62.

[0330] 64. A method for preparing an immunogenic composition according to paragraph 63, comprising the step of mixing a modified Als3 protein according to any of paragraphs 1 to 37 and 56, a conjugate according to any of paragraphs 38 to 54, or a bioconjugate according to any of paragraphs 55 and 62 with a pharmaceutically acceptable excipient or carrier.

[0331] 65. A vaccine comprising the immunogenic composition of paragraph 63 and optionally pharmaceutically acceptable excipients or carriers.

[0332] 66. A Candida albicans vaccine comprising (1) a modified Als3 protein according to any of paragraphs 1 to 35, (2) at least one Candida albicans glycoantigen linked to the modified Als3 protein, and optionally (3) a pharmaceutically acceptable carrier or adjuvant.

[0333] 67. A method for treating or preventing Candida albicans infection in a subject requiring treatment or prevention of Candida albicans infection, comprising administering a therapeutically effective dose to the subject of any modified Als3 protein from paragraphs 1 to 37 and 56, any conjugate from paragraphs 38 to 55, any bioconjugate from paragraphs 55 and 62, any immunogenic composition from paragraph 63, or any vaccine from paragraphs 65 and 66.

[0334] 68. Candida albicans infection causes recurrent vulvovaginal candidiasis (RVVC) in the subject, as described in paragraph 54.

[0335] 69. A method for immunizing a subject against Candida albicans infection, comprising administering an immunoprotective dose to the subject of any modified Als3 protein from paragraphs 1 to 37 and 56, any conjugate from paragraphs 38 to 55, any bioconjugate from paragraphs 55 and 62, any immunogenic composition from paragraph 63, or any vaccine from paragraphs 65 and 66.

[0336] 70. A method for inducing an immune response to Candida albicans infection in a subject, comprising administering a therapeutic or prophylactic dose to the subject of any modified Als3 protein from paragraphs 1 to 37 and 56, any conjugate from paragraphs 38 to 55, any bioconjugate from paragraphs 55 and 62, any immunogenic composition from paragraph 63, or any vaccine from paragraphs 65 and 66.

[0337] 71. Any method described in paragraphs 66 to 70, wherein the subject is human.

[0338] 72. A modified Als3 protein from any of paragraphs 1 to 37 and 56, a conjugate from any of paragraphs 38 to 55, a bioconjugate from any of paragraphs 55 and 62, an immunogenic composition from paragraph 63, or a vaccine from any of paragraphs 65 and 66, for use in the treatment or prevention of diseases caused by Candida albicans infection.

[0339] 73. A modified Als3 protein from any of paragraphs 1 to 37 and 56, a conjugate from any of paragraphs 38 to 55, a bioconjugate from any of paragraphs 55 and 62, an immunogenic composition from paragraph 63, or a vaccine from any of paragraphs 65 and 66, for use in the manufacture of a pharmaceutical product for the treatment or prevention of a disease caused by Candida albicans infection.

[0340] 74. A method for improving the expression level of any of the modified Als3 proteins described in paragraphs 1 to 37, comprising substituting an amino acid between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 with one or more consensus sequences 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 18-316 of SEQ ID NO: 1, wherein the modified Als3 protein exhibits an improved expression level compared to a control Als3 protein that does not contain one or more consensus sequences that are substituted between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1, or at equivalent positions within an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 18-316 of SEQ ID NO: 1.

[0341] 75. The method of paragraph 74, wherein the expression level of the modified Als3 protein is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold compared to the control Als3 protein.

[0342] 76. The method of paragraph 75, wherein the expression level of the modified Als3 protein is increased by approximately 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold compared to the control Als3 protein.

[0343] 77. A host cell, i. A nucleotide sequence encoding one or more heterologous glycosyltransferases capable of synthesizing β-1,3-glucan polymers. ii. A nucleotide sequence encoding a glycosyltransferase that can covalently bond a glucose molecule to an N-acetylglucosamine (GlcNac) molecule. iii. Nucleotide sequences encoding heterologous oligosaccharide transferases, and iv. A nucleotide sequence encoding a modified carrier protein that includes 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.

[0344] 78. The host cells of paragraph 77, containing one or more heterologous glycosyltransferases from rhizobia, possibly from the genus Sinorhizobium, possibly from Sinorhizobium melilotii 1021, including exoL, exoM, exoO, exoU, and exoW.

[0345] 79. The host cells of paragraph 77, containing one or more heterologous glycosyltransferases from rhizobia, possibly from the genus Agrobacterium, and possibly from the species Agrobacterium ZX09, including SleC, SleE, SleF, SleU, and SleW.

[0346] 80. i. Host cells of paragraph 79, wherein one or more heterologous glycosyltransferases have amino acid sequences that are at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleC of Agrobacterium species ZX09, including SEQ ID NO: 12.

[0347] 81. i. A host cell of any of paragraphs 79 and 80, wherein one or more heterologous glycosyltransferases have an amino acid sequence at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleE of Agrobacterium species ZX09, including SEQ ID NO: 13.

[0348] 82. One or more heterologous glycosyltransferases from paragraphs 79 to 81 have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleF of Agrobacterium species ZX09, including SEQ ID NO: 14, in any one host cell from paragraphs 79 to 81.

[0349] 83. One or more heterologous glycosyltransferases from paragraphs 79 to 82 have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of SleU of Agrobacterium species ZX09, including SEQ ID NO: 15, in any one host cell from paragraphs 79 to 82.

[0350] 84. One or more heterologous glycosyltransferases from paragraphs 79 to 83 have an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the amino acid sequence of Agrobacterium species ZX09 SleW, including SEQ ID NO: 16, in any one host cell from paragraphs 79 to 83.

[0351] 85. One host cell from any of paragraphs 79 to 84 that produces more SleW than SleC, SleE, SleF, or SleU.

[0352] 86. A host cell from any one of paragraphs 79 to 85 containing at least two gene copies of SleW of Agrobacterium species ZX09.

[0353] 87. Host cells from any of paragraphs 79 to 86 containing at least one copy of SleW from the Agrobacterium species ZX09, under the control of a potent promoter that enables higher expression of SleW compared to SleC, SleE, SleF, or SelU.

[0354] 88. ii. The glycosyltransferase capable of covalently bonding glucose to GlcNac is WfaP from E. coli O56, in any one host cell from paragraphs 77 to 87.

[0355] 89. Host cells of paragraph 88 in which WfaP has an amino acid sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 17.

[0356] 90. A host cell from any one of paragraphs 77 to 89, comprising (i) a wzm gene comprising the nucleotide sequence of SEQ ID NO: 36, and optionally comprising a nucleotide sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 36, and (ii) a nucleotide sequence comprising a wzt gene comprising the nucleotide sequence of SEQ ID NO: 37, and optionally comprising a nucleotide sequence at least 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 37.

[0357] 91. iii. The oligosaccharide transferase is PglB in any one host cell from paragraphs 77 to 90.

[0358] 92. Host cells of paragraph 91 in which PglB is derived from Campylobacter, possibly Campylobacter jejuni or Campylobacter coli containing the amino acid sequence of SEQ ID NO: 20.

[0359] 93. iv. The modified carrier protein is selected from the group consisting of Als3, Sap2, 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, thermolabile enterotoxin of Escherichia coli, detoxified variant of thermolabile enterotoxin of Escherichia coli, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin of Escherichia coli, sat protein of Escherichia coli, passenger domain of sat protein of Escherichia coli, C. jejuni AcrA, and C. jejuni natural glycoprotein in any one host cell from paragraphs 77 to 92.

[0360] 94. Host cells of paragraph 93 in which the modified carrier protein is one of the modified Als3 proteins from paragraphs 1 to 35.

[0361] 95. A host cell from any one of paragraphs 77 to 94, which is a species of Escherichia, Shigella, Klebsiella, Xanthomonas, Salmonella, Yersinia, Lactococcus, Lactobacillus, Pseudomonas, Corynebacterium, Streptomyces, Streptococcus, Staphylococcus, Bacillus, or Clostridium.

[0362] 96. The host cell of paragraph 95, which is a species of E. coli.

[0363] 97. A method for producing a glycoconjugate comprising a modified carrier protein and β-1,3-glucan, comprising culturing any one of the host cells described in paragraphs 77 to 96 under conditions suitable for protein production.

[0364] 98. A bioconjugate produced by the method of paragraph 97.

[0365] 99. Structure:

[0366] [ka] A sugar that is a glucan having, n is 2-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25. sugar.

[0367] 100. Structure:

[0368] [ka] It has, Paragraph 99 sugars where n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25.

[0369] 101. Structure: [ka] A sugar that is a glucan having, n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25. sugar.

[0370] 102. One of the sugars from paragraphs 99 to 101, linked to a lipid carrier.

[0371] 103. The sugar of paragraph 102, in which the lipid carrier is undecaprenyl.

[0372] 104. A conjugate containing one of the sugars from paragraphs 99 to 101 linked to an asparagine residue of a modified carrier protein.

[0373] 105. The conjugate of paragraph 104, wherein the asparagine residue is in the consensus sequence D / EXNZS / T, and X and Z are independently any amino acid other than proline.

[0374] 106. A conjugate of either paragraph 104 or 105, wherein the modified carrier protein is selected from the group consisting of Als3, Sap2, 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, thermolabile enterotoxin of Escherichia coli, detoxified variant of thermolabile enterotoxin of Escherichia coli, cholera toxin B subunit (CTB), cholera toxin, detoxified variant of cholera toxin of Escherichia coli, sat protein of Escherichia coli, passenger domain of sat protein of Escherichia coli, C. jejuni AcrA, and C. jejuni natural glycoprotein.

[0375] 107. A conjugate of any one of paragraphs 104 to 106, wherein the carrier protein is any one of the modified Als3 proteins from paragraphs 1 to 35.

[0376] 108. A bioconjugate, which is one of the conjugates from paragraphs 104 through 107.

[0377] 109. A method for producing β-1,3-glucan polymers in prokaryotic host cells, In host cells, i. A nucleotide sequence encoding a first glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetylglucosamine (GlcNAc) molecule, wherein the first glycosyltransferase is WfaP derived from E. coli O56. ii. A nucleotide sequence encoding a further glycosyltransferase capable of synthesizing fungal β-1,3-glucan, wherein the further glycosyltransferase comprises SleC, SleE, SleF, SleU, and SleW derived from rhizobia, possibly from the genus Agrobacterium, and possibly from the species Agrobacterium ZX09, wherein the host cell produces more SleW than SleC, SleE, SleF, or SleU, and iii. A nucleotide sequence encoding a translocase that can, in some cases, transfer β-1,3-glucan to the periplasmic side of the inner membrane of a prokaryotic host cell, wherein the translocase contains Wzm-Wzt derived from a species of the genus Klebsiella, and in some cases from Klebsiella pneumoniae. Steps to introduce and express A method comprising a β-1,3-glucan polymer linked to a lipid carrier via GlcNAc, wherein the β-1,3-glucan polymer contains at least four β-1,3-linked glucose molecules.

[0378] 110. The method of paragraph 109, where the prokaryotic host cell is E. coli.

[0379] 111. The method of paragraph 110, wherein GlcNAc is linked to a lipid carrier by WecA derived from host E. coli cells.

[0380] 112. Any method of paragraphs 109 to 111, wherein the β-1,3-glucan polymer is a fungal, possibly Candida, possibly Candida albicans β-1,3-glucan polymer.

[0381] 113. A β-1,3-glucan polymer having the structure of any one sugar from paragraphs 99 to 101, according to any one method from paragraphs 109 to 112.

[0382] 114. Any method of paragraphs 109 to 113, wherein the lipid carrier is undecaprenyl.

[0383] 115. A method for generating a bioconjugate in a prokaryotic host cell, a. The step of obtaining a prokaryotic host cell that produces a β-1,3-glucan polymer, as described in any of paragraphs 109 to 114, b. Furthermore, in host cells, i. A nucleotide sequence encoding a modified carrier protein comprising a glycosylation site including the consensus sequence D / EXNZS / T, wherein X and Z are any amino acids other than proline, and 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. A nucleotide sequence encoding an oligosaccharide transferase capable of generating a bioconjugate by transferring a β-1,3-glucan polymer from a lipid carrier to a modified carrier protein, wherein the oligosaccharide transferase is PglB derived from the genus Campylobacter, and possibly from Campylobacter jejuni or Campylobacter coli. Steps to introduce and express A method that includes this.

[0384] 116. The method of paragraph 115, wherein the bacterial signal sequence is selected from the group consisting of Flgl, MalE, OmpA, and OmpC.

[0385] 117. The method of paragraph 116, where the bacterial signal sequence is Flgl.

[0386] 118. The method of paragraph 116, wherein Flgl contains the amino acid sequence of sequence number 21.

[0387] 119. 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, by any method of paragraphs 115 to 118.

[0388] 120. Any method of paragraphs 115 to 119, wherein the modified carrier protein is any modified Als3 protein of paragraphs 1 to 35.

[0389] 121. A method for improving the expression level of any of the modified Als3 proteins described in paragraphs 1 to 37 in a host cell, comprising substituting an amino acid between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 with one or more consensus sequences 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 18-316 of SEQ ID NO: 1, wherein the modified Als3 protein, when expressed in a host cell, exhibits an improved expression level compared to a control Als3 protein that does not contain one or more consensus sequences, in which an amino acid between amino acid residues 104-108 of amino acid residues 18-316 of SEQ ID NO: 1 is substituted with one or more consensus sequences 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 18-316 of SEQ ID NO: 1.

[0390] 122. A method for generating a bioconjugate in a prokaryotic host cell, a. The step of obtaining a prokaryotic host cell that produces a β-1,3-glucan polymer, as described in any of paragraphs 109 to 114, b. Furthermore, in host cells, i. A nucleotide sequence encoding a modified carrier protein comprising a glycosylation site including the consensus sequence D / EXNZS / T, wherein X and Z are any amino acids other than proline (optionally the polynucleotide sequence of paragraph 57), and the modified carrier protein further comprises an N-terminal bacterial signaling sequence capable of transporting the modified carrier protein to the periplasmic side of the inner membrane of a prokaryotic host cell, and ii. A nucleotide sequence encoding an oligosaccharide transferase capable of generating a bioconjugate by transferring a β-1,3-glucan polymer from a lipid carrier to a modified carrier protein, wherein the oligosaccharide transferase is PglB derived from the genus Campylobacter, and possibly from Campylobacter jejuni or Campylobacter coli. Steps to introduce and express A method that includes this.

[0391] 123. A modified Als3 protein according to any of paragraphs 26 to 28, wherein the modified Als3 protein comprises at least one further consensus sequence containing the amino acid sequence JUBD / EXNZS / TJUB, where 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 amino acid residues 18 to 316 of SEQ ID NO: 3.

[0392] 124. A host cell comprising (1) one or more polynucleotide sequences encoding one or more heterologous glycosyltransferases, (2) a polynucleotide sequence encoding a glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetylglucosamine (GlcNac) molecule, (3) a polynucleotide sequence encoding a heterologous oligosaccharide transferase, (4) a polynucleotide sequence encoding a modified A1s3 protein according to any of paragraphs 1 to 35, and optionally a polynucleotide sequence encoding a polymerase.

[0393] 125. A host cell, i. A nucleotide sequence encoding one or more heterologous glycosyltransferases capable of synthesizing β-1,3-glucan polymers. ii. A nucleotide sequence encoding a glycosyltransferase that can covalently bond a glucose molecule to an N-acetylglucosamine (GlcNac) molecule. 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 of paragraph 57. Host cells containing these cells.

[0394] 126. A method for inhibiting the attachment of Candida albicans hyphae to vaginal epithelial cells in a subject (e.g., human), comprising administering a therapeutic or prophylactic dose to the subject of any modified Als3 protein from paragraphs 1 to 37 and 56, any conjugate from paragraphs 38 to 55, any bioconjugate from paragraphs 55 and 62, any immunogenic composition from paragraph 63, or any vaccine from paragraphs 65 and 66.

[0395] 127. A method for mediating neutrophil-killing by Candida albicans hyphae in a subject (e.g., human), comprising administering a therapeutic or prophylactic dose to the subject of any modified Als3 protein from paragraphs 1 to 37 and 56, any conjugate from paragraphs 38 to 55, any bioconjugate from paragraphs 55 and 62, any immunogenic composition from paragraph 63, or any vaccine from paragraphs 65 and 66.

[0396] 128. A method for inhibiting the formation of a Candida albicans biofilm, comprising (i) administering a therapeutically effective or prophylactic dose to a subject (e.g., a human) of a modified Als3 protein from any of paragraphs 1 to 37 and 56, a conjugate from any of paragraphs 38 to 55, a bioconjugate from any of paragraphs 55 and 62, an immunogenic composition from paragraph 63, or a vaccine from any of paragraphs 65 and 66.

[0397] Any publications referenced herein are incorporated herein by reference in their entirety. The term “or” as used herein indicates alternatives that can be combined, where appropriate; that is, the term “or” includes each of the listed alternatives individually, as well as any combination thereof. Where used herein, unless the context explicitly indicates otherwise, references to the singular, such as the singular forms “a,” “an,” and “the,” include their plurals, and references to the plural include their singular forms.

[0398] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0399] [Examples] Materials and methods Operation of Als3-NT for glycosylation by antigenic glycans To predict suitable insertion sites for glycosites, the crystal structure of the Als3 protein of the present invention ("Als3-NT") having the amino acid sequence of SEQ ID NO: 27 was analyzed using various software.

[0400] Sequence ID 27 - Candida albicans Als3-NT (amino acid sequence 18-316): [ka]

[0401] A total of 20 positions were selected for the insertion of a consensus sequence for glycosylation, i.e., site-directed mutagenesis of a glycosite (e.g., D / EXNZS / T) (Table 1 and Figure 1). These positions included the N-terminus and C-terminus of the Als3-NT domain, as well as its solvent-accessible loop and several beta strands. Modified Als3-NT proteins were created by substituting one or more (up to five) amino acids with the glycosite sequence. In some cases, the glycosite was inserted between two amino acids of the Als3-NT protein without creating any sequence substitutions. Modified Als3-NT proteins containing a single glycosite were tested for glycosylation using the Klebsiella pneumoniae O5 antigen. By combining the best-performing glycosites, modified Als3-NT proteins containing a total of 2 to 6 glycosites were generated. The six positions selected for the combination are 33-37 (Mut1), 104-108 (Mut4), 163-164 (Mut8), 220 (Mut12), 299-300 (Mut18), and 316 (C-terminus), and their numbering corresponds to residues in the native Candida albicans Als3 sequence (SEQ ID NO: 1).

[0402] Glycosylation test using manipulated Als3-NT containing one or more glycosites Modified Als3-NT proteins containing a single insertion glycosite were tested for in vivo glycosylation efficiency using the Klebsiella pneumoniae O5 antigen. In the dataset presented in this study, the E. coli W3110 derivative strain used contained a deletion of the LPS-O antigen ligase waaL and contained a gene cluster for Klebsiella pneumoniae O5 glycan biosynthesis, replacing the native O antigen cluster rfbO16. E. coli strains producing KpO5 glycan were transformed with the pEC415 plasmid containing the modified Als3-NT protein and a plasmid expressing PglB. To prepare precultures, colonies were streaked from transformation plates and seeded in 5 ml of TB medium containing 10 mM MgCl2 and appropriate antibiotics, and grown overnight at 37°C. Using the precultures, seeding was performed in 50 ml of supplemental TB medium in a shaking flask to obtain an initial OD600 = 0.1. The culture was grown at 37°C with shaking at 200 rpm until an OD600 of 0.8-1 was reached, and then induced by adding 0.001% arabinose (Als3) and 0.1 mM IPTG (PglB). Expression and glycosylation of the modified Als3-NT protein were continued overnight at 25°C.

[0403] Selection criteria for modified Als3-NT proteins with a single glycosite included total expression level and the level of generated glycoconjugate, the latter indicating the suitability of the glycosite location for modification by PglB.

[0404] Preparation of periplasm extract OD 600 The amount of cells from the overnight culture corresponding to =60 (measured using a 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.

[0405] Enrichment of periplasm extracts by immobilized metal affinity chromatography (IMAC) To enrich periplasm extracts containing modified Als3-NT protein and enable more direct reading by SDS-PAGE, the His-tagged modified Als3-NT 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-equilibrated Ni-NTA slurry and incubated for 30 minutes with gentle shaking. 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). IMAC-enriched PPE was analyzed by SDS-PAGE. (“Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4”, Nature, 227 (5259): pp. 680-685. Bibcode:1970Natur.227..680L. doi:10.1038 / 227680a0. ISSN 0028-0836. PMID 5432063). Non-glycosylated Als3-NT proteins and glycoconjugates glycosylated at one or more positions (i.e., modified Als3-NT proteins linked to one or more polysaccharide chains) were detected on gels by Coomassie 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: pp. 377-391. doi:10.1016 / 0006-3002(63)91092-8. PMID 18421828).

[0406] Western blot analysis of periplasm extract Periplasm extracts were also analyzed by immunoblotting against the His tag and polysaccharides bound to the modified Als3-NT protein. For the detection of KpO5, antiserum against the K-capsule mutant of Klebsiella pneumoniae O5 strain was used.

[0407] Construction of a modified Als3-NT-β-1,3-glucan-producing strain The E. coli K12 W3110 derivative strain was constructed by sequentially replacing the target gene cluster with the target gene, followed, if present, by removing an FRT site adjacent selection marker via λ-Red homologous recombination, and then by removing the FLP recombinase-catalyzed marker described in (TE Kuhlman and EC Cox. Nucleic Acids Res. April 2010; 38(6): e92). Five homologous recombination / marker removal steps were performed to remove the following genomic sequences: i. O16 O-antigen cluster (rfb or wb, GenBank NC_007779, positions 2'114'113~2'103'814), ii. cholanaic acid cluster (wca, GenBank NC_007779, positions 2'138'241~2'118'033), iii. ECA cluster retaining wecA (wec, GenBank NC_007779, positions 3'666'604~3'656'725), iv. O16wzz2 or cld (GenBank NC_007779, positions 2'099'458~2'100'438), v. gtrABS or yfdGHI (GenBank NC_007779 (2'473'301~2'475'908), vi. araBA (GenBank NC_007779 66'835~70'048). Furthermore, a codon-optimized version of C. Jejuni pglB (GenBank WP_002866139) was introduced to replace the LPS-O antigen ligase waaL (GenBank NC_007779 3'842'208~3'843'467).

[0408] Expression plasmids containing the selected modified Als3-NT protein gene, followed by genes necessary for β-1,3-glucan biosynthesis in the pEC415 backbone (Schulz et al. J Biol Chem. August 1998; 281(5380): pp. 1197-200), were constructed starting from a synthetic DNA template using different steps of classical restriction enzyme cloning and Gibson assembly (Gibson, DG, et al. (2009) Nat. Methods 6, pp. 343-345). The final plasmids, from 5' to 3', contained the following genes under an arabinose-inducible promoter: als3-NT, sleW, sleU, sleF, sleE, sleC, wfaP, wzm, and wzt. The modified Als3-NT protein is described above. The sequences sleW, sleU, sleF, sleE, and sleC are derived from the succinoglycan-like extracellular polysaccharide biosynthesis gene cluster of Agrobacterium species ZX09 (GenBank KT780309; Xu et al. Appl Microbiol Biotechnol. (2017) 101:585~598), the wfaP sequence is derived from E. coli O56 (GenBank DQ220293), and wzm and wzt are derived from K. pneumoniae (GenBank CP052562, positions 1'695'622~1'697'129).

[0409] The second expression plasmid was constructed by cloning a codon-optimized version of C. jejuni pglB (GenBank WP_002866139) into the vector pEXT21 (Dykxhoorn et al. Gene. 1996. 177(1-2):133~136) under an IPTG-inducible promoter.

[0410] Conjugate-producing strains were obtained by transforming the manipulated strain with two plasmids. The roles of each gene in bioconjugate generation and the structure of the glycan are illustrated in Figure 2.

[0411] Production and analysis of modified Als3-NT-β-1,3-glucan bioconjugates The conjugate-producing strain was grown in buffered rich medium at 35°C and pH 7 in a fed-batch 10L bioreactor. The OD of the culture was measured. 600nm When the temperature recorded a value of 20-25, the temperature was switched to 30°C, bioconjugate formation was induced with IPTG and arabinose, and the supply of rich medium was started. Cells were harvested by centrifugation 18 hours after induction, washed, and resuspended in TBSE buffer. An osmotic shock protocol was applied for 1 hour to dilute the cells with 5 times the volume of H2O, and the contents of the periplasm were obtained in the supernatant. Cells were separated from the supernatant by centrifugation, and cell residue was removed by filtration through 0.45 μm and 0.2 μm filters. Purification of the bioconjugate from the filtered supernatant consisted of four chromatographic steps: i. anion exchange, ii. anion exchange, iii. hydrophobic interaction, and iv. size exclusion. Elution profiles were followed by absorbance at 280 nm and SDS-PAGE / Coomsie staining. The purified bioconjugate was analyzed using SDS-PAGE gel, followed by i. Coomassie staining, ii. anti-Als3 immunoblotting, iii. anti-Fba immunoblotting, iv. anti-β-1,3-glucan immunoblotting, and dectin-1 blotting, as reported in Figure 5.

[0412] The results of these experiments are shown in Figures 3-16, as described in the following examples.

[0413] [Example 1] SDS-PAGE analysis of modified Als3-NT protein-glycosytic mutants purified from PPE by IMAC SDS-PAGE analysis (Figure 3) was performed on IMAC-enriched periplasm extracts of E. coli strains that produce KpO5 polysaccharide and express a modified Als3-NT protein containing PglB and the glycosite D / EXNZS / T introduced at the following position in SEQ ID NO: 1.

[0414] [Table 1]

[0415] The bands shown in Figure 3 correspond to the non-glycosylated modified Als3-NT protein ("u-carrier") and the KpO5 modified Als3-NT bioconjugate ("conjugate") which has one occupied glycosite.

[0416] Conclusion: As shown in Figures 3 and 4, 19 out of 20 modified Als3-NT proteins containing a single glycosite are expressed at levels comparable to or higher than the wild-type (wt) Als3-NT protein (SEQ ID NO: 27). Surprisingly, the Mut4 mutant shows a >2-fold improvement in expression compared to wt Als3-NT. Figure 4A shows the relative expression levels of modified Als3-NT proteins containing a single glycosite compared to the expression level of wt Als3-NT. Figure 4B shows the glycosylation efficiency of modified Als3-NT proteins containing a single glycosite.

[0417] Glycosylation of the modified Als3-NT protein by KpO5 at 17 of the 19 positions was found to be equally good or better than that of the non-glycosylated wt Als3-NT control (Figure 3, lane 2). In particular, Als3-NT mutants with glycosite D / EXNZS / T introduced at positions 33-37 (Mut1), 104-108 (Mut4), 163-164 (Mut8), 220 (Mut12), 299-300 (Mut18), or 316 (C-terminal glycotag) appeared to be superior to the non-glycosylated wt Als3-NT control. The modified Als3-NT protein used in this analysis contained a histidine tag, but those skilled in the art will understand that modified Als3-NT protein without the histidine tag can also be used for analysis. Therefore, in certain embodiments, the present invention provides a modified Als3-NT protein in which the histidine tag has been removed.

[0418] [Example 2] Western blot analysis of purified modified Als3-NT protein-glucan conjugate Immunoblot analysis was performed on periplasmic extracts of *E. coli* strains producing the KpO5 antigen polysaccharide and expressing a modified Als3-NT protein with PglB and one glycosite introduced at the position shown in Table 1 above. Figure 5A shows SDS-PAGE analysis of purified non-glycosylated Als3-NT-Fba (SEQ ID NO: 10) (lane 1) and purified Als3-NT-glucan conjugate (lane 2). M represents the protein standard. Figures 5B-5D show immunoblots probed using anti-Als3 antibody (Figure 5B), anti-Fba antibody (Figure 5C), and anti-glucan antibody (Figure 5D), respectively. Figure 5E shows the recognition of glucan conjugated to the modified Als3-NT protein by the Dectin-1 receptor. The bands in Figures 5B-5E correspond to a modified Als3-NT protein-glucan conjugate (lane 2) that extends to and occupies three glycosites of the non-glycosylated wt Als3-NT protein (lane 1).

[0419] Conclusion: SDS-PAGE demonstrates that the modified Als3-NT protein-glucan conjugate could be purified to high purity and that a significant level of glycosylation at the β-glucan is indicated by an increased molecular weight compared to non-glycosylated Als3-NT-Fba. Antigen identity was confirmed by four independent Western blot analyses, using specific antibodies against Als3, Fba peptide, and β-1,3-glucan. The Western blot in Figure 5E shows that the glucan chain contains at least 11 β-1,3-linked glucose units, as such a minimum β-1,3-glucan length is required for recognition by the Dectin-1 receptor (Palma et al., 2006, J Biol Chem., 281(9):5771-9).

[0420] [Example 3] Assay demonstrating the functionality of modified Als3-NT-SPR using fibronectin The attachment function of Als3 is located within the N-terminal (NT) region, which has a peptide-binding cavity to which various peptide ligands can be bound (Lin, J et al., J. Biol. Chem. 2014; 2. Coleman, DA et al., J. Mol. Meth. 2009; 3. Schmidt, CS A et al., Vaccine 2012). The objective was to demonstrate that the modified Als3-NT protein expressed in E. coli periplasm is conserved and has the same structure and function as its native source, Candida albicans. A further objective was to confirm that manipulation and glycosylation of the Als3-NT glycosite do not affect protein function.

[0421] Assay setup: The surface plasma resonance (SPR) assay is intended to test the binding of the modified Als3-NT protein to its native ligand, fibronectin, based on a modified version of the published protocol (Ielasi et al. 2016, mBio 7(4): e00584-16). The modified Als3-NT protein is subjected to C-terminal His 10 The protein was captured on an NTA chip via a tag (SEQ ID NO: 72). Fibronectin was added to the mobile phase, and binding was tested at eight concentrations (from 450 nM to 3.5 nM). The interaction between the modified Als3-NT protein and fibronectin was analyzed by multicycle kinetics. A 1:1 binding mode was applied to the kinetic fitting. SPR sensorgrams and measurement parameters are shown in Figure 6.

[0422] Conclusion: As shown in Figure 6, the manipulated nonglycosylation modified Als3-NT protein ("uAls 318-316 -3S" (central panel) or glycosylated Als3-NT protein ("β-glucan-Als 318-316 -3S (right panel) has 264, 272, and 280 nM of K, respectively. D The value is for the unmodified wt Als3-NT protein ("Als 318-316The "wt" (left panel) shows a very good resemblance to the binding to fibronectin. Therefore, the structure and function of Als3 are conserved upon manipulation and glycosylation.

[0423] [Example 4] Immunogenicity of Modified Als3-NT Protein-Glucan Bioconjugates Figure 7 shows the preclinical trial of the modified Als3-NT protein-glucan bioconjugate (Als3-NT-3S-Fba_bgluc d+ , Als3-3FG) in rabbits. Figure 7A shows the properties of the bioconjugate. Figure 7B shows the 3D representation of the modified Als3-NT protein-glucan bioconjugate. The structure of the modified Als3-NT protein is shown as a schematic diagram. The spheres represent the positions of the three introduced glycosites. The conjugated beta-glucan chains are represented schematically, and the position and sequence of the Fba peptide sequence are shown in red. Figure 7C shows the immunization scheme of rabbits using the modified Als3-NT protein-glucan bioconjugate.

[0424] Figures 8A and 8B show the immunogenicity of the modified Als3-NT protein-glucan bioconjugate in rabbits. Modified Als3-NT protein-glucan bioconjugate: Als3 18-316 -3S-Fba-bgluc d+ , the control shows animals immunized with buffer, and the whole group was tested with AS03. New Zealand White (NZW) rabbits were immunized three times at two-week intervals. Coating ELISA: 8A) Modified Als3 not engineered with His tag and 8B) Fba peptide. Als3 or Fba-specific serum IgG concentrations (arbitrary units, AU) in rabbit sera before, after-II, after-III (d0, d28, d42) for each treatment group. The lines show the geometric mean concentration (GMC) + / - 95% confidence interval. ****: p < 0.0001, one-way ANOVA. Ref: 36_010.

[0425] Conclusion: 1) The modified Als3-NT protein-glucan bioconjugate is immunogenic. Significant titer increases were shown in pre / post-II and pre / post-III (Figure 8A). 2) The Fba peptide (part of the modified Als3-NT protein-glucan bioconjugate) is immunogenic. Significant titer increases were shown in pre / post-II and pre / post-III (Figure 8B).

[0426] [Example 5] Immunogenicity of modified Als3-NT protein-glucan bioconjugate (glycan) Figure 9 shows the immunogenicity of the modified Als3-NT protein-glucan bioconjugate in rabbits. Modified Als3-NT protein-glucan bioconjugate: Als3 18-316 -3S-Fba-bgluc d+ The control group consisted of animals immunized with buffer, and all groups were tested with AS03. NZW rabbits were immunized three times at 2-week intervals. Coated ELISA: β-glucan extract. β-glucan-specific serum IgG concentrations (arbitrary units, AU) in rabbit serum before, after-II, and after-III (d0, d28, d42) for each treatment group. Lines indicate the GMC+ / - 95% confidence interval. ****: p<0.0001, one-way ANOVA.

[0427] Conclusion: As shown in Figure 9, the modified Als3-NT protein-glucan bioconjugate containing β-glucan is immunogenic. Significant increases in titer are shown in pre / post-II and pre / post-III.

[0428] [Example 6] Quantitative adhesion inhibition assay for plastics Figure 10 shows the ability of antibodies against a modified Als3-NT protein-glucan bioconjugate to inhibit the adhesion of C. albicans mycelium to plastic. Serum was mixed with C. albicans mycelium (ATCC90028), added to plastic wells, and incubated for 4 hours. After washing, viable cells were measured using CellTiter Glo®. Modified Als3-NT protein-glucan bioconjugate: Als3 18-316 -3S-Fba-bgluc d+ The control group consisted of animals immunized with buffer, and all groups were tested with AS03. NZW rabbits were immunized three times at 2-week intervals. Graphs depict mean + SD. ****: p<0.0001, one-way ANOVA.

[0429] Conclusion: As shown in Figure 10, serum containing a modified Als3-NT protein-glucan bioconjugate inhibits the adhesion of C. albicans hyphae to plastic. A significant reduction in adhesion to plastic wells is shown in pre / post-III.

[0430] [Example 7] Quantitative adhesion assay of C. albicans mycelium Figures 11A and 11B show the ability of antibodies against a modified Als3-NT protein-glucan bioconjugate to inhibit the adhesion of C. albicans to vaginal epithelial cells. Figure 11A: Quantification of adhesion, Figure 11B: Microscopic images of Candida species attached to epithelial cells. Serum was mixed with C. albicans (SC5314) and added to epithelial cells (A431), and incubated for 1.5 hours. After washing, adherent cells were measured using concavalin A - Alexa fluor 488. Modified Als3-NT protein-glucan bioconjugate: Als3 18-316 -3S-Fba-bgluc d+ The control group consisted of animals immunized with buffer, and all groups were tested with AS03. NZW rabbits were immunized three times at 2-week intervals. Graphs depict mean + SD. ****: p<0.0001, one-way ANOVA.

[0431] Conclusion: As shown in Figures 11A and 11B, serum containing a modified Als3-NT protein-glucan bioconjugate inhibits the adhesion of C. albicans hyphae to vaginal epithelial cells. A significant reduction in adhesion to epithelial cells is shown in post-III compared to control serum.

[0432] [Example 8] Antibody binding to C. albicans mycelium Figures 12 and 13 show the ability of antibodies against a modified Als3-NT protein-glucan bioconjugate to bind to C. albicans cells. Figure 12 shows antibody binding to C. albicans hyphae using whole-cell ELISA. Coated ELISA: Clinical isolate of C. albicans hyphae (SC5314) grown in RPMI.

[0433] Figure 13 shows microscopic images of antibodies bound to C. albicans cells. Cells were observed using differential interference contrast microscopy (DIC) and fluorescence microscopy (secondary antibody Alexa 488). Serum was mixed with C. albicans (SC5314) and stained with concavalin A - Alexa fluor 488. Modified Als3-NT protein-glucan bioconjugate: Als3 18-316 -3S-Fba-bgluc d+ The control group consisted of animals immunized with buffer, and all groups were tested with AS03. NZW rabbits were immunized three times at two-week intervals.

[0434] Conclusion: As shown in Figures 12 and 13, antibodies against the modified Als3-NT protein-glucan bioconjugate can bind to C. albicans cells coated on a plate. Using laser confocal microscopy, it is possible to confirm the binding of antibodies to yeast and hyphae segments of Candida cells.

[0435] [Example 9] Antibody binding to C. auris Figure 14 shows microscopic images of antibodies bound to C. auris VPCI479 / P / 13 cells. Cells were observed using differential interference contrast microscopy (DIC) and fluorescence microscopy (secondary antibody Alexa 488). Serum was mixed with C. auris VPCI479 / P / 13 (South Asian lineage) and stained with concavalin A - Alexa fluor 488. Modified Als3-NT protein-glucan bioconjugate: Als3 18-316 -3S-Fba-bgluc d+ The control group consisted of animals immunized with buffer, and all groups were tested with AS03. NZW rabbits were immunized three times at two-week intervals.

[0436] Conclusion: As shown in Figure 14, antibodies against the modified Als3-NT protein-glucan bioconjugate can bind to C. auris cells coated on a plate and in Figure 13. Laser confocal microscopy can be used to confirm the binding of antibodies to C. auris cells.

[0437] [Example 10] Inhibition of biofilm formation on 96-well plates Figure 15 shows the ability of an antibody against a modified Als3-NT protein-glucan bioconjugate to inhibit biofilm formation of C. albicans mycelium on a 96-well plate. Serum was added to plastic wells with C. albicans mycelium (SC5314) and incubated at 37°C for 24 hours. After washing, viable cells were measured using XTT. Modified Als3-NT protein-glucan bioconjugate: Als3 18-316 -3S-Fba-bgluc d+ The control group consisted of animals immunized with buffer, and all groups were tested with AS03. NZW rabbits were immunized three times at 2-week intervals. Graphs depict mean + SD. *: p<0.05, one-way ANOVA.

[0438] Conclusion: As shown in Figure 15, serum containing a modified Als3-NT protein-glucan bioconjugate inhibits C. albicans biofilm formation on plastics. A significant reduction in biofilm formation was shown compared to pre- or post-III control serum.

[0439] [Example 11] Neutrophil killing assay Figure 16 shows the ability of antibodies against a modified Als3-NT protein-glucan bioconjugate to mediate neutrophil killing by C. albicans mycelium. C. albicans mycelium (SC5314) was mixed with modified Als3-NT protein-glucan bioconjugate serum, added to neutrophils, and incubated. The neutrophils were then lysed and incubated on YPD agar, after which Candida CFUs were counted. Modified Als3-NT protein-glucan bioconjugate: Als3 18-316 -3S-Fba-bgluc d+ The control group consisted of animals immunized with buffer, and a pre-immunization control was created by pooling the pre-serum of all animals. All groups were tested with AS03. NZW rabbits were immunized three times at 2-week intervals. Graphs show mean + SD. ***: p<0.001, *: p<0.05, one-way ANOVA.

[0440] Conclusion: As shown in Figure 16, serum containing a modified Als3-NT protein-glucan bioconjugate mediates neutrophil killing of C. albicans mycelium. A significant improvement in killing (reduction in CFU) was shown compared to pre- or post-III control serum.

[0441] [Sequence List] Sequence ID 1: Full-length wild-type Als3 sequence from Candida albicans (with wild-type leader sequence) [ka]

[0442] Sequence ID 2: Consensus Sequence (Artificial Sequence) GSGGGD / EXNZS / TGSGG

[0443] Sequence ID 3 Fba sequence YGKDVKDLFDYAQE

[0444] Sequence ID 4: Consensus Sequence (Artificial Sequence) KD / EXNZS / T X is Q (glutamine) and Z is A (alanine).

[0445] Sequence ID 5: Consensus Sequence (Artificial Sequence) KDQNAT

[0446] Sequence ID 6: Consensus Sequence (Artificial Sequence) KDQNAS

[0447] Sequence ID 7: Consensus Sequence (Artificial Sequence) DQNAT

[0448] Sequence ID 8: Consensus Sequence (Artificial Sequence) JUBD / EXNZS / TJUB 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.

[0449] Sequence ID 9: Consensus Sequence (Artificial Sequence) GSGGGDQNATGSGGG

[0450] Sequence ID No. 10 Modified Als3-NT sequence (containing amino acids 18-316 of Sequence ID No. 1, with an underlined insertion glycosite and a double-underlined fba sequence) (artificial sequence) [ka]

[0451] Sequence ID 11 Modified Als3-NT sequence (containing amino acids 18-329 of Sequence ID 1, with an underlined insertion glycosite and a double-underlined fba sequence) (artificial sequence) [ka]

[0452] Sequence ID 12: ZX09 SleC sequence for Agrobacterium species. [ka]

[0453] Sequence ID 13: ZX09 SleE sequence for species of the genus Agrobacterium. [ka] [ka]

[0454] Sequence ID 14: ZX09 SleF sequence for species of the genus Agrobacterium. [ka]

[0455] Sequence ID 15: Agrobacterium species ZX09 SleU sequence [ka]

[0456] Sequence ID 16: Agrobacterium species ZX09 SleW sequence [ka]

[0457] Sequence ID No. 17: E. coli O56 WfaP sequence [ka]

[0458] Sequence ID 18: Klebsiella pneumoniae Wzm sequence [ka]

[0459] Sequence ID 19: Klebsiella pneumoniae Wzt sequence [ka]

[0460] Sequence ID 20: Campylobacter PglB sequence [ka]

[0461] Sequence ID No. 21: Flagellin (FlgI) signaling sequence of Escherichia coli. MIKFLSALILLLVTTAAQA

[0462] Sequence ID No. 22: E. coli outer membrane porin A (OmpA) signal sequence MKKTAIAIAVALAGFATVAQA

[0463] Sequence ID No. 23: E. coli maltose-binding protein (MalE) signal sequence MKIKTGARILALSALTTMMFSASALA

[0464] Sequence ID No. 24: E. coli outer membrane porin A (OmpC) signal sequence MKVKVLSLLVPALLVAGAANA

[0465] Sequence ID 25: Als3-NT(1-316) sequence derived from Candida albicans (with wild-type leader sequence) [ka]

[0466] Sequence ID 26: Als3-NT(1-329) sequence derived from Candida albicans (with wild-type leader sequence) [ka]

[0467] Sequence ID 27: Als3-NT (18-316) sequence from Candida albicans. [ka]

[0468] Sequence ID 28: Als3-NT (18-329) sequence from Candida albicans. [ka]

[0469] Sequence ID 29: Nucleotide sequence of Campylobacter PglB (codon optimized) [ka] [ka]

[0470] Sequence ID 30: Nucleotide sequence of sleW from Agrobacterium species ZX09 [ka]

[0471] Sequence ID 31: Nucleotide sequence of sleU from Agrobacterium species ZX09 [ka]

[0472] Sequence ID 32: Nucleotide sequence of sleF from Agrobacterium species ZX09 [ka]

[0473] Sequence ID 33: Nucleotide sequence of sleE from Agrobacterium species ZX09 [ka] [ka]

[0474] Sequence ID 34: Nucleotide sequence of sleC from Agrobacterium species ZX09 [ka]

[0475] Sequence ID No. 35: Nucleotide sequence of wfaP from E. coli O56 [ka] [ka]

[0476] Sequence ID 36: Nucleotide sequence of wzm from Klebsiella pneumoniae [ka]

[0477] Sequence ID 37: Nucleotide sequence of wzt from Klebsiella pneumoniae [ka]

[0478] Sequence ID 38: Als3-NT(1-316) sequence derived from Candida albicans (containing wild-type leader sequence and Fba sequence) [ka]

[0479] Sequence ID 39: Candida albicans-derived Als3-NT(1-329) sequence (containing wild-type leader sequence and Fba sequence) [ka]

[0480] Sequence ID 40: Als3-NT(18-316) sequence (containing the Fba sequence) derived from Candida albicans. [ka]

[0481] Sequence ID 41: Als3-NT(18-329) sequence (containing the Fba sequence) derived from Candida albicans. [ka]

[0482] Sequence ID 42: Full-length wild-type Als3 sequence from Candida albicans (without leader sequence) [ka] [ka]

[0483] Sequence ID 43 (Als3_18-316-N) [ka]

[0484] Sequence ID 44 (Als3_18-316-C) [ka]

[0485] Sequence ID 45 (Als3_18-316-Mut1) [ka]

[0486] Sequence ID 46 (Als3_18-316-Mut2) [ka]

[0487] Sequence ID 47 (Als3_18-316-Mut3) [ka]

[0488] Sequence ID 48 (Als3_18-316-Mut4) [ka]

[0489] Sequence ID 49 (Als3_18-316-Mut5) [ka]

[0490] Sequence ID 50 (Als3_18-316-Mut6) [ka]

[0491] Sequence ID 51 (Als3_18-316-Mut7) [ka]

[0492] Sequence ID 52 (Als3_18-316-Mut8) [ka]

[0493] Sequence ID 53 (Als3_18-316-Mut9) [ka]

[0494] Sequence ID 54 (Als3_18-316-Mut10) [ka]

[0495] Sequence ID 55 (Als3_18-316-Mut11) [ka]

[0496] Sequence ID 56 (Als3_18-316-Mut12) [ka]

[0497] Sequence ID 57 (Als3_18-316-Mut13) [ka]

[0498] Sequence ID 58 (Als3_18-316-Mut14) [ka]

[0499] Sequence ID 59 (Als3_18-316-Mut15) [ka]

[0500] Sequence ID 60 (Als3_18-316-Mut16) [ka]

[0501] Sequence ID 61 (Als3_18-316-Mut17) [ka]

[0502] Sequence ID 62 (Als3_18-316-Mut18) [ka]

[0503] Sequence ID 63: Ervinia carotoborapectinate lyase (PelB) MKYLLPTAAAGLLLLAAQPAMA

[0504] Sequence ID No. 64: Enterotoxin LTIIb of Heat-Long-Blooming Escherichia coli MSFKKIIKAFVIMAALVSVQAHA

[0505] Sequence ID No. 65: Bacillus subtilis endoxylanase XynA MFKFKKKFLVGLTAAFMSISMFSATASA

[0506] Sequence ID No. 66 E. coli DsbA MKKIWLALAGLVLAFSASA

[0507] Sequence ID 67 TolB MKQALRVAFGFLILWASVLHA

[0508] Sequence ID 68 SipA MKMNKKVLLTSTMAASLLSVASVQAS

[0509] Nucleotide sequence of the modified Als3-NT protein (sequence number 69, containing amino acids 18-316 of sequence number 1) [ka] [ka]

[0510] Nucleotide sequence of the modified Als3-NT protein (sequence number 70, including amino acids 18-329 of sequence number 1) [ka]

[0511] Consensus sequence (artificial sequence) KD / EXNZS / T

[0512] Consensus sequence (artificial sequence) JUBD / EXNZS / TJUB

Claims

1. A modified aglutinin-like sequence 3 (Als3) protein comprising an amino acid sequence identical to amino acid residues 18-316 of SEQ ID NO: 1, or at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of amino acid residues 18-316 of SEQ ID NO: 1, wherein the amino acid sequence is modified in that it comprises one or more consensus sequences including the amino acid sequence D / EXNZS / T, and X and Z are independently any amino acid other than proline.

2. The modified Als3 protein according to claim 1, further comprising at least one fructose diphosphate aldolase-1 (Fba) peptide of Candida albicans having the amino acid sequence YGKDVKDLFDYAQE (SEQ ID NO: 3), or having an amino acid sequence identical to SEQ ID NO: 3 by at least 70%, 80%, 85%, 90%, or 92%.

3. The modified Als3 protein according to either claim 1 or 2, wherein X is Q (glutamine), Z is A (alanine), one or more consensus sequences are selected from the group consisting of KDQNAT (SEQ ID NO: 5), KDQNAS (SEQ ID NO: 6), and DQNAT (SEQ ID NO: 7), the modified Als3 protein comprises at least one further consensus sequence comprising the amino acid sequence JUBD / EXNZS / TJUB, X and Z are independently any amino acid other than proline, J, U, and B independently comprise 1 to 5 naturally occurring amino acid residues, and at least one 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 amino acid residues 18 to 316 of SEQ ID NO:

3.

4. A modified Als3 protein containing the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:

11.

5. A conjugate comprising the modified Als3 protein and at least one sugar antigen according to any one of claims 1 to 4, which is optionally a bioconjugate.

6. A modified Als3 protein of Candida albicans comprising (1) the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11 and (2) at least one sugar antigen of the genus Candida, wherein the at least one sugar antigen is a β-1,3-glucan polymer consisting of at least six consecutive β-1,3-linked glucose molecules, and the at least one sugar antigen is linked to at least one of three asparagine residues at positions 20, 92 and 324 of SEQ ID NO: 10 or positions 20, 92 and 337 of SEQ ID NO:

11.

7. A polynucleotide sequence encoding the modified Als3 protein according to any one of claims 1 to 4.

8. A vector comprising the polynucleotide sequence described in claim 7.

9. An immunogenic composition comprising a modified Als3 protein according to any one of claims 1 to 4 and 6, a conjugate according to claim 5, or a bioconjugate according to claim 5.

10. A Candida albicans vaccine comprising (1) a modified Als3 protein according to any one of claims 1 to 4, (2) at least one Candida albicans glycoantigen linked to the modified Als3 protein, and optionally (3) a pharmaceutically acceptable carrier or adjuvant.

11. A method for treating or preventing Candida albicans infection in a subject requiring treatment or prevention of Candida albicans infection, comprising administering a therapeutically effective amount of a modified Als3 protein according to any one of claims 1 to 4 and 6, a conjugate according to claim 5, a bioconjugate according to claim 5, an immunogenic composition according to claim 9, or a vaccine according to claim 10 to the subject.

12. A method for immunizing a subject against Candida albicans infection, comprising administering to the subject an immunoprotective dose of a modified Als3 protein according to any one of claims 1 to 4 and 6, a conjugate according to claim 5, a bioconjugate according to claim 5, an immunogenic composition according to claim 9, or a vaccine according to claim 10.

13. A method for inducing an immune response to Candida albicans infection in a subject, comprising administering a therapeutically effective or prophylactically effective amount of a modified Als3 protein according to any one of claims 1 to 4 and 6, a conjugate according to claim 5, a bioconjugate according to claim 5, an immunogenic composition according to claim 9, or a vaccine according to claim 10 to the subject.

14. A modified Als3 protein according to any one of claims 1 to 4 and 6, a conjugate according to claim 5, a bioconjugate according to claim 5, an immunogenic composition according to claim 9, or a vaccine according to claim 10, for use in the treatment or prevention of diseases caused by Candida albicans infection.

15. A method for improving the expression level of a modified Als3 protein according to any one of claims 1 to 4 and 6, comprising substituting an amino acid between amino acid residues 104 to 108 of amino acid residues 18 to 316 of SEQ ID NO: 1 with one or more consensus sequences 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 18 to 316 of SEQ ID NO: 1, wherein the modified Als3 protein exhibits an improved expression level compared to a control Als3 protein that does not contain one or more consensus sequences that are substituted between amino acid residues 104 to 108 of amino acid residues 18 to 316 of SEQ ID NO: 1, or 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 18 to 316 of SEQ ID NO:

1.

16. It is a host cell, i. A nucleotide sequence encoding one or more heterologous glycosyltransferases capable of synthesizing β-1,3-glucan polymers. ii. A nucleotide sequence encoding a glycosyltransferase that can covalently bond a glucose molecule to an N-acetylglucosamine (GlcNac) molecule. iii. Nucleotide sequences encoding heterologous oligosaccharide transferases, and iv. 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. Host cells containing these cells.

17. structure: 【Chemistry 1】 A sugar that is a glucan having, n is 2-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25. sugar.

18. structure: 【Chemistry 2】 A sugar that is a glucan having, n is 4-100, 4-50, 4-35, 4-25, 6-100, 6-50, 6-35, or 6-25. sugar.

19. A method for producing β-1,3-glucan polymers in prokaryotic host cells, In host cells, i. A nucleotide sequence encoding a first glycosyltransferase capable of covalently bonding a glucose molecule to an N-acetylglucosamine (GlcNAc) molecule, wherein the first glycosyltransferase is WfaP derived from E. coli O56. ii. A nucleotide sequence encoding a further glycosyltransferase capable of synthesizing fungal β-1,3-glucan, wherein the further glycosyltransferase comprises SleC, SleE, SleF, SleU, and SleW derived from rhizobia, possibly from the genus Agrobacterium, and possibly from the species Agrobacterium ZX09, wherein the host cell produces more SleW than SleC, SleE, SleF, or SleU, and iii. A nucleotide sequence encoding a translocase that can, in some cases, transfer β-1,3-glucan to the periplasmic side of the inner membrane of a prokaryotic host cell, wherein the translocase contains Wzm-Wzt derived from a species of the genus Klebsiella, and in some cases from Klebsiella pneumoniae. The steps of introducing and expressing A method comprising a β-1,3-glucan polymer linked to a lipid carrier via GlcNAc, wherein the β-1,3-glucan polymer contains at least four β-1,3-linked glucose molecules.

20. A method for generating a bioconjugate in a prokaryotic host cell, a. The step of obtaining a prokaryotic host cell according to claim 16 that produces a β-1,3-glucan polymer, and b. Furthermore, in host cells, i. A nucleotide sequence encoding a modified carrier protein comprising a glycosylation site including the consensus sequence D / EXNZS / T, wherein X and Z are any amino acids other than proline, and 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. A nucleotide sequence encoding an oligosaccharide transferase capable of generating a bioconjugate by transferring a β-1,3-glucan polymer from a lipid carrier to a modified carrier protein, wherein the oligosaccharide transferase is PglB derived from the genus Campylobacter, and optionally from Campylobacter jejuni or Campylobacter coli. Steps to introduce and express A method that includes this.

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