Modified proteins and methods

Abstract

The present invention relates to modified nanoparticles, immunogenic compositions and vaccines comprising modified nanoparticles, and to their preparation and use of such compositions in medicine.

Description

sequence list This application includes a sequence list, which is submitted electronically in XML file format, the entire contents of which are incorporated herein by reference. The XML copy was created on November 9, 2023, and is named 70277US01P_SL.xml, with a size of 60,947 bytes. Technical Field

[0001] This invention relates to the fields of modified proteins, immunogenic compositions comprising modified proteins, and vaccines, as well as methods for their production and preparation, and the use of such compositions in medicine. More particularly, it relates to bioconjugated modified nanoparticle (NP) subunits (or bioconjugated modified carrier proteins linked to optionally modified nanoparticle (NP) subunits) and methods for preparing assembled bioconjugated modified nanoparticles (or assembled bioconjugated modified carrier proteins linked to optionally modified nanoparticles (NP)) in host cells. It also relates to a method for preparing assembled bioconjugated modified nanoparticles (or assembled bioconjugated modified carrier proteins linked to optionally modified nanoparticles (NPs)) in host cells (e.g., bacterial host cells), wherein the host cells express modified nanoparticle subunit proteins (or modified carrier proteins linked to optionally modified nanoparticle (NP) subunits) and glycosyltransferases (e.g., glycosyltransferase pglB) for attaching sugars (e.g., oligosaccharides, polysaccharides) to glycosylation sites of the modified nanoparticle subunit proteins or the modified carrier proteins to produce assembled bioconjugated modified glycoprotein nanoparticles (or assembled bioconjugated modified carrier proteins linked to optionally modified nanoparticles (NPs)) in a single step in the host cells. In some aspects, the modified nanoparticle subunit proteins (or modified carrier proteins linked to optionally modified nanoparticle (NP) subunits) and glycosyltransferases are expressed in the periplasm. Background Technology

[0002] Protein nanoparticles (NPs) possess the ability to self-assemble into highly ordered, symmetrical, and stable structures. NPs can be used to display target antigens on their surfaces in high-density, defined patterns, similar to natural pathogen-associated molecular patterns (PAMPs), which can simultaneously induce both innate and adaptive immune responses. For example, protein NPs can act as scaffolds to present antigens in highly ordered arrays of repetitive antigens (see, for example, WO02 / 056905). Therefore, the ability of self-assembled protein nanoparticles to elicit immune responses has been demonstrated, and they can be used in technologies for developing and generating novel vaccines.

[0003] Glycoconjugate vaccines are an effective technology for controlling bacterial diseases. Traditional glycoconjugate vaccines are produced by covalently linking one or more bacterial polysaccharides to a carrier protein. Glycosylation is a post-translational modification in bacteria through which glycans covalently attach to surface proteins (e.g., flagella or pili). Glycoproteins may play roles in adhesion, stabilizing proteins against proteolysis, or evading the host's immune response.

[0004] Different protein glycosylation mechanisms can be distinguished by the pattern of glycan transfer to proteins. One mechanism involves the direct transfer of carbohydrates from nucleotide-activated glycosyltransfers to receptor proteins (e.g., O-glycosylation of proteins in the Golgi apparatus of eukaryotic cells, and O-glycosylation of flagellated proteins in certain types of bacteria). A second mechanism involves the pre-assembly of polysaccharides on lipid carriers (e.g., via glycosyltransferases), followed by transfer to protein receptors via oligosaccharide transferases (OTases) (Faridmoayer et al., J. Bacteriology, pp. 8088-8098, 2007). This mechanism is used, for example, in N-glycosylation in the endoplasmic reticulum of eukaryotic cells and in Campylobacter jejuni (…). Campylobacter jejuni Well-characterized N-linked glycosylation systems and recently characterized Neisseria meningitidis ( Neisseria meningitidis ), Neisseria gonorrhoeae ( Neisseria gonococcus ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa The O-linked glycosylation system of the protein receptor. For O-linked glycosylation (O-glycosylation), the glycan is usually attached to a serine or threonine residue of the protein receptor. For N-linked glycosylation (N-glycosylation), the glycan is usually attached to an asparagine residue of the protein receptor. N-glycosylation of Campylobacter jejuni protein can be reconstructed by simultaneously recombinantly expressing the pgl locus and the receptor glycoprotein in Escherichia coli (Wacker et al., (2002) Science 298, 1790-1793).

[0005] Recently, bacterial cells have been used to produce glycoconjugated vaccines. For example, genetically modified bacterial cells (such as *E. coli*) can be used to link glycans to glycosylation sites on carrier proteins to produce glycoconjugated vaccines. The method of linking glycans to glycosylation sites on carrier proteins is called bioconjugation, which provides a low-cost option for producing glycoconjugated vaccines. This method offers flexibility in producing different glycan / carrier recombinants for different vaccines. Bioconjugation can rely on conjugation enzymes, such as the oligosaccharide transferase pgIB, to transfer sugars (e.g., oligosaccharides, polysaccharides, etc.) to glycosylation sites on different carrier proteins (e.g., optionally modified CRMs (e.g., CRM-197), optionally modified EPAs, optionally modified diphtheria toxoids, optionally modified OMPCs, optionally modified tetanus toxoids, etc.).

[0006] WO2006 / 119987 (Aebi et al.) describes a protein, and means and methods for producing the protein, which is capable of efficient N-glycosylation in prokaryotes. It further describes the introduction of N-glycans into recombinant proteins to alter the immunogenicity, stability, biological activity, preventative and / or therapeutic activity of said proteins, and the provision of host cells displaying the recombinant N-glycosylated protein of this embodiment on their surface. Furthermore, it describes a recombinant N-glycosylated protein comprising one or more of the following optimized amino acid sequences: D / EXNZS / T (SEQ ID NO: 49), where X and Z can be any native amino acid other than Pro. Introducing such optimized amino acid sequences into a protein results in the protein being N-glycosylated by oligosaccharide transferases at these introduced sites.

[0007] Conjugated vaccines (vaccines containing a carrier protein covalently linked to an immunogenic antigen) have become a successful method for vaccinating against a variety of bacterial infections. Conjugating T-cell-independent antigens (e.g., sugars) to carrier proteins has long been established as a method to enable T-cell assistance as part of a normal T-cell-independent antigen-based immune response. In this way, the immune response can be enhanced by allowing the development of immune memory and boosting the response. To improve the production efficiency of conjugated vaccines, in vivo methods (for the production of “biological conjugated vaccines”) have been developed. These in vivo methods utilize the N-glycosylation and O-glycosylation systems discussed above (see WO2009 / 104074 and WO2017 / 035181).

[0008] Peng et al. (Adv. Sci. (2021) vol. 8, 2100549) reported the production of biosynthetic nanoconjugated vaccines using the Nano-B5 self-assembly platform to generate conjugated vaccines against Klebsiella pneumoniae. In this study, Peng et al. used an oligosaccharide transferase (PglL) to add O2 polysaccharides to specific glycosylation sites.

[0009] There is a need for improved vaccine technologies. In particular, there is a need for adaptable bioconjugated nanoparticle platforms capable of preparing self-assembled bioconjugated nanoparticle subunits (or bioconjugated carrier proteins directly or indirectly fused to self-assembled nanoparticle subunits). Such platforms are suitable for preparing immunogenic compositions and / or vaccines targeting different nanoparticles and different sugars (e.g., polysaccharide antigens, oligosaccharide antigens, etc.). Suitably, such platforms can produce bioconjugated nanoparticles (or bioconjugated carrier proteins directly or indirectly fused to nanoparticles) in a single step. Summary of the Invention

[0010] In one aspect, a nanoparticle-based (NP) platform is provided for the production of bioconjugated nanoparticles (or bioconjugated carrier proteins fused directly or indirectly to nanoparticles). Modified nanoparticle subunits are translocated together with a glycosyltransferase (e.g., glycosyltransferase pglB) into or expressed therein the periplasm of a host cell (e.g., a bacterial host cell, such as *E. coli*). The glycosyltransferase conjugates an antigen (e.g., bacterial glycosyl, such as a polysaccharide or oligosaccharide) to a specific region of a protein containing a glycosylation site motif / shared sequence introduced into the nanoparticle subunit or carrier protein sequence. In some aspects, the shared sequence comprises or consists of D / EXNZS / T (SEQ ID NO: 49), wherein X and Z can independently be any native amino acid other than proline (e.g., the shared sequence could be DQNXT, where X can be A or R (e.g., SEQ ID NO: 53)). Advantageously, assembled glycoconjugated -NPs or glycoconjugated -carrier protein -NPs (e.g., modified NP subunits or modified carrier protein -NP subunits undergo bioconjugation and self-assembly in the periplasm) are generated in a single step to form assembled bioconjugated nanoparticles (or assembled bioconjugated carrier proteins directly or indirectly fused to nanoparticles). Therefore, in one aspect, this embodiment relates to self-assembled nanoparticles (NPs) displaying antigens (e.g., bacterial polysaccharides / oligosaccharides) (or carrier proteins directly or indirectly fused to self-assembled nanoparticles), to compositions comprising such nanoparticles, and to methods for preparing and using such nanoparticles and compositions.

[0011] Individual NP subunit proteins can self-assemble to form nanoparticles, for example, with sizes ranging from about 5 to 100 nm, from about 5 nm to about 75 nm, from about 6 nm to about 75 nm, from about 6 nm to about 50 nm, from about 6 nm to about 25 nm, from about 6 nm to about 24 nm, etc. NP self-assembly refers to the oligomerization or aggregation of NP subunits or carrier proteins fused to NP subunits into an ordered arrangement driven by non-covalent interactions.

[0012] In one embodiment, the modified NP subunit, the antigen (e.g., bacterial antigen), and the glycosyltransferase (e.g., pglB) are expressed or translocated to the periplasm of a bacterial host cell (e.g., *E. coli*). In one embodiment, the glycosyltransferase conjugates the antigen (e.g., bacterial polysaccharide or oligosaccharide) to the modified NP subunit (or to a modified carrier protein directly or indirectly fused to the nanoparticle subunit), which self-assembles into a bioconjugated nanoparticle.

[0013] In one embodiment, multiple copies of the antigenic epitope are displayed on the outer surface of the assembled NP. In a further embodiment, the assembled NP displays bacterial antigens (e.g., from Gram-positive or Gram-negative bacteria).

[0014] Bioconjugated NPs can be used for any suitable purpose, such as inducing an immune response in subjects and / or preventing or treating a condition or disease. In one aspect, this embodiment provides a glycoconjugated nanoparticle vaccine against a variety of bacterial pathogens that present cell surface sugars, wherein an effective immune response can be achieved after one or more administrations of the immunogenic composition or vaccine. NPs may include any suitable protein or virus-like particle capable of self-assembly, including but not limited to dodecin, ferritin (e.g., ferritin Hp, ferritin Pa, etc.), E2p, EPA-ferritin, etc.

[0015] In one aspect, this embodiment provides a modified nanoparticle subunit modified to include one or more N-glycosylation sites (e.g., by introducing a consensual sequence). In other aspects, this embodiment provides a modified carrier protein fused to an optionally modified nanoparticle subunit, wherein the carrier protein is modified to include N-glycosylation sites. For N-linked glycosylation (N-glycosylation), glycans are typically attached to asparagine residues on a protein receptor. In some aspects, this embodiment provides a modified protein comprising at least one consensual sequence for N-glycosylation (e.g., D / EXNZS / T (SEQ ID NO: 49)) for conjugation to an antigen (e.g., a bacterial polysaccharide / oligosaccharide). In some aspects, a modified carrier protein comprising at least one consensual sequence for N-glycosylation (e.g., D / EXNZS / T (SEQ ID NO: 49)) may be fused to or linked to a nanoparticle subunit. Carrier proteins include, but are not limited to, modified CRM (e.g., CRM-197), modified EPA, modified diphtheria toxoid, modified OMPC, modified tetanus toxoid, etc.

[0016] In the modified NP of this embodiment, a glycosylated consensual sequence is introduced into a specific region of the NP subunit or a carrier protein fused to the NP subunit. The location of the consensual sequence in the modified NP subunit can improve glycosylation efficiency and / or optimize the manipulation of N-glycosylation sites.

[0017] Therefore, a method for preparing glycosylated nanoparticles (NPs) is provided, comprising the steps of: providing a host cell; translocating or expressing a modified nanoparticle subunit into the periplasm of the host cell; translocating or expressing a glycosyltransferase into the periplasm of the host cell; glycosylating the modified nanoparticle subunit in the periplasm by the glycosyltransferase; and producing assembled glycoprotein nanoparticles from the glycosylated modified nanoparticle subunit. In some aspects, the method comprises the steps of: providing a host cell; translocating or expressing a modified nanoparticle subunit into the periplasm of the host cell, wherein the modified nanoparticle subunit contains one or more glycosylation sites (e.g., D / EXNZS / T (SEQ ID NO: 49)); translocating or expressing a glycosyltransferase pglB into the periplasm of the host cell; glycosylating the modified nanoparticle subunit by the glycosyltransferase pglB; and producing assembled glycosylated nanoparticles from the glycosylated modified nanoparticle subunit in the periplasm. In a further aspect, the method comprises a host cell, which is a bacterial host cell. In other respects, the method includes a host cell, which is a Gram-negative bacterial host cell (e.g., Escherichia coli).

[0018] In a further aspect, the method includes conjugating a capsular polysaccharide or oligosaccharide to a modified nanoparticle subunit (or a modified carrier protein fused to an optionally modified nanoparticle subunit) at one or more glycosylation motifs. For example, the capsular polysaccharide or oligosaccharide may be selected from Escherichia coli spp. (…). Escherichia ) species, Shigella genus ( Shigella ) species, Klebsiella genus ( Klebsiella ) species, Salmonella genus ( Salmonella ) species, Yersinia genus ( Yersinia ) species, Helicobacter genus ( Helicobacter ) species, Proteus genus ( Proteus ) species, Pseudomonas genus ( Pseudomonas ) species, Corynebacterium genus ( Corynebacterium ) species, Streptomyces genus ( Streptomyces ) species, Streptococcus genus ( Streptococcus ) species, Enterococcus genus ( Enterococcus ) species, Staphylococcus genus ( Staphylococcus ) species, Bacillus genus ( Bacillus ) species, Clostridium ( Clostridium ) species, Listeria genus ( Listeria ) species, Campylobacter genus ( Campylobacter ) species, genus Neisseria meningitidis ( Meningococcal Capsular polysaccharides and Candida spp. Candida Polysaccharides.

[0019] Therefore, in a second aspect, a method for preparing a glycosylated carrier protein conjugated to nanoparticles (NPs) is provided, comprising the steps of: providing a host cell; translocating or expressing a modified carrier protein conjugated to optionally modified nanoparticle subunits into or therein the periplasm of the host cell, wherein the modified carrier protein comprises one or more glycosylation sites (e.g., D / EXNZS / T (SEQ ID NO: 49)); translocating or expressing a glycosyltransferase (e.g., pglB) in the periplasm of the host cell; glycosylating the modified carrier protein by means of the glycosyltransferase pglB; and generating in the periplasm assembled nanoparticles formed by direct or indirect fusion of the glycoprotein carrier protein to nanoparticle subunits.

[0020] In a further aspect, the method includes translocating or expressing a modified carrier protein, which is directly or indirectly fused to optionally modified nanoparticle subunits, in the periplasm, wherein the carrier protein includes, but is not limited to: modified CRM (e.g., CRM197), modified diphtheria toxoid (DT), modified tetanus toxoid (TT), modified OMPC, or modified EPA. In some aspects, the method includes conjugating a capsular polysaccharide or oligosaccharide to the modified carrier protein (e.g., at one or more glycosylation motifs). For example, capsular polysaccharides or oligosaccharides may include, but are not limited to, Escherichia coli spp. (…). Escherichia ) species, Shigella genus ( Shigella ) species, Klebsiella genus ( Klebsiella ) species, Salmonella genus ( Salmonella ) species, Yersinia genus ( Yersinia ) species, Helicobacter genus ( Helicobacter ) species, Proteus genus ( Proteus ) species, Pseudomonas genus ( Pseudomonas ) species, Corynebacterium genus ( Corynebacterium ) species, Streptomyces genus ( Streptomyces ) species, Streptococcus genus ( Streptococcus ) species, Enterococcus genus ( Enterococcus ) species, Staphylococcus genus ( Staphylococcus ) species, Bacillus genus ( Bacillus ) species, Clostridium ( Clostridium ) species, Listeria genus ( Listeria ) species, Campylobacter genus ( Campylobacter ) species, genus Neisseria meningitidis ( Meningococcal Capsular polysaccharides and Candida spp. Candida Polysaccharides.

[0021] In a third aspect, a method for producing assembled bioconjugated nanoparticles comprising modified nanoparticle subunits conjugated with capsular polysaccharides or oligosaccharides is provided, the method comprising: (i) culturing host cells under conditions suitable for the production of glycoproteins, nanoparticle subunits and glycosyltransferases, and (ii) isolating the assembled glycoprotein nanoparticles, optionally isolating the assembled glycoprotein nanoparticles from a periplasmic extract of the host cells.

[0022] In a fourth aspect, a method for producing assembled nanoparticles comprising a bioconjugated carrier protein directly or indirectly fused to an optionally modified nanoparticle subunit, wherein the modified carrier protein is conjugated to a capsular polysaccharide or oligosaccharide fused to the nanoparticle subunit, the method comprising: (i) culturing host cells under conditions suitable for the production of glycoproteins, nanoparticle subunits, and glycosyltransferases; and (ii) isolating the assembled bioconjugated carrier protein fused to the optionally modified nanoparticles, optionally isolating the assembled bioconjugated carrier protein fused to the optionally modified nanoparticles from a periplasmic extract of the host cells.

[0023] In a fifth aspect, a method for inducing an immune response in a subject (e.g., a human) is provided, the method comprising administering to a subject (e.g., a human) a therapeutically or prophylactically effective amount of assembled glycoprotein nanoparticles (as an immunogenic composition or as a vaccine). In another aspect, a method for inducing an immune response in a subject (e.g., a human) is provided, the method comprising administering to a subject (e.g., a human) a therapeutically or prophylactically effective amount of assembled bioconjugated nanoparticles (as an immunogenic composition or as a vaccine), wherein the modified bioconjugated nanoparticles comprise a glycoprotein carrier protein directly or indirectly fused to optionally modified nanoparticle subunits.

[0024] In a sixth aspect, a modified NP or a modified carrier protein is provided that is fused directly or indirectly to an optionally modified NP, wherein the amino acid sequence of the modified NP subunit or the carrier protein comprises one or more common sequences of D / EXNZS / T (SEQ ID NO:49), wherein X and Z are independently any amino acid other than proline.

[0025] In a seventh aspect, a modified dodecin NP subunit is provided comprising an amino acid sequence of amino acid residues 2-70 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 2-70 of SEQ ID NO: 1, the modification being that the amino acid sequence comprises one or more consensual sequences of D / EXNZS / T (SEQ ID NO: 49), wherein X and Z are independently any amino acid other than proline, wherein each of the one or more consensual sequences is added to or replaces one or more amino acids immediately adjacent to or at the following positions: (a) at the N-terminus, (b) at the C-terminus (wherein a histidine tag is optionally preceding or following the consensual sequence) and / or (c) replacing one or more amino acids that are independently selected from one or more amino acids between amino acid residues 49-55 of SEQ ID NO: 1 (e.g., one or more amino acids between amino acid residues 49-55, e.g., amino acid residue 51) or identical to amino acid residues 2-70 of SEQ ID NO: 1. Amino acid residues 2-70 of SEQ ID NO: 1 occupy at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence at equivalent positions. In some aspects, SEQ ID NO: 1 optionally comprises one or more substitutions selected from G25N, V50T, and A53T.

[0026] In another aspect, a modified dodecin NP subunit is provided, comprising the amino acid sequence of amino acid residues 2-70 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 2-70 of SEQ ID NO: 1. In some aspects, SEQ ID NO: 1 comprises one or more of the following substitutions: G25N, V50T, and A53T.

[0027] This document provides an eighth aspect of the present embodiments, providing a modified ferritin NP (e.g., ferritin Hp) comprising an amino acid sequence of amino acid residues 1-167 of SEQ ID NO: 11 or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-167 of SEQ ID NO: 11, the modification being that the amino acid sequence comprises one (or more) concordant sequences of D / EXNZS / T (SEQ ID NO: 49), wherein X and Z are independently any amino acid other than proline, wherein each of the one (or more) concordant sequences is added to or replaces one or more amino acids immediately adjacent to: (a) at the N-terminus (wherein optionally a histidine tag precedes or follows the concordant sequence), (b) replacing one or more amino acids independently selected from one or more amino acid residues between amino acid residues 65-81 of SEQ ID NO: 11 (e.g., K79 or E81 replaced by DQNAT (SEQ ID NO: 49)). 32) substitution), and / or (c) substitution of one or more amino acids, independently selected from one or more amino acid residues between 145-149 of SEQ ID NO: 11 (e.g., residues 145-149 or 146-148 are substituted with GDQNATG (SEQ ID NO: 35)) or equivalent positions within the same amino acid sequence as amino acid residues 1-167 of SEQ ID NO: 11, which are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%. For example, (a) and (b); (b) and (c); or (a), (b), and (c) can be combined.

[0028] In another aspect, a modified ferritin NP subunit is provided, comprising an amino acid sequence of amino acid residues 1-167 or 2-167 of SEQ ID NO: 11 or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-167 of SEQ ID NO: 11.

[0029] In a ninth aspect of the embodiments provided herein, a modified ferritin NP (e.g., ferritin Pa) is provided, comprising an amino acid sequence of amino acid residues 1-154 of SEQ ID NO: 20 or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-154 of SEQ ID NO: 20, the modification being that the amino acid sequence comprises one (or more) common sequences of D / EXNZS / T (SEQ ID NO: 49), wherein X and Z are independently any amino acid other than proline, wherein each of the one (or more) common sequences is added to or replaces one or more amino acids immediately adjacent to: (a) at the N-terminus (wherein optionally a histidine tag precedes or follows the common sequence), (b) replacing one or more amino acids independently selected from one or more amino acid residues between amino acid residues 65-81 of SEQ ID NO: 20 (e.g., T80 replaced by GSGDQNATGSG (SEQ ID NO: 49)). 31) substitution), and / or (c) substitution of one or more amino acids, which are independently selected from one or more amino acids between amino acid residues 145-149 of SEQ ID NO: 20 (e.g., G145 is substituted by GSGDQNATGSG (SEQ ID NO: 31)) or equivalent positions within the same amino acid sequence as amino acid residues 1-154 of SEQ ID NO: 20, which are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%, wherein SEQ ID NO: 20 optionally contains one or more substitutions selected from the following: M31I, K120L, A124R, M144I and I154M.

[0030] In another aspect, a modified ferritin NP subunit is provided, comprising an amino acid sequence of amino acid residues 1-154 or 2-154 of SEQ ID NO: 20, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 1-154 of SEQ ID NO: 20. SEQ ID NO: 20 optionally comprises one or more substitutions selected from the group consisting of M31I, K120L, A124R, M144I, and I154M.

[0031] In a tenth aspect of the embodiments provided herein, a modified E2p NP is provided, comprising an amino acid sequence of amino acid residues 185-426 of SEQ ID NO: 26 or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 185-426 of SEQ ID NO: 26, the modification being that the amino acid sequence comprises one or more common sequences of D / EXNZS / T (SEQ ID NO: 49), wherein X and Z are independently any amino acid other than proline, wherein each of the one or more common sequences is added to or replaces one or more amino acids immediately adjacent to or replacing the following positions: at the N-terminus (wherein a histidine tag is optionally preceding or following the common sequence), 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 185-426 of SEQ ID NO: 26. In some respects, SEQ ID NO: 26 optionally includes one or more substitutions selected from the following: A187T, F196Y, T281N, P314S, A352V, L425I and Δ427-428.

[0032] In another aspect, a modified E2p NP subunit is provided, comprising the amino acid sequence of amino acid residues 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof. SEQ ID NO: 26 optionally comprises one or more substitutions selected from the group consisting of A187T, F196Y, T281N, P314S, A35V, L425I, and Δ427-428.

[0033] In the eleventh aspect of this embodiment provided herein, a modified carrier protein fused with NP is provided, wherein the modified glycoconjugated carrier protein NP is fused with ferritin and is *Pseudomonas aeruginosa* (…). Pseudomonas aeruginosaThe modified exotoxin A of the EPA protein has the amino acid sequence of SEQ ID NO: 52 or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 52, and the modification is that the amino acid sequence contains one (or more) common sequences of D / EXNZS / T (SEQ ID NO: 49), wherein X and Z are independently any amino acid other than proline, wherein the one (or more) common sequences are each added to or replace one or more adjacent amino acids.

[0034] In another aspect, a modified EPA ferritin NP subunit is provided, comprising the amino acid sequence of amino acid residues 35-855 of SEQ ID NO: 52 or a self-assembled fragment thereof, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acid residues 35-855 of SEQ ID NO: 52 or a self-assembled fragment thereof. SEQ ID NO: 52 optionally comprises one or more substitutions selected from L552V and Δ553.

[0035] In a twelfth aspect, a conjugate (e.g., a bioconjugate) is provided comprising a modified NP or a modified carrier protein fused to an NP and conjugated to an antigen (e.g., a glycoantigen, optionally a bacterial polysaccharide or oligosaccharide). According to a further aspect, a conjugate (e.g., a bioconjugate) is provided comprising modified ferritin nanoparticles linked to an antigen (e.g., a glycoantigen, optionally a bacterial polysaccharide or oligosaccharide). According to a further aspect of the embodiments provided herein, a conjugate (e.g., a bioconjugate) is provided comprising modified dodecin nanoparticles linked to an antigen (e.g., a glycoantigen, optionally a bacterial polysaccharide or oligosaccharide). According to a further aspect of the embodiments provided herein, a conjugate (e.g., a bioconjugate) is provided comprising modified E2p nanoparticles linked to an antigen (e.g., a glycoantigen, optionally a bacterial polysaccharide or oligosaccharide). According to a further aspect of the embodiments provided herein, a conjugate (e.g., a bioconjugate) nanoparticle is provided comprising a modified carrier protein fused to an optionally modified ferritin nanoparticle subunit linked to an antigen (e.g., a glycoantigen, optionally a bacterial polysaccharide or oligosaccharide).

[0036] In a thirteenth aspect of the embodiments provided herein, a polynucleotide is provided that encodes a modified NP or a modified carrier protein fused to an NP. According to a further aspect, a polynucleotide is provided that encodes optionally modified dodecin nanoparticles. According to a further aspect, an oligonucleotide is provided that encodes modified ferritin nanoparticles. According to a further aspect, an oligonucleotide is provided that encodes modified E2p nanoparticles. According to a further aspect, an oligonucleotide is provided that encodes modified EPA fused to ferritin nanoparticles.

[0037] In a fourteenth aspect, a first vector is provided comprising a nucleotide encoding a modified NP, or encoding a modified carrier protein fused to an optionally modified NP (e.g., a modified dodecin monomer subunit, a modified ferritin monomer subunit, a modified E2p monomer subunit, a modified EPA fused to a ferritin monomer subunit, etc.). According to a further aspect, a second vector is provided comprising a polynucleotide encoding an oligosaccharide or polysaccharide gene cluster. For example, said nucleotide may encode a pneumococcal capsular polysaccharide or Klebsiella pneumoniae (e.g., Sp12F, Sp33F) from any suitable serotype. Klebsiella pneumoniae The polynucleotide (e.g., KpO3b) is provided. According to a further aspect, a third vector is provided comprising a polynucleotide encoding a glycosyltransferase (e.g., pglB). According to a further aspect, the polynucleotide encoding a modified NP or a modified protein vector fused to an NP, the polynucleotide encoding an oligosaccharide or polysaccharide gene cluster, and the polynucleotide encoding the glycosyltransferase can be provided in one, two, or three vectors. In another aspect, the vector may comprise any two or more nucleotides encoding a glycosyltransferase, an oligosaccharide or polysaccharide gene cluster, or a modified NP or a modified vector protein fused to an NP.

[0038] According to a further aspect, a vector is provided that comprises an oligonucleotide encoding a dodecin protein (e.g., a modified dodecin protein). According to a further aspect, a second vector is provided that comprises a polynucleotide encoding an oligosaccharide or polysaccharide gene cluster. For example, the vector may encode a pneumococcal capsular polysaccharide or Klebsiella pneumoniae (e.g., Sp12F, Sp33F) from any suitable serotype. Klebsiella pneumoniae Polysaccharides (e.g., KpO3b). According to a further aspect, a third carrier is provided which contains a polynucleotide encoding a glycosyltransferase (e.g., pglB).

[0039] According to a further aspect, a vector is provided that contains encoding information derived from Helicobacter pylori (H. pylori). H.pylori The vector contains polynucleotides encoding ferritin (e.g., modified ferritin Hp protein). According to a further aspect, a second vector is provided that contains polynucleotides encoding oligosaccharide or polysaccharide gene clusters. For example, the vector may encode capsular polysaccharides from any suitable serotype (e.g., Sp12F, Sp33F) or Klebsiella pneumoniae (…). Klebsiella pneumoniae Polysaccharides (e.g., KpO3b). According to a further aspect, a third carrier is provided which contains a polynucleotide encoding a glycosyltransferase (e.g., pglB).

[0040] According to a further aspect, a vector is provided that contains encoding of Pseudomonas aeruginosa (… P. aeruginosa The vector contains polynucleotides encoding ferritin (e.g., modified ferritin Pa protein). According to a further aspect, a second vector is provided that contains polynucleotides encoding oligosaccharide or polysaccharide gene clusters. For example, the vector may encode a pneumococcal capsular polysaccharide from any suitable serotype (e.g., Sp12F, Sp33F) or Klebsiella pneumoniae (…). Klebsiella pneumoniae Polysaccharides (e.g., KpO3b). According to a further aspect, a third carrier is provided which contains a polynucleotide encoding a glycosyltransferase (e.g., pglB).

[0041] According to a further aspect, a vector is provided that contains encoding of Bacillus steatophilus (…). G. stearothermophilus The vector contains polynucleotides encoding E2p proteins (e.g., modified E2p proteins). According to a further aspect, a second vector is provided that contains polynucleotides encoding oligosaccharide or polysaccharide gene clusters. For example, the vector may encode capsular polysaccharides from any suitable serotype (e.g., Sp12F, Sp33F) or Klebsiella pneumoniae (…). Klebsiella pneumoniae Polysaccharides (e.g., KpO3b). According to a further aspect, a third carrier is provided which contains a polynucleotide encoding a glycosyltransferase (e.g., pglB).

[0042] According to a further aspect, a vector is provided that comprises encoding an EPA-ferritin protein (e.g., a modified EPA-ferritin fusion or a carrier protein nanoparticle fusion protein) from Helicobacter pylori. According to a further aspect, a second vector is provided that comprises a polynucleotide encoding an oligosaccharide or polysaccharide gene cluster. For example, the vector may encode a pneumococcal capsular polysaccharide or Klebsiella pneumoniae (e.g., Sp12F, Sp33F) from any suitable serotype. Klebsiella pneumoniaePolysaccharides (e.g., KpO3b). According to a further aspect, a third carrier is provided which contains a polynucleotide encoding a glycosyltransferase (e.g., pglB).

[0043] In the fifteenth aspect, a host cell is provided, comprising: i) One or more nucleotide sequences containing a polysaccharide synthesis gene, optionally used to produce bacterial polysaccharide antigens (e.g., N antigen, derived from Gram-negative bacteria, optionally from pneumococcal capsular polysaccharide (e.g., serotype Sp12F); O antigen, derived from Gram-negative bacteria, optionally from Shigella dysenteriae). Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa (e.g., KpO3b), Klebsiella pneumoniae ( Klebsiella pneumoniae ) or capsular polysaccharides, which are derived from Gram-positive bacteria, optionally from Streptococcus pneumoniae ( Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylcoccus aureus Yeast polysaccharide antigens or mammalian polysaccharide antigens may optionally be integrated into the host cell genome; ii) A nucleotide sequence encoding a heteroglycosyltransferase, optionally within a plasmid; iii) A nucleotide sequence encoding a modified nanoparticle subunit or a modified carrier protein fused to a nanoparticle, optionally within a plasmid.

[0044] In a sixteenth aspect, an immunogenic composition is provided comprising a conjugate (e.g., a bioconjugate) and optionally a pharmaceutically acceptable excipient and / or carrier (optionally using an adjuvant). According to a further aspect, a vaccine is provided comprising an immunogenic composition and optionally an adjuvant.

[0045] In a seventeenth aspect, a method for producing bioconjugated nanoparticles comprising nanoparticles linked to polysaccharides or oligosaccharides (e.g., modified nanoparticles or modified carrier proteins fused to nanoparticles) is provided, the method comprising: (i) culturing host cells under conditions suitable for producing bioconjugated nanoparticles (or bioconjugated carrier proteins fused to nanoparticles) and (ii) isolating the bioconjugated nanoparticles (or carrier proteins fused to nanoparticles) produced by the host cells, optionally isolating the bioconjugated nanoparticles from a periplasmic extract of the host cells.

[0046] In the eighteenth aspect, a method for inducing an immune response in a subject (e.g., a human) is provided, the method comprising administering to a subject (e.g., a human) a therapeutically or preventively effective amount of a conjugate (e.g., a biological conjugate), an immunogenic composition, or a vaccine.

[0047] In the nineteenth aspect, a conjugate (e.g., a biological conjugate), an immunogenic composition, or a vaccine is provided for inducing an immune response in a subject (e.g., a human).

[0048] In a twentieth aspect of the embodiments provided herein, a conjugate (e.g., a biological conjugate), an immunogenic composition, or a vaccine is provided for the preparation of a medicament that induces an immune response in a subject (e.g., a human).

[0049] In a twenty-first aspect, a nanoparticle monomer subunit is provided, comprising amino acids 1 to 79 or 2 to 73 of SEQ ID NO: 2. Typically, a carrier module can be fused to the N-terminus or C-terminus of SEQ ID NO: 2.

[0050] In a twenty-second aspect, a protein nanoparticle is provided having one or more sites (e.g., one or more amino acid residues within a monomer sequence) on its outer or inner surface suitable for conjugating a display molecule (e.g., an antigen or an immunostimulatory molecule).

[0051] One or more amino acid residues within a peptide subunit sequence can be modified using methods known in the art to provide sites suitable for chemical / biochemical conjugation to heterologous molecules (e.g., antigens or immunostimulatory molecules) on the outer or inner surface of nanoparticles, wherein such modifications do not prevent peptide monomers from self-assembling into nanoparticles.

[0052] Another aspect is a protein nanoparticle that contains one or more sites (e.g., one or more amino acid residues within a monomer sequence) on its outer or inner surface suitable for conjugating display molecules (e.g., antigens or immunostimulatory molecules).

[0053] In one embodiment, the amino acid residues on the modified nanoparticles or the modified carrier protein fused with the nanoparticle subunit protein are selected from the following: Ala, Arg, Asp, Cys, Gly, Glu, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. Optionally, the amino acid is an amino acid containing a terminal amino group, lysine, arginine, glutamic acid, aspartic acid, cysteine, tyrosine, histidine, or tryptophan. In one aspect, the antigen is conjugated to an amino acid selected from the following on the modified nanoparticles or the modified carrier protein: asparagine, aspartic acid, glutamic acid, lysine, cysteine, tyrosine, histidine, arginine, or tryptophan (e.g., asparagine), and in the case of asparagine, the conjugate can be a biological conjugate (e.g., enzymatic conjugation using an oligosaccharide transferase (such as PglB)). In one embodiment, the amino acid residues on the modified nanoparticles or the modified carrier protein linked to the antigen are asparagine residues. In some respects, the amino acid residues on the modified nanoparticles or modified carrier proteins linked to the antigen are part of a common sequence, such as asparagine in the common sequences of D / EXNZS / T (SEQ ID NO: 49), KD / EXNZS / TK (SEQ ID NO: 50), or JD / EXNZS / TU (SEQ ID NO: 51).

[0054] The conjugate can be a bioconjugate fused to, recombinantly modified nanoparticles, or recombinantly modified carrier proteins. Alternatively, the conjugate can be a bioconjugate fused to or isolated from a nanoparticle subunit, or recombinantly modified carrier protein. Attached Figure Description

[0055] Figure 1A Helicobacter pylori was shown. Helicobacter pylori The wild-type amino acid sequence of ferritin was used to mark loop 1 (L1), loop 2 (L2), loop 3 (L3), and loop 4 (L4).

[0056] Figure 1B It showed Pseudomonas aeruginosa ( Pseudomonas aeruginosa The wild-type amino acid sequence of ferritin was used to mark loop 1 (L1), loop 2 (L2), loop 3 (L3), and loop 4 (L4).

[0057] Figure 1C Helicobacter pylori was shown. Helicobacter pylori Ferritin subunit (Hp, light gray) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa Structural alignment of ferritin subunits (Pa, dark gray). Although the sequence conservation among ferritins is low (e.g.Figure 1D Sequence alignment between Pa ferritin and Hp ferritin is shown), but single subunits (such as...) Figure 1C The structure shown is very similar to that of nanoparticles. The locations of glycosylation sites introduced in Pa ferritin are represented by hollow spheres, and they correspond to... Figure 1D The residues highlighted in the text.

[0058] Figure 1D It showed Pseudomonas aeruginosa ( Pseudomonas aeruginosa The amino acid sequence of ferritin was compared with that of Helicobacter pylori. Therefore, this embodiment is extended to all such ferritins having a suitable tertiary structure, having a single subunit and N-terminal and / or loop domains (e.g., L1, L2, L3 and L4) exposed on the surface of the corresponding nanoparticles.

[0059] In one aspect, the insertion sites of the Hp ferritin glycosylation sites are as follows: (1) N-terminus - "glycosylation site and His tag" GGSKDQNRTKDGSGHHHHHH (SEQ ID NO: 58); (2) loop 2 (L2) - K79 is replaced with GDQNATG (SEQ ID NO: 35); (3) loop 3 (L3) - D113 is replaced with DQNAT (SEQ ID NO: 32); (4) loop 4 (L4) - residues 146-149 are replaced with GDQNATG (SEQ ID NO: 35).

[0060] The insertion sites of the glycosylation sites of Hp ferritin are also designed for Pa ferritin.

[0061] Figure 2 SDS-PAGE analysis of ferritin expression in the periplasm is shown, with each ferritin subunit exhibiting a distinct signal sequence attached to the N-terminus. Except for sample 10, all samples contained a histidine tag at the N-terminus following the signal sequence. Sample 1 contained a glycosylation site between the DsbA signal sequence and the histidine tag (this construct corresponds to SEQ ID NO: 13). Ferritin is expressed at high levels in the E. coli periplasm, with the DsbA signal sequence producing the highest periplasmic expression, followed by the XynA signal sequence. The combination of the DsbA signal sequence with the N-terminal glycosylation site and the histidine tag (SEQ ID NO: 13) results in particularly high expression levels.

[0062] Figure 3A A 3D schematic diagram of ferritin nanoparticles is shown, in which magnified individual subunits are highlighted in dark gray. According to various aspects of this embodiment, the sites of glycosylation site insertion (N-terminus, loop 1, loop 2, loop 3, and loop 4) are represented by black spheres in the figure.

[0063] Figure 3BThe results of glycosylation site screening are shown, which was performed by analyzing IMAC (immobilized metal affinity chromatography) enriched periplasmic extracts of *E. coli* strains that produce Sp12F antigenic polysaccharide and express ferritin variants with PglB and the introduced glycosylation site DQNAT (SEQ ID NO: 32). In this example, the upper figure shows SDS-PAGE analysis of the Sp12F-ferritin bioconjugate. The lower figure shows Western blot analysis of the Sp12F-ferritin bioconjugate (using anti-Sp12F antibody). In this example, the following constructs confirmed suitable expression and glycosylation: N-GS-His (SEQ ID NO: 13), His-L2-GS5 (SEQ ID NO: 15), His-L3-GS3 (SEQ ID NO: 55), and His-L4-GS4 (SEQ ID NO: 16). Here, the N-terminus and three internal sites (L2, L3, and L4) are shown as suitable for glycosylation. Glycosylation sites can be evaluated at different locations, and such as Figure 3D The combination shown is performed.

[0064] Figure 3C Analysis of the periplasmic extract of an IMAC-enriched *E. coli* strain that produces KpO3b polysaccharide and expresses PglB and ferritin glycosylation site variants is shown (Coomassie-stained SDS-PA gel - top; anti-His Western blot - middle; anti-KpO3b Western blot - bottom). Bands corresponding to the unglycosylated ferritin carrier and KpO3b-ferritin bioconjugates with occupied glycosylation sites are labeled. The KpO3b ferritin glycosylation site variants exhibit more efficient glycosylation compared to Sp12F. SEQ ID NO is specified in the figure.

[0065] Figure 3D Selected sites identified as suitable for engineering glycosylation sites for ferritin nanoparticle formation are shown. This example demonstrates that glycosylation sites at the N-terminus and two internal sites (N, L2, L4, and combinations thereof) can be glycosylated using Sp12F and KpO5. In the case of KpO5, glycoforms with one, two, or three glycosylation sites can be resolved. SEQ ID NO is specified in the figure.

[0066] Figure 4AThe final purification steps of the Sp12F-ferritin bioconjugate are shown. Impurities remaining after anion exchange chromatography were effectively removed by SEC using a HiPrep 26 / 60 Sephacryl S 500 HR column. Sp12F was eluted at a volume of 180 mL (first peak), while the second peak contained impurities. Fractions 2-11 (labeled on the chromatogram) containing enriched or purified Sp12F-ferritin were collected and combined.

[0067] Figure 4B Showing Figure 4A SDS-PAGE analysis of the collected SEC fractions. Fractions 2-11 were enriched with Sp12F-ferritin.

[0068] Figure 5A SDS-PAGE analysis of Helicobacter pylori ferritin bioconjugated with Sp12F capsular polysaccharide is shown. The ferritin variant used in this experiment contains engineered glycosylation sites at the N-terminus and loop 4 (SEQ ID NO: 18). Fractions of collected Sp12F-ferritin-rich nanoparticles were mixed together to form a final mixture. Sp12F-ferritin bioconjugation and unglycosylated ferritin (uferritin) were labeled on an SDS-PAGE gel.

[0069] Figure 5B This is a 3D schematic diagram of Helicobacter pylori ferritin nanoparticles. The spheres represent the locations of glycosylation sites. This results in a total of 48 glycosylation sites per nanoparticle (SEQ ID NO: 18). The structure of the Sp12F repeating unit is also shown.

[0070] Figure 5C This demonstrates the effect of dynamic light scattering (DLS) on... Figure 5A Characterization of the Sp12F-ferritin bioconjugate. The measured average particle size was 30 nm, indicating a high loading of Sp12F glycans onto the ferritin nanoparticles.

[0071] Figure 5D The assembled Sp12F-ferritin nanoparticles based on negatively stained EM are shown. The measured particle size is approximately 12 nm.

[0072] Figure 6A The final purification steps of the KpO3b-ferritin-Hp bioconjugate are shown. The ferritin variant used in this experiment contains engineered glycosylation sites at the N-terminus and in loop 4 (SEQ ID NO: 18). The ferritin bioconjugate was treated with a SECon Superose 6 10 / 300 column. Fractions 12-16 of the glycosylated KpO3b-ferritin-Hp bioconjugate were mixed to prepare the final mixture according to SDS-PAGE analysis.

[0073] Figure 6B Density analysis of the final KpO3b-ferritin mixture is shown (in) Figure 7A (as shown in the figure) to determine the percentage of conjugated to unconjugated ferritin. It was found that approximately 90% of the ferritin was in the conjugated form.

[0074] Figure 7A SDS-PAGE analysis of the purified KpO3b-ferritin bioconjugate is shown, identifying glycoforms where the KpO3b glycan occupies one and two glycosylation sites. The ferritin variant used in this experiment contains engineered glycosylation sites at the N-terminus and in loop 4 (SEQ ID NO: 18).

[0075] Figure 7B This is a 3D schematic diagram of Helicobacter pylori ferritin nanoparticles. The spheres represent the locations of glycosylation sites. This results in a total of 48 glycosylation sites per nanoparticle (SEQ ID NO: 18). The structure of the KpO3b repeating unit is also shown.

[0076] Figure 7C This demonstrates the effect of dynamic light scattering (DLS) on... Figure 7A Characterization of the KpO3b-ferritin bioconjugate. The measured average particle size was 25 nm, indicating a high loading of KpO3b glycans onto the ferritin nanoparticles.

[0077] Figure 7D The assembled KpO3b-ferritin nanoparticles based on negatively stained EM are shown. The measured particle size is approximately 12 nm.

[0078] Figure 8 The images show Pseudomonas aeruginosa obtained by SDS-PAGE (top) and Western blot analysis of Sp12F glycans (bottom). Pseudomonas aeruginosaAnalysis of periplasmic expression and glycosylation of ferritin. Engineered Helicobacter pylori ferritin variants (SEQ ID NO: 13 and 16) were used as controls. Each Pa ferritin subunit was fused with a different signal sequence attached to the N-terminus and contained glycosylation sites in the N-terminus (samples labeled N on the left) or in the loops (samples labeled L1, L2, and L4 on the right). Pa ferritin was expressed at high levels in the E. pylori periplasm, with the DsbA signal sequence producing the highest periplasmic expression (SEQ ID NO: 23), followed by FlgI (obtaining a good ratio of glycosylated to unglycosylated ferritin with a single Sp12F repeat unit), MalE, and RBP signal sequences. The introduction of glycosylation sites in loop 4 (L4, SEQ ID NO: 25) resulted in more efficient glycosylation than the glycosylation sites in loop 2 (L2, SEQ ID NO: 24). However, the glycosylation sites in L4 of Pa ferritin are not as well tolerated as those in L4 of Hp ferritin because the expression level of this variant is reduced.

[0079] Figure 9A The images show Mycobacterium tuberculosis obtained by SDS-PAGE (top) and Western blot analysis of Sp12F glycans (bottom). Mycobacterium tuberculosis Analysis of periplasmic expression and glycosylation of dodecin. Constructs with C-terminal glycosylation sites and the following signal sequences confirmed suitable expression: DsbA, TolB, PelB, FlgI, and LtIIb. In the right figure, the insertion of glycosylation sites at the N-terminus and in the three loops (Mut1, Mut2, Mut3) was evaluated using the DsbA signal sequence. In addition to the C-terminus, the N-terminus and Mut3 (loop 3) also showed suitability for glycosylation. As shown in the example constructs, glycosylation sites can also be combined (e.g., DsbAss – dodecin-Mut3+C) (SEQ ID NO: 8).

[0080] The SDS-PAGE gel showed a band at approximately 66 kD, corresponding to the dodecin hexamer. The dodecin subunit (monomer) appeared at approximately 11 kD on the SDS-PAGE gel. The SDS-PAGE behavior for dodecin has been reported in the literature (Liu et al., 2011). Suitable signal sequences include, but are not limited to, DsbA, TolB, PelB, Flgl, and Ltllb. Suitable locations for glycosylation site insertion include the N-terminus (SEQ ID NO:4), C-terminus (SEQ ID NO:5), and Mut3 (SEQ ID NO:56), or the C-terminus and Mut3 (SEQ ID NO:8). The glycosylation site combination Mut3+C showed higher glycosylation than other constructs. Constructs with suitable expression and glycosylation (e.g., Sp12F glycosylation) include: DsbAss-dodecin-N (SEQ ID NO: 4), DsbAss-dodecin-C (SEQ ID NO: 5), DsbAss-dodecin-Mut3 (SEQ ID NO: 56), and DsbAss-dodecin-Mut3+C (SEQ ID NO: 8).

[0081] Figure 9B The structure of the dodecin subunit is shown. The locations of the glycosylation sites were indicated by spheres and labeled as: N-terminus, C-terminus, Mut1-ring 1, Mut2-ring 2, Mut3-ring 3. These sites are exposed on the surface of the assembled dodecimal nanoparticles.

[0082] Figure 9C The SEC chromatogram is shown on a Sephacryl S-500 HR size size exclusion column as the final purification step of Sp12F-dodecin. The dodecin variant used in this experiment contains engineered glycosylation sites at the N-terminus and C-terminus (SEQ ID NO: 7).

[0083] Figure 9D SDS-PAGE analysis of fractions from a Sephacryl S-500 HR size size exclusion column is shown; fractions 24 to 32 correspond to peak fractions containing Sp12F-dodecin bioconjugates.

[0084] Figure 10A SDS-PAGE analysis of the purified KpO3b-dodecin bioconjugate is shown. The dodecin variant contains engineered glycosylation sites at the N-terminus and C-terminus (SEQ ID NO: 7).

[0085] Figure 10BA 3D schematic diagram of dodecin nanoparticles from Mycobacterium tuberculosis is shown. The spheres represent the N-terminus and C-terminus where glycosylation sites are inserted (only 6 frontal subunits are shown). This results in a total of 24 glycosylation sites per nanoparticle (SEQ ID NO: 7). The structure of the KpO3b repeating unit is also shown.

[0086] Figure 10C This demonstrates the effect of dynamic light scattering (DLS) on... Figure 10A Characterization of the KpO3b-dodecin bioconjugate. The measured average particle size was 16.9 nm, indicating a high amount of KpO3b glycan loaded onto the dodecin nanoparticles.

[0087] Figure 10D The assembled KpO3b-ferritin nanoparticles based on negatively stained EM are shown. The measured particle size is approximately 6 nm.

[0088] Figure 11A The analysis shows the periplasmic expression and glycosylation of E2p by SDS-PAGE (left panel) and Western blot targeting histidine tags (right panel). Each E2p subunit was fused with a different signal sequence attached to the N-terminus, followed by a glycosylation site. E2p is derived from *Bacillus stearothermophilus* (…). Geobacillus stearothermophilus E2p is a dihydrolipoyltransferase. It is expressed at high levels in the periplasm of E. coli, with the TolB signal sequence producing the highest periplasmic expression (SEQ ID NO: 29), followed by the MalE signal sequence. Figure 11A The results show that sp12F-E2p exists at a high level when using TolB-N and MalE-N signal sequences.

[0089] Figure 11B The SEC chromatogram is shown on a Sephacryl S-500 HR size size exclusion column as the final purification step of KpO3b-E2p. The E2p variant used in this experiment contains an engineered glycosylation site at the N-terminus (SEQ ID NO:29).

[0090] Figure 11C The SDS-PAGE analysis of fractions from a Sephacryl S-500 HR size size exclusion column is shown; fractions 12 to 21 correspond to peak fractions containing KpO3b-E2p bioconjugates.

[0091] Figure 12A SDS-PAGE analysis of the purified KpO3b-E2p bioconjugate is shown. The E2p variant contains an engineered glycosylation site at the N-terminus (SEQ ID NO: 29).

[0092] Figure 12B This is a 3D schematic diagram of E2p nanoparticles from *Bacillus stearothermophilus*. The E2p nanoparticles are 60 mers in size and have a particle size of 24 nm. The N-terminus of each subunit is indicated by a black sphere. The structure of the KpO3b repeating unit is also shown.

[0093] Figure 12C This demonstrates the effect of dynamic light scattering (DLS) on... Figure 12A Characterization of the KpO3b-E2p bioconjugate. The measured average particle size was 42 nm, indicating the presence of glycans on the nanoparticles.

[0094] Figure 12D The KpO3b-E2p nanoparticles assembled according to negatively stained EM are shown. The measured particle size is approximately 24 nm.

[0095] Figure 13A Elution profiles of KpO3b nanoparticles on a Superose 6 10 / 300 column are shown. The KpO3b bioconjugates eluted according to their size, with KpO3b-E2p being the largest sugar-nanoparticle, followed by KpO3b-ferritin in the middle, and finally KpO3b-dodecin being the smallest. Elution volumes are indicated on the chromatograms.

[0096] Figure 13B The size comparison of the prepared unglycosylated and KpO3b-glycosylated nanoparticles, measured by DLS, is shown. An increase in size due to glycosylation was observed in all three types of nanoparticles.

[0097] Figure 14A The illustration shows a nanoparticle in which EPA is fused to the N-terminus of Helicobacter pylori ferritin (the N-terminus of the ferritin subunit is indicated by a black sphere). Only one EPA molecule fused to a single ferritin subunit (labeled in dark gray) is shown in the figure. The linker between EPA and ferritin is 30 amino acids long. This carrier protein nanoparticle fusion molecule corresponds to SEQ ID NO: 52.

[0098] Figure 14B The SEC chromatogram of a Sephacryl S-500 HR size size exclusion column used for the purification of Sp12F-EPA-ferritin is shown.

[0099] Figure 14C Showing the use of Figure 14B SDS-PAGE analysis of the fractions of Sp12F-EPA-ferritin purified and eluted from SEC. Sp12F-EPA-ferritin nanoparticles were enriched in fractions A24-A34.

[0100] Figure 15AThe table shows samples used in preclinical studies to test the immunogenicity of Sp12F in mice, with different protein carriers used for bioconjugation. Nanoparticle-based carriers included the Sp12F-ferritin bioconjugate (Group 4, SEQ ID NO: 18), the Sp12F-dodecin bioconjugate (Group 5, SEC ID NO: 7), and the Sp12F-EPA-ferritin bioconjugate (Group 6, SEQ ID NO: 52). The following were used as controls: PBS (Group 1, negative control), the Sp12F-EPA bioconjugate (Group 2, previously shown to be non-immunogenic to Sp12F), and Sp12F-CRM197 (Group 3, previously shown to induce high Sp12F immunogenicity). The table also shows the mass properties of each conjugate, including the sugar-to-protein ratio and particle size. While all groups used an equal amount of 0.22 µg of polysaccharide, the protein dosage varied. Aluminum phosphate was used as an adjuvant in all groups.

[0101] Figure 15B Showing the use Figure 15A The IgG titers of the samples listed are shown after immunization. For each sample, serum IgG titers were measured before the first immunization (pre-immunization), after the second immunization (post-II), and after the third immunization (post-III). The highest IgG titers and response rates were obtained using Sp12F-ferritin (SEQ ID NO: 18), even higher than those obtained using a chemical conjugate of Sp12F with CRM197. Significant Sp12F immunogenicity was obtained when EPA with five engineered glycosylation sites fused with ferritin (SEQ ID NO: 52) was used as a carrier. Therefore, the EPA-ferritin fusion molecule is superior to EPA as a single protein, indicating that particle size plays an important role in generating a potent immune response. In the case of Sp12F-dodecin, the response rate was lower, but still better than when EPA was used as a carrier.

[0102] Figure 16AThe samples used in preclinical studies to test the immunogenicity of KpO3b in mice are shown, with different protein carriers used for bioconjugation. Nanoparticle-based carriers included the KpO3b-ferritin bioconjugate (Group 1, SEQ ID NO: 18), the KpO3b-dodecin bioconjugate (Group 2, SEC ID NO: 7), and the KpO3b-E2p bioconjugate (Group 3, SEQ ID NO: 29). The following were used as controls: the KpO3b-EPA bioconjugate (Group 4) and PBS buffer (Group 5). The table also shows the mass properties of each conjugate, including the sugar-to-protein ratio and particle size. While all groups used an equal amount of 0.22 µg of polysaccharide, the protein dosage differed. AS03 was used as an adjuvant in all groups.

[0103] Figure 16B The IgG titers following immunization with KpO3b-ferritin (Group 1, SEQ ID NO: 18) are shown compared to the control group (Group 5), as measured before the first immunization (pre-immunization) and after the third immunization (post-III). No immunogenicity against KpO3b was induced when Dodecin, E2p, and EPA were used as carriers for the KpO3b glycan (data not shown).

[0104] Figure 17A Characterization of the purified Sp33F-ferritin bioconjugates is shown by SDS-PAGE, anti-Sp33F Western blot, DLS, and negative staining EM. The ferritin variant used in this example corresponds to SEQ ID NO: 18. The average particle size measured by DLS was 21 nm, indicating the presence of Sp33F glycans on the nanoparticles. EM analysis showed that the assembled Sp33F-ferritin nanoparticles had a measured particle size of approximately 12 nm.

[0105] Figure 17B Characterization of the purified Sp33F-dodecin bioconjugates by SDS-PAGE, anti-Sp33F Western blot, DLS, and negative staining EM is shown. The dodecin variant used in this example corresponds to SEQ ID NO: 7. The average particle size measured by DLS was 15 nm, indicating the presence of Sp33F glycans on the nanoparticles. EM analysis showed that the assembled Sp33F-dodecin nanoparticles had a measured particle size of approximately 6 nm. Detailed Implementation

[0106] This article provides an expression system in which modified nanoparticle subunits, transferases (e.g., PglB), optional carrier proteins, and sugar-producing glycosylation units are expressed and / or translocated to the periplasm of host cells to produce self-assembled, bioconjugated nanoparticles (e.g., in a single step).

[0107] definition As used herein, the term “any amino acid other than proline (Pro, P)” refers to an amino acid selected from the following: 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).

[0108] As used herein, the term "naturally occurring amino acid residue" refers to an amino acid naturally introduced into a polypeptide. Specifically, it refers to 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).

[0109] As used herein, the term "glycosyltransferase (GTF, Gtf)" refers to an enzyme capable of forming glycosidic bonds. Glycosyltransferases are enzymes that catalyze the formation of glycosidic bonds to form glycosides, for example, catalyzing the transfer of a sugar moiety from an activated nucleotide sugar (also known as a "glycosyl donor") to a nucleophilic glycosyl acceptor molecule, whose nucleophilic reagent can be based on oxygen, carbon, nitrogen, or sulfur.

[0110] As used herein, the term "O-antigen (also known as O-specific polysaccharide or O-side chain)" refers to a component of the surface lipopolysaccharide (LPS) of Gram-negative bacteria. Examples include those from *Pseudomonas aeruginosa* (…). Pseudomonas aeruginosa) and Klebsiella pneumoniae ( Klebsiella pneumoniae O-antigen of ).

[0111] As used herein, the term "capsular polysaccharide (CP)" refers to polysaccharides found on the cell walls of bacteria. Examples include, but are not limited to, those from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae Haemophilus influenzae ( ) Haemophilus influenzae ), Neisseria meningitidis ( Neisseria meningitidis ) and Staphylococcus aureus ( Staphylcoccus aureus Capsular polysaccharides.

[0112] As used herein, the term " wzy "This refers to the gene encoding a polysaccharide polymerase, an enzyme that catalyzes the polymerization of polysaccharides. The encoded enzyme transfers oligosaccharide units to non-reducing ends to form glycosidic bonds."

[0113] As used herein, the term " waaL "This refers to the O antigen ligase gene encoding a membrane-bound enzyme. The encoded enzyme transfers the O antigen bound to undecylene diphosphate (UPP) to the lipid A core oligosaccharide, forming lipopolysaccharide."

[0114] "Modified" refers to modifying a protein sequence compared to the wild-type (wt) sequence, such as linking a glycosylated common sequence to the N-terminus or C-terminus of a nanoparticle monomer subunit, or linking it to the amino acid sequence of the nanoparticle monomer subunit itself; modification also refers to mutating an amino acid sequence to improve its stability.

[0115] "Stabilization" refers to introducing one or more mutations into the amino acid sequence (e.g., in nanoparticle monomer subunits) to improve the stability of nanoparticles (e.g., in terms of self-assembly).

[0116] "Nanoparticles" refer to three-dimensional structures that are self-assembled from multiple nanoparticle monomer subunits. A nanoparticle monomer or subunit is one of the subunits of a nanoparticle. The term "self-assembled fragment" of a nanoparticle monomer subunit refers to a fragment that assembles into a nanoparticle and displays a target peptide antigen to generate an immune response against the target antigen.

[0117] In some respects, a nanoparticle can refer to a single nanoparticle or multiple nanoparticles. In other respects, a nanoparticle can refer to multiple assembled monomeric subunits, for example, where each monomeric subunit contains a carrier protein fused to a single nanoparticle monomeric subunit.

[0118] As used herein, the term "immunogenic fragment" refers to a portion of an antigen that is smaller than the complete antigen and capable of eliciting a humoral and / or cellular immune response against that fragment in a host animal (e.g., a human). Fragments of proteins can be produced using techniques known in the art, such as recombinant techniques, proteolytic digestion, or chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for terminal fragments) or both ends (for internal fragments) of the nucleic acid encoding the polypeptide. Typically, fragments contain at least 10, 20, 30, 40, or 50 consecutive amino acids of the full-length sequence. Fragments can be readily modified by adding or removing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 amino acids from either or both of the N-terminus and C-terminus.

[0119] As used herein, the term "conservative amino acid substitution" refers to the substitution of a native amino acid residue with a non-native amino acid residue such that the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position is little or no affected, and does not result in a decrease in immunogenicity. Examples of such substitutions include: 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 modifications to the encoding nucleotides) of a polypeptide sequence can produce polypeptides with similar functions and chemical properties to the parent polypeptide.

[0120] As used herein, the term “deletion” refers to the removal of one or more amino acid residues from a protein sequence. Typically, no more than about 1 to 10 residues (e.g., 1 to 7 residues, 1 to 6 residues, or 1 to 4 residues) are deleted at any site within a protein molecule.

[0121] As used herein, the terms “insertion” or “addition” (including its other tenses, such as “inserted”) refer to the addition of one or more non-natural amino acid residues to a protein sequence, or, as the context requires, the addition of one or more non-natural nucleotides to a polynucleotide sequence. Typically, insertions at any site within a protein molecule sequence do not exceed about 1 to 10 residues (e.g., 1 to 7 residues, 1 to 6 residues, or 1 to 4 residues).

[0122] As used herein, the term “adjacent addition” refers to the addition of one or more non-natural amino acid residues in a protein sequence at a position adjacent to a reference amino acid or amino acid region. For example, “one or more amino acid additions between amino acid residues 198-218” means an addition at any position adjacent to amino acid residues 198-218 (including adjacent to amino acid residues 198 or 218).

[0123] As used herein, the term "glycosylation site" refers to an amino acid sequence recognized by bacterial oligosaccharide transferases (e.g., PglB of Campylobacter jejuni).

[0124] A “shared sequence” is a sequence with a specific structure and / or function. As used herein, the term “shared sequence” is a sequence containing a glycosylation site. Shared sequences may be selected from: a five-amino acid shared sequence D / EXNZS / T (SEQ ID NO: 49), a seven-amino acid shared sequence KD / EXNZS / TK (SEQ ID NO: 50), or an extended shared sequence (e.g., JD / EXNZS / TU (SEQ ID NO: 51)).

[0125] As used herein, the term “introduced at…” refers to the location and manner in which a common sequence is inserted into an amino acid sequence. A glycosylation site introduced at the N-terminus or C-terminus of a protein may be added next to an amino acid sequence immediately adjacent to the N-terminus or C-terminus, while a common sequence (or glycosylation site) introduced at a specific amino acid residue within the protein (e.g., Y208) may replace that amino acid.

[0126] Unless otherwise explicitly stated, numerical ranges (e.g., "25-30") are provided including the endpoints (i.e., including values ​​25 and 30). For example, "between amino acids 198 to 218 of SEQ ID NO: X" refers to the position of the amino acid sequence between amino acid 198 and amino acid 218 of SEQ ID NO: X, including both amino acids 198 and 218.

[0127] The term "identical" or percentage "identity" refers to nucleotide or amino acid sequences that are identical or have a specified percentage of identical nucleotide or amino acid residues (e.g., 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identity within a specified region) when compared and aligned using, for example, sequence comparison algorithms or through manual alignment and visual inspection to achieve maximum correspondence. Identity between peptides can be calculated using various algorithms. Typically, when calculating percentage identity, the two sequences being compared are aligned to obtain the maximum correlation between them. This may involve inserting "gaps" in one or both sequences to enhance the alignment accuracy. For example, the Needleman-Wunsch algorithm (Needleman and Wunsch 1970, J. Mol. Biol. 48: 443-453) for global alignment can be used, or the Smith-Waterman algorithm (Smith and Waterman 1981, J. Mol. Biol. 147: 195-197) for local alignment can be used, for example with default parameters (Smith-Waterman uses a BLOSUM 62 scoring matrix, with a gap opening penalty of 10 and a gap extension penalty of 1). A preferred algorithm was described by Dufresne et al. in Nature Biotechnology (vol. 20, pp. 1269-71) in 2002 and is used in the GenePAST software (Genome Quest Life Sciences, Inc., Boston, MA). GenePAST's "percentage identity" algorithm finds the best match between the query sequence and the topic sequence and expresses the alignment as a precise percentage. GenePAST does not adjust alignment scores based on biological relevance between the query and subject sequences. Identification between two sequences is calculated over their entire length and expressed as a percentage of the reference sequence.

[0128] As used herein, the term "recombinant" means artificial or synthetic. In some embodiments, "recombinant protein" refers to a protein made using a recombinant nucleotide sequence (a nucleotide sequence introduced into the host cell). In some embodiments, the nucleotide sequence encoding a "recombinant polypeptide" is heterologous to the host cell.

[0129] As used herein, the terms “isolated” or “purified” refer to proteins, conjugates (e.g., bioconjugates), polynucleotides, or carriers that are present in a form not found in nature. This includes, for example, proteins, conjugates (e.g., bioconjugates), polynucleotides, or carriers that have been isolated from host cells or organisms (including crude extracts) or otherwise isolated from their natural environment. In some embodiments, an isolated or purified protein is a protein that is substantially free of all other polypeptides that are inherently associated with (or inherently contacted) by that protein.

[0130] As used herein, the term “subject” refers to an animal, particularly a mammal such as a primate (e.g., a human).

[0131] As used herein, in the context of administering a therapy (e.g., the immunogenic composition or vaccine of this embodiment) to a subject, the term "effective amount" refers to the amount of therapy that has a preventive and / or therapeutic effect. In some implementations, an "effective amount" means an amount of therapy sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of a bacterial infection or related symptoms; (ii) shortening the duration of a bacterial infection or related symptoms; (iii) preventing the progression of a bacterial infection or related symptoms; (iv) causing the resolution of a bacterial infection or related symptoms; (v) preventing the occurrence or onset of a bacterial infection or related symptoms; (vi) preventing the recurrence of a bacterial infection or related symptoms; (vii) reducing organ failure associated with a bacterial infection; (viii) reducing hospitalization in subjects with a bacterial infection; (ix) shortening the length of hospital stay in subjects with a bacterial infection; (x) improving the survival rate of subjects with a bacterial infection; (xi) eliminating a bacterial infection in a subject; (xii) inhibiting or reducing bacterial replication in a subject; and / or (xiii) enhancing or improving the preventive or therapeutic effect of another therapy.

[0132] The term “comprising” is open-ended, meaning “including.” Therefore, unless the context requires otherwise, the words “comprising” or “having” and their variations (including “containing” and “comprising” or “have” and “having”, respectively) should be understood to imply inclusion of the stated compound, molecule, composition, or step, but not to exclude any other compound, molecule, composition, or step. The terms “comprising” and “having” are open-ended when used as transitional phrases herein, while the term “consisting of” is closed-ended (i.e., limited to what is listed, nothing more) when used as transitional phrases herein. The abbreviation “eg” originates from Latin. exempli gratia In this document, "eg" is used to refer to non-restrictive instances. Therefore, the abbreviation "eg" is synonymous with the term "for example".

[0133] "Modified carrier proteins" are carrier proteins modified to include one or more glycosylation sites. Modified carrier proteins can be fused to nanoparticle subunits and may optionally include modifications for stability.

[0134] "Nanoparticle subunit" refers to a nanoparticle protein monomer that self-assembles with other nanoparticle protein monomers to form nanoparticles. Nanoparticle subunits can conjugate with antigens or fuse with carrier proteins that conjugate with the same antigen.

[0135] "Modified nanoparticle subunits" refer to nanoparticle subunits capable of being modified for stability (e.g., one or more mutations) and / or containing one or more glycosylation sites, wherein the glycosylation sites can be bioconjugated to one or more antigen molecules (e.g., polysaccharides or oligosaccharides) as provided herein. Bioconjugated nanoparticle subunits (or bioconjugated carrier proteins fused to nanoparticle subunits) self-assemble with other subunits to form self-assembled nanoparticles (e.g., composed of multiple subunits). Modified nanoparticle subunits bioconjugated to antigens (or modified carrier proteins bioconjugated to antigens) may be part of a pharmaceutical composition designed to elicit an immune response against one or more antigen molecules bioconjugated to a carrier protein or nanoparticle subunit. In some aspects, the nanoparticles may be used to display polysaccharide antigens to induce host responses, such as effective B cell and / or T cell responses. In some aspects, the modified nanoparticle subunits may optionally contain one or more stabilizing mutations, and may optionally contain one or more insertions, substitutions, or deletions to introduce one or more glycosylation sites.

[0136] The assembled nanoparticles can display multiple molecules (one or more antigens and / or one or more immunostimulants / carrier proteins) in an ordered array. Regarding nanoparticles displaying one or more antigens and / or immunostimulants, it is believed that the ordered diversity of antigens presented on the outer surface of the nanoparticles allows for multiple binding events to occur simultaneously between the nanoparticles and host cells, which is conducive to inducing a strong host immune response (see, for example, Lopez-Sagaseta et al., Comput StructBiotechnol J, 14:58-68 (2016); see also Perotti and Perez 2020 Viruses 12(35): doi:10.3390 / v12010035 (17 pages); Ueda et al., 2020 eLife 9: e57659 (30 pages)) and conjugation with polysaccharides (see Polonskaya et al., 2017 J. Clin. Invest. 127(4):1492-1504; Pan et al., 2020 Adv. Mater. 32:2002940). Nanoparticles can self-assemble into highly symmetrical, stable, and ordered structures, and can be modified to display one or more antigens to mimic the surface of viruses or bacteria.

[0137] As used herein, the term "carrier protein" refers to an immunogenic protein (e.g., CRM, DT, TT, EPA, etc.) that, when conjugated to an antigen (e.g., a glycoantigen, such as a bacterial polysaccharide antigen or oligosaccharide antigen) and administered to an animal, will enhance the immune response in the animal, particularly producing antibodies that specifically bind to the conjugated polysaccharide or oligosaccharide. In some aspects, the carrier protein may be directly or covalently linked to the N-terminal amino acid of the nanoparticle subunit (peptide monomer) via a short (20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid long) peptide linker sequence located between the carrier protein and the nanoparticle subunit. The linkers between nanoparticles and conjugated carrier proteins include, for example, glycine / serine / alanine linkers (8 to 14 amino acid residues containing glycine, serine, or alanine repeats (see WO2009 / 109428 (PCT / EP2009 / 050996)).

[0138] As used herein, the term "conjugate" refers to a carrier protein or nanoparticle covalently linked to an antigen.

[0139] As used herein, the term "bioconjugate" refers to a conjugate prepared in a host cell context between a protein (e.g., a carrier protein or nanoparticle subunit fused to a nanoparticle subunit) and an antigen (e.g., a glycoantigen, such as a bacterial polysaccharide antigen), wherein a host cell mechanism links the antigen to the protein (e.g., N-linked glycosylation). In one embodiment, nanoparticles already bioconjugated to polysaccharides or oligosaccharides are provided. In another embodiment, a carrier protein fused to nanoparticles is provided, wherein the carrier protein has been bioconjugated to polysaccharides or oligosaccharides.

[0140] As used herein, the term “modified protein” refers to a protein that has been altered (in one or more ways) compared to the wild type (e.g., “modified protein” does not include wild-type protein).

[0141] Some embodiments provide a nucleic acid molecule comprising a polynucleotide encoding such modified nanoparticles (containing glycosylation sites) or fusion carrier protein nanoparticle molecules (e.g., containing a modified carrier protein having one or more biological conjugation sites fused to a nanoparticle subunit).

[0142] Some embodiments provide a pharmaceutical composition comprising one or more nanoparticle monomers. Other embodiments provide a composition comprising nanoparticle subunits linked to one or more antigens (e.g., sugars) or nanoparticles linked to a carrier protein conjugated to the same antigen. The nanoparticle subunits can be assembled into nanoparticles.

[0143] Computational Design Computational and structural design has been applied to design suitable nanoparticles and identify locations within nanoparticle subunits and / or carrier proteins for insertion into glycosylation sites. For example, Rosetta and MOE can be used to identify suitable sites for modification (e.g., modification / mutation to insert glycosylation sites and / or to improve stability). Once the lead design is determined, these lead sequences can be cloned and expressed to evaluate their expression in the periplasm and their suitability for bioconjugation.

[0144] Glycosylcompetent *E. coli* strains transformed with different plasmids carrying different nanoparticle genes and leader sequences have been screened to achieve efficient expression, bioconjugation, and assembly of nanoparticles in the periplasm. These experiments demonstrate that, technically, it is feasible to successfully obtain PglB glycosylated nanoparticles and correctly assemble them in the bacterial periplasm to produce glycosyl nanoparticles for bioconjugated vaccines.

[0145] Modified nanoparticle subunits One object of this embodiment is to provide a nanoparticle (NP) platform for producing bioconjugated nanoparticles (e.g., bioconjugated vaccines). Modified nanoparticle subunits (or modified carrier proteins attached to optionally modified nanoparticle subunits) are translocated together with a glycosyltransferase (e.g., glycosyltransferase pglB) into or expressed therein the periplasm of a host cell (e.g., a bacterial host cell, such as *E. coli*). The glycosyltransferase conjugates an antigen (e.g., a bacterial polysaccharide) to a glycosylation site containing a glycosylation site motif (e.g., an N-glycosylation site: D / EXNZS / T (SEQ ID NO: 49), where X and Z can be any native amino acid other than Pro; for example, a concordant sequence could be DQNXT, where X can be A or R (SEQ ID NO: 53)) to introduce the nanoparticle subunit.

[0146] The inventors have made a surprising discovery that assembled glycoconjugated -NP or glycoconjugated carrier proteins fused with NP can advantageously be generated in a single step (e.g., peptides undergo expression, bioconjugation, and self-assembly to form self-assembled nanoparticles in the periplasm). Therefore, in one aspect, this embodiment relates to self-assembled nanoparticles generated in the periplasm, which exhibit polysaccharide / antigen molecules on their outer surface, to compositions comprising such nanoparticles, and to methods for preparing and using such nanoparticles and compositions.

[0147] consensus sequence Modified nanoparticle subunit proteins may contain one or more consensual sequences (D / EXNZS / T (SEQ ID NO: 49)), where X and Z are independently any amino acid other than proline, and J and U (see below) are independently 1 to 5 naturally occurring amino acid residues. For example, the classic 5-amino acid glycosylation consensual sequence (D / EXNZS / T (SEQ ID NO: 49)) can be extended with 1-5 other amino acid residues on either side of the consensual sequence to achieve more efficient glycosylation, such as... KD / EXNZS / TK(SEQ ID NO: 50) JD / EXNZS / TU(SEQ ID NO: 51) The classic 5-amino acid glycosylation concordance sequence (D / EXNZS / T (SEQ ID NO: 49)) can be extended with lysine residues to achieve more efficient glycosylation (e.g., KD / EXNZS / TK (SEQ ID NO: 50), see also (SEQ ID NO: 37)). Therefore, the concordance sequence in the modified nanoparticle subunit protein of this embodiment may include (or consist of) a D / EXNZS / T (SEQ ID NO: 49) concordance sequence.

[0148] In the modified nanoparticle subunit protein of this embodiment, the shared sequence may be selected from: D / EXNZS / T (SEQ ID NO: 49) or KD / EXNZS / TK (SEQ ID NO: 50), where X is Q (glutamine) and Z is A (alanine) or R (arginine). In another embodiment, the shared sequence is D / EXNZS / T (SEQ ID NO: 49), where Q is Q (glutamine) and Z is A (alanine) or R (arginine), for example, DQNAT (SEQ ID NO: 32) is also called "DQNAT". In another embodiment, the shared sequence is KD / EXNZS / TK (SEQ ID NO: 50), where XQ (glutamine) and Z are A (alanine), for example, KDQNATK (SEQ ID NO: 37) is also called "KDQNATK". In another embodiment, the common sequence may be selected from: D / EXNZS / T (SEQ ID NO: 49), KD / EXNZS / TK (SEQ ID NO: 50) or JD / EXNZS / TU (SEQ ID NO: 51), wherein X is Q (glutamine), Z is A (alanine) or R (arginine), and J and U are independently 1 to 5 amino acid residues, which are independently selected from glycine and / or serine.

[0149] Examples of concordant sequences include the classic 5-amino acid glycosylation concordant sequence (D / EXNZS / T (SEQ ID NO: 49)), which has been extended by 1-5 amino acids on either side: KDQNATK SEQ ID NO: 37 GDQNATG SEQ ID NO: 35 GGDQNATGG SEQ ID NO: 39 GSGDQNATGSG SEQ ID NO: 31 GGSKDQNRTKDGSG SEQ ID NO: 33 GGSKDQNATKDGSG SEQ ID NO: 34 GSGKDQNRTKDGSG SEQ ID NO: 30 GSGGGDQNATGSGGG SEQ ID NO: 36 In some aspects, the modified nanoparticle subunit protein of this embodiment comprises one, two, three, four, five, six, seven or more of any one of SEQ ID NO: 30-39.

[0150] In one embodiment, the modified nanoparticle subunit protein of this embodiment contains at least two D / EXNZS / T (SEQ ID NO: 49) or KD / EXNZS / TK (SEQ ID NO: 50) co-occurring sequences. In one embodiment, the modified nanoparticle subunit protein of this embodiment contains at least three D / EXNZS / T (SEQ ID NO: 49) or KD / EXNZS / TK (SEQ ID NO: 50) co-occurring sequences. In one embodiment, the modified nanoparticle subunit protein of this embodiment contains at least four D / EXNZS / T (SEQ ID NO: 49) or KD / EXNZS / TK (SEQ ID NO: 50) co-occurring sequences. In one embodiment, the modified nanoparticle subunit protein of this embodiment contains at least five D / EXNZS / T (SEQ ID NO: 49) or KD / EXNZS / TK (SEQ ID NO: 50) co-occurring sequences. In one embodiment, the modified nanoparticle subunit protein of this embodiment contains at least six D / EXNZS / T (SEQ ID NO: 49) or KD / EXNZS / TK (SEQ ID NO: 50) co-occurring sequences. In one embodiment, the modified nanoparticle subunit protein of this embodiment contains at least seven D / EXNZS / T (SEQ ID NO: 49) or KD / EXNZS / TK (SEQ ID NO: 50) co-occurring sequences. In one embodiment, the modified nanoparticle subunit protein contains three to seven D / EXNZS / T (SEQ ID NO: 49) or KD / EXNZS / TK (SEQ ID NO: 50) co-occurring sequences. In one embodiment, the modified nanoparticle subunit protein contains four to seven D / EXNZS / T (SEQ ID NO: 49) or KD / EXNZS / TK (SEQ ID NO: 50) co-occurring sequences. In one embodiment, the modified nanoparticle subunit protein comprises five to seven D / EXNZS / T (SEQ ID NO: 49) or KD / EXNZS / TK (SEQ ID NO: 50) concordant sequences. In some aspects, any of these sequences may be extended by 1-5 amino acids to one or both sides of the concordant sequence.

[0151] The introduction of such glycosylation sites can be achieved, for example, by adding new amino acids to the primary structure of the nanoparticle subunit protein (i.e., adding glycosylation sites completely or partially), or by mutating existing amino acids in the protein to create glycosylation sites (i.e., without adding amino acids to the protein, but mutating selected amino acids of the protein to form glycosylation sites). In one embodiment, a common sequence recombination is introduced into the nanoparticle subunit amino acid sequence or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to said sequence.

[0152] signal sequence In one embodiment, the modified nanoparticle subunit protein includes a signal sequence capable of guiding the carrier protein nanoparticle subunit fusion molecule 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 during protein translocation to, for example, the periplasm (i.e., a mature protein is a protein for which at least the signal sequence has been removed). The signal sequences can originate from *Escherichia coli* DsbA [MKKIWLALAGLVLAFSASA (SEQ ID NO: 10)], TolB [MKQALRVAFGFLILWASVLHA (SEQ ID NO: 41)], *Escherichia coli* flagellin (FlgI) [MIKFLSALILLLVTTAAQA (SEQ ID NO: 42)], *Escherichia coli* outer membrane pore protein A (OmpA) [MKKTAIAIAVALAGFATVAQA (SEQ ID NO: 43)], *Escherichia coli* maltose-binding protein (MalE) [MKIKTGARILALSALTTMMFSASALA (SEQ ID NO: 44)], and *Erwinia carotene* (…). Erwinia carotovorans Pectic acid lyase (PelB) [MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 45)], thermostable Escherichia coli enterotoxin LTIIb [MSFKKIIKAFVIMAALVSVQAHA (SEQ ID NO: 46)], Bacillus subtilis ( Bacillus subtilis Endoxylanase XynA [MFKFKKKFLVGLTAAFMSISMFSATASA (SEQ ID NO:47)] or SipA [MKMNKKVLLTSTMAASLLSVASVQAS (SEQ ID NO:48)]. In one particular embodiment, the signal sequence is derived from Escherichia coli DsbA [MKKIWLALAGLVLAFSASA (SEQ ID NO:10)].

[0153] Therefore, this embodiment provides a modified nanoparticle subunit protein, wherein the amino acid sequence further includes a signal sequence capable of guiding the nanoparticle carrier protein molecule into the periplasm of a host cell (e.g., bacteria), said signal sequence being any suitable sequence. Techniques for fusing signal peptides to nanoparticle subunits are well known in the art (e.g., a signal peptide gene from the E. coli protein DsbA can be fused to the N-terminus of a mature nanoparticle subunit protein sequence) (see Schulz, H., Hennecke, H., and Thony-Meyer, L., Science, 281, 1197-1200, 1998).

[0154] Those skilled in the art will understand that references to "between amino acids" (e.g., "between amino acids 198-218") refer to the number of amino acids counted consecutively from the N-terminus of the amino acid sequence. For example, "between amino acids 49-55 of SEQ ID NO: X" refers to the position in the amino acid sequence between amino acids 49 and 55 of SEQ ID NO: X, including amino acids 49 and 55. Therefore, regarding nanoparticle subunit proteins, in one embodiment, a common sequence selected from D / EXNZS / T (SEQ ID NO: 49) and KD / EXNZS / TK (SEQ ID NO: 50) (e.g., KDQNATK (SEQ ID NO: 37)) is added to or substitutes one or more amino acids immediately adjacent to or substituted between amino acid residues 49-55. The common sequence may be added to or substitute any one (or more) of amino acid numbers 49, 50, 51, 52, 53, 54, and 55 in SEQ ID NO: X.

[0155] The amino acid numbers cited herein correspond to the amino acids in SEQ ID NO: X, and as described above, those skilled in the art can determine, by comparison, the equivalent amino acid positions in the amino acid sequence that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to those in SEQ ID NO: X. Addition or deletion of amino acids from variants and / or fragments of SEQ ID NO: X may result in differences in the actual amino acid positions of the common sequence in the mutated sequence; however, by comparing the mutated sequence with a reference sequence, amino acids at equivalent positions to the corresponding amino acids in the reference sequence can be identified, thereby determining the appropriate positions for addition or substitution of the common sequence. In some aspects, the mutated position listed in the sequence may refer to the first sequence aligned with the reference sequence (e.g., the wt sequence) (e.g., having the mutation).

[0156] Specific examples are shown below.

[0157] peptide monomers and protein nanoparticles Nanoparticles prepared from non-viral protein subunits can display antigen molecules on their outer surface. Such nanoparticles include those made from bacterial, insect, and mammalian proteins that can naturally self-assemble into nanoparticles. In this paper, cell engineering is used to generate bioconjugated nanoparticles. In one aspect, nanoparticles containing glycosylation sites are translocated to or expressed therein in the periplasm, where they undergo glycosylation and assembly to form nanoparticles. In other aspects, carrier proteins containing glycosylation sites are conjugated with nanoparticle monomers and translocated to or expressed therein in the periplasm, where they undergo glycosylation and assembly. In some aspects, a leader sequence is present. Modified nanoparticle subunits may contain one, two, three, four, or more mutations for stabilization.

[0158] As presented herein, the nanoparticles include, but are not limited to, ferritin, E2p, dodecin, and EPA. These nanoparticles have been shown to undergo bioconjugation and assembly into nanoparticles within the periplasm.

[0159] Dodecin subunit protein The Dodecin subunit protein (also known as mtDod) is a soluble secretory protein of approximately 8 kDa with a βαββ topological structure (Bourdeaux et al., Nature Scientific Reports, 2020, 10: 13297), produced by Mycobacterium tuberculosis (…). Mycobacterium tuberculosis The gene Rv1498a encodes (Liu et al., Journal of Structural Biology, 2011, 175:31-18), which can self-assemble into spherical dodecomers. In some respects, the N-terminus and C-terminus are exposed on the protein surface (Bourdeaux et al., Nature Scientific Reports, 2020, 10: 13297), facilitating its use as a carrier molecule. Dodecin nanoparticles can be assembled from 12 subunits to form spheres with a diameter of approximately 6 nm.

[0160] The dodecin subunit polypeptide of this embodiment can be prepared by the methods provided herein. The amino acid sequence of wild-type dodecin is shown in SEQ ID NO: 1 (Uniprot: Q8VK10). Dodecin is modified to improve stability, said modification being located at one, two, or three of positions G25, V50, and A53 (e.g., G25N, V50T, A53T), where the positions are relative to the wt sequence. The nucleotide sequence of the modified amino acid sequence of dodecin is provided in SEQ ID NO: 3.

[0161] In one aspect of this embodiment, a dodecin subunit protein is modified to introduce a glycosylation site at a designed location in the amino acid sequence. The modification to introduce the glycosylation site can be one or more at the N-terminus, C-terminus, or within the dodecin sequence (e.g., amino acid residues 49-55, 50-54, 50-53, 50-52, 51) to produce a modified dodecin subunit protein expressed in the periplasm, where it undergoes glycosylation. The modified glycosylated dodecin subunit protein further undergoes self-assembly in the periplasm to form self-assembled glycosylated nanoparticles. In some aspects, dodecin is modified to include one or more glycosylation sites (e.g., at the N-terminus and / or at the C-terminus and / or within the amino acid sequence) and can optionally be linked to a signal sequence at the N-terminus and / or a histidine tag for purification (e.g., at the N-terminus or C-terminus).

[0162] The modified dodecin subunit protein may contain one or more stabilizing mutations of glycine to asparagine (G25N), valine to threonine (V50T), and alanine to threonine (A53T) relative to the amino acid sequence of SEQ ID NO: 1 (or an equivalent position in the amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 1). The modified dodecin subunit protein may be 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% of SEQ ID NO: 1) and contain a glycine to asparagine (G25N) substitution. The modified dodecin subunit protein of this embodiment may be 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 SEQ ID NO: 1) and contains a substitution from valine to threonine (V50T). The modified dodecin subunit protein of the present invention may be 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 SEQ ID NO: 1) and contains a substitution from alanine to threonine (A53T).

[0163] In some aspects, the modified dodecin subunit protein of this embodiment comprises amino acids 2-70 of SEQ ID NO: 2 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 2-70 of SEQ ID NO: 1 or a self-assembled fragment thereof), comprising one or more substitutions of glycine to asparagine (G25N), valine to threonine (V50T), and alanine to threonine (A53T). In some aspects, the modified dodecin subunit protein of this embodiment comprises amino acids 2-70 of SEQ ID NO: 2 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 2-70 of SEQ ID NO: 1 or a self-assembled fragment thereof), comprising two or more of G25N, V50T, and A53T. In some aspects, the modified dodecin subunit protein of this embodiment comprises amino acids 2-70 of SEQ ID NO: 2 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 2-70 of SEQ ID NO: 1 or a self-assembled fragment thereof), and comprises all of G25N, V50T, and A53T, as shown in SEQ ID NO: 2.

[0164] Therefore, in some aspects, stable mutations may include (i) G25N, (ii) V50T, (iii) A53T, (iv) G25N and V50T, (v) G25N and A53T, (vi) V50T and A53T, or (vii) G25N, V50T and A53T. In a preferred embodiment, specific mutations may include G25N, V50T and A53T.

[0165] In some aspects, the dodecin subunit can be modified to include a glycosylation site by attaching or substituting one or more residues (e.g., methionine) at the N-terminus, as shown in SEQ ID NO: 4. In other aspects, the dodecin subunit can be modified to include a glycosylation site by substituting one or more residues at the C-terminus or by attaching a glycosylation site at the C-terminus (e.g., SEQ ID NO: 5), or both. In still other aspects, dodecin can be modified to include a glycosylation site within the amino acid sequence of ring 3 (Mut3) (e.g., residues 49-55, 51), as shown in SEQ ID NO: 6. In other aspects, dodecin can be modified to include glycosylation sites at the N-terminus and C-terminus, as shown in SEQ ID NO: 7; to include glycosylation sites within the amino acid sequence of the C-terminus and loop 3 (Mut3), as shown in SEQ ID NO: 8; or to include glycosylation sites within the amino acid sequence of the N-terminus, C-terminus, and loop 3 (Mut3), as shown in SEQ ID NO: 9. In some aspects, the modified dodecin subunit protein of this embodiment can be an amino acid sequence of any one of SEQ ID NO: 4-9 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4, 5, 6, 7, 8, or 9).

[0166] In other aspects, the signal sequence may replace one or more N-terminal residues or be attached to the N-terminus, for example, upstream of a glycosylation site. Any suitable signal sequence may be used (e.g., DsbA (SEQ ID NO: 10), TolB (SEQ ID NO: 41), PelB (SEQ ID NO: 45), FlgI (SEQ ID NO: 42), LtIIB (SEQ ID NO: 46)). In some aspects, the signal sequence may be attached to the N-terminus (e.g., upstream of a glycosylation site) 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 that of SEQ ID NO: 1).

[0167] In some aspects, the modified sequence may optionally contain a Poly His tag (e.g., a sequence consisting of 5 to 7 or 6 histidine residues) at the N-terminus or C-terminus to facilitate the purification of the amino acid sequence SEQ ID NO:4-9 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4-9).

[0168] Depending on the aspects, assembled glycosylated nanoparticles are provided in a single step. The method includes the following steps: providing host cells; expressing a modified protein (e.g., a modified dodecin subunit containing an antigen) and the glycosyltransferase pglB in the host cells to generate subunits that self-assemble in the periplasm to form glycoprotein nanoparticles. The polysaccharide is produced by the host cells along with any other proteins required for the transfer of the polysaccharide to the periplasm, based on the techniques provided herein.

[0169] Those skilled in the art will understand that for fragments of the amino acid sequence of the variant dodecin relative to SEQ ID NO:1 and / or the amino acid sequence of SEQ ID NO:1, such amino acid sequences are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, and the reference to "ammonia...between" means that if the sequence is aligned with the amino acid sequence of SEQ ID NO:1 to maximize the sequence identity between the two sequences, the position will be equivalent to the defined position (sequence alignment tools are not limited to Clustal Omega (www.ebi.ac.ac.uk), MUSCLE (www.ebi.ac.uk), or T-coffee (www.tcoffee.org)). In one aspect, the sequence alignment tool used is Clustal Omega (www.ebi.ac.ac.uk).

[0170] Ferritin subunit protein Ferritin is found in a variety of organisms, including vertebrates, amphibians, plants, bacteria, and humans. Ferritin is a globular protein with a molecular weight of approximately 474 kDa that functions as an intracellular iron storage protein and is secreted into serum as an iron transporter. Previous literature describes nanoparticles based on insect and human ferritin for displaying antigens on the surface of NPs (see, for example, WO2018 / 005558; Kwong et al. (2018)). Li et al. described nanoparticles composed of recombinant fusion peptides containing human ferritin light chain subunits and short HIV-1 antigenic peptides linked to the amino termini of the ferritin light chain sequence. The self-assembly of these fusion peptides allowed the HIV-1 antigenic peptides to be localized on the outer surface of NPs (see Li et al.). Ind. Biotechnol. 2:143-47 (2006)).

[0171] Ferritin can be modified by introducing one or more glycosylation sites into the amino acid residues exposed on the surface of nanoparticles. The selection of modification sites should prioritize non-interference with nanoparticle formation, their presence on the nanoparticle surface, and suitability for antigen display. Suitable locations for introducing glycosylation sites include the N-terminus, loop 1 (L1), the long loop between helices 2 and 3 (L2), the short loop between helices 3 and 4 (L3), and the loop between helices 4 and 5 (L4). Ferritin nanoparticles can self-assemble from 24 subunits into spherical structures with a diameter of approximately 12 nm. When each ferritin subunit contains 2 and 3 glycosylation sites, each ferritin nanoparticle has 48 and 72 glycan-linking sites, respectively. This embodiment covers any ferritin molecule from any species; representative examples are described below.

[0172] Ferritin has been demonstrated to self-assemble into nanoparticles during recombinant expression (e.g., in prokaryotic expression systems). The ferritin sequence described herein can be recombinantly modified to include short amino acid tags for purification assistance, such as histidine tags known in the art, which can optionally be linked to the ferritin sequence via short peptide linkers. Lysine or asparagine residues exposed on the NP surface can be used to conjugate glycans to the NP surface.

[0173] Suitable signal peptide sequences include DsbA (SEQ ID NO: 10), TolB (SEQ ID NO: 41), MalE (SEQ ID NO: 44), and XynA (SEQ ID NO: 47).

[0174] Ferritin HP Helicobacter pylori bacterial ferritin (see Protein Database (PDB) accession number Q9ZLI1) has been studied as a pharmaceutically acceptable carrier. Helicobacter pylori bacterial ferritin consists of 24 identical polypeptide subunits that can self-assemble into spherical nanoparticles. Li et al. reported the preparation of nucleotide sequences encoding a fusion protein of bacterial (Helicobacter pylori) ferritin subunit polypeptides and rotavirus antigens, and its expression in a prokaryotic (Escherichia coli) system. The expressed fusion polypeptides were described as being able to self-assemble into spherical NPs displaying rotavirus capsid proteins and inducing an immune response in mice (Li et al., J Nanobiotechnol 17:13 (2019)). Wang et al. designed a mixture containing Helicobacter pylori ferritin and Neisseria gonorrhoeae ( N. gonorrhoeae Chimeric polypeptides of antigenic peptides; these chimeric polypeptides are described as being able to assemble into 24-mer nanoparticles displaying antigenic peptides on the outer surface of NPs (Wang et al.). FEBS Open Bio7(8):1196 (2017)). Kanekiyo et al. described a self-assembled recombinant bacterial (Helicobacter pylori) ferritin nanoparticle (24-mer) containing a fusion protein of ferritin subunit polypeptides and influenza antigen peptides, displaying influenza trimers on its surface (Kanekiyo et al., Nature 499(7456):102 (2013)).

[0175] This article provides the amino acid sequence (SEQ ID NO: 11) and nucleotide sequence (SEQ ID NO: 12) of Helicobacter pylori ferritin (e.g., Uniprot: Q9ZLI1).

[0176] In one aspect, ferritin subunits are modified to produce modified ferritin subunits, which are then expressed and glycosylated (e.g., Sp12). The modified glycosylated ferritin subunits further self-assemble in the periplasm to form assembled glycosylated nanoparticles. In some aspects, ferritin has been modified to include one or more glycosylation sites (e.g., at the N-terminus and / or C-terminus and / or within the amino acid sequence). In some aspects, ferritin may have a signal peptide sequence linked to its N-terminus and / or a histidine tag linked to its N-terminus for purification.

[0177] In some aspects, the modified ferritin subunit protein comprises amino acids 1-167 of SEQ ID NO: 11 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1-167 of SEQ ID NO: 11) or fragments thereof. In some aspects, a glycosylation site (SEQ ID NO: 13) may be introduced by modification at the N-terminus of ferritin. In some aspects, the glycosylation site is SEQ ID NO: 33 or SEQ ID NO: 34. In some aspects, the modified ferritin subunit protein may comprise a glycosylation site, optionally followed by a histidine tag and an optional linker (e.g., QDP), followed by ferritin. For example, the modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 or 14).

[0178] In other respects, the modified ferritin subunit protein includes a glycosylation site introduced into the ferritin sequence. For example, a glycosylation site may be introduced at K79 of loop 2, wherein a glycosylation site (SEQ ID NO: 32) (SEQ ID NO: 15) is introduced. As another example, a glycosylation site may be introduced at 146-149 of loop 4, wherein a glycosylation site (SEQ ID NO: 35) (SEQ ID NO: 16) is introduced. For example, the modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15 or 16).

[0179] In some aspects, ferritin may be modified to include one or more of the following: a glycosylation site in L2, a glycosylation site in L4, and an N-terminal glycosylation site. For example, ferritin may be modified to include glycosylation sites in both the N-terminus and L2. Ferritin may be modified to include glycosylation sites in both the N-terminus and L4. In some aspects, ferritin may be modified to include glycosylation sites in the N-terminus, L2, and L4. The modified ferritin subunit may be the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19, or a self-assembled fragment thereof (or an amino acid sequence or a self-assembled fragment thereof that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17, 18, or 19).

[0180] Optionally, the ferritin subunit protein may include a signal peptide sequence at its N-terminus. Any suitable signal peptide sequence may be attached to the N-terminus of the ferritin subunit protein (e.g., DsbA (SEQ ID NO: 10), TolB (SEQ ID NO: 41), PelB (SEQ ID NO: 45), FlgI (SEQ ID NO: 42), LtIIB (SEQ ID NO: 46)). In some aspects, the signal peptide sequence may be attached to the N-terminus (e.g., upstream of the glycosylation site) and linked to the modified ferritin subunit amino acid sequence shown in SEQ ID NO: 14-19 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14-19). In some aspects, the modified ferritin subunit protein may comprise a leader / signal peptide sequence followed by a glycosylation site, optionally followed by a histidine tag and an optional linker (QDP), followed by ferritin (optionally having one or more internal glycosylation sites in L2 and / or L4). In some aspects, the signal peptide sequence may replace the starting methionine of SEQ ID NO: 11 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 11).

[0181] In some aspects, the modified sequence may optionally include a Poly His tail (e.g., a tandem of 5 to 7 histidine residues, or 6 histidine residues) appended to the N-terminus for purifying the amino acid sequence of SEQ ID NO: 13-19 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13-19).

[0182] According to the aspects provided herein, assembled glycosylated nanoparticles can be obtained in a single step. The method includes the following steps: providing host cells; expressing a modifying protein (e.g., a modified ferritin subunit containing a glycosylation site) and the glycosyltransferase pglB to produce glycoprotein nanoparticles. The host cells produce the polysaccharide based on the techniques provided herein, along with any other proteins required for expression and / or transport of the polysaccharide to the periplasm.

[0183] For example, those skilled in the art will understand that, for a fragment of the variant ferritin amino acid sequence relative to SEQ ID NO: 11 and / or the amino acid sequence of SEQ ID NO: 11, i.e., an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11, when referring to "between amino acids," it means the position corresponding to the defined position when the sequence is aligned with the amino acid sequence of SEQ ID NO: 11 to maximize the sequence identity between the two sequences (sequence alignment tools include, but are not limited to, Clustal Omega (www.ebi.ac.ac.uk), MUSCLE (www.ebi.ac.uk), or T-coffee (www.tcoffee.org). In one aspect, the sequence alignment tool used is Clustal Omega (www.ebi.ac.ac.uk).

[0184] Ferritin PA Pseudomonas aeruginosa bacterial ferritin (see Protein Database (PDB) accession number Q9HWF9) has been studied as a pharmaceutically acceptable carrier. Pseudomonas aeruginosa bacterial ferritin consists of 24 identical polypeptide subunits and can self-assemble into spherical nanoparticles.

[0185] This article provides the amino acid sequence (SEQ ID NO: 20) of Pseudomonas aeruginosa bacterial ferritin (e.g., Uniprot: Q9HW59) (see, for example, Joyce 2021 (Science Translational Medicine) vol. 14: 632). In some aspects, the amino acid sequence encoding Pseudomonas aeruginosa ferritin has been stabilized. The modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20). The modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20 or a self-assembled fragment thereof), which contains a substitution from methionine to isoleucine (M31I). The modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20 or its self-assembled fragment), containing a substitution from lysine to leucine (K120L). The modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20 or its self-assembled fragment), containing a substitution from alanine to arginine (A124R). The modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20 or a self-assembled fragment thereof), containing a substitution from methionine to isoleucine (M144I). The modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20 or a self-assembled fragment thereof), containing a substitution from isoleucine to methionine (I154M). In some aspects, the modified ferritin subunit protein may contain one, two, three, four, or more of the mutant M31I, K120L, A124R, M144I, and / or I154M, or a self-assembled fragment thereof.

[0186] In some respects, the modified ferritin subunit protein may contain all of the mutants M31I, K120L, A124R, M144I and I154M, as shown in the amino acid sequence (SEQ ID NO: 21) and corresponding nucleotide sequence (SEQ ID NO: 22) of Pseudomonas aeruginosa bacterial ferritin.

[0187] In other aspects, the modified ferritin subunit comprises a signal peptide sequence, followed by a glycosylation sequence, then a histidine tag, and then ferritin (SEQ ID NO: 23), or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23. In some aspects, the modified ferritin subunit comprises a signal peptide sequence, followed by a histidine tag, and then ferritin, with a glycosylation site inserted at T80 of ferritin ring 2 (SEQ ID NO: 24), or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 24. In some respects, the modified ferritin subunit contains a signal peptide sequence followed by a histidine tag, then ferritin, with a glycosylation site inserted at G145 of ferritin ring 4 (SEQ ID NO: 25), or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25.

[0188] In some aspects, the modified ferritin subunit protein of this embodiment comprises amino acids 1-154 of SEQ ID NO: 21 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1-167 of SEQ ID NO: 21) or fragments thereof. In some aspects, the N-terminus of ferritin may be modified to introduce a glycosylation site (SEQ ID NO: 23). In some aspects, the glycosylation site is SEQ ID NO: 34. In some aspects, the modified ferritin subunit protein may include an internal glycosylation site (e.g., SEQ ID NO: 24, SEQ ID NO: 25). For example, the modified ferritin subunit protein may include a modification of residue 80 in ring 2 (counting from methionine in the native ferritin sequence UNIPROT ID: Q9HWF9). In some aspects, T80 is replaced by a sugar tag (SEQ ID NO: 31) or by an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 24. In other aspects, G145 is replaced by a sugar tag (SEQ ID NO: 31) or by an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 25. In some aspects, ferritin may be modified as described herein to include one or more of the glycosylation sites in L2, L4, and N-terminal glycosylation sites.

[0189] Optionally, the ferritin subunit protein may include a signal peptide sequence at its N-terminus. Any suitable signal peptide sequence may be appended to the N-terminus of the ferritin subunit protein (e.g., DsbA (SEQ ID NO: 10), TolB (SEQ ID NO: 41), PelB (SEQ ID NO: 45), FlgI (SEQ ID NO: 42), LtIIB (SEQ ID NO: 46)). In some aspects, the signal peptide sequence (e.g., SEQ ID NO: 10) may be appended to the N-terminus (e.g., upstream of the glycosylation site) and linked to the modified ferritin subunit amino acid sequence shown in SEQ ID NO: 23-25 ​​(or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23-25). In some aspects, the modified ferritin subunit protein may comprise a signal peptide sequence followed by a glycosylation site, optionally followed by a histidine tag, and then ferritin (optionally having one or more internal glycosylation sites in L2 and / or L4). In some aspects, the signal peptide sequence may replace the starting methionine of SEQ ID NO: 21 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 23).

[0190] In some aspects, the modified sequence may optionally include a Poly His tail (e.g., a tandem of 5 to 7 histidine residues, or 6 histidine residues) appended to the N-terminus for the purpose of purifying the amino acid sequence (see SEQ ID NO: 23-25 ​​(or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 23-25)).

[0191] Depending on the aspects, assembled glycosylated nanoparticles can be obtained in a single step. The method includes the following steps: providing host cells; expressing a modifying protein (e.g., a modified ferritin subunit containing an antigen) and the glycosyltransferase pglB to produce glycoprotein nanoparticles. The host cells produce the polysaccharide based on the techniques provided herein, along with any other proteins required for expression and / or transport of the polysaccharide to the periplasm.

[0192] For example, those skilled in the art will understand that, for a fragment of the variant ferritin amino acid sequence relative to SEQ ID NO: 20 and / or the amino acid sequence of SEQ ID NO: 20, i.e., an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20, when referring to "between amino acids," it means the position corresponding to the defined position when the sequence is aligned with the amino acid sequence of SEQ ID NO: 20 to maximize the sequence identity between the two sequences (sequence alignment tools include, but are not limited to, Clustal Omega (www.ebi.ac.ac.uk), MUSCLE (www.ebi.ac.uk), or T-coffee (www.tcoffee.org). In one aspect, the sequence alignment tool used is Clustal Omega (www.ebi.ac.ac.uk).

[0193] E2p E2p is a protein found in *Bacillus stearothermophilus*. E2p nanoparticles can assemble to form 60-mer hollow dodecahedrons with a diameter of approximately 24 nm. Recombinant E2p nanoparticles generated by expression of recombinant genes in bacterial expression systems can be purified from bacterial homogenates by size exclusion chromatography (Kozlovska et al., 1993) or by a combination of stepwise ammonium sulfate precipitation and size exclusion chromatography (Vasiljeva et al. (1998); Cilens et al. (2000)).

[0194] In some aspects of this paper, E2p nanoparticle subunits can be linked to sugars and self-assemble to form nanoparticles. The wild-type amino acid sequence (SEQ ID NO: 26) and nucleotide sequence (SEQ ID NO: 28) of E2p are provided in this paper (e.g., Uniprot: P11961).

[0195] In one aspect, the E2p subunit protein is modified to produce a modified E2p subunit protein, which is expressed in a host cell and undergoes glycosylation (e.g., with Sp12F). The modified glycosylated E2p subunit protein further self-assembles in the periplasm to form assembled glycosylated nanoparticles. In some aspects, E2p has been modified to include one or more glycosylation sites (e.g., at the N-terminus and / or C-terminus and / or within an amino acid sequence). In some aspects, E2p may have a signal peptide sequence linked to its N-terminus and / or a histidine tag linked for purification (e.g., at the N-terminus).

[0196] In some aspects, the modified E2p subunit protein comprises amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof) or a fragment thereof. In some aspects, the amino acid sequence encoding E2p has been stabilized. The modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof), comprising a substitution from alanine to threonine (A187T). The modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof), containing a substitution from phenylalanine to tyrosine (F196Y). The modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof), containing a substitution from threonine to asparagine (T281N). The modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof), containing a proline to serine substitution (P314S). The modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof), containing alanine to valine substitution (A352V).The modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof), containing a substitution from leucine to isoleucine (L425I). The modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or a self-assembled fragment thereof), containing an amino acid deletion at positions 427 and 428 (Δ427-428).

[0197] In some aspects, the modified E2p subunit protein may comprise one or more of the following mutations of amino acids 185-426 of SEQ ID NO: 26: A187T, F196Y, T281N, P314S, A352V, L425I, and Δ427-428. In some aspects, the modified E2p subunit protein may comprise all the mutations of amino acids 185-426 of SEQ ID NO: 26: A187T, F196Y, T281N, P314S, A352V, L425I, and Δ427-428.

[0198] In some aspects, E2p may be modified to include a glycosylation site at the N-terminus. In some aspects, the consortium sequence includes D / EXNZS / T (SEQ ID NO: 49), wherein X and Z may independently be any native amino acid other than proline (e.g., the consortium sequence may be DQNXT, wherein X may be A or R (SEQ ID NO: 53)). In some aspects, the glycosylation site is SEQ ID NO: 36. In some aspects, the modified ferritin subunit protein may include a signal peptide sequence, optionally followed by a linker, optionally followed by a histidine tag and optionally a linker, followed by amino acids 185-426 of SEQ ID NO: 26 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29).

[0199] Optionally, the E2p subunit protein may include a signal peptide sequence at its N-terminus. Any suitable signal peptide sequence may be appended to the N-terminus of the E2p subunit protein (e.g., DsbA (SEQ ID NO: 10), TolB (SEQ ID NO: 41), PelB (SEQ ID NO: 45), FlgI (SEQ ID NO: 42), LtIIB (SEQ ID NO: 46)). In some aspects, the signal peptide sequence (e.g., SEQ ID NO: 10) may be appended to the N-terminus (e.g., upstream of the glycosylation site) and linked to the modified E2p subunit amino acid sequence shown in SEQ ID NO: 29 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29). In some aspects, the modified E2p subunit protein may include a signal peptide sequence, optionally followed by a histidine tag, then a glycosylation site and optionally a linker, and then E2p.

[0200] In some aspects, the modified sequence may optionally include a polyHis tail (e.g., a tandem of 5 to 7 histidine residues, or 6 histidine residues) attached to the N-terminus for purifying the amino acid sequence of SEQ ID NO: 29 (or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29).

[0201] Depending on the aspects, assembled glycosylated nanoparticles can be obtained in a single step. The method includes the following steps: providing host cells; expressing a modified protein (e.g., a modified E2p subunit) and the glycosyltransferase pglB in the host cells, wherein the subunit undergoes glycosylation and self-assembly to produce glycoprotein nanoparticles. The host cells produce the polysaccharide based on the techniques provided herein and any other proteins required for the transport of the polysaccharide to the periplasm.

[0202] In one aspect, the E2p subunit protein is modified to include glycosylation sites, thereby producing a modified E2p subunit protein for expression and glycosylation in the periplasm.

[0203] For example, those skilled in the art will understand that, for a fragment of the variant E2p amino acid sequence relative to SEQ ID NO: 26 and / or the amino acid sequence of SEQ ID NO: 26, i.e., an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26, when referring to "between amino acids," it means the position corresponding to the defined position when the sequence is aligned with the amino acid sequence of SEQ ID NO: 26 to maximize the sequence identity between the two sequences (sequence alignment tools include, but are not limited to, Clustal Omega (www.ebi.ac.ac.uk), MUSCLE (www.ebi.ac.uk), or T-coffee (www.tcoffee.org). In one aspect, the sequence alignment tool used is Clustal Omega (www.ebi.ac.ac.uk).

[0204] Gene fusion In other respects, nanoparticle subunits (such as ferritin) can be fused with carrier proteins (such as EPA) using techniques known in the art. The fusion of carrier protein and ferritin can undergo expression and glycosylation in cells and can self-assemble in the periplasm.

[0205] The modified EPA carrier protein in this embodiment can be a recombinant modified EPA carrier protein fused to a ferritin nanoparticle subunit (see, for example, SEQ ID NO: 52). The modified EPA protein in this embodiment can be a separate recombinant modified EPA protein fused to a ferritin nanoparticle subunit.

[0206] In some aspects, the modified nanoparticle subunit protein may further include a "peptide tag" or "tag," which allows for the isolation and / or identification of the amino acid sequence of the modified subunit protein. For example, adding a tag to a modified nanoparticle subunit protein can be used for the purification of the protein, and thus for the purification of conjugates (e.g., bioconjugates) vaccines containing the tagged modified nanoparticle subunit protein. Exemplary tags available herein include, but are not limited to, histidine (HIS) tags (e.g., hexahistidine tags or 6Xhis-Tag), FLAG-TAG, and HA tags. In one embodiment, the tag is a hexahistidine tag. The tags used herein are removable, for example, removed by chemical reagents or enzymatic methods when no longer needed (e.g., after protein purification). Therefore, the modified nanoparticle subunit protein may further include a peptide tag. Optionally, the peptide tag is located at the C-terminus of the amino acid sequence. Optionally, the tag comprises six histidine residues located at the N-terminus or C-terminus of the amino acid sequence. In one aspect, the modified nanoparticle subunit protein comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to any nanoparticle subunit sequence provided herein, and a peptide tag (e.g., six histidine residues at the N-terminus or C-terminus of the amino acid sequence).

[0207] antigen One embodiment includes modified nanoparticles (or modified carrier proteins fused with optionally modified nanoparticles) displaying one or more polysaccharide or oligosaccharide antigens on the outer surface of the modified nanoparticles (or modified carrier proteins). In one aspect of the invention, the polysaccharide or oligosaccharide antigens may be conjugated to glycosylation sites on the nanoparticles, wherein the nanoparticles are not conjugated to another polypeptide. In other embodiments of the invention, the polysaccharide or oligosaccharide antigens are conjugated to glycosylation sites on a modified carrier protein fused with the nanoparticles, such as modified CRM (e.g., CRM-197), modified EPA, modified diphtheria toxoid (DT), modified OMPC, or modified tetanus toxoid (TT), etc.

[0208] Antigens bioconjugated to modified nanoparticles or modified carrier proteins via glycosylation sites can be glycoantigens, such as bacterial polysaccharides or oligosaccharides, yeast polysaccharides, or mammalian polysaccharides. Polysaccharides may contain two or more monosaccharides, typically more than ten. Oligosaccharides may contain a few monosaccharides, for example, fewer than ten. In one embodiment, the antigen is a bacterial polysaccharide antigen, such as an O-antigen from Gram-negative bacteria or a capsular polysaccharide from Gram-positive bacteria. In one embodiment, the antigen displayed on the nanoparticles is any O-antigen or capsular polysaccharide or its immunogenic fragment, or a combination of such antigens suitable for a particular serotype.

[0209] In a further embodiment, the antigen (e.g., a bioconjugate) displayed on the surface of the modified nanoparticles is a polysaccharide antigen derived from Escherichia coli. Escherichia ) species, Shigella genus ( Shigella ) species, Klebsiella genus ( Klebsiella ) species, Salmonella genus ( Salmonella ) species, Yersinia genus ( Yersinia ) species, Helicobacter genus ( Helicobacter ) species, Proteus genus ( Proteus ) species, Pseudomonas genus ( Pseudomonas ) species, Corynebacterium genus ( Corynebacterium ) species, Streptomyces genus ( Streptomyces ) species, Streptococcus genus ( Streptococcus ) species, Enterococcus genus ( Enterococcus ) species, Staphylococcus genus ( Staphylococcus ) species, Bacillus genus ( Bacillus ) species, Clostridium ( Clostridium ) species, Listeria genus ( Listeria ) species or Campylobacter genus ( Campylobacter ) species. Examples include, but are not limited to, Shigella dysenteriae ( Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae Streptococcus pneumoniae () Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylcoccus aureus In one embodiment, the antigen in the conjugate (e.g., a biological conjugate) of this embodiment is selected from Shigella flexneri (…). Shigella flexneri ), Klebsiella pneumoniae ( Klebsiella pneumoniae ) or Streptococcus pneumoniae ( Streptococcus pneumoniae Bacterial polysaccharides.

[0210] In some embodiments, the antigen is an O-antigen, for example, from Gram-negative bacteria (e.g., Salmonella spp.). Salmonella ) species, Shigella genus ( Shigella ) species, Pseudomonas genus ( Pseudomonas ) species or Klebsiella genus ( Klebsiella ) species, such as Shigella dysenteriae ( Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) or Klebsiella pneumoniae ( Klebsiella pneumoniae(See also Dmitriev, BA et al., Somatic Antigensof) Shigella , Eur J. Biochem, 1979. 98: p. 8; Liu et al., Structure and geneticsof Shigella O antigens, FEMS Microbiology Review, 2008. 32: p. 27). In one embodiment, the antigen is from Pseudomonas aeruginosa (O antigens, FEMS Microbiology Review, 2008. 32: p. 27). Pseudomonas aeruginosa The O-antigen of ). For example, the antigen may be from Pseudomonas aeruginosa ( Pseudomonas aeruginosa The O-antigen of serotypes 1-20 (Raymond et al., J Bacteriol. 2002 184(13):3614-22). In one embodiment, the antigen is derived from Klebsiella pneumoniae ( Klebsiella pneumoniae O-antigen of ).

[0211] In some embodiments, the antigen is a capsular polysaccharide derived from Gram-negative or Gram-positive bacteria, such as Neisseria meningitidis (…). Neisseria meningitidis ) serogroup A (MenA), Neisseria meningitidis ( Neisseria meningitidis ) serogroup C (MenC), Neisseria meningitidis ( Neisseria meningitidis ) serogroup Y (MenY), Neisseria meningitidis ( Neisseria meningitidis ) serogroup W (MenW), Haemophilus influenzae type b (Hib), group B streptococci (GBS), Streptococcus pneumoniae ( Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus In some embodiments, the antigen is derived from Streptococcus spp. ( ). Streptococcus ) species or genus Staphylococcus ( Staphylococcus ) species (e.g., Streptococcus pneumoniae) Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus Capsular polysaccharides of Staphylococcus aureus. In one embodiment, the antigen is derived from Staphylococcus aureus. Staphylococcus aureus Capsular polysaccharides. For example, antigens may be derived from Staphylococcus aureus (Staphylococcus aureus). Staphylococcus aureus Capsular polysaccharides of Streptococcus pneumoniae types 5 and 8. In one embodiment, the antigen is derived from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae Capsular polysaccharides (e.g., any of the 90+ serotypes (see, Shoji et al., Infection and Drug Resistance (2018) vol. 11, pp 1387-1400).

[0212] Antigen display Molecules (including antigenic molecules such as carbohydrates) attached to the outer surface of modified nanoparticles or modified carrier proteins are referred to herein as “displayed” or “displayed” molecules. Modified nanoparticles (or modified carrier proteins) that display antigens preferably display multiple copies of the antigen molecule in an ordered arrangement displayed on the nanoparticle surface to allow multiple binding events to occur simultaneously between the nanoparticle and the host cell, thereby facilitating the induction of a robust host immune response (see, for example, Lopez-Sagaseta et al. (2016)). Displaying antigens on nanoparticles has been used to improve the immunogenicity of subunit protein antigens (see, Jardine et al. (2013); Correira et al. (2014)).

[0213] Vector Vectors containing such polynucleotides are another aspect of this embodiment. In a specific embodiment, a plasmid is used to introduce a heterologous nucleotide sequence into a host cell, for example, the heterologous nucleotide sequence is expressed in the host cell via a plasmid (e.g., an expression vector).

[0214] host cells In some respects, a host cell is provided that comprises: i) One or more nucleotide sequences containing a polysaccharide synthesis gene, optionally used to produce bacterial polysaccharide antigens (e.g., O-antigens from Gram-negative bacteria, optionally from Shigella dysenteriae). Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), or capsular polysaccharides from Gram-positive bacteria, optionally from Streptococcus pneumoniae ( Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylcoccus aureus ), or from Neisseria meningitidis ( Neisseria meningitidis (capsular polysaccharide), or yeast polysaccharide antigen or mammalian polysaccharide antigen, which optionally integrate into the host cell genome; ii) A nucleotide sequence encoding a heterooligosaccharide transferase (e.g., PglB), optionally within a plasmid; iii) The nucleotide sequence encoding the modified nanoparticle subunit protein of this embodiment, optionally within a plasmid.

[0215] Host cells that can be used to generate the bioconjugated nanoparticles of this embodiment or the bioconjugated carrier protein fused with the nanoparticles include archaea, prokaryotic host cells, and eukaryotic host cells. In some embodiments, the host cell is a non-human host cell. Exemplary prokaryotic host cells used to generate the bioconjugations of this embodiment include Escherichia coli (Escherichia coli). Escherichia ) species, Shigella genus ( Shigella ) species, Klebsiella genus ( Klebsiella ) species, Xanthomonas genus ( Xhantomonas ) species, Salmonella genus ( Salmonella ) species, Yersinia genus ( Yersinia ) species, Lactococcus genus ( Lactococcus ) species, Lactobacillus genus ( Lactobacillus ) species, Pseudomonas genus ( Pseudomonas ) species, Corynebacterium genus ( Corynebacterium ) species, Streptomyces genus ( Streptomyces ) species, Streptococcus genus ( Streptococcus ) species, Staphylococcus genus ( Staphylococcus ) species, Bacillus genus ( Bacillus ) species and Clostridium ( Clostridium ) species. In some respects, the host cell is Escherichia coli.

[0216] Publications regarding methods for preparing such host cells suitable for the methods provided herein are found in WO 06 / 119987, WO 09 / 104074, WO 11 / 62615, WO 11 / 138361, WO 14 / 57109, WO14 / 72405 and WO16 / 20499.

[0217] The host cell can be modified to delete or modify genes in the host cell's genetic background (genome) that compete with or interfere with the synthesis of the target polysaccharide (e.g., genes that compete with or interfere with one or more heteropolysaccharide synthesis genes introduced into the host cell through recombination). These genes can be deleted or modified in the host cell background (genome) in a manner that inactivates or eliminates their function (i.e., the deleted / modified host cell nucleotide sequence does not encode a functional protein or does not encode a protein at all). In one embodiment, when a nucleotide sequence is deleted from the host cell genome of this embodiment, it is replaced with a desired sequence, such as a sequence for glycoprotein production. Exemplary genes that can be deleted in the host cell (and in some cases replaced with other desired nucleotide sequences) include host cell genes involved in glycolipid biosynthesis, such as... waaL (See, for example, Feldman et al. 2005, PNAS USA 102:3016-3021), O-antigen cluster ( rfb or wb ), Enterobacterial common antigen cluster (wec ), lipid A core biosynthetic cluster ( waa ), galactose clusters ( gal ), arabinose clusters ( ara ), colonic acid clusters ( wc Capsular polysaccharide clusters, and undecadienol pyrophosphate biosynthesis genes (e.g., uppS (undecadienyl pyrophosphate synthase)). uppP (Undecadienyl bisphosphatase), Und-P cycle genes, metabolic enzymes involved in nucleotide activation and sugar biosynthesis, common antigenic clusters of intestinal bacteria, and prophage O-antigen modification clusters, such as gtrABS Cluster. In one embodiment, the cluster is deleted or functionally inactivated from the prokaryotic host cell genome of this embodiment. waaL Gene, gtrA Gene, gtrB Gene, gtrS One or more of the genes, or from wec One or more genes of the cluster, or from rfb One or more genes in a gene cluster.

[0218] The host cell in this embodiment is *Escherichia coli*, which contains the naturally occurring enterobacterial common antigen cluster (ECA). wec )remove wecA External and colonic acid clusters ( wca The O16-antigen cluster has been missing. Furthermore, the native lipopolysaccharide O-antigen ligase is missing from the host cells of this embodiment. waaL Furthermore, the host cells of this embodiment may be deficient in natural [elements / materials]. gtrA Gene, gtrB Genes and gtrS Gene.

[0219] The host cell of the present invention is engineered to contain heterologous nucleotide sequences. The host cell of the present invention is engineered to contain nucleotide sequences encoding modified nanoparticle subunit proteins (or modified carrier proteins fused to nanoparticle subunit proteins), optionally within a plasmid. The host cell of this embodiment also contains one or more nucleotide sequences containing polysaccharide synthesis genes. Therefore, the host cell of this embodiment can produce bioconjugates containing antigens, such as glycoantigens (e.g., bacterial, yeast, or mammalian polysaccharide antigens) linked to modified nanoparticle subunit proteins (or modified carrier proteins fused to nanoparticle subunit proteins). One or more heterologous nucleotide sequences may encode polysaccharide synthesis proteins to produce bacterial polysaccharide antigens, yeast polysaccharide antigens, or mammalian polysaccharide antigens. Therefore, the present invention also provides a host cell comprising: i) One or more heteronucleotide sequences containing a bacterial polysaccharide antigen (e.g., an O-antigen from a Gram-negative bacterium, optionally from Shigella dysenteriae). Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), or capsular polysaccharides from Gram-positive bacteria, optionally from Streptococcus pneumoniae ( Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylcoccus aureus ), or from Neisseria meningitidis ( Neisseria meningitidis The polysaccharide synthesis gene of the capsular polysaccharide (or yeast polysaccharide antigen or mammalian polysaccharide antigen), which is optionally integrated into the host cell genome; ii) A nucleotide sequence encoding a heterooligosaccharide transferase (e.g., pglB), optionally within a plasmid; iii) The nucleotide sequence encoding a modified nanoparticle subunit protein (or a modified carrier protein fused to a nanoparticle subunit protein), optionally within a plasmid.

[0220] The host cell of this embodiment may contain one or more nucleotide sequences sufficient to produce glycoantigens (e.g., bacterial polysaccharide antigens), particularly glycoantigens heterologous to the host cell (e.g., bacterial polysaccharide antigens). For example, if the host cell is *E. coli*, the host cell may contain one or more nucleotide sequences including a polysaccharide synthesis gene sufficient to produce bacterial polysaccharide antigens that are not *E. coli* polysaccharide antigens. The bacterial polysaccharide antigen may be an O-antigen or a capsular polysaccharide antigen. Therefore, this embodiment also provides a host cell comprising: i) One or more nucleotide sequences containing a bacterial polysaccharide antigen (e.g., an O-antigen from Gram-negative bacteria, optionally from Shigella dysenteriae). Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), or capsular polysaccharides from Gram-positive bacteria, optionally from Streptococcus pneumoniae ( Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylcoccus aureus The polysaccharide synthesis gene, which is optionally integrated into the host cell genome; ii) A nucleotide sequence encoding a heterooligosaccharide transferase (e.g., pglB), optionally within a plasmid; iii) A nucleotide sequence encoding the modified nanoparticle subunit protein (or a modified carrier protein fused to the nanoparticle subunit protein) of this embodiment, optionally within a plasmid.

[0221] The polysaccharide synthesis gene encodes a protein involved in polysaccharide synthesis (polysaccharide synthesis protein). In one embodiment, the host cell may contain one or more nucleotide sequences containing a polysaccharide synthesis gene for producing proteins derived from Shigella dysenteriae (…). Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and Klebsiella pneumoniae ( Klebsiella pneumoniae The O-antigen of Gram-negative bacteria, or from Streptococcus pneumoniae (Streptococcus pneumoniae) Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylcoccus aureus Capsular polysaccharides from Gram-positive bacteria (such as Shigella flexneri). In one embodiment, the host cell may contain one or more nucleotide sequences comprising a polysaccharide synthesis gene for producing polysaccharide from bacteria selected from Shigella flexneri. Shigella flexneri ) and Klebsiella pneumoniae ( Klebsiella pneumoniae The O-antigen of Gram-negative bacteria, or from Streptococcus pneumoniae (Streptococcus pneumoniae) Streptococcus pneumoniae ) and Staphylococcus aureus ( Staphylcoccus aureus Capsular polysaccharides of Gram-positive bacteria.

[0222] Host cells for producing bacterial polysaccharide antigens The host cell in this embodiment may contain one or more nucleotide sequences that include a polysaccharide synthesis gene for producing the O-antigen. In some embodiments, the host cell contains one or more nucleotide sequences that include a gene for producing a polysaccharide synthesis gene from Salmonella spp. Salmonella ) species, Shigella genus ( Shigella ) species, Pseudomonas genus ( Pseudomonas ) species or Klebsiella genus ( Klebsiella The O-antigen polysaccharide synthesis gene of the species Shigella. In some embodiments, the host cell contains one or more nucleotide sequences that contain the gene for producing the O-antigen polysaccharide from Shigella species. Shigella ) species, Pseudomonas genus ( Pseudomonas ) species or Klebsiella genus ( Klebsiella ) species (e.g., Shigella dysenteriae) Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) or Klebsiella pneumoniae ( Klebsiella pneumoniaeThe O-antigen polysaccharide synthesis gene from Shigella spp. is described. In some embodiments, the host cell contains one or more nucleotide sequences that contain the polysaccharide synthesis gene for the O-antigen from Shigella spp. Shigella ) species or Klebsiella genus ( Klebsiella ) species (e.g., Shigella dysenteriae) Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ) or Klebsiella pneumoniae ( Klebsiella pneumoniae The O-antigen polysaccharide synthesis gene from Shigella dysenteriae. In one embodiment, the host cell contains one or more nucleotide sequences that contain the polysaccharide synthesis gene for producing the O-antigen from Shigella dysenteriae. Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri ) or Shigella sonnei ( Shigella sonnei The host cell may contain one or more nucleotide sequences that contain polysaccharide synthesis genes for producing O-antigens from Shigella dysenteriae serotype 1, Shigella sonnei, and Shigella flexneri serotype 6, as well as Shigella flexneri 2a and 3a 0 (Dmitriev, BA et al., Somatic Antigens of 0). Shigella , Eur J. Biochem, 1979. 98: p. 8; Liu et al., Structure and geneticsof Shigella O antigens, FEMS Microbiology Review, 2008. 32: p. 27). In one embodiment, the host cell contains one or more nucleotide sequences that contain the information for producing antigens from Pseudomonas aeruginosa (O antigens, FEMS Microbiology Review, 2008. 32: p. 27). Pseudomonas aeruginosa (For example, Pseudomonas aeruginosa) Pseudomonas aeruginosa The polysaccharide synthesis gene for the O-antigen of serotypes 1-20 is described. In one embodiment, the host cell contains one or more nucleotide sequences that include the gene for producing the polysaccharide from Klebsiella pneumoniae (serotypes 1-20). Klebsiella pneumoniae The polysaccharide synthesis gene of the O-antigen.

[0223] The host cell in this embodiment may contain one or more nucleotide sequences that include a polysaccharide synthesis gene for producing capsular polysaccharides. In some embodiments, the host cell contains one or more nucleotide sequences that include genes for producing polysaccharide synthesis from Neisseria meningitidis serogroup A (MenA), Neisseria meningitidis serogroup C (MenC), Neisseria meningitidis serogroup Y (MenY), Neisseria meningitidis serogroup W (MenW), Haemophilus influenzae type b (Hib), Group B Streptococcus (GBS), and Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureusA polysaccharide synthesis gene for capsular polysaccharides from Streptococcus ( ). In some embodiments, the host cell contains one or more nucleotide sequences that contain the gene for producing polysaccharides from Streptococcus ( ). Streptococcus ) species or genus Staphylococcus ( Staphylococcus ) species (e.g., Streptococcus pneumoniae) Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus A polysaccharide synthesis gene for capsular polysaccharides from Staphylococcus aureus. In one embodiment, the host cell contains one or more nucleotide sequences that include the gene for producing polysaccharide from Staphylococcus aureus. Staphylococcus aureus (For example, Staphylococcus aureus) Staphylococcus aureus Polysaccharide synthesis genes for capsular polysaccharides of types 5 and 8. In one embodiment, the host cell contains one or more nucleotide sequences that include genes for producing polysaccharides from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae The polysaccharide synthesis gene of capsular polysaccharide.

[0224] Host cell comprising heterologous nucleotide sequence for producing bacterial polysaccharide antigen The host cells of this invention can naturally express one or more nucleotide sequences containing polysaccharide synthesis genes for the production of carbohydrate antigens (e.g., bacterial polysaccharide antigens); or the host cells can be engineered to express one or more such nucleotide sequences. For example, the host cells of this invention can sequentially assemble oligosaccharides (cytoplasmic glycosyltransferases) in the cytoplasm using endogenous or heteroglycosyltransferases. Heteronucleotide sequences (e.g., nucleotide sequences encoding carrier proteins and / or nucleotide sequences encoding other proteins (e.g., proteins involved in glycosylation)) can be introduced into the host cells of embodiments of this invention using methods such as electroporation, heat shock chemical transformation, natural transformation, phage transduction, and conjugation. In a specific embodiment, heteronucleotide sequences are introduced into the host cells of this embodiment using plasmids; for example, the heteronucleotide sequences are expressed in the host cells via plasmids (e.g., expression vectors). In another specific embodiment, heteronucleotide sequences are introduced into the host cells of this embodiment using the method described in WO14 / 037585. In some embodiments, the host cells contain one or more nucleotide sequences containing polysaccharide synthesis genes derived from the host cells. In some embodiments, one or more nucleotide sequences comprising a heterologous polysaccharide synthesis gene from the host cell are integrated into the host cell genome. The heterologous nucleotide sequence may encode, but is not limited to, glycosyltransferases, oligosaccharide transferases, epimerases, flippers, and / or polymerases. In some embodiments, the host cell of this embodiment contains one or more heterologous nucleotide sequences encoding glycosyltransferases, which may be derived, for example, from Escherichia coli spp. Escherichia ) species, Shigella genus ( Shigella ) species, Klebsiella genus ( Klebsiella ) species, Salmonella genus (Salmonella ) species, Pseudomonas genus ( Pseudomonas ) species, Streptococcus genus ( Streptococcus ) species or genus Staphylococcus ( Staphylococcus ) species.

[0225] The host cell in this embodiment may contain one or more heterologous nucleotide sequences including a polysaccharide synthesis gene for producing the O-antigen. In some embodiments, the host cell contains one or more sequences derived from Salmonella spp. Salmonella ) species, Shigella genus ( Shigella ) species, Pseudomonas genus ( Pseudomonas ) species or Klebsiella genus ( Klebsiella The nucleotide sequence encoding a polysaccharide-synthesizing protein from the species is used to produce the O-antigen. In some embodiments, the host cell contains one or more nucleotide sequences from the genus Shigella. Shigella ) species, Pseudomonas genus ( Pseudomonas ) species or Klebsiella genus ( Klebsiella ) species (e.g., Shigella dysenteriae) Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) or Klebsiella pneumoniae ( Klebsiella pneumoniae The nucleotide sequence encoding a polysaccharide-synthesizing protein is used to generate the O-antigen. In some embodiments, the host cell contains one or more nucleotide sequences from the genus Shigella. Shigella ) species or Klebsiella genus ( Klebsiella ) species (e.g., Shigella dysenteriae) Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ) or Klebsiella pneumoniae ( Klebsiella pneumoniae The nucleotide sequence encoding a polysaccharide-synthesizing protein is used to generate the O-antigen. In one embodiment, the host cell contains one or more nucleotide sequences derived from Shigella dysenteriae. Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri ) or Shigella sonnei ( Shigella sonnei The host cell may contain one or more nucleotide sequences encoding polysaccharide-synthetic proteins from Shigella dysenteriae type 1, Shigella sonnei, and Shigella flexneri type 6, as well as Shigella flexneri 2a and 3a, for the production of the O-antigen. In one embodiment, the host cell may contain one or more nucleotide sequences encoding polysaccharide-synthetic proteins from Pseudomonas aeruginosa. Pseudomonas aeruginosa (For example, Pseudomonas aeruginosa) Pseudomonas aeruginosa The nucleotide sequence encoding a polysaccharide synthetic protein (serotypes 1-20) for producing the O-antigen. In one embodiment, the host cell contains one or more nucleotide sequences from Klebsiella pneumoniae (serotypes 1-20). Klebsiella pneumoniae The nucleotide sequence encoding the polysaccharide-synthesizing protein used to produce the O-antigen is described. The nucleotide sequence encoding the O-antigen can be... rfb Cluster. As used in this article, rfb A cluster refers to a gene cluster encoding an enzymatic mechanism capable of synthesizing the O-antigen. Host cells may contain genes from Shigella dysenteriae (…). Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae )of rfb Gene clusters.

[0226] The host cell in this embodiment may contain one or more heterologous nucleotide sequences including a polysaccharide synthesis gene for producing capsular sugars. In some embodiments, the host cell contains one or more of the following: Neisseria meningitidis serogroup A (MenA), Neisseria meningitidis serogroup C (MenC), Neisseria meningitidis serogroup Y (MenY), Neisseria meningitidis serogroup W (MenW), Haemophilus influenzae type b (Hib), Group B Streptococcus (GBS), and Streptococcus pneumoniae (…). Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus The nucleotide sequence encoding a polysaccharide-synthesizing protein is used to produce capsular sugars. In some embodiments, the host cell contains one or more nucleotide sequences from the genus Streptococcus. Streptococcus ) species or genus Staphylococcus ( Staphylococcus ) species (e.g., Streptococcus pneumoniae) Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus The nucleotide sequence encoding a polysaccharide-synthesizing protein is used to produce capsular polysaccharides. In one embodiment, the host cell contains one or more nucleotide sequences derived from Staphylococcus aureus. Staphylococcus aureus (For example, Staphylococcus aureus) Staphylococcus aureus Nucleotide sequences encoding polysaccharide synthesis proteins (types 5 and 8) for producing capsular polysaccharides. In one embodiment, the host cell contains one or more nucleotide sequences comprising polysaccharide synthesis genes for producing polysaccharide from Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae Capsular polysaccharides from Streptococcus. The nucleotide sequence may be a capsular polysaccharide gene cluster. Host cells may contain polysaccharides from the genus Streptococcus (…). Streptococcus ) species (e.g., Streptococcus pneumoniae, S. pyrogenes , S. agalacticae ) or Staphylococcus spp. Staphylococcus ) strains (e.g. S. aureus Capsular polysaccharide gene clusters of Streptococcus pneumoniae. Streptococcus pneumoniae The capsular polysaccharide gene cluster is located on the Streptococcus pneumoniae chromosome between dexB and aliA (see, Llull et al., 1999, J. Exp. Med. 190, 241-251). There are typically four relatively conserved genes at the 5′ end of the capsular polysaccharide gene cluster: ( wzg ), ( wzh ), ( wzd ), ( wze (Jiang et al., 2001, Infect. Immun. 69, 1244-1255). The capsular polysaccharide gene cluster of Streptococcus pneumoniae also includes... wzx (polysaccharide invertase gene) and wzy (Polysaccharide polymerase gene). The Sanger Institute has sequenced the CP gene clusters of at least 90 Streptococcus pneumoniae serotypes (http: / / www.sanger.ac.uk / Projects / S_pneumoniae / CPS / ), and sequenced the CP gene clusters of at least 89 serotypes. wzx and wzy Annotations and analyses were performed (Kong et al., 2005, J. Med. Microbiol. 54, 351-356). The capsule biosynthesis genes of *Streptococcus pneumoniae* are further described in Bentley et al. (PloS Genet. 2006 Mar; 2(3): e31), and their sequences are available in GenBank. Therefore, in one embodiment, the host cell of the present invention may further comprise an enzyme encoding a polymerase (e.g., ...). wzy ), flip enzyme (e.g., wzx The nucleotide sequence of ), and optionally encoding chain length regulators (e.g., wzz The nucleotide sequence of ).

[0227] In some implementations, the host cell may also contain cells located at... rfb Heteronucleotide sequences outside of clusters or capsular polysaccharide clusters. For example, sequences encoding polysaccharides present in host cells can be introduced. rfb Nucleotide sequences of glycosyltransferases and acetyltransferases that are outside of clusters or capsular polysaccharide clusters and can modify recombinant polysaccharides.

[0228] Oligosaccharide transferase N-linked protein glycosylation (the addition of a sugar molecule to an asparagine residue in the target protein polypeptide chain) is the most common type of post-translational modification in the endoplasmic reticulum of eukaryotes. This process is accomplished by the enzymatic oligosaccharide transferase complex (OST), which is responsible for transferring pre-assembled oligosaccharides from a lipid carrier (polyterpene phosphate) to an asparagine residue in the conserved sequence Asn-X-Ser / Thr (where X is any amino acid except proline) in the nascent protein. This process occurs within the endoplasmic reticulum.

[0229] Previous studies have shown that a bacterium—the foodborne pathogen Campylobacter jejuni (…) Campylobacter jejuni Campylobacter jejuni can also N-glycosylate its proteins (Wacker et al., Science. 2002; 298(5599):1790-3), because it possesses its own glycosylation mechanism. The mechanism responsible for this reaction is encoded by a cluster called "pgl" (protein glycosylation). The glycosylation mechanism of Campylobacter jejuni can be transferred to Escherichia coli to allow glycosylation of recombinant proteins expressed in E. coli cells. Previous studies have demonstrated how to generate E. coli strains capable of N-glycosylation (see, for example, 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 Publication Nos. WO2003 / 074687, WO2006 / 119987, WO 2009 / 104074, WO / 2011 / 06261 and WO2011 / 138361).

[0230] The host cell of the present invention contains a nucleotide sequence encoding a heterologous oligosaccharide transferase, optionally within a plasmid. In one particular embodiment, the oligosaccharide transferase is derived from the genus Campylobacter (…). Campylobacter The oligosaccharide transferase is pglB, optionally derived from Campylobacter jejuni (…). In another specific embodiment, the oligosaccharide transferase is pglB, optionally derived from Campylobacter jejuni (…). Campylobacter jejuni (i.e., pglB; see, for example, Wacker et al. 2002, Science 298:1790-1793; also see, for example, NCBI Gene ID: 3231775, UniProt accession number O86154) SEQ ID NO: 54: MLKKEYLKNPYLVLFAMIILAYVFSVFCRFYWVWWASEFNEYFFNNQLMIISNDGYAFAEGARDMIAGFHQPNDLSYYGSSLSALTYWLYKITPFSFESIILYMSTFLSSLVVIPTILLANEYKRPLMGFVAALLASIANSYYNRTMSGYYDTDMLVIVLPMFILFFMVRMILKKDFF SLIALPLFIGIYLWWYPSSYTLNVALIGLFLIYTLIFHRKEKIFYIAVILSSLTLSNIAWFYQSAIIVILFALFALEQKRLNFMIIGILGSATLIFLILSGGVDPILYQLKFYIFRSDESANLTQGFMYFNVNQTIQEVENVDLSEFMRRISGSEIVFLFSLFGFVWLLRKHKSMIMA LPILVLGFLALKGGLRFTIYSVPVMALGFGFLLSEFKAIMVKKYSQLTSNVCIVFATILTLAPVFIHIYNYKAPTVFSQNEASLLNQLKNIANREDYVVTWWDYGYPVRYYSDVKTLVDGGKHLGKDNFFPSFALSKDEQAAANMARLSVEYTEKSFYAPQNDILKTDILQAMMKDYN QSNVDLFLASLSKPDFKIDTPKTRDIYLYMPARMSLIFSTVASFSFINLDTGVLDKPFTFSTAYPLDVKNGEIYLSNGVVLSDDFRSFKIGDNVVSVNSIVEINSIKQGEYKITPIDDKAQFYIFYLKDSAIPYAQFILMDKTMFNSAYVQMFFLGNYDKNLFDLVINSRDAKVFKLKI Therefore, the host cell of the present invention may contain a nucleotide sequence encoding pglB, optionally derived from Campylobacter jejuni (… Campylobacter jejuni pglB, optionally encoding Campylobacter jejuni ( Campylobacter jejuni The nucleotide sequence of pglB, which is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54, optionally within a plasmid.

[0231] polymerase The host cell of the present invention may also contain encoding a polymerase (e.g., wzy The nucleotide sequence of ). In one embodiment, a polymerase (e.g., wzy The polymerase is introduced into the host cell of an embodiment of the invention (i.e., the polymerase is heterologous to the host cell). In one embodiment, the polymerase is a bacterial polymerase. In another embodiment, the polymerase is a capsular polysaccharide polymerase (e.g., wzy ) or O-antigen polymerase (e.g., wzy In one embodiment, the polymerase is an O-antigen polysaccharide polymerase (such as wzy, for example, wzy ), for example, from the genus Shigella ( Shigella ) species, Pseudomonas genus ( Pseudomonas ) species or Escherichia genus ( Escherichia ) species (e.g., Shigella dysenteriae) Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) or E. coli ( E. coli In one embodiment, the polymerase is a capsular polysaccharide polymerase (e.g., wzy For example, from Neisseria meningitidis serogroup A (MenA), Neisseria meningitidis serogroup C (MenC), Neisseria meningitidis serogroup Y (MenY), Neisseria meningitidis serogroup W (MenW), Haemophilus influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae (… Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus In one embodiment, the polymerase is Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae Capsular polysaccharide polymerase (e.g., wzy The aforementioned wzy Polymerase can be used as rfb A cluster or a portion of a capsular polysaccharide cluster is introduced (e.g., inserted into the genome or expressed by a plasmid) into the host cell. Therefore, the host cell of this embodiment may further contain encoding heterologous... wzy The nucleotide sequence of the polymerase.

[0232] flip enzyme The host cell in this embodiment may also contain an enzyme encoding a flip enzyme (e.g., wzxThe nucleotide sequence of the enzyme, for example, a heterologous flippant enzyme. The flippant enzyme translocates wild-type repeat units and / or their corresponding engineered (hybrid) repeat units from the cytoplasm to the periplasm of the host cell (e.g., *E. coli*). In one embodiment, the flippant enzyme is a bacterial flippant enzyme, for example, a flippant enzyme of the target polysaccharide biosynthesis pathway. In a specific embodiment, the host cell of the present invention comprises a genera encoding *Streptococcus* (…). Streptococcus ) species, Shigella genus ( Shigella ) species, Escherichia coli genus ( Escherichia ) species, Pseudomonas genus ( Pseudomonas ) species or genus Staphylococcus ( Staphylococcus ) species (e.g., Streptococcus pneumoniae) Streptococcus pneumoniae ), Shigella dysenteriae ( Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Escherichia coli, Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) or Staphylococcus aureus ( Staphylcoccus aureus The polysaccharide biosynthesis pathway inverted enzymes (e.g., wzx The nucleotide sequence of ). In one embodiment, the flipase is Streptococcus pneumoniae (Streptococcus pneumoniae) Streptococcus pneumoniae Capsular polysaccharide invertase (e.g., wzx Other flipping enzymes that can be introduced into the host cells of this embodiment, such as those from Campylobacter jejuni (…), can be introduced. Campylobacter jejuni )(For example, pglK ).

[0233] coenzyme In one embodiment, a nucleotide sequence encoding one or more coenzymes is introduced into the host cell of this embodiment. Therefore, the host cell of this embodiment may further contain one or more of these coenzymes. Such nucleotide sequences encoding one or more coenzymes may be carried by a plasmid or integrated into the host cell genome. Exemplary coenzymes include, but are not limited to, epimerases (see, for example, WO2011 / 062615), branching enzymes, modifying enzymes (e.g., adding choline, glycerophosphate, pyruvate), amidases, chain length regulating enzymes, acetyltransferases, formylates, and polymerases. Therefore, the host cell of this embodiment may also contain factors encoding chain length regulating factors (e.g., wzz The nucleotide sequence of , for example, a heterologous chain length regulator. In one embodiment, the chain length regulator is Streptococcus pneumoniae (Streptococcus pneumoniae). Streptococcus pneumoniae Capsular polysaccharide chain length regulators (e.g., wzz ).

[0234] Biological conjugates This embodiment provides a bioconjugate comprising a modified nanoparticle subunit (or a modified carrier protein fused to a nanoparticle subunit) linked to an antigen (e.g., a bacterial polysaccharide antigen, a yeast polysaccharide antigen, or a mammalian polysaccharide antigen). In specific embodiments, the antigen is an O-antigen or a capsular polysaccharide. In one embodiment, the antigen is an O-antigen from Gram-negative bacteria. In some aspects, a bioconjugate is provided comprising a modified nanoparticle subunit (or a modified carrier protein fused to a nanoparticle subunit) conjugated to an antigen, wherein the antigen is a sugar, optionally a bacterial polysaccharide (e.g., from Shigella dysenteriae). Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae Streptococcus pneumoniae () Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylcoccus aureus In another embodiment, the present invention provides a bioconjugate comprising a modified nanoparticle subunit linked to an antigen (or a modified carrier protein fused to a nanoparticle subunit), wherein the antigen is derived from Shigella flexneri. Shigella flexneri ), Klebsiella pneumoniae ( Klebsiella pneumoniae ) or Streptococcus pneumoniae ( Streptococcus pneumoniae The antigen is a bacterial polysaccharide. The antigen is linked to a modified nanoparticle subunit (or a modified carrier protein fused to a nanoparticle subunit) to an amino acid selected from asparagine, aspartic acid, glutamic acid, lysine, cysteine, tyrosine, histidine, arginine, or tryptophan (e.g., asparagine). As described herein, bioconjugates have advantageous properties compared to chemical conjugates of antigen-carrier proteins because they require fewer chemicals in their manufacture and are easier to reliably produce consistent end products.

[0235] Another aspect of this embodiment is a method for generating bioconjugated nanoparticle subunits, the subunits comprising or consisting of modified nanoparticle subunits linked to glycosides, the method comprising (i) culturing host cells of this embodiment under conditions suitable for generating glycoproteins, glycosyltransferases and nanoparticle subunits, and (ii) isolating the bioconjugated nanoparticles generated by the host cells, optionally isolating the bioconjugated nanoparticles from a periplasmic extract of the host cells.

[0236] Another aspect of this embodiment is a method for generating bioconjugated carrier nanoparticle subunits, the subunits comprising or consisting of a fusion of a modified carrier protein linked to a glycoside and a nanoparticle subunit, the method comprising (i) culturing host cells of this embodiment under conditions suitable for generating glycoproteins, glycosyltransferases and nanoparticle carrier proteins fused to the subunits, and (ii) isolating the bioconjugated carrier nanoparticle product generated by the host cells, optionally isolating the bioconjugated carrier nanoparticles from a periplasmic extract of the host cells.

[0237] For example, shake-flask methods (e.g., in LB shake flasks) can be used to prepare bioconjugated nanoparticles. In some aspects, fed-batch methods can be used to produce recombinant glycosylated proteins in bacteria to prepare bioconjugated nanoparticles. The aim is to improve glycosylation efficiency and recombinant protein yield per cell while maintaining process simplicity and reproducibility. By developing optimized manufacturing methods using typical E. coli production processes, bioconjugated nanoparticles can be manufactured on a commercial scale. Various types of fed-batch strategies, such as batch, chemostat, and fed-batch cultures, can be used.

[0238] The bioconjugated nanoparticles of this embodiment can be purified by, for example, chromatography (e.g., ion exchange, anion exchange, affinity chromatography, and volumetric column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification (see, for example, Saraswat et al. 2013, Biomed. Res. Int. ID#312709 (pp. 1-18); also see the methods described in WO2009 / 104074). Furthermore, the bioconjugated nanoparticles can be fused with the heterologous polypeptide sequences described herein or other known in the art to facilitate purification.

[0239] Conjugation of nanoparticles or nanoparticle fusion proteins The nanoparticles (or carrier proteins fused to nanoparticle subunits) described herein can be conjugated by any suitable method. Demonstrating molecules can be incorporated into or attached to nanoparticles (or carrier proteins fused to nanoparticle subunits) by any suitable means.

[0240] Alternative methods for preparing the nanoparticles described herein include chemical conjugation.

[0241] In chemical conjugation, functional groups present on the nanoparticle subunit peptide (or carrier protein fused to the nanoparticle subunit) can be used for conjugation of the display molecule. Amino acid side chain groups used for conjugation include the amino group on lysine, the thiol group on cysteine, the carboxyl group on aspartic acid and glutamic acid, and the hydroxyl group on tyrosine, and can be used with various chemical methods known in the art. Homo- or hetero-bifunctional crosslinking agents can be used for conjugation. The amino group on the side chain of lysine residues is a nucleophilic group; therefore, lysine residues exposed on the surface of nanoparticles have high solvent accessibility and can be used as sites for conjugation with the display molecule.

[0242] One or more selected amino acid residues within the subunit polypeptide sequence (or carrier protein sequence) of a nanoparticle can be modified using methods known in the art to provide sites suitable for chemobioconjugation on the outer surface of the nanoparticle (or on a carrier protein attached to the outer surface of the nanoparticle), wherein such modification does not disrupt the assembly of the nanoparticle.

[0243] In one aspect, a modified nanoparticle is provided, wherein one or more display molecules (e.g., antigens) are chemically conjugated to residues present on the outer surface of the nanoparticle. The display molecule may be an oligosaccharide, polysaccharide, glycan, or glycoconjugate, or a combination thereof.

[0244] Covalent conjugation of carbohydrates to modified carrier proteins or modified nanoparticles enhances the immunogenicity of carbohydrates by converting them from T-cell-independent antigens to T-cell-dependent antigens, thereby allowing the initiation of immune memory. Conjugation has been useful for pediatric and adult vaccines (see Ramsay et al. (2001); Lindberg (1999); Buttery & Moxon (2000); Ahmad & Chapnick (1999); Goldblatt (1998); European Patent 0477508; US Patent No. 5,306,492; WO98 / 42721; Dick et al., Conjugate Vaccines (1989); Hermanson, (1996)).

[0245] One alternative conjugation method involves using the –NH2 group in the sugar (from de-N-acetylation or after the introduction of an amino group) to bind a bifunctional linker, as described in (WO2006 / 082530). Another alternative method is found in WO96 / 40795 and Michon et al. (2006). In this method, conjugation is performed by reductive amination using the free aldehyde group of the terminal 2,5-dehydrated-D-mannose residue, resulting from the mild cleavage depolymerization of type II or III capsular sugars via de-N-acetylation / nitrosation.

[0246] A conjugate (e.g., a bioconjugate) is also provided, comprising or consisting of modified nanoparticles or a modified carrier protein fused to the nanoparticles (e.g., modified EPA fused to a ferritin subunit), the nanoparticles being linked to an antigen (e.g., a glycoantigen, optionally a bacterial polysaccharide antigen). The antigen may be a bacterial polysaccharide antigen, or a yeast polysaccharide antigen, or a mammalian polysaccharide antigen.

[0247] In one embodiment, a conjugate (e.g., a bioconjugate) is provided comprising, or consisting of, a modified nanoparticle covalently linked to, an antigen (e.g., a glycoantigen, optionally a bacterial polysaccharide antigen), or a modified carrier protein fused to the nanoparticle, wherein the antigen is linked (directly or via a linker) to the modified carrier protein or the modified nanoparticle. In one embodiment, the antigen is directly linked to the modified nanoparticle or the modified carrier protein fused to the nanoparticle. In another embodiment, the antigen is directly linked to amino acid residues of the modified nanoparticle or the modified carrier protein fused to the nanoparticle.

[0248] Display molecules (e.g., antigens and / or immunostimulants) can be conjugated to nanoparticles (e.g., protein nanoparticles) or carrier proteins fused to nanoparticles by any suitable means, including spontaneous isopeptide bond formation, chemical conjugation, gene fusion, biological conjugation, or bioorthogonal chemistry using non-natural amino acids. Those skilled in the art can choose from a variety of known conjugation techniques suitable for specific nanoparticles and specific display molecules, including those described and / or demonstrated in the following literature (in no particular order): Aubin-Tam, “ Conjugation of Nanoparticles to Proteins "Nanomaterial Interfaces in Biology: Methods and Protocols – Methods in Molecule Biology 2013 (Bergese and Hamad-Schifferli, eds.); Bruun et al., 2018 ACS Nano 12(9):8855-8866; Ma et al., 2018 Nat. Comm. 9: 1489 (DOI: 10.1038 / s41467-018-03931-4); Micoli et al., 2011 Vaccine 29(4): 712-720; Scaria et al., 2017 PloS ONE 12(12):e0190312; JM Carter, " Conjugation of Peptides to Carrier Proteins via GlutaraldenhydeThe Protein Protocols Handbook 1997 (Ed. JM Walker); King et al., 1978 Biochemistry 17(8): 1499-1506; Kuan et al., 2016 Chem. Eur. J. 22: 17112-17129; Farkas and Bystricky 2010 Chemical Papers 64(6): 683-695; Lauster et al., 2020 Nat. Nanotech. 15: 373-379.

[0249] Examples of conjugating antigen molecules to nanoparticles can be found in the literature (e.g., severe acute respiratory syndrome (SARS) (Zhang et al., 2020 bioRxiv (doi: 10.1101 / 2020.06.11.147496)), influenza, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), respiratory syncytial virus (RSV), and human cytomegalovirus (HCMV) (see, Perotti and Perez 2020 Viruses 12(35): doi:10.3390 / v12010035 (total 17 pages)).

[0250] Methods for producing glycoconjugates typically involve the reductive amination of purified sugars with a carrier protein (such as tetanus toxoid (TT) or CRM197) (Wessels et al. (1990)). Reductive amination involves the amino group on the side chain of the amino acid in the carrier protein and the aldehyde group in the sugar. Since some capsular sugars do not contain an aldehyde group in their natural form, the aldehyde group is usually generated prior to conjugation by oxidizing (e.g., periodate oxidation) a portion (e.g., between 5% and 40%) of the sialic acid residues of the sugar [Wessels et al. (1990); U.S. Patent No. 4,356,170].

[0251] In the literature, previously obtained biosynthetic conjugates (NPs) include: Pan, C.; Wu, J.; Qing, S.; Zhang, X.; Zhang, LL; Yue, H.; Zeng, M.; Wang, B.; Yuan, Z.; Qiu, YF et al., Biosynthesis of self-assembled proteinaceous nanoparticles for vaccination. Adv. Mater. 2020, 32, 2002940; and Peng, ZH; Wu, J.; Wang, KF; Li, X.; Sun, P.; Zhang, LL; Huang, J.; Liu, Y.; Hua, XT; Yu, YS et al., Production of a promising biosynthetic self-assembled nanoconjugate vaccine against Klebsiella pneumoniae serotype O2 in a general Escherichia coli host. Adv. Sci., forthcoming, DOI: 10.1002 / advs.202100549.

[0252] Chemical conjugation In one embodiment, the modified nanoparticles or the modified carrier protein fused to the nanoparticles can be covalently linked to the antigen via chemical bonds obtained using a chemical conjugation method (i.e., the conjugate is generated via chemical conjugation). Chemical conjugation methods can be selected from the group consisting of: carbodiimide chemistry, reductive amination, cyanidation chemistry (e.g., CDAP chemistry), maleimide chemistry, hydrazide chemistry, ester chemistry, and N-hydroxysuccinimide chemistry. Conjugates can be prepared by direct reductive amination methods, as described in US200710184072 (Hausdorff), US 4365170 (Jennings), and US 4673574 (Anderson). Other methods are described in EP-0-161-188, EP-208375, and EP-0-477508. Alternatively, the conjugation method may rely on activating sugars with 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) to form cyanate esters. Such conjugates are described in PCT public applications WO93 / 15760 Uniformed Services University and WO 95 / 08348 and WO 96 / 29094. See also Chu C. et al., Infect. Immunity, 1983 245 256.

[0253] Functional groups of one or more amino acids on a polypeptide monomer (modified nanoparticle monomer or a modified carrier protein fused to a nanoparticle monomer) can be used for site-specific conjugation to display molecules (e.g., antigens or immunostimulants). Amino acid side chain groups used for conjugation include an amino group on lysine, a thiol group on cysteine, a carboxyl group on aspartic acid and glutamic acid, and a hydroxyl group on tyrosine. Heteromorphic bifunctional crosslinking agents can be used for protein conjugation. The primary amine on the first polypeptide can be conjugated to a carboxylic acid on the second polypeptide using a 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide (EDC) crosslinking agent, typically in combination with N-hydroxysuccinimide (NHS). The side chain amino group of the lysine residue is a nucleophilic group, thus lysine residues exposed on the outer surface of nanoparticles have high solvent accessibility and can be used as sites for conjugation to display molecules. Chemical methods also include site-specific chemical conjugation via engineered cysteine ​​or selenocysteine ​​(see Siegmund et al., 2016 Scientific Reports 6(39291)). Typically, the following types of chemical groups on modified carrier proteins or nanoparticle subunits can be used for conjugation / linking: A) Carboxyl group (e.g., via aspartic acid or glutamic acid). In one embodiment, this group is directly attached to an amino group on a sugar or to an amino group at a linker via carbodiimide chemistry (e.g., EDAC); B) Amino group (e.g., via lysine). In one embodiment, the group is directly attached to a carboxyl group on a sugar or to a carboxyl group on a linker via carbodiimide chemistry (e.g., EDAC). In another embodiment, the group is directly attached to a hydroxyl group on a sugar activated with CDAP or CNBr or to a group on such a linker; to a sugar or linker having an aldehyde group; or to a sugar or linker having a succinimide ester group. C) A thiol group (e.g., via cysteine). In one embodiment, this group is linked to a brominated or chloroacetylated sugar or a linker via maleimide chemistry. In one embodiment, this group is activated / modified with diazinon. D) Hydroxyl group (e.g., via tyrosine). In one embodiment, this group is activated / modified with diazinon; E) Imidazole group (e.g., via histidine). In one embodiment, this group is activated / modified with bis(azinobenzene); F) Guanidin (e.g., via arginine); G) Indole group (e.g., via tryptophan).

[0254] On carbohydrates, the following groups are commonly used for conjugation: OH, COOH, or NH2. Aldehyde groups can be generated after various treatments, such as periodate oxidation, acid hydrolysis, and hydrogen peroxide.

[0255] Biological conjugation In a preferred embodiment, one or more nucleotide constructs are prepared for recombinant expression comprising a monomeric amino acid sequence (e.g., a modified nanoparticle subunit) and a polypeptide sequence (e.g., a continuous amino acid sequence) representing a display molecule (e.g., a display of an antigen or an immunostimulatory glycoside). When expressed as a modified monomeric subunit, the modified nanoparticles self-assemble into nanoparticles (e.g., as shown in Pan et al. 2020 Adv. Mater. 32:2002940). When expressed as a modified carrier protein fusion construct (i.e., a modified carrier protein fused to a nanoparticle subunit), the modified carrier protein fusion construct self-assembles into nanoparticles. In both cases, either the modified monomeric subunit or the modified carrier protein fusion construct self-assembles into nanoparticles.

[0256] Through spontaneous isopeptide conjugation The use of peptide “tags” and binding chaperones (or “captures” or “anchors”) to spontaneously form isopeptide bonds between heterologous molecules (“tag-capture systems”) is known (see, for example, Zakeri et al., 2012 PNAS 109(12): E690-E697 of “SpyTag-SpyCatcher” systems; WO2011 / 098772 (PCT / GB2011 / 000188); M. HOWARTH and Haterm et al., 2019 Int. J. Mol. Sci. 20(9): 2129, 19 pages; Ma et al., 2018 Nat. Comm. 9: 1489 (DOI: 10.1038 / s41467-018-03931-4); Zhang et al., 2020 bioRxiv (doi: 10.1101 / 2020.06.11.147496); US 63 / 038237 related literature, involving the “GalacTag-GalacDock” system; Veggiani et al., 2016 PNAS 113(5): 1202-1207, involving the “SnoopTag-SnoopCatcher” system and its combination).

[0257] Therefore, one embodiment of the invention is a nanoparticle covalently linked to a carrier (e.g., on its outer surface) and undergoing bioconjugation. The nanoparticle and carrier can be conjugated using a tag-capture system (e.g., the SpyTag-SpyCatcher binding system (SPYBiotech, Oxford, England); see Hatlem et al., 2019 Int.J. Mol. Sci. 20(9): 2129, 19 pages; Zhang et al. 2020 bioRxiv (doi: 10.1101 / 2020.06.11.147496)). Thus, one aspect is a nanoparticle conjugated to a tag molecule of a tag-capture system (optionally, the nanoparticle monomer (e.g., a polypeptide subunit monomer) is conjugated to a tag molecule of the tag-capture system). In some aspects, the nanoparticle is linked to a tag molecule, which in turn is linked to a bioconjugated carrier molecule connected to a carrier molecule. Alternatively, one aspect is a nanoparticle conjugated to a capture molecule of a tag-capture system (optionally wherein a monomer (such as a peptide monomer) is conjugated to a capture molecule of the tag-capture system). In some aspects, the nanoparticle is linked to a capture molecule, which in turn is linked to a bioconjugated carrier attached to a tag molecule. When such a nanoparticle comes into contact with a heterologous molecule (e.g., an antigen / carrier or immunostimulant) that is itself conjugated to the corresponding capture molecule or tag molecule (i.e., the heterologous molecule contains another sequence of the binding pair), a heteropeptide bond is formed, and the heterologous molecule is covalently attached to the nanoparticle. See, for example, Brune et al. Frontiers in Immunology 9:1432 (2018); Reddington et al., Curr Opinion Chem Biol 29:94-99 (2015); Brune et al. Bioconjugate Chem 28(5):1544-51 (2017); Tan et al., PLOS One 11(10):e0165074(2016); WO2015 / 156870 (PCT / US2015 / 011534, DENG Z.); Bruun et al., 2018 ACS Nano 12(9):8855-8866.

[0258] In addition, various enzymatic and chemical enzymatic conjugation methods have been reported, including the use of engineered galactosyl and sialyl transferases, formylglycine synthase (FGE), phosphate pantothenic thioethylamine transferase (PPTase), sorting enzyme A, and microbial transglutaminase (an enzyme that forms an isopeptide bond between the glutamine side chain and the amine donor substrate) (see Siegmund et al., 2016 Scientific Reports 6(39291)).

[0259] Conjugates (e.g., chemical conjugates, biological conjugates, etc.) can be purified by any method known in the art for purifying proteins, such as chromatography (e.g., ion exchange, anion exchange, affinity chromatography, and size exclusion chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. See, for example, Saraswat et al. 2013, Biomed. Res. Int. ID0312709 (pp. 1-18); also see the methods described in WO 2009 / 104074. The actual conditions used to purify a particular conjugate will depend in part on the synthetic strategy (e.g., synthetic production vs. recombinant production) and factors such as the net charge, hydrophobicity, and / or hydrophilicity of the biological conjugate.

[0260] Analytical methods Various methods can be used to analyze the composition and glycan length of the bioconjugates of this embodiment and to determine the glycosylation sites.

[0261] Hydrazinolysis can be used to analyze glycans. First, following the manufacturer's instructions (Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK), the glycan is released from the protein carrier by incubation with hydrazine. The nucleophilic hydrazine attacks the glycosidic bond between the glycan and the nanoparticle subunit protein or carrier protein, thereby releasing the attached glycan. During this process, the N-acetyl group is lost and must be recovered by re-N-acetylation. The free glycan is purified on a carbon column and then labeled at the reduced end with the fluorophore 2-aminobenzamide (see Bigge et al., Nonselective and efficient fluorescent labeling of glycans using 2-aminobenzamide and anthranilic acid. Anal Biochem, 1995, 230(2):229-238). The labeled polysaccharides were separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al. (see, Royle et al., Ananalytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins, Anal Biochem, 2002, 304(1):70-90). The resulting fluorescence chromatograms showed the length of the polysaccharides and the number of repeating units. Structural information could be obtained by collecting each peak and then performing MS / MS analysis. This confirmed the monosaccharide composition and sequence of the repeating units and determined the homogeneity of the polysaccharide composition. Alternatively, high-resolution intact mass spectrometry and size-exclusion HPLC could be used to measure the size of intact bioconjugates.

[0262] Yield can be expressed as the amount of carbohydrates produced by one liter of bacterial production culture grown in a bioreactor under controlled and optimized conditions. After purification of the bioconjugated nanoparticles, carbohydrate yield can be measured directly by anthrone assay or using an ELISA with carbohydrate-specific antiserum. Indirect measurements can also be made by calculating the theoretical carbohydrate content per gram of protein using protein mass (determined by the BCA, Lowry, or Bradford methods) and glycan length and structure. Furthermore, yield can be measured by drying the glycoprotein product in a volatile buffer and weighing it using a balance.

[0263] Various methods can be used to analyze the bioconjugated nanoparticles of this embodiment, including, for example, SDS-PAGE or capillary gel electrophoresis. Polymer length is defined by the number of repeating units in a linear assembly. This means that the typical ladder-like banding pattern is a result of the different numbers of repeating units that make up the glycan. Therefore, in SDS-PAGE (or other size-separated techniques), adjacent bands differ by only one repeating unit. These discrete differences are utilized when analyzing glycoproteins for glycan size: unglycosylated nanoparticle subunits and bioconjugated nanoparticle subunits 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 (n) on the bioconjugated nanoparticles are measured. 平均 For example, these parameters can be used to demonstrate batch-to-batch consistency or polysaccharide stability.

[0264] Glycosylation site utilization can be quantified, for example, by LC-MS / MS of glycopeptides: the conjugate is digested with a protease, the peptides are separated by a suitable chromatographic method (C18, hydrophilic interaction HPLC HILIC, GlycoSepN column, SE HPLC, AE HPLC), and the different peptides are identified using MS / MS. This method may or may not use prior chemical (Smith degradation) or enzymatic methods to shorten the glycan chain. Quantification of glycopeptide peaks using UV at 215 to 280 nm allows for relative determination of glycosylation site utilization. In another embodiment, site utilization can be quantified by size exclusion HPLC. Higher glycosylation site utilization is reflected in earlier elution times from the SE HPLC column. In yet another embodiment, site utilization can be quantified by determining the quantitative density of purified bioconjugates stained with Coomassie Brilliant Blue after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0265] purification As used herein, the term "purified" means the separation or isolation of a defined product (e.g., a bioconjugate of recombinant expressed nanoparticles (or a recombinant expressed carrier protein fused to nanoparticles)) from a composition containing other components (e.g., host cells or host cell culture medium). A composition that has been fractionated to remove unwanted components and retains its biological activity is considered purified. Purified bioconjugate nanoparticles (or purified carrier proteins fused to nanoparticles) retain their biological activity. "Purified" means removed from its natural environment and substantially free of impurities from that natural environment (such as other extrachromosomal and extrachromosomal DNA and RNA, organelles, and proteins, including other proteins, lipids, or polysaccharides also secreted into the culture medium or produced by host cell lysis). For clarity, and as will be fully understood by those skilled in the art, molecules (such as antigens or immunostimulants) within or suitable for pharmaceutical, immunogenic, vaccine, or adjuvant compositions herein are purified molecules (such as purified antigens or purified immunostimulants), whether or not the term "purified" is used. It is understood in the art that for a molecule (such as an antigen, reagent, immunostimulant, additive, carrier or other compound) or composition to be suitable (i.e., safe) for pharmaceutical or vaccine use (e.g., administration) in humans or non-human mammals (i.e., the molecule is pharmaceutically acceptable), it must be at least purified (i.e., not crude).

[0266] "Purified" is a relative term and does not require absolute (100%) purity for use in pharmaceutical or vaccine applications. The molecule may constitute at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% of the total protein content or mass of the composition (determined by assessing the number of peptides in the fractions, for example, by gel electrophoresis, such as by SDS / PAGE analysis). In other words, "purification" or "performing purification" refers to the process of removing unwanted components from the composition or host cells or cultures. Various methods for purifying peptides and nanoparticles are known in the art, including, for example, various types of chromatography such as high-performance liquid chromatography (HPLC), ion exchange chromatography and size exclusion chromatography, hydrophobic interactions, ion exchange, affinity, chelation, and size exclusion; electrophoresis such as gel electrophoresis; centrifugation such as density gradient centrifugation; dialysis; filtration; precipitation; antibody capture; solvent extraction; affinity purification; and combinations thereof. The peptide NP may be expressed with a tag suitable for affinity purification, such as the 6x histidine tag known in the art. His-tagged peptides can be purified using, for example, Ni-NTA column chromatography or anti-6xHis antibodies fused to a solid phase.

[0267] A "substantially pure" article of polypeptides (or nanoparticles, or carrier proteins fused to nanoparticles) or nucleic acid molecules means that the desired component comprises at least 50% of the total polypeptide (or nucleic acid) content of the article. In some embodiments, a substantially pure article will contain at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more of the total polypeptide (or nucleic acid) content of the article. Methods for quantifying the degree of purification of the expressed polypeptide are known in the art, including, for example, determining the specific activity of the active fraction, or assessing the number of polypeptides in the fraction by SDS / PAGE analysis. As used herein, the terms "purification" or "performing purification" refer to the process of removing unwanted components present in the composition or host cells or culture. Molecules that have not undergone any purification steps (i.e., naturally occurring molecules or "crude" molecules) are not suitable for pharmaceutical use (i.e., not suitable for administration to a subject).

[0268] A one-step method for preparing bioconjugated nanoparticles. The nanoparticle subunit peptides or carrier proteins fused with nanoparticle subunit peptides of this embodiment can be generated by any suitable means, including recombinant expression or chemical synthesis, and purified using any suitable method known in the art (if necessary). The nanoparticle products can be analyzed using methods known in the art, such as crystallography, dynamic light scattering (DLS), nano-differential scanning fluorescence (Nano-DSF), and electron microscopy, to confirm the generation of suitable nanoparticles.

[0269] Recombinant expression methods suitable for producing nanoparticle subunit peptides or carrier proteins fused with nanoparticle subunit peptides are known in the art. The expressed peptide may contain a purification tag and / or a protease site. Various expression systems are known in the art, including systems using human (e.g., HeLa) host cells, mammalian (e.g., Chinese hamster ovary (CHO)) host cells, prokaryotic host cells (e.g., *E. coli*), or insect host cells. Typically, host cells are transformed with a recombinant nucleic acid sequence encoding the desired peptide product, cultured under conditions suitable for product expression, and the product is purified from the cells or culture medium. Cell culture conditions vary depending on the cell type and expression vector, as is known in the art.

[0270] Host cells can be cultured in conventional nutrient media, appropriately modified as will be clear to those skilled in the art (e.g., for promoter activation). Culture conditions (e.g., temperature, pH, etc.) can be determined using knowledge in the art, see, for example, Freshney (1994) and its cited references. In bacterial host cell systems, a variety of expression vectors are available, including but not limited to multifunctional E. coli clones and expression vectors such as BLUESCRIPT (Stratagene) or pET vectors (Novagen, Madison WI). In mammalian host cell systems, a variety of expression systems, including plasmid-based and virus-based systems, are commercially available.

[0271] Eukaryotic or microbial host cells expressing nanoparticle subunit peptides or carrier proteins fused with nanoparticle subunit peptides can be disrupted by any convenient method (including freeze-thaw cycles, sonication, mechanical disruption), and the peptides and / or self-assembled nanoparticles or carrier proteins fused with nanoparticle subunit peptides can be recovered and purified from recombinant cell cultures by any suitable method known in the art (including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g., using any labeling system described herein), hydroxyapatite chromatography, and lectin chromatography). High-performance liquid chromatography (HPLC) can be used for the final purification step.

[0272] Typically, the expression of recombinantly encoded nanoparticle subunit peptides (or recombinantly encoded carrier proteins fused to nanoparticle subunits) using methods known in the art involves preparing an expression vector containing a recombinant polynucleotide under the control of one or more promoters, such that the promoters stimulate transcription of the polynucleotide and promote the expression of the encoded peptide. As used herein, "recombinant expression" refers to such methods.

[0273] A "recombinant expression vector" contains a recombinant nucleic acid sequence operatively linked to a control sequence that enables the expression of a gene product. The "control sequence" is a nucleic acid sequence that enables the expression of the nucleic acid molecule and does not need to be contiguous with the nucleic acid sequence, as long as it can direct its expression. "Recombinant host cells" contain such recombinant expression vectors.

[0274] Another embodiment is a method for generating nanoparticles containing bacterial polysaccharide or oligosaccharide antigens on the outer surface of nanoparticles. The method includes the following steps: (a) culturing recombinant host cells under the following conditions: (1) expressing or transporting modified nanoparticle subunit polypeptides, sugars, and glycosyltransferase pglB in the periplasm of the host cells, wherein the glycosyltransferase bioconjugates the optionally modified nanoparticles at glycoconjugate sites, and under conditions favorable to nanoparticle self-assembly; (b) recovering or purifying the assembled bioconjugated nanoparticles from the host cells or the culture medium in which the host cells are grown, as applicable.

[0275] Another embodiment is a method for generating nanoparticles containing bacterial polysaccharide or oligosaccharide antigens on the outer surface of a carrier protein fused with nanoparticles. The method includes the following steps: (a) culturing recombinant host cells under the following conditions: (1) expressing or transporting a modified carrier protein fused with a nanoparticle subunit polypeptide, sugars, and a glycosyltransferase pglB fused to the periplasm of the host cells, wherein the glycosyltransferase glycoconjugates the modified carrier protein at a glycoconjugation site, and under conditions favorable to nanoparticle self-assembly; (b) recovering or purifying the assembled, bioconjugated carrier protein fused with nanoparticles from the host cells or the culture medium in which the host cells are grown, as applicable.

[0276] In one aspect, a method for preparing assembled glycoprotein nanoparticles (NPs) is provided, comprising the steps of: providing a host cell; expressing a nanoparticle subunit or a modified carrier protein fused to a nanoparticle subunit in the periplasm of the host cell, wherein the optionally modified nanoparticle subunit or carrier protein contains one or more glycosylation sites (e.g., D / EXNZS / T SEQ ID NO: 49); expressing or transporting a glycosyltransferase pglB in the periplasm of the host cell; glycosylating the optionally modified nanoparticle subunit (or the modified carrier protein fused to the optionally modified nanoparticle subunit) with glycosyltransferase pglB in the periplasm using glycosyltransferase pglB; and generating assembled glycoprotein nanoparticles from the glycosylated nanoparticle subunit (or the glycosylated carrier protein fused to the nanoparticle subunit). In some aspects, the method includes expressing modified nanoparticle subunits such as ferritin, dodecin, E2p, etc.

[0277] Another embodiment is a method for generating nanoparticles containing bacterial polysaccharide or oligosaccharide antigens on the outer surface of nanoparticles. The method includes the following steps: (a) culturing recombinant host cells under the following conditions: (1) expressing modified nanoparticle subunit polypeptides and glycosyltransferase pglB in the host cells, wherein the glycosyltransferase bioconjugates sugars to the optionally modified nanoparticles at sugar conjugation sites, and under conditions favorable to nanoparticle self-assembly; (b) recovering or purifying the assembled bioconjugated nanoparticles from the periplasm of the host cells or the culture medium in which the host cells are grown, as applicable.

[0278] Another embodiment is a method for generating nanoparticles containing bacterial polysaccharide or oligosaccharide antigens on the outer surface of a carrier protein fused with nanoparticles. The method includes the following steps: (a) culturing recombinant host cells under the following conditions: (1) expressing a modified carrier protein fused with a nanoparticle subunit polypeptide and a glycosyltransferase pglB in the host cells, wherein the glycosyltransferase conjugates sugars to the modified carrier protein at sugar conjugation sites, and wherein the nanoparticle subunits are under conditions conducive to nanoparticle self-assembly; (b) recovering or purifying the assembled, bioconjugated carrier protein fused with nanoparticles from the periplasm of the host cells or the culture medium in which the host cells are grown, as applicable.

[0279] In one aspect, a method for preparing assembled glycoprotein nanoparticles (NPs) is also provided, comprising the steps of: providing host cells; expressing nanoparticle subunits or modified carrier proteins fused to nanoparticle subunits in the host cells, wherein the optionally modified nanoparticle subunits or carrier proteins contain one or more glycosylation sites (e.g., D / EXNZS / T SEQ ID NO: 49); expressing glycosyltransferase pglB in the host cells; glycosylating the optionally modified nanoparticle subunits (or modified carrier proteins fused to optionally modified nanoparticle subunits) with glycosyltransferase pglB using glycosyltransferase pglB; and generating assembled glycoprotein nanoparticles from the glycosylated nanoparticle subunits (or glycosylated carrier proteins fused to nanoparticle subunits) in the periplasm. In some aspects, the method includes expressing modified nanoparticle subunits, such as modified ferritin, modified dodecin, or modified E2p, etc.

[0280] Composition A further embodiment relates to an immunogenic composition or pharmaceutical composition, such as a vaccine, comprising nanoparticles displaying bacterial oligosaccharide or polysaccharide antigens (or a carrier protein fused to the nanoparticles), and a pharmaceutically acceptable diluent or excipient. In some cases, administration of an immunogenic composition to a subject is intended to elicit an immune response that protects the subject from infection by a pathogen or alleviates symptoms or conditions caused by the pathogen. Within the context of this disclosure, the term immunogenic composition will be understood to encompass compositions intended for administration to a subject or group of subjects to elicit a protective or alleviating immune response against said bacteria.

[0281] An "immunogenic composition" is a composition of substances suitable for administration to human or non-human mammalian subjects, and which, upon administration of an immunologically effective amount, elicits a specific immune response, for example, against an antigen displayed on nanoparticles or a carrier protein fused to nanoparticles. The immunogenic compositions of the present invention may contain one or more other components, such as excipients and / or adjuvants. Although administration of an antigen displayed on nanoparticles (or a carrier protein fused to nanoparticles) can enhance the subject's immune response to the antigen (compared to administration of the antigen in the absence of nanoparticles), nanoparticles used as a scaffold, as used herein, are not defined as adjuvants.

[0282] For example, a variety of pharmaceutically acceptable diluents and / or pharmaceutically acceptable excipients known in the art are described in Remington's Pharmaceutical Sciences, EW Martin, Mack Publishing Co., Easton, PA, 15th edition (1975). The adjective “pharmaceuticalally acceptable” means that the diluent or excipient is suitable for administration to a subject (e.g., a human or non-human mammalian subject). Generally, the nature of the diluent and / or excipient and / or carrier will depend on the specific administration method used. For example, parenteral preparations typically contain injectable fluids, including pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, dextran aqueous solutions, glycerol, etc., as carriers. In some preparations (e.g., solid compositions, such as powder forms), liquid diluents are not used. In such preparations, non-toxic solid ingredients may be used, including, for example, pharmaceutical-grade trehalose, mannitol, lactose, starch, or magnesium stearate. Suitable solid components are typically large, slowly metabolizing macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive viral particles.

[0283] Therefore, those skilled in the art can select suitable excipients to prepare formulations suitable for delivery to subjects via a selected route of administration.

[0284] In one embodiment, the immunogenic or pharmaceutical composition comprising nanoparticles does not further comprise an adjuvant. In another embodiment, the immunogenic or pharmaceutical composition comprising nanoparticles further comprises an adjuvant.

[0285] The preparation of immunogenic compositions, such as vaccines, including those intended for administration to human subjects, is generally described in Pharmaceutical Biotechnology, vol.61 Vaccine Design - the subunit and adjuvant approach, edited by Powell and Newman, Plenum Press, 1995 (see also New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, USA, 1978).

[0286] The bioconjugates of this embodiment are particularly suitable for inclusion in immunogenic compositions and vaccines.

[0287] This embodiment provides an immunogenic composition comprising nanoparticles and optionally pharmaceutically acceptable excipients and / or carriers.

[0288] The immunogenic composition comprises an immunologically effective amount of bioconjugated nanoparticles (or a bioconjugated carrier protein fused to the nanoparticles), and any other components. "Immunologically effective amount" means that such an amount, administered as a single dose or as part of a series, is effective for treatment or prevention in an individual. This amount varies depending on the individual's health and physical condition, age, desired level of protection, vaccine formulation, and other relevant factors.

[0289] 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 buffers (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., TWEEN 80). Pharmaceutically acceptable excipients may include preservatives, such as 2-phenoxyethanol or thimerosal. Pharmaceutically acceptable excipients may include carriers, such as water or saline solution.

[0290] Compositions containing nanoparticles may further comprise one or more pharmaceutically acceptable additives, such as buffers, carriers, excipients, tensiating agents, wetting agents or emulsifiers, detergents, antimicrobial agents, and diluents. Pharmaceutically acceptable additives are well known in the art (e.g., in Remington's Pharmaceutical Sciences, EWMartin, Mack Publishing Co., Easton, PA, 15th edition (1975)). Therefore, those skilled in the art can select suitable compositional components to prepare suitable formulations (e.g., suitable for pharmaceutical use).

[0291] Additives may include pharmaceutically acceptable diluents (e.g., sterile water), saline, glycerin, etc. Furthermore, pharmaceutically acceptable additives may include excipients such as wetting agents, emulsifiers, or pH buffers.

[0292] Additives may include pharmaceutically acceptable excipients. Such excipients include, but are not limited to: glycerol, polyethylene glycol (PEG), glass-forming polyols (such as sorbitol, trehalose), N-lauroyl sarcosine (e.g., sodium salt), L-proline, non-detergent sulfobetaine, guanidine hydrochloride, urea, trimethylamine oxide, KCl, Ca2+, Mg2+, Mn2+, Zn2+ (and other divalent cation-related salts), dithiothreitol (DTT), dithioerythritol, β-mercaptoethanol, detergents (including, for example, Tween 80, Tween 20, Triton X-100, NP-40, Empigen BB, octyl glucoside, lauroyl maltodextrin, Zwittergent 3-08, Zwittergent 3-10, Zwittergent 3-12, Zwittergent 3-14, Zwittergent... 3-16, CHAPS, sodium deoxycholate, sodium dodecyl sulfate, and hexadecyltrimethylammonium bromide.

[0293] Pharmaceutically acceptable additives used in this article may be detergents, such as TWEEN (polysorbate), like TWEEN 80. Detergents are typically present in low doses, for example, <0.01%.

[0294] The composition containing protein nanoparticles has a pH between 6 and 8, for example, between 6.5 and 7.5 (e.g., about 7). A stable pH can be maintained by using a buffer solution (e.g., acetate buffer, citrate buffer, histidine buffer, maleate buffer, phosphate buffer, succinate buffer, tartrate buffer, or Tris buffer). Therefore, the composition will typically contain a buffer solution. The composition can be sterile and / or pyrogen-free. The composition can be isotonic relative to humans.

[0295] A method for preparing an immunogenic composition is also provided, comprising the step of mixing nanoparticles with a pharmaceutically acceptable excipient and / or carrier.

[0296] An immunogenic composition (e.g., a vaccine composition) is also provided, which optionally contains an adjuvant.

[0297] The term "adjuvant" refers to a compound that, when administered in combination with or as part of the immunogenic composition or vaccine of this embodiment, enhances, improves, and / or promotes an immune response to nanoparticles, but does not produce an immune response to nanoparticles when the compound is administered alone. Adjuvants can enhance immune responses through several mechanisms, including, for example, lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. Specific examples of adjuvants include, but are not limited to, aluminum salts (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3-de-O-acylated monophospholipid A (MPL) (see British Patent GB2220211), MF59 (Novartis), AS01 (GlaxoSmithKline), AS03 (GlaxoSmithKline), and saponins, such as QS21 (see, Kensil et al., Vaccine Design: The Subunit and Adjuvant Approach (edited by Powell & Newman, Plenum Press, NY, 1995); US Patent No. 5,057,540). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil-in-water emulsions (such as squalene or peanut oil), optionally in combination with an immunostimulant (such as monophospholipid A) (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)).

[0298] The immunogenic compositions of the present invention may additionally contain one or more adjuvants. An "adjuvant" is an agent that enhances the production of an immune response in a non-specific manner. Common adjuvants include mineral suspensions (e.g., aluminum salts, aluminum hydroxide, aluminum phosphate); saponins, such as QS21; emulsions, including water-in-oil and oil-in-water (and their variants, including double emulsions and reversible emulsions), lipoglycosides, lipopolysaccharides, immunostimulatory nucleic acid molecules (such as CpG oligonucleotides), liposomes, Toll receptor agonists, Toll-like receptor agonists (particularly TLR2, TLR4, TLR7 / 8, and TLR9 agonists), and various combinations of such components. For the purposes of this embodiment, assembled nanoparticles are not considered adjuvants.

[0299] A method for preparing an immunogenic composition is also provided, comprising the steps of mixing nanoparticles (NPs) or carrier proteins fused with nanoparticles (NPs) with a pharmaceutically acceptable excipient and / or carrier and adjuvant. Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (edited by Powell MF & Newman MJ) (1995) Plenum Press New York).

[0300] The immunogenic composition of this embodiment may be included in a container, package, or dispenser along with the instructions for use.

[0301] The immunogenic composition or vaccine thereof may be stored prior to use, for example, the composition may be frozen (e.g., at about -20°C or about -70°C); stored under refrigerated conditions (e.g., at about 4°C); or stored at room temperature. The immunogenic composition or vaccine of this embodiment may be stored in solution or lyophilized. In one embodiment, the solution is lyophilized in the presence of sugars (such as sucrose, trehalose, or lactose). In another embodiment, the vaccine of this embodiment is lyophilized and temporarily reconstituted prior to use.

[0302] Application and dosage The immunogenic composition or vaccine of this embodiment can be administered via systemic or mucosal routes for the protection or treatment of subjects (e.g., mammals). These administrations may include intramuscular (IM), intraperitoneal, intradermal (ID), or subcutaneous (SC) injections; or administration via mucosal routes through the oral / gastrointestinal tract, respiratory tract, or genitourinary tract.

[0303] In one aspect, the immunogenic composition or vaccine of this embodiment is administered via intramuscular delivery. Intramuscular administration may be in the thigh or upper arm. Injection is typically performed with a needle (e.g., a hypodermic needle), but needleless injection may also be used. A typical intramuscular dose is about 0.5 mL.

[0304] In another aspect, the immunogenic composition or vaccine of this embodiment is administered intradermally. Human skin comprises an outer “horny” epidermis called the stratum corneum, which covers the epidermis. Below the epidermis is a layer called the dermis, which in turn covers the subcutaneous tissue. The conventional intradermal injection technique, the “mantoux procedure,” involves cleaning the skin, then stretching the skin with one hand and inserting a narrow-gauge needle (26 to 31 gauge) at a 10 to 15-degree angle with the bevel facing upwards. Once the bevel is inserted, the syringe is lowered and advanced further while applying slight pressure to elevate it above the skin. The liquid is then injected very slowly, creating a blister or bulge on the skin surface, followed by slow withdrawal of the needle.

[0305] In another aspect, the immunogenic composition or vaccine of this embodiment is administered intranasally. Typically, the immunogenic composition or vaccine is applied topically to the nasopharyngeal region, for example, without being inhaled into the lungs. It is desirable to use an intranasal delivery device that delivers the immunogenic composition or vaccine formulation to the nasopharyngeal region without entering or substantially not entering the lungs. A suitable device for intranasal vaccine administration according to this embodiment is a spray device. Suitable commercially available nasal spray devices include ACCUSPRAY™ (Becton Dickinson).

[0306] The amount of bioconjugate in each immunogenic composition or vaccine dose is selected to induce an immune protective response in typical vaccinated individuals without producing significant adverse side effects. This amount will vary depending on the specific immunogen used and its delivery method. The content of the bioconjugate will typically be in the range of 1–100 µg (suitably 5–50 µg).

[0307] Preventive and therapeutic uses The present invention also provides the immunogenic composition of this embodiment or the vaccine of this embodiment for use in medicine.

[0308] A method for inducing an immune response in a subject (e.g., a human) is provided, the method comprising administering to a subject (e.g., a human) a therapeutically or prophylactically effective amount of a bioconjugated nanoparticle (optionally fused with a carrier protein), an immunogenic composition thereof, or a vaccine thereof. A bioconjugated nanoparticle, an immunogenic composition thereof, or a vaccine thereof is also provided for inducing an immune response in a subject (e.g., a human). Bioconjugated nanoparticles (optionally fused with a carrier protein), an immunogenic composition thereof, or a vaccine thereof are also provided for preparing a medicament for inducing an immune response in a subject (e.g., a human).

[0309] A further aspect is a method for inducing an immune response for the purpose of treating and / or preventing bacterial infection in a subject, comprising administering to the subject an immunologically effective amount of a bacterial antigen molecule displayed on the surface of a nanoparticle or a carrier molecule fused to a nanoparticle displaying an antigen molecule, which is required for an immune response, wherein said antigen is capable of inducing a protective or therapeutic immune response. Such bacterial antigen molecules displayed on the surface of a nanoparticle or a carrier molecule fused to a nanoparticle may be in an immunogenic or pharmaceutical composition as described herein. In one embodiment of this invention, a single dose is administered to the subject. This dose may be adjuvant-free, or it may further contain an adjuvant.

[0310] This article also provides a method for inducing an immune response against bacteria in a subject, comprising administering to the subject the bioconjugated nanoparticles of this embodiment, an immunogenic composition thereof, or a vaccine thereof. The conjugated (e.g., bioconjugated) nanoparticles of this embodiment are immunogenic compositions or vaccines that can be used to induce an immune response against bacteria, such as Shigella spp. Shigella ) species, Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), Neisseria meningitidis, Haemophilus influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae ( Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus In one embodiment, the bioconjugated nanoparticles of this embodiment are immunogenic compositions or vaccines that can be used to induce an immune response against bacteria, such as Streptococcus spp. Streptococcus ) species, Shigella genus ( Shigella ) species, Pseudomonas genus ( Pseudomonas ) species, Klebsiella genus ( Klebsiella ) species or genus Staphylococcus ( Staphylococcus ) species. (For example, Shigella dysenteriae ( Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei (Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae Streptococcus pneumoniae () Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus In one embodiment, the subject has a bacterial infection at the time of administration. In another embodiment, the subject does not have a bacterial infection at the time of administration.

[0311] A method for treating and / or preventing yeast or bacterial infections in a subject is also provided, comprising administering bioconjugated nanoparticles to the subject. The bioconjugated nanoparticles may be in the form of an immunogenic composition or a vaccine. Therefore, a method for treating and / or preventing yeast or bacterial infections in a subject (e.g., a human) is provided, the method comprising administering a therapeutically or preventively effective amount of bioconjugated nanoparticles, their immunogenic composition, or a vaccine thereof to a subject (e.g., a human) in need of this treatment or prevention. A bioconjugated nanoparticle, its immunogenic composition, or a vaccine thereof is provided for the treatment and / or prevention of yeast or bacterial infections in a subject (e.g., a human). A bioconjugated nanoparticle, its immunogenic composition, or a vaccine thereof is also provided for the preparation of a medicament for the treatment and / or prevention of yeast or bacterial infections in a subject (e.g., a human).

[0312] In one particular embodiment, the immunogenic compositions or vaccines provided herein are used to prevent a subject from contracting a bacterial infection. Bacterial infections that can be treated and / or prevented using the bioconjugated nanoparticles provided herein include those caused by Neisseria meningitidis, Haemophilus influenzae type b (Hib), and Streptococcus spp. Streptococcus ) species, Shigella genus ( Shigella ) species, Pseudomonas genus ( Pseudomonas ) species, Klebsiella genus ( Klebsiella ) species or genus Staphylococcus ( Staphylococcus Those caused by certain species. (For example, Shigella dysenteriae) Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae Streptococcus pneumoniae () Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus )).

[0313] A further aspect is a method for inducing an immune response in mammalian subjects (such as human subjects), wherein the immune response is specific to bacterial antigen molecules displayed on the surface of nanoparticles provided herein or carrier molecules fused to nanoparticles. The method comprises administering to the subject an immunologically effective amount of bacterial antigen molecules displayed on the surface of nanoparticles or carrier proteins fused to nanoparticles, which is required for the immune response. The subject may have a bacterial infection at the time of administration, or the administration may be prophylactic for subjects without a bacterial infection at the time of administration. In one embodiment, the applied nanoparticles display antigens from at least two pathogenic serotypes. This can be achieved by administering a mixture of nanoparticles, wherein each nanoparticle displays a single serotype antigen, or by administering nanoparticles displaying multiple serotype antigens. The antigen may be a capsular polysaccharide or an immunogenic fragment thereof, an oligosaccharide, a glycoconjugate, or a mixture thereof.

[0314] This article also provides a method for inducing opsonophagocytic antibodies against bacteria in subjects, comprising administering to subjects conjugate (e.g., bioconjugate) nanoparticles, their immunogenic compositions, or vaccines thereof. Bioconjugate nanoparticles are immunogenic compositions or vaccines capable of inducing opsonophagocytic antibodies against bacteria in subjects, such as those against Shigella spp. Shigella ) species, Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ), Neisseria meningitidis Haemophilus influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae ( Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus In one embodiment, the bioconjugate nanoparticles of the immunogenic composition or vaccine can be used to induce the production of opsonization antibodies against bacteria, such as Streptococcus spp., in a subject. Streptococcus ) species, Shigella genus ( Shigella ) species, Pseudomonas genus ( Pseudomonas ) species, Klebsiella genus ( Klebsiella ) species or genus Staphylococcus ( Staphylococcus ) species. (For example, Shigella dysenteriae ( Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae Streptococcus pneumoniae () Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus )).

[0315] Another embodiment is a method of immunizing a human subject to reduce disease risk, wherein the subject receives a primary immunizing dose and a booster dose of the composition, and wherein both the primary and booster doses can elicit IgG antibodies specific against the same pathogenic serotype in the subject. In one embodiment, the booster dose is administered more than 14 days, 21 days, 30 days, 2 months, 3 months, 6 months, 1 year, or longer after the primary immunizing dose. The primary immunizing and / or booster doses may be adjuvant-free, or one or both may further contain an adjuvant.

[0316] In one embodiment, bioconjugated nanoparticles (optionally including a carrier protein) and compositions thereof are used in a method of immunizing a subject to achieve a protective (preventive) immune response in the subject and the fetus born to the subject (through placental transfer of maternal antibodies).

[0317] The immunogenic compositions provided herein can be administered parenterally as usual, for example, via subcutaneous, intraperitoneal, percutaneous, or intramuscular injection. Dosage treatment can be a single-dose regimen or a multiple-dose regimen.

[0318] The various features mentioned in the individual chapters above may be applied to other chapters where appropriate. Therefore, a feature explicitly described in one chapter may be combined with features explicitly described in other chapters where suitable. Those skilled in the art will recognize or be able to determine many equivalents to the specific embodiments described herein (or aspects of this disclosure) using no more than conventional experiments.

[0319] The implementation method is further described in the following numbered paragraphs:

[0320] 1. A method for preparing glycoprotein nanoparticles (NPs), comprising the following steps: Provide host cells; Modified nanoparticle subunits are expressed in the host cells; Glycosyltransferases are expressed in the host cells; The modified nanoparticles are subunit glycosylated by the glycosyltransferase in the host cells; and Glycoprotein nanoparticles are assembled from glycosylated modified nanoparticle subunits in the periplasm of the host cell.

[0321] 2. The method according to paragraph 1 further comprises one or more of the following: (i) transporting the modified nanoparticle subunit to or expressing therein the periplasm of the host cell; (ii) transporting the glycosyltransferase to or expressing therein the periplasm of the host cell; (iii) glycosylating the modified nanoparticle subunit in the periplasm of the host cell by means of the glycosyltransferase.

[0322] 3. The method according to paragraph 1 or paragraph 2, wherein the modified nanoparticle subunit contains one or more glycosylation sites (e.g., SEQ ID NO: 49; D / EXNZS / T).

[0323] 4. The method according to any of the preceding paragraphs, wherein the glycosyltransferase is glycosyltransferase pglB (optionally derived from Campylobacter jejuni). Campylobacter jejuni )).

[0324] 5. The method according to any of the preceding paragraphs further comprises glycosylation of the modified nanoparticle subunits by means of the glycosyltransferase pglB.

[0325] 6. The method according to any of the preceding paragraphs, wherein the host cell is a Gram-negative bacterial host cell.

[0326] 7. The method described in any of the preceding paragraphs, wherein the host cell is Escherichia coli.

[0327] 8. The method according to any of the preceding paragraphs, wherein the modified nanoparticle monomer subunit is selected from the following: E2p monomer subunit, ferritin monomer subunit, and dodecin monomer subunit.

[0328] 9. The method according to any of the preceding paragraphs, wherein a capsular polysaccharide or oligosaccharide is conjugated to the modified nanoparticle monomer subunit at one or more glycosylation sites.

[0329] 10. The method according to any of the preceding paragraphs, wherein the capsular polysaccharide or oligosaccharide is selected from the following: Escherichia spp. ( Escherichia ) species, Shigella genus ( Shigella ) species, Klebsiella genus ( Klebsiella ) species, Salmonella genus ( Salmonella ) species, Yersinia genus ( Yersinia ) species, Helicobacter genus ( Helicobacter ) species, Proteus genus ( Proteus ) species, Pseudomonas genus ( Pseudomonas ) species, Corynebacterium genus ( Corynebacterium ) species, Streptomyces genus ( Streptomyces ) species, Streptococcus genus (Streptococcus ) species, Enterococcus genus ( Enterococcus ) species, Staphylococcus genus ( Staphylococcus ) species, Bacillus genus ( Bacillus ) species, Clostridium ( Clostridium ) species, Listeria genus ( Listeria ) species, Campylobacter genus ( Campylobacter ) species, genus Neisseria meningitidis ( Meningococcal Capsular polysaccharides and Candida spp. Candida Polysaccharides.

[0330] 11. The method according to paragraph 10, wherein the capsular polysaccharide or oligosaccharide is selected from: Streptococcus spp. ( Streptococcus ) species or Klebsiella genus ( Klebsiella ) species.

[0331] 12. A method for preparing glycoprotein nanoparticles (NPs), comprising the following steps: Provide host cells; Nanoparticle subunits fused with a modified carrier protein expressed in the host cells; Glycosyltransferases (e.g., pglB) are expressed in the host cells. The modified carrier protein is glycosylated in the host cell by the glycosyltransferase (e.g., pglB); and Glycoprotein nanoparticles were prepared and assembled from multiple fused nanoparticle subunits in the periplasm of the host cell.

[0332] 13. The method according to paragraph 12 further comprises one or more of the following: (i) transporting a nanoparticle subunit fused with the modified carrier protein to or expressing therein the periplasm of the host cell; (ii) transporting a glycosyltransferase (e.g., pglB) to or expressing therein the periplasm of the host cell; (iii) glycosylating the modified carrier protein in the periplasm of the host cell by means of the glycosyltransferase (e.g., pglB).

[0333] 14. The method according to paragraph 12 or paragraph 13, wherein the modified carrier protein comprises one or more glycosylation sites (e.g., SEQ ID NO:49; D / EXNZS / T).

[0334] 15. The method according to any one of paragraphs 12 to 14, wherein the glycosyltransferase is glycosyltransferase pglB (optionally derived from Campylobacter jejuni). Campylobacter jejuni )).

[0335] 16. The method according to any one of paragraphs 12 to 15, further comprising optionally glycosylating the modified nanoparticle subunits in the periplasm by means of the glycosyltransferase pglB.

[0336] 17. The method according to any one of paragraphs 12 to 16, wherein a capsular polysaccharide or oligosaccharide is conjugated to the modified carrier protein.

[0337] 18. The method according to any one of paragraphs 12 to 17, wherein the capsular polysaccharide or oligosaccharide is selected from the genus *Escherichia* (…). Escherichia ) species, Shigella genus ( Shigella ) species, Klebsiella genus ( Klebsiella ) species, Salmonella genus ( Salmonella ) species, Yersinia genus ( Yersinia ) species, Helicobacter genus ( Helicobacter ) species, Proteus genus ( Proteus ) species, Pseudomonas genus ( Pseudomonas ) species, Corynebacterium genus ( Corynebacterium ) species, Streptomyces genus ( Streptomyces ) species, Streptococcus genus ( Streptococcus ) species, Enterococcus genus ( Enterococcus ) species, Staphylococcus genus ( Staphylococcus ) species, Bacillus genus ( Bacillus ) species, Clostridium ( Clostridium ) species, Listeria genus ( Listeria ) species, Campylobacter genus ( Campylobacter ) species, genus Neisseria meningitidis ( Meningococcal Capsular polysaccharides and Candida spp. Candida Polysaccharides.

[0338] 19. The method according to paragraph 18, wherein the capsular polysaccharide or oligosaccharide is selected from: Streptococcus spp. ( Streptococcus ) species or Klebsiella genus ( Klebsiella ) species.

[0339] 20. The method according to any one of paragraphs 12 to 19, wherein the nanoparticle subunit is selected from the following: E2p monomer subunit, ferritin monomer subunit, and dodecin monomer subunit.

[0340] 21. The method according to any one of paragraphs 12 to 20, wherein the nanoparticle subunit is fused with a carrier protein.

[0341] 22. The method according to any one of paragraphs 12 to 21, wherein the carrier protein is selected from the following: diphtheria toxin cross-reactive substance (CRM), exoprotein A (EPA), diphtheria toxoid (DT), or tetanus toxoid (TT).

[0342] 23. A composition comprising dodecin nanoparticles (NP) (optionally modified), said dodecin NP comprising one or more dodecin subunits, each dodecin subunit comprising: Amino acids 2-70 of SEQ ID NO: 1 or their self-assembled fragments, or amino acid sequences that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 2-70 of SEQ ID NO: 1 or their self-assembled fragments; at least one glycosylated common sequence.

[0343] 24. The composition according to paragraph 23, wherein the dodecin subunit comprises one or more substitution mutations selected from positions G25, V50 and A53, wherein the position numbering is relative to SEQ ID NO: 1.

[0344] 25. The composition according to paragraph 22 or paragraph 23, wherein the dodecin subunit comprises one (or more) glycosylated concordant sequences of D / EXNZS / T (SEQ ID NO: 49), wherein X and Z are independently any amino acid other than proline, wherein the one (or more) concordant sequences are (a) added to the N-terminus, (b) added to the C-terminus, and / or (c) substituted for one or more amino acids independently selected from amino acid residues 49-55 of SEQ ID NO: 1 or at an equivalent position within the same amino acid sequence as at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 1.

[0345] 26. The composition according to any one of paragraphs 23 to 25, wherein the modified dodecin subunit further comprises one or more substitution mutations selected from G25N, V50T and A53T, wherein the position number is relative to SEQ ID NO: 1.

[0346] 27. The composition according to any one of paragraphs 23 to 26, wherein the dodecin subunit comprises each of the substitution mutations selected from G25N, V50T and A53T, wherein the position number is relative to SEQ ID NO: 1.

[0347] 28. The composition according to any one of paragraphs 23 to 27, wherein: (i) At the N-terminus, any one of amino acid residues 1-5 of SEQ ID NO: 1 (e.g., amino acid residues 1, 1-2, 1-3, 1-4 or 1-5 of SEQ ID NO: 1) is replaced by a glycosylation common sequence, or the glycosylation common sequence is added immediately adjacent to or upstream of amino acid residue 2 of SEQ ID NO: 1. (ii) At the C-terminus, any one of amino acid residues 60-70 of SEQ ID NO: 1 (e.g., amino acid residues 70, 69-70, 68-70, 67-70, 66-70 or 65-70 of SEQ ID NO: 1) is substituted by a glycosylation common sequence, or the glycosylation common sequence is added immediately adjacent to or downstream of amino acid residue 65 of SEQ ID NO: 1; (iii) The one (or more) glycosylation sequences replace one or more amino acids (e.g., amino acid residue D51 from SEQ ID NO: 1), or A combination of two or more of (iv), (i), (ii), and (iii).

[0348] 29. The composition according to any one of paragraphs 23 to 28, wherein the glycosylation concordant sequence is upstream of amino acid residue position 2 of SEQ IDNO: 1 (e.g., within 1-6 amino acids, or within 3 amino acids).

[0349] 30. The composition according to any one of paragraphs 23 to 29, wherein the dodecin subunit comprises the sequence of SEQ ID NO: 4.

[0350] 31. The composition according to any one of paragraphs 23 to 30, wherein the glycosylation concordant sequence is downstream of amino acid residue position 65 of SEQ ID NO: 1 (e.g., within 1-10 amino acids, within 5 amino acids, or within 3 amino acids).

[0351] 32. The composition according to any one of paragraphs 23 to 31, wherein the dodecin subunit comprises the sequence of amino acid residues 21-100 of SEQ ID NO: 5 or a self-assembled fragment thereof, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 21-100 of SEQ ID NO: 5 or a self-assembled fragment thereof.

[0352] 33. The composition according to any one of paragraphs 23 to 32, wherein the glycosylation concordant sequence is a substitution at amino acid residue position 51 of SEQ ID NO: 1.

[0353] 34. The composition according to any one of paragraphs 23 to 33, wherein the first glycosylation common sequence is upstream of amino acid residue position 2 of SEQ ID NO: 1 (e.g., within 1-6 amino acids, or within 3 amino acids) and the second glycosylation common sequence is downstream of amino acid residue position 65 of SEQ ID NO: 1 (e.g., within 1-10 amino acids, or within 3 amino acids).

[0354] 35. The composition according to any one of paragraphs 23 to 34, wherein the dodecin subunit comprises the sequence of amino acid residues 21-111 of SEQ ID NO: 7.

[0355] 36. The composition according to any one of paragraphs 23 to 35, wherein the first glycosylation concordant sequence is a substitution at amino acid residue position 51 of SEQ ID NO: 1 and the second glycosylation concordant sequence is downstream of amino acid residue position 65 of SEQ ID NO: 8 (e.g., within 1-10 amino acids, or within 3 amino acids).

[0356] 37. The composition according to any one of paragraphs 23 to 36, wherein the dodecin subunit comprises the sequence of amino acid residues 21-104 of SEQ ID NO: 8.

[0357] 38. The composition according to any one of paragraphs 23 to 37, wherein the first glycosylation concordant sequence is upstream of amino acid residue position 2 of SEQ ID NO: 1 (e.g., within 1-6 amino acids, or within 3 amino acids), the second glycosylation concordant sequence is a substitution at amino acid residue position 51 of SEQ ID NO: 1, and the third glycosylation concordant sequence is downstream of amino acid residue position 65 of SEQ ID NO: 1 (e.g., within 1-10 amino acids, or within 3 amino acids).

[0358] 39. The composition according to any one of paragraphs 23 to 38, wherein the dodecin subunit comprises the sequence of amino acid residues 21-115 of SEQ ID NO: 9.

[0359] 40. The composition according to any one of paragraphs 23 to 39, further comprising a signal sequence of any one of SEQ ID NO: 10, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45 or SEQ ID NO: 46.

[0360] 41. The composition according to any one of paragraphs 23 to 40, wherein the signal sequence is SEQ ID NO:10.

[0361] 42. The composition according to any one of paragraphs 23 to 41, wherein the dodecin subunit (optionally modified) comprises the amino acid sequence SEQ ID NO: 5.

[0362] 43. The composition according to any one of paragraphs 23 to 41, wherein the dodecin subunit (optionally modified) comprises the amino acid sequence SEQ ID NO: 6.

[0363] 44. The composition according to any one of paragraphs 23 to 41, wherein the dodecin subunit (optionally modified) comprises the amino acid sequence SEQ ID NO: 7.

[0364] 45. The composition according to any one of paragraphs 23 to 41, wherein the dodecin subunit (optionally modified) comprises the amino acid sequence SEQ ID NO: 8.

[0365] 46. ​​The composition according to any one of paragraphs 23 to 41, wherein the dodecin subunit (optionally modified) comprises the amino acid sequence SEQ ID NO: 9.

[0366] 47. The composition according to any one of paragraphs 23 to 46, wherein the dodecin subunit (optionally modified) is conjugated to a glycobiological sequence at one or more glycosylated concordant sequences, optionally a bacterial polysaccharide (e.g., from Shigella dysenteriae). Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae Streptococcus pneumoniae () Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus )).

[0367] 48. The composition according to any one of paragraphs 23 to 47, wherein the dodecin subunits self-assemble to form dodecin nanoparticles.

[0368] 49. The composition according to any one of paragraphs 23 to 48, which is produced by any one of paragraphs 1 to 22.

[0369] 50. The composition according to any one of claims 23 to 49, wherein the dodecin subunit is encoded by a nucleic acid comprising SEQ ID NO: 3.

[0370] 51. A composition comprising modified ferritin nanoparticles (NP), said modified ferritin nanoparticles comprising one or more modified ferritin subunits, each ferritin subunit comprising: (a) Amino acids 1-167 of SEQ ID NO: 11 or their self-assembled fragments, or amino acid sequences that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1-167 of SEQ ID NO: 11 or their self-assembled fragments; and (b) One or more glycosylation sequences, wherein the one or more glycosylation sequences are added to or replace one or more amino acids of the modified amino acid sequence immediately adjacent to the ferritin subunit.

[0371] 52. The composition according to paragraph 51, wherein: (i) The one (or more) glycosylated sequences have been added to or substituted at the N-terminus to amino acid residues 1-10 of SEQ ID NO: 11 immediately adjacent to (e.g., within about 1-15 amino acids) (e.g., amino acid residues 1-10, 1-5, 1-4, 1-3, 1-2, 1 of SEQ ID NO: 11). (ii) The one (or more) glycosylation sequences have been added to or substituted for one or more amino acids immediately adjacent to amino acid residues 74-84 of SEQ ID NO: 11 (e.g., amino acid residues 76-82 or 78-80 of SEQ ID NO: 11, or residue K79 of SEQ ID NO: 11); (iii) The one (or more) glycosylated sequences have been added to or substituted for one or more amino acids immediately adjacent to residues 141-154 of SEQ ID NO: 11 (e.g., amino acid residues 143-152 or 145-150 of SEQ ID NO: 11, amino acid residues 146-149 of SEQ ID NO: 11). (iv) Combinations of two or more of (i), (ii), and (iii); The modification may be at an equivalent position within the same amino acid sequence as SEQ ID NO: 11, which is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%.

[0372] 53. The composition according to paragraph 51 or paragraph 52, wherein the glycosylation concordant sequence is selected from GSGDQNATGSG (SEQ ID NO: 33), GGSKDQNRTKDGSG (SEQ ID NO: 34), DQNAT (SEQ ID NO: 32) and / or GDQNATG (SEQ ID NO: 35).

[0373] 54. The composition according to any one of paragraphs 51 to 53, further comprising the signal sequence SEQ ID NO:10.

[0374] 55. The composition according to any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence SEQ ID NO: 13.

[0375] 56. The composition according to any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence SEQ ID NO: 14.

[0376] 57. The composition according to any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence SEQ ID NO: 15.

[0377] 58. The composition according to any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence SEQ ID NO: 16.

[0378] 59. The composition according to any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence SEQ ID NO: 17.

[0379] 60. The composition according to any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence SEQ ID NO: 18.

[0380] 61. The composition according to any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence SEQ ID NO: 19.

[0381] 62. The composition according to any one of paragraphs 51 to 61, wherein the modified amino acid sequence of the ferritin subunit self-assembles to form ferritin nanoparticles.

[0382] 63. The composition according to any one of paragraphs 51 to 62, which is produced by the method according to any one of paragraphs 1 to 22.

[0383] 64. A composition comprising modified ferritin nanoparticles, said ferritin NP comprising one or more ferritin subunits, each ferritin nanoparticle subunit comprising: (a) Amino acids 1-153 of SEQ ID NO: 20 or their self-assembled fragments, or amino acid sequences that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1-153 of SEQ ID NO: 20 or their self-assembled fragments; and (b) One or more glycosylation sequences (e.g., GSGDQNATGSG (SEQ ID NO: 33), GGSKDQNATKDGSG (SEQ ID NO: 34)), wherein the one or more glycosylation sequences are added to or replace one or more amino acids immediately adjacent to the modified amino acid sequence.

[0384] 65. The composition according to paragraph 64, wherein the modified amino acid sequence comprises one or more mutations selected from the group consisting of M31I, K120L, A124R, M144I, and I154M, wherein the position number is relative to SEQ ID NO:20.

[0385] 66. The composition according to paragraph 64 or paragraph 65, wherein the modified amino acid sequence comprises each of the mutants M31I, K120L, A124R, M144I and I154M, wherein the position numbering is relative to SEQ ID NO:20.

[0386] 67. The composition according to any one of paragraphs 64 to 66, wherein: (i) The one (or more) glycosylated sequences have been added to or substituted at the N-terminus to amino acid residues 1-10 of SEQ ID NO: 20 immediately adjacent to (e.g., within about 10-25 amino acids) (e.g., amino acid residues 1-10, 1-5, 1-4, 1-3, 1-2, 1 of SEQ ID NO: 20). (ii) The one (or more) glycosylation sequences have been added to or substituted for one or more amino acids immediately adjacent to amino acid residues 62-64 of SEQ ID NO: 20 (e.g., amino acid residues 77-83 or 79-81 of SEQ ID NO: 20, or residue T80 of SEQ ID NO: 20); (iii) The one (or more) glycosylated sequences have been added to or substituted for one or more amino acids immediately adjacent to residues 140-150 of SEQ ID NO: 20 (e.g., amino acid residues 144-146 of SEQ ID NO: 20, or amino acid residue 145 of SEQ ID NO: 20), and A combination of two or more of (iv), (i), (ii), and (iii).

[0387] 68. The composition according to any one of paragraphs 64 to 67, further comprising the signal sequence SEQ ID NO:10.

[0388] 69. The composition according to paragraph 64, wherein the modified ferritin subunit comprises the amino acid sequence SEQ ID NO: 23.

[0389] 70. The composition according to paragraph 64, wherein the modified ferritin subunit comprises the amino acid sequence SEQ ID NO: 24.

[0390] 71. The composition according to paragraph 64, wherein the modified ferritin subunit comprises the amino acid sequence SEQ ID NO: 25.

[0391] 72. The composition according to any one of paragraphs 64 to 71, wherein the modified amino acid sequence self-assembles to form ferritin nanoparticles.

[0392] 73. The modified amino acid sequence according to any one of paragraphs 64 to 72, which is generated by any one of paragraphs 1 to 16.

[0393] 74. The composition according to any one of paragraphs 51 to 78, further comprising the signal sequence SEQ ID NO: 10, SEQ ID NO: 41, SEQ ID NO: 44 or SEQ ID NO: 47.

[0394] 75. A composition comprising a modified amino acid sequence of E2p nanoparticles, wherein the nanoparticles comprise E2p nanoparticle subunits comprising: (a) Amino acids 185-426 of SEQ ID NO: 26 or their self-assembled fragments, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or their self-assembled fragments; and (b) One or more glycosylation sequences GSGGGDQNATGSGGG (SEQ ID NO: 36), wherein the one or more glycosylation sequences are added to or replace one or more amino acids immediately adjacent to the modified amino acid sequence.

[0395] 76. The composition according to paragraph 75, wherein the modified amino acid sequence comprises one or more of the following mutations relative to SEQ ID NO: 26: A187T, F196Y, T281N, P314S, A352V, L425I, Δ427-428, or mutations at positions equivalent to A187T, F196Y, T281N, P314S, A352V, L425I, Δ427-428 of SEQ ID NO: 26.

[0396] 77. The composition according to paragraph 75 or paragraph 76, wherein the modified amino acid sequence comprises each of the following mutations relative to SEQ ID NO: 26: A187T, F196Y, T281N, P314S, A352V, L425I, Δ427-428, or mutations at positions equivalent to A187T, F196Y, T281N, P314S, A352V, L425I, Δ427-428 of SEQ ID NO: 26.

[0397] 78. The composition according to any one of paragraphs 75 to 77, wherein: The one (or more) glycosylated sequences have been added to or substituted at the N-terminus of amino acid residues 185-426 of SEQ ID NO: 26, either immediately adjacent to (e.g., within about 10-20 amino acids) of SEQ ID NO: 26, wherein the modification may be at an equivalent position within the same amino acid sequence as at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of SEQ ID NO: 26.

[0398] 79. The composition according to any one of paragraphs 75 to 78, further comprising the signal sequence SEQ ID NO: 10, SEQ ID NO: 41, SEQ ID NO: 44 or SEQ ID NO: 47.

[0399] 80. The composition according to any one of paragraphs 75 to 79, wherein the modified E2p subunit comprises the amino acid sequence SEQ ID NO: 29.

[0400] 81. The composition according to any one of paragraphs 75 to 80, wherein the modified amino acid sequence self-assembles to form nanoparticles.

[0401] 82. The composition according to any one of paragraphs 75 to 81, which is produced by the method according to any one of paragraphs 1 to 22.

[0402] 83. A composition comprising modified exotoxin protein A (EPA) ferritin nanoparticles, wherein each subunit comprises modified EPA fused directly or indirectly to ferritin.

[0403] 84. The composition according to paragraph 83 further comprises one or more sequences selected from: (i) One, two, or three glycosylation sequences within the modified EPA; (ii) The concordant glycosylation sequence at the N-terminus of EPA; (iii) Shared glycosylation sequences between EPA and ferritin; and (iv), (i), (ii), or one or more of (ii).

[0404] 85. The composition according to paragraph 83 or paragraph 84, wherein the modified EPA-ferritin subunit comprises amino acids 36-663 of SEQ ID NO: 52 or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 36-663 of SEQ ID NO: 52.

[0405] 86. The composition according to paragraph 85, wherein the modified EPA-ferritin subunit comprises the amino acid sequence SEQ ID NO: 52.

[0406] 87. The composition according to any one of paragraphs 83 to 86, wherein the modified amino acid sequence self-assembles to form nanoparticles.

[0407] 88. The composition according to any one of paragraphs 83 to 87, which is produced by any one of paragraphs 1 to 22.

[0408] 89. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a modified nanoparticle subunit as described in any of the preceding paragraphs, or said nanoparticle subunit fused with a modified carrier protein.

[0409] 90. A vector comprising isolated nucleic acid molecules as described in paragraph 89.

[0410] 91. A host cell comprising an isolated nucleic acid molecule as described in paragraph 89 or a vector as described in paragraph 90, wherein the host cell is an Escherichia coli cell (e.g., Escherichia coli K12 W3110).

[0411] 92. The host cell according to paragraph 91, comprising: i) One or more nucleotide sequences containing a polysaccharide synthesis gene, optionally used to produce bacterial polysaccharide antigens (e.g., O-antigens from Gram-negative bacteria, optionally from Shigella dysenteriae). Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ) or capsular polysaccharides derived from Gram-positive bacteria, optionally from Streptococcus pneumoniae ( Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus () or yeast polysaccharide antigen or mammalian polysaccharide antigen, which optionally integrate into the host cell genome; ii) A nucleotide sequence encoding a heterologous oligosaccharide transferase (such as glycosyltransferase pglB), optionally within a plasmid; iii) A nucleotide sequence encoding a modified or unmodified nanoparticle subunit or a nanoparticle subunit fused to a modified carrier protein, as described in any one of paragraphs 1 to 90, optionally within a plasmid.

[0412] 93. The host cell according to paragraph 91 or 92 further comprises encoding a polymerase (e.g., wzy ), flip enzyme (e.g., wzx The nucleotide sequence of ) and optionally encodes chain length regulators (e.g., wzz The nucleotide sequence of ).

[0413] 94. The host cell according to any one of paragraphs 91 to 93, wherein the oligosaccharide transferase is PglB, optionally derived from Campylobacter jejuni ( Campylobacter jejuni ).

[0414] 95. An immunogenic composition comprising glycoprotein nanoparticles assembled according to any of the preceding paragraphs.

[0415] 96. The immunogenic composition according to paragraph 95, further comprising an adjuvant.

[0416] 97. The immunogenic composition according to paragraph 95 or paragraph 96, wherein the adjuvant is selected from the following: aluminum salts, aluminum hydroxide, aluminum phosphate, saponins, water-in-oil emulsions, oil-in-water emulsions, lipoglycosides, lipopolysaccharides, immunostimulatory nucleic acid molecules, liposomes, and Toll receptors or Toll-like receptor agonists.

[0417] 98. The immunogenic composition according to any one of paragraphs 95 to 97, which does not further contain an adjuvant.

[0418] 99. The immunogenic composition according to any one of paragraphs 95 to 98, optionally comprising a pharmaceutically acceptable diluent or excipient polypeptide.

[0419] 100. A vaccine comprising the immunogenic composition described in any one of paragraphs 95 to 99, and optionally an adjuvant.

[0420] 101. Use of the assembled glycoprotein nanoparticles according to any one of paragraphs 1 to 22, the immunogenic composition according to any one of paragraphs 95 to 99, or the vaccine according to paragraph 100 in the preparation of a medicament for inducing an immune response.

[0421] 102. Use of the assembled glycoprotein nanoparticles according to any one of paragraphs 1 to 22, the immunogenic composition according to any one of paragraphs 95 to 99, or the vaccine according to paragraph 100 in the preparation of a medicament for inducing an immune response in a subject.

[0422] 103. Use of the assembled glycoprotein nanoparticles according to any one of paragraphs 1 to 22, the immunogenic composition according to any one of paragraphs 95 to 99, or the vaccine according to paragraph 100 in the preparation of a medicament for the prevention or treatment of a disease.

[0423] 104. A method for preparing assembled glycoprotein nanoparticles, said assembled glycoprotein nanoparticles comprising modified nanoparticle subunits conjugated to capsular polysaccharides or oligosaccharides or nanoparticle subunits fused to modified carrier proteins, said method comprising: (i) Culturing the host cells described in any one of paragraphs 91 to 94 under conditions suitable for glycoprotein production, and (ii) Separate the assembled glycoprotein nanoparticles, optionally from the periplasmic extract of the host cells.

[0424] 105. A method for inducing an immune response in a human subject, comprising administering to the subject an immunologically effective amount of an assembled glycoprotein nanoparticle according to any one of paragraphs 1 to 22, an immunogenic composition according to any one of paragraphs 95 to 99, or a vaccine according to paragraph 100, for the production of a pharmaceutical.

[0425] 106. A method for inducing an immune response in a subject (e.g., a human), the method comprising administering to a subject (e.g., a human) in need of a therapeutic or prophylactic amount of assembled glycoprotein nanoparticles as described in any of the preceding paragraphs, an immunogenic composition as described in any one of paragraphs 95 to 99, or a vaccine as described in paragraph 100.

[0426] 107. The method according to paragraph 105 or paragraph 106, wherein the subject receives a single administration of the assembled glycoprotein nanoparticles, the immunogenic composition, or the vaccine.

[0427] 108. The method according to paragraph 107, wherein the subject receives intramuscular administration.

[0428] 109. A method for treating and / or preventing yeast or bacterial infection in a subject (e.g., a human), the method comprising administering to a subject (e.g., a human) in need of treatment or prevention an effective amount of an immunogenic composition according to any one of paragraphs 95 to 99 or a vaccine according to paragraph 100.

[0429] 110. An immunogenic composition according to any one of paragraphs 95 to 99 or a vaccine according to paragraph 100, for use in treating and / or preventing yeast or bacterial infections in a subject (e.g., a human).

[0430] 111. The immunogenic composition according to any of the preceding paragraphs, wherein the concordant sequence is selected from D / EXNZS / T (SEQ ID NO: 49) and JD / EXNZS / TU (SEQ ID NO: 51), wherein X and Z are independently any amino acid other than proline, and J and U are independently 1 to 5 naturally occurring amino acid residues, wherein the concordant sequence has been added at the N-terminus to one or more amino acids immediately adjacent to the nanoparticle subunit protein or to an equivalent position within the same amino acid sequence as at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% of the nanoparticle subunit protein, or has been substituted therein.

[0431] 112. The immunogenic composition according to paragraph 111, wherein X is Q (glutamine) and Z is A (alanine).

[0432] 113. The immunogenic composition according to paragraph 111 or paragraph 112, wherein the amino acid sequence further comprises a peptide tag, optionally the peptide tag comprising six histidine residues and optionally the peptide tag being located at the C-terminus of the amino acid sequence.

[0433] 114. The immunogenic composition according to any one of paragraphs 111 to 113, further comprising a conjugate (e.g., a bioconjugate) which optionally comprises a modified nanoparticle subunit linked to an antigen (e.g., a glycoantigen, optionally a bacterial polysaccharide antigen).

[0434] 115. The immunogenic composition according to any one of paragraphs 111 to 114, wherein the optionally modified nanoparticle subunits are covalently linked to the antigen.

[0435] 116. The immunogenic composition according to any one of paragraphs 111 to 115, wherein said antigen is a sugar, optionally a bacterial polysaccharide (e.g., from Shigella dysenteriae). Shigella dysenteriae ), Shigella flexneri ( Shigella flexneri Shigella sonnei ( Shigella sonnei ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Klebsiella pneumoniae ( Klebsiella pneumoniae ) or capsular polysaccharides derived from Gram-positive bacteria, optionally from Streptococcus pneumoniae ( Streptococcus pneumoniae ) or Staphylococcus aureus ( Staphylococcus aureus ), optionally, is the O-antigen from Gram-negative bacteria.

[0436] Unless the context clearly indicates otherwise, the singular terms “a,” “an,” and “the” include plural references. Similarly, unless the context clearly indicates otherwise, the word “or” is intended to include “and.” The term “multiple” refers to two or more. It should be further understood that all base or amino acid dimensions and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and for descriptive purposes only.

[0437] Furthermore, numerical limits given regarding the concentration or level of a substance (such as an antigen) are intended to be approximate. Therefore, when a concentration is indicated as at least (for example) 200 pg, it is intended to be understood as at least approximately (or “about” or “~”) 200 pg.

[0438] The term “comprises” means “includes”. Therefore, unless the context requires otherwise, the word “comprise” and its variations, such as “comprise” and “comprising”, will be understood to include the compound or composition shown (e.g., nucleic acid, polypeptide, antigen) or step or group of compounds or steps, but does not exclude any other compound, composition, step, or group thereof. The abbreviation “eg” is used herein to indicate a non-limiting example and is synonymous with the term “for example”.

[0439] It should be further understood that all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acid molecules or peptides are approximate values ​​and are for descriptive purposes only. Furthermore, numerical limits given regarding the concentration or level of a substance (such as an antigen) are intended to be approximate values. Therefore, when a concentration is indicated as at least (e.g.) 200 pg, it is intended to be understood as at least approximately (or “about” or “~”) 200 pg.

[0440] The term "and / or" as used in the phrase, such as "A and / or B", is intended to include "A and B", "A or B", "A" and "B". Similarly, the term "and / or" as used in the phrase, such as "A, B and / or C", is intended to cover each of the following implementations: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0441] Unless otherwise specified, a process that includes the step of mixing two or more ingredients does not require any specific mixing order. Therefore, the ingredients can be mixed in any order. If there are three ingredients, two ingredients can be combined with each other, and that combination can then be combined with a third ingredient, and so on. Similarly, while the steps of a method can be numbered (e.g., (1), (2), (3), etc., or (i), (ii), (iii)), the numbering of the steps does not imply that the steps must be performed in that order (i.e., step 1, then step 2, then step 3, and so on). The words “then” or “followed by” can be used to specify the order of the method steps.

[0442] This invention is not limited to the specific embodiments described herein. It should be understood that, for clarity, certain features of the invention described in the context of individual embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the invention described in the context of individual embodiments may also be provided individually or in any suitable sub-combination. The invention specifically covers and discloses all combinations of embodiments herein as if each combination were individually and explicitly disclosed. Furthermore, all sub-combinations are also specifically covered by the invention and disclosed herein as if each such sub-combination were individually and explicitly disclosed herein.

[0443] Although similar or equivalent methods and materials to those described herein may be used in practice or testing of this disclosure, suitable methods and materials are described below.

[0444] All published references, patents, and disclosed patent applications cited in this article are incorporated herein by reference in their entirety.

[0445] The following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of this disclosure in any way.

[0446] Example Example 1: Summary of Nanoparticles Table 1:

[0447] The periplasmic expression, glycosylation, and assembly of constructs with inserted glycosylation sites were designed, prepared, and evaluated. The nanoparticles were characterized and examined under an electron microscope, and in vitro studies were performed. Representative examples are provided below. Example 2: Engineering modification of nanoparticles using antigenic glycan-glycosylated nanoparticles To predict suitable locations for glycosylation site insertion, the crystal structure of the nanoparticles was analyzed using various software programs (e.g., Rosetta). Sites that would not interfere with nanoparticle formation were identified. These sites were determined to be suitable for modification (e.g., insertion, deletion, mutation) to make their sequences suitable for glycosylation (e.g., allowing glycosylation site insertion at a specified location). Generally, suitable sites are also exposed on the surface (e.g., recognizable by the humoral immune system for antigen recognition). This criterion allows for the identification of sites suitable for glycosylation site insertion. In some cases, the number of identified sites is limited. In other cases, identified sites are internal, N-terminal, or C-terminal sites, or combinations thereof. Solvent-accessible amino acid residues are selected for site-directed mutagenesis or gene synthesis.

[0448] In some cases, suitable sites for stabilization were identified, and one or more stabilization mutations were added to stabilize the nanoparticles.

[0449] Generally, when engineering glycosylation sites at internal locations (surface exposed loops), the introduced sequence is DQNAT (SEQ ID NO: 32). After testing glycosylation efficiency, this common sequence is extended in some cases by adding flanking lysine residues (KDQNATK (SEQ ID NO: 37)), flanking glycine residues (GDQNATG (SEQ ID NO: 35)), or longer flanking sequences (e.g., GSGDQNATGSG (SEQ ID NO: 31) or GSGGGDQNATGSGGG (SEQ ID NO: 36)). Furthermore, when engineering sequences for glycosylation (glycosylation sites) at the N-terminus or C-terminus, the following example sequences are added: GSGGGDQNATGSGGG (SEQ ID NO: 36) or GSGDQNATGSG (SEQ ID NO: 31) (see also SEQ ID NO: 30-39). To generate nanoparticle variants containing combinations of two or more glycosylation sites, additional rounds of mutagenesis are performed on available and selected unit site variants as needed.

[0450] Generally, when adding internally, at the N-terminus or C-terminus, a flanking sequence of 1-5 amino acid residues (e.g., G, S residues) can be added to either side or both sides of the common sequence DQNAT (SEQ ID NO: 32).

[0451] mi3 and E2p Structural analysis revealed that glycosylation site insertion in the mi3 and E2p nanoparticle subunits was feasible only at their N-termini. For both nanoparticles, a flexible linker sequence carrying a shared glycosylation sequence was inserted between the signal sequence and the first amino acid of the nanoparticle protein subunit. For example, for mi3, the inserted sequence was GSG. KDQNRTK DGSG (SEQ ID NO:30), while for E2p, the inserted sequence is GSGGG. DQNAT GSGGG (SEQ ID NO: 36). Among them, the common glycosylation sequences are DQNAT (SEQ ID NO: 32) and KDQNRTK (SEQ ID NO: 37).

[0452] For E2p, representative examples of expression, assembly, and glycosylation in a single step are shown (e.g., based on SDS-PAGE and Western blot characterization). Figure 11A The results for Sp12F are shown in [the image / image]. Figure 12A-12D The results for KpO3b are shown in the figure.

[0453] Ferritin For those from Helicobacter pylori ( Helicobacter pylori ) and Pseudomonas aeruginosa ( Pseudomonas aeruginosa Structural analysis of the ferritin nanoparticles revealed that suitable positions for engineered glycosylation sites were the N-terminus and the four loops connecting the helices: A and B (loop 1), B and C (loop 2), C and D (loop 3), and D and E (loop 4). The C-terminus of ferritin is solvent-inaccessible and therefore unsuitable for glycosylation site addition. For each loop, a set of 4–6 glycosylation site variants were designed by altering the residues mutated to glycosylation sites. Preliminary glycosylation tests using Sp12F glycans indicated that optimal glycosylation sites included the N-terminus, loop 2 (Helicobacter pylori ferritin residue range 64–82, Pseudomonas aeruginosa ferritin residue range 65–81), and loop 4 (Helicobacter pylori ferritin residue range 145–149, Pseudomonas aeruginosa ferritin residue range 143–146). For example, favorable positions for glycosylation site insertion in loop 2 of Helicobacter pylori ferritin include K79 and E81 when replaced with DQNAT (SEQ ID NO: 32).

[0454] Glycosylation of Helicobacter pylori ferritin in loops 1 (residues 34-36) and 3 (residues 111-113) was tested using Sp12F, but the glycosylation efficiency was low. When using another glycan, KpO3b, the glycosylation efficiency of Helicobacter pylori ferritin was significantly improved. Significant amounts of glycoconjugates were obtained by inserting glycosylation sites in all four loops. Unbound by theory, the glycosylation site in loop 1 may decrease ferritin expression levels and stability; however, the amount of glycoconjugates is no less than with other variants. Optimal KpO3b glycosylation was obtained with a variant containing a glycosylation site in loop 3, specifically a D113 mutation to DQNAT. Most glycosylation site variants in loops 2 and 4 achieved efficient KpO3b glycosylation. The selection of combinations of glycosylation sites was based on the Sp12F glycosylation test because this glycan is more challenging.

[0455] Results: Representative examples of expression, assembly, and glycosylation in a single step (e.g., based on SDS-PAGE and Western blot characterization) were shown to demonstrate [the effectiveness / significance] in [the context of the process]. Figure 3A-8 As shown in 13A-14C. These examples demonstrate the assembly and glycosylation of nanoparticles with N-based glycosylation sites (including Sp12F and KpO3b) of different glycoantigens expressed in E. coli.

[0456] Dodecin For dodecin, suitable glycosylation sites were identified at the N-terminus and C-terminus, as well as in the hairpin loop between amino acid residues 49-55. For example, efficient glycosylation was achieved when D51 was substituted with DQNAT (SEQ ID NO: 32).

[0457] Results: Representative examples of expression, assembly, and glycosylation in a single step (e.g., based on SDS-PAGE and Western blot characterization) are shown throughout the specification, for example, see, Figure 9A-10D These examples demonstrate the assembly and glycosylation of nanoparticles with N-based glycosylation sites (including Sp12F and KpO3b) of different glycoantigens (e.g., polysaccharides) expressed in Escherichia coli.

[0458] IGPD For IGPD, glycosylation sites were designed at multiple locations, but glycosylation characterization showed that only the N-terminal glycosylation site achieved effective glycosylation. Periplasmic expression was reduced compared to other constructs. Results not shown.

[0459] I5350A Component A of nanoparticle I5350A was screened for glycosylation site insertion at 12 positions. Variants containing glycosylation sites in the loop between the N-terminus and residues 41-44 (where FTPV was replaced by DQNAT (SEQ ID NO: 32)) received weak glycosylation. Results not shown.

[0460] Qbeta For Qbeta, the glycosylation sites were engineered at the N-terminus and three internal positions (including residues 12-16, 75-79, and 118). These residues were substituted with DQNAT (SEQ ID NO: 32). These residues appear to have undergone weak glycosylation. Results not shown. Example 3: Glycosylation test of engineered nanoparticles containing one or more glycosylation sites For constructs encoding variant nanoparticle subunits containing a single glycosylation site or a combination of several glycosylation sites, in vivo glycosylation efficiency was tested using a variety of antigenic glycans. For the dataset presented in this paper, glycosylation was tested using the following glycan antigens: Streptococcus pneumoniae (Streptococcus pneumoniae) Streptococcus pneumoniae Capsular polysaccharide 12F (Sp12F), Klebsiella pneumoniae ( Klebsiella pneumoniae O antigens O3b (KpO3b) and O5 (KpO5), as well as Sp33F.

[0461] In some cases, glycosylation assays are performed using *E. coli* strains in which polysaccharide biosynthesis gene clusters have been integrated into the *E. coli* genome (see WO2014 / 057109 and WO2015 / 052344 for more details on integration), which allows for transformation with only two plasmids expressing nanoparticle subunits and PglB. For the dataset presented in this work, the *E. coli* strain used was a derivative of strain W3110, which includes the lipopolysaccharide O antigen ligase gene. waaL The absence of O16O antigen cluster rfb The absence or substitution of [something], and the use of gene clusters responsible for the biosynthesis of the desired recombinant polysaccharide (e.g., Klebsiella pneumoniae). Klebsiella pneumoniae O antigen ( KpO antigen), Sp12F and Sp33F Capsular polysaccharide) replacement gene cluster.

[0462] KpO5 The glycan biosynthesis gene cluster was integrated into the O16 locus. KpO3b The glycan biosynthesis gene cluster was also integrated into the O16 locus. For KpO3b The biosynthesis of GDP-mannose also requires the manB (phosphogannatase) and manC (mannose-1-phosphogguanylate transferase) genes, which are essential for GDP-mannose biosynthesis. The manBC gene is expressed from a high-copy plasmid.

[0463] In strains expressing Sp12F capsular polysaccharide, the gene cluster responsible for the biosynthesis of O16O antigen polysaccharide ( rbf ) has been infected with Streptococcus pneumoniae ( S. pneumoniae Serum type 12F capsular polysaccharide biosynthetic cluster ( cpsSp 12F) replacement. O antigen ligase encoding gene waaL The cassette encoding the IPTG-inducible oligosaccharide transferase PglB has been replaced. The gene cluster responsible for the biosynthesis of intestinal bacterial common antigens (ECA) ( wec ) has been found to be Campylobacter jejuni ( Campylobacter jejuni (Source) gne Gene replacement. The gene cluster responsible for the biosynthesis of colanic acid (…). wca ) has been from cpsSp 12F genes wciJ-wcxB-wcxD-wcxE-wcxF Additional copy and another gne Copy and replace.

[0464] In strains expressing Sp33F capsular polysaccharide, the gene cluster responsible for kolanovic acid biosynthesis ( wca ) has been identified as a biosynthetic cluster of capsular polysaccharides from serotype 33F of Streptococcus pneumoniae ( cpsSp 33F) replacement. O antigen ligase encoding gene waaL Already wchA Gene substitution, the gene cluster responsible for the biosynthesis of O16 O antigen polysaccharide ( rbf ) has been found to contain genes wbbH-gnd The box was replaced.

[0465] Selection criteria for nanoparticle subunit variants containing one or more glycosylation sites included: the total expression level of the nanoparticle subunit and the level of glycoconjugates produced (the latter indicating that the glycosylation site location is suitable for PglB modification), as well as the self-assembly ability of the nanoparticle subunit. Therefore, the method for determining glycosylation levels was similar to that for determining expression levels. *E. coli* strains producing Sp12F, KpO3b, or KpO5 glycans were transformed with the pEC415 plasmid encoding the nanoparticle subunit variant and a plasmid expressing PglB. To prepare a preculture, colonies from transformation plates were streaked with 5 mL of TB medium containing 10 mM MgCl2 and appropriate antibiotics and incubated overnight at 37°C. The preculture was then inoculated into shake flasks with 50 mL of supplemented TB medium to achieve the initial OD. 600 = 0.1. The culture was incubated at 37℃ with shaking at 200 rpm until OD reached... 600 = 0.8-1, then arabinose (concentration ranging from 0.001% to 0.1% depending on the nanoparticle subunit) and 0.1 mM IPTG (for inducing PglB) were added for induction. Variant expression and glycosylation continued overnight at 30°C.

[0466] Periplasmic extracts and IMAC-enriched periplasmic extracts were prepared using the same protocol as for determining expression levels. For example, samples were analyzed by SDS-PAGE, anti-His Western blot, and anti-polysaccharide Western blot. Western blot or SDS-PAGE readings showed an increase in the molecular weight of the glycoconjugates, indicating a higher polysaccharide-to-protein ratio due to an increased number of glycosylation sites.

[0467] Results: Representative examples of SDS-PAGE and Western blot maps showing glycosylation of ferritin, E2p, and dodecin glycosylation site variants (single glycosylation sites and combinations of glycosylation sites) are shown in the figures. These examples illustrate the assembly and glycosylation of different glycoantigens (including Sp12F, KpO3b, and KpO5) with nanoparticles. Example 4: Screening of signal sequences expressed by nanoparticles in the periplasm of E. coli clone To target the expression of nanoparticle subunits in the periplasm of *E. coli*, different signal sequences were screened using a library containing signal sequences from 23 bacteria. This library contained the following signal sequences: ArgT, BtuB, DsbA (SEQ ID NO: 10), FlgI (SEQ ID NO: 42), OmpAV1, OmpAV2, OmpC, OmpT, PhoA, TolB (SEQ ID NO: 41), DegP, FhuA, Hla, LtIIb (SEQ ID NO: 46), LutA, MalE (SEQ ID NO: 44), OmpA (SEQ ID NO: 43), PelB (SEQ ID NO: 45), RBP, SipA (SEQ ID NO: 48), SufI, TorA, and XynA (SEQ ID NO: 47). The codon-optimized nanoparticle subunit gene was cloned into a plasmid derived from pEC415 (see Schulz, H., Hennecke, H., Thony-Meyer, L., "Prototype of a heme chaperone essential for cytochrome c maturation"). Science 281, 1197-1200, 1998), used NheI and XhoI The restriction site was fused within the N-terminal signal sequence frame. To facilitate Western blotting and IMAC purification, a His tag was introduced at the N-terminus or C-terminus of the nanoparticle protein subunit during cloning. Clones containing the target insert were verified by plasmid sequencing.

[0468] Expression cultivation To test the effects of different signal sequences on nanoparticle subunit expression, a derivative of *E. coli* strain W3110 lacking the araBA gene was used. The deletion of the araBA gene allows for better control of arabinose induction, as arabinose cannot be metabolized in this strain. Therefore, a lower arabinose concentration can be used to titrate the ara promoter, thus finding the optimal inducer concentration. Competent *E. coli* cells were transformed with the pEC415 plasmid encoding the nanoparticle subunit. To prepare a preculture, colonies from transformation plates were streaked with 5 mL of TB medium containing 10 mM MgCl2 and kanamycin and incubated overnight at 37°C. The preculture was then inoculated into shake flasks with 50 mL of supplemented TB medium to induce OD. 600 = 0.1. The culture was incubated at 37℃ with shaking at 200 rpm until OD reached... 600= 0.8-1, then arabinose at concentrations ranging from 0.001% to 1% was added for induction. Nanoparticle expression continued overnight at different temperatures (e.g., 25, 30, and 37 °C for testing). Example 5: Periplasmic protein extraction Harvesting equivalent to OD from overnight culture by centrifugation 600 = 60 cells (measured using a spectrophotometer). The cell pellet was resuspended in 1.5 mL of lysis buffer (30 mM Tris-HCl pH 8.5, 1 mM EDTA, 20% sucrose), and lysozyme was added to a final concentration of 1 mg / mL. The suspension was incubated with gentle shaking at 4°C for 25 min, followed by centrifugation at 16,000 rcf for 10 min. After centrifugation, the supernatant corresponding to the periplasmic extract (PPE) was transferred to a new tube. The periplasmic extract was used directly for SDS-PAGE and Western blot analysis, or for small-scale IMAC purification. Example 6: Enrichment of periplasmic extract by immobilized metal affinity chromatography (IMAC) To enrich periplasmic extracts containing nanoparticle variants and allow for more direct readings via SDS-PAGE, His-tagged nanoparticle proteins were purified using a one-step purification process on Ni-NTA (nickel-triacetic acid) agarose. 1 mL of PPE was mixed with 200 µL of pre-equilibrated Ni-NTA slurry and incubated with gentle shaking for 30 min. Subsequently, the resin was washed and the bound proteins were eluted with elution buffer (30 mM Tris pH 8.0, 500 mM imidazole, 50 mM NaCl). IMAC-enriched PPE was analyzed by SDS-PAGE (see, Laemmli, UK (1970), "Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4". Nature. 227 (5259):680-685. Bibcode:1970Natur.227.680L. doi:10.1038 / 227680a0. ISSN 0028-0836.PMID 5432063). The purified nanoparticles were detected on Coomassie Brilliant Blue stained gels (see, Fazekas de St. Groth, S.; Webster, RG; Datyner, A. (1963). "Two new staining procedures for quantitative estimation of proteins on electrophoretic strips". Biochimica et Biophysica Acta. 71: 377-391. doi:10.1016 / 0006-3002(63)91092-8.PMID 18421828). Example 7: Western blot analysis of peritoneal extract Peripheral extracts were also analyzed using immunoblotting (Qiagen, catalog number 34660) with an anti-His tag.

[0469] result: Representative SDS-PAGE and / or Western blots of Sp12F ferritin Hp nanoparticles are shown below. Figure 3B , Figure 3D , Figure 4B , Figure 5A .

[0470] Representative SDS-PAGE and / or Western blots of KpO3b ferritin Hp nanoparticles are shown below. Figure 3C , Figure 6Aand Figure 7A .

[0471] Representative SDS-PAGE and / or Western blots of Sp12F ferritin Pa nanoparticles are shown below. Figure 8 .

[0472] Representative SDS-PAGE and / or Western blots of Sp12F dodecin nanoparticles are shown below. Figure 9A and Figure 9D Representative SDS-PAGE and / or Western blots of KpO3b dodecin nanoparticles are shown in [reference needed]. Figure 10A .

[0473] Representative SDS-PAGE and / or Western blots of Sp12F E2p nanoparticles are shown below. Figure 11A .

[0474] Representative SDS-PAGE and / or Western blots of KpO3b E2p nanoparticles are shown below. Figure 11C and Figure 12A . Example 8: Development of Fermentation and Purification Process for High-Quality Ferritin Glycoside Nanoparticles Optimal fermentation conditions were screened on a 250 mL fermenter scale in a DASbox microbioreactor system (Eppendorf). The optimal conditions for glycan production were combined with the optimal induction conditions for nanoparticle expression (e.g., Sp12F-ferritin).

[0475] The developed purification procedure is based on the following 5 steps: 1. Preparation of peritoneal extract 2.65℃ heat precipitation of other proteins 3. Use 100 kDa membrane filtration (TFF) 4. Anion exchange chromatography (AIEX) was performed on a Q Ceramic HyperD F 200 mL column. 5. Perform chromatographic inhibition (SEC) analysis on a HiPrep 26 / 60 Sephacryl S 500 HR (320 mL) column. The yield obtained after this process is approximately 100 mg ferritin / L BR and approximately 100 mg Sp12F / L BR.

[0476] result: For Sp12F ferritin, representative SEC chromatograms and SDS-PAGE of the SEC fractions are shown below. Figure 4A and Figure 4B . Figure 4B Fraction 2-11 in the sample shows glycosylated products.

[0477] For KpO3b ferritin Hp, representative SEC chromatograms and SDS-PAGE of SEC fractions are shown below. Figure 6A . Figure 6A Fractions 12-16 in the sample show glycosylated products.

[0478] For dodecin Sp12F, representative SEC chromatograms and SDS-PAGE of the SEC fractions are shown below. Figure 9C and 9D . Figure 9D Fractions A24-A32 in the sample show glycosylated products.

[0479] For KpO3b-E2p, representative SEC chromatograms and SDS-PAGE of SEC fractions are shown below. Figure 11B and 11C . Figure 11C Fractions 12-21 in the sample show glycosylated products.

[0480] For Sp12F-EPA-ferritin, representative SEC chromatograms and SDS-PAGE of the SEC fractions are shown below. Figure 14B and 14C . Figure 14B Fractions A24-A34 in the sample show glycosylated products. Example 9: Analysis of purified sugar nanoparticles The purity of the purified NP-bioconjugates was analyzed by SDS-PAGE, and particle size was analyzed by dynamic light scattering (DLS) and negative staining electron microscopy. The polysaccharide content of each bioconjugate was determined by HPAEC-PAD (high performance anion exchange chromatography-pulse amperometric detection).

[0481] result: For Sp12F ferritin Hp, representative examples include SDS-PAGE ( Figure 5A ), DLS ( Figure 5C (showing an average particle size of 30 nm), and electron micrographs ( Figure 5D ).

[0482] Regarding KpO5-ferritin Hp, introducing a glycosylation site at the N-terminus and combining this N-terminal glycosylation site with glycosylation sites in loops 2 and 4 (N+L2, N+L4, or N+L2+L4 positions) successfully achieved expression, assembly, and glycosylation. Figure 3D ).

[0483] For KpO3b ferritin Hp, representative examples include SDS-PAGE ( Figure 7A ), DLS ( Figure 7C(showing an average particle size of approximately 25 nm), and electron micrographs ( Figure 7D ).

[0484] For Sp12F-dodecin, representative embodiments include SDS-PAGE ( Figure 9D For KpO3b-dodecin, representative embodiments include SDS-PAGE (…). Figure 10A ), DLS ( Figure 10C (showing an average particle size of approximately 17 nm), and electron micrographs ( Figure 10D ).

[0485] For KpO3b-E2p, representative embodiments include SDS-PAGE ( Figure 12A ), DLS ( Figure 12C (showing an average particle size of approximately 42 nm), and electron micrographs ( Figure 12D ).

[0486] For Sp33F-ferritin, representative examples include SDS-PAGE ( Figure 17A DLS (showing an average particle size of approximately 21 nm) Figure 17A ), and electron micrographs ( Figure 17A ).

[0487] For Sp33F-dodecin, representative embodiments include SDS-PAGE ( Figure 17B DLS (showing an average particle size of approximately 15 nm) Figure 17B ), and electron micrographs ( Figure 17B ).

[0488] The polysaccharide / protein ratios of the generated sugar nanoparticles, as determined by HPAEC-PAD, are as follows: Sp12F-ferritin 109%, Sp12F-dodecin 45%, Sp12F-EPA-ferritin 36%, KpO3b-ferritin 40%, KpO3b-dodecin 64%, KpO3b-E2p 18%, Sp33F-ferritin 22%, and Sp33F-dodecin 70%. Example 10: Animal Research CD1 mice were administered 0.22 µg of Sp12F capsular polysaccharide via intramuscular injection (im) three times every two weeks (d0, d14, d28). Antibody responses in serum were monitored before immunization (day 0 / pre), two weeks after the second injection (day 28 / post-II), and two weeks after the third injection (day 42 / post-III). AlPO4 was used as an adjuvant. Animal studies of KpO3b glycan followed the same design and schedule, except that AS03 was used as an adjuvant.

[0489] result: ELISA readings: Sp12F-specific IgG titers measured in individual serum samples pre, (after II), and after III (see [reference]). Figure 15B Compared to the chemical conjugate of Sp12F-CRM, the Sp12F IgG titer of the bioconjugated nanoparticle 12F-ferritin (SEQ ID NO:18) increased after stage III. The Sp12F IgG titer of the bioconjugated nanoparticle 12F-Dodecin (SEQ ID NO:7) increased after stage III compared to the control group immunized with PBS and Sp12F-EPA. The Sp12F IgG titer of the bioconjugated nanoparticle using EPA-ferritin (SEQ ID NO:52) fusion protein as a carrier was comparable to that of the chemical conjugate of Sp12F-CRM after stage III. In the preclinical evaluation of KpO3b-nanoparticles ( Figure 16B Significant immunogenicity against KpO3b was only achieved when ferritin was used as a carrier. In summary, these results demonstrate that nanoparticles (such as ferritin) can achieve a superior immune response against conjugated polysaccharides compared to non-particulate carrier systems (e.g., EPA). Example 11: Analysis Method electron microscope Negative staining electron microscopy was used to observe the assembled NPs. The EM protocol is known in the art.

[0490] Western blot of peritoneal extract Periplasmic extracts were analyzed by immunoblotting to target polysaccharides attached to modified nanoparticle subunits. SDS-PAGE analysis was performed on IMAC-enriched periplasmic extracts from *E. coli* strains that produce antigenic polysaccharides and express PglB.

[0491] Dynamic light scattering (DLS) The hydrodynamic diameter of assembled nanoparticles in solution was measured using dynamic light scattering (DLS). The DLS protocol is known in the art. Table 2: Direct glycosylation of NPs or carriers

[0492] Table 3: Direct glycosylation of NPs or carriers

[0493] Table 4: Direct glycosylation of NPs or carriers

[0494] sequence list SEQ ID NO: 1 Dodecin wild-type amino acid sequence (Q8VK10) MSNHTYRVIEIVGTSPDGVDAAIQGGLARAAQTMRALDWFEVQSIRGHLVDGAVAHFQVTMKVGFRLEDS SEQ ID NO: 2 Dodecin-modified amino acid sequences (G25N, V50T, A53T) and His tag MSNHTYRVIEIVGTSPDGVDAAIQ N GLARAAQTMRALDWFEVQSIRGHL T DG T VAHFQVTMKVGFRLEDSGSGHHHHHH SEQ ID NO: 3 Dodecin-modified nucleic acid sequences ATGTCTAACCATACTTACCGTGTTATCGAAATCGTGGGAACTTCGCCGGACGGAGTGGACGCCGCGATTCAGAACGGCTTAGCCCGTGCTGCCCAAACCATGCGTGCGCTTGATTGGTTTGAGGTGCAGTCAATTCGCGGACATCTGACA GATGGCACGGTCGCTCACTTCCAAGTAACCATGAAAGTGGGGTTTCGCCTGGAGGACTCTGGCAGTGGCCACCACCACCACCATCACTAACGTTGCGCGGAGTTTCTGCGTATGCACAACCTGGAAGACAGCTGCTTCAGCTTTCTGTAA SEQ ID NO: 4 The dodecin-modified amino acid sequence (signal sequence, N-terminal sugar tag sequence (corresponding to G25N, V50T, and A53T of SEQ ID NO:2)) and histone tag MKKIWLALAGLVLAFSASA SGSGDQNATGGSGSNHTYRVIEIVGTSPDGVDAAIQ N GLARAAQTMRALDWFEVQSIRGHL T DG T VAHFQVTMKVGFRLEDSGSGHHHHHH SEQ ID NO: 5 The dodecin-modified amino acid sequence (signal sequence, corresponding to G25N, V50T, A53T in SEQ ID NO:2, G44N, V69T, A72T), C-terminal sugar tag sequence) and histidine tag. MKKIWLALAGLVLAFSASA SSNHTYRVIEIVGTSPDGVDAAIQ N GLARAAQTMRALDWFEVQSIRGHL T DG T VAHFQVTMKVGFRLEDSGSGDQNATGSGHHHHHH SEQ ID NO: 6 The dodecin-modified amino acid sequence (signal sequence, G44N, V69T, A76T (corresponding to G25N, V50T, A53T in SEQ ID NO:2) and D51 are replaced by DQNAT (Mut3)) and the histidine tag. MKKIWLALAGLVLAFSASA SSNHTYRVIEIVGTSPDGVDAAIQ N GLARAAQTMRALDWFEVQSIRGHL T DQNATG T VAHFQVTMKVGFRLEDSGSGHHHHHH SEQ ID NO: 7 Dodecin-modified amino acid sequences (signal sequence, N+C sugar tag sequence) and histidine tags MKKIWLALAGLVLAFSASA SGSGDQN...

Claims

1. A method for preparing glycoprotein nanoparticles (NPs), comprising the following steps: Provide host cells; Optionally, modified nanoparticle subunits are expressed in the periplasm of the host cells; Optionally, glycosyltransferases are expressed in the periplasm of the host cells; Optionally, the modified nanoparticles are subunit glycosylated in the periplasm of the host cell by the glycosyltransferase; as well as Glycoprotein nanoparticles are assembled from glycosylated modified nanoparticle subunits in the periplasm of the host cell.

2. The method of claim 1, wherein the modified nanoparticle subunit comprises one or more glycosylation sites (e.g., SEQ ID NO: 49; D / EXNZS / T).

3. The method according to claim 1 or claim 2, wherein the glycosyltransferase is glycosyltransferase pglB (optionally derived from Campylobacter jejuni).

4. The method according to any one of claims 1 to 3, further comprising glycosylation of the modified nanoparticle subunits in the periplasm by means of the glycosyltransferase pglB.

5. The method according to any of the preceding claims, wherein the host cell is a Gram-negative bacterial host cell.

6. The method according to any of the preceding claims, wherein the host cell is Escherichia coli.

7. The method according to any of the preceding claims, wherein the modified nanoparticle subunit is selected from the following: E2p subunit, ferritin subunit, and dodecin subunit.

8. The method according to any of the preceding claims, wherein a capsular polysaccharide or oligosaccharide is conjugated to the modified nanoparticle subunit at one or more glycosylation sites.

9. The method according to any of the preceding claims, wherein the capsular polysaccharide or oligosaccharide is selected from: Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptococcus species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Neisseria meningitidis capsular polysaccharide, and Candida species polysaccharide.

10. The method according to claim 9, wherein the capsular polysaccharide or oligosaccharide is selected from Streptococcus species or Klebsiella species.

11. A method for preparing glycoprotein nanoparticles (NPs), comprising the following steps: Provide host cells; Nanoparticle subunits fused with a modified carrier protein are expressed in the periplasm of the host cells; Glycosyltransferases were expressed in the periplasm of the host cells; In the periplasm, one or more glycosylation sites of the modified carrier protein are glycosylated by the glycosyltransferase; as well as Glycoprotein nanoparticles were prepared and assembled from multiple nanoparticle subunits in the periplasm.

12. The method of claim 11, wherein the glycosyltransferase is glycosyltransferase pglB (optionally derived from Campylobacter jejuni).

13. The method according to claim 11 or claim 12, further comprising glycosylation of the modified nanoparticle subunits in the periplasm by means of the glycosyltransferase pglB.

14. The method according to any one of claims 11 to 13, wherein the modified carrier protein is selected from: modified CRM197, modified diphtheria toxoid (DT), modified tetanus toxoid (TT), or modified EPA.

15. The method according to any one of claims 11 to 14, wherein the capsular polysaccharide or oligosaccharide is selected from: species of Escherichia, Shigella, Klebsiella, Salmonella, Yersinia, Helicobacter, Proteus, Pseudomonas, Corynebacterium, Streptomyces, Streptococcus, Enterococcus, Staphylococcus, Bacillus, Clostridium, Listeria, Campylobacter, Neisseria meningitidis capsular polysaccharide, and Candida polysaccharide.

16. The method according to any one of claims 11 to 15, wherein the capsular polysaccharide or oligosaccharide is selected from Streptococcus species or Klebsiella species.

17. The method according to any one of claims 11 to 16, wherein the nanoparticle monomer subunit is selected from: E2p subunit, ferritin subunit, and dodecin subunit.

18. A composition comprising modified dodecin nanoparticles (NPs), said dodecin NPs comprising one or more dodecin subunits, each dodecin subunit comprising amino acids 2-70 of SEQ ID NO: 1 or a self-assembled fragment thereof, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 2-70 of SEQ ID NO: 1 or a self-assembled fragment thereof; and one or more modifications selected from: (i) One or more substitution mutations selected from positions G25, V50, and A53, wherein the position number is relative to SEQ ID NO: 1, and (ii) At least one glycosylated common sequence.

19. A composition comprising a modified amino acid sequence of ferritin nanoparticle subunits, said modified amino acid sequence comprising: (i) Amino acids 1-167 of SEQ ID NO: 11 or their self-assembled fragments, or amino acid sequences that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1-167 of SEQ ID NO: 11 or their self-assembled fragments; and (ii) One or more glycosylated sequences (e.g., GSGDQNATGSG (SEQ ID NO: 33), GGSKDQNRTKDGSG (SEQ ID NO: 34), DQNAT (SEQ ID NO: 32) and / or GDQNATG (SEQ ID NO: 35)), wherein the one or more glycosylated sequences are adjacent to the modified amino acid sequence and add or replace one or more amino acids of the modified amino acid sequence.

20. A composition comprising a modified amino acid sequence of ferritin nanoparticle subunits, said modified amino acid sequence comprising: (a) Amino acids 1-154 of SEQ ID NO: 20 or their self-assembled fragments, or amino acid sequences that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 1-154 of SEQ ID NO: 20 or their self-assembled fragments; and (b) One or more glycosylation sequences (e.g., GSGDQNATGSG (SEQ ID NO: 33), GGSKDQNRTKDGSG (SEQ ID NO: 34)), wherein the one or more glycosylation sequences are adjacent to the modified amino acid sequence and add or replace one or more amino acids of the modified amino acid sequence.

21. A composition comprising a modified amino acid sequence of E2p nanoparticles, wherein the nanoparticles comprise E2p nanoparticle subunits comprising: (a) Amino acids 185-426 of SEQ ID NO: 26 or their self-assembled fragments, or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or their self-assembled fragments; and (b) One or more glycosylation sequences (e.g., GSGGGDQNATGSGGG (SEQ ID NO: 36)), wherein the one or more glycosylation sequences are adjacent to the modified amino acid sequence and add or replace one or more amino acids of the modified amino acid sequence.

22. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a modified nanoparticle subunit or a nanoparticle subunit fused with a modified carrier protein according to any of the preceding claims.

23. A carrier comprising the isolated nucleic acid molecule according to claim 22.

24. A host cell comprising the isolated nucleic acid molecule according to claim 22 or the vector according to claim 23.

25. An immunogenic composition comprising glycoprotein nanoparticles assembled according to any of the preceding claims.

26. The immunogenic composition of claim 25, further comprising an adjuvant, wherein the adjuvant is selected from: aluminum salts, aluminum hydroxide, aluminum phosphate, saponins, water-in-oil emulsions, oil-in-water emulsions, lipoglycosides, lipopolysaccharides, immunostimulatory nucleic acid molecules, liposomes, and Toll receptors or Toll-like receptor agonists.

27. A vaccine comprising the immunogenic composition according to claim 25 or claim 26 and optionally comprising an adjuvant.

28. A method for producing assembled glycoprotein nanoparticles, said assembled glycoprotein nanoparticles comprising modified nanoparticle subunits conjugated to capsular polysaccharides or oligosaccharides or nanoparticle subunits fused to modified carrier proteins, said method comprising: (i) The host cells of claim 24 are cultured under conditions suitable for the production of glycoproteins; as well as (ii) Isolate the assembled glycoprotein nanoparticles, optionally from a periplasmic extract of the host cells.

29. A method for inducing an immune response in a human subject, comprising administering to the subject an immunologically effective amount of an assembled glycoprotein nanoparticle produced according to any one of claims 1-17, or an immunogenic composition according to any one of claims 25-26, or a vaccine according to claim 27, for use in the production of a pharmaceutical.

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