Modified proteins and methods

Abstract

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

Description

[0001] MODIFIED PROTEINS AND METHODS

[0002] SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on November 9, 2023, is named 70277US01P_SL.xml and is 60,947 bytes in size.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to the field of modified proteins, immunogenic compositions and vaccines comprising the modified proteins, their manufacture and method of making and the use of such compositions in medicine. More particularly, it relates to a bioconjugated modified nanoparticle (NP) subunit (or a bioconjugated modified carrier protein linked to an optionally modified nanoparticle (NP) subunit) and methods for preparing an assembled bioconjugated modified nanoparticle in a host cell (or an assembled bioconjugated modified carrier protein linked to an optionally modified nanoparticle (NP)). It also relates to methods for preparing an assembled bioconjugated modified nanoparticle (or an assembled bioconjugated modified carrier protein linked to an optionally modified nanoparticle (NP)) in a host cell (e.g. a bacterial host cell), wherein the host cell expresses a modified nanoparticle subunit protein (or a modified carrier protein linked to an optionally modified nanoparticle (NP) subunit), a glycosyltransferase (e.g. glycosyltransferase pglB) for attaching a saccharide (e.g. an oligosaccharide, a polysaccharide) to a glycosite of the modified nanoparticle subunit protein or the modified carrier protein to produce, in a single step, an assembled bioconjugated modified glycoprotein nanoparticle (or an assembled bioconjugated modified carrier protein linked to an optionally modified nanoparticle (NP)) in a host cell. In aspects, the modified nanoparticle subunit protein (or a modified carrier protein linked to the optionally modified nanoparticle (NP) subunit) and glycosyltransferase are expressed in the periplasm.

[0006] BACKGROUND

[0007] Protein nanoparticles (NPs) have the ability to self-assemble into highly ordered symmetric and stable structures. NPs can be used for displaying target antigens in a defined pattern at high density on their surface, which resembles pathogen-associated molecular patterns (PAMPs) in nature, inducing both innate and adaptive immune responses. For instance, protein NPs may act as scaffolds to present antigens in highly ordered repetitive antigen arrays (see e.g. W002 / 056905). Accordingly, self-assembling protein nanoparticles have demonstrated the ability to evoke an immune response and may be used in the development and generation of new types of vaccine technologies.

[0008] Glycoconjugate vaccines represent an effective technology for controlling the occurrence of bacterial diseases. A traditional glycoconjugate vaccine is produced by covalently linking one or more bacterial polysaccharides to a carrier protein. Protein glycosylation is a posttranslational modification in bacteria by which glycans are covalently attached to surface proteins, e.g. flagella, or pili. Glycoproteins may have roles in adhesion, stabilization of proteins against proteolysis, or evasion of the host immune response.

[0009] Different protein glycosylation mechanisms are distinguishable by the mode in which the glycans are transferred to proteins. One mechanism involves the transfer of carbohydrates directly from nucleotide-activated sugars to acceptor proteins (e.g. protein O-glycosylation in the Golgi apparatus of eukaryotic cells, and flagellin O-glycosylation in certain types of bacteria). A second mechanism involves the preassembly of a polysaccharide onto a lipid-carrier (e.g. by glycosyltransferases), which is then transferred to a protein acceptor by an oligosaccharyltransferase (OTase) (Faridmoayer et al., J. Bacteriology, pp. 8088-8098, 2007). This mechanism is used in, e.g. N-glycosylation in the endoplasmic reticulum of eukaryotic cells, the well-characterized N-linked glycosylation system of Campylobacter jejuni, and the more recently characterized O-linked glycosylation systems of Neisseria meningitidis, Neisseria gonococcus, and Pseudomonas aeruginosa. For O-linked glycosylation (O-glycosylation), glycans are generally attached to a serine or threonine residue on the protein acceptor. For N-linked glycosylation (N-glycosylation), glycans are generally attached to an asparagine residue on the protein acceptor. The N-glycosylation of C. jejuni proteins may be reconstituted by recombinantly expressing the pgl locus and acceptor glycoprotein in E. coli at the same time (W acker et al . (2002) Science 298, 1790-1793).

[0010] More recently, bacterial cells have been used to produce glycoconjugate vaccines. For example, a genetically modified bacterial cell such as E. coli may be used to link glycans to glycosites on a carrier protein to produce a glycoconjugate vaccine . The process of linking glycans to glycosites on a carrier protein, termed bioconjugation, provides a low-cost option for production of glycoconjugate vaccines. This approach offers the flexibility of producing different glycan / carrier recombinants for different vaccines. Bioconjugation may rely on a conjugating enzyme, e.g. oligosaccharyltransferase pgIB, to transfer a saccharide (e.g. an oligosaccharide, a polysaccharide, etc.) to glycosites on different carrier proteins (e.g. an optionally modified CRM (e.g. CRM-197), an optionally modified EPA, an optionally modified Diptheria Toxoid, an optionally modified OMPC, an optionally modified Tetanis Toxoid, etc.).

[0011] W02006 / 119987 (Aebi et al.) describes proteins, as well as means and methods for producing proteins, with efficiency for N-glycosylation in prokaryotic organisms in vivo. It further describes the introduction of N-glycans into recombinant proteins for modifying immunogenicity, stability, biological, prophylactic and / or therapeutic activity of said proteins, and the provision of a host cell that displays recombinant N-glycosylated proteins of present embodiments on its surface. In addition, it describes a recombinant N-glycosylated protein comprising one or more of the following optimized amino acid sequence(s): D / E-X-N-Z-S / T (SEQ ID NO: 49), wherein X and Z may be any natural amino acid except Pro. The introduction of such optimized amino acid sequence(s) into proteins leads to proteins that may be N-glycosylated by an oligosaccharyl transferase in these introduced positions.

[0012] Conjugate vaccines (vaccines comprising a carrier protein covalently linked to an immunogenic antigen) have been a successful approach for vaccination against a variety of bacterial infections. Conjugation of T-independent antigens, for example saccharides, to carrier proteins has long been established as a way of enabling T-cell to help to become part of the immune response for a normally T-independent antigen. In this way, an immune response can be enhanced by allowing the development of immune memory and boostability of the response. To increase conjugate vaccine production efficiency, in vivo methods (to produce a “bioconjugate vaccine”) have been in development. These in vivo methods leverage the N-glycosylation and O- glycosylation systems discussed above (see, W02009 / 104074 and W02017 / 035181).

[0013] Peng et al. (Adv. Sci. (2021) vol. 8, 2100549) report production of a biosynthetic nanoconjugate vaccine using a Nano-B5 self-assembly platform, to generate a conjugate vaccine against K. pneumoniae . In this study, Peng et al. utilize an enzyme, an oligosaccharyl transferase (PglL), to add O2-polysaccharides to specific glycosites.

[0014] There exists a need for improved vaccine technologies. In particular, there exists a need for an adaptable bioconjugate nanoparticle platform, capable of producing a bioconjugated nanoparticle subunit that self-assembles (or a bioconjugated carrier protein fused directly or indirectly to a nanoparticle subunit that self-assembles) . Such a platform is suitable for preparation of immunogenic compositions and / or vaccines for different nanoparticles and different saccharides (e.g. polysaccharide antigens, oligosaccharide antigens, etc.). Suitably, such a platform would be capable of producing a bioconjugated nanoparticle (or a bioconjugated carrier protein fused directly or indirectly to a nanoparticle) in a single step.

[0015] SUMMARY

[0016] In an aspect, a nanoparticle (NP) based platform is provided for producing a bioconjugated nanoparticle (or a bioconjugated carrier protein fused directly or indirectly to a nanoparticle). Modified nanoparticle subunits are translocated to or expressed in the periplasm of a host cell (e.g. a bacterial host cell such as E. coli) along with a glycosyltransferase (e.g. glycosyltransferase pglB). The glycosyltransferase conjugates antigens (e.g. bacterial saccharides such as polysaccharides or oligosaccharides) to specific regions of proteins comprising a glycosite motif / consensus sequence introduced into the nanoparticle subunit or carrier protein sequence. In aspects, the consensus sequence comprises or consists of D / E-X-N-Z-S / T (SEQ ID NO: 49), wherein X and Z may be independently any natural amino acid except proline (e.g. the consensus sequence may be D-Q-N-X-T, wherein X may be A or R (e.g. SEQ ID NO:53)). Advantageously, the assembled glycoconjugated-NP or glycoconjugated-carrier protein-NP are generated in a single step (e.g. the modified NP subunits or modified carrier protein-NP subunits undergo bioconjugation and self assembly in the periplasm of a cell) to form assembled bioconjugated nanoparticles (or assembled bioconjugated carrier proteins fused directly or indirectly to nanoparticles). Thus, in an aspect, the present embodiments relate to self-assembling nanoparticles (NPs) (or carrier proteins fused directly or indirectly to self-assembling nanoparticles) that display antigens (e.g. bacterial polysaccharides / oligosaccharides), to compositions comprising such nanoparticles, and to methods of making and using such nanoparticles and compositions.

[0017] Individual NP subunit proteins are capable of self-assembly to form nanoparticles, e .g . with a size 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. Self-assembly of NPs refers to the oligomerization or aggregation ofNP subunits or carrier proteins fused to NP subunits into an ordered arrangement driven by non-covalent interactions.

[0018] In an embodiment, the modified NP subunits, the antigen (e.g. bacterial antigen), and the glycosyltransferase (e.g. pglB) are expressed in or translocated to the periplasm of a bacterial host cell (e.g., E. col ). In an embodiment, the glycosyltransferase conjugates the antigen (e.g. the bacterial polysaccharide or oligosaccharide) to the modified NP subunit (or to the modified carrier protein fused directly or indirectly to a nanoparticle subunit), which self-assembles into a bioconjugated nanoparticle.

[0019] In an embodiment, multiple copies of antigenic epitopes are displayed on the exterior surface of the assembled NP. In a further embodiment, the assembled NPs display bacterial antigens (e.g. from gram-positive bacteria or gram-negative bacteria).

[0020] The bioconjugated NPs may be used for any suitable purpose, such as for inducing an immune response in a subject, and / or preventing or treating a disorder or disease. In an aspect, the present embodiments provide glycoconjugate nanoparticle vaccines against a variety of bacterial pathogens that present cell surface carbohydrates, where an effective immune response may 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.

[0021] In one aspect, the present embodiments provide modified nanoparticle subunits, modified to include one or more N-glycosylation sites (e.g. by introduction of a consensus sequence). In other aspects, the present embodiments provide modified carrier proteins fused to optionally modified nanoparticle subunits, wherein the carrier protein is modified to include N-glycosylation sites. For N-linked glycosylation (N-glycosylation), glycans are generally attached to an asparagine residue on the protein acceptor. In aspects, the present embodiments provide modified proteins comprising at least one consensus sequence for N-glycosylation (e.g. D / E-X-N-Z-S / T (SEQ ID NO: 49)) for use in conjugation to an antigen (e.g. bacterial polysaccharide / oligosaccharide). In aspects, a modified carrier protein comprising at least one consensus sequence for N-glycosylation (e.g. D / E-X-N-Z-S / T (SEQ ID NO: 49)) may be fused or joined with a nanoparticle subunit. Carrier proteins include but are not limited to a modified CRM (e.g. CRM- 197), a modified EPA, a modified Diptheria Toxoid, a modified OMPC, a modified Tetanis Toxoid, etc.

[0022] In the modified NP of present embodiments, glycosylation consensus sequences are introduced into specific regions of the NP subunit or carrier protein fused to the NP subunit. The position of the consensus sequence in the modified NP subunit may increase glycosylation efficiency and / or optimize the operation of the N-glycosylation site. Accordingly, there is provided a method for preparing a glycosylated nanoparticle (NP) 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 in the periplasm of the host cell; glycosylating, via the glycosyltransferase, the modified nanoparticle subunit in the periplasm; and producing an assembled glycoprotein nanoparticle from the glycosylated modified nanoparticle subunits. In 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 comprises one or more glycosylation sites (e.g. D / E-X-N-Z-S / T (SEQ ID NO: 49)); translocating or expressing glycosyltransferase pglB in the periplasm of the host cell; glycosylating, via glycosyltransferase pglB, the modified nanoparticle subunit; and producing an assembled glycosylated nanoparticle from the glycosylated modified nanoparticle subunits in the periplasm. In further aspects, the method comprises a host cell that is a bacterial host cell. In other aspects, the method comprises a host cell that is a gram -negative bacterial host cell (e.g. E. coli).

[0023] In a further aspect, the method comprises conjugating a capsular polysaccharide or oligosaccharide to the modified nanoparticle subunit (or modified carrier protein fused to an optionally modified nanoparticle subunit) at the one or more glycosylation motifs. For example, a capsular polysaccharide or oligosaccharide may be selected from the group consisting of Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Meningococcal capsular saccharides, and Candida saccharides.

[0024] Accordingly, there is provided in a second aspect, a method for preparing a glycosylated carrier protein conjugated to a nanoparticle (NP) comprising the steps of: providing a host cell; translocating or expressing a modified carrier protein conjugated to an optionally modified nanoparticle subunit into the periplasm of the host cell, wherein the modified carrier protein comprises one or more glycosylation sites (e.g. D / E-X-N-Z-S / T (SEQ ID NO: 49)); translocating or expressing a glycosyltransferase (e.g. pglB) in the periplasm of the host cell; glycosylating, via glycosyltransferase pglB, the modified carrier protein; and producing an assembled nanoparticle, formed of glycoprotein carrier proteins fused directly or indirectly to nanoparticle subunits, in the periplasm.

[0025] In further aspects, the method comprises translocating or expressing a modified carrier protein fused directly or indirectly to an optionally modified nanoparticle subunit in the periplasm, wherein the carrier protein includes but is not limited to: a modified CRM (e.g. CRM197), a modified Diphtheria Toxoid (DT), a modified Tetanus Toxoid (TT), a modified OMPC, or a modified EPA. In aspects, the method comprises conjugating a capsular polysaccharide or oligosaccharide to the modified carrier protein (e.g. at the one or more glycosylation motifs). For example, a capsular polysaccharide or oligosaccharide may include but is not limited to Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Meningococcal capsular saccharides, and Candida saccharides.

[0026] In a third aspect, there is provided a process for producing an assembled bioconjugated nanoparticle that comprises a modified nanoparticle subunit conjugated to a capsular polysaccharide or oligosaccharide, said process comprising: (i) culturing the host cell under conditions suitable for the production of glycoproteins, nanoparticle subunits and glycosyltransferase, and (ii) isolating the assembled glycoprotein nanoparticle, optionally isolating the assembled glycoprotein nanoparticle from a periplasmic extract from the host cell.

[0027] In a fourth aspect, there is provided a process for producing an assembled nanoparticle comprising a bioconjugated carrier protein fused directly or indirectly to an optionally modified nanoparticle subunit, wherein the modified carrier protein is conjugated to a capsular polysaccharide or oligosaccharide fused to a nanoparticle subunit, said process comprising: (i) culturing the host cell under conditions suitable for the production of glycoproteins, nanoparticle subunits and a glycosyltransferase, and (ii) isolating the assembled bioconjugated carrier protein fused to an optionally modified nanoparticle, optionally isolating the assembled bioconjugated carrier protein fused to an optionally modified nanoparticle from a periplasmic extract of the host cell.

[0028] In a fifth aspect, there is provided a method of inducing an immune response in a subject (e.g. human), the method comprising administering a therapeutically or prophylactically effective amount of the assembled glycoprotein nanoparticle, as an immunogenic composition or as a vaccine to a subject (e.g. human) in need thereof. In another aspect, there is provided a method of inducing an immune response in a subject (e.g. human), the method comprising administering a therapeutically or prophylactically effective amount of the assembled bioconjugated nanoparticle, wherein the assembled bioconjugated nanoparticle, comprising a glycoprotein carrier protein fused directly or indirectly to an optionally modified nanoparticle subunit, as an immunogenic composition or as a vaccine to a subject (e.g. human) in need thereof.

[0029] In a sixth aspect, there is provided a modified NP or a modified carrier protein fused directly or indirectly to an optionally modified NP, modified in that the amino acid sequence of the NP subunit or carrier protein comprises one or more consensus sequence(s) of D / E-X-N-Z-S / T (SEQ ID NO: 49), wherein X and Z are independently any amino acid except proline.

[0030] 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 at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 2-70 of SEQ ID NO: 1, modified in that the amino acid sequence comprises one (or more) consensus sequence(s) of D / E-X-N-Z-S / T (SEQ ID NO: 49), wherein X and Z are independently any amino acid except proline, wherein the one (or more) consensus sequences have each been added next to or substituted for one or more amino acids (a) at the N-terminus, (b) at the C-terminus (wherein the consensus sequence is optionally preceded or followed by a histidine tag), and / or (c) substituted for one or more amino acids, 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 at equivalent position(s) within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 2-70 of SEQ ID NO: 1. In aspects, SEQ ID NO: 1 optionally comprises one or more substitutions selected from G25N, V50T, and A53T.

[0031] In another 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 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 aspects, SEQ ID NO: 1 comprises one or more of the following substitutions: G25N, V50T, and A53T.

[0032] In an eighth aspect of the present embodiments provided herein, a modified ferritin NP is provided (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 at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues amino acid residues 1-167 of SEQ ID NO: 11, modified in that the amino acid sequence comprises one (or more) consensus sequence(s) of D / E-X-N-Z- S / T (SEQ ID NO: 49), wherein X and Z are independently any amino acid except proline, wherein the one (or more) consensus sequences have each been added next to or substituted for one or more amino acids at (a) the N-terminus (wherein the consensus sequence is optionally preceded or followed by a histidine tag), (b) substituted for one or more amino acids, independently selected from one or more amino acids between amino acid residues 65-81 of SEQ ID NO: 11 (e.g. K79 or E81 substituted with DQNAT (SEQ ID NO: 32)), and / or (c) substituted for one or more amino acids, independently selected from one or more amino acids between amino acid residues 145-149 of SEQ ID NO: 11 (e.g. residues 145-149, or 146-148 substituted with GDQNATG (SEQ ID NO: 35)), or at equivalent position(s) within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 1-167 of SEQ ID NO: 11. For example, (a) and (b); (b) and (c); or (a), (b) and (c) may be combined.

[0033] 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 at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 1-167 of SEQ ID NO: 11.

[0034] 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 at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 1- 154 of SEQ ID NO: 20, modified in that the amino acid sequence comprises one (or more) consensus sequence(s) of D / E-X-N-Z-S / T (SEQ ID NO:49), wherein X and Z are independently any amino acid except proline, wherein the one (or more) consensus sequences have each been added next to or substituted for one or more amino acids at (a) the N- terminus (wherein the consensus sequence is optionally preceded or followed by a histidine tag), (b) substituted for one or more amino acids, independently selected from one or more amino acids between amino acid residues 65-81 of SEQ ID NO: 20 (e.g. T80 substituted with GSGDQNATGSG (SEQ ID NO: 31)), and / or (c) substituted for one or more amino acids, independently selected from one or more amino acids between amino acid residues 145-149 of SEQ ID NO: 20 (e.g. G145 substituted with GSGDQNATGSG (SEQ ID NO: 31)), or at equivalent position(s) within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 1-154 of SEQ ID NO: 20, wherein SEQ ID NO: 20 optionally comprises one or more substitutions selected from: M3 II, K120L, A124R, M144I, and I154M.

[0035] 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 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: M3 II, K120L, A124R, M144I, and I154M.

[0036] 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 at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 185-426 of SEQ ID NO: 26, modified in that the amino acid sequence comprises one (or more) consensus sequence(s) of D / E-X-N-Z-S / T (SEQ ID NO: 49), wherein X and Z are independently any amino acid except proline, wherein the one (or more) consensus sequences have each been added next to or substituted for one or more amino acids at the N-terminus (wherein the consensus sequence is optionally preceded or followed by a histidine tag), or at equivalent position(s) within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 185-426 of SEQ ID NO: 26. In aspects, SEQ ID NO: 26 optionally comprises one or more substitutions selected from: A187T, F196Y, T281N, P314S, A352V, L425I, and A427-428.

[0037] In another aspect, a modified E2p NP subunit is provided comprising an amino acid sequence of amino acid residues 185-426 of SEQ ID NO: 26 of SEQ ID NO: 26 or a selfassembling fragment thereof or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acid residues 185-426 ofSEQ ID NO: 26 or a self-assembling fragment thereof. SEQ ID NO: 26 optionally comprises one or more substitutions selected from: A187T, F196Y, T281N, P314S, A35V, L425I, and A427-428.

[0038] In an eleventh aspect of the present embodiments provided herein, a modified carrier protein fused to a NP is provided, wherein the modified glycoconjugated carrier protein NP is a modified Exotoxin A of Pseudomonas aeruginosa (EP A) protein fused to ferritin having an amino acid sequence of SEQ ID NO: 52 or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 52, modified in that the amino acid sequence comprises one (or more) consensus sequence(s) of D / E-X-N-Z-S / T (SEQ ID NO: 49), wherein X and Z are independently any amino acid except proline, wherein the one (or more) consensus sequences have each been added next to or substituted for one or more amino acids.

[0039] In another aspect, a modified EPA ferritin NP subunit is provided comprising an amino acid sequence of amino acid residues 35-855 of SEQ ID NO: 52 or a self-assembling fragment thereof or an amino acid sequence 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-assembling fragment thereof. SEQ ID NO: 52 optionally comprises one or more substitutions selected from: L552V and delta553. In a twelfth aspect, there is provided a conjugate (e.g. bioconjugate) comprising a modified NP or a modified carrier protein fused to a NP and conjugated to an antigen (e.g. a saccharide antigen, optionally a bacterial polysaccharide or oligosaccharide). According to a further aspect, there is provided a conjugate (e.g. bioconjugate) comprising a modified ferritin nanoparticle linked to an antigen (e.g. a saccharide antigen, optionally a bacterial polysaccharide or oligosaccharide). According to a further aspect of the embodiments provided herein, there is provided a conjugate (e.g. bioconjugate) comprising a modified dodecin nanoparticle linked to an antigen (e.g. a saccharide antigen, optionally a bacterial polysaccharide or oligosaccharide). According to a further aspect of the embodiments provided herein, there is provided a conjugate (e.g. bioconjugate) comprising a modified E2p nanoparticle linked to an antigen (e.g. a saccharide antigen, optionally a bacterial polysaccharide or oligosaccharide). According to a further aspect of the embodiments provided herein, there is provided a conjugate (e.g. bioconjugate) nanoparticle comprising a modified carrier protein fused to an optionally modified ferritin nanoparticle subunit linked to an antigen (e.g. a saccharide antigen, optionally a bacterial polysaccharide or oligosaccharide).

[0040] In a thirteenth aspect of the embodiments provided herein, there is provided a polynucleotide encoding a modified NP or a modified carrier protein fused to an NP. According to a further aspect, there is provided a polynucleotide encoding an optionally modified dodecin nanoparticle . According to a further aspect, there is provided a polynucleotide encoding a modified ferritin nanoparticle. According to a further aspect, there is provided a polynucleotide encoding a modified E2p nanoparticle. According to a further aspect, there is provided a polynucleotide encoding a modified EPA fused to a ferritin nanoparticle.

[0041] In a fourteenth aspect, there is provided a first vector 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, there is provided a second vector comprising a polynucleotide encoding an oligosaccharide or polysaccharide gene cluster. For example, the nucleotide may encode a pneumococcal capsular polysaccharide from any suitable serotype (e.g. Spl2F, Sp33F) or a Klebsiella pneumoniae polysaccharide (e.g. KpO3b). According to a further aspect, there is provided a third vector comprising a polynucleotide encoding a glycosyltransferase (e.g. pglB). According to a further aspect, the polynucleotide encoding the modified NP or modified carrier protein fused to a NP, the polynucleotide encoding an oligosaccharide or polysaccharide gene cluster, and the polynucleotide encoding a glycosyltransferase may be provided in one, two or three vectors. In another aspect, the vector may comprise any two or more of the nucleotides encoding a glycosyltransferase, an oligosaccharide or polysaccharide gene cluster, or a modified NP or modified carrier protein fused to a NP.

[0042] According to a further aspect, there is provided a vector comprising a polynucleotide encoding a dodecin protein (e.g. a modified dodecin protein). According to a further aspect, there is provided a second vector comprising a polynucleotide encoding an oligosaccharide or polysaccharide gene cluster. For example, the vector may encode a pneumococcal capsular polysaccharide from any suitable serotype (e.g. Spl2F, Sp33F) or a Klebsiella pneumoniae polysaccharide (e.g. KpO3b). According to a further aspect, there is provided a third vector comprising a polynucleotide encoding glycosyltransferase (e.g. pglB).

[0043] According to a further aspect, there is provided a vector comprising a polynucleotide encoding a ferritin protein from H. pylori (e.g. a modified ferritin Hp protein). According to a further aspect, there is provided a second vector comprising a polynucleotide encoding an oligosaccharide or polysaccharide gene cluster. For example, the vector may encode a pneumococcal capsular polysaccharide from any suitable serotype (e.g. Spl2F, Sp33F) or a Klebsiella pneumoniae polysaccharide (e.g. KpO3b). According to a further aspect, there is provided a third vector comprising a polynucleotide encoding glycosyltransferase (e.g. pglB).

[0044] According to a further aspect, there is provided a vector comprising a polynucleotide encoding a ferritin protein from P. aeruginosa (e.g. a modified ferritin Pa protein). According to a further aspect, there is provided a second vector comprising a polynucleotide encoding an oligosaccharide or polysaccharide gene cluster. For example, the vector may encode a pneumococcal capsular polysaccharide from any suitable serotype (e.g. Spl2F, Sp33F) or a Klebsiella pneumoniae polysaccharide (e.g. KpO3b). According to a further aspect, there is provided a third vector comprising a polynucleotide encoding glycosyltransferase (e.g. pglB).

[0045] According to a further aspect, there is provided a vector comprising a polynucleotide encoding a E2p protein from G. stearothermophilus (e.g. a modified E2p protein). According to a further aspect, there is provided a second vector comprising a polynucleotide encoding an oligosaccharide or polysaccharide gene cluster. For example, the vector may encode a pneumococcal capsular polysaccharide from any suitable serotype (e.g. Spl2F, Sp33F) or a Klebsiella pneumoniae polysaccharide (e.g. KpO3b). According to a further aspect, there is provided a third vector comprising a polynucleotide encoding glycosyltransferase (e.g. pglB). According to a further aspect, there is provided a vector comprising a polynucleotide encoding an EPA-ferritin protein from H. pylori (e.g. a modified EPA-ferritin fusion or a carrier protein nanoparticle fusion protein). According to a further aspect, there is provided a second vector comprising a polynucleotide encoding an oligosaccharide or polysaccharide gene cluster. For example, the vector may encode a pneumococcal capsular polysaccharide from any suitable serotype (e.g. Spl2F, Sp33F) or a Klebsie Ila pneumoniae polysaccharide (e.g. KpO3b). According to a further aspect, there is provided a third vector comprising a polynucleotide encoding glycosyltransferase (e.g. pglB).

[0046] In a fifteenth aspect, there is provided a host cell comprising: i) one or more nucleotide sequences comprising polysaccharide synthesis genes, optionally for producing a bacterial polysaccharide antigen (e.g. an N-antigen from a gramnegative bacterium optionally from pneumococcal capsular polysaccharide (e.g. serotype Spl2F); an O-antigen from a gram-negative bacterium optionally from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa (e.g. KpO3b). Klebsiella pneumoniae, pneumoniae or a capsular polysaccharide from a gram-positive bacterium optionally from Streptococcus pneumoniae or Staphylcoccus aureus) or a yeast polysaccharide antigen or a mammalian polysaccharide antigen, optionally integrated into the host cell genome; ii) a nucleotide sequence encoding a heterologous glycotransferase, optionally within a plasmid; iii) a nucleotide sequence that encodes a modified nanoparticle subunit or a modified carrier protein fused to a nanoparticle, optionally within a plasmid.

[0047] In a sixteenth aspect, there is provided an immunogenic composition comprising a conjugate (e.g. bioconjugate) and optionally a pharmaceutically acceptable excipient and / or carrier (optionally with an adjuvant). According to a further aspect, there is provided a vaccine comprising an immunogenic composition and optionally an adjuvant.

[0048] In a seventeenth aspect, there is provided a process for producing a bioconjugate nanoparticle that comprises (or consists of) a nanoparticle (e.g. a modified nanoparticle or a modified carrier protein fused to a nanoparticle) linked to a polysaccharide or oligosaccharide, said process comprising: (i) culturing the host cell under conditions suitable for the production of a bioconjugate nanoparticle (or bioconjugate carrier protein fused to a nanoparticle) and (ii) isolating the bioconjugate nanoparticle (or carrier protein fused to a nanoparticle) produced by said host cell, optionally isolating the bioconjugate nanoparticle from a periplasmic extract from the host cell. In an eighteenth aspect, there is provided a method of inducing an immune response in a subject (e.g. human), the method comprising administering a therapeutically or prophylactically effective amount of a conjugate (e.g. bioconjugate), an immunogenic composition or a vaccine to a subject (e.g. human) in need thereof.

[0049] In a ninteenth aspect, there is provided a conjugate (e.g. bioconjugate), an immunogenic composition, or a vaccine, for use in inducing an immune response in a subject (e.g. human).

[0050] In a twentieth aspect of the embodiments provided herein, there is provided a conjugate (e.g. bioconjugate), an immunogenic composition, or a vaccine, for use in the manufacture of a medicament inducing an immune response in a subject (e.g. human).

[0051] In a twenty-first aspect, there is provided a nanoparticle monomer subunit comprising amino acids 1 to 79 or 2 to 73 of SEQ ID NO: 2. In general, carrier modules may be fused to the N or C terminus of SEQ ID NO: 2.

[0052] In a twenty-second aspect, there is provided a protein nanoparticle comprising one or more sites (e.g. one or more amino acid residues within a monomer sequence) on its exterior or interior surface suitable for conjugation of a display molecule (e.g. an antigenic or immunostimulating molecule).

[0053] One or more amino acid residues within a polypeptide subunit sequence may be modified using methods known in the art to provide a site suitable for chemical / biochemical conjugation to a heterologous molecule (e.g. to an antigenic or immunostimulating molecule) at a nanoparticle exterior or interior surface, where such modification does not prevent the polypeptide monomer from self-assembling into a nanoparticle.

[0054] Another aspect is a protein nanoparticle comprising one or more sites (e.g. one or more amino acid residues within a monomer sequence) on its exterior or interior surface suitable for conjugation of a display molecule (e.g. an antigenic or immunostimulating molecule).

[0055] In an embodiment, the amino acid residue on the modified nanoparticle or modified carrier protein fused to the nanoparticle subunit protein is selected from the group consisting of: Ala, Arg, Asp, Cys, Gly, Glu, Gin, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai. Optionally, the amino acid is: an amino acid containing a terminal amine group, a lysine, an arginine, a glutaminic acid, an aspartic acid, a cysteine, a tyrosine, a histidine or a tryptophan. In an aspect, the antigen is conjugated to an amino acid on the modified nanoparticle or modified carrier protein selected from asparagine, aspartic acid, glutamic acid, lysine, cysteine, tyrosine, histidine, arginine or tryptophan (e.g. asparagine) and in the case of asparagine, the conjugate may be a bioconjugate (for example an enzymatic conjugation using an oligosaccharyltransferase such as PglB). In an embodiment, the amino acid residue on the modified nanoparticle or modified carrier protein to which the antigen is linked is an asparagine residue. In aspects, the amino acid residue on the modified nanoparticle or modified carrier protein to which the antigen is linked is part of the consensus sequence, e.g. the asparagine in D / E-X-N-Z-S / T (SEQ ID NO: 49), K-D / E-X-N-Z-S / T- K (SEQ ID NO: 50) or J-D / E-X-N-Z-S / T-U (SEQ ID NO: 51) consensus sequence.

[0056] The conjugate may be a bioconjugate of a recombinant modified nanoparticle or a recombinant modified carrier protein fused to an optionally modified nanoparticle. The conjugate may be a bioconjugate of an isolated recombinant modified nanoparticle or recombinant modified carrier protein fused to a nanoparticle subunit.

[0057] DESCRIPTION OF DRAWINGS / FIGURES

[0058] FIG. 1A shows a wild type amino acid sequence of ferritin from Helicobacter pylori, with loop 1 (LI), loop 2 (L2), loop 3 (L3), and loop 4 (L4) labeled.

[0059] FIG. IB shows a wild type amino acid sequence of ferritin from Pseudomonas aeruginosa, with loop 1 (LI), loop 2 (L2), loop 3 (L3), and loop 4 (L4) labeled.

[0060] FIG. 1C shows a structural alignment of ferritin subunit from Helicobacter pylori (Hp, light gray) with ferritin subunit from Pseudomonas aeruginosa (Pa, dark gray). Despite a low level of sequence conservation between ferritins (as shown by the sequence alignment between Pa ferritin and Hp ferritin in FIG. ID), the tertiary structures of the single subunits (as shown by FIG. 1C) and nanoparticles are very similar. The positions for introducing glycosites in Pa ferritin are represented by open spheres and correspond to the highlighted residues in FIG. ID.

[0061] FIG. ID shows an amino acid sequence alignment between ferritin from Pseudomonas aeruginosa and from H pylori. Accordingly, the present embodiments extend to all such ferritins with suitable tertiary structures with a surface exposed N-terminus and / or loop domains (e.g., LI, L2, L3 and L4) of single subunits and corresponding nanoparticles.

[0062] In an aspect, the positions for glycosite insertion with Hp ferritin are as follows: (1) N-terminus

[0063] - “glycosite and His tag” GGSKDQNRTKDGSGHHHHHH (SEQ ID NO: 58); (2) Loop 2 (L2)

[0064] - K79 replaced with GDQNATG (SEQ ID NO: 35); (3) Loop 3 (L3) - DI 13 replaced with DQNAT (SEQ ID NO: 32); (4) Loop 4 (L4) - residues 146-149 replaced with GDQNATG (SEQ ID NO: 35).

[0065] The positions for glycosite insertion with Hp ferritin were also designed for Pa ferritin. FIG. 2 shows SDS-PAGE analysis of ferritin expression in the periplasm, each ferritin subunit having a different signal sequence attached at the N terminus. All samples, besides sample 10, contain a histidine tag at the N-terminus, behind the signal sequence. Sample 1 contains a glycosite between the DsbA signal sequence and the Histidine tag (this construct corresponds to SEQ ID NO: 13). Ferritin is expressed at a high level in E.coli periplasm, with the DsbA signal sequence yielding the highest periplasmic expression, followed by the XynA signal sequences. The combination of the DsbA signal sequence with N-terminal glycosite and histidine tag (SEQ ID NO: 13) leads to a particularly high expression level.

[0066] FIG. 3A shows a 3D representation of a ferritin nanoparticle with the zoom on one subunit highlighted in dark gray, showing sites (N terminus, Loop 1, Loop 2, Loop 3 and Loop 4) for glycosite insertion represented as black spheres according to aspects of the present embodiments.

[0067] FIG. 3B shows results of a glycosite screen by analysing IMAC (Immobilized Metal Affinity Chromatography) enriched periplasmic extract of E.coli strains producing Spl2F-antigen polysaccharide and expressing PglB and Ferritin variants with glycosite DQNAT (SEQ ID NO: 32) introduced. In this example, the upper panel shows SDS-PAGE analysis of the Spl2F-ferritin bioconjugate. The bottom panel shows Western Blot analysis (using an anti-Spl2F antibody) of Spl2F-ferritin bioconjugate. In this example, the following constructs demonstrated 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 3 internal positions (L2, L3 and L4) are shown as being suitable for glycosylation. Glycosylation sites may be evaluated at different positions and combined as shown in FIG. 3D.

[0068] FIG. 3C shows analysis of IMAC enriched periplasmic extract of E.coli strains producing / ?O3b polysaccharide and expressing PglB and Ferritin glycosite variants (Coomassie stained SDS-PA gel - upper panel; anti -His Western blot - middle panel; anti-KpO3b Western blot - lower panel). The bands corresponding to the unglycosylated Ferritin carrier, and to XpO3b-Ferritin bioconjugates with occupied glycosite are labelled. Glycosylation of ferritin glycosite variants is more efficient with KpO3b than with Spl2F. SEQ ID NOs are designated in the figure.

[0069] FIG. 3D shows selected positions for engineering of glycosites identified as suitable for ferritin nanoparticle formation. This example shows that glycosites at the N-terminus and two internal positions (N, L2, L4, and combinations thereof) can be glycosylated with Spl2F and KpO5. In the case of KpO5, it is possible to resolve glycoforms having one, two, or three glycosites occupied. SEQ ID NOs. are designated in the figure. FIG. 4A shows the last purification step of an Spl2F-ferritin bioconjugate. The impurities remaining after the anion exchange chromatography can be efficiently removed by SEC on HiPrep 26 / 60 Sephacryl S 500 HR column. Spl2F elutes at the elution volume of 180 m (first peak), while the second peak contains impurities. The fractions 2-11 containing enriched or pure Spl2F- ferritin were collected and pooled (labelled on the chromatogram).

[0070] FIG. 4B shows SDS-PAGE analysis of the SEC fractions collected from FIG. 4A. Fractions 2-11 were enriched in Spl2F-ferritin.

[0071] FIG. 5A shows SDS-PAGE analysis of ferritin from H. pylori bioconjugated to Spl2F capsular polysaccharide. The ferritin variant used in this experiment contains engineered glycosites at the N-terminus and in loop 4 (SEQ ID NO: 18). Collected fractions enriched in an Spl2F-ferritin nanoparticle were pooled together to form a final pool. The Spl2F-ferritin bioconjugate and the unglycosylated ferritin (uFerritin) are labeled on the SDS-PAGE gel.

[0072] FIG. 5B is a 3D representation of the ferritin nanoparticle from H. pylori. The spheres represent the positions where glycosites were inserted. This leads to 48 glycosites in total per nanoparticle (SEQ ID NO: 18). The structure of Spl2F repeat unit is shown as well.

[0073] FIG. 5C shows characterization of Spl2F -ferritin bioconjugate from FIG. 5 A by Dynamic Light Scattering (DLS). The measured average particle diameter is 30 nm, which indicates high Spl2F glycan load on the ferritin nanoparticle.

[0074] FIG. 5D shows assembled Spl2F-ferritin nanoparticles according to a negative stain EM. The measured particle size is around 12 nm.

[0075] FIG. 6A shows the last purification step of a KpO3b-ferritin Hp bioconjugate. The ferritin variant used in this experiment contains engineered glycosites at the N-terminus and in loop 4 (SEQ ID NO: 18). The ferritin bioconjugate was subjected to SECon Superose 6 10 / 300 column . According to SDS-PAGE analysis, fractions 12-16 contained the glycosylated KpO3b-ferritin Hp bioconjugate and were mixed to make a final pool.

[0076] FIG. 6B shows densitometry analysis of the final KpO3b-ferritin pool (shown in FIG. 7A) to determine the percentage of conjugate versus unconjugated ferritin. Around 90% of ferritin is found in the conjugated form.

[0077] FIG. 7A shows SDS-PAGE analysis of a purified KpO3b-ferritin bioconjugate, with labeling of the glycoforms with one and two glycosites occupied by KpO3b glycan. The ferritin variant used in this experiment contains engineered glycosites at the N-terminus and in loop 4 (SEQ ID NO: 18).

[0078] FIG. 7B is a 3D representation of the ferritin nanoparticle from H. pylori. The spheres represent the positions where glycosites were inserted. This leads to 48 glycosites in total per nanoparticle (SEQ ID NO: 18). The structure of the KpO3b repeat unit is shown as well.

[0079] FIG. 7C shows characterization of KpO3b-ferritin bioconjugate from FIG. 7A by Dynamic Light Scattering (DLS). The measured average particle diameter is 25 nm which indicates high KpO3b glycan load on ferritin nanoparticle.

[0080] FIG. 7D shows assembled KpO3b-ferritin nanoparticles according to a negative stain EM. The measured particle size is around 12 nm.

[0081] FIG. 8 shows analysis of periplasmic expression and glycosylation for ferritin from Pseudomonas aeruginosa by SDS-PAGE (upper panel) and Western blot against Spl2F glycan (lower panel). Engineered H.pylori ferritin variants (SEQ ID NO: 13 and 16) are used as controls. Each Pa ferritin subunit is fused with a different signal sequence attached at the N terminus and contains a glycosite either at the N-terminus (samples on the left side labeled with N) or in the loops (samples on the right side labeled with LI, L2 and L4). Pa ferritin is expressed at a high level in E.coli periplasm, with the DsbA signal sequence yielding the highest periplasmic expression (SEQ ID NO: 23), followed by the Flgl (gives a good ratio of glycosylated with single Spl2F repeat unit versus unglycosylated ferritin), MalE and RBP signal sequences. Glycosite introduced in loop 4 (L4, SEQ ID NO: 25) is more efficiently glycosylated than the glycosite in loop 2 (L2, SEQ ID NO: 24). However, the glycosite in L4 in Pa ferritin is not tolerated as well as L4 in Hp ferritin, since the expression level of this variant is decreased.

[0082] FIG. 9A shows analysis of periplasmic expression and glycosylation for dodecin from Mycobacterium tuberculosis by SDS-PAGE (upper panel) and Western blot against Spl2F glycan (lower panel). The constructs with the glycosite at the C-terminus and the following signal sequences demonstrated suitable expression: DsbA, TolB, PelB, Flgl, and Ltllb. On the right panel, glycosite insertion at the N-terminus and in the 3 loops (Mutl, Mut2, Mut3) is evaluated with DsbA signal sequence. Besides the C terminus, the N-terminus and Mut3 (loop 3) are shown as being suitable for glycosylation. Glycosites may also be combined as shown in the examples of the construct (e.g. DsbAss - dodecin-Mut3+C) (SEQ ID NO: 8).

[0083] This SDS-PAGE gel shows bands at about 66 kD corresponding to dodecin hexamers. Dodecin subunits (monomers) appear at about l lkD on SDS-PAGE gel. This SDS-PAGE running behaviour for dodecin has been reported in literature (Liu et al. 2011). Suitable signal sequences include but are not limited to DsbA, TolB, PelB, Flgl and Ltllb. Suitable positions for glycosite insertion include the N-terminus (SEQ ID NO:4), the C-terminus (SEQ ID NO: 5) and Mut3 (SEQ ID NO: 56), or the C-terminus with Mut3 (SEQ ID NO: 8). The glycosite combination Mut3+C shows a higher glycosylation than other constructs. Constructs with suitable expression and glycosylation (e.g. Spl2F glycosylation) included: 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).

[0084] FIG. 9B shows the structure of dodecin subunit. The positions tested for glycosite insertion are represented by spheres and labelled: N - N-terminus, C - C-terminus, Mutl - loop 1, Mut2 - loop 2, Mut3 - loop 3. These positions are exposed on the surface of the assembled dodecameric nanoparticle.

[0085] FIG. 9C shows a SEC chromatogram on Sephacryl S-500 HR size exclusion column as the last purification step of Spl2F-dodecin. The dodecin variant used in this experiment contains engineered glycosites at the N- and C-terminus (SEQ ID NO: 7).

[0086] FIG. 9D shows an SDS-PAGE analysis of fractions from the Sephacryl S-500 HR size exclusion column; fractions 24 to 32 correspond to the peak fractions containing Spl2F-dodecin bioconjugate.

[0087] FIG. 10A shows SDS-PAGE analysis of a purified KpO3b-dodecin bioconjugate. The dodecin variant contains engineered glycosites at the N- and C-terminus (SEQ ID NO: 7).

[0088] FIG. 10B is a 3D representation of the dodecin nanoparticle from M. tuberculosis . The spheres represent the N- and C-termini where glycosites were inserted (only shown for the 6 frontal subunits). This leads to 24 glycosites in total per nanoparticle (SEQ ID NO: 7). The structure of KpO3b repeat unit is shown as well.

[0089] FIG. IOC shows characterization of KpO3b-dodecin bioconjugate from FIG. 10A by Dynamic Light Scattering (DLS). The measured average particle diameter is 16.9 nm which indicates high KpO3b glycan load on dodecin nanoparticle.

[0090] FIG. 10D shows assembled KpO3b-ferritin nanoparticles according to a negative stain EM. The measured particle size is around 6 nm.

[0091] FIG. HA shows analysis of E2p periplasmic expression and glycosylation with Spl2F glycan by SDS-PAGE (left panel) and Western blot against histidine tag (right panel). Each E2p subunit is fused with a different signal sequence attached at the N terminus, followed by a glycosite. E2p is dihydrolipoyl transacetylase from Geobacillus stearothermophilus . E2p can be expressed at a high level in E.coli periplasm, with the TolB signal sequence yielding the highest periplasmic expression (SEQ ID NO: 29), followed by the MalE signal sequence. FIG. 11 A shows the presence of the spl2F-E2p bioconjugate at high levels with TolB-N and MalE-N signal sequence.

[0092] FIG. 11B shows a SEC chromatogram on Sephacryl S-500 HR size exclusion column as the last purification step of KpO3b-E2p. The E2p variant used in this experiment contains engineered glycosite at the N-terminus (SEQ ID NO: 29).

[0093] FIG. 11C shows an SDS-PAGE analysis of fractions from the Sephacryl S-500 HR size exclusion column; fractions 12 to 21 correspond to the peak fractions containing KpO3b-E2p bioconjugate.

[0094] FIG. 12A shows SDS-PAGE analysis of the purified KpO3b-E2p bioconjugate. The E2p variant contains engineered glycosite at the N -terminus (SEQ ID NO: 29).

[0095] FIG. 12B is a 3D representation of the E2p nanoparticle from G. stearothermophilus . E2p is a 60mer and has particle size of 24 nm. The N-terminus of each subunit is represented by a black sphere. The structure of KpO3b repeat unit is shown as well.

[0096] FIG. 12C shows characterization of KpO3b-E2p bioconjugate from FIG. 12A by Dynamic Light Scattering (DLS) . The measured average particle diameter is 42 nm which indicates that the glycan is present on the nanoparticle.

[0097] FIG. 12D shows assembled KpO3b-E2p nanoparticles according to a negative stain EM. The measured particle size is around 24 nm.

[0098] FIG. 13A shows elution profiles of the KpO3b nanoparticles on Superose 6 10 / 300 column. KpO3b bioconjugates elute according to their size, first KpO3b-E2p as the biggest glyco- nanoparticle, then KpO3b-ferritin as intermediate and as last KpO3b-dodecin as the smallest glyco-nanoparticle. The elution volumes are indicated on the chromatogram.

[0099] FIG. 13B shows a size comparison of the produced unglycosylated and KpO3b-glycosylated nanoparticles as measured by DLS. Size increase due to glycosylation can be observed for all three nanoparticles.

[0100] FIG. 14A shows an illustration of a nanoparticle in which EPA is fused to the N-terminus of ferritin from H. pylori (N-termini of ferritin subunits are shown as black spheres). In this figure, only one EPA molecule fused to one ferritin subunit (colored in dark gray) is presented. The linker between EPA and ferritin is 30 amino acids long. This carrier protein nanoparticle fusion molecule corresponds to SEQ ID NO: 52.

[0101] FIG. 14B shows a SEC chromatogram on Sephacryl S-500 HR size exclusion column for purifying Spl2F-EPA-Ferritin.

[0102] FIG. 14C shows SDS-PAGE analysis of the fractions eluted from SEC for purifying Spl2F-EPA - Ferritin in FIG. 14B. Fractions A24-A34 were enriched in the Spl2F-EPA-Ferritin nanoparticle.

[0103] FIG. 15A shows samples used in a preclinical study for testing Spl2F immunogenicity in mice using different carrier proteins for bioconjugation. Nanoparticle based carriers include an Spl2F- ferritin bioconjugate (group 4, SEQ ID NO: 18), a Spl2F -dodecin bioconjugate (group 5, SEC ID NO: 7), and an Spl2F-EPA-ferritin bioconjugate (group 6, SEQ ID NO: 52). As controls, the following have been used: PBS (group 1, negative control), Spl2F-EPA bioconjugate (group 2, previously shown not to be immunogenic for Spl2F) and Spl2F-CRM197 (group 3, previously shown to give high Spl2F immunogenicity). The quality attributes of each conjugate including sugar to protein ratio and particle size are shown in the table as well. While an equal polysaccharide of 0.22 pg was used for all groups, the protein dose was different. Aluminium -phosphate was used as adjuvant in all groups.

[0104] FIG. 15B shows IgG titers after immunization with the samples listed in FIG. 15 A. For each sample, IgG serum titers before the first immunization (pre) and after the second (post-II) and third immunization (post-III) have been measured. The highest IgG titers and responder rate was obtained using Spl2F -ferritin (SEQ ID NO: 18), even higher than when using chemical conjugate of Spl2F to CRM 197. Significant Spl2F immunogenicity was obtained when using EPA with 5 engineered glycosites fused to Ferritin (SEQ ID NO: 52) as a carrier. EPA-Ferritin fusion molecule is therefore superior to EPA as a stand-alone protein, suggesting the importance of particle size in generating potent immune response. In the case of Spl2F-dodecin the responder rate was low, but better than when using EPA as carrier.

[0105] FIG. 16A shows samples used in a preclinical study for testing KpO3b immunogenicity in mice using different carrier proteins for bioconjugation. Nanoparticle based carriers include a KpO3b- ferritin bioconjugate (group 1, SEQ ID NO: 18), a KpO3b-dodecin bioconjugate (group 2, SEQ ID NO: 7), and a KpO3b-E2p bioconjugate (group 3, SEQ ID NO: 29). As controls the following have been used: KpO3b-EPA bioconjugate (group 4) and PBS buffer (group 5). The quality attributes of each conjugate including sugar to protein ratio and particle size are shown in the table as well. While an equal polysaccharide of 0.22 pg was used for all groups, the protein dose was different. AS03 was used as adjuvant in all groups.

[0106] FIG. 16B shows IgG titers after immunization with KpO3b-ferritn (group 1, SEQ ID NO: 18) compared to the control group (group 5) as measured before the first immunization (pre) and after the third immunization (post-III). When using Dodecin, E2p and EPA as carriers for KpO3b glycan immunogenicity against KpO3b was not induced (data not shown).

[0107] FIG. 17A shows characterization of a purified Sp33F-ferritin bioconjugate by SDS-PAGE, anti- Sp33F Western blot, DLS and negative stain EM. The ferritin variant used in this example corresponds to SEQ ID NO: 18. The measured average particle diameter by DLS is 21 nm which indicates that the Sp33F glycan is present on the nanoparticle. The EM analysis indicates assembled Sp33F-ferritin nanoparticles with the measured particle size of around 12 nm.

[0108] FIG. 17B shows characterization of a purified Sp33F-dodecin bioconjugate by SDS-PAGE, anti- Sp33F Western blot, DLS and negative stain EM. The dodecin variant used in this example corresponds to SEQ ID NO: 7. The measured average particle diameter by DLS is 15 nm which indicates that the Sp33F glycan is present on the nanoparticle. The EM analysis indicates assembled Sp33F-dodecin nanoparticles with the measured particle size of around 6 nm.

[0109] DETAILED DESCRIPTION

[0110] An expression system in which a modified nanoparticle subunit, a transferase (e.g. PglB), optionally a carrier protein, and a glycosylation unit to produce saccharides is expressed in and / or translocated to the periplasm of the host cell, to produce a self-assembling bioconjugated nanoparticle (e.g. in a single step), is provided herein.

[0111] DEFINITIONS

[0112] As used herein, the term “any amino acid except proline (pro, P)” refers to an amino acid selected from the group consisting of alanine (ala, A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, 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).

[0113] As used herein, the term “naturally occurring amino acid residues” refers to amino acids that are naturally incorporated into polypeptides. In particular, 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 (gin, 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).

[0114] As used herein, the term “glycosyltransferases (GTFs, Gtfs)” refers to enzymes that establish glycosidic linkages. Glycosyltransferases are enzymes that catalyze the formation of the glycosidic linkage to form a glycoside, for example, by catalyzing the transfer of saccharide moieties from an activated nucleotide sugar (also known as the “glycosyl donor”) to a nucleophilic glycosyl acceptor molecule, the nucleophile of which can be oxygen-, carbon-, nitrogen-, or sulfurbased.

[0115] As used herein, the term “O-Antigens (also known as O-specific polysaccharides or O-side chains)” refers to a component of the surface lipopolysaccharide (LPS) of gram-negative bacteria. Examples include O-antigens from Pseudomonas aeruginosa and Klebsiella pneumoniae.

[0116] As used herein, the term “capsular polysaccharide (CP)” refers to a polysaccharide found on the bacterial cell wall. Examples include but are not limited to capsular polysaccharide from Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis and Staphylcoccus aureus.

[0117] As used herein, the term "wzy" refers to a polysaccharide polymerase gene encoding an enzyme which catalyzes polysaccharide polymerization. The encoded enzyme transfers oligosaccharide units to the non-reducing end forming a glycosidic bond.

[0118] As used herein, the term “waaL ” refers to an O antigen ligase gene encoding a membrane bound enzyme. The encoded enzyme transfers undecaprenyl-diphosphate (UPP)-bound O antigen to the lipid A core oligosaccharide, forming lipopolysaccharide.

[0119] “Modified” refers to a modification made to a protein sequence as compared to a wild type (wt) sequence, such as linking a glycosylation consensus sequence to the N-terminus or the C- terminus of a nanoparticle monomer subunit or within the amino acid sequence of the nanoparticle monomer subunit itself; modified also refers to mutations made to an amino acid sequence to improve its stability.

[0120] “Stabilized” refers to introducing one or more mutations into an amino acid sequence, for example, of a nanoparticle monomer subunit, in order to improve the stability of the nanoparticle (e.g. with regards to self assembly).

[0121] “Nanoparticle” refers to a three-dimensional structure that self assembles from a plurality of nanoparticle monomer subunits. A nanoparticle monomer or subunit is 1 subunit of a nanoparticle. A “self-assembling fragment” of a nanoparticle monomer subunit is a fragment that assembles into a nanoparticle and displays target antigens to generate an immune response against the target antigen.

[0122] In aspects, a nanoparticle may refer to one nanoparticle or a plurality of nanoparticles. In aspects, a nanoparticle may refer to a plurality of assembled monomer subunits, e.g. wherein each monomer subunit comprises one carrier protein fused to a single nanoparticle monomer subunit.

[0123] As used herein, the term "immunogenic fragment" means a portion of an antigen smaller than the whole, that is capable of eliciting a humoral and / or cellular immune response in a host animal, e.g. human, specific for that fragment. Fragments of a protein can be produced using techniques known in the art, e.g. recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Typically, fragments comprise at least 10, 20, 30, 40 or 50 contiguous amino acids of the full-length sequence. Fragments may be readily modified by adding or removing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40 or 50 amino acids from either or both of the N- and C- termini.

[0124] As used herein, the term “conservative amino acid substitution” involves substitution of a native amino acid residue with a non-native residue such that there is little or no effect on the size, polarity, charge, hydrophobicity, or hydrophilicity of the amino acid residue at that position, and without resulting in decreased immunogenicity. For example, these may be substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Conservative amino acid modifications to the sequence of a polypeptide (and the corresponding modifications to the encoding nucleotides) may produce polypeptides having functional and chemical characteristics similar to those of a parental polypeptide.

[0125] As used herein, the term “deletion” refers to the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 1 to 10 (e.g. 1 to 7 residues, 1 to 6 residues or 1 to 4 residues) are deleted at any one site within the protein molecule.

[0126] As used herein, the terms “insertion” or “addition” (including other tenses thereof such as “inserted”) means the addition of one or more non-native amino acid residues in the protein sequence or, as the context requires, addition of one or more non-native nucleotides in the polynucleotide sequence. Typically, no more than about from 1 to 10 residues, (e.g. 1 to 7 residues, 1 to 6 residues, or 1 to 4 residues) are inserted at any one site within the protein molecule.

[0127] As used herein, the term “added next to” refers to the addition of one or more non-native amino acid residues in the protein sequence at a position adjacent to the referenced amino acid or amino acid region. For example, “added next to one or more amino acids between amino acid residues 198-218” means the addition at a position adjacent to any one of amino acid residues 198- 218 (including adjacent to amino acid residues 198 or 218).

[0128] As used herein, the term “glycosite” refers to an amino acid sequence recognized by a bacterial oligosaccharyltransferase, e.g. PglB of C. jejuni.

[0129] A “consensus sequence” is a sequence have a specific structure and / or function. As used herein, the term “consensus sequence” is a sequence comprising a glycosite. A consensus sequence may be selected from: a five amino acid consensus sequence D / E-X-N-Z-S / T (SEQ ID NO: 49), a seven amino acid consensus sequence K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) or an extended consensus sequence (e.g. J-D / E-X-N-Z-S / T-U (SEQ ID NO: 51)).

[0130] As used herein, the term “introduced at” is used herein to reference the location and manner of inserting a consensus sequence into an amino acid sequence. A glycosite which is introduced at an N-terminal or C-terminal position of a protein may be added next to the amino acid sequence at the N-terminus or C-terminus, whereas a consensus sequence (or glycosite) which is introduced at a specific amino acid residue within the protein, e.g. Y208, may be substituted for that amino acid.

[0131] Unless specifically stated otherwise, providing a numeric range (e.g. “25-30”) is inclusive of endpoints (i.e. includes the values 25 and 30). For example, “between amino acids 198 to 218...of SEQ ID NO: X” refers to a position in the amino acid sequence between amino acid 198 and amino acid 218 of SEQ ID NO: X including both amino acids 198 and 218.

[0132] The terms “identical” or percent “identity” refer to nucleotide sequences or amino acid sequences that are the same or have a specified percentage of nucleotide residues or amino acid residues that are the same (e.g. 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identity over a specified region), when compared and aligned for maximum correspondence using, for example, sequence comparison algorithms or by manual alignment and visual inspection. Identity between polypeptides may be calculated by various algorithms. In general, when calculating percentage identity the two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. For example, the Needleman Wunsch algorithm (Needleman and Wunsch 1970, J. Mol. Biol. 48: 443-453) for global alignment, or the Smith Waterman algorithm (Smith and Waterman 1981 , J. Mol. Biol. 147: 195- 197) for local alignment may be used, e.g. using the default parameters (Smith Waterman uses BLOSUM 62 scoring matrix with a Gap opening penalty of 10 and a Gap extension penalty of 1). A preferred algorithm is described by Dufresne et al. in Nature Biotechnology in 2002 (vol. 20, pp. 1269-71) and is used in the software GenePAST (Genome Quest Life Sciences, Inc. Boston, MA). The GenePAST “percent identity” algorithm finds the best fit between the query sequence and the subject sequence and expresses the alignment as an exact percentage. GenePAST makes no alignment scoring adjustments based on considerations of biological relevance between query and subject sequences. Identity between two sequences is calculated across the entire length of both sequences and is expressed as a percentage of the reference sequence.

[0133] As used herein the term “recombinant” means artificial or synthetic. In certain embodiments, a “recombinant protein” refers to a protein that has been made using recombinant nucleotide sequences (nucleotide sequences introduced into a host cell). In certain embodiments, the nucleotide sequence that encodes a “recombinant protein” is heterologous to the host cell.

[0134] As used herein the terms “isolated” or “purified” mean a protein, conjugate (e.g. bioconjugate), polynucleotide, or vector in a form not found in nature. This includes, for example, a protein, conjugate (e.g. bioconjugate), polynucleotide, or vector having been separated from host cell or organism (including crude extracts) or otherwise removed from its natural environment. In certain embodiments, an isolated or purified protein is a protein essentially free from all other polypeptides with which the protein is innately associated (or innately in contact with).

[0135] As used herein, the term “subject” refers to an animal, in particular a mammal such as a primate (e.g. human).

[0136] As used herein, the term “effective amount,” in the context of administering a therapy (e.g. an immunogenic composition or vaccine of present embodiments) to a subject refers to the amount of a therapy which has a prophylactic and / or therapeutic effect(s). In certain embodiments, an “effective amount” refers to the amount of a therapy which is sufficient to achieve one, two, three, four, or more of the following effects: (i) reduce or ameliorate the severity of a bacterial infection or symptom associated therewith; (ii) reduce the duration of a bacterial infection or symptom associated therewith; (iii) prevent the progression of a bacterial infection or symptom associated therewith; (iv) cause regression of a bacterial infection or symptom associated therewith; (v) prevent the development or onset of a bacterial infection, or symptom associated therewith; (vi) prevent the recurrence of a bacterial infection or symptom associated therewith; (vii) reduce organ failure associated with a bacterial infection; (viii) reduce hospitalization of a subject having a bacterial infection; (ix) reduce hospitalization length of a subject having a bacterial infection; (x) increase the survival of a subject with a bacterial infection; (xi) eliminate a bacterial infection in a subject; (xii) inhibit or reduce a bacterial replication in a subject; and / or (xiii) enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0137] The term “comprises” is open-ended and means “includes.” Thus, unless the context requires otherwise, the word “comprises” or “has”, and variations thereof (including “comprise” and “comprising” or “have” and “having,” respectively), will be understood to imply the inclusion of a stated compound(s), molecule(s), composition(s), or steps, but not to the exclusion of any other compound(s), molecule(s), composition(s), or steps. The terms “comprising” and “having” when used as a transition phrase herein are open-ended whereas the term “consisting of’ when used as a transition phrase herein is closed (i.e., limited to that which is listed and nothing more). The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” “Modified carrier protein” is a carrier protein modified to include one or more glycosites. Modified carrier proteins may be fused to nanoparticle subunits and optionally may comprise modifications for stability.

[0138] “Nanoparticle subunit” is a nanoparticle protein monomer that self-assembles with other nanoparticle protein monomers to form a nanoparticle. The nanoparticle subunit may be conjugated to an antigen or may be fused to a carrier protein that is conjugated to an antigen.

[0139] A “modified nanoparticle subunit” refers to a nanoparticle subunit that has been modified for stability (e.g., one or more mutations) and / or to include one or more glycosites, wherein the glycosites may be bioconjugated to one or more antigenic molecules (e.g. polysaccharides or oligosaccharides) as provided herein. The bioconjugated nanoparticle subunit (or bioconjugated carrier protein fused to a nanoparticle subunit) self-assembles with other subunits to form an assembled nanoparticle (e.g. composed of multiple subunits). A modified nanoparticle subunit that is bioconjugated to an antigen (or a modified carrier protein bioconjugated to an antigen) may be part of a pharmaceutical composition designed to elicit an immune response against the one or more antigen molecules bioconjugated to the carrier protein or nanoparticle subunit. In aspects, nanoparticles may be used to display polysaccharide antigens to induce a host response such as an effective B cell response and / or a T cell response. In aspects, the modified nanoparticle subunit may optionally include one or more stabilizing mutations, and optionally may include one or more insertions, substitution or deletions to introduce one or more glycosites. Assembled nanoparticles may display multiple molecules (one or more antigens and / or one or more immunostimulants / carrier proteins) in an ordered array. With respect to a nanoparticle displaying one or more antigen(s) and / or immunostimulant(s), it is thought that an ordered multiplicity of antigens presented on a nanoparticle exterior surface allows multiple binding events to occur simultaneously between the nanoparticle and host cell, which favors the induction of a potent host immune response (see e.g. Lopez-Sagaseta et al., Comput Struct Biotechnol J, 14:58- 68 (2016); see also Perotti and Perez 2020 Viruses 12(35): doi: 10.3390 / vl2010035 (17 total pages); Ueda et al. 2020 eLife 9: e57659 (30 total pages)) along with conjugation to a polysaccharide (see Polonskaya et al. 2017 J. Clin. Invest. 127(4): 1492-1504; Pan etal. 2020 Adv. Mater. 32:2002940)). Nanoparticles may undergo self-assembly into highly symmetric stable and organized structures and may be modified to display antigen(s) in order to mimic the surface of a virus or bacteria.

[0140] As used herein, the term “carrier protein” refers to an immunogenic protein (e.g. such as CRM, DT, TT, EPA, etc.) which, when conjugated to an antigen (e.g. a saccharide antigen, such as a bacterial polysaccharide antigen or oligosaccharide antigen) and administered to an animal, will enhance an immune response in the animal, particularly the production of antibodies that bind specifically to the conjugated polysaccharide or oligosaccharide. In aspects, the carrier protein may be covalently linked directly to the N-terminal amino acid of the nanoparticle subunit (polypeptide monomer) or 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 placed between the carrier protein and the nanoparticle subunit. Linkers between a nanoparticle and a conjugated carrier protein include, for example, Glycine / Serine / Alanine linkers (8 to 14 amino acid residues containing repeats of Glycine, Serine, or Alanine (see W02009 / 109428 (PCI7EP2009 / 050996)).

[0141] As used herein, the term “conjugate” refers to a carrier protein or a nanoparticle covalently linked to an antigen.

[0142] As used herein, the term “bioconjugate” refers to a conjugate between a protein (e.g. a carrier protein fused to a nanoparticle subunit, or a nanoparticle subunit) and an antigen (e.g. a saccharide antigen, such as a bacterial polysaccharide antigen) prepared in a host cell background, wherein host cell machinery links the antigen to the protein (e.g. N-linked glycosylation). In an embodiment, a nanoparticle is provided which has undergone bioconjugation to a polysaccharide or oligosaccharide. In another embodiment, a carrier protein fused to a nanoparticle is provided, wherein the carrier protein has undergone bioconjugation to a polysaccharide or oligosaccharide. As used herein, the term “modified protein” means a protein that is altered (in one or more way) as compared to wild type (e.g. a “modified protein” excludes a wild type protein).

[0143] Certain embodiments provide a nucleic acid molecule comprising a polynucleotide that encodes such modified nanoparticles (comprising glycosylation sites) or fusion carrier protein nanoparticle molecules (e.g. comprising a modified carrier protein, with one or more bioconjugation sites fused to a nanoparticle subunit.

[0144] Certain embodiments provide a pharmaceutical composition comprising one or more nanoparticle monomers. Certain other embodiments provide a composition comprising a nanoparticle subunit linked to one or more antigens (e.g., saccharides) or a nanoparticle subunit linked to a carrier protein that is conjugated to an antigen). The nanoparticle subunits may assemble into nanoparticles.

[0145] Computational Design

[0146] Computational and structure-based design have been applied to design suitable nanoparticles and to identify positions within nanoparticle subunits and / or carrier proteins for glycosite insertion. For example, Rosetta and MOE may be used to identify suitable sites for modification (e.g. modifications / mutations to insert a site capable of undergoing glycosylation and / or to improve stability). Once a lead design is identified, these leads may be cloned and expressed to evaluate their expression in the periplasm as well as suitability for bioconjugation.

[0147] Glyco-competent E. coli strains transformed with different plasmids carrying different nanoparticle genes and leader sequences have been screened for efficient nanoparticle expression, bioconjugation and assembly in the periplasm. Such experiments revealed that it is technically feasible to obtain nanoparticles successfully glycosylated by PglB and correctly assembled in the bacterial periplasm to generate glyco-nanoparticles for use as bioconjugate vaccines.

[0148] Modified Nanoparticle Subunits

[0149] It is an object of present embodiments to provide a nanoparticle (NP) platform for producing bioconjugated nanoparticles (e.g. bioconjugated vaccines). A modified nanoparticle subunit (or a modified carrier protein attached to an optionally modified nanoparticle subunit) is translocated to or expressed in the periplasm of a host cell (e.g. a bacterial host cell such as E. coli) along with a glycosyltransferase (e.g. glycosyltransferase pglB). The glycosyltransferase conjugates antigens (e.g. bacterial polysaccharides) to glycosites comprising a glycosite motif (e.g. N glycosylation site: D / E-X-N-Z-S / T (SEQ ID NO: 49) wherein X and Z may be any natural amino acid except Pro; for example, the consensus sequence may be D-Q-N-X-T, wherein X may be A or R (SEQ ID NO: 53)), introduced into the nanoparticle subunit.

[0150] The present inventors have surprisingly found that the assembled glycoconjugated-NP or glycoconjugated carrier proteins fused to NPs are generated, advantageously in a single step (e.g. the polypeptides undergo expression, bioconjugation and self-assembly to form assembled nanoparticles in the periplasm). Thus, in an aspect, present embodiments relate to self-assembling nanoparticles produced in the periplasm that display polysaccharides / antigenic molecules on the external surface of the nanoparticle, to compositions comprising such nanoparticles, and to methods of making and using such nanoparticles and compositions.

[0151] Consensus sequences

[0152] The modified nanoparticle subunit proteins may comprise one or more consensus sequences (D / E-X-N-Z-S / T (SEQ ID NO: 49)), wherein X and Z are independently any amino acid except proline and J and U (see below) are independently 1 to 5 naturally occurring amino acid residues. For example, the classical 5 amino acid glycosylation consensus sequence (D / E-X- N-Z-S / T (SEQ ID NO: 49)) may be extended by 1-5 other amino acid residues on either side of the consensus sequence for more efficient glycosylation, such as

[0153] K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50)

[0154] J-D / E-X-N-Z-S / T-U (SEQ ID NO: 51)

[0155] The classical 5 amino acid glycosylation consensus sequence (D / E-X-N-Z-S / T (SEQ ID NO: 49)) may be extended by lysine residues for more efficient glycosylation (e.g. K-D / E-X-N- Z-S / T-K (SEQ ID NO: 50) see also (SEQ ID NO: 37)). Thus, consensus sequences in the modified nanoparticle subunit protein of present embodiments may comprise (or consist) of a D / E-X-N-Z- S / T (SEQ ID NO: 49) consensus sequence.

[0156] In the modified nanoparticle subunit protein of present embodiments, the consensus sequence(s) may be selected from: D / E-X-N-Z-S / T (SEQ ID NO: 49) or K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) wherein X is Q (glutamine) and Z is A (alanine) or R (arginine). In another embodiment, the consensus sequence is D / E-X-N-Z-S / T (SEQ ID NO: 49), wherein X is Q (glutamine) and Z is A (alanine) or R (arginine), e.g. D-Q-N-A-T (SEQ ID NO: 32) also referred to as “DQNAT”. In another embodiment, the consensus sequence is K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50), wherein X is Q (glutamine) and Z is A (alanine), e.g. K-D-Q-N-A-T-K (SEQ ID NO: 37) also referred to as “KDQNATK”. In another embodiment, the consensus sequence(s) may be selected from: D / E-X-N-Z-S / T (SEQ ID NO: 49), K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) or J- D / E-X-N-Z-S / T-U (SEQ ID NO: 51) wherein X is Q (glutamine), Z is A (alanine) or R (arginine), J and U are independently 1 to 5 amino acid residues independently selected from glycine and / or serine.

[0157] Examples of consensus sequences that include the classical 5 amino acid glycosylation consensus sequence (D / E-X-N-Z-S / T (SEQ ID NO: 49)) that has been extended by 1-5 amino acids on either side:

[0158] KDQNATK SEQ ID NO: 37

[0159] GDQNATG SEQ ID NO: 35

[0160] GGDQNATGG SEQ ID NO: 39 GSGDQNATGSG SEQ ID NO: 31

[0161] GGSKDQNRTKDGSG SEQ ID NO: 33

[0162] GGSKDQNATKDGSG SEQ ID NO: 34

[0163] GSGKDQNRTKDGSG SEQ ID NO: 30

[0164] GSGGGDQNATGSGGG SEQ ID NO: 36

[0165] In aspects, the modified nanoparticle subunit protein of present embodiments comprises one, two, three, four, five, six, or seven or more of any of SEQ ID NOs: 30 - 39.

[0166] In an embodiment, the modified nanoparticle subunit protein of present embodiments comprises at least two D / E-X-N-Z-S / T (SEQ ID NO: 49) or K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) consensus sequences. In an embodiment, the modified nanoparticle subunit protein comprises at least three D / E-X-N-Z-S / T (SEQ ID NO: 49) or K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) consensus sequences. In an embodiment, the modified nanoparticle subunit protein comprises at least four D / E-X-N-Z-S / T (SEQ ID NO: 49) or K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) consensus sequences. In an embodiment, the modified nanoparticle subunit protein comprises at least five D / E-X-N-Z-S / T (SEQ ID NO: 49) or K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) consensus sequences. In an embodiment, the modified nanoparticle subunit protein comprises at least six D / E-X-N-Z-S / T (SEQ ID NO: 49 or K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) consensus sequences. In an embodiment, the modified nanoparticle subunit protein comprises at least seven D / E-X-N- Z-S / T (SEQ ID NO: 49) or K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) consensus sequences. In an embodiment, the modified nanoparticle subunit protein contains three to seven D / E-X-N-Z-S / T (SEQ ID NO: 49) or K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) consensus sequences. In an embodiment, the modified nanoparticle subunit protein contains four to seven D / E-X-N-Z-S / T (SEQ ID NO: 49) or K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) consensus sequences. In an embodiment, the modified nanoparticle subunit protein contains five to seven D / E-X-N-Z-S / T (SEQ ID NO: 49) or K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) consensus sequences. In aspects, any of these sequences may be extended by 1 -5 amino acids on either or both sides of the consensus sequence.

[0167] Introduction of such glycosylation sites can be accomplished by, e.g. adding new amino acids to the primary structure of the nanoparticle subunit protein (i. e. the glycosylation sites are added, in full or in part), or by mutating existing amino acids in the protein in order to generate the glycosylation sites (i.e. amino acids are not added to the protein, but selected amino acids of the protein are mutated so as to form glycosylation sites). In an embodiment, the consensus sequence(s) are recombinantly introduced into the nanoparticle subunit amino acid sequence or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to said sequence.

[0168] Signal sequences

[0169] In an embodiment, the modified nanoparticle subunit protein comprises a signal sequence which is capable of directing the carrier protein nanoparticle subunit fusion molecule to the periplasm ofa host cell (e.g. bacterium). Signal sequences, including periplasmic signal sequences, are usually removed during translocation of the protein into, for example, the periplasm by signal peptidases (i.e. a mature protein is a protein from which at least the signal sequence has been removed). The signal sequence may be from E. coli DsbA [MKKIWLALAGLVLAFSASA (SEQ ID NO: 10)], TolB [MKQALRVAFGFLILWASVLHA (SEQ ID NO: 41)], E. coli flagellin (Figi) [MIKFLSALILLLVTTAAQA (SEQ ID NO: 42)], E. coli outer membrane porin A (OmpA) [MKKTAIAIAVALAGFATVAQA (SEQ ID NO: 43)], E. coli maltose binding protein (MalE) [MKIKTGARILALSALTTMMFSASALA (SEQ ID NO: 44)], Erwinia carotovorans pectate lyase (PelB) [MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 45)], heat labile E. coli enterotoxin LTIIb [MSFKKIIKAFVIMAALVSVQAHA (SEQ ID NO: 46)], Bacillus subtilis endoxylanase XynA [MFKFKKKFLVGLTAAFMSISMFSATASA (SEQ ID NO: 47], or SipA [MKMNKKVLLTSTMAASLLSVASVQAS (SEQ ID NO: 48], In a specific embodiment, the signal sequence is from E. coli DsbA [MKKIWLALAGLVLAFSASA (SEQ ID NO: 10)].

[0170] Thus, present embodiments provide a modified nanoparticle subunit protein, wherein the amino acid sequence further comprises a signal sequence which is capable of directing the nanoparticle carrier protein molecule to the periplasm of a host cell (e.g. bacterium), said signal sequence being any suitable sequence. Techniques for fusing signal peptide to a nanoparticle subunit are known in the art (e.g. a signal peptide of the protein DsbA from E. coli can be genetically fused to the N-terminus of the mature nanoparticle subunit protein sequence) (see, Schulz, H., Hennecke, H., and Thony-Meyer, L., Science, 281, 1197-1200, 1998).

[0171] It will be understood by a person skilled in the art, that reference to “between amino acids ...” (for example “between amino acids 198-218”) is referring to the amino acid number counting consecutively from the N-terminus of the amino acid sequence, for example “between amino acids 49-55 of SEQ ID NO: X” refers to position in the amino acid sequence between amino acid 49 and amino acid 55 of SEQ ID NO: X including both amino acids 49 and 55. Thus, with respect to a nanoparticle subunit protein and in an embodiment where a consensus sequence selected from D / E-X-N-Z-S / T (SEQ ID NO: 49) and K-D / E-X-N-Z-S / T-K (SEQ ID NO: 50) (e.g. K-D-Q-N-A- T-K (SEQ ID NO: 37)) has been added next to or substituted for one or more amino acids between amino acid residues 49-55, the consensus sequence may have been added next to or substituted for any one (or more) of amino acid numbers 49, 50, 51, 52, 53, 54 and 55 in SEQ ID NO: X.

[0172] The amino acid numbers referred to herein correspond to the amino acids in SEQ ID NO: X and as described above, a person skilled in the art can determine equivalent amino acid positions in an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: X by alignment. The addition or deletion of amino acids from the variant and / or fragment of SEQ ID NO: X could lead to a difference in the actual amino acid position of the consensus sequence in the mutated sequence, however, by lining the mutated sequence up with the reference sequence, the amino acid in an equivalent position to the corresponding amino acid in the reference sequence can be identified and hence the appropriate position for addition or substitution of the consensus sequence can be established. In aspects, mutation positions listed with respect to a sequence may refer to a first sequence (e.g. having mutations) aligned with a reference sequence (e.g. wt sequence).

[0173] Specific examples follow.

[0174] Polypeptide Monomers & Protein Nanoparticles

[0175] Nanoparticles made of non-viral protein subunits may display antigenic molecules on the exterior surface. Such nanoparticles include those made of bacterial, insect, and mammalian proteins that naturally self-assemble into nanoparticles. Herein, cells were engineered to produce bioconjugate nanoparticles. In an aspect, the nanoparticle monomer, containing the glycosylation site, is translocated to or expressed in the periplasm, wherein it undergoes glycosylation and assembly to form a nanoparticle. In other aspects, a carrier protein containing the glycosylation site(s) is conjugated to the nanoparticle monomer and translocated to or expressed in the periplasm, wherein it undergoes glycosylation and assembly. In aspects, a leader sequence is present. The modified nanoparticle subunit may include one, two, three, four or more mutations for stability.

[0176] As provided herein, nanoparticles include but are not limited to ferritin, E2p, dodecin, and EPA. These nanoparticles were shown to undergo bioconjugation and assembly into nanoparticles in the periplasm.

[0177] Dodecin Subunit Protein

[0178] Dodecin subunit protein (also known as mtDod) is a soluble secretory protein of about 8 kDa with a p«pp topology (Bourdeaux et al., Nature Scientific Reports, 2020, 10: 13297), encoded by the gene Rvl498a of Mycobacterium tuberculosis (Liu et al. Journal of Structural Biology, 2011, 175:31-18), which self-assembles into a dodecamer of spherical shape. In aspects, N- and C-termini are exposed at the protein surface (Bourdeaux et al., Nature Scientific Reports, 2020, 10: 13297), facilitating its use as a carrier molecule. A dodecin nanoparticle may assemble from 12 subunits to form a spherical shape with about a 6 nm diameter.

[0179] A dodecin subunit protein of present embodiments can be produced by methods provided herein. The amino acid sequence of wildtype dodecin is shown in SEQ ID NO: 1 (Uniprot: Q8VK10). Modifications to dodecin were made to improve stability, with said modifications at one, two or three of positions G25, V50 and A53 (e.g., G25N, V50T, A53T), wherein the positions are with respect to the wt sequence. A nucleotide sequence for the modified amino acid sequence of dodecin is provided at SEQ ID NO: 3.

[0180] In one aspect of present embodiments, the dodecin subunit protein is modified to introduce glycosylation sites at designated positions in the amino acid sequence. Modifications to introduce a glycosylation site may be at one or more of the N-terminus, the C terminus, or internally 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, which is expressed in the periplasm wherein it undergoes glycosylation. The modified glycosylated dodecin subunit protein further undergoes selfassembly in the periplasm to form an assembled glycosylated nanoparticle. In aspects, dodecin has been 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 may optionally be linked to a signal sequence at the N- terminus and / or a histidine tag for purification (e.g. at the N- or C- terminus). The modified dodecin subunit protein may comprise stabilizing mutations at one or more of glycine to asparagine (G25N), valine to threonine (V50T), and alanine to threonine (A53T) with reference to the amino acid sequence of SEQ ID NO: 1 (or an equivalent position in an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to 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 at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1) and comprising substitution of glycine to asparagine (G25N). The modified dodecin subunit protein of present embodiments may be the amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1) and comprising substitution of valine to threonine (V50T). The modified dodecin subunit protein of the invention may be the amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1) and comprising substitution of alanine to threonine (A53T).

[0181] In aspects, the modified dodecin subunit protein of present embodiments includes amino acids 2-70 of SEQ ID NO: 2 or a self-assembling fragment thereof (or an amino acid sequence 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-assembling fragment thereof) comprising one or more of substitution of glycine to asparagine (G25N), valine to threonine (V50T), and alanine to threonine (A53T). In aspects, the modified dodecin subunit protein of present embodiments includes amino acids 2-70 of SEQ ID NO: 2 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 2-70 of SEQ ID NO: 1 comprising two or more of G25N, V50T, and A53T. In aspects, the modified dodecin subunit protein of present embodiments includes amino acids 2-70 of SEQ ID NO: 2 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 2-70 of SEQ ID NO: 1 comprising all of G25N, V50T, and A53T as shown by SEQ ID NO: 2.

[0182] Thus, in aspects, stability 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, the specific mutations may include G25N, V50T, and A53T.

[0183] In aspects, the dodecin subunit may be modified to include a glycosylation site by appending or replacing one or more residues (e.g. methionine) at the N terminus as shown by SEQ ID NO: 4. In other aspects, dodecin subunit may be modified to include a glycosylation site by replacing one or more residues at the C terminus or by appending the glycosylation site to the C- terminus (e.g. SEQ ID NO: 5) or both. In still other aspects, dodecin may be modified to include a glycosylation site within the amino acid sequence of loop 3 (Mut3) (e.g. residues 49-55, 51) as shown by SEQ ID NO: 6. In still other aspects, dodecin may be modified to include a glycosylation site at the N-terminus and the C-terminus as shown by SEQ ID NO: 7; to include a glycosylation site at the C-terminus and within the amino acid sequence of loop 3 (Mut3) as shown by SEQ ID NO: 8; or to include a glycosylation site at the N-terminus and C-terminus and within the amino acid sequence of loop 3 (Mut3) as shown by SEQ ID NO: 9. In aspects, the modified dodecin subunit protein of present embodiments may be the amino acid sequence of any of SEQ ID NOs: 4-9 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 4, 5, 6, 7, 8, or 9).

[0184] In still other aspects, a signal sequence may replace one or more N-terminal residues or be appended at the N terminus, e.g. upstream of the glycosylation site. Any suitable signal sequence (e g. DsbA (SEQ ID NO: 10), TolB (SEQ ID NO: 41), PelB (SEQ ID NO: 45), Figi (SEQ ID NO: 42), LtllB (SEQ ID NO: 46)) may be used. In aspects, a signal sequence may be appended at the N terminus (e.g. upstream of the glycosylation site) of the amino acid sequence of SEQ ID NO: 1 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 1)).

[0185] In aspects, the modified sequence optionally may include a Poly His tag (e.g. a string of 5 to 7 histidine residues, or 6 histidine resides) at the N or C terminus for purification of the amino acid sequences of SEQ ID NOs: 4-9 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 4-9).

[0186] According to aspects, an assembled glycosylated nanoparticle is provided in a single step. The method comprises the steps of providing a host cell; expressing a modified protein (e.g. a modified dodecin subunit comprising an antigen) and glycosyltransferase pglB in the host cell to produce subunits that self-assemble in the periplasm to form a glycoprotein nanoparticle. Polysaccharides are produced by the host cell based on the techniques provided herein along with any other proteins needed to translocate the polysaccharides to the periplasm.

[0187] A person skilled in the art will understand that for variant dodecin amino acid sequences with respect to SEQ ID NO: 1 and / or fragments of an amino acid sequence of SEQ ID NO: 1, such an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, and with reference to “between amino acids ...” refers to the position that would be equivalent to the defined position, if this sequence was lined up with an amino acid sequence of SEQ ID NO: 1 in order to maximise the sequence identity between the two sequences (Sequence alignment tools are not limited to Clustal Omega (www(.)ebi(.)ac(.)ac(.)uk) MUSCLE (www(.)ebi(.)ac(.)uk), or T-coffee (www(.)tcoffee(.)org). In one aspect, the sequence alignment tool used is Clustal Omega (www(.)ebi(.)ac(.)ac(.)uk).

[0188] Ferritin Subunit Protein

[0189] Ferritin may be found in several different organisms, including vertebrates, amphibians, plants, bacteria and humans. Ferritin is a globular protein with a mass of about 474 kDa and acts as an intracellular iron storage protein, and may be secreted into serum to act as an iron transporter. Nanoparticles based on insect and human ferritin have been described for use in displaying, on the NP surface, antigens (see, e.g. W02018 / 005558; Kwong et al. (2018)). Li et al. described a nanoparticle made of recombinant fusion polypeptides comprising a human ferritin light-chain subunit and a short HIV-1 antigenic peptide attached to the amino terminus of the ferritin lightchain sequence, with self-assembly of these fusion polypeptides resulting in placement of the HIV- 1 antigenic peptide at the exterior surface of the NP (see Li et al., Ind. Biotechnol. 2: 143-47 (2006)).

[0190] Ferritin may be modified by introducing one or more glycosites into amino acid residue(s) exposed at the nanoparticle surface. Sites for modification are selected so as to not interfere with nanoparticle formation but are present on the nanoparticle surface and suitable for antigen display. For example, suitable positions for introduction of glycosites include the N-terminus, loop 1 (LI), the long loop between helix 2 and 3 (L2), the short loop between helix 3 and 4 (L3), and the loop between helix 4 and (5) L4. A ferritin nanoparticle may assemble from 24 subunits to form a spherical shape with a 12 nm diameter. For 2 and 3 glycosites per ferritin subunit, this leads to 48 and 72 positions for glycan attachment per ferritin nanoparticle. Present embodiments encompass any ferritin molecule from any species; representative examples are below.

[0191] Ferritin has been shown to self-assemble into a nanoparticle when recombinantly expressed for example, in a prokaryotic expression system. The ferritin sequences herein may be recombinantly modified to contain a short amino acid tag to aid in purification, such as a histidine tag as is known in the art, which may optionally be joined to the ferritin sequence via a short peptide linker. Lysine or asparagine residues exposed at the surface of the NP may be used in conjugating glycans to the NP surface.

[0192] Suitable signal sequences include DsbA (SEQ ID NO: 10), TolB (SEQ ID NO: 41), MalE (SEQ ID NO: 44), XynA (SEQ ID NO: 47).

[0193] Ferritin HP H. pylori bacterial ferritin (see Protein Data Bank (PDB) Accession Number Q9ZLI1) has been investigated for use as a pharmaceutically acceptable carrier. H. pylori bacterial ferritin consists of 24 identical polypeptide subunits that self-assemble into a spherical nanoparticle. Li et al. reported preparation of a nucleotide sequence encoding a fusion of bacterial (H. pylori) ferritin subunit polypeptide and a rotavirus antigen, with expression in a prokaryotic (E. coli) system. The expressed fusion polypeptides are described as self-assembling into spherical NPs displaying the rotavirus capsid protein, and capable of inducing an immune response in mice (Li et al., J Nanobiotechnol 17: 13 (2019)). Wang et al. designed chimeric polypeptides comprising H. pylori ferritin and antigenic peptides from N. gonorrhoeae,' the chimeric polypeptide is described as assembling into a 24-mer nanoparticle displaying the antigenic peptides on the NP exterior surface. (Wang et al., FEBS Open Bio 7(8): 1196 (2017)). Kanekiyo et al. described a self-assembling recombinant bacterial (H. pylori) ferritin nanoparticle (24-mer), comprising fusions of the ferritin subunit polypeptide and influenza antigenic peptides, which displayed influenza trimers on its surface (Kanekiyo et al., Nature 499(7456): 102 (2013)).

[0194] The amino acid sequence (SEQ ID NO: 11) and nucleotide sequence (SEQ ID NO: 12) for ferritin H. pylori (e.g. Uniprot: Q9ZLI1) are provided herein.

[0195] In one aspect, the ferritin subunit protein is modified to produce a modified ferritin subunit protein, which is expressed and undergoes glycosylation (e.g. Spl2). The modified glycosylated ferritin subunit protein further undergoes self-assembly in the periplasm to form an assembled glycosylated nanoparticle. In aspects, ferritin has been 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). In aspects, ferritin may be linked to a signal sequence at its N- terminus and / or linked to a histidine tag for purification at the N-terminus.

[0196] In aspects, the modified ferritin subunit protein includes amino acids 1-167 of SEQ ID NO: 11 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 1-167 of SEQ ID NO: 11) or a fragment thereof. In aspects, ferritin may be modified to include a glycosylation site at the N-terminus (SEQ ID NO: 13). In aspects, the glycosylation site is SEQ ID NO:33 or SEQ ID NO: 34. In 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 at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 13 or 14).

[0197] In other aspects, the modified ferritin subunit protein includes glycosylation sites introduced in the ferritin sequence. For example, the glycosylation site may be introduced at K79 in Loop 2, wherein glycosylation site (SEQ ID NO: 32) is introduced (SEQ ID NO: 15). As another example, the glycosylation site may be introduced at 146-149 in Loop 4, wherein glycosylation site (SEQ ID NO: 35) is introduced (SEQ ID NO: 16). 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 at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 15 or 16).

[0198] In aspects, ferritin may be modified to include one or more of a glycosite in L2, a glycosite in L4, and a glycosite at the N-terminus. For example, ferritin may be modified to include a glycosite at the N-terminus and in L2. Ferritin may be modified to include a glycosite at the N- terminus and in L4. In aspects, ferritin may be modified to include a glycosite at the N-terminus, L2 and in L4. The modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19 or a self-assembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 17, 18, or 19 or a self-assembling fragment thereof).

[0199] Optionally, the ferritin subunit protein may comprise a signal sequence at the N- terminus. Any suitable signal 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), Figi (SEQ ID NO: 42), LtllB (SEQ ID NO: 46)). In aspects, a signal sequence may be appended at the N terminus (e.g. upstream of the glycosylation site) to a modified ferritin subunit amino acid sequence as shown in SEQ ID NOs: 14-19 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 14-19). In aspects, the modified ferritin subunit protein may comprise a leader / signal sequence followed by a glycosylation site, optionally followed by a histidine tag and an optional linker (QDP), followed by ferritin (optionally with one or more internal glycosylation sites in L2 and / or L4). In aspects, a signal sequence may replace a start methionine of SEQ ID NO: 11 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11).

[0200] In aspects, the modified sequence optionally may include a Poly His tail (e.g. a string of 5 to 7 histidine residues, or 6 histidine resides) appended to the N terminus for purification of the amino acid sequences of SEQ ID NOs: 13-19 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 13-19).

[0201] According to aspects provided herein, an assembled glycosylated nanoparticle is provided in a single step. The method comprises the steps of providing a host cell: expressing a modified protein (e.g. a modified ferritin subunit comprising glycosylation site(s)) and glycosyltransferase pglB to produce the glycoprotein nanoparticle. Polysaccharides are produced by the host cell based on the techniques provided herein along with any other proteins needed to express and / or translocate the polysaccharides to the periplasm.

[0202] For example, a person skilled in the art will understand that for variant ferritin amino acid sequences with respect to SEQ ID NO: 11 and / or fragments of an amino acid sequence of SEQ ID NO: 11, such an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11, and with reference to “between amino acids ...” refers to the position that would be equivalent to the defined position, if this sequence was lined up with an amino acid sequence of SEQ ID NO: 11 in order to maximise the sequence identity between the two sequences (Sequence alignment tools are not limited to Clustal Omega (www(.)ebi(.)ac(.)ac(.)uk) MUSCLE (www(.)ebi(.)ac(.)uk), or T-coffee (www(.)tcoffee(.)org). In one aspect, the sequence alignment tool used is Clustal Omega (www(.)ebi(.)ac(.)ac(.)uk).

[0203] Ferritin PA

[0204] P. aeruginosa bacterial ferritin (see Protein Data Bank (PDB) Accession Number Q9HWF9) has been investigated for use as a pharmaceutically acceptable carrier. P. aeruginosa bacterial ferritin consists of 24 identical polypeptide subunits that self-assemble into a spherical nanoparticle.

[0205] The amino acid sequence (SEQ ID NO: 20) for P. aeruginosa bacterial ferritin (e.g. Uniprot: Q9HW59) is provided herein (see, e.g. Joyce 2021 (Science Translational Medicine) vol. 14: 632). In aspects, the amino acid sequence encoding P. aeruginosa ferritin has been modified for stability. The modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 (or an amino acid sequence 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-assembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20 or a selfassembling fragment thereof) comprising substitution of methionine to isoleucine (M3 II). The modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 or a selfassembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20 or a self-assembling fragment thereof) comprising substitution of lysine to leucine (K120L). The modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 or a self-assembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20 or a selfassembling fragment thereof) comprising substitution of alanine to arginine (A124R). The modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 or a selfassembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20 or a self-assembling fragment thereof) comprising substitution of methionine to isoleucine (Ml 441). The modified ferritin subunit protein may be the amino acid sequence of SEQ ID NO: 20 or a self-assembling fragment thereof (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20 or a self-assembling fragment thereof) comprising substitution of isoleucine to methionine (I154M). In aspects, the modified ferritin subunit protein may comprise one, two, three, four or more of mutations M3 II, K120L, A124R, M144I and / or I154M or a self-assembling fragment thereof.

[0206] In aspects, the modified ferritin subunit protein may comprise all the mutations M3 II, K120L, A124R, M144I and I154M, as shown by amino acid sequence (SEQ ID NO: 21) and corresponding nucleotide sequence (SEQ ID NO: 22) for P. aeruginosa bacterial ferritin.

[0207] In other aspects, the modified ferritin subunit comprises a signal sequence followed by a glycosylation sequence, followed by a histidine tag followed by ferritin (SEQ ID NO:23), or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 23. In aspects, the modified ferritin subunit comprises a signal sequence followed by a histidine tag followed by ferritin, with insertion of a glycosylation site at T80 in Loop 2 of ferritin (SEQ ID NO:24), or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24. In aspects, the modified ferritin subunit comprises a signal sequence followed by a histidine tag followed by ferritin, with insertion of a glycosylation site at G145 in Loop 4 of ferritin (SEQ ID NO:25), or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25.

[0208] In aspects, the modified ferritin subunit protein of present embodiments includes amino acids 1-154 of SEQ ID NO: 21 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 1-167 of SEQ ID NO: 21) or a fragment thereof. In aspects, ferritin may be modified to include a glycosylation site at the N-terminus (SEQ ID NO: 23). In aspects, the glycosylation site is SEQ ID NO: 34. In aspects, the modified ferritin subunit protein may comprise 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 in Loop 2 to residue 80 (counting from the methionine of native ferritin sequence UNIPROT ID: Q9HWF9). In aspects, T80 is substituted with a glycotag (SEQ ID NO: 31) or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 24. In other aspects, G145 is substituted with a glycotag (SEQ ID NO: 31) or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25. In aspects, ferritin may be modified to include one or more of a glycosite in L2, a glycosite in L4, and a glycosite at the N- terminus as recited herein.

[0209] Optionally, the ferritin subunit protein may comprise a signal sequence at the N-terminus. Any suitable signal sequence may be appended to the N terminus of ferritin subunit protein (e.g. DsbA (SEQ ID NO: 10), TolB (SEQ ID NO: 41), PelB (SEQ ID NO: 45), Figi (SEQ ID NO: 42), LtIIB_(SEQ ID NO: 46)). In aspects, a signal sequence (e.g. SEQ ID NO: 10) may be appended at the N-terminus (e.g. upstream of the glycosylation site) to the modified ferritin subunit amino acid sequence as shown in SEQ ID NOs: 23-25 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 23-25). In aspects, the modified ferritin subunit protein may comprise a signal sequence followed by a glycosylation site, optionally followed by a histidine tag, and followed by ferritin (optionally with one or more internal glycosylation sites in L2 and / or L4). In aspects, a signal sequence may replace a start methionine of SEQ ID NO: 21 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 23).

[0210] In aspects, the modified sequence optionally may include a Poly His tail (e.g. a string of 5 to 7 histidine residues, or 6 histidine resides) appended to the N-terminus for purification of the amino acid sequences (see, SEQ ID NOs: 23-25 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOs: 23-25)).

[0211] According to aspects, an assembled glycosylated nanoparticle is provided in a single step. The method comprises the steps of providing a host cell; expressing a modified protein (e.g. a modified ferritin subunit comprising an antigen) and glycosyltransferase pglB to produce the glycoprotein nanoparticle. Polysaccharides are produced by the host cell based on the techniques provided herein along with any other proteins needed to express and / or translocate the polysaccharides to the periplasm.

[0212] For example, a person skilled in the art will understand that for variant ferritin amino acid sequences with respect to SEQ ID NO:20 and / or fragments of an amino acid sequence of SEQ ID NO: 20, such an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 20, and with reference to “between amino acids ...” refers to the position that would be equivalent to the defined position, if this sequence was lined up with an amino acid sequence of SEQ ID NO: 20 in order to maximise the sequence identity between the two sequences (Sequence alignment tools are not limited to Clustal Omega (www(.)ebi(.)ac(.)ac(.)uk) MUSCLE (www(.)ebi(.)ac(.)uk), or T-coffee (www(.)tcoffee(.)org). In one aspect, the sequence alignment tool used is Clustal Omega (www(.)ebi(.)ac(.)ac(.)uk).

[0213] E2y

[0214] E2p is a protein found in G. stearothermophilus . An E2p nanoparticle may assemble to form a 60-mer hollow dodecahedron with about a 24 nm diameter. Recombinant E2p nanoparticles produced using recombinant gene expression in a bacterial expression system may be purified from bacterial homogenate by size exclusion chromatography (Kozlovska et al. 1993) or by a combination of fractionated ammonium sulphate precipitation and size exclusion chromatography (Vasiljeva et al (1998); Ciliens et al. (2000)).

[0215] In aspects herein, E2p nanoparticle subunits may be linked to saccharides and selfassemble to form nanoparticles. The wild type amino acid sequence (SEQ ID NO: 26) and nucleotide sequence (SEQ ID NO: 28) for E2p (e.g. Uniprot: Pl 1961) are provided herein.

[0216] In one aspect, the E2p subunit protein is modified to produce a modified E2p subunit protein, which is expressed in the host cell and undergoes glycosylation (e.g. with Spl2F). The modified glycosylated E2p subunit protein further undergoes self-assembly in the periplasm to form an assembled glycosylated nanoparticle. In aspects, E2p has been 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). In aspects, E2p may be linked to a signal sequence at its N-terminus and / or linked to a histidine tag for purification (e.g. at the N-terminus).

[0217] In aspects, the modified E2p subunit protein includes amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof (or an amino acid sequence 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 selfassembling fragment thereof) or a fragment thereof. In aspects, the amino acid sequence encoding E2p has been modified for stability. The modified E2p subunit protein may be the amino acid sequence of amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof (or an amino acid sequence 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-assembling fragment thereof) comprising substitution of 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-assembling fragment thereof (or an amino acid sequence 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-assembling fragment thereof) comprising substitution of 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-assembling fragment thereof (or an amino acid sequence 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-assembling fragment thereof) comprising substitution of 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-assembling fragment thereof (or an amino acid sequence 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-assembling fragment thereof) comprising substitution of proline to serine (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-assembling fragment thereof (or an amino acid sequence 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-assembling fragment thereof) comprising substitution of alanine to valine (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-assembling fragment thereof (or an amino acid sequence 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-assembling fragment thereof) comprising substitution of 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-assembling fragment thereof (or an amino acid sequence 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-assembling fragment thereof) comprising deletion of amino acids at positions 427 and 428 (A427-428).

[0218] In aspects, the modified E2p subunit protein may comprise one or more of mutations A187T, F196Y, T281N, P314S, A352V, L425I, A427-428 of amino acids 185-426 of SEQ ID NO: 26. In aspects, the modified E2p subunit protein may comprise all the mutations A187T, F196Y, T281N, P314S, A352V, L425I, A427-428 of amino acids 185-426 of SEQ ID NO: 26.

[0219] In aspects, E2p may be modified to include a glycosylation site at the N-terminus. In aspects, the consensus sequence comprises D / E-X-N-Z-S / T (SEQ ID NO: 49), wherein X and Z may be independently any natural amino acid except proline (e.g. the consensus sequence may be D-Q-N-X-T, wherein X may be A or R (SEQ ID NO:53)). In aspects, the glycosylation site is SEQ ID NO:36. In aspects, the modified ferritin subunit protein may comprise a signal sequence, optionally followed by a linker, optionally followed by a histidine tag and an optional linker, followed by amino acids 185-426 of SEQ ID NO: 26 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29).

[0220] Optionally, the E2p subunit protein may comprise a signal sequence at the N-terminus. Any suitable signal sequence may be appended to the N-terminus of E2p subunit protein (e.g. DsbA (SEQ ID NO: 10), TolB (SEQ ID NO: 41), PelB (SEQ ID NO: 45), Figi (SEQ ID NO: 42), LtllB (SEQ ID NO: 46)). In aspects, a signal sequence (e.g. SEQ ID NO: 10) may be appended at the N-terminus (e.g. upstream of the glycosylation site) to the modified E2p subunit amino acid sequence as shown in SEQ ID NOs: 29 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29). In aspects, the modified E2p subunit protein may comprise a signal sequence, optionally followed by a histidine tag, followed by a glycosylation site and optional linker, followed by E2p.

[0221] In aspects, the modified sequence optionally may include a poly His tail (e.g. a string of 5 to 7 histidine residues, or 6 histidine resides) appended to the N-terminus for purification of the amino acid sequence of SEQ ID NO: 29 (or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29).

[0222] According to aspects, an assembled glycosylated nanoparticle is provided in a single step. The method comprises the steps of providing a host cell; expressing a modified protein (e.g. a modified E2p subunit) and glycosyltransferase pglB in the host cell, wherein the subunit undergoes glycosylation and self-assembly to produce the glycoprotein nanoparticle. Polysaccharides are produced by the host cell based on the techniques provided herein along with any other proteins needed to translocate the polysaccharides to the periplasm.

[0223] In one aspect, the E2p subunit protein is modified to include a glycosite thereby producing a modified E2p subunit protein, for expression and glycosylation in the periplasm.

[0224] For example, a person skilled in the art will understand that for variant E2p amino acid sequences with respect to SEQ ID NO: 26 and / or fragments of an amino acid sequence of SEQ ID NO: 26, such an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 26, and with reference to “between amino acids ...” refers to the position that would be equivalent to the defined position, if this sequence was lined up with an amino acid sequence of SEQ ID NO: 26 in order to maximise the sequence identity between the two sequences (Sequence alignment tools are not limited to Clustal Omega (www(.)ebi(.)ac(.)ac(.)uk) MUSCLE (www(.)ebi(.)ac(.)uk), or T-coffee (www(.)tcoffee(.)org). In one aspect, the sequence alignment tool used is Clustal Omega (www(.)ebi(.)ac(.)ac(.)uk).

[0225] Genetic fusions In other aspects, nanoparticle subunits such as ferritin may be fused to a carrier protein such as EPA, according to techniques known in the art. The fusion of the carrier protein with ferritin may undergo expression and glycosylation in the cell and may self-assemble in the periplasm.

[0226] The modified EPA carrier protein of present embodiments may be a recombinant modified EPA carrier protein fused to a ferritin nanoparticle subunit (see, e.g. SEQ ID NO: 52). The modified EPA protein of present embodiments may be an isolated recombinant modified EPA protein fused to a ferritin nanoparticle subunit.

[0227] In aspects, the modified nanoparticle subunit protein may further comprise a “peptide tag” or “tag”, i.e. a sequence of amino acids that allows for the isolation and / or identification of the modified subunit protein. For example, adding a tag to a modified nanoparticle subunit protein can be useful in the purification of that protein and, hence, the purification of conjugate (e.g. bioconjugate) vaccines comprising the tagged modified nanoparticle subunit protein. Exemplary tags that can be used herein include, without limitation, histidine (HIS) tags (e.g. hexa histidine- tag, or 6Xhis-Tag), FLAG-TAG, and HA tags. In one embodiment, the tag is a hexa-histidine tag. The tags used herein are removable, e.g. removal by chemical agents or by enzymatic means, once they are no longer needed, e.g. after the protein has been purified. Thus, the modified nanoparticle subunit protein may further comprise 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 at the N- terminus or at the C-terminus of the amino acid sequence. In one aspect, the modified nanoparticle subunit protein comprises (or consists of) an amino acid sequence which is at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% identical to any one of the nanoparticle subunit sequences provided herein and a peptide tag (e.g. six histidine residues at the N- or C-terminus of the amino acid sequence).

[0228] Antigens

[0229] An embodiment comprises a modified nanoparticle (or a modified carrier protein fused to an optionally modified nanoparticle) displaying one or more poly- or oligo- saccharide antigens on the exterior surface of the modified nanoparticle (or modified carrier protein). In one aspect of the present invention, the poly- or oligo- saccharide antigens may be conjugated at glycosylation sites of the nanoparticle, wherein the nanoparticle is not conjugated to another polypeptide. In other embodiments of the present invention, the poly- or oligo- saccharide antigens are conjugated at glycosylation sites of a modified carrier protein fused to the nanoparticle, such as a modified CRM (e.g. CRM-197), a modified EPA, a modified Diptheria Toxoid (DT), a modified OMPC, or a modified Tetanis Toxoid (TT), etc.

[0230] The antigen bioconjugated to the modified nanoparticle or modified carrier protein via a glycosylation site may be a saccharide antigen, for example, a bacterial poly- or oligo- saccharide, a yeast polysaccharide or a mammalian polysaccharide. Polysaccharides may comprise two or more monosaccharides, typically greater than ten monosaccharides. Oligo- saccharides may comprise a few monosaccharides, for example, less than ten monosaccharides. In an embodiment, the antigen is a bacterial polysaccharide antigen such as O-antigen from a gram-negative bacterium, or a capsular polysaccharide from a gram -positive bacterium. In an embodiment, the antigen displayed on the nanoparticle is any O- antigen or capsular polysaccharide or immunogenic fragment thereof, or combinations of such of any suitable serotype.

[0231] In a further embodiment, the antigen (e.g. bioconjugate) displayed on the modified nanoparticle surface is a polysaccharide antigen from Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, or Campylobacter species. Examples include but are not limited to Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylcoccus aureus. In an embodiment, the antigen in a conjugate (e.g. bioconjugate) of present embodiments is a bacterial polysaccharide selected from Shigella flexneri, Klebsiella pneumoniae and Streptococcus pneumoniae.

[0232] In certain embodiments, the antigen is an O-antigen e.g. from a gram-negative bacterium (e.g. Salmonella species, Shigella species, Pseudomonas species or Klebsiella species such as Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, or Klebsiella pneumoniae (see, Dmitriev, B.A., et al. Somatic Antigens of Shigella Eur J. Biochem, 1979. 98: p. 8; Liu et al Structure and genetics of Shigella O antigens FEMS Microbiology Review, 2008. 32: p. 27)). In an embodiment, the antigen is an O-antigen from Pseudomonas aeruginosa. For example, the antigen may be an O-antigen from Pseudomonas aeruginosa serotypes 1-20 (Raymond et al., J Bacteriol. 2002 184( 13) :3614-22). In an embodiment, the antigen is an O- antigen from Klebsiella pneumoniae.

[0233] In certain embodiments, the antigen is a capsular polysaccharide from a gram-negative or gram-positive bacteria such as Neisseria meningitidis serogroup A (MenA), N. meningitidis serogroup C (MenC), N. meningitidis serogroup Y (MenY), N. meningitidis serogroup W (MenW), H. influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In certain embodiments, the antigen is a capsular polysaccharide from Streptococcus species or Staphylococcus species (e.g. Streptococcus pneumoniae or Staphylcoccus aureus). In an embodiment, the antigen is a capsular polysaccharide from Staphylococcus aureus. For example, the antigen may be a capsular polysaccharide from Staphylococcus aureus type 5 and 8. In an embodiment, the antigen is a capsular polysaccharide from Streptococcus pneumoniae (e.g. any of the 90+ serotypes (see, Shoji et al., Infection and Drug Resistance (2018) vol. 11, pp 1387-1400).

[0234] Antigenic display

[0235] Molecules, including antigenic molecules (e.g. saccharides), attached to the exterior surface of a modified nanoparticle or modified carrier protein may be referred to herein as “display” or “displayed” molecules. Antigen-displaying modified nanoparticles (or modified carrier proteins) preferably display multiple copies of antigenic molecules in an ordered array, such an ordered array presented on a nanoparticle surface, to allow multiple binding events to occur simultaneously between the nanoparticle and host cell, which may favor the induction of a potent host immune response (see e.g. Lopcz-Sagascta <++ / / .. (2016)). Presentation of antigens on nanoparticles has been exploited to improve the immunogenicity of subunit protein antigens (see, Jardine et al, (2013); Correira et al, (2014)).

[0236] Vectors

[0237] A vector comprising such a polynucleotide is a further aspect of present embodiments. In specific embodiments, heterologous nucleotide sequences are introduced into the host cells using a plasmid, e.g. the heterologous nucleotide sequences are expressed in the host cells by a plasmid (e.g. an expression vector).

[0238] Host cell

[0239] In aspects, a host cell is provided comprising: i) one or more nucleotide sequences comprising polysaccharide synthesis genes, optionally for producing a bacterial polysaccharide antigen (e.g. an O-antigen from a gramnegative bacterium optionally from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or a capsular polysaccharide from a gram- positive bacterium optionally from Streptococcus pneumoniae or Staphylcoccus aureus or a capsular polysaccharide from Neisseria meningitidis) or a yeast polysaccharide antigen or a mammalian polysaccharide antigen, optionally integrated into the host cell genome; ii) a nucleotide sequence encoding a heterologous oligosaccharyl transferase (e.g. PglB), optionally within a plasmid; iii) a nucleotide sequence that encodes a modified nanoparticle subunit protein of present embodiments, optionally within a plasmid.

[0240] Host cells that can be used to produce the bioconjugate nanoparticles or bioconjugate carrier proteins fused to nanoparticles of present embodiments, include archea, prokaryotic host cells, and eukaryotic host cells. In certain embodiments, the host cell is a non-human host cell. Exemplary prokaryotic host cells for use in production of the bioconjugates of present embodiments include Escherichia species, Shigella species, Klebsiella species, Xhantomonas species, Salmonella species, Yersinia species, Lactococcus species, Lactobacillus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Staphylococcus species, Bacillus species, and Clostridium species. In aspects, the host cell is E. coli.

[0241] Disclosures of methods for making such host cells suitable for use with the methods provided herein are WO 06 / 119987, WO 09 / 104074, WO 11 / 62615, WO 11 / 138361, WO 14 / 57109, WO14 / 72405 and WO 16 / 20499.

[0242] Host cells may be modified to delete or modify genes in the host cell genetic background (genome) that compete or interfere with the synthesis of the polysaccharide of interest (e.g. compete or interfere with one or more heterologous polysaccharide synthesis genes that are recombinantly introduced into the host cell). These genes can be deleted or modified in the host cell background (genome) in a manner that makes them inactive / dysfunctional (i.e. the host cell nucleotide sequences that are deleted / modified do not encode a functional protein or do not encode a protein whatsoever) . In an embodiment, when nucleotide sequences are deleted from the genome of the host cells of present embodiments, they are replaced by a desirable sequence, e.g. a sequence that is useful for glycoprotein production. Exemplary genes that can be deleted in host cells (and, in some cases, replaced with other desired nucleotide sequences) include genes of host cells involved in glycolipid biosynthesis, such as waaL (see, e.g. Feldman et al. 2005, PNAS USA 102:3016-3021), the O antigen cluster (rft> or wb), enterobacterial common antigen cluster (wee), the lipid A core biosynthesis cluster (waa), galactose cluster (gal), arabinose cluster (ara), colonic acid cluster (wc), capsular polysaccharide cluster, undecaprenol-pyrophosphate biosynthesis genes (e.g. uppS (Undecaprenyl pyrophosphate synthase), uppP (Undecaprenyl diphosphatase)), Und-P recycling genes, metabolic enzymes involved in nucleotide activated sugar biosynthesis, enterobacterial common antigen cluster, and prophage O antigen modification clusters like the gtrABS cluster. In an embodiment, one or more of the waciL gene, gtrA gene, gtrB gene, gtrS gene, or a gene or genes from the wee cluster or a gene or genes from the rfb gene cluster are deleted or functionally inactivated from the genome of a prokaryotic host cell of present embodiments.

[0243] The host cell of present embodiments is E. coli, wherein the native enterobacterial common antigen cluster (ECA, wee) with the exception of wecA. the colanic acid cluster (wca), and the 016-antigen cluster have been deleted. In addition, the native lipopolysaccharide O-antigen ligase waaL may be deleted from the host cell of present embodiments. In addition, the native gtrA gene, gtrB gene and gtrS gene, may be deleted from the host cell of present embodiments.

[0244] The host cells of the present invention are engineered to comprise heterologous nucleotide sequences. The host cells of the present invention are engineered to comprise a nucleotide sequence that encodes a modified nanoparticle subunit protein (or a modified carrier protein fused to a nanoparticle subunit protein), optionally within a plasmid. The host cells of present embodiments also comprise one or more nucleotide sequences comprising polysaccharide synthesis genes. Thus, host cells of present embodiments can produce a bioconjugate comprising an antigen, for example a saccharide antigen (e.g. a bacterial, yeast or mammalian polysaccharide antigen) which is attached to a modified nanoparticle subunit protein (or a modified carrier protein fused to a nanoparticle subunit protein). One or more heterologous nucleotide sequences may encode for the polysaccharide synthesis proteins to produce the bacterial polysaccharide antigen, yeast polysaccharide antigen or mammalian polysaccharide antigen. Thus, the present invention also provides a host cell comprising: i) one or more heterologous nucleotide sequences comprising polysaccharide synthesis genes for producing a bacterial polysaccharide antigen (e.g. an O-antigen from a gramnegative bacterium optionally from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or a capsular polysaccharide from a grampositive bacterium optionally from Streptococcus pneumoniae or Staphylcoccus aureus, or a capsular polysaccharide from N. meningitidis) or a yeast polysaccharide antigen or a mammalian polysaccharide antigen, optionally integrated into the host cell genome; ii) a nucleotide sequence encoding a heterologous oligosaccharyl transferase (e.g. pglB), optionally within a plasmid; iii) a nucleotide sequence that encodes a modified nanoparticle subunit protein (or a modified carrier protein fused to a nanoparticle subunit protein), optionally within a plasmid.

[0245] The host cells of present embodiments may comprise one or more nucleotide sequences sufficient for producing a saccharide antigen (e.g. abacterial polysaccharide antigen), in particular for producing a saccharide antigen (e.g. a bacterial polysaccharide antigen) that is heterologous to the host cell. For example, if the host cell is E. coli, the host cell may comprise one or more nucleotide sequences comprising polysaccharide synthesis genes sufficient for producing a bacterial polysaccharide antigen of a bacteria which is not an E. coli polysaccharide antigen. The bacterial polysaccharide antigen may be an O-antigen or a capsular polysaccharide antigen. Thus, present embodiments also provide a host cell comprising: i) one or more nucleotide sequences comprising polysaccharide synthesis genes, for producing a bacterial polysaccharide antigen (e.g. an O-antigen from a gram-negative bacterium, optionally from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, or a capsular polysaccharide from a gram-positive bacterium optionally from Streptococcus pneumoniae or Staphylcoccus aureus), optionally integrated into the host cell genome; ii) a nucleotide sequence encoding a heterologous oligosaccharyl transferase (e.g. pglB), optionally within a plasmid; iii) a nucleotide sequence that encodes a modified nanoparticle subunit protein (or a modified carrier protein fused to a nanoparticle subunit protein) of present embodiments, optionally within a plasmid.

[0246] Polysaccharide synthesis genes encode proteins involved in synthesis of a polysaccharide (polysaccharide synthesis proteins). In an embodiment, the host cells may comprise one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from a gram-negative bacterium selected from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa and Klebsiella pneumoniae, or a capsular polysaccharide from a grampositive bacterium selected from Streptococcus pneumoniae and Staphylcoccus aureus. In another embodiment, the host cells may comprise one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from a gram-negative bacterium selected from Shigella flexneri and Klebsiella pneumoniae, or a capsular polysaccharide from a gram-positive bacterium selected from Streptococcus pneumoniae and Staphylcoccus aureus. Host cells for production of a bacterial polysaccharide antigen

[0247] The host cells of present embodiments may comprise one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen. In certain embodiments, the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from Salmonella species, Shigella species, Pseudomonas species or Klebsiella species. In certain embodiments, the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from Shigella species, Pseudomonas species or Klebsiella species (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, or Klebsiella pneumoniae). In certain embodiments, the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from Shigella species or Klebsiella species, (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei or Klebsiella pneumoniae) . In an embodiment, the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from Shigella dysenteriae, Shigella flexneri or Shigella sonnei. For example, the host cell may comprise one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from .S', dysenteriae type 1, .S', sonnei, and .S', flexneri type 6, and .S', flexneri 2a and 3a 0 (Dmitriev, B.A., et al Somatic Antigens of Shigella Eur J. Biochem, 1979. 98: p. 8; Liu et al Structure and genetics of Shigella O antigens FEMS Microbiology Review, 2008. 32: p. 27). In an embodiment, the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from Pseudomonas aeruginosa, e.g. Pseudomonas aeruginosa serotypes 1-20. In an embodiment, the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen from Klebsiella pneumoniae.

[0248] The host cells of present embodiments may comprise one or more nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular polysaccharide. In certain embodiments, the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular polysaccharide from N. meningitidis serogroup A (MenA), N. meningitidis serogroup C (MenC), N. meningitidis serogroup Y (MenY), N. meningitidis serogroup W (MenW), H. influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In certain embodiments, the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular polysaccharide from Streptococcus species, or Staphylococcus species (e.g. Streptococcus pneumoniae or Staphylcoccus aureus) . In an embodiment, the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular polysaccharide from Staphylococcus aureus, e.g. from Staphylococcus aureus type 5 and 8. In an embodiment, the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular polysaccharide from Streptococcus pneumoniae.

[0249] Host cells comprising heterologous nucleotide sequences for producing a bacterial polysaccharide antigen

[0250] The host cells of the present invention may naturally express one or more nucleotide sequences comprising polysaccharide synthesis genes for production of a saccharide antigen (e.g. a bacterial polysaccharide antigen), or the host cells may be engineered to express one or more such nucleotide sequences. For example, host cells of the present invention may utilize endogenous or heterologous glycosyltransferases for sequential assembly of oligosaccharides in the cytosol (cytosolic glycosyltransferases). Heterologous nucleotide sequences (e.g. nucleotide sequences that encode carrier proteins and / or nucleotide sequences that encode other proteins, e.g. proteins involved in glycosylation) can be introduced into the host cells of present embodiments using methods such as electroporation, chemical transformation by heat shock, natural transformation, phage transduction, and conjugation. In specific embodiments, heterologous nucleotide sequences are introduced into the host cells of present embodiments using a plasmid, e.g. the heterologous nucleotide sequences are expressed in the host cells by a plasmid (e.g. an expression vector). In another specific embodiment, heterologous nucleotide sequences are introduced into the host cells of present embodiments using the method described in WO14 / 037585. In certain embodiments, the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes which are heterologous to the host cell. In certain embodiments, one or more of said nucleotide sequences comprising polysaccharide synthesis genes which are heterologous to the host cell are integrated into the genome of the host cell. The heterologous nucleotide sequences may encode, without limitation, glycosyltransferases, oligosaccharyl transferases, epimerases, flippases, and / or polymerases. In certain embodiments, the host cells of present embodiments comprise one or more heterologous nucleotide sequences encoding glycosyltransferase(s), which can be derived from, e.g. Escherichia species, Shigella species, Klebsiella species, Salmonella species, Pseudomonas species, Streptococcus species, or Staphylococcus species.

[0251] The host cells of present embodiments may comprise one or more heterologous nucleotide sequences comprising polysaccharide synthesis genes for producing an O-antigen. In certain embodiments, the host cell comprises one or more nucleotide sequences from Salmonella species, Shigella species, Pseudomonas species or Klebsiella species that encode polysaccharide synthesis proteins for producing an O-antigen. In certain embodiments, the host cell comprises one or more nucleotide sequences from Shigella species, Pseudomonas species or Klebsiella species (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, or Klebsiella pneumoniae) that encode polysaccharide synthesis proteins for producing an O-antigen. In certain embodiments, the host cell comprises one or more nucleotide sequences from Shigella species or Klebsiella species (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei, or Klebsiella pneumoniae) that encode polysaccharide synthesis proteins for producing an O-antigen. In an embodiment, the host cell comprises one or more nucleotide sequences from Shigella dysenteriae, Shigella flexneri or Shigella sonnei that encode polysaccharide synthesis proteins for producing an O-antigen. For example, the host cell may comprise one or more nucleotide sequences from .S'. dysenteriae type 1, .S', sonnei, and .S', flexneri type 6, and .S', flexneri 2a and 3a that encode polysaccharide synthesis proteins for producing an O-antigen. In an embodiment, the host cell comprises one or more nucleotide sequences from Pseudomonas aeruginosa, e.g. Pseudomonas aeruginosa serotypes 1-20, that encode polysaccharide synthesis proteins for producing an O- antigen. In an embodiment, the host cell comprises one or more nucleotide sequences from Klebsiella pneumoniae that encode polysaccharide synthesis proteins for producing an O-antigen. The nucleotide sequences that encode an O-antigen may be a rft> cluster. As used herein, rft> cluster refers to a gene cluster that encodes enzymatic machinery capable of synthesis of an O antigen. The host cells may comprise a rfb gene cluster from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae.

[0252] The host cells of present embodiments may comprise one or more heterologous nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular saccharide. In certain embodiments, the host cell comprises one or more nucleotide sequences from N. meningitidis serogroup A (MenA), N. meningitidis serogroup C (MenC), N. meningitidis serogroup Y (MenY), N. meningitidis serogroup W (MenW), H. influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus that encode polysaccharide synthesis proteins for producing a capsular saccharide. In certain embodiments, the host cell comprises one or more nucleotide sequences from Streptococcus species, or Staphylococcus species (e.g. Streptococcus pneumoniae or Staphylcoccus aureus) that encode polysaccharide synthesis proteins for producing a capsular polysaccharide. In an embodiment, the host cell comprises one or more nucleotide sequences from Staphylococcus aureus, e.g. from Staphylococcus aureus type 5 and 8, that encode polysaccharide synthesis proteins for producing a capsular polysaccharide. In an embodiment, the host cell comprises one or more nucleotide sequences comprising polysaccharide synthesis genes for producing a capsular polysaccharide from Streptococcus pneumoniae. The nucleotide sequences may be a capsular polysaccharide gene cluster. The host cells may comprise a capsular polysaccharide gene cluster from a Streptococcus strain (e.g. S. pneumoniae, S. pyrogenes, S. agalacticae), a Staphylococcus strain (e.g. S. aureus). The capsular polysaccharide gene cluster for Streptococcus pneumoniae maps between dexB and aliA in the pneumococcal chromosome (see, Llull et al., 1999, J. Exp. Med. 190, 241-251). There are typically four relatively conserved genes: (wzg), (wzh), (wzd), (wze) at the 5' end of the capsular polysaccharide gene cluster (Jiang et al., 2001, Infect. Immun. 69, 1244-1255). Also included in the capsular polysaccharide gene cluster of .S', pneumoniae are wzx (polysaccharide flippase gene) and wzy (polysaccharide polymerase gene). The CP gene clusters of at least 90 .S', pneumoniae serotypes have been sequenced by Sanger Institute (http: / / www.sanger.ac.uk / Projects / S_pneumoniae / CPS / ), and wzx and wzy of at least 89 serotypes have been annotated and analyzed (Kong et al., 2005, J. Med. Microbiol. 54, 351-356). The capsular biosynthetic genes of .S', pneumoniae are further described in Bentley et al. (PloS Genet. 2006 Mar; 2(3): e31 and the sequences are provided in GenBank. Thus, in an embodiment the host cells of present embodiments may further comprise a nucleotide sequence encoding a polymerase (e.g. wzy), a flippase (e.g. wzx) and optionally a nucleotide sequence encoding and / or a chain length regulator (e.g. wzz).

[0253] In certain embodiments, the host cells may also comprise heterologous nucleotide sequences that are located outside of a rft> cluster or a capsular polysaccharide cluster. For example, nucleotide sequences encoding glycosyltransferases and acetyltransferases that are found outside of rfb clusters or capsular polysaccharide clusters and that modify recombinant polysaccharides can be introduced into the host cells.

[0254] Oligosaccharyl Transferase

[0255] N-linked protein glycosylation (the addition of carbohydrate molecules to an asparagine residue in the polypeptide chain of the target protein) is the most common type of post-translational modification occurring in the endoplasmic reticulum of eukaryotic organisms. The process is accomplished by the enzymatic oligosaccharyltransferase complex (OST) responsible for the transfer of a preassembled oligosaccharide from a lipid carrier (dolichol phosphate) to an asparagine residue of a nascent protein within the conserved sequence Asn-X-Ser / Thr (where X is any amino acid except proline) in the endoplasmic reticulum.

[0256] It has been shown that a bacterium, the food-borne pathogen Campylobacter jejuni, can also N-glycosylate its proteins (Wacker et al. Science. 2002; 298(5599): 1790-3) due to the fact that it possesses its own glycosylation machinery. The machinery responsible of this reaction is encoded by a cluster called “pgl” (for protein glycosylation). The C. jejuni glycosylation machinery can be transferred to E. coli to allow for the glycosylation of recombinant proteins expressed by the E. coli cells. Previous studies have demonstrated how to generate E. coli strains that can perform N-glycosylation (see, e.g. Wacker etal. Science. 2002; 298 (5599): 1790-3; Nita- Lazar et al. Glycobiology. 2005; 15(4):361 -7; Feldman et al. Proc Natl Acad Sci U S A. 2005; 102(8):3016-21; Kowarik et al. EMBO J. 2006; 25(9): 1957-66; Wacker et al. Proc Natl Acad Sci U S A. 2006; 103(18):7088-93; International Patent Application Publication Nos. W02003 / 074687, W02006 / 119987, WO 2009 / 104074, and WO / 2011 / 06261, and WO2011 / 138361).

[0257] The host cells of the present invention comprise a nucleotide sequence encoding a heterologous oligosaccharyl transferase, optionally within a plasmid. In a specific embodiment, the oligosaccharyl transferase is an oligosaccharyl transferase from Campylobacter. In another specific embodiment, the oligosaccharyl transferase is a pglB, optionally from Campylobacter jejuni (i.e. pglB; see, e.g. Wacker et al. 2002, Science 298: 1790-1793; see also, e.g. NCBI Gene ID: 3231775, UniProt Accession No. 086154) SEQ ID NO: 54:

[0258] MLKKEYLKNPYLVLFAMIILAYVFSVFCRFYWVWWASEFNEYFFNNQL

[0259] MIISNDGYAFAEGARDMIAGFHQPNDLSYYGSSLSALTYWLYKITPFSFES IILYMSTFLSSLVVIPTILLANEYKRPLMGFVAALLASIANSYYNRTMSGY YDTDMLVIVLPMFILFFMVRMILKKDFFSLIALPLFIGIYLWWYPSSYTLN VALIGLFLIYTLIFHRKEKIFYIAVILSSLTLSNIAWFYQSAIIVILFALFALEQ KRLNFMIIGILGSATLIFLILSGGVDPILYQLKFYIFRSDESANLTQGFMYFN VNQTIQEVENVDLSEFMRRISGSEIVFLFSLFGFVWLLRKHKSMIMALPIL VLGFLALKGGLRFTIYSVPVMALGFGFLLSEFKAIMVKKYSQLTSNVCIVF ATILTLAPVFIHIYNYKAPTVFSQNEASLLNQLKNIANREDYVVTWWDYG YPVRYYSDVKTLVDGGKHLGKDNFFPSFALSKDEQAAANMARLSVEYT EKSFYAPQNDILKTDILQAMMKDYNQSNVDLFLASLSKPDFKIDTPKTRDI YLYMPARMSLIFSTVASFSFINLDTGVLDKPFTFSTAYPLDVKNGEIYLSN GVVLSDDFRSFKIGDNVVSVNSIVEINSIKQGEYKITPIDDKAQFYIFYLKD

[0260] SAIPYAQFILMDKTMFNSAYVQMFFLGNYDKNLFDLVINSRDAKVFKLKI

[0261] Thus host cells of the present invention may comprise a nucleotide sequence encoding pglB, optionally pglB from Campylobacter jejuni, optionally a nucleotide sequence encoding pglB from Campylobacter jejuni having a sequence at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 54, optionally within a plasmid.

[0262] Polymerase

[0263] Host cells of the present invention may also comprise a nucleotide sequence that encodes a polymerase (e.g. wzy). In an embodiment, the polymerase (e.g. wzy) is introduced into a host cell of present embodiments (i.e. the polymerase is heterologous to the host cell). In an embodiment, the polymerase is a bacterial polymerase. In an embodiment, the polymerase is a capsular polysaccharide polymerase (e.g. wzy) or an O antigen polymerase (e.g. wzy). In an embodiment, the polymerase is an O-antigen polysaccharide polymerase (e.g. wzy), e.g. from Shigella species, Pseudomonas species or Escherichia species, (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, or E. coli). In an embodiment, the polymerase is a capsular polysaccharide polymerase (e.g. wzy), e.g. from N. meningitidis serogroup A (MenA), N. meningitidis serogroup C (MenC), N. meningitidis serogroup Y (MenY), N. meningitidis serogroup W (MenW), H. influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In an embodiment, the polymerase is a capsular polysaccharide polymerase (e.g. wzy) of Streptococcus pneumoniae. Said wzy polymerase may be incorporated (e.g. inserted into the genome or expressed by a plasmid) in said host cell as part of a rfb cluster or capsular polysaccharide cluster. Thus, a host cell of present embodiments may further comprise a nucleotide sequence encoding a heterologous wzy polymerase.

[0264] Flippases

[0265] A host cell of present embodiments may also comprise a nucleotide sequence encoding a flippase (e.g. wzx), e.g. a heterologous flippase. Flippases translocate wild type repeating units and / or their corresponding engineered (hybrid) repeat units from the cytoplasm into the periplasm of host cells (e.g. E. coli). In an embodiment, the flippase is a bacterial flippase, e.g. a flippase of the polysaccharide biosynthetic pathway of interest. In a specific embodiment, the host cell of present embodiments comprises a nucleotide sequence encoding a flippase (e.g. wzx gene) of a polysaccharide biosynthetic pathway of a Streptococcus species, Shigella species, Escherichia species, Pseudomonas species, or Staphylococcus species, (e.g. Streptococcus pneumoniae, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, E. coli, Pseudomonas aeruginosa, or Staphylcoccus aureus. In an embodiment, the flippase is a capsular polysaccharide flippase (e.g. wzx) of Streptococcus pneumoniae. Other flippases that can be introduced into the host cells of present embodiments are for example from Campylobacter jejuni (e.g. pglK).

[0266] Accessory Enzymes

[0267] In an embodiment, nucleotide sequences encoding one or more accessory enzymes are introduced into the host cells of present embodiments. Thus, a host cell of present embodiments may further comprise one or more of these accessory enzymes. Such nucleotide sequences encoding one or more accessory enzymes can be either plasmid-borne or integrated into the genome of the host cells of present embodiments. Exemplary accessory enzymes include, without limitation, epimerases (see e.g. WO2011 / 062615), branching, modifying (e.g. to add cholins, glycerolphosphates, pyruvates), amidating, chain length regulating, acetylating, formylating, polymerizing enzymes. Thus, a host cell of present embodiments may also comprise a nucleotide sequence encoding a chain length regulator (e.g. wzz), e.g. a heterologous chain length regulator. In an embodiment, the chain length regulator is a capsular polysaccharide chain length regulator (e.g. wzz) of Streptococcus pneumoniae.

[0268] Bioconjugates

[0269] Present embodiments provide 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 or a yeast polysaccharide antigen or a mammalian polysaccharide antigen). In a specific embodiment, said antigen is an O-antigen or a capsular polysaccharide. In an embodiment, the antigen is an O-antigen from a gram-negative bacterium. In aspects, a bioconjugate comprising a modified nanoparticle subunit (or a modified carrier protein fused to a nanoparticle subunit) conjugated to an antigen is provided wherein the antigen is a saccharide, optionally a bacterial polysaccharide (e.g. from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae or Staphylcoccus aureus). In an embodiment, the present invention provides a bioconjugate comprising a modified nanoparticle subunit (or a modified carrier protein fused to a nanoparticle subunit) linked to an antigen wherein the antigen is a bacterial polysaccharide (e.g. from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Klebsiella pneumoniae, or Streptococcus pneumoniae) . In another embodiment, the present invention provides a bioconjugate comprising a modified nanoparticle subunit (or a modified carrier protein fused to a nanoparticle subunit) linked to an antigen wherein the antigen is a bacterial polysaccharide from Shigella flexneri, Klebsiella pneumoniae or Streptococcus pneumoniae. The antigen is linked to an amino acid on the modified nanoparticle subunit (or a modified carrier protein fused to a nanoparticle subunit) selected from asparagine, aspartic acid, glutamic acid, lysine, cysteine, tyrosine, histidine, arginine or tryptophan (e.g. asparagine). Bioconjugates, as described herein, have advantageous properties over chemical conjugates of antigen-carrier protein, in that they require less chemicals in manufacture and are more consistent in terms of the final product generated.

[0270] A further aspect of present embodiments is a process for producing a bioconjugate nanoparticle subunit that comprises or consists of a modified nanoparticle subunit linked to a saccharide, said process comprising (i) culturing the host cell of present embodiments under conditions suitable for the production of glycoproteins, glycotransferases, and nanoparticle subunits, and (ii) isolating the bioconjugate nanoparticle produced by said host cell, optionally isolating the bioconjugate nanoparticle from a periplasmic extract from the host cell.

[0271] A further aspect of present embodiments is a process for producing a bioconjugate carrier nanoparticle subunit that comprises (or consists of) a modified carrier protein (linked to a saccharide) fused to a nanoparticle subunit, said process comprising (i) culturing the host cell of present embodiments under conditions suitable for the production of glycoproteins, glycotransferases, and nanoparticle carrier proteins fused to subunits, and (ii) isolating the bioconjugate carrier nanoparticle product produced by said host cell, optionally isolating the bioconjugate carrier nanoparticle from a periplasmic extract from the host cell.

[0272] For example, bioconjugate nanoparticles can be made using the shakeflask process, e.g. in a LB shake flask. In aspects, a fed-batch process for the production of recombinant glycosylated proteins in bacteria can be used to produce bioconjugate nanoparticles. The aim is to increase glycosylation efficiency and recombinant protein yield per cell and while maintaining simplicity and reproducibility in the process. Bioconjugate nanoparticles can be manufactured on a commercial scale by developing an optimized manufacturing method using typical E. coli production processes. Various types of feed strategies, such as batch, chemostat and fed-batch can be used.

[0273] The bioconjugate nanoparticles of present embodiments can be purified for example, by chromatography (e.g. ion exchange, anionic exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins (see, e.g. Saraswat et al. 2013, Biomed. Res. Int. ID#312709 (p. 1-18); see also the methods described in WO 2009 / 104074). Further, the bioconjugates may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0274] Conjugation of Nanoparticles or Nanoparticle Fusion Proteins

[0275] The nanoparticles (or carrier proteins fused to nanoparticle subunits) herein may be conjugated by any suitable method. Displayed molecules may be incorporated onto or attached to the nanoparticles (or carrier proteins fused to nanoparticle subunits) by any suitable means.

[0276] Alternative methods for producing the nanoparticles provided herein include chemical conjugation.

[0277] In chemical conjugation, functional groups present on the nanoparticle subunit polypeptides (or carrier proteins fused to nanoparticle subunits) can be used for conjugation of display molecules. Amino acid side-chain groups used for conjugation include an amino group on lysine, thiol on cysteine, carboxylic acid on aspartic acids and glutamic acids, and hydroxyl moiety on tyrosine with different chemistries known in the art. Homo- or hetero-bifunctional crosslinkers are available for conjugation. The side-chain amino groups of lysine residues are nucleophiles, so lysine residues exposed at the nanoparticle surface have large solvent accessibility and can be used as sites for conjugation to display molecules.

[0278] One or more selected amino acid residues within a nanoparticle subunit polypeptide sequence (or carrier protein sequence) may be modified using methods known in the art to provide a site suitable for chemical bioconjugation at the nanoparticle exterior surface (or carrier protein attached to the nanoparticle exterior surface), where such modification does not disrupt nanoparticle assembly.

[0279] In one aspect, a modified nanoparticle is provided, wherein one or more display molecules (e.g. antigens) are chemically conjugated to residues present at the exterior surface of the nanoparticle. The display molecule(s) may be an oligosaccharide, a polysaccharide, a glycan, or a glycoconjugate, or combinations thereof.

[0280] Covalent conjugation of saccharides to modified carrier proteins or modified nanoparticles enhances the immunogenicity of saccharides as it converts them from T-independent antigens to T-dependent antigens, thus allowing priming for immunological memory. Conjugation is 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. in Conjugate Vaccines (1989); Hermanson, (1996)).

[0281] An alternative conjugation process involves the use of -NH2 groups in the saccharide (either from de-N-acetylation, or after introduction of amines) in conjunction with bifunctional linkers, as described (W02006 / 082530). A further alternative process is described in WO96 / 40795 and Michon et al. (2006). In this process, the free aldehydes groups of terminal 2,5-anhydro-D- mannose residues from depolymerization of type II or type III capsular saccharides by mild cleavage through de-N-acetylation / nitrosation are used for conjugation by reductive amination.

[0282] Also provided is a conjugate (e.g. bioconjugate) comprising (or consisting of) a modified nanoparticle or modified carrier protein fused to a nanoparticle (e.g. a modified EPA fused to a ferritin subunit) linked to an antigen (e.g. a saccharide antigen, optionally a bacterial polysaccharide antigen). The antigen may be a bacterial polysaccharide antigen, or a yeast polysaccharide antigen, or a mammalian polysaccharide antigen.

[0283] In an embodiment, a conjugate (e.g. bioconjugate) is provided comprising (or consisting of) a modified nanoparticle or modified carrier protein fused to a nanoparticle covalently linked to an antigen (e.g. a saccharide antigen, optionally a bacterial polysaccharide antigen), wherein the antigen is linked (either directly or through a linker) to the modified carrier protein or modified nanoparticle. In an embodiment, the antigen is directly linked to the modified nanoparticle or modified carrier protein fused to a nanoparticle. In an embodiment, the antigen is directly linked to an amino acid residue of the modified nanoparticle or modified carrier protein fused to a nanoparticle.

[0284] Displayed molecules (e.g. antigen(s) and / or immunostimulant(s)) may be conjugated to a nanoparticle (e.g. a protein nanoparticle) or a carrier protein fused to a nanoparticle by any suitable means including spontaneous isopeptide bond formation, chemical conjugation, genetic fusion, bioconjugation, or bio-orthogonal chemistry with unnatural amino acids. An artisan may choose from a variety of known conjugation techniques for use with a particular nanoparticle and particular displayed molecule(s), such techniques include those described and / or demonstrated by the following (in no particular order)'. Aubin-Tam, “Conjugation of Nanoparticles to Proteins ” IN NANOMATERIAL INTERFACES IN BIOLOGY: METHODS AND PROTOCOLS - METHODS IN MOLECULE BIOLOGY 2013 (eds. Bergese and Hamad-Schifferli); 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; J. M. Carter, “Conjugation of Peptides to Carrier Proteins via Glutaraldenhyde ” in THE PROTEIN PROTOCOLS HANDBOOK 1997 (Ed. J. M. 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; Laustcr c / « / . 2020 Nat. Nanotech. 15: 373-379.

[0285] Examples of conjugating an antigenic molecule to a nanoparticle may 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 / vl2010035 (17 total pages)).

[0286] A process for production of glycoconjugates typically involves reductive amination of a purified saccharide to a carrier protein such as tetanus toxoid (TT) or CRM 197 (Wessels et al. (1990)). The reductive amination involves an amine group on the side chain of an amino acid in the carrier and an aldehyde group in the saccharide. As some capsular saccharides do not include an aldehyde group in their natural form then this is typically generated before conjugation by oxidation (e.g. periodate oxidation) of a portion (e.g. between 5 and 40%) of the saccharide’s sialic acid residues [Wessels et al. (1990); US Patent No. 4,356,170],

[0287] In literature, bioconjugated NPs have been obtained previously in: Pan, C.; Wu, J.; Qing, S.; Zhang, X.; Zhang, L. L.; Yue, H.; Zeng, M.; Wang, B.; Yuan, Z.; Qiu, Y. F. et al. Biosynthesis of self-assembled proteinaceous nanoparticles for vaccination. Adv. Mater. 2020, 32, 2002940; and Peng, Z. H.; Wu, J.; Wang, K. F.; Li, X.; Sun, P.; Zhang, L. L.; Huang, J.; Liu, Y.; Hua, X. T.; Yu, Y. S. et al. Production of a promising biosynthetic self-assembled nanoconjugate vaccine against Klebsiella pneumoniae serotype 02 in a general Escherichia coli host. Adv. Sci., in press, DOI: 10.1002 / advs.202100549.

[0288] Chemical conjugation

[0289] In an embodiment, the modified nanoparticle or modified carrier protein fused to a nanoparticle may be covalently linked to the antigen through a chemical linkage obtainable using a chemical conjugation method (i.e. the conjugate is produced by chemical conjugation). The chemical conjugation method may be selected from the group consisting of carbodiimide chemistry, reductive animation, cyanylation chemistry (for example CDAP chemistry), maleimide chemistry, hydrazide chemistry, ester chemistry, and N-hydroysuccinimide chemistry. Conjugates can be prepared by direct reductive amination methods as described in, US200710184072 (Hausdorff) US 4365170 (Jennings) and US 4673574 (Anderson). Other methods are described in EP-0-161- 188, EP-208375 and EP-0-477508. The conjugation method may alternatively rely on activation of the saccharide with l-cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. Such conjugates are described in PCT published application WO 93 / 15760 Uniformed Services University and WO 95 / 08348 and WO 96 / 29094. See also Chu C. etal. Infect. Immunity, 1983 245 256.

[0290] The functional groups on one or more amino acids of the polypeptide monomer(s) (of the modified nanoparticle monomers or the modified carrier protein fused to the nanoparticle monomer) can be used for site-specific conjugation to a display molecule(s) (e.g. to an antigen or an immunostimulant). Amino acid side-chain groups used for conjugation include amino group on lysine, thiol on cysteine, carboxylic acid on aspartic acids and glutamic acids, and hydroxyl moiety on tyrosine. Heterobifunctional crosslinkers are available for protein conjugation. Primary amines on a first polypeptide can be conjugated to carboxylic acids on a second polypeptide using 1 -ethyl - 3-(-3-dimethylaminopropyl) carbodiimide (EDC) crosslinkers, typically in combination with N- hydroxysuccinimide (NHS). The side-chain amino groups of lysine residues are nucleophiles, so lysine residues exposed at the nanoparticle exterior surface have large solvent accessibility and can be used as sites for conjugation to display molecules. Chemical methods also include sitespecific chemical conjugation through engineered cysteines or selenocysteines (see Siegmund et al. 2016 Scientific Reports 6(39291)). In general, the following types of chemical groups on a modified carrier protein or nanoparticle subunit can be used for coupling / conjugation:

[0291] A) Carboxyl (for instance via aspartic acid or glutamic acid). In one embodiment this group is linked to amino groups on saccharides directly or to an amino group on a linker with carbodiimide chemistry e.g. with EDAC.

[0292] B) Amino group (for instance via lysine). In one embodiment this group is linked to carboxyl groups on saccharides directly or to a carboxyl group on a linker with carbodiimide chemistry e.g. with EDAC. In another embodiment this group is linked to hydroxyl groups activated with CDAP or CNBr on saccharides directly or to such groups on a linker; to saccharides or linkers having an aldehyde group; to saccharides or linkers having a succinimide ester group.

[0293] C) Sulphydryl (for instance via cysteine). In one embodiment this group is linked to a bromo or chloro acetylated saccharide or linker with maleimide chemistry. In one embodiment this group is activated / modified with bis diazobenzidine.

[0294] D) Hydroxyl group (for instance via tyrosine). In one embodiment this group is activated / modified with bis diazobenzidine.

[0295] E) Imidazolyl group (for instance via histidine). In one embodiment this group is activated / modified with bis diazobenzidine.

[0296] F) Guanidyl group (for instance via arginine).

[0297] G) Indolyl group (for instance via tryptophan).

[0298] On a saccharide, in general the following groups can be used for a coupling: OH, COOH or NH2. Aldehyde groups can be generated after different treatments such as: periodate, acid hydrolysis, hydrogen peroxide, etc.

[0299] Bioconjugation

[0300] In a preferred embodiment, one or more nucleotide constructs are prepared that recombinantly express a polypeptide sequence (e.g. a contiguous amino acid sequence) comprising the monomer amino acid sequence (e.g. a modified nanoparticle subunit) and the display molecule (e.g. display antigen or display immunostimulant saccharide). Expressed as a modified monomeric subunit, the modified nanoparticles self-assemble into a nanoparticle (as shown, for example, by Pan et al. 2020 Adv. Mater. 32:2002940). Expressed as a modified carrier protein fusion construct (i.e., a modified carrier protein fused to the nanoparticle subunit), the modified carrier protein fusion construct self-assembles into a nanoparticle. In both cases, the modified monomeric subunit or the modified carrier protein fusion construct self-assemble into a nanoparticle.

[0301] Conjugation through spontaneous isopeptide bonds

[0302] The use of peptide “tag” and binding partner (or “capture” or “dock”) pairs for the spontaneous formation of isopeptide bonds between heterologous molecules (“tag-capture system”) is known (see, e.g. Zakeri et al. 2012 PNAS 109(12): E690-E697 of “SpyTag-SpyCatcher” system; WO2011 / 098772 (PCT / GB2011 / 000188), M. HOWARTH and Hatlem etal. 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); relatives of US 63 / 038237 for “GalacTag-GalacDock” system; Veggiani et al. 2016 PNAS 113(5): 1202-1207 of “SnoopTag- SnoopCatcher” system and combinations thereof).

[0303] Therefore, one embodiment of the present invention is a nanoparticle covalently linked to an carrier (e.g. on its exterior surface) that has undergone bioconjugation. The nanoparticles and the carrier may be conjugated using a tag-capture system (e.g. wherein the tag-capture system is 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)). Accordingly, one aspect is a nanoparticle conjugated to a tag molecule of a tag-capture system (optionally wherein the nanoparticle monomer, such as the polypeptide subunit monomer, is conjugated to a tag molecule of a tag -capture system). In aspects, a nanoparticle is attached to a tag molecule, and the tag molecule attaches to a bioconjugate carrier attached to a carrier molecule. Alternatively, one aspect is a nanoparticle conjugated to a capture molecule of a tag -capture system (optionally wherein the monomer, such as the polypeptide monomer, is conjugated to a capture molecule of a tag -capture system). In aspects, a nanoparticle is attached to a capture molecule, and the capture molecule attaches to a bioconjugate carrier attached to a tag molecule. When one such nanoparticle is contacted with a heterologous molecule (e.g. an antigen / carrier or an immunostimulant) that is itself conjugated to the respective capture molecule or tag molecule (i.e., the heterologous molecule contains the other sequence of the binding pair), an isopeptide bond forms and the heterologous molecule becomes covalently attached to the nanoparticle. See, e.g. 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 1 l(10):e0165074 (2016); WO2015 / 156870 (PCT / US2015 / 011534, DENG Z ); Bruun et al. 2018 ACS Nano 12(9):8855-8866.

[0304] In addition, several enzymatic and chemoenzymatic conjugation approaches have been reported including the use of engineered galactosyl- and sialyltransferases, formyl glycine generating enzyme (FGE), phosphopantetheinyl transferases (PPTases), sortase A, and microbial transglutaminase (an enzyme forming an isopeptide bond between a glutamine side -chain and an amine-donor substrate) (see Siegmund et al. 2016 Scientific Reports 6(39291)).

[0305] Conjugates (e.g. chemical conjugates, bioconjugates, etc.) can be purified by any method known in the art for purification of a protein, for example, by chromatography (e.g. ion exchange, anionic exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. See, e.g. Saraswat et al., 2013, Biomed. Res. Int. ID0312709 (p. 1-18); see also the methods described in WO 2009 / 104074. The actual conditions used to purify a particular conjugate will depend, in past, on the synthesis strategy (e.g. synthetic production vs. recombinant production) and on factors such as net charge, hydrophobicity, and / or hydrophilicity of the bioconjugate.

[0306] Analytical Methods

[0307] Various methods can be used to analyze the compositions and sugar chain lengths of the bioconjugates of present embodiments and to determine glycosylation site usage.

[0308] Hydrazinolysis can be used to analyze glycans. First, polysaccharides are released from their protein carriers by incubation with hydrazine according to the manufacturer’s instructions (Ludger Liberate Hydrazinolysis Glycan Release Kit, Oxfordshire, UK). The nucleophile hydrazine attacks the glycosidic bond between the polysaccharide and the nanoparticle subunit protein or the carrier protein and allows release of the attached glycans. N-acetyl groups are lost during this treatment and have to be reconstituted by re-N-acetylation. The free glycans are purified on carbon columns and subsequently labeled at the reducing end with the fluorophor 2- amino benzamide (see, Bigge et al., Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Anal Biochem, 1995, 230(2):229-238). The labeled polysaccharides are separated on a GlycoSep-N column (GL Sciences) according to the HPLC protocol of Royle et al. (see, Royle et al., An analytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins, (see, Anal Biochem, 2002, 304( 1) :70-90). The resulting fluorescence chromatogram indicates the polysaccharide length and number of repeating units. Structural information can be gathered by collecting individual peaks and subsequently performing MS / MS analysis. Thereby, the monosaccharide composition and sequence of the repeating unit can be confirmed and additionally the homogeneity of the polysaccharide composition can be determined. Alternatively, high resolution intact mass measurements and size exclusion HPLC can be applied to measure the size of the complete bioconjugates.

[0309] Yield may be measured as carbohydrate amount derived from a liter of bacterial production culture grown in a bioreactor under controlled and optimized conditions. After purification of bioconjugate nanoparticles, the carbohydrate yields can be directly measured by either the anthrone assay or ELISA using carbohydrate specific antisera. Indirect measurements are possible by using the protein amount (measured by BCA, Lowry, or Bradford assays) and the glycan length and structure to calculate a theoretical carbohydrate amount per gram of protein. In addition, yield can also be measured by drying the glycoprotein preparation from a volatile buffer and using a balance to measure the weight.

[0310] Various methods can be used to analyze the bioconjugate nanoparticles of present embodiments including, for example, SDS-PAGE or capillary gel electrophoresis. Polymer length is defined by the number of repeat units that are linearly assembled. This means that the typical ladder-like pattern is a consequence of different repeat unit numbers that compose the glycan. Thus, two bands next to each other in SDS PAGE (or other techniques that separate by size) differ by only a single repeat unit. These discrete differences are exploited when analyzing glycoproteins for glycan size: the unglycosylated nanoparticle subunit and the bioconjugate nanoparticle subunit with different polymer chain lengths separate according to their electrophoretic mobilities. The first detectable repeat unit number (m) and the average repeat unit number (naverage) present on a bioconjugate nanoparticle are measured. These parameters can be used to demonstrate batch to batch consistency or polysaccharide stability, for example.

[0311] Glycosylation site usage may be quantified by, for example, glycopeptide LC-MS / MS: conjugates are digested with protease(s), and the peptides are separated by a suitable chromatographic method (Cl 8, Hydrophilic interaction HPLC HILIC, GlycoSepN columns, SE HPLC, AE HPLC), and the different peptides are identified using MS / MS. This method can be used with or without previous sugar chain shortening by chemical (Smith degradation) or enzymatic methods. Quantification of glycopeptide peaks using UV detection at 215 to 280nm allows relative determination of glycosylation site usage. In another embodiment, site usage may be quantified by size exclusion HPLC. Higher glycosylation site usage is reflected by an earlier elution time from a SE HPLC column. In yet another embodiment, site usage may be quantified by quantitative densitometry of purified bioconjugates stained with Coomassie Brilliant Blue following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0312] Purification

[0313] The term “purified” as used herein refers to the separation or isolation of a defined product (e.g. a recombinantly expressed nanoparticle (or a recombinantly expressed carrier protein fused to a nanoparticle) that is bioconjugated from a composition containing other components (e.g. a host cell or host cell medium). A composition that has been fractionated to remove undesired components, and which composition retains its biological activity, is considered purified. A purified bioconjugate nanoparticle (or purified carrier protein fused to a nanoparticle) retains its biological activity. Purified means removed from its natural environment and substantially free of impurities from that natural environment (such as other chromosomal and extra-chromosomal DNA and RNA, organelles, and proteins (including other proteins, lipids, or polysaccharides which are also secreted into culture medium or result from lysis of host cells)). For clarity and as would be well understood by a person in the field, herein a molecule (such as an antigen or immunostimulant) within, or suitable for use within, a pharmaceutical, immunogenic, vaccine, or adjuvant composition is a purified molecule (such as a purified antigen or purified immunostimulant) whether or not the word “purified” is recited. It is understood in the field that for a molecule (such as an antigen, agent, immunostimulant, additive, vector, or other compound) or composition to be suitable (i.e., safe) for pharmaceutical or vaccine use (e.g. administration) with a human or non-human mammal (i.e., for the molecule to be pharmaceutically acceptable), it is at least purified (i.e., not crude).

[0314] Purified is a relative term and that absolute (100%) purity is not required for, e.g. pharmaceutical or vaccine use. A molecule may be at a purity of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% or 95% of a composition’s total proteinaceous content or mass (determined by, e.g. gel electrophoresis such as assessing the number of polypeptides within a fraction by SDS / PAGE analysis). Stated another way, “purification” or “purifying” refers to the process of removing undesired components from a composition or host cell or culture. Various methods for use in purifying polypeptides and NPs are known in the art and include, e.g. various types of chromatography such as High Performance Liquid Chromatography (HPLC), ionexchange chromatography, and size -exclusion chromatography, hydrophobic interaction, ion exchange, affinity, chelating, 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. Polypeptides NPs may be expressed with a tag operable for affinity purification, such as a 6xHistidine tag as is known in the art. A His-tagged polypeptide may be purified using, for example, Ni-NTA column chromatography or using anti-6xHis antibody fused to a solid support.

[0315] A “substantially pure” preparation of polypeptides (or nanoparticles, or carrier proteins fused to nanoparticles) or nucleic acid molecules is one in which the desired component represents at least 50% of the total polypeptide (or nucleic acid) content of the preparation. In certain embodiments, a substantially pure preparation 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 preparation. Methods for quantifying the degree of purification of expressed polypeptides are known in the art and include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by SDS / PAGE analysis. The term “purification” or “purifying” herein refers to the process of removing components from a composition or host cell or culture, the presence of which is not desired. Molecules which have not been subjected to any purification steps (i.e., the molecule as it is found in nature or a “crude” molecule) are not suitable for pharmaceutical use (i.e., not suitable for administration to a subject). Methods

[0316] The nanoparticle subunit polypeptide or carrier protein fused to nanoparticle subunit polypeptide of present embodiments may be produced by any suitable means, including by recombinant expression or by chemical synthesis, and purified (if necessary) using any suitable method known in the art. The nanoparticle product may be analyzed using methods known in the art, e.g. by crystallography, Dynamic Light Scattering (DLS), Nano-Differential Scanning Fluorimetry (Nano-DSF), and Electron Microscopy, to confirm production of suitable nanoparticles.

[0317] Methods of recombinant expression suitable for the production of the nanoparticle subunit polypeptides or carrier proteins fused to nanoparticle subunit polypeptides are known in the art. The expressed polypeptide may include a purification tag and / or a protease site. Various expression systems are known in the art, including those 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. The host cell is typically transformed with the recombinant nucleic acid sequence encoding the desired polypeptide product, cultured under conditions suitable for expression of the product, and the product purified from the cell or culture medium. Cell culture conditions are particular to the cell type and expression vector, as is known in the art.

[0318] Host cells can be cultured in conventional nutrient media modified as appropriate and as will be apparent to those skilled in the art (e.g. for activating promoters). Culture conditions, such as temperature, pH and the like, may be determined using knowledge in the art, see e.g. Freshney (1994) and the references cited therein. In bacterial host cell systems, a number of expression vectors are available including, but not limited to, multifunctional E. coll cloning and expression vectors such as BLUESCRIPT (Stratagene) or pET vectors (Novagen, Madison WI). In mammalian host cell systems, a number of expression systems, including both plasmids and viralbased systems, are available commercially.

[0319] Eukaryotic or microbial host cells expressing nanoparticle subunit polypeptides or carrier proteins fused to nanoparticle subunit polypeptides can be disrupted by any convenient method (including freeze-thaw cycling, sonication, mechanical disruption), and polypeptides and / or selfassembled nanoparticles or carrier proteins fused to nanoparticle subunit polypeptides can be recovered and purified from recombinant cell culture by any suitable method known in the art (including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography (e.g. using any of the tagging systems noted herein), hydroxyapatite chromatography, and lectin chromatography)). High performance liquid chromatography (HPLC) can be employed in the final purification steps.

[0320] In general, and using methods as are known in the art, expression of a recombinantly encoded nanoparticle subunit polypeptide (or a recombinantly encoded carrier protein fused to a nanoparticle subunit) involves preparation of an expression vector comprising a recombinant polynucleotide under the control of one or more promoters, such that the promoter stimulates transcription of the polynucleotide and promotes expression of the encoded polypeptide. “Recombinant Expression” as used herein refers to such a method.

[0321] “Recombinant expression vectors” comprise a recombinant nucleic acid sequence operatively linked to control sequences capable of effecting expression of the gene product. “Control sequences” are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules and need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. “Recombinant host cells” comprise such recombinant expression vectors.

[0322] A further embodiment is a method of producing a nanoparticle comprising bacterial poly- or oligosaccharide antigens on the exterior surface of the nanoparticle. The method comprises the steps of (a) culturing a recombinant host cell under conditions conducive to (1) expressing or translocating the modified nanoparticle subunit polypeptide(s), saccharides, and glycosyltransferase pglB in the periplasm of the host cell, wherein the glycosyltransferase bioconjugates the optionally modified nanoparticle at glycoconjugation sites and under conditions conducive to self-assembly of the nanoparticle; (b) recovering or purifying assembled bioconjugated nanoparticles from the host cell or the culture medium in which the host cell is grown, as is suitable.

[0323] A further embodiment is a method of producing a nanoparticle comprising bacterial poly- or oligosaccharide antigens on the exterior surface of a carrier protein fused to a nanoparticle. The method comprises the steps of (a) culturing a recombinant host cell under conditions conducive to (1) expressing or translocating the modified carrier protein fused to the nanoparticle subunit polypeptide(s), saccharides, and glycosyltransferase pglB in the periplasm of the host cell, wherein the glycosyltransferase glycoconjugates the modified carrier protein at glycoconjugation sites and under conditions conducive to self-assembly of the nanoparticle; (b) recovering or purifying an assembled, bioconjugated carrier protein fused to a nanoparticle, from the host cell or the culture medium in which the host cell is grown, as is suitable.

[0324] There is provided in one aspect, a method for preparing an assembled glycoprotein nanoparticle (NP) comprising the steps of providing a host cell; expressing a nanoparticle subunit or modified carrier protein fused to a nanoparticle subunit in the periplasm of the host cell, wherein the optionally modified nanoparticle subunit or carrier protein comprises one or more glycosylation sites (e.g. D / E-X-N-Z-S / T SEQ ID NO: 49); expressing or translocating glycosyltransferase pglB in the periplasm of the host cell; glycosylating, via glycosyltransferase pglB, the optionally modified nanoparticle subunit (or modified carrier protein fused to the optionally modified nanoparticle subunit) with a saccharide in the periplasm; and producing an assembled glycoprotein nanoparticle from the glycosylated nanoparticle subunits (or glycosylated carrier protein fused to nanoparticle subunits). In aspects, the method comprises expressing a modified nanoparticle subunit such as ferritin, dodecin, E2p, etc.

[0325] A further embodiment is a method of producing a nanoparticle comprising bacterial poly- or oligosaccharide antigens on the exterior surface of the nanoparticle. The method comprises the steps of (a) culturing a recombinant host cell under conditions conducive to (1) expressing the modified nanoparticle subunit polypeptide (s) and glycosyltransferase pglB in the host cell, wherein the glycosyltransferase bioconjugates saccharides to the optionally modified nanoparticle at glycoconjugation sites and under conditions conducive to self-assembly of the nanoparticle; (b) recovering or purifying assembled bioconjugated nanoparticles from the periplasm of the host cell or the culture medium in which the host cell is grown, as is suitable.

[0326] A further embodiment is a method of producing a nanoparticle comprising bacterial poly- or oligosaccharide antigens on the exterior surface of a carrier protein fused to a nanoparticle. The method comprises the steps of (a) culturing a recombinant host cell under conditions conducive to (1) expressing the modified carrier protein fused to the nanoparticle subunit polypeptide(s), and glycosyltransferase pglB in the host cell, wherein the glycosyltransferase glycoconjugates saccharides to the modified carrier protein at glycoconjugation sites and wherein the nanoparticle subunits are under conditions conducive to self-assembly of the nanoparticle; (b) recovering or purifying an assembled, bioconjugated carrier protein fused to a nanoparticle, from the periplasm of the host cell or the culture medium in which the host cell is grown, as is suitable.

[0327] There is also provided in one aspect, a method for preparing an assembled glycoprotein nanoparticle (NP) comprising the steps of providing a host cell; expressing a nanoparticle subunit or modified carrier protein fused to a nanoparticle subunit in the host cell, wherein the optionally modified nanoparticle subunit or carrier protein comprises one or more glycosylation sites (e.g. D / E-X-N-Z-S / T SEQ ID NO: 49); expressing glycosyltransferase pglB in the host cell; glycosylating, via glycosyltransferase pglB, the optionally modified nanoparticle subunit (or modified carrier protein fused to the optionally modified nanoparticle subunit) with a saccharide; and producing an assembled glycoprotein nanoparticle from the glycosylated nanoparticle subunits (or glycosylated carrier protein fused to nanoparticle subunits) in the periplasm. In aspects, the method comprises expressing a modified nanoparticle subunit such as a modified ferritin, a modified dodecin, or a modified E2p, etc.

[0328] Compositions

[0329] A further embodiment is immunogenic compositions or pharmaceutical compositions, such as vaccines, which comprise nanoparticles (or carrier proteins fused to nanoparticles) displaying bacterial oligo- or polysaccharide antigens, and a pharmaceutically acceptable diluent, or excipient. In certain instances, immunogenic compositions are administered to subjects to elicit an immune response that protects the subject against infection by a pathogen, or decreases symptoms or conditions induced by a pathogen. In the context of this disclosure, the term immunogenic composition will be understood to encompass compositions that are intended for administration to a subject or population of subjects for the purpose of eliciting a protective or palliative immune response against said bacteria.

[0330] An “immunogenic composition” is a composition of matter suitable for administration to a human or non-human mammalian subject and which, upon administration of an immunologically effective amount, elicits a specific immune response, e.g. against an antigen displayed on the nanoparticle or a carrier protein fused to the nanoparticle. An immunogenic composition of the present invention can include one or more additional components, such as an excipient, and / or adjuvant. While administration of an antigen displayed on nanoparticles (or carrier proteins fused to nanoparticles) may enhance a subject’s immune response to the antigen (as compared to administration of the antigen in the absence of the nanoparticle), as used herein, the nanoparticle scaffolds are not defined as an adjuvant.

[0331] Numerous pharmaceutically acceptable diluents and / or pharmaceutically acceptable excipients are known in the art and are described, e.g. in Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975). The adjective “pharmaceutically acceptable” indicates that the diluent or excipient is suitable for administration to a subject (e.g. a human or non-human mammalian subject). In general, the nature of the diluent and / or excipient will depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In certain formulations (for example, solid compositions, such as powder forms), a liquid diluent is not employed. In such formulations, nontoxic solid components can be used, including for example, pharmaceutical grades of trehalose, mannitol, lactose, starch or magnesium stearate. Suitable solid components are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.

[0332] Accordingly, suitable excipients can be selected by those of skill in the art to produce a formulation suitable for delivery to a subject by a selected route of administration.

[0333] In one embodiment, the immunogenic or pharmaceutical compositions comprising nanoparticles do not further comprise an adjuvant. In another embodiment, the immunogenic or pharmaceutical compositions comprising nanoparticles do further comprise an adjuvant.

[0334] Preparation of immunogenic compositions, such as vaccines, including those 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, U.S.A. 1978).

[0335] The bioconjugates of present embodiments are particularly suited for inclusion in immunogenic compositions and vaccines.

[0336] The present embodiments provide an immunogenic composition comprising a nanoparticle, and optionally a pharmaceutically acceptable excipient and / or carrier.

[0337] Immunogenic compositions comprise an immunologically effective amount of the bioconjugate nanoparticle (or bioconjugate carrier protein fused to a nanoparticle), as well as any other components. By “immunologically effective amount,” it is meant that the administration of that amount to an individual, either as a single dose or as part of a series is effective for treatment or prevention. This amount varies depending on the health and physical condition of the individual to be treated, age, the degree of protection desired, the formulation of the vaccine and other relevant factors.

[0338] Pharmaceutically acceptable excipients and carriers are described, for example, in Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co. Easton, PA, 5th Edition (1975). Pharmaceutically acceptable excipients can include a buffer, such as a phosphate buffer (e.g. sodium phosphate). Pharmaceutically acceptable excipients can include a salt, for example, sodium chloride. Pharmaceutically acceptable excipients can include a solubilizing / stabilizing agent, for example, polysorbate (e.g. TWEEN 80). Pharmaceutically acceptable excipients can include a preservative, for example 2-phenoxyethanol or thiomersal. Pharmaceutically acceptable excipients can include a carrier such as water or saline.

[0339] Compositions comprising a nanoparticle may further comprise one or more pharmaceutically acceptable additives such as buffers, carriers, excipients, tonicity agents, wetting or emulsifying agents, detergents, antimicrobials, and diluents. Pharmaceutically acceptable additives are known in the field (e.g. in Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975)). Accordingly, suitable composition components can be selected by an artisan to produce a suitable formulation (e.g. suitable for pharmaceutical use).

[0340] The additive may comprise a pharmaceutically acceptable diluent (e.g. sterile water), saline, glycerol, etc. Additionally, a pharmaceutically acceptable additive may comprise auxiliary substances, such as wetting or emulsifying agents, or pH buffering substances.

[0341] The additive may comprise a pharmaceutically acceptable excipient. Such excipients include, without limitation: glycerol, polyethylene glycol (PEG), glass forming polyols (such as, sorbitol, trehalose) N-lauroylsarcosine (e.g. sodium salt), L-proline, non-detergent sulfobetaine, guanidine hydrochloride, urea, trimethylamine oxide, KC1, Ca2+, Mg2+ , Mn2+ , Zn2+ (and other divalent cation related salts), dithiothreitol (DTT), dithioerytrol, B-mercaptoethanol, detergents (including, e.g. Tween 80, Tween 20, Triton X-100, NP-40, Empigen BB, Octylglucoside, Lauroyl maltoside, Zwittergent 3-08, Zwittergent 3-10, Zwittergent 3-12, Zwittergent 3-14, Zwittergent 3- 16, CHAPS, sodium deoxycholate, sodium dodecyl sulphate, and cetyltrimethylammonium bromide).

[0342] A pharmaceutically acceptable additive for use herein may be a detergent, e.g. a TWEEN (polysorbate), such as TWEEN 80. Detergents are generally present at low levels e.g. <0.01%.

[0343] The pH of a composition comprising a protein nanoparticle may be between 6 and 8, for example between 6.5 and 7.5 (e.g. about 7). Stable pH may be maintained by the use of a buffer (e.g. an acetate buffer, citrate buffer, histidine buffer, maleate buffer, phosphate buffer, succinate buffer, tartrate buffer, or Tris buffer). Thus, a composition will generally include a buffer. A composition may be sterile and / or pyrogen-free. Compositions may be isotonic with respect to humans. Also provided is a method of making the immunogenic composition comprising the step of mixing the nanoparticle with a pharmaceutically acceptable excipient and / or carrier.

[0344] Also provided is an immunogenic composition (e.g. a vaccine composition) optionally comprising an adjuvant.

[0345] The term “adjuvant” refers to a compound that when administered in conjunction with or as part of an immunogenic composition of the vaccine of present embodiments augments, enhances and / or boosts the immune response to nanoparticles, but when the compound is administered alone does not generate an immune response to the nanoparticles. Adjuvants can enhance an immune response by several mechanisms including, e.g. lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. Specific examples of adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (see United Kingdom Patent GB2220211), MF59 (Novartis), AS01 (GlaxoSmithKline), AS03 (GlaxoSmithKline) and saponins, such as QS21 (see Kensil et al. in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540). 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 immune stimulants, such as monophosphoryl lipid A (see Stoute et al. N. Engl. J. Med. 336, 86-91 (1997)).

[0346] Immunogenic compositions of the present invention may additionally include 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 suspensions of minerals (e.g. alum, aluminum hydroxide, aluminum phosphate); saponins such as QS21; emulsions, including water-in-oil, and oil-in-water (and variants thereof, including double emulsions and reversible emulsions), liposaccharides, lipopolysaccharides, immunostimulatory nucleic acid molecules (such as CpG oligonucleotides), liposomes, Toll Receptor agonists, Toll-like Receptor agonists (particularly, TLR2, TER4, TER7 / 8 and TER9 agonists), and various combinations of such components. For the purposes of the present embodiments, the assembled nanoparticle is not considered an adjuvant.

[0347] Also provided is a method of making the immunogenic composition comprising the step of mixing the nanoparticle (NP) or the carrier protein fused to the nanoparticle (NP) with a pharmaceutically acceptable excipient and / or carrier and an adjuvant. Vaccine preparation is generally described in Vaccine Design (“The subunit and adjuvant approach” (eds Powell M.F. & Newman M.J.) (1995) Plenum Press New York).

[0348] The immunogenic compositions of present embodiments can be included in a container, pack, or dispenser together with instructions for administration.

[0349] The immunogenic compositions or vaccines of thereof can be stored before use, e.g. the compositions can be stored frozen (e.g. at about -20°C or at about -70°C); stored in refrigerated conditions (e.g. at about 4°C); or stored at room temperature. The immunogenic compositions or vaccines of present embodiments may be stored in solution or lyophilized. In an embodiment, the solution is lyophilized in the presence of a sugar such as sucrose, trehalose or lactose. In another embodiment, the vaccines of present embodiments are lyophilized and extemporaneously reconstituted prior to use.

[0350] Administration and Dosage

[0351] Immunogenic compositions or vaccines of present embodiments may be used to protect or treat a subject (e.g. mammal), by means of administering said immunogenic composition or vaccine via systemic or mucosal route. These administrations may include injection via the intramuscular (IM), intraperitoneal, intradermal (ID) or subcutaneous (SC) routes; or via mucosal administration to the oral / alimentary, respiratory, genitourinary tracts.

[0352] In one aspect, the immunogenic composition or vaccine of present embodiments is administered by the intramuscular delivery route. Intramuscular administration may be to the thigh or the upper arm. Injection is typically via a needle (e.g. a hypodermic needle), but needle-free injection may alternatively be used. A typical intramuscular dose is 0.5 mb.

[0353] In another aspect, the immunogenic composition or vaccine of present embodiments is administered by the intradermal administration. Human skin comprises an outer "homy" cuticle, called the stratum comeum, which overlays the epidermis. Underneath this epidermis is a layer called the dermis, which in turn overlays the subcutaneous tissue. The conventional technique of intradermal injection, the "mantoux procedure," comprises steps of cleaning the skin, and then stretching with one hand, and with the bevel of a narrow-gauge needle (26 to 31 gauge) facing upwards the needle is inserted at an angle of between 10 to 15°. Once the bevel of the needle is inserted, the barrel of the needle is lowered and further advanced whilst providing a slight pressure to elevate it under the skin. The liquid is then injected very slowly thereby forming a bleb or bump on the skin surface, followed by slow withdrawal of the needle.

[0354] In another aspect, the immunogenic composition or vaccine of present embodiments is administered by the intranasal administration. Typically, the immunogenic composition or vaccine is administered locally to the nasopharyngeal area, e.g. without being inhaled into the lungs. It is desirable to use an intranasal delivery device which delivers the immunogenic composition or vaccine formulation to the nasopharyngeal area, without or substantially without it entering the lungs. Suitable devices for intranasal administration of the vaccines according to present embodiments are spray devices. Suitable commercially available nasal spray devices include ACCUSPRAY™ (Becton Dickinson).

[0355] The amount of bioconjugate in each immunogenic composition or vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed and how it is presented. The content of bioconjugate will typically be in the range 1-100 pg, suitably 5-50 pg.

[0356] Prophylactic and Therapeutic Uses

[0357] The present invention also provides an immunogenic composition of present embodiments, or the vaccine of present embodiments, for use in medicine.

[0358] A method is provided of inducing an immune response in a subject (e.g. human), the method comprising administering a therapeutically or prophylactically effective amount of bioconjugate nanoparticle (optionally fused to a carrier protein), an immunogenic composition thereof, or a vaccine thereof, to a subject (e.g. human) in need thereof. Also provided is a bioconjugate nanoparticle thereof, an immunogenic composition thereof or a vaccine thereof, for use in inducing an immune response in a subject (e.g. human). Also provided is a bioconjugate nanoparticle (optionally fused to a carrier protein), the immunogenic composition thereof, or the vaccine thereof for use in the manufacture of a medicament for inducing an immune response in a subject (e.g. human).

[0359] A further aspect is a method of inducing an immune response for the purpose of treating and / or preventing a bacterial infection of a subject, comprising administering to the subject an immunologically effective amount of the bacterial antigenic molecule displayed on the surface of nanoparticles or carrier molecules fused to nanoparticles that display the antigenic molecule to which an immune response is desired, where said antigens can induce a protective or therapeutic immune response. Such bacterial antigenic molecules displayed on the surface of nanoparticles or carrier molecules fused to nanoparticles may be within an immunogenic or pharmaceutical composition as described herein. In one embodiment of present embodiments, a single dose is administered to the subject. The dose may be adjuvant-free, or it may further comprise an adjuvant.

[0360] Also provided herein are methods of inducing an immune response in a subject against a bacterium, comprising administering to the subject bioconjugate nanoparticle of present embodiments, an immunogenic composition thereof or a vaccine thereof. The conjugate (e.g. bioconjugate) nanoparticle of present embodiments is an immunogenic composition or a vaccine that can be used to induce an immune response against a bacterium, e.g. Shigella species, Pseudomonas aeruginosa, Klebsiella pneumoniae, N. meningitidis, H. influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In an embodiment, the bioconjugate nanoparticle of present embodiments is an immunogenic composition or a vaccine that can be used to induce an immune response against a bacterium, e.g. Streptococcus species, Shigella species, Pseudomonas species, Klebsiella species, or Staphylococcus species, (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, or Staphylcoccus aureus). In one embodiment, said subject has a bacterial infection at the time of administration. In another embodiment, said subject does not have a bacterial infection at the time of administration.

[0361] Also provided are methods of treating and / or preventing a yeast or bacterial infection in a subject comprising administering to the subject a bioconjugate nanoparticle. The bioconjugate nanoparticle may be in the form of an immunogenic composition or vaccine. Thus a method of treating and / or preventing a yeast or bacterial infection in a subject (e.g. humanjis provided, the method comprising administering a therapeutically or prophylactically effective amount of a bioconjugate nanoparticle, an immunogenic composition thereof or a vaccine thereof, to a subject (e.g. human) in need thereof. A bioconjugate nanoparticle, an immunogenic composition thereof or a vaccine thereof, is provided for use in treating and / or preventing a yeast or bacterial infection in a subject (e.g. human). Also provided is a bioconjugate nanoparticle, the immunogenic composition thereof or the vaccine thereof for use in the manufacture of a medicament for treating and / or preventing yeast or bacterial infections in a subject (e.g. human).

[0362] In a specific embodiment, the immunogenic composition or vaccine provided herein is used in the prevention of infection of a subject by a bacterium. Bacteria infections that can be treated and / or prevented using the bioconjugate nanoparticle provided herein including those caused by N. meningitidis, H. influenzae type b (Hib), Streptococcus species, Shigella species, Pseudomonas species, Klebsiella species, or Staphylococcus species, (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae or Staphylcoccus aureus) .

[0363] A further aspect is a method of inducing an immune response in a mammalian subject, such as a human subject, wherein said immune response is specific for a bacterial antigenic molecule displayed on the surface of nanoparticles or carrier molecules fused to nanoparticles provided herein. The method comprises administering to a subject an immunologically effective amount of the bacterial antigenic molecule displayed on the surface of nanoparticles or carrier molecules fused to nanoparticles to which an immune response is desired. The subject may have a bacterial infection at the time of administration, or the administration may be given prophylactically to a subject who does not have a bacterial infection at the time of administration. In one embodiment, the nanoparticles administered display antigens from at least two diseasecausing serotypes. This may be achieved by administering a mixture of nanoparticles where each nanoparticle displays a single serotype antigen, or by administering nanoparticles that display multiple serotype antigens. The antigens may be capsular polysaccharides or immunogenic fragments thereof, oligosaccharides, glycoconjugates, or a mixture thereof.

[0364] Also provided herein are methods of inducing the production of opsonophagocytic antibodies in a subject against a bacterium, comprising administering to the subject a conjugate (e.g. bioconjugate) nanoparticle, an immunogenic composition thereof, or a vaccine thereof. The bioconjugate nanoparticle is an immunogenic composition or a vaccine that can be used to induce the production of opsonophagocytic antibodies in a subject against a bacterium, e.g. Shigella species, Pseudomonas aeruginosa, Klebsiella pneumoniae, N. meningitidis, H. influenzae type b (Hib), Group B Streptococcus (GBS), Streptococcus pneumoniae, or Staphylococcus aureus. In an embodiment, the bioconjugate nanoparticle which is an immunogenic composition, or a vaccine can be used to induce the production of opsonophagocytic antibodies in a subject against a bacterium, e.g. Streptococcus species, Shigella species, Pseudomonas species, Klebsiella species, or Staphylococcus species, (e.g. Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae or Staphylcoccus aureus) .

[0365] Another embodiment is a method of immunising a human subject in order to decrease the risk of disease, wherein the subject receives both a priming dose and a boosting dose of a composition, and where the priming and the boosting dose each elicit in the subject IgG antibodies specific for the same disease-causing serotype(s). In one embodiment, the boosting dose is administered more than 14 days, 21 days, 30 days, 2 months, 3 months, 6 months, 1 year or more after the priming dose. The priming and / or boosting dose may be adjuvant-free, or either or both may further comprise an adjuvant.

[0366] In one embodiment, the bioconjugate nanoparticles (optionally including a carrier protein) and compositions thereof are utilized in methods of immunizing a subject to achieve a protective (prophylactic) immune response in both the subject and (via transplacental transfer of maternal antibodies) to an infant bom to the subject.

[0367] The immunogenic compositions provided herein are conventionally administered parenterally, e.g. by injection, either subcutaneously, intraperitoneally, transdermally, or intramuscularly. Dosage treatment may be a single dose schedule or a multiple dose schedule.

[0368] The various features which are referred to in individual sections above apply, as appropriate, to other sections. Consequently, features specified in one section may be combined with features specified in other sections, as appropriate. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments (or aspects of the disclosure) described herein.

[0369] Embodiments are further described in the subsequent numbered paragraphs:

[0370] 1. A method for preparing a glycoprotein nanoparticle (NP) comprising the steps of: providing a host cell; expressing a modified nanoparticle subunit, in the host cell; expressing a glycosyltransferase, in the host cell; glycosylating, via the glycosyltransferase, the modified nanoparticle subunit, in the host cell; and producing an assembled glycoprotein nanoparticle from the glycosylated modified nanoparticle subunits in the periplasm of the host cell.

[0371] 2. The method of paragraph 1, further comprising one or more of (i) transporting to or expressing the modified nanoparticle subunit in the periplasm of the host cell; (ii) transporting to or expressing the glycosyltransferase in the periplasm of the host cell; (iii) glycosylating, via the glycosyltransferase, the modified nanoparticle subunit in the periplasm of the host cell. 3. The method of paragraph 1 or 2, wherein the modified nanoparticle subunit comprises one or more glycosylation sites (e.g. SEQ ID NO: 49; D / E-X-N-Z-S / T).

[0372] 4. The method of any preceding paragraph, wherein the glycosyltransferase is glycosyltransferase pglB (optionally derived from Campylobacter jejuni).

[0373] 5. The method of any preceding paragraph, further comprising glycosylating, via glycosyltransferase pglB, the modified nanoparticle subunit.

[0374] 6. The method of any preceding paragraph, wherein the host cell is a gram-negative bacterial host cell.

[0375] 7. The method of any preceding paragraph, wherein the host cell is E. colt.

[0376] 8. The method of any preceding paragraph, wherein the modified nanoparticle monomer subunit is selected from the group consisting of an E2p monomer subunit, a ferritin monomer subunit, and a dodecin monomer subunit.

[0377] 9. The method of any preceding paragraph, wherein a capsular polysaccharide or oligosaccharide is conjugated to the modified nanoparticle monomer subunit at the one or more glycosylation sites.

[0378] 10. The method of any preceding paragraph, wherein the capsular polysaccharide or oligosaccharide is selected from the group consisting of Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Meningococcal capsular saccharides, and Candida saccharides.

[0379] 11. The method of paragraph 10, wherein the capsular polysaccharide or oligosaccharide is selected from Streptococcus or Klebsiella species. 12. A method for preparing a glycoprotein nanoparticle (NP) comprising the steps of: providing a host cell; expressing a nanoparticle subunit fused to a modified carrier protein in the host cell; expressing a glycosyltransferase (e.g. pglB) in the host cell; glycosylating, via the glycosyltransferase (e.g. pglB), the modified carrier protein in the host cell; and producing an assembled glycoprotein nanoparticle from the plurality of fused nanoparticle subunits in the periplasm of the host cell.

[0380] 13. The method of paragraph 12, further comprising one or more of (i) transporting to or expressing a nanoparticle subunit fused to a modified carrier protein in the periplasm of the host cell; (ii) transporting or expressing a glycosyltransferase (e.g. pglB) in the periplasm of the host cell; (iii) glycosylating, via the glycosyltransferase (e.g. pglB), the modified carrier protein in the periplasm of the host cell.

[0381] 14. The method of paragraph 12 or 13, wherein the modified carrier protein comprises one or more glycosylation sites (e.g. SEQ ID NO:49; D / E-X-N-Z-S / T).

[0382] 15. The method of any one of paragraphs 12 to 14, wherein the glycosyltransferase is glycosyltransferase pglB (optionally derived from Campylobacter jejuni).

[0383] 16. The method of any one of paragraphs 12 to 15, further comprising glycosylating, via glycosyltransferase pglB, the modified nanoparticle subunit optionally in the periplasm.

[0384] 17. The method of any one of paragraphs 12-16, wherein a capsular polysaccharide or oligosaccharide is conjugated to the modified carrier protein.

[0385] 18. The method of any one of paragraphs 12-17, wherein the capsular polysaccharide or oligosaccharide is selected from the group consisting of Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species, Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Meningococcal capsular saccharides, and Candida saccharides. The method of paragraph 18, wherein the capsular polysaccharide or oligosaccharide is selected from Streptococcus or Klebsiella species. The method of any one of paragraphs 12 to 19, wherein the nanoparticle subunit is selected from the group consisting of: an E2p monomer subunit, a ferritin monomer subunit, and a dodecin monomer subunit. The method of any one of paragraphs 12 to 20, wherein the nanoparticle subunit is fused to a carrier protein. The method of any one of paragraphs 12 to 21, wherein the carrier protein is selected from the group consisting of cross-reacting material of diptheria toxin (CRM), ExoProtein A (EP A), diptheria toxoid (DT) or tetanus toxoid (TT). A composition comprising a dodecin nanoparticle (NP) (optionally modified), the dodecin NP comprising one or more dodecin subunits, each dodecin subunit comprising: amino acids 2-70 of SEQ ID NO: 1 or a self-assembling fragment thereof, or an amino acid sequence 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-assembling fragment thereof; at least one glycosylation consensus sequence. The composition of paragraph 23, wherein the dodecin subunit comprises one or more substitution mutation(s) selected from positions G25, V50 and A53 wherein the numbering of positions is relative to SEQ ID NO: 1. The composition of paragraph 22 or 23, wherein the dodecin subunit comprises one (or more) glycosylation consensus sequence(s) of D / E-X-N-Z-S / T (SEQ ID NO: 49), wherein X and Z are independently any amino acid except proline, wherein the one (or more) consensus sequence(s) are (a) added at the N-terminus, (b) added at the C- terminus, and / or (c) substituted for one or more amino acids, independently selected from one or more amino acids between amino acid residues 49-55 of SEQ ID NO: 1 or at equivalent position(s) within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. The composition of any one of paragraphs 23 to 25, wherein the modified dodecin subunit further comprises one or more substitution mutation(s) selected from G25N, V50T and A53T, wherein the numbering of positions is relative to SEQ ID NO: 1. The composition of any one of paragraphs 23 to 26, wherein the dodecin subunit comprises each of the substitution mutation(s) selected from G25N, V50T and A53T wherein the numbering of positions is relative to SEQ ID NO: 1. The composition of any one of paragraphs 23 to 27, wherein:

[0386] (i) at the N terminus, any of the 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) are substituted for the glycosylation consensus sequence(s), or the glycosylation consensus sequence is next to or upstream of amino acid residue 2 of SEQ ID NO: 1;

[0387] (ii) at the C terminus, any of the 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), are substituted for the glycosylation consensus sequence(s), or the glycosylation consensus sequence is next to or downstream of amino acid residue 65 of SEQ ID NO: 1;

[0388] (iii) the one (or more) glycosylation sequence(s) have been substituted for one or more amino acids (e.g. from amino acid residue D51 of SEQ ID NO: 1), or

[0389] (iv) a combination of two or more of (i), (ii), and (iii). The composition of any one of paragraphs 23 to 28, wherein the glycosylation consensus sequence(s) is upstream (e.g. within 1-6 amino acids, or within 3 amino acids) of amino acid residue position 2 of SEQ ID NO: 1. The composition of any one of paragraphs 23 to 29, wherein the dodecin subunit comprises the sequence of SEQ ID NO: 4.

[0390] 31. The composition of any one of paragraphs 23 to 30, wherein the glycosylation consensus sequence(s) is downstream (e.g. within 1-10 amino acids, within 5 amino acids or within 3 amino acids) of amino acid residue position 65 of SEQ ID NO: 1.

[0391] 32. The composition of paragraph of 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 selfassembling fragment thereof or an amino acid sequence 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-assembling fragment thereof.

[0392] 33. The composition of any of one paragraphs 23 to 32, wherein the glycosylation consensus sequence(s) is a substitution at amino acid residue position 51 of SEQ ID NO: 1.

[0393] 34. The composition of any one of paragraphs 23 to 33, wherein the first glycosylation consensus sequence(s) is upstream (e.g. within 1-6 amino acids, or within 3 amino acids) of amino acid residue position 2 of SEQ ID NO: 1 and the second glycosylation consensus sequence(s) is downstream (e.g. within 1-10 amino acids, or within 3 amino acids) of amino acid residue position 65 of SEQ ID NO: 1.

[0394] 35. The composition of 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.

[0395] 36. The composition of any one of paragraphs 23 to 35, wherein the first glycosylation consensus sequence(s) is a substitution at amino acid residue position 51 of SEQ ID NO: 1, and the second glycosylation consensus sequence(s) is downstream (e.g. within 1-10 amino acids, or within 3 amino acids) of amino acid residue position 65 of SEQ ID NO: 8.

[0396] 37. The composition of 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.

[0397] 38. The composition of any one of paragraphs 23 to 37, wherein the first glycosylation consensus sequence(s) is upstream (e.g. within 1-6 amino acids, or within 3 amino acids) of amino acid residue position 2 of SEQ ID NO: 1, the second glycosylation consensus sequence(s) is a substitution at amino acid residue position 51 of SEQ ID NO: 1, and the third glycosylation consensus sequence(s) is downstream (e.g. within 1-10 amino acids, or within 3 amino acids) of amino acid residue position 65 of SEQ ID NO: 1.

[0398] 39. The composition of 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.

[0399] 40. The composition of any one of paragraphs 23 to 39, further comprising a signal sequence of any of SEQ ID NO: 10, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45 or SEQ ID NO: 46.

[0400] 41. The composition of any one of paragraphs 23 to 40, wherein the signal sequence is SEQ ID NO: 10.

[0401] 42. The composition of any one of paragraphs 23 to 41, wherein the dodecin subunit (optionally modified) comprises the amino acid sequence of SEQ ID NO: 5.

[0402] 43. The composition of any one of paragraphs 23 to 41, wherein the dodecin subunit (optionally modified) comprises the amino acid sequence of SEQ ID NO: 6.

[0403] 44. The composition of any one of paragraphs 23 to 41, wherein the dodecin subunit (optionally modified) comprises the amino acid sequence of SEQ ID NO: 7.

[0404] 45. The composition of any one of paragraphs 23 to 41, wherein the dodecin subunit (optionally modified) comprises the amino acid sequence of SEQ ID NO: 8.

[0405] 46. The composition of any one of paragraphs 23 to 41, wherein the dodecin subunit (optionally modified) comprises the amino acid sequence of SEQ ID NO: 9.

[0406] 47. The composition of any one of paragraphs 23 to 46, wherein the dodecin subunit (optionally modified) is bioconjugated to a saccharide, optionally a bacterial polysaccharide (e.g. from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae or Staphylcoccus aureus) at the one or more glycosylation consensus sequence(s).

[0407] 48. The composition of any one of paragraphs 23 to 47, wherein the dodecin subunits selfassemble to form a dodecin nanoparticle.

[0408] 49. The composition of any one of paragraphs 23 to 48, produced by the method of any one of paragraphs 1-22.

[0409] 50. The composition of any one of paragraphs 23 to 49, wherein the dodecin subunit is encoded by the nucleic acid comprising SEQ ID NO: 3.

[0410] 51. A composition comprising a modified ferritin nanoparticle (NP), the modified ferritin nanoparticle comprising one or more modified ferritin subunits, each ferritin subunit comprising:

[0411] (a) amino acids 1-167 of SEQ ID NO: 11 or a self-assembling fragment thereof, or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 1-167 of SEQ ID NO: 11 or a self-assembling fragment thereof; and

[0412] (b) one or more glycosylation sequence(s), wherein the one or more glycosylation sequence(s) are added next to or substituted for one or more amino acids of the modified amino acid sequence for the ferritin subunit.

[0413] 52. The composition of paragraph 51, wherein:

[0414] (i) the one (or more) glycosylation sequence(s) have been added next to (e.g. within about 1-15 amino acids) or substituted for, at the N terminus, amino acid residues 1-10 of SEQ ID NO: 11 (e.g. amino acid residues 1-10, 1-5, 1-4, 1-3, 1-2, 1 of SEQ ID NO: 11),

[0415] (ii) the one (or more) glycosylation sequence(s) have been added next to or substituted for one or more amino acids from 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) glycosylation sequence(s) have been added next to or substituted for one or more amino acids from amino acid 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),

[0416] (iv) a combination of two or more of (i), (ii), and (iii); wherein modifications may be at equivalent position(s) within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 11.

[0417] 53. The composition of paragraph 51 or 52, wherein the glycosylation consensus sequence(s) 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).

[0418] 54. The composition of any one of paragraphs 51 to 53, further comprising a signal sequence of SEQ ID NO: 10.

[0419] 55. The composition of any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence of SEQ ID NO: 13.

[0420] 56. The composition of any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence of SEQ ID NO: 14.

[0421] 57. The composition of any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence of SEQ ID NO: 15.

[0422] 58. The composition of any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence of SEQ ID NO: 16.

[0423] 59. The composition of any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence of SEQ ID NO: 17.

[0424] 60. The composition of any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence of SEQ ID NO: 18. 61. The composition of any one of paragraphs 51 to 53, wherein the modified ferritin subunit comprises the amino acid sequence of SEQ ID NO: 19.

[0425] 62. The composition of any one of paragraphs 51 to 61, wherein the modified amino acid sequence for a ferritin subunit self-assembles to form a ferritin nanoparticle.

[0426] 63. The composition of any one of paragraphs 51 to 62, produced by the method of any one of paragraphs 1-22.

[0427] 64. A composition comprising a modified ferritin nanoparticle, the ferritin NP comprising one or more ferritin subunits, each ferritin nanoparticle subunit comprising:

[0428] (a) amino acids 1-153 of SEQ ID NO: 20 or a self-assembling fragment thereof, or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 1-153 of SEQ ID NO: 20 or a self-assembling fragment thereof; and

[0429] (b) one or more glycosylation sequence(s) (e.g. GSGDQNATGSG (SEQ ID NO: 33), GGSKDQNATKDGSG (SEQ ID NO: 34)), wherein the one or more glycosylation sequence(s) are added next to or substituted for one or more amino acids of the modified amino acid sequence.

[0430] 65. The composition of paragraph 64, wherein the modified amino acid sequence comprises one or more of the following mutations selected from: M3 II, K120L, A124R, M144I, and I154M wherein the numbering of the positions is relative to SEQ ID NO:20.

[0431] 66. The composition of paragraph 64 or paragraph 65, wherein the modified amino acid sequence comprises each of mutations M3 II, K120L, A124R, M144I, and I154M, wherein the numbering of the positions is relative to SEQ ID NO:20.

[0432] 67. The composition of any one of paragraphs 64 to 66, wherein:

[0433] (i) the one (or more) glycosylation sequence(s) have been added next to (e.g. within about 10 -25 amino acids) or substituted for, at the N terminus, amino acid residues 1-10 of SEQ ID NO: 20 (e.g. amino acid residues 1-10, 1-5, 1-4, 1-3, 1-2, or 1 of SEQ ID NO: 20),

[0434] (ii) the one (or more) glycosylation sequence(s) have been added next to or substituted for one or more amino acids from 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);

[0435] (iii)the one (or more) glycosylation sequence(s) have been added next to or substituted for one or more amino acids from amino acid 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

[0436] (iv)a combination of two or more of (i), (ii), and (iii).

[0437] 68. The composition of any one of paragraphs 64 to 67, further comprising a signal sequence of SEQ ID NO: 10.

[0438] 69. The composition of paragraph 64, wherein the modified ferritin subunit comprises the amino acid sequence of SEQ ID NO: 23.

[0439] 70. The composition of paragraph 64, wherein the modified ferritin subunit comprises the amino acid sequence of SEQ ID NO: 24.

[0440] 71. The composition of paragraph 64, wherein the modified ferritin subunit comprises the amino acid sequence of SEQ ID NO: 25.

[0441] 72. The composition of any one of paragraphs 64 to 71, wherein the modified amino acid sequence self-assembles to form a ferritin nanoparticle.

[0442] 73. The modified amino acid sequence of any one of paragraphs 64 to 72, produced by the method of any one of paragraphs 1-16.

[0443] 74. The composition of any one of paragraphs 51 to 78, further comprising a signal sequence of SEQ ID NO: 10; SEQ ID NO: 41, SEQ ID NO: 44, or SEQ ID NO: 47.

[0444] 75. A composition comprising a modified amino acid sequence of a E2p nanoparticle, wherein the nanoparticle comprises E2p nanoparticle subunits comprising:

[0445] (a) amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof, or an amino acid sequence 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-assembling fragment thereof; and

[0446] (b) one or more glycosylation sequence(s) of GSGGGDQNATGSGGG (SEQ ID NO: 36), wherein the one or more glycosylation sequence(s) are added next to or substituted for one or more amino acids of the modified amino acid sequence.

[0447] 76. The composition of paragraph 75, wherein the modified amino acid sequence comprises one ormore ofthe following mutations relative to SEQ IDNO: 26: A187T, F196Y, T281N, P314S, A352V, L425I, A427-428 or at a position equivalent to A187T, F196Y, T281N, P314S, A352V, L425I, A427-428 of SEQ ID NO:26.

[0448] 77. The composition of 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, A427-428 or at a position equivalent to A187T, F196Y, T281N, P314S, A352V, L425I, A427-428 of SEQ ID NO:26.

[0449] 78. The composition of any one of paragraphs 75 to 77, wherein: the one (or more) glycosylation sequence(s) have been added next to (e.g. within about 10 - 20 amino acids) or substituted for, at the N terminus, amino acid residues 185-426 of SEQ ID NO: 26, wherein modifications may be at equivalent position(s) within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 26.

[0450] 79. The composition of any one of paragraphs 75 to 78, further comprising a signal sequence of SEQ ID NO: 10; SEQ ID NO: 41, SEQ ID NO: 44, or SEQ ID NO: 47.

[0451] 80. The composition of any one of paragraphs 75 to 79, wherein the modified E2p subunit comprises the amino acid sequence of SEQ ID NO: 29.

[0452] 81. The composition of any one of paragraphs 75 to 80, wherein the modified amino acid sequence self-assembles to form a nanoparticle.

[0453] 82. The composition of any one of paragraphs 75 to 81, produced by the method of any of paragraphs 1 to 22.

[0454] 83. A composition comprising a modified exotoxin protein A (EP A) ferritin nanoparticle, wherein each subunit comprises a modified EPA fused directly or indirectly to ferritin.

[0455] 84. The composition of paragraph 83, further comprising one or more sequences selected from the group consisting of:

[0456] (i) one, two or three glycosylation sequences within the modified EPA;

[0457] (ii) a glycosylation consensus sequence at the N terminus of EPA;

[0458] (iii) a glycosylation consensus sequence between EPA and ferritin; and

[0459] (iv) one or more of (i), (ii), or (iii).

[0460] 85. The composition of paragraph 83 or 84, wherein the modified EPA-ferritin subunit comprises amino acids 36-663 of SEQ ID NO: 52, or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 36-663 of SEQ ID NO: 52.

[0461] 86. The composition of paragraph 85, wherein the modified EPA-ferritin subunit comprises the amino acid sequence of SEQ ID NO: 52.

[0462] 87. The composition of any one of paragraphs 83 to 86, wherein the modified amino acid sequence self-assembles to form a nanoparticle.

[0463] 88. The composition of any one of paragraphs 83 to 87, produced by the method of any of paragraphs 1 to 22.

[0464] 89. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding the modified nanoparticle subunit or the nanoparticle subunit fused to a modified carrier protein according to any preceding paragraph. 90. A vector, comprising the isolated nucleic acid molecule according to paragraph 89.

[0465] 91. A host cell, comprising the isolated nucleic acid molecule according to paragraph 89 or the vector according to paragraph 90, wherein the host cell is an E. Colt cell (e.g. E. coli K12 W3110).

[0466] 92. The host cell of paragraph 91 comprising: i) one or more nucleotide sequences comprising polysaccharide synthesis genes, optionally for producing a bacterial polysaccharide antigen (e.g. an O-antigen from a gram-negative bacterium optionally from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae or a capsular polysaccharide from a gram-positive bacterium optionally from Streptococcus pneumoniae or Staphylcoccus aureus) or a yeast polysaccharide antigen or a mammalian polysaccharide antigen, optionally integrated into the host cell genome; ii) a nucleotide sequence encoding a heterologous oligosaccharyl transferase, such as glycosyltransferase pglB, optionally within a plasmid; iii) a nucleotide sequence that encodes a modified or unmodified nanoparticle subunit or a nanoparticle subunit fused to a modified carrier protein according to any one of paragraphs 1 to 90, optionally within a plasmid.

[0467] 93. A host cell according to paragraph 91 or 92, further comprising a nucleotide sequence encoding a polymerase (e.g. wzy), a flippase (e.g. wzx) and optionally a nucleotide sequence encoding a chain length regulator (e.g. wzz).

[0468] 94. The host cell according to any one of paragraphs 91 to 93, wherein the oligosaccharyl transferase is a PglB, optionally derived from Campylobacter jejuni.

[0469] 95. An immunogenic composition comprising an assembled glycoprotein nanoparticle according to any preceding paragraph.

[0470] 96. The immunogenic composition of paragraph 95, further comprising an adjuvant. The immunogenic composition of paragraph 95 or 96, wherein said adjuvant is selected from the group consisting of alum, aluminium hydroxide, aluminium phosphate, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a liposaccharide, a lipopolysaccharide, an immunostimulatory nucleic acid molecules, a liposome, and a Toll Receptor or Toll- Like Receptor agonist. The immunogenic composition of any one of paragraphs 95 to 97 which does not further comprise an adjuvant. The immunogenic composition of any one of paragraphs 95 to 98, and optionally comprising a pharmaceutically acceptable diluent or excipient polypeptide. . A vaccine comprising the immunogenic composition of any one of paragraphs 95 to 99, and optionally an adjuvant. . Use 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 manufacture of a medicament for inducing an immune response. . Use 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 manufacture of a medicament, for inducing an immune response in a subject. . Use of an assembled glycoprotein nanoparticle according to any one of paragraphs 1-22, an immunogenic composition according to any one of paragraphs 95 to 99, or a vaccine according to paragraph 100, for the manufacture of a medicament, in the prevention or treatment of disease. . A process for producing an assembled glycoprotein nanoparticle that comprises a modified nanoparticle subunit conjugated to a capsular polysaccharide or oligosaccharide or a nanoparticle subunit fused to a modified carrier protein, said process comprising:

[0471] (i) culturing the host cell of any one of paragraphs 91 to 94 under conditions suitable for the production of glycoproteins, and

[0472] (ii) isolating the assembled glycoprotein nanoparticle, optionally isolating the assembled glycoprotein nanoparticle from a periplasmic extract from the host cell. . A method of inducing an immune response in a human subject, comprising administering to the subject an immunologically effective amount of the 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 manufacture of a medicament. . A method of inducing an immune response in a subject (e.g. human), the method comprising administering a therapeutically or prophylactically effective amount of the assembled glycoprotein nanoparticle of any preceding paragraph, the immunogenic composition of any one of paragraphs 95 to 99 or the vaccine of paragraph 100, to a subject (e.g. human) in need thereof. . The method according to paragraph 105 or 106, where said subject receives a single administration of said assembled glycoprotein nanoparticle, said immunogenic composition, or said vaccine. . The method according to paragraph 107, where said subject receives an intramuscular administration. . A method of treating and / or preventing a yeast or bacterial infection in a subject (e.g. human), the method comprising administering a therapeutically or prophylactically effective amount of the immunogenic composition of any one of paragraphs 95 to 99 or the vaccine of paragraph 100, to a subject (e.g. human) in need thereof. . The immunogenic composition of any one of paragraphs 95 to 99 or the vaccine of paragraph 100, for use in treating and / or preventing a yeast or bacterial infection in a subject (e.g. human). . The immunogenic composition of any preceding paragraph, wherein the consensus sequence(s) selected from D / E-X-N-Z-S / T (SEQ ID NO: 49) and J-D / E-X-N- Z-S / T-U (SEQ ID NO: 51), wherein X and Z are independently any amino acid except proline and J and U are independently 1 to 5 naturally occurring amino acid residues, has been added next to, or substituted for, one or more amino acids, at the N-terminus of a nanoparticle subunit protein or at an equivalent position within an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to a nanoparticle subunit protein. . The immunogenic composition of paragraph 111, wherein X is Q (glutamine) and Z is A (alanine). . The immunogenic composition of paragraph 111 or 112, wherein the amino acid sequence further comprises a peptide tag, optionally said peptide tag comprises six histidine residues and optionally said peptide tag is located at the C-terminus of the amino acid sequence. . The immunogenic composition of any one of paragraphs 111 to 113 further comprising a conjugate (e.g. a bioconjugate) comprising a optionally modified nanoparticle subunit linked to an antigen (e.g. a saccharide antigen, optionally a bacterial polysaccharide antigen). . The immunogenic composition of any one of paragraphs 111 to 114, wherein the optionally modified nanoparticle subunit is covalently linked to an antigen. . The immunogenic composition of any one of paragraphs 111 to 115, wherein the antigen is a saccharide, optionally a bacterial polysaccharide (e.g. from Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae or Staphylcoccus aureus), optionally an O- antigen from a gram -negative bacterium. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “plurality” refers to two or more. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

[0473] Additionally, numerical limitations given with respect to concentrations or levels of a substance, such as an antigen, are intended to be approximate. Thus, where a concentration is indicated to be at least (for example) 200 pg, it is intended that the concentration be understood to be at least approximately (or “about” or “~”) 200 pg.

[0474] The term “comprises” means “includes.” Thus, unless the context requires otherwise, the word “comprises,” and variations such as “comprise” and “comprising” will be understood to imply the inclusion of a stated compound or composition (e.g. nucleic acid, polypeptide, antigen) or step, or group of compounds or steps, but not to the exclusion of any other compounds, composition, steps, or groups thereof. The abbreviation, “e.g.” is used herein to indicate a nonlimiting example and is synonymous with the term “for example.”

[0475] It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acid molecules or polypeptides are approximate and are provided for description. It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Additionally, numerical limitations given with respect to concentrations or levels of a substance, such as an antigen, are intended to be approximate. Thus, where a concentration is indicated to be at least (for example) 200 pg, it is intended that the concentration be understood to be at least approximately (or “about” or “~”) 200 Pg-

[0476] The term "and / or" as used in a phrase such as "A and / or B" is intended to include “A and B," "A or B," "A," and "B." Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: 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).

[0477] Unless specifically stated, a process comprising a step of mixing two or more components does not require any specific order of mixing. Thus, components can be mixed in any order. Where there are three components, then two components can be combined with each other, and then the combination may be combined with the third component, etc. Similarly, while steps of a method may be numbered (such as (1), (2), (3), etc. or (i), (ii), (iii)), the numbering of the steps does not mean that the steps must be performed in that order (i.e., step 1 then step 2 then step 3, etc.). The word “then” or “followed by” may be used to specify the order of a method’s steps.

[0478] The present invention is not limited to particular embodiments described herein. It is appreciated that certain features of the invention which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0479] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0480] The entire disclosure of published references, patents, and published patent applications cited herein are incorporated herein by reference in their entirety.

[0481] The following examples are set forth. These examples are for purposes of illustration only and are not to be construed as limiting the scope of the present embodiments in any manner.

[0482] EXAMPLES

[0483] Example 1. Summary of nanoparticles

[0484] Table 1.

[0485] Constructs for insertion of glycosites were designed, made, and evaluated for periplasmic expression, glycosylation, and assembly. Nanoparticles were characterized and examined under electron microscopy and in vitro studies were conducted. Representative examples are provided below.

[0486] Example 2, Engineering of nanoparticles for glycosylation with antigenic glycans

[0487] To predict suitable positions for insertion of glycosites, the crystal structures of nanoparticles were analyzed using various software (e.g. Rosetta). Positions were identified that were predicted to not interfere with nanoparticle formation. These positions were identified as suitable for modification (e.g. insertion, deletion, mutation) to a sequence suitable for glycosylation (e.g. to allow glycosite insertion at specified locations). In general, suitable positions are also surface exposed (e.g. to be accessible for antigen recognition by the humoral immune system). This criterion allowed identification of positions for glycosite insertion. In some cases, identified positions were limited. In other cases, identified positions were identified as internal positions, N- terminus positions or C- terminus positions, or combinations of the aforementioned. Solvent accessible amino acid residues were selected for site directed mutagenesis or gene synthesis.

[0488] In some cases, positions were identified that were suitable for stabilization, and one or more stabilizing mutations were added to stabilize the nanoparticles. In general, when engineering glycosites at internal positions (surface exposed loops) the sequence introduced was DQNAT (SEQ ID NO: 32). After testing glycosylation efficiency, this consensus sequence was in some cases extended either by adding flanking lysine residues (KDQNATK (SEQ ID NO: 37)), flanking glycine residues (GDQNATG (SEQ ID NO: 35)) or a longer flanking sequence (for example GSGDQNATGSG (SEQ ID NO: 31) or GSGGGDQNATGSGGG (SEQ ID NO: 36)). Additionally, when engineering sequences for glycosylation (glycosites) at the N- or C- terminus, the following example sequences were added: GSGGGDQNATGSGGG (SEQ ID NO: 36) or GSGDQNATGSG (SEQ ID NO: 31) (see also, SEQ ID NOs: 30-39). For generation of nanoparticle variants containing a combination of two or more glycosites, additional rounds of mutagenesis in the available and selected single site variants were performed as needed.

[0489] In general, a flanking sequence of between 1-5 amino acid residues (e.g. of G, S residues) may be added to either or both sides of the consensus sequence DQNAT (SEQ ID NO: 32), when added internally, or at the N- or C- terminus. mi3 and E2p

[0490] Structural analysis revealed that glycosite insertion in mi3 and E2p nanoparticle subunits is possible only at their N-terminus. For these two nanoparticles, a stretch of flexible linker sequence carrying a consensus glycosylation sequence was inserted between the signal sequence and the first amino acid of the nanoparticle protein subunit. For example, with reference to mi3, the sequence GSGKDQNRTKDGSG (SEQ ID NO: 30) was inserted, while for E2p the sequence GSGGGDQNATGSGGG (SEQ ID NO: 36) was inserted. Here, the consensus glycosylation sequence was DQNAT (SEQ ID NO: 32) and KDQNRTK (SEQ ID NO: 37).

[0491] For E2p, representative examples showing expression, assembly, and glycosylation in a single step (e.g. based on SDS-PAGE and Western blot characterization) are shown in FIG. 11A for Spl2F and in FIG. 12A-12D for KpO3b.

[0492] Ferritins

[0493] The structural analysis of ferritin nanoparticles from Helicobacter pylori and Pseudomonas aeruginosa revealed that suitable positions for engineering glycosites are the N-terminus and the 4 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). Ferritin C-terminus is not solvent accessible and therefore not suitable for addition of glycosites. For each loop a set of 4-6 glycosite variants was designed by varying the residues that were mutated to a glycosite. Initial glycosylation tests were done using Spl2F glycan and showed that the best positions for glycosylation include the N-terminus, loop 2 (residue range 64-82 of H. pylori ferritin and residue range 65-81 of / ' aeruginosa ferritin) and the loop 4 (residue range 145- 149 of H. pylori ferritin and residue range 143-146 of P. aeruginosa ferritin). For example, favorable positions for glycosite insertion within loop 2 of H. pylori ferritin included K79 and E81 when substituted with DQNAT (SEQ ID NO: 32).

[0494] For / / . pylori ferritin loop 1 (residues 34-36) and loop 3 (residues 111-113) were tested for glycosylation with Spl2F, but glycosylation was not efficient. When using another glycan, namely KpO3b, the glycosylation efficiency of H. pylori ferritin was much higher. A significant amount of glycoconjugate can be obtained by inserting glycosylation sites in all 4 loops. Without being bound by theory, the glycosites in loop 1 potentially reduce ferritin expression level and stability; however, the amount of glycoconjugate is not lower than for other variants. The best KpO3b glycosylation is obtained with a variant containing a glycosite in loop 3, specifically the mutation of DI 13 to DQNAT. Most of the glycosite variants in loop 2 and loop 4 enable efficient glycosylation with KpO3b. Selection of glycosites for combination was based on Spl2F glycosylation tests, since this glycan is more challenging.

[0495] Results: Representative examples showing expression, assembly, and glycosylation in a single step (e.g. based on SDS-PAGE and Western blot characterization) are shown in FIGs. 3A - 8 and 13A-14C. These examples show assembly and glycosylation of nanoparticles expressed in E. coli for different glyco-antigens, for N- based glycosylation sites, including Sp 12F and KpO3b.

[0496] Dodecin

[0497] In the case of dodecin, suitable glycosites were identified at the N- and C-terminus as well as the hairpin loop between amino acid residues 49-55. For example, D51 when substituted with DQNAT (SEQ ID NO: 32) yielded efficient glycosylation.

[0498] Results: Representative examples showing expression, assembly, and glycosylation in a single step (e.g. based on SDS-PAGE and Western blot characterization) are shown throughout the specification, e.g. see, FIGs. 9A - 10D. These examples show assembly and glycosylation of nanoparticles expressed in E. coli for different glyco-antigens (e.g. polysaccharides), for N based glycosylation sites, including Spl2F and KpO3b.

[0499] IGPD

[0500] For IGPD, glycosites were designed at several positions, but glycosylation characterization showed efficient glycosylation only for the N-terminal glycosite. Periplasmic expression was reduced compared to other constructs. Results are not shown.

[0501] I5350A

[0502] Component A of the nanoparticle I5350A was screened for glycosite insertion at 12 positions. Weak glycosylation was obtained with variants containing a glycosite at the N-terminus and in the loop between residues 41-44 (wherein FTPV was substituted with DQNAT (SEQ ID NO: 32)). Results are not shown.

[0503] Qbeta

[0504] For Qbeta, glycosites were engineered at the N-terminus and in 3 internal positions including residues 12-16, 75-79 and 118. These residues were substituted with DQNAT (SEQ ID NO: 32). These residues appeared to undergo weak glycosylation. Results are not shown.

[0505] Example 3, Glycosylation tests with engineered nanoparticles containing one or more glycosites

[0506] The constructs encoding variant nanoparticle subunits containing a single inserted glycosite or a combination of several glycosites were tested for in vivo glycosylation efficiency using various antigenic glycans. For the data set presented herein, glycosylation was tested with the following glycan antigens: Streptococcus pneumoniae capsular polysaccharide 12F (.S / ? 12F). Klebsiella pneumoniae O-antigens O3b ( pO3b) and 05 ( pO5), and 5p33F.

[0507] In some cases, a glycosylation test was performed with an E. coli strain in which the cluster of genes for polysaccharide biosynthesis was integrated into the E. coli genome (see W02014 / 057109 and WO2015 / 052344 for further details relating to integration), which allowed transformation only with two plasmids expressing the nanoparticle subunit and PglB. For the data set presented in this work, the used E. coli strains are derivatives of strain W3110, which include a deletion in the lipopolysaccharide O-antigen ligase gene waaL, the deletion or replacement of the 016 O-antigen cluster rfb and the replacement of a genomic cluster with the cluster responsible for the biosynthesis of the wanted recombinant glycan (e.g. Klebsiella pneumoniae O-antigen (KpO-antigen), 5pl2F, and 5p33F capsular polysaccharides.

[0508] The cluster of genes for the biosynthesis of Kp05 glycan was integrated in the 016 locus. The cluster of genes for the biosynthesis of / ?O3b glycan was integrated into the 016 locus as well. For biosynthesis of / ?O3b it was in addition necessary to provide manB (phosphomannomutase) and manC (mannose-l-phosphate guanyly transferase) genes that are needed forthe biosynthesis of GDP-mannose. The manBC genes were expressed from ahigh copy plasmid.

[0509] In the strain expressing Spl2F capsular polysaccharide, the cluster responsible for the biosynthesis of the 016 O-antigen polysaccharide (rbf) has been replaced by the .S', pneumoniae serotype 12F capsular polysaccharide biosynthesis cluster (cpsSpllF). The O-antigen ligase- encoding gene waaL has been replaced by a cassette encoding IPTG-inducible oligosaccharyltransferase PglB. The genomic cluster responsible for the biosynthesis of the enterobacterial common antigen, ECA (wee) has been replaced by the gene gne. of Camplyobacter jejuni origin. The cluster responsible for the biosynthesis of colanic acid (wca) has been replaced with an additional copy of genes wciJ-wcxB-wcxD-wcxE-wcxF from cps.Sp \ 2F and another copy of gne.

[0510] In the strain expressing Sp33F capsular polysaccharide, the cluster responsible for the biosynthesis of colanic acid (wca) has been replaced by the S.pneumoniae serotype 33F capsular polysaccharide biosynthesis cluster (cpsSp33Y). The O-antigen ligase-encoding gene waaL has been replaced by the wchA gene and the cluster responsible for the biosynthesis of the 016 O- antigen polysaccharide rbf) has been replaced by the cassette containing the genes wbbH-gnd.

[0511] The selection criteria for nanoparticle subunit variants with one or more glycosites included the total expression level of the nanoparticle subunit and the level of produced glycoconjugate, the later indicating suitability of glycosite position for modification by PglB, as well as the ability of the nanoparticle subunit to self-assemble. Thus, the procedure for determining a level of glycosylation is similar to procedures for determining a level of expression. The E. coli strain producing Spl2F, KpO3b, or KpO5 glycan was transformed with a pEC415 plasmid encoding a nanoparticle subunit variant and a plasmid expressing PglB. To prepare a preculture, 5 mL TB medium containing 10 mM MgCh and appropriate antibiotic(s) were inoculated with a streak of colonies from the transformation plate and grown at 37°C o / n. The pre-culture was used to inoculate 50 mL of supplemented TB medium in a shake flask to give a starting ODeoo = 0.1. The cultures were grown at 37°C, with 200 rpm shaking until reaching ODeoo = 0.8-1 and then induced by addition of arabinose (depending on the nanoparticle subunit, the concentration was in the range of 0.001 to 0.1%) with 0.1 mM IPTG (for induction of PglB). The expression and glycosylation of variants was continued at 30°C o / n.

[0512] Periplasmic extracts and IMAC-enriched periplasmic extracts were prepared using the same protocol also used for determining a level of expression. For example, the samples were analyzed by SDS-PAGE, anti-His Western blot and anti-glycan Western blots. The read-out by Western blot or SDS-PAGE showed increased molecular weight of glyco-conjugates suggesting higher sugar to protein ratio due to increased number of glycosites.

[0513] Results: Representative examples showing SDS-PAGE and Western blot figures for glycosylation of ferritin, E2p and dodecin glycosite variants (single and combined glycosites) are shown in the figures. These examples show assembly and glycosylation of nanoparticles with different glycoantigens, including Spl2F, KpO3b, and KpO5.

[0514] Example 4, Signal sequence screen for nanoparticle expression in E.coli periplasm

[0515] Cloning

[0516] For screening different signal sequences for expression of nanoparticle subunits in E. colt periplasm, a library of 23 bacterial signal sequences was used. The library contained the following signal sequences: ArgT, BtuB, DsbA (SEQ ID NO: 10), Figi (SEQ ID NO: 42), OmpAVl, 0mpAV2, OmpC, OmpT, PhoA, TolB (SEQ ID NO: 41), DegP, FhuA, Hla, Ltllb (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). Codon optimized nanoparticle subunit genes were 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) in frame with N-terminal signal sequence using restriction sites Nhel and Xho\. To enable detection in Western blot and purification by IMAC, a His-tag was introduced at N- or C- terminus of a nanoparticle protein subunit during cloning. Clones containing the desired insert were verified by plasmid sequencing.

[0517] Expression cultures

[0518] For testing expression of nanoparticle subunits with various signal sequences, a derivative of the E. col strain W3110 with deleted araBA genes was used. The deletion of araBA genes allows better control of induction with arabinose, since in such strain arabinose cannot be metabolized. Therefore, lower arabinose concentration can be used for titrating the ara promoter and an optimal inducer concentration can be found. The competent E.coli cells were transformed with a pEC415 plasmid encoding a nanoparticle subunit. To prepare a pre-culture, 5 mL TB medium containing 10 mM MgCh and kanamycin antibiotic was inoculated with a streak of colonies from the transformation plate and grown at 37°C o / n. The pre-culture was used to inoculate 50 mL of supplemented TB medium in a shake flask to give a starting ODeoo = 0.1. The cultures were grown at 37°C, with 200 rpm shaking until reaching ODeoo = 0.8-1 and then induced by addition of arabinose in the concentration range between 0.001% to 1%. The expression of nanoparticles was continued at o / n at different temperatures (e.g. 25, 30 and 37°C tested).

[0519] Example 5 , Periplasmic protein extraction

[0520] The amount of cells from o / n cultures corresponding to ODeoo = 60 (measured using a spectrophotometer) was harvested by centrifugation. The cell pellets were resuspended in 1.5 mL of lysis buffer (30 mM Tris-HCl pH 8.5, 1 mM EDTA (Ethylenediaminetetraacetic acid), 20% sucrose) and lysozyme was added to a final concentration of 1 mg / mL. The suspensions were incubated with slight shaking for 25 minutes at 4°C and then centrifuged at 16’000 ref for 10 min. After centrifugation, the supernatant corresponding to periplasmic extract (PPE) was transferred to a fresh tube. Periplasmic extracts were either directly used for SDS-PAGE and Western blot analysis or submitted to small-scale purification by IMAC.

[0521] Example 6, Enrichment of periplasmic extract by immobilized metal affinity chromatography (IMAC)

[0522] To enrich periplasmic extracts with nanoparticle variants and allow more direct read-out by SDS-PAGE, the His-tagged nanoparticle proteins were purified using one-step purification on Ni-NTA (Nickel Nitrilo-triacetic Acid) agarose. 1 mL of PPE was mixed with 200 pl of preequilibrated Ni-NTA slurry and incubated with slight shaking for 30 min. After that, the resin was washed and the bound protein eluted with elution buffer (30 mM Tris pH 8.0, 500 mM imidazole, 50 mMNaCl). The IMAC enriched PPE was analysed by SDS-PAGE (see, Laemmli, U. K. (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). Purified nanoparticles were detected on the gel by Coomassie staining (see, Fazekas de St. Groth, S.; Webster, R. G.; 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).

[0523] Example 7. Western blot analysis of periplasmic extract

[0524] Periplasmic extracts were also analysed by immunoblots against His-tag (Qiagen, Cat. number 34660).

[0525] Results: Representative SDS-PAGE and / or Western blots for Spl2F ferritin Hp nanoparticles are shown in FIG. 3B, 3D, 4B, 5A.

[0526] Representative SDS-PAGE and / or Western blots for KpO3b ferritin Hp nanoparticles are shown in FIGs. 3C, 6A and 7A.

[0527] Representative SDS-PAGE and / or Western blots for Spl2F ferritin Pa nanoparticles are shown in FIG. 8.

[0528] Representative SDS-PAGE and / or Western blots for Spl2F dodecin nanoparticles are shown in FIGs. 9A and 9D. Representative SDS-PAGE and / or Western blots for KpO3b dodecin nanoparticles are shown in FIG. 10A.

[0529] Representative SDS-PAGE and / or Western blots for Spl2F E2p nanoparticles are shown in FIG. 11A.

[0530] Representative SDS-PAGE and / or Western blots for KpO3b E2p nanoparticles are shown in FIGs. 11C and 12A. for high-quality ferritin

[0531] Screening for optimal fermentation conditions was performed on a 250 mb fermenter scale in the DASbox Mini Bioreactor System (Eppendorf) . The optimal conditions for glycan production were combined with optimal induction of nanoparticle expression (e.g. Spl2F-Ferritin).

[0532] The purification procedure that was developed was based on 5 steps that included:

[0533] 1. Periplasmic extract preparation

[0534] 2. Heat precipitation of other proteins at 65°C

[0535] 3. Filtration (TFF) using 100 kDa membrane

[0536] 4. Anion exchange chromatography (AIEX) on Q Ceramic HyperD F 200 mb column 5. Size exclusion chromatography (SEC) on HiPrep 26 / 60 Sephacryl S 500 HR (320 mL) column

[0537] The obtained yield after such process was: -100 mg Ferritin / L BR and -100 mg 5pl2F / L BR.

[0538] Results: For Spl2F ferritin, representative SEC chromatogram and SDS-PAGE of SEC fractions are shown in FIG. 4A and FIG. 4B. Fractions 2-11 in FIG. 4B show a glycosylated product.

[0539] For KpO3b ferritin Hp, representative SEC chromatogram and SDS-PAGE of SEC fractions are shown in FIG. 6A. Fractions 12-16 in FIG. 6A show a glycosylated product.

[0540] For dodecin Spl2F, representative SEC chromatogram and SDS-PAGE of SEC fractions are shown in FIG. 9C and 9D. Fractions A24 - A32 in FIG. 9D show a glycosylated product.

[0541] For KpO3b-E2p, representative SEC chromatogram and SDS-PAGE of SEC fractions are shown in FIG. 1 IB and 11C. Fractions 12-21 in FIG. 11C show a glycosylated product.

[0542] For Spl2F-EPA-ferritin, representative SEC chromatogram and SDS-PAGE of SEC fractions are shown in FIG. 14B and 14C. Fractions A24-A34 in FIG. 14B show a glycosylated product.

[0543] Purified NP-bioconjugates were analysed for purity by SDS-PAGE, for particle size by dynamic light scattering (DLS) and by negative staining electron microscopy. The amount of glycan in each bioconjugate was determined by HPAEC-PAD (high-performance anion-exchange chromatography with pulsed amperometric detection).

[0544] Results: For Spl2F ferritin Hp, representative examples include SDS-PAGE (FIG. 5A), DLS (FIG. 5C) showing an average particle diameter of 30 nm and an electron micrograph (FIG. 5D).

[0545] Regarding KpO5-ferritin Hp, introducing glycosites at the N-terminus, and combining the glycosite at the N-terminus with the one in loop 2 and in loop 4 (N+L2 position, at N+L4 position, or at N+L2+L4) led to successfully expression and assembly as well as glycosylation (FIG. 3D).

[0546] For KpO3b ferritin Hp, representative examples include SDS-PAGE (FIG. 7A), DLS (FIG. 7C) showing an average particle diameter of about 25 nm, and an electron micrograph (FIG. 7D).

[0547] For Spl2F-dodecin, representative examples include SDS-PAGE (FIG. 9D). For KpO3b- dodecin representative examples include SDS-PAGE (FIG. 10A), DLS (FIG. 10C) showing an average particle diameter of about 17 nm, and an electron micrograph (FIG. 10D).

[0548] For KpO3b-E2p, representative examples include SDS-PAGE (FIG. 12A), DLS (FIG. 12C) showing an average particle diameter of about 42 nm and an electron micrograph (FIG. 12D).

[0549] For Sp33F-ferritin, representative examples include SDS-PAGE (FIG. 17A), DLS showing an average particle diameter of about 21 nm (FIG. 17A) and an electron micrograph (FIG. 17A).

[0550] For Sp33F-dodecin, representative examples include SDS-PAGE (FIG. 17B), DLS showing an average particle diameter of about 15 nm (FIG. 17B) and an electron micrograph (FIG. 17B).

[0551] Glycan / protein ratios for produced glyco-nanoparticles were measured by HPAEC-PAD and had the following values: Spl2F-ferritin 109%, Spl2F-dodecin 45%, Spl2F-EPA-ferritin 36%, KpO3b-ferritin 40%, KpO3b-dodecin 64%, KpO3b-E2p 18%, Sp33F-ferritin 22%, and Sp33F-dodecin 70%.

[0552] Example 10. Animal studies

[0553] CD1 mice were administrated intra-muscular (i.m.) with a 0.22 pg Spl2F capsular polysaccharide per dose three times, every two weeks (dO, dl4, d28). Antibody responses in serum were monitored prior to immunization (day 0 / pre) and 2 weeks after the second (day 28 / post-II) and third injection (day 42 / post-III). A1PO4 was used as an adjuvant. The animal study with KpO3b glycan had the same design and schedule except for using AS03 as an adjuvant.

[0554] Results: ELISA read-out: Spl2F specific IgG titers pre, (post-II) and post-III, measured in individual serum samples (see, FIG. 15B). Spl2F IgG titers at post-III were improved for bioconjugate nanoparticle 12F-Ferritin (SEQ ID NO: 18) compared to the chemical conjugate of Spl2F-CRM. Spl2F IgG titers for a bioconjugate nanoparticle 12F-Dodecin (SEQ ID NO:7) post- III were elevated as compared to the control groups immunized with PBS and Sp 12F-EPA. Sp 12F IgG titers at post-III for a bioconjugate nanoparticle in which EPA-Ferritin (SEQ ID NO: 52) fusion was used as a carrier were comparable to the chemical conjugate of Spl2F-CRM. In the case of preclinical evaluation of KpO3b-nanoparticles (FIG. 16B), significant immunogenicity against KpO3b was obtained only by using Ferritin as a carrier. All together, these results show that nanoparticles (as e.g. Ferritin) enable superior immune response against conjugated polysaccharide over non-particulate carrier systems (as e.g. EP A). Example 11. Analytical Methods

[0555] Electron Microscopy

[0556] To visualize the assembled NPs, negative staining electron microscopy was used. Protocols for EM are known in the art.

[0557] Western blot of periplasmic extract

[0558] Periplasmic extracts were analysed by immunoblots against polysaccharide attached to the modified nanoparticle subunit. SDS-PAGE analysis was carried out on IMAC enriched periplasmic extract of E.coli strains producing antigen polysaccharide and expressing PglB.

[0559] Dynamic light scattering (DLS)

[0560] The hydrodynamic diameter of the assembled nanoparticles in solution was measured with dynamic light scattering (DLS). Protocols for DLS are known in the art.

[0561] Table 2. Direct Glycosylation of NP or carrier

[0562] Table 3. Direct Glycosylation of NP or carrier

[0563] Table 4. Direct Glycosylation of NP or carrier

[0564] SEQUENCE LISTING

[0565] SEQ ID NO: 1

[0566] Dodecin wild type amino acid sequence (Q8VK10)

[0567] MSNHTYRVI EIVGTSPDGVDAAIQGGLARAAQTM RALDWFEVQSIRG HLVDGAVAH FQVTM KVGFRLEDS

[0568] SEQ ID NO: 2

[0569] Dodecin modified amino acid sequence (G25N, V50T, A53T) and His tag

[0570] MSNHTYRVI EIVGTSPDGVDAAIQNGLARAAQTM RALDWFEVQSIRG HLTDGTVAHFQVTMKVG FRLEDSGSGHHH

[0571] HHH

[0572] SEQ ID NO: 3

[0573] Dodecin modified nucleic acid sequence

[0574] ATGTCTAACCATACTTACCGTGTTATCGAAATCGTGGGAACTTCGCCGGACGGAGTGGACGCCGCGATTCAGAAC

[0575] GGCTTAGCCCGTGCTGCCCAAACCATGCGTGCGCTTGATTGGTTTGAGGTGCAGTCAATTCGCGGACATCTGACA

[0576] GATGGCACGGTCGCTCACTTCCAAGTAACCATGAAAGTGGGGTTTCGCCTGGAGGACTCTGGCAGTGGCCACCAC

[0577] CACCACCATCACTAACGTTGCGCGGAGTTTCTGCGTATGCACAACCTGGAAGACAGCTGCTTCAGCTTTCTGTAA

[0578] SEQ ID NO: 4

[0579] Dodecin modified amino acid sequence (signal sequence, N term glycotag sequence, (G55N, V80T, A83T corresponding to G25N, V50T, A53T of SEQ I D NO:2)) and Histidine tag

[0580] H LTDGTVAH FQVTM KVG F R LE DSGSG H H H H H H

[0581] SEQ ID NO: 5

[0582] Dodecin modified amino acid sequence (signal sequence, (G44N, V69T, A72T corresponding to G25N,

[0583] V50T, A53T of SEQ I D NO:2), C terminus glycotag sequence) and Histidine tag

[0584] M KKIWLALAG LVLAFSASASSNHTYRVI EIVGTSPDGVDAAIQNGLARAAQTM RALDWFEVQSI RG HLTDGTVAHFQV

[0585] TM KVG FRLEDSGSGDQNATGSGHHHHH H SEQ ID NO: 6

[0586] Dodecin modified amino acid sequence (signal sequence, G44N, V69T, A76T (corresponding to G25N,

[0587] V50T, A53T of SEQ I D NO:2) and D51 substituted with DQNAT(Mut3)) and Histidine tag

[0588] AHFQVTM KVGFRLEDSGSG HHHHHH

[0589] SEQ ID NO: 7

[0590] Dodecin modified amino acid sequence (signal sequence, N + C glycotag sequences) and Histidine tag

[0591] H LTDGTVAH FQVTM KVG F R LE DSGSG DQN ATGSG H H H H H H

[0592] SEQ ID NO: 8

[0593] Dodecin modified amino acid sequence (signal sequence, Mut3+C glycotag sequence) and Histidine tag

[0594] AH FQVTM KVG F RLE DSGSG DQN ATGSG H H H H H H

[0595] SEQ ID NO: 9

[0596] Dodecin modified amino acid sequence (signal sequence, N+Mut3+C glycotag sequence) and Histidine tag

[0597] H LTDQN ATGTVAH FQVTM KVG F R LE DSGSG DQN ATGSG H H H H H H

[0598] SEQ ID NO: 10

[0599] Signal Sequence DsbA

[0600] MKKIWLALAG LVLAFSASA

[0601] ***

[0602] SEQ ID NO: 11

[0603] Ferritin H. pylori wild type amino acid sequence (also Q9ZLI1)

[0604] MLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKF

[0605] EGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGI

[0606] AKSRKS

[0607] SEQ ID NO: 12

[0608] Ferritin H. pylori wild type nucleic acid sequence

[0609] ATGCTGAGCAAAGATATTATCAAACTGCTGAACGAACAGGTGAACAAAGAAATGAATAGCAGCAACCTGTATATG

[0610] AGCATGAGCAGCTGGTGTTATACCCATAGCCTGGATGGTGCAGGTCTGTTTCTGTTTGATCATGCAGCCGAAGAAT

[0611] ATGAGCACGCAAAAAAACTGATCATCTTCCTGAATGAAAATAACGTTCCGGTTCAGCTGACCAGCATTAGCGCACC

[0612] GGAACATAAATTTGAAGGTCTGACCCAGATTTTCCAGAAAGCCTATGAACATGAACAGCATATTAGCGAGAGCAT

[0613] CAACAACATTGTGGATCATGCCATCAAATCCAAAGATCATGCGACCTTTAACTTTCTGCAGTGGTATGTTGCAGAA

[0614] CAGCATGAAGAAGAAGTGCTGTTTAAAGACATCCTGGATAAAATTGAACTGATCGGCAATGAAAACCATGGTCTG

[0615] TATCTGGCAGATCAGTATGTTAAAGGTATTGCCAAAAGCCGCAAAAGCTGATGA

[0616] SEQ ID NO: 13

[0617] Ferritin H. pylori modified amino acid sequence (N-terminus glycotag sequence) and Histidine tag

[0618] GGSKDQNRTKDGSGHHHHHHSQDPMLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYE

[0619] HAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLF

[0620] KDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0621] SEQ ID NO: 14

[0622] Ferritin H. pylori modified amino acid sequence (signal sequence, N-terminus glycotag sequence)

[0623] MKKIWLALAGLVLAFSASAGGSKDQNATKDGSGSQDPMLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGA

[0624] GLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQ

[0625] WYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0626] SEQ ID NO: 15

[0627] Ferritin H. pylori modified amino acid sequence (signal sequence, wt K79 position in Loop 2 substituted with DQNAT glycosite) and Histidine tag

[0628] MKKIWLALAGLVLPAFSASAHHHHHHSQDPMLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHA

[0629] AEEYEHAKKLIIFLNENNVPVQLTSISAPEHDQNATFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYV

[0630] AEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS SEQ ID NO: 16

[0631] Ferritin H. pylori modified amino acid sequence (signal sequence, wt residues 146-149 (NEN) in Loop 4 substituted with GDQNATG glycosite) and Histidine tag

[0632] MKKIWLALAGLVLAFSASAHHHHHHSQDPMLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHA

[0633] AEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHE

[0634] EEVLFKDILDKIELIGGDQNATGHGLYLADQYVKGIAKSRKS

[0635] SEQ ID NO: 17

[0636] Ferritin H. pylori modified amino acid sequence (signal sequence, N + L2 glycosites) and Histidine tag

[0637] MKKIWLALAGLVLAFSASAGGSKDQNATKDGSGHHHHHHSQDPMLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYT

[0638] HSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHDQNATFEGLTQIFQKAYEHEQHISESINNIVDHAIKS

[0639] KDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0640] SEQ ID NO: 18

[0641] Ferritin H. pylori modified amino acid sequence (signal sequence, N + L4 glycosites) and Histidine tag

[0642] MKKIWLALAGLVLAFSASAGGSKDQNATKDGSGHHHHHHSQDPMLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYT

[0643] HSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHA

[0644] TFNFLQWYVAEQHEEEVLFKDILDKIELIGGDQNATGHGLYLADQYVKGIAKSRKS

[0645] SEQ ID NO: 19

[0646] Ferritin H. pylori modified amino acid sequence (signal sequence, N + L2 + L4 glycosites) and Histidine tag

[0647] MKKIWLALAGLVLAFSASAGGSKDQNATKDGSGHHHHHHSQDPMLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYT

[0648] HSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHDQNATFEGLTQIFQKAYEHEQHISESINNIVDHAIKS

[0649] KDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGGDQNATGHGLYLADQYVKGIAKSRKS

[0650] SEQ ID NO: 20

[0651] Ferritin P. aeruginosa wild type amino acid sequence (Q9HWF9) MQGHPEVIDYLNTLLTGELAARDQYFIHSRMYEDWGFSKLYERLNHEMEEETQHADALLRRILLLEGTPRMRPDDIHP

[0652] GTTVPEMLEADLKLERHVRAALAKGIALCEQHKDFVSRDILKAQLADTEEDHAYWLEQQLGLIARMGLENYLQSQI

[0653] SEQID NO: 21

[0654] Ferritin P. aeruginosa modified amino acid sequence (with stabilizing substitutions M31I, K120L, A124R,

[0655] M144I, I154M)

[0656] MQGHPEVIDYLNTLLTGELAARDQYFIHSRIYEDWGFSKLYERLNHEMEEETQHADALLRRILLLEGTPRMRPDDIHPGT

[0657] TVPEMLEADLKLERHVRAALAKGIALCEQHKDFVSRDILLAQLRDTEEDHAYWLEQQLGLIARIGLENYLQSQM

[0658] SEQID NO: 22

[0659] Ferritin P. aeruginosa modified nucleic acid sequence

[0660] ATGGGCTCGAGCCACCATCATCATCACCATTCGATGCAGGGACACCCTGAAGTTATTGACTATCTGAACACCCTGC

[0661] TGACGGGGGAACTCGCAGCGCGCGACCAGTATTTTATCCATTCTCGTATTTACGAGGACTGGGGCTTCTCGAAGTT

[0662] GTATGAACGGCTGAATCACGAAATGGAGGAAGAAACACAGCATGCCGACGCGCTGTTACGCCGCATCCTCCTGTT AGAAGGTACCCCGCGCATGCGTCCAGATGATATTCATCCCGGCACCACGGTGCCCGAAATGCTGGAGGCGGATCT TAAACTGGAGCGCCACGTGCGCGCAGCATTGGCCAAGGGTATTGCTCTGTGCGAGCAGCATAAAGATTTTGTAAG

[0663] CCGTG ATATCCTG CTG GCG CAG CTCCG CG ATACAG AAG AGG ACCATGCTTACTGG CTG G AACAG CAG CTG G GTTT AATCGCGCGCATCGGTCTGGAAAACTACCTGCAGAGTCAGATGTGA

[0664] SEQID NO: 23

[0665] Ferritin P. aeruginosa modified amino acid sequence (signal sequence, stabilizing substitutions, N terminus glycotag)

[0666] MKKIWLALAGLVLAFSASASGGSKDQNATKDGSGHHHHHHSMQGHPEVIDYLNTLLTGELAARDQYFIHSRIYEDWG

[0667] FSKLYERLNHEMEEETQHADALLRRILLLEGTPRMRPDDIHPGTTVPEMLEADLKLERHVRAALAKGIALCEQHKDFVSR DILLAQLRDTEEDHAYWLEQQLGLIARIGLENYLQSQM

[0668] SEQID NO: 24

[0669] Ferritin P. aeruginosa modified amino acid sequence (signal sequence, stabilizing substitutions, wt T80 position in Loop 2 substituted with GSGDQNATGSG glycosite) and Histidine tag

[0670] MKKIWLALAGLVLAFSASASHHHHHSMQGHPEVIDYLNTLLTGELAARDQYFIHSRIYEDWGFSKLYERLNHEMEEET QHADALLRRILLLEGTPRMRPDDIHPGGSGDQNATGSGTVPEMLEADLKLERHVRAALAKGIALCEQHKDFVSRDILLA QLRDTEEDHAYWLEQQLGLIARIGLENYLQSQM SEQ ID NO: 25

[0671] Ferritin P. aeruginosa modified amino acid sequence (signal sequence, stabilizing substitutions, wt G145 position in Loop 4 substituted with GSGDQNATGSG glycosite) and Histidine tag

[0672] MKKIWLALAGLVLAFSASASHHHHHSMQGHPEVIDYLNTLLTGELAARDQYFIHSRIYEDWGFSKLYERLNHEMEEET

[0673] QHADALLRRILLLEGTPRMRPDDIHPGTTVPEMLEADLKLERHVRAALAKGIALCEQHKDFVSRDILLAQLRDTEEDHAY

[0674] WLEQQLGLIARIGSGDQNATGSGLENYLQSQM

[0675] SEQ ID NO: 26

[0676] E2p wild type amino acid sequence ( Pl 1961)

[0677] MAFEFKLPDIGEGIHEGEIVKWFVKPGDEVNEDDVLCEVQNDKAVVEIPSPVKGKVLEILVPEGTVATVGQTLITLDAPG

[0678] YENMTFKGQEQEEAKKEEKTETVSKEEKVDAVAPNAPAAEAEAGPNRRVIAMPSVRKYAREKGVDIRLVQGTGKNGR

[0679] VLKEDIDAFLAGGAKPAPAAAEEKAAPAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKL

[0680] VAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALA

[0681] QEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHR

[0682] MIDGATAQKALNHIKRLLSDPELLLMEA

[0683] SEQ ID NO: 27

[0684] E2p modified amino acid sequence (with mutations at wt positions A187T, F196Y, T281N, P314S, A352V,

[0685] L425I, A427-428) and Histidine tag

[0686] MGSSHHHHHHSAATKPATTEGEYPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGI

[0687] KLTFLPYVVKALVSALREYPVLNTSIDDENEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKSIFALAQEINELAEKARDGKL

[0688] TPGEMKGASCTITNIGSVGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHI

[0689] KRLLSDPELLIM

[0690] SEQ ID NO: 28

[0691] E2p modified nucleic acid sequence

[0692] ATGGGAAGCAGCCACCACCATCACCATCACAGCGCAGCCACCAAACCAGCCACTACCGAAGGTGAATATCCTGAA

[0693] ACCCGGGAAAAAATGTCTGGAATCCGTCGCGCGATCGCGAAAGCGATGGTGCATTCCAAACATACTGCACCGCAC

[0694] GTGACCCTGATGGACGAAGCTGATGTCACAAAACTGGTTGCTCATCGCAAAAAATTCAAAGCCATTGCAGCCGAG

[0695] AAAGGCATTAAACTGACCTTTCTGCCGTACGTAGTAAAAGCACTGGTATCCGCGCTTCGTGAATATCCCGTACTGA

[0696] ACACAAGTATCGATGATGAAAATGAAGAAATTATTCAAAAGCATTACTACAACATTGGTATTGCCGCAGACACCGA TCGTGGGTTGCTCGTCCCGGTAATTAAACATGCTGATCGGAAAAGCATTTTCGCACTGGCGCAGGAAATTAACGA

[0697] ACTGGCGGAAAAAGCTCGCGATGGCAAACTGACGCCGGGTGAAATGAAAGGCGCCTCGTGTACCATTACCAACA

[0698] TTGGATCTGTTGGAGGTCAATGGTTTACCCCGGTGATTAATCATCCGGAAGTTGCCATTTTGGGGATCGGCCGCAT

[0699] TGCAGAAAAACCGATCGTGCGTGATGGGGAAATTGTAGCGGCGCCAATGCTCGCCTTATCCCTTAGTTTTGACCAC

[0700] CGTATGATTGACGGAGCCACCGCGCAGAAGGCCCTTAATCATATCAAACGTTTGCTTTCTGATCCCGAACTGCTCA

[0701] TCATGTGA

[0702] SEQ ID NO: 29

[0703] E2p modified amino acid sequence (signal sequence N-terminus glycotag sequence) and Histidine tag

[0704] MKQALRVAFGFLILWASVLHASHHHHHHGSGGGDQNATGSGGGSAATKPATTEGEYPETREKMSGIRRAIAKAMVH

[0705] SKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTSIDDENEEIIQKHYYNIGIAADT

[0706] DRGLLVPVIKHADRKSIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSVGGQWFTPVINHPEVAILGIGRIAEKPIV

[0707] RDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLIM

[0708] SEQ ID NO: 30

[0709] Glycotag amino acid sequence

[0710] GSGKDQNRTKDGSG

[0711] SEQ ID NO: 31

[0712] Glycotag amino acid sequence

[0713] GSGDQNATGSG

[0714] SEQ ID NO: 32

[0715] Glycotag amino acid sequence

[0716] DQNAT

[0717] SEQ ID NO: 33

[0718] Glycotag amino acid sequence

[0719] GGSKDQNRTKDGSG

[0720] SEQ ID NO: 34

[0721] Glycotag amino acid sequence

[0722] GGSKDQNATKDGSG SEQ ID NO: 35

[0723] Glycotag amino acid sequence

[0724] GDQNATG

[0725] SEQ ID NO: 36

[0726] Glycotag amino acid sequence

[0727] GSGGGDQNATGSGGG

[0728] SEQ ID NO: 37

[0729] Glycotag amino acid sequence

[0730] KDQNATK

[0731] SEQ ID NO: 38

[0732] Repeating

[0733] Glycotag amino acid sequence

[0734] GSGGGDQNATGSDQNATGSDQNATGSKDQNATKDGSG

[0735] SEQ ID NO: 39

[0736] Glycotag amino acid sequence

[0737] GGDQNATGG

[0738] SEQ ID NO: 40

[0739] Histidine tag

[0740] MGSSHHHHHHS

[0741] SEQ ID NO: 41

[0742] Signal sequence (TolB)

[0743] M KQALRVAFG FLI LWASVLHA

[0744] SEQ ID NO: 42

[0745] Signal sequence (Flgl)

[0746] MIKFLSALILLLVTTAAQA SEQ ID NO: 43

[0747] Signal sequence (OmpA)

[0748] M KKTAI Al AVALAG FATVAQA

[0749] SEQ ID NO: 44

[0750] Signal sequence (MalE)

[0751] MKIKTGARILALSALTTMMFSASALA

[0752] SEQ ID NO: 45

[0753] Signal sequence (PelB)

[0754] MKYLLPTAAAGLLLLAAQPAMA

[0755] SEQ ID NO: 46

[0756] Signal sequence (LTIIb)

[0757] MSFKKI I KAFVIMAALVSVQAHA

[0758] SEQ ID NO: 47

[0759] Signal sequence (XynA)

[0760] MFKFKKKFLVGLTAAFMSISMFSATASA

[0761] SEQ ID NO: 48

[0762] Signal sequence (SipA)

[0763] MKMNKKVLLTSTMAASLLSVASVQAS

[0764] SEQ ID NO: 49

[0765] Consensus sequence

[0766] D / E-X-N-Z-S / T

[0767] SEQ ID NO: 50

[0768] Consensus sequence

[0769] K-D / E-X-N-Z-S / T-K

[0770] SEQ ID NO: 51 Consensus sequence

[0771] J-D / E-X-N-Z-S / T-U

[0772] SEQ ID NO: 52

[0773] EPA-5s-Ferritin modified amino acid sequence (signal sequence, glycosites, histidine tag) with mutations

[0774] L552V, deltaE553

[0775] MKKIWLALAGLVLAFSASAGSGGGDQNATGSGGGAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG

[0776] VLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELN

[0777] AGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVS VMAQAQPRREKRWSEWASGKVLCL

[0778] LDPLDGVYNKDQNATKLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLE

[0779] AFTKDQNATKHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLAL

[0780] TLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQN

[0781] WTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIR

[0782] NGALLRVYVPRWSLPGFYRTGLTLKDQNATKAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRVTILGWPLAERTVVIPS

[0783] AIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLGSGGGDQNATASGGGHHHHHHSQDPMLSKDIIKLL

[0784] NEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQK

[0785] AYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0786] SEQ ID NO: 53

[0787] Consensus glycotag amino acid sequence {X = R, A}

[0788] DQNXT

[0789] SEQ ID NO: 54

[0790] Sequence for glycotransferase PgIB

[0791] MLKKEYLKNPYLVLFAMIILAYVFSVFCRFYWVWWASEFNEYFFNNQLMIISNDGYAFAEGARDMIAGFHQP

[0792] NDLSYYGSSLSALTYWLYKITPFSFESIILYMSTFLSSLVVIPTILLANEYKRPLMGFVAALLASIANSYYNRTMSGY

[0793] YDTDMLVIVLPMFILFFMVRMILKKDFFSLIALPLFIGIYLWWYPSSYTLNVALIGLFLIYTLIFHRKEKIFYIAVILSS

[0794] LTLSNIAWFYQSAIIVILFALFALEQKRLNFMIIGILGSATLIFLILSGGVDPILYQLKFYIFRSDESANLTQGFMYFN

[0795] VNQTIQEVENVDLSEFMRRISGSEIVFLFSLFGFVWLLRKHKSMIMALPILVLGFLALKGGLRFTIYSVPVMALG

[0796] FGFLLSEFKAIMVKKYSQLTSNVCIVFATILTLAPVFIHIYNYKAPTVFSQNEASLLNQLKNIANREDYVVTWWD

[0797] YGYPVRYYSDVKTLVDGGKHLGKDNFFPSFALSKDEQAAANMARLSVEYTEKSFYAPQNDILKTDILQAMMK

[0798] DYNQSNVDLFLASLSKPDFKIDTPKTRDIYLYMPARMSLIFSTVASFSFINLDTGVLDKPFTFSTAYPLDVKNGEI YLSNGVVLSDDFRSFKIGDNVVSVNSIVEINSIKQGEYKITPIDDKAQFYIFYLKDSAIPYAQFILMDKTMFNSAY

[0799] VQMFFLGNYDKNLFDLVINSRDAKVFKLKI

[0800] SEQID NO: 55

[0801] His-L3-GS3

[0802] MKKIWLALAGLVLAFSASAHHHHHHSQDPMLSKDIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHA

[0803] AEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDQNATHATFNFLQWYV

[0804] AEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS

[0805] SEQID NO: 56

[0806] DsbAss-modified dodecin-Mut3

[0807] MKKIWLALAGLVLAFSASASSNHTYRVIEIVGTSPDGVDAAIQNGLARAAQTMRALDWFEVQSIRGHLTDQNATGTV

[0808] AHFQVTMKVGFRLEDSGSGHHHHHH

[0809] SEQID NO: 57

[0810] Ferritin P. aeruginosa modified amino acid sequence

[0811] MGSSHHHHHHSMQGHPEVIDYLNTLLTGELAARDQYFIHSRIYEDWGFSKLYERLNHEMEEETQHADALLRRILLLEGTPRM

[0812] RPDDIHPGTTVPEMLEADLKLERHVRAALAKGIALCEQHKDFVSRDILLAQLRDTEEDHAYWLEQQLGLIARIGLENYLQSQM

[0813] SEQID NO: 58

[0814] Glycotag sequence

[0815] GGSKDQNRTKDGSGHHHHHH

Claims

CLAIMS1. A method for preparing a glycoprotein nanoparticle (NP) comprising the steps of: providing a host cell; expressing a modified nanoparticle subunit, optionally in the periplasm of the host cell; expressing a glycosyltransferase, optionally in the periplasm of the host cell; glycosylating, via the glycosyltransferase, the modified nanoparticle subunit, optionally in the periplasm of the host cell; and producing an assembled glycoprotein nanoparticle from the 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 / E-X-N-Z-S / T).

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

4. The method of any one of claims 1 to 3, further comprising glycosylating, via glycosyltransferase pglB, the modified nanoparticle subunit in the periplasm.

5. The method of any preceding claim, wherein the host cell is a gram-negative bacterial host cell.

6. The method of any preceding claim, wherein the host cell is E. coli.

7. The method of any preceding claim, wherein the modified nanoparticle subunit is selected from the group consisting of an E2p subunit, a ferritin subunit, and a dodecin subunit.

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

9. The method of any preceding claim wherein the capsular polysaccharide or oligosaccharide is selected from the group consisting of Escherichia species, Shigella species, Klebsiella species, Salmonella species, Yersinia species, Helicobacter species, Proteus species,Pseudomonas species, Corynebacterium species, Streptomyces species, Streptococcus species, Enterococcus species, Staphylococcus species, Bacillus species, Clostridium species, Listeria species, Campylobacter species, Meningococcal capsular saccharides, and Candida saccharides.

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

11. A method for preparing a glycoprotein nanoparticle (NP) comprising the steps of: providing a host cell; expressing a nanoparticle subunit fused to a modified carrier protein in the periplasm of the host cell; expressing a glycosyltransferase in the periplasm of the host cell; glycosylating, via the glycosyltransferase, one or more glycosylation sites of the modified carrier protein in the periplasm; and producing an assembled glycoprotein nanoparticle from the plurality of 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 of claim 11 or claim 12, further comprising glycosylating, via glycosyltransferase pglB, the modified nanoparticle subunit in the periplasm.

14. The method of any one of claims 11 to 13, wherein the modified carrier protein is selected from the group consisting of: a modified CRM 197, a modified Diphtheria Toxoid (DT), a modified Tetanus Toxoid (TT), or a modified EPA.

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

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

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

18. A composition comprising a modified dodecin nanoparticle (NP), the dodecin NP comprising one or more dodecin subunits, each dodecin subunit comprising amino acids 2-70 of SEQ ID NO: 1 or a self-assembling fragment thereof, or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 2-70 of SEQ ID NO: 1 or the self-assembling fragment thereof; and one or more modifications selected from the group consisting of:(i) one or more substitution mutation(s) selected from positions G25, V50 and A53 wherein the numbering of positions is relative to SEQ ID NO: 1, and(ii) at least one glycosylation consensus sequence.

19. A composition comprising a modified amino acid sequence of a ferritin nanoparticle subunit, the modified amino acid sequence comprising:(i) amino acids 1-167 of SEQ ID NO: 11 or a self-assembling fragment thereof, or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 1-167 of SEQ ID NO: 11 or the self-assembling fragment thereof; and(ii) one or more glycosylation sequence(s) (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 glycosylation sequence(s) are added next to or substituted for one or more amino acids of the modified amino acid sequence.

20. A composition comprising a modified amino acid sequence of a ferritin nanoparticlesubunit, the modified amino acid sequence comprising:(a) amino acids 1-154 of SEQ ID NO: 20 or a self-assembling fragment thereof, or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 1-154 of SEQ ID NO: 20 or the self-assembling fragment thereof; and(b) one or more glycosylation sequence(s) (e.g., GSGDQNATGSG (SEQ ID NO: 33), GGSKDQNATKDGSG (SEQ ID NO: 34)), wherein the one or more glycosylation sequence(s) are added next to or substituted for one or more amino acids of the modified amino acid sequence.

21. A composition comprising a modified amino acid sequence of a E2p nanoparticle, wherein the nanoparticle comprises E2p nanoparticle subunits comprising:(a) amino acids 185-426 of SEQ ID NO: 26 or a self-assembling fragment thereof, or an amino acid sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to amino acids 185-426 of SEQ ID NO: 26 or the self-assembling fragment thereof; and(b) one or more glycosylation sequence(s) (e.g., GSGGGDQNATGSGGG (SEQ ID NO: 36)), wherein the one or more glycosylation sequence(s) are added next to or substituted for one or more amino acids of the modified amino acid sequence.

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

23. A vector, 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 an assembled glycoprotein nanoparticle according to any preceding claim.

26. The immunogenic composition of claim 25, further comprising an adjuvant,wherein said adjuvant is selected from the group consisting of alum, aluminium hydroxide, aluminium phosphate, a saponin, a water-in-oil emulsion, an oil-in-water emulsion, a liposaccharide, a lipopolysaccharide, an immunostimulatory nucleic acid molecule, a liposome, and a Toll Receptor or Toll-Like Receptor agonist.

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

28. A process for producing an assembled glycoprotein nanoparticle that comprises a modified nanoparticle subunit conjugated to a capsular polysaccharide or oligosaccharide, or a nanoparticle subunit fused to a modified carrier protein, said process comprising:(i) culturing the host cell of claim 24 under conditions suitable for the production of glycoproteins; and(ii) isolating the assembled glycoprotein nanoparticle, optionally isolating the assembled glycoprotein nanoparticle from a periplasmic extract from the host cell.

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

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