A minimal sequon sufficient for O-linked glycosylation

Abstract

Provided herein are short ComP glycosylation fragments (sequons) and glycoconjugates comprising ComP glycosylation fragments, as well as methods of making and using them, for example, for use in producing glycoconjugate vaccines.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of U.S. Provisional Application No. 63 / 181,014, filed April 28, 2021.

[0002] This application is related to U.S. Application No. 15 / 553,733, filed August 25, 2017, which is a U.S. national stage application of PCT / CA2016 / 050208, filed February 16, 2016, which claims the benefit of U.S. Provisional Application No. 62 / 121,439, filed February 26, 2015.

[0003] This application is also related to PCT / US2019 / 037251, filed June 14, 2019, which claims the benefit of U.S. Provisional Application No. 62 / 685,970, filed June 16, 2018, and U.S. Provisional Application No. 62 / 783,971, filed December 21, 2018.

[0004] This application is also related to PCT / US2019 / 059893, filed November 5, 2019, which claims the benefit of U.S. Provisional Application No. 62 / 783,971, filed December 21, 2018.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Description based on MPEP 310. This invention was made with Government support under Grant R44AI131742 awarded by the National Institute of Allergy and Infectious Diseases (NIAID). The Government has certain rights in this invention. [Background technology]

[0006] The present disclosure is directed to the field of protein glycosylation, in particular glycosylation of very short glycosylated fragments of ComP, and glycoconjugates comprising very short glycosylated fragments of ComP. Also provided are methods of manufacture for use, for example, in producing glycoconjugate vaccines.

[0007] Protein glycosylation is the most common post-translational modification found in nature. Evidence for prokaryotic glycosylation was first reported in Campylobacter jejuni just over 20 years ago (Szymanski, C. M. et al., 1999), and was functionally introduced into E. coli shortly thereafter (Wacker, M. et al., 2002). Prokaryotic protein glycosylation is largely either O-linked or N-linked, with the O-linked system attaching glycans to the side chains of serine or threonine residues and the N-linked system attaching glycans to asparagine side chains (Nothaft, H. & Szymanski, C. M., 2010; Schaffer, C. & Messner, 2017). Both O-linked and N-linked systems can be further classified as oligosaccharyltransferase (OTase)-independent or OTase-dependent (Harding, CM & Feldman, 2019). OTase-independent glycosylation occurs in the cytoplasm and relies on dedicated glycosyltransferases to glycosylate the cognate receptor protein. OTase-dependent glycosylation relies on oligosaccharyltransferases to transfer preassembled oligosaccharides en bloc to the periplasmic receptor protein. The OTase-dependent protein glycosylation pathway shares many similarities with O-antigen polysaccharide biosynthesis, beginning with the transfer of phosphorylated monosaccharides from nucleotide-activated precursors to the lipid carrier undecaprenyl phosphate in the inner leaflet of the plasma membrane (Valvano, MA, 2003; Hug, I. & Feldman, 2011). The lipid-linked monosaccharides are sequentially elongated by the action of specific glycosyltransferases to give lipid-linked oligosaccharides, which are flipped into the periplasmic leaflet by flippases (Raetz, CR & Whitfield, 2002) and then transferred to acceptor proteins by OTases, which are promiscuous and transfer a variety of different glycans, including long-chain polysaccharides, from various bacterial species to acceptor proteins (Wacker, M. et al. 2006; Faridmoayer, A. et al. 2008).This attractive property led to the development of OTases that transfer bacterial surface polysaccharides, such as O antigens and capsular polysaccharides (CPS), to specific periplasmic carrier proteins, thereby generating polysaccharide-protein conjugates used as conjugate vaccines (Feldman, MF et al. 2005). This glycoengineering process is called bioconjugation, and to date, three different OTases, named PglB, PglL and PglS, have been characterized and used to develop bioconjugate vaccines.

[0008] PglB is a general N-linked OTase from C. jejuni and is the first bacterial OTase to be characterized and used for the production of glycoengineered bioconjugates in E. coli (Szymanski, CM, et al. 1999; Feldman, MF et al. 2005). PglB naturally transfers polysaccharides with C2-acetamido sugars at the reducing end to acceptor proteins (Wacker, M. et al. 2006). The natural glycan substrate versatility of PglB is the most limited of all OTases, with only N-linked sequons, [ka] ( NThe bacterial N-linked sequon is the shortest (X1 and X2 are glycosylated and neither X1 nor X2 are proline) (Kowarik, M. et al. 2006). The bacterial N-linked sequon is similar to that recognized by Stt3, the catalytic subunit of the eukaryotic N-linked OTase complex (Kowarik, M. et al. 2006). PglL (also known as PglO) is the first characterized general O-linked OTase from Neisseria species, which transfers glycans bearing either C2-acetamido sugars or galactose at the reducing end to acceptor proteins (Faridmoayer, A., Fentabil, et al., 2007). In contrast to PglB, there is no obvious conserved sequon for PglL, but glycosylation occurs mainly in regions of low amino acid complexity, rich in alanine, proline, and glycine residues (Vik, A. et al. 2009). Recently, an optimized PglL sequon [ka] was derived from PilE, one of the natural pilin substrates for PglL (Pan, C. et al. 2016), but the hydrophilic amino acid sequences DPRNVGGDLD (SEQ ID NO: 180) and QPGKPPR (SEQ ID NO: 181) were required on either side of the optimized sequon for PglL to efficiently glycosylate proteins containing this tag. ComP PglS, previously called PglS, is an O-linked OTase that specifically glycosylates the bacterial pilin protein ComP of Acinetobacter species (Harding, CM et al., 2015). Importantly, PglS is the only OTase known that is capable of naturally transferring glycans with glucose at the reducing end in addition to glycans containing either galactose or C2-acetamido sugars at the reducing end (Harding, CM et al., 2019). Thus, PglS has the broadest polysaccharide substrate versatility of the three OTases used in the development of bioconjugate vaccines.

[0009] Over the past decade, PglB and, to a lesser extent, PglL have been used to develop bioconjugate vaccines against Staphylococcus aureus, Shigella dysenteria and flexneri, extraintestinal pathogenic E. coli, Salmonella species, etc. (Wacker, M. et al., 2014; Hatz, CF et al., 2015; Huttner, A. et al., 2017; Sun, P. et al., 2018; van den Dobbelsteen, G. et al., 2016). However, due to the inability of PglB and PglL to naturally transfer polysaccharides with glucose at the reducing end, PglB and PglL cannot be used to produce bioconjugate vaccines against several prominent bacterial threats (Harding, CM et al., 2019). For example, approximately 75% of the capsules of Streptococcus pneumoniae, >50% of the capsules of Klebsiella pneumoniae, and all ten species of group B Streptococcus agalactiae (GBS) contain glucose as the reducing end sugar (Geno, KA et al., 2015; Pan, YJ et al., 2015; Berti, F. et al., 2014). The natural ability of PglS to transfer polysaccharides with glucose at the reducing end is therefore particularly suitable for developing vaccines against the common pneumococci K. pneumoniae and GBS, targeting their capsular polysaccharides. Indeed, the production of bioconjugate vaccines against current non-vaccine serotypes of S. pneumoniae and the highly virulent K. pneumoniae has been reported using PglS and ComP as carrier proteins or engineered ComP fusion proteins (Harding, CM et al., 2019; Feldman, MF et al., 2019).

[0010] The versatility of PglS's polysaccharide substrates makes it an attractive OTase for the production of next-generation bioconjugate vaccines, but the minimal ComP sequon sufficient for PglS-dependent glycosylation has yet to be identified. Previous bioconjugate vaccines developed using PglS have relied on the use of either the full-length native ComP protein, a naturally membrane-bound protein, as a carrier protein, or an N-terminally truncated 117 amino acid ComP variant translationally fused to the C-terminus of exotoxin A (EPA) from Pseudomonas aeruginosa. It would be preferable to identify a shorter, more modular ComP sequon that can be efficiently glycosylated by PglS. This is because previous repeats containing the 117 amino acid ComP fragment are only suitable for glycosylation when translationally fused to the C-terminus of the carrier protein, limiting their utility. For example, in PglB, knowledge of the short N-linked sequon allowed multiple glycosylation sites to be engineered onto the surface of the carrier protein, resulting in mono- and polyglycosylated bioconjugates (Ihssen, J. et al., 2010), which also allowed for more sophisticated in vitro studies involving different PglB peptide substrate variants and their effect on peptide binding and catalysis (Gerber, S. et al., 2013).

[0011] Thus, there remains a need to provide insight into the structural determinants of acceptor protein specificity in the PglS OTase family, to facilitate comparison with sequons recognized by other O-linked OTases such as PglL, and to identify short or minimal ComP sequons that could help guide improved glycoengineering design.

[0012] Summary of the Invention Provided herein is a sugar conjugate comprising an oligosaccharide or polysaccharide covalently linked to a fusion protein, the fusion protein comprising a ComP protein (ComP) glycosylation fragment, the fusion protein being glycosylated with the oligosaccharide or polysaccharide at a serine residue corresponding to the conserved serine residue at position 82 of ComP110264 (SEQ ID NO: 1) in the ComP glycosylation fragment. In certain embodiments, the ComP glycosylation fragment does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 71 of ComP110264 (SEQ ID NO: 1) and / or does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 93 of ComP110264 (SEQ ID NO: 1). In certain embodiments, the ComP glycosylation fragment is located internally in the fusion protein. In certain embodiments, the ComP glycosylation fragment is solvent (or surface) exposed. In certain embodiments, the ComP glycosylation fragment is incorporated into a C10 β-turn, β-turn, β-twist, β-loop, U-turn, reverse turn, strand reversal, or hairpin loop of the fusion protein.

[0013] Provided herein is a ComP glycosylation fragment that comprises or consists of a fragment of an isolated ComP protein, the ComP glycosylation fragment being a ComP glycosylation fragment. 110264 (SEQ ID NO: 1), and / or 110264 (SEQ ID NO: 1), and the ComP glycosylated fragment does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 93 of ComP. 110264 (SEQ ID NO:1) contains a serine residue corresponding to the conserved serine residue at position 82 of (SEQ ID NO:1).

[0014] Provided herein is a fusion protein comprising a ComP glycosylation fragment of the present disclosure, wherein the ComP glycosylation fragment is located within the fusion protein. In certain embodiments, the fusion protein comprises SEQ ID NO: 1 (ComP 110264) is glycosylated with an oligosaccharide or polysaccharide at a serine residue in the glycosylated fragment corresponding to the serine residue in the ComP glycosylated fragment at position 82 of the ComP glycosylated fragment.

[0015] Provided herein are methods for in vivo conjugation of an oligo- or polysaccharide to a receptor polypeptide, the method comprising covalently attaching the oligo- or polysaccharide to the receptor polypeptide by PglS oligosaccharyltransferase (OTase), wherein the receptor polypeptide comprises a ComP glycosylated fragment of the disclosure.

[0016] Provided herein is a method of inducing a host immune response against a bacterial pathogen, the method comprising administering to a subject in need thereof an effective amount of a conjugate vaccine, fusion protein, or composition of the present disclosure. Also provided herein is a method of preventing or treating bacterial disease and / or infection in a subject, the method comprising administering to a subject in need thereof a conjugate vaccine, fusion protein, or composition of the present disclosure.

[0017] Further provided herein is a method of producing a pneumococcal conjugate vaccine against pneumococcal infection, the method comprising: (a) isolating a glycoconjugate or a glycosylated fusion protein of the present disclosure, and (b) combining the isolated glycoconjugate or the isolated glycosylated fusion protein with an adjuvant.

[0018] Further provided is a glycoconjugate, a glycosylated fusion protein, or a conjugate vaccine for use in inducing a host immune response against a bacterial pathogen and / or in preventing or treating bacterial disease and / or infection in a subject. [Brief description of the drawings]

[0019] [Figure 1]A shows a schematic diagram of the EPA-ComP110264 fusion protein, with the ComP glycosylation fragment fused at the C-terminus of the fusion protein. "ssDsbA" corresponds to the DsbA Sec secretion signal. GGGS (SEQ ID NO: 182) is the flexible linker between the EPA and ComP110264 fragments. B shows the different amino acid sequences for the ComP glycosylation fragment fused to the C-terminus of the EPA fusion protein. The bold underlined serine residue in each sequence corresponds to the conserved serine 82 of ComP110264, which is the site of glycosylation. The bold underlined cysteine ​​residues corresponding to Cys71 and Cys93 are also highlighted. (C2, SEQ ID NO:183; D2, SEQ ID NO:184; E2, SEQ ID NO:185; F2, SEQ ID NO:186; G2, SEQ ID NO:187; H2, SEQ ID NO:188; A3, SEQ ID NO:189; B3, SEQ ID NO:190; C3, SEQ ID NO:191; D3, SEQ ID NO:192; E3, SEQ ID NO:193; F3, SEQ ID NO:194; and C1, SEQ ID NO:195). C, D, and E show Western blot analysis of periplasmic extracts from E. coli SDB1 expressing PglS, CPS8 glycans, and the EPA-ComP110264 variant. Each lane of the Western blot panel corresponds to an SDB1 strain expressing a different EPA-ComP variant with a ComP glycosylation fragment corresponding to the sequence shown in B. C shows proteins reacting with anti-EPA antiserum. D shows proteins reacting with anti-His antiserum. E shows a merged Western blot image of C and D. Equal amounts of periplasmic extracts were loaded per lane based on OD600. g0 on the right of panels C-E indicates unglycosylated EPA-ComP110264, and gn indicates EPA-ComP110264 glycosylated with different numbers of CPS8 repeat units. Protein mass markers (in kDa) are shown on the left of panels C-E. [Diagram 2]A shows a schematic diagram of the CRM197-ComPC1 fusion protein. "ssFlgI" corresponds to the FlgI SRP secretion signal. GGGS (SEQ ID NO: 182) is the flexible linker between CRM197 and ComPC1. B, C, and D show Western blot analysis of purified CRM197-ComPC1-CPS8 glycoconjugate. B shows proteins reacting with anti-CPS8 antiserum. C shows proteins reacting with anti-CRM197 antiserum. D shows merged Western blot images of B and C. Loss of CRM197 and CPS8 signals in proteinase K (PK) treated samples demonstrates that this pneumococcal serotype 8 signal is CRM197-bound and not the result of contamination with free polysaccharide or lipid-linked polysaccharide precursors. Protein mass markers (in kDa) are shown on the left of panels B-D. [Diagram 3] A shows a schematic diagram of C-terminal and N-terminal CRM197 variants, including C1ComP glycosylation fragments. B shows Western blot analysis of periplasmic extracts of E. coli SDB1 expressing CRM197-ComPC1 or ComPC1-CRM197 and CPS8 glycans in the presence (+) or absence (-) of PglS. Equal amounts of periplasmic extracts were loaded per lane based on OD600. Protein mass markers (in kDa) are shown on the left. GGGS (SEQ ID NO: 182). [Figure 4]A shows a schematic of an EPA fusion protein containing a ComP glycosylation fragment integrated within the EPA amino acid sequence. B shows the amino acid sequences of two iGT ComP glycosylation fragments inserted between EPA residues Ala489 and Arg489. These have either two terminal cysteines ("iGCC", SEQ ID NO:30) or serines ("iGSS", SEQ ID NO:31). C and D show Western blots of periplasmic extracts of E. coli SDB1 expressing the CPS8 glycans, EPAiGTcc or EPAiGTss, in the presence (+) or absence (-) of PglS. C shows proteins reacting with anti-EPA antiserum. D shows proteins reacting with anti-His antiserum. E shows a merged Western blot image of C and D. Equal amounts of periplasmic extracts were loaded into the lanes based on OD600. Protein mass markers (in kDa) are shown on the left side of the panels. [Diagram 5]A shows a schematic of the EPA constructs containing ComP glycosylation fragments (from top to bottom, SEQ ID NOs: 6-28) used in these experiments. Twenty-two 5 amino acid truncated variants of the iGTCC ComP glycosylation fragment were inserted between Ala489 and Arg489 in the EPA coding sequence. B shows the amino acid sequences of the 22 truncated iGT ComP glycosylation fragments, with name designations given on the left. The underlined, bold serine is the glycosylation site. (iGTcc SEQ ID NO:30, Δ0-1 SEQ ID NO:32, Δ1-0 SEQ ID NO:43, Δ1-2 SEQ ID NO:45, Δ2-1 SEQ ID NO:56, Δ2-3 SEQ ID NO:58, Δ3-2 SEQ ID NO:69, Δ3-4 SEQ ID NO:71, Δ4-3 SEQ ID NO:82, Δ4-5 SEQ ID NO:84, Δ5-4 SEQ ID NO:95, Δ5-6 SEQ ID NO:97, Δ6-5 SEQ ID NO:108, Δ6-6 SEQ ID NO:109, Δ6-7 SEQ ID NO:110, Δ7-6 SEQ ID NO:121, Δ7-7 SEQ ID NO:122, Δ7-8 SEQ ID NO:123, Δ8-7 SEQ ID NO:134, Δ8-8 SEQ ID NO:135, Δ8-9 SEQ ID NO:136, Δ9-8 SEQ ID NO:146, Δ9-9 SEQ ID NO:147). C shows Western blot analysis of periplasmic extracts of E. coli SDB1 expressing EPAiGT fusion proteins containing PglS, CPS8 and truncated glycosylated fragments. Each lane of the Western blot panel corresponds to an SDB1 strain expressing a different EPAiGT fusion protein containing a truncated ComP glycosylation fragment with the ComP glycosylation fragment corresponding to the sequence shown in B. C shows proteins reacting with anti-EPA antisera probed with an anti-EPA antibody. EPAiGTcc is shown for comparison. The "EPA" lane corresponds to EPA lacking ComP-derived sequences and serves as a negative control. Equal amounts of periplasmic extracts were loaded into the lanes based on OD600. D shows the same Western blot analysis as above with the intensity of the anti-EPA signal increased to indicate low-level glycosylation for the smallest ComP glycosylation fragment. [Figure 6]Western blot analysis of Ni-affinity chromatography purified EPA fusion protein containing the iGTΔ6-6 ComP glycosylation fragment incorporated between residues Ala489 and Arg490 of EPA. The fusion protein was purified in the presence (+) or absence (-) of PglS from SDB1 cells expressing CPS8 glycans. A shows proteins reacting with anti-His antiserum. B shows proteins reacting with anti-CPS8 antiserum. C shows a merge of A and B. Protein mass markers (in kDa) are indicated on the left of panels A-C. [Figure 7] A shows a schematic diagram of an EPA fusion protein containing an iGTΔ3-4 ComP glycosylated fragment incorporated between residues Glu548 and Gly549 of EPA. The iGTΔ3-4 amino acid sequence is shown below the figure (SEQ ID NO:71). B shows a Western blot analysis of periplasmic extracts of E. coli SDB1 expressing PglS, CPS8 and an EPA fusion protein containing an iGTΔ3-4 ComP glycosylated fragment incorporated between residues Glu548 and Gly549. Proteins reacting with anti-EPA antisera probed with anti-EPA antibody are shown. [Figure 8] Western blot analysis of Ni-affinity chromatography purified EPA fusion protein containing the iGTΔ3-4 ComP glycosylation fragment incorporated between residues Glu548 and Gly549 of EPA. The fusion protein was purified in the presence (+) or absence (-) of PglS from SDB cells expressing CPS8 glycans. A shows proteins reacting with anti-His antiserum. B shows proteins reacting with anti-CPS8 antiserum. C shows a merge of A and B. Protein mass markers (in kDa) are indicated on the left of panels A-C. [Figure 9] The amino acid sequences of ComP orthologues are shown, with predicted glycosylation sites in bold. [Figure 10]The amino acid sequence of the ComPΔ28 orthologue is shown, in which amino acids corresponding to the 28 N-terminal amino acids of ComPADP1:AAC45886.1 have been removed. Predicted glycosylation sites are shown in bold. [Figure 11] An alignment of the ComP sequence region containing a serine (S) residue (boxed) that corresponds to the serine residue at position 82 of ComP110264 (SEQ ID NO: 1) and also corresponds to the serine residue at position 84 of ComPADP1 (SEQ ID NO: 2) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] To the extent necessary to provide explanatory support, the subject matter and / or documents of the appended claims are incorporated herein by reference in their entirety.

[0021] All readers of this written description will understand that the exemplary aspects and embodiments described and claimed herein may suitably be practiced in the absence of the recitation of any feature, element or step that is or is not specifically disclosed herein.

[0022] definition It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a polysaccharide" is understood to refer to one or more polysaccharides. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0023] Furthermore, as used herein, "and / or" should be considered as a specific disclosure of each of the specified features or components with or without the other. Thus, as used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases 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 (single), B (single), and "C" (single).

[0024] Whenever an embodiment is described herein in conjunction with the phrase "comprising" or "comprises," it is understood that other similar embodiments described in terms such as "consisting of," "consists of," "consisting essentially of," and / or "consists essentially of" are also provided.

[0025] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0026] Numeric ranges are inclusive of the numbers defining the range. Even if a list of values, e.g., 1, 2, 3, or 4, is recited and not expressly specified by "and any range therebetween," etc., the disclosure specifically includes any range between those values, e.g., 1-3, 1-4, 2-4, etc., unless otherwise stated.

[0027] The headings provided herein are intended merely for ease of reference and do not limit the various aspects or aspects of the disclosure, which can be made by reference to the specification as a whole.

[0028] As used herein, the term "non-naturally occurring" material, composition, entity, and / or any combination of materials, compositions, or entities, or any grammatical variations thereof, is a qualified term that expressly excludes, but only excludes, forms of material, composition, entity, and / or any combination of materials, compositions, or entities that are well understood by those of skill in the art as being "naturally occurring" or that are or may be determined or construed at any time by a determiner or administrative or judicial body as being "naturally occurring."

[0029] As used herein, the term "polypeptide" encompasses the singular "polypeptide" and the plural "polypeptides" and is intended to refer to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain(s) of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other term used to refer to a chain(s) of two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" may be used in place of or synonymously with any of these terms. The term "polypeptide" is also intended to refer to the product of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-standard amino acids. A polypeptide may be derived from a natural biological source or may be produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

[0030] As used herein, "protein" can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides linked together, for example, by disulfide bonds, hydrogen bonds, or hydrophobic interactions to produce a multimeric protein.

[0031] By "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0032] As used herein, the term "non-naturally occurring" polypeptide, or any grammatical variant thereof, is a qualified term that expressly excludes, but only excludes, forms of the polypeptide that are well understood by those of skill in the art as being "naturally occurring" or that have been or may be determined or construed at any time by an adjudicator or administrative or judicial body as being "naturally occurring."

[0033] Disclosed herein are certain binding molecules, or antigen-binding fragments, variants, or derivatives thereof. Unless a full-sized antibody, e.g., a naturally occurring antibody, is specifically referenced, the term "binding molecule" encompasses full-sized antibodies and antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g., engineered antibody molecules or fragments that bind to antigen in a manner similar to a naturally occurring antibody or immunoglobulin or antibody molecule.

[0034] As used herein, the term "binding molecule" in its broadest sense refers to a molecule that specifically binds to an antigenic determinant. As further described herein, a binding molecule may contain one or more "binding domains". As used herein, a "binding domain" is a two-dimensional or three-dimensional polypeptide structure that can specifically bind to a given antigenic determinant or epitope. A non-limiting example of a binding molecule is an antibody or a fragment thereof that contains a binding domain that specifically binds to an antigenic determinant or epitope. Another example of a binding molecule is a bispecific antibody that contains a first binding domain that binds to a first epitope and a second binding domain that binds to a second epitope.

[0035] The terms "antibody" and "immunoglobulin" may be used interchangeably herein. As disclosed herein, an antibody (or a fragment, variant, or derivative thereof) comprises at least a variable domain of a heavy chain and at least variable domains of a heavy chain and a light chain. Basic immunoglobulin structure in vertebrate systems is relatively well understood. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

[0036] Binding molecules, e.g., antibodies or antigen-binding fragments, variants, or derivatives thereof, include, but are not limited to, polyclonal, monoclonal, human, humanized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fv, single chain Fv (scFv), single chain antibodies, disulfide-linked Fv (sdFv), fragments containing either the VL or VH domains, fragments produced by a Fab expression library. ScFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules encompassed by the present disclosure can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.

[0037] "Specifically binds" means that a binding molecule, such as an antibody or a fragment, variant, or derivative thereof, binds to an epitope via its antigen-binding domain, and that the binding requires a degree of complementarity between the antigen-binding domain and the epitope. According to this definition, a binding molecule is said to "specifically bind" to an epitope when it binds to that epitope via its antigen-binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular binding molecule binds to a particular epitope. For example, binding molecule "A" may be considered to have a higher specificity for a given epitope than binding molecule "B," or binding molecule "A" may be said to bind epitope "C" with a higher specificity than it has for related epitope "D."

[0038] The term "polynucleotide" is intended to encompass single and multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds as found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide. An "isolated" nucleic acid or polynucleotide contemplates a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide subunit contained in a vector is considered isolated as disclosed herein. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides contained in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of a polynucleotide. Isolated polynucleotides or nucleic acids further include synthetically produced molecules. In addition, a polynucleotide or nucleic acid may be, or may include, a regulatory element, such as a promoter, a ribosome binding site, or a transcription terminator.

[0039] As used herein, a "non-naturally occurring" polynucleotide, or any grammatical variant thereof, is a qualified definition that expressly excludes, but only excludes, forms of polynucleotides that are well understood by those of skill in the art as being "naturally occurring" or that are or may be determined or interpreted at any time by a determiner or administrative or judicial body as being "naturally occurring."

[0040] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide may be RNA.

[0041] A "vector" is a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector may contain nucleic acid sequences that allow it to replicate in the host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements known in the art.

[0042] A "transformed" cell, or a "host" cell, is a cell into which a nucleic acid molecule has been introduced by molecular biological techniques. As used herein, the term transformation encompasses techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with a viral vector, transformation with a plasmid vector, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. A transformed or host cell can be a bacterial cell or a eukaryotic cell.

[0043] As used herein, the term "expression" refers to the process by which a gene produces a biochemical, e.g., a polypeptide. This process includes any expression of the functional presence of a gene in a cell, including, but not limited to, gene knockdown, and both transient and stable expression. This includes, but is not limited to, transcription of a gene into messenger RNA (mRNA) and translation of such mRNA into a polypeptide(s). When the final desired product is a biochemical, expression includes the creation of that biochemical and any precursors. Expression of a gene produces a "gene product." As used herein, a gene product can be a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide translated from a transcript. Gene products as described herein further include nucleic acids with post-transcriptional modifications, e.g., polyadenylation, or polypeptides with post-translational modifications, e.g., methylation, glycosylation, lipidation, association with other protein subunits, proteolytic cleavage, and the like.

[0044] As used herein, the terms "treat", "treatment" or "treatment of" (e.g., in the phrase "treating a subject") refer to reducing the likelihood of a disease condition, reducing the occurrence of a disease symptom, e.g., to the extent that the subject has a longer survival rate or reduced discomfort. For example, treatment can refer to the ability of a treatment to reduce a disease symptom, sign, or cause when administered to a subject. Treatment also refers to alleviating or reducing at least one clinical symptom, and / or inhibiting or delaying the progression of the symptom, and / or preventing or delaying the onset of a disease or condition.

[0045] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, farm animals, sport animals, and zoo animals, including, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, etc.

[0046] The term "pharmaceutical composition" refers to a preparation in a form that effectively utilizes the biological activity of an active ingredient and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered. Such compositions may be sterile.

[0047] An "effective amount" of an antibody disclosed herein is an amount sufficient to achieve a specifically stated purpose. An "effective amount" can be determined empirically and routinely in relation to the stated purpose.

[0048] As used herein, "sequon" refers to a specific sequence of amino acids that consists of amino acid residues for recognition and subsequent glycosylation by a specific oligosaccharyltransferase.

[0049] As used herein, a "glycoconjugate" refers to a polypeptide covalently attached to a carbohydrate moiety. It is understood that the carbohydrate moiety can be a monosaccharide, oligosaccharide, or polysaccharide. For purposes of this disclosure, a "glycoconjugate" is a specific type of "bioconjugate" as referred to herein.

[0050] overview Conjugate vaccines consisting of polysaccharides linked to proteins are life-saving prophylactic drugs. Traditionally, conjugate vaccines are produced using chemical methods. However, in vivo bacterial conjugation has emerged as an alternative production method. In vivo conjugation (bioconjugation) relies on oligosaccharyltransferases to attach polysaccharides to proteins. Currently, oligosaccharyltransferases used for bioconjugation are not suitable for the production of conjugate vaccines when the polysaccharide contains glucose at the reducing end. This limitation is significant since approximately 75% of the capsule of Streptococcus pneumoniae contains glucose as the reducing end sugar. Disclosed herein is the use of O-linked oligosaccharyltransferases to generate the first multivalent pneumococcal bioconjugate vaccine with polysaccharides containing glucose at the reducing end. Pneumococcal bioconjugates were immunogenic, prophylactic, and rapidly produced by recombinant technology. Certain embodiments disclosed herein provide the engineering, characterization, and immunological responses to multivalent pneumococcal bioconjugate vaccines using the native receptor protein ComP as a vaccine carrier, as well as monovalent pneumococcal bioconjugate vaccines using traditional vaccine carriers, such as, in certain embodiments, those that include Pseudomonas aeruginosa exotoxin A protein, establishing a platform to overcome the limitations of other conjugating enzymes and enabling the development of bioconjugate vaccines against many important human and animal pathogens.

[0051] Over the past two decades, even with the introduction and implementation of pneumococcal conjugate vaccines, approximately 1.5 million deaths are attributable to S.pneumoniae each year. This is due in part to the more than 90 serotypes of S.pneumoniae and the complex manufacturing methods required to synthesize pneumococcal conjugate vaccines. These factors combine to hinder global distribution and development of a broader, more preventative variety of vaccines. To expedite development and lower production costs, disclosed herein is a platform for developing conjugate vaccines, e.g., pneumococcal conjugate vaccines, using in vivo conjugation. This streamlined process has the potential to complement existing manufacturing pipelines or completely avoid reliance on chemical conjugation methods, allowing for the production of more comprehensive conjugate vaccines.

[0052] Traditional chemical conjugate vaccine synthesis is considered complex, costly, and laborious (Frasch, CEVaccine 27, 6468-6470 (2009)); however, in vivo conjugation has been thoroughly advanced as a viable biosynthetic alternative (Huttner, A. et al. Lancet Infect Dis 17, 528-537 (2017)). These advances are best highlighted by the success of GlycoVaxyn (LimmaTech Biologics AG, now an independent company with direct ties to GlaxoSmithKline). This is a clinical-stage biopharmaceutical company with multiple bioconjugate vaccines in various phases of clinical trials, one of which (Flexyn2a) has just completed a phase 2b challenge trial. GlycoVaxyn is at the forefront of the in vivo conjugation revolution, but the ability to glycosylate carrier / acceptor proteins with polysaccharides containing glucose (Glc) as the reducing end sugar has been elusive and, as expected, has hindered the development of pneumococcal bioconjugate vaccines.

[0053] Previously called PglL by Schulz et al. (PMID 23658772) and previously called PglL by Harding et al. 2015 (PMID 26727908). ComP The oligosaccharyltransferase PglS, previously called PglS, was the only recently characterized functional OTase (Schulz, BL et al. PLoS One 8, e62768 (2013)). Subsequent mass spectrometry studies on total glycopeptides showed that PglS does not act as a general PglL-like OTase, but glycosylates multiple periplasmic and outer membrane proteins (Harding, CM et al. Mol Microbiol 96, 1023-1041 (2015)). Indeed, the genome of A. baylyi ADP1 encodes two OTases, a PglL-like orthologue (UniProtKB / Swiss-Prot:Q6FFS6.1) that acts as a general OTase, and a single protein, PglS (UniProtKB / Swiss-Prot:Q6F7F9.1), which glycosylates ComP (Harding, CM et al. Mol Microbiol 96, 1023-1041 (2015)).

[0054] ComP is orthologous to type IV pilin proteins such as PilA from Pseudomonas aeruginosa and PilE from Neisseria meningiditis, both of which are glycosylated by the OTases TfpO (Castric, P. Microbiology 141(Pt 5), 1247-1254 (1995)) and PglL (Power, PM et al. Mol Microbiol 49, 833-847 (2003)), respectively. TfpO and PglL also glycosylate their cognate pilins at serine residues, but the sites of glycosylation differ between each system. TfpO glycosylates the cognate pilin at a C-terminal serine residue that is not present in ComP (Comer, JE, Marshall, MA, Blanch, VJ, Deal, CD & Castric, P. Infect Immun 70, 2837-2845 (2002)). PglL glycosylates PilE at an internal serine at position 63 (Stimson, E. et al. Mol Microbiol 17, 1201-1214 (1995)). ComP also contains a serine residue near position 63, and the surrounding residues show moderate conservation with PilE from N. meningiditis. However, comprehensive glycopeptide analysis revealed that this serine and surrounding residues are not the glycosylation site of ComP. PglS glycosylates PilE at an internal serine at position 63 (Stimson, E. et al. Mol Microbiol 17, 1201-1214 (1995)). ComP also contains a serine residue near position 63, and the surrounding residues show moderate conservation with PilE from N. meningiditis. However, comprehensive glycopeptide analysis revealed that this serine and surrounding residues are not the glycosylation site of ComP. 110264 : Corresponding to the conserved serine at position 82 of ENV58402.1 (SEQ ID NO: 1) (ComP ADP1 PglS glycosylates ComP at a single serine residue at position 84 of TfpO (which also corresponds to the conserved serine residue at position 84 of AAC4588631 (SEQ ID NO:2)). This is a novel glycosylation site not previously seen within the type IV pilin superfamily. The identification of a novel glycosylation site within the pilin superfamily, as well as the ability of PglS to transfer polysaccharides containing glucose as the reducing end sugar, demonstrates that PglS is a functionally distinct OTase from PglL and TfpO.

[0055] Bioinformatic characterization of ComP pilin orthologues ComP was first described as a factor required for natural transformation of Acinetobacter baylyi ADP1 (Porstendorfer, D., Drotschmann, U. & Averhoff, B. Appl Environ Microbiol 63, 4150-4157 (1997)). Subsequent studies have demonstrated that ComP from A. baylyi ADP1 (referred to herein as ComP ADP1 It was demonstrated that the OTase PglS, which is located immediately downstream of ComP, is glycosylated by the novel OTase PglS, which is located not only in the chromosome but also in the chromosome, which is a general OTase located elsewhere (Harding, CM et al. Mol Microbiol 96, 1023-1041 (2015)). ADP1 The protein (NCBI identifier AAC45886.1) belongs to a family of proteins called type IV pilins. Specifically, ComP shares homology with the major pilins of type IVa (Giltner, CL, Nguyen, Y. & Burrows, LLMicrobiol Mol Biol Rev 76, 740-772 (2012)). Type IVa pilins share high sequence homology at the N-terminus, which encodes a highly conserved leader sequence and an N-terminal alpha helix, but their C-termini show significant divergence between genera and even within species (Giltner, CL, Nguyen, Y. & Burrows, LLMicrobiol Mol Biol Rev 76, 740-772 (2012)). To help distinguish the ComP orthologues from other type IVa pilin proteins, such as PilA from A. baumannii, P. aeruginosa, and Haemophilus influenzae and PilE from Neisseria species (Pelicic, V. Mol Microbiol 68, 827-837 (2008)), BLASTp analysis was performed to identify the ComP orthologues. ADP1 The primary amino acid sequence of ComP was compared with all proteins from bacteria of the genus Acinetobacter. ADP1Although many Acinetobacter type IVa pilin orthologues, including those from Acinetobacter truncatula , share high homology at the N-terminus, few proteins show high sequence conservation across the entire amino acid sequence of ComP. At least six ComP orthologues (Figure 9) are identified in the ComP ADP1 ComP pilin variants were identified based on the presence of a conserved serine at position 84 relative to the comP pilin variant and a conserved disulfide bond adjacent to a site of predicted glycosylation connecting the predicted alpha-beta loop to the beta strand region (Giltner, CL, Nguyen, Y. & Burrows, LL Microbiol Mol Biol Rev 76, 740-772 (2012)). In addition, all six ComP orthologs have both a pglS homolog immediately downstream of the comP gene and a pglL homolog elsewhere on the chromosome. Taken together, the presence of at least a conserved serine at position 84, a disulfide loop adjacent to a site of glycosylation, the presence of a pglS gene immediately downstream of comP, and the presence of a pglL homolog elsewhere on the chromosome distinguish ComP pilin variants from other type IVa pilin variants.

[0056] Thus, disclosed herein are common features of ComP proteins that identify ComP orthologues of different Acinetobacter species. ComP proteins can be distinguished from other pilins by the presence of a conserved glycosylated serine located at position 84 relative to the ADP1 ComP protein, and the presence of a disulfide loop adjacent to the glycosylation site. In addition, the presence of a pglS homologue immediately downstream of ComP is indicative of ComP. Furthermore, to be classified as a PglS OTase protein rather than a PglL OTase protein, the OTase downstream of ComP must show high sequence conservation with PglS (ACIAD3337) when compared to PglL (ACIAD0103) of A. baylyi ADP1. One of skill in the art will appreciate that in any embodiment disclosed herein, the ComP protein may be identified as SEQ ID NO: 1 (ComP 110264It will also be apparent that the ribozyme contains a serine residue corresponding to the conserved serine residue at position 82 of ENV58402.1) and is capable of being glycosylated thereat.

[0057] ComP protein glycosylation fragment The PglS orthologue from Acinetobacter baylyi strain ADP1 was previously demonstrated to glycosylate the ComP orthologue from A. soli strain CIP110264 at a single serine residue located at position 82 (Harding, CM et al., 2019, WO / 2019 / 241672, incorporated herein by reference in its entirety). PglS was engineered to functionally glycosylate heterologous proteins by translationally fusing a large fragment of ComP (117 amino acids) to the C-terminus of a known carrier protein. Specifically, the 117 amino acid ComP 110264 The fragment was fused at the C-terminus of genetically inactivated exotoxin A (EPA) from Pseudomonas aeruginosa (EPA) between a flexible GGGS linker (SEQ ID NO: 182). This chimeric carrier protein also had an N-terminal DsbA signal sequence (ssDsbA) for translocation to the periplasm via the Sec pathway and a C-terminal hexahistidine tag for detection.

[0058] Additionally, a shorter ComP glycosylation fragment sufficient for glycosylation by PglS has been identified (WO / 2020 / 131236, incorporated herein by reference in its entirety). 110264 The glycosylated fragment was also capable of glycosylation by PglS, but the ComP glycosylated fragment was not glycosylated by ComP. 110264These observations were confirmed in a series of experiments aimed at identifying even shorter ComP glycosylation fragments. Figures 1A and 1B show the ComP glycosylation fragments designed to shift one amino acid N-terminal to C-terminal with respect to the glycosylation site of PglS, serine 82, when fused to the extreme C-terminus of the EPA carrier protein. 110264 Fragments are shown. ComP glycosylation fragments were PCR amplified and cloned into the C-terminus of EPA to examine bioconjugation with PglS. In these and all experiments described below, we used serotype 8 pneumococcal capsular polysaccharide (CPS8) expressed from the pB-8 plasmid (Kay, EJ, et al., 2016) as the glycan source. CPS8 glycans were chosen because they contain glucose as the reducing end sugar and were previously demonstrated to be efficiently transferred to ComP by PglS (Harding, CM et al., 2019). Furthermore, in these and all experiments described below, bioconjugation was performed in E. coli strain SDB1. SDB1 has a deletion of WecA, which initiates the biosynthesis of enterobacterial common antigen and O-antigen polysaccharides, and a deletion of WaaL, which translocates undecaprenyl pyrophosphate-linked glycan precursors to the outer core of lipid A (Garcia-Quintanilla, F., et al., 2014). Collectively, these mutations promote the accumulation of heterologously expressed lipid-linked glycan precursors, such as the CPS8 polysaccharide lipid-linked precursor, for exclusive use by PglS. The expression of CPS8 glycan, PglS and the fused EPA-ComP from an IPTG-inducible vector was suppressed by the addition of the EPA-ComP to the fusion protein. 110264 The SDB1 strain expressing the construct was cultured in LB medium, induced at mid-log phase, and grown overnight. Samples were taken approximately 20 hours after induction for Western blot analysis of periplasmic extracts and EPA-ComP. 110264Expression of the fusion protein and glycosylation of the protein were evaluated. Western blots were probed with antibodies against EPA (anti-EPA) and against the hexahistidine tag (anti-His). Probing with both antibodies confirmed whether the EPA protein and / or the C-terminal ComP fragment remained intact.

[0059] 1C, 1D, and 1E show the ComP 110264 The presence of Cys71 and Cys93 residues on either side of Ser82 in EPA-ComP when this ComP glycosylated fragment was fused at the C-terminus. 110264 This reaffirms that PglS is essential for glycosylation. As seen in Figure 1C, 1D, and 1E, fusion proteins containing ComP glycosylation fragments lacking either Cys71 or Cys93 were not glycosylated. Transfer of CPS8 glycans was observed only in fusion proteins containing ComP glycosylation fragments with both cysteine ​​residues. The efficiency of glycosylation by PglS and the average number of transferred CPS8 repeat units were similar for all fusion proteins containing ComP glycosylation fragments containing both Cys71 and Cys93. Further examination of the Western blots revealed that the chimeric EPA-ComP 110264 The variants (designated as C2, D2, E3, and F3 in Fig. 1C, D, and E) were observed to react poorly with anti-His antibody when compared with the anti-EPA signal (Fig. 1D). Furthermore, the anti-EPA channel showed that these variants were non-glycosylated EPA-ComPs containing both Cys71 and Cys93. 110264 The ComP fragment lacking both cysteine ​​residues was found to migrate at a slightly lower molecular weight compared to the variant (Figure 1C). Taken together, these observations indicate that the ComP fragment lacking both cysteine ​​residues is unstable and prone to C-terminal degradation, thereby preventing glycosylation by PglS. Without being bound by theory, Cys71 and Cys93 may mediate the glycosylation of ComP by forming a covalent disulfide bridge. 110264 It is believed that this can stabilize the

[0060] A variety of proteins from different organisms, usually inactivated bacterial toxins, have been used as carriers for conjugate and bioconjugate vaccines. Cross-reactive substances 197 (CRM 197 CRM is a genetically inactivated form of diphtheria toxin that is widely used as a carrier protein in several conjugate vaccines against Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenza type b (Berti, F. & Adamo, R., 2018). 197 Considering the frequent use of PglS, we used this PglS bioconjugation system as a CRM 197 In these experiments, we expanded the previously identified 25 amino acid "C1" ComP glycosylation fragment (ComP C1 ) to which the GGGS sequence (SEQ ID NO: 182) is linked 197 The SRP-dependent FlgI secretion sequence (ssFlgI) was translationally fused to the C-terminus of CRMP for transport to the periplasm. 197 (Goffin, P., et al., 2017). Finally, a C-terminal hexahistidine tag was added to aid in purification (Figure 2A). 197 -ComP C1 E. coli SDB1 cells expressing the carrier (expected size 61.8 kDa) were cultured in shake flasks and harvested after 24 hours. 197 -ComP C1 The -CPS8 glycoconjugate was purified by three successive chromatographic steps. Nickel-affinity chromatography was used first because the glycoconjugate contains a C-terminal hexahistidine tag. Fractions containing the glycoconjugate were pooled and the glycosylated glycoconjugate was concentrated using a MonoQ column and eluted with a linear salt gradient. A final purification step to remove large aggregates was performed on a Superdex200 Increase column. As can be seen in Figures 2B, 2C, and 2D, anti-CRM 197and pneumococcal CPS8 antiserum were used to detect the CRM 197 -ComP C1 The carrier was demonstrated to be glycosylated with CPS8. The CRMP was isolated by digesting the purified glycoconjugates with proteinase K prior to separation on SDS-PAGE. 197 The complete disappearance of the polysaccharide-specific signal indicates that the CPS8 glycan is a CRM 197 -ComP C1 It was shown to be covalently bound to the protein.

[0061] Next, this ComP C1 The glycotag is a CRM 197 We investigated whether the ComP gene could be moved to another site in the fusion. C1 CRM 197 The new construct was designed by placing the FlgI secretion signal at the N-terminus of the coding region (Figure 3A). C1 Directly placed at the N-terminus of the glycosylated fragment, CRM 197 The C-terminus of was tagged with hexahistidine. C1 -CRM 197 E. coli SDB1 cells expressing the carrier were cultured in shake flasks and harvested after 24 hours. As seen in Fig. 3B, Western blot analysis of periplasmic extracts probed with anti-His antibody revealed that ComP C1 -CRM 197 ComP was also shown to be glycosylated by PglS. The average number of CPS8 repeat units and glycosylation efficiency of both fusions were similar, indicating that ComP C1 It has been shown that the glycotag can be placed either at the N-terminus or the C-terminus of the carrier protein.

[0062] The 11-amino acid ComP is sufficient for PglS glycosylation 110263 Identification of sequons. In a previous report, Cys71 and Cys93 were fused to the C-terminus of EPA during the translational stage in ComP. 110264Although shown to be required for glycosylation of fusion proteins containing glycosylated fragments (e.g., Figure 1C, Figure 1D, and Figure 1E), these data do not establish whether these two cysteine ​​residues and the putative disulfide bridge formed between them are essential for glycosylation by PglS in all circumstances. The N-linked sequon recognized by PglB has been engineered into multiple sites in the surface loops of EPA and used as an "internal" glycotag (Ihssen, J. et al., 2010). ComP 110264 To determine whether Cys71 and Cys93 of PglS are required for PglS glycosylation, we use the internal glycotag iGT, which stands for internal glycotag-cysteine-cysteine. CC A 23-amino acid sequence spanning Cys71 to Cys93, called ComP 110264 The entire glycosylation fragment was incorporated within the EPA amino acid sequence. 110264 iGT CC was inserted between residues Ala489 and Arg490 of EPA, which is in a β-turn structure on the surface of the catalytic domain (Fig. 4A). As a control, an iGT ss (called iGTss) was used, which contains serine residues instead of cysteine ​​residues at positions 71 and 93 of ComP. CC A variant of the ComP glycosylation fragment was also incorporated. This iGTSS ComP glycosylation fragment was similarly incorporated between residues Ala489 and Arg490 of EPA. The serine residue is hypothesized to contribute steric bulk similar to that of a cysteine ​​residue, but cannot be oxidized and form disulfide bonds (Figure 4B). iGTcc or EPA iGTss The ability of PglS to translocate EPA to the EPA β-lactamase was evaluated in the three-plasmid system described above. As can be seen in Figures 4C and 4D, iGT Glycosylation of both the cysteine-cysteine ​​and serine-serine variants of ComP demonstrated that Cys71 and Cys93 (and the putative disulfide bond formed between them) are not required for glycosylation by PglS when the ComP fragment is introduced into the EPA protein.

[0063] Since these cysteine ​​residues are not required for PglS-dependent glycosylation, but only when the ComP glycosylation fragment is incorporated inside the fusion protein, it was hypothesized that a shorter ComP glycosylation fragment, representing a minimal O-linked ComP sequon, might be found within the 23 amino acid ComP glycosylation fragment spanning Cys71 to Cys93. To test this, we used a shorter iGT glycosylation fragment that is incorporated between residues Ala489 and Arg490 of EPA. CC We generated variants of the ComP glycosylation fragment and identified the ComP residues required for glycosylation. Alternating single amino acids were inserted into this 23 amino acid iGT CC We generated 22 truncation variants, each of which contains the glycosylation site Ser82 of PglS (Fig. 5A and 5B). CC The names refer to the number of residues deleted from either side of the iGT CC These truncated EPA-iGT variants correspond to the deletion of three amino acids from the N-terminus and four amino acids from the C-terminus. The shortest variant generated was five amino acids long. CC The variants were tested for bioconjugation with CPS8 and PglS in shake flasks under the same conditions as in previous experiments. As negative controls, we included DsbA secretion and a construct expressing only the EPA coding sequence with a hexahistidine tag.

[0064] Figure 5C shows that strong glycosylation was observed in all EPA fusion proteins containing ComP glycosylated fragments at least 11 amino acids in length, with the glycosylation ratios ranging from the 23 amino acid iGT CCThe glycosylation efficiency of the iGT ComP glycosylated fragments was comparable to that of the ComP glycosylated fragments, suggesting that moderate truncations on either side of Ser82 do not significantly affect glycosylation efficiency by PglS. Although these fusion proteins were glycosylated, a mild decrease in glycosylation efficiency was observed as the amino acid sequence of the iGT ComP glycosylated fragments was shortened. The shortest internal ComP glycosylated fragment that was efficiently glycosylated was iGTΔ6-6, which has the sequence IASGASAATTN (SEQ ID NO:109) (Figure 5C). Removal of either the N-terminal isoleucine residue (iGTΔ7-6, SEQ ID NO:121) or the C-terminal asparagine residue (iGTΔ6-7, SEQ ID NO:110) dramatically reduced glycosylation efficiency of the carrier protein, suggesting that these residues play an important role in glycosylation by PglS. Most of the variants smaller than iGTΔ6-6 showed minimal glycosylation, the best of these being the sequence ASGASAATTN (SEQ ID NO: 121) of iGTΔ7-6. Interestingly, small amounts of higher molecular weight ladders were also observed in fusion proteins containing the smallest ComP glycosylated fragments, iGTΔ9-8 (SEQ ID NO: 146) and iGTΔ9-9 (SEQ ID NO: 147) (Figure 5D), suggesting that these variants of 6 and 5 amino acids, respectively, were glycosylated at very low levels by PglS. This suggests that the ComP fragments recognized by PglS are 110264 This means that the glycosylation sequon can be as small as five amino acids in size.

[0065] Next, the CPS8-glycosylated EPA fusion protein containing the iGTΔ6-6 ComP glycosylation fragment located between residues Ala489 and Arg490 was purified from whole cell lysates using Ni-affinity chromatography, and the eluates were subjected to Western blot analysis using antisera specific for either the EPA protein or the CPS8 glycan. The results of these experiments clearly show that the EPA fusion protein containing the iGTΔ6-6 ComP glycosylation fragment located between residues Ala489 and Arg490 is glycosylated with CPS8 by PglS (Figures 6A, 6B, and 6C). Overall, these experiments demonstrate that the ComP 110264 The glycosylated fragment is the 117 amino acid ComP 110264 These results unexpectedly demonstrate that the ComP sequon previously shown to be required when fused at the C-terminus can be shortened to as little as 11 amino acids or even shorter while still maintaining glycosylation. 110264 This indicates that the cysteine ​​residues corresponding to Cys71 and Cys93 in are not required for PglS-dependent glycosylation when the ComP glycosylation fragment is incorporated within a fusion protein.

[0066] The iGT truncation series described above was examined at one internal site of EPA between residues Ala489 and Arg490. Next, a second site between residues Glu548 and Gly549 of EPA and the introduced iGTΔ3-4 ComP glycosylation fragment (SEQ ID NO: 71) was tested. Similar to the first site, the second site is found on a surface-exposed loop of the catalytic domain of EPA. This alternatively tagged variant for bioconjugation with CPS8 and PglS was tested under the same conditions as the other truncations. This construct was observed to be glycosylated by CPS8 with similar efficiency as when iGTΔ3-4 was placed at the first site of EPA. Next, the CPS8-glycosylated EPA fusion protein containing the iGTΔ3-4 ComP glycosylation fragment located between residues Glu548 and Gly549 was purified from whole cell lysates using Ni-affinity chromatography, and the eluates were subjected to Western blot analysis using antisera specific for either the EPA protein or the CPS8 glycan. The results of these experiments similarly indicate that the EPA fusion protein containing the iGTΔ3-4 ComP glycosylation fragment located between residues Glu548 and Gly549 is glycosylated with CPS8 by PglS. Overall, these experiments suggest that the ComP 110264 The glycosylated fragment is the 117 amino acid ComP 110264 These results unexpectedly demonstrate that the ComP sequence can be shortened to sequons as short as 11 amino acids while maintaining glycosylation. 110264 This indicates that the cysteine ​​residues corresponding to Cys71 and Cys93 in are not required for PglS-dependent glycosylation when the ComP glycosylation fragment is incorporated within a fusion protein.

[0067] Provided herein is a saccharide conjugate comprising an oligosaccharide or polysaccharide linked to a fusion protein.In certain embodiments, the oligosaccharide or polysaccharide is covalently bound to the fusion protein.The fusion protein comprises a glycosylated fragment of ComP protein (described in detail elsewhere herein).In certain embodiments of the saccharide conjugate of the present disclosure, the oligosaccharide or polysaccharide comprises glucose at its reducing end.

[0068] ComP is glycosylated at a serine (S) residue, which is located at SEQ ID NO:1 (ComP 110264 This serine residue is conserved in ComP proteins and corresponds to position 82 of SEQ ID NO:2 (ComP ADP1 :AAC45886.1). Thus, in certain embodiments, the fusion protein (and thus the glycoconjugate) corresponds to position 84 of SEQ ID NO:2 (ComP ADP1 : AAC45886.1), or SEQ ID NO: 1 (ComP 110264 11 shows that the ComP glycosylated fragment is glycosylated with an oligosaccharide or polysaccharide at a serine residue corresponding to the serine residue at position 82 of ComP Glycosylated Fragment (SEQ ID NO: 1 (ComP Glycosylated Fragment)) that is conserved across ComP sequences. 110264 5 shows an alignment of the ComP sequence region including the serine (S) residue (boxed) corresponding to the serine residue at position 82 of ENV58402.1.

[0069] One of skill in the art will recognize that by aligning a ComP sequence with SEQ ID NO:1 (e.g., full length or partial sequence), one can identify a conserved serine residue in a non-SEQ ID NO:1 ComP protein that corresponds to the serine residue at position 82 in SEQ ID NO:1. Furthermore, one of skill in the art will understand that by aligning a ComP sequence with SEQ ID NO:1, other residues, regions, and / or features that correspond to residues, regions, and / or features of SEQ ID NO:1 referred to herein can be identified in a non-SEQ ID NO:1 ComP sequence and referenced in relation to SEQ ID NO:1. Also, although reference is made generally herein to SEQ ID NO:1, by analogy, any residue, region, feature, etc. of any ComP sequence disclosed herein can similarly be referenced in relation to, for example, SEQ ID NO:2.

[0070] ComP protein is a protein that is identified as a ComP protein consistent with the description provided herein. For example, representative examples of ComP proteins include, but are not limited to, AAC45886.1 ComP [Acinetobacter sp. ADP1], ENV58402.1 hypothetical protein F951_00736 [Acinetobacter soli CIP 110264], APV36638.1 competence protein [Acinetobacter soli GFJ-2], PKD82822.1 competence protein [Acinetobacter radioresistens 50v1], SNX44537.1 type IV pilus assembly protein PilA [Acinetobacter puyangensis ANC 4466], OAL75955.1 competence protein [Acinetobacter sp. SFC], ComP P5312 , and ComP ANT_H59 In certain embodiments, the ComP protein is represented by SEQ ID NO:2 (ComP ADP1 ) or SEQ ID NO:1 (ComP 110264), and includes a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO:2 or position 82 of SEQ ID NO:1. SEQ ID NO:2 includes a leader sequence of 28 amino acids. In certain embodiments, the ComP protein does not include the amino acid leader sequence but includes the amino acid sequence of SEQ ID NO:1 (ComP 110264 SEQ ID NO: 10 (ComPΔ28) containing a serine residue corresponding to the conserved serine residue at position 82 of AAC45886.1 ADP1 ), SEQ ID NO: 9 (ComPΔ28 110264 ), SEQ ID NO: 11 (ComPΔ28 GFJ-2 ), SEQ ID NO: 12 (ComPΔ28 P50v1 ), SEQ ID NO: 13 (ComPΔ28 4466 ), SEQ ID NO: 14 (ComPΔ28 SFC ), SEQ ID NO: 15 (ComPΔ28 P5312 ), or SEQ ID NO: 16 (ComPΔ29 ANT_H59 In certain embodiments, the ComP protein does not include the 28 amino acid leader sequence, but comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1 (ComP 110264 SEQ ID NO: 9 (ComPΔ28), which contains a serine residue corresponding to the conserved serine residue at position 82 of 110264 In certain embodiments, the ComP protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10 (ComPΔ28). ADP1 ), SEQ ID NO: 9 (ComPΔ28 110264 ), SEQ ID NO: 11 (ComPΔ28 GFJ-2 ), SEQ ID NO: 12 (ComPΔ28 P50v1 ), SEQ ID NO: 13 (ComPΔ28 4466 ), SEQ ID NO: 14 (ComPΔ28 SFC ), SEQ ID NO: 15 (ComPΔ28P 5312 ), or SEQ ID NO: 16 (ComPΔ29ANT_H59 In certain embodiments, the ComP protein comprises SEQ ID NO:2 (ComP ADP1 :AAC45886.1), SEQ ID NO: 1 (ComP 110264 :ENV58402.1), sequence number 3 (ComP GFJ-2 :APV36638.1), SEQ ID NO:4 (ComP 50v1 :PKD82822.1), SEQ ID NO:5 (ComP 4466 :SNX44537.1), SEQ ID NO:6 (ComP SFC :OAL75955.1), SEQ ID NO: 7 (ComP P5312 ), or SEQ ID NO:8 (ComP ANT_H59 ).

[0071] Provided herein is a glycoconjugate comprising an oligosaccharide or polysaccharide covalently attached to a fusion protein, the fusion protein comprising a ComP protein (ComP) glycosylation fragment. In certain embodiments, the ComP glycosylation fragment is a ComP glycosylation fragment. 110264 In certain embodiments, the ComP glycosylation fragment does not contain a cysteine ​​(C) residue corresponding to the conserved cysteine ​​(C) residue at position 71 of (SEQ ID NO: 1). 110264 (SEQ ID NO: 1). As described in more detail herein, the fusion protein does not contain a cysteine ​​(C) residue corresponding to the conserved cysteine ​​(C) residue at position 93 of ComP. 110264 (SEQ ID NO:1). In certain embodiments, the ComP glycosylation fragment is located internally in the fusion protein. Moreover, in certain embodiments, the ComP glycosylation fragment portion of the fusion protein is solvent (or surface) exposed and / or is glycosylated at a serine residue of the ComP glycosylation fragment corresponding to the conserved serine residue at position 82 of (SEQ ID NO:1). In certain embodiments, the ComP glycosylation fragment is located internally in the fusion protein. Moreover, in certain embodiments, the ComP glycosylation fragment portion of the fusion protein is solvent (or surface) exposed and / or is glycosylated at a serine residue of the fusion protein. 10 It may incorporate a β-turn, β-turn, β-twist, β-loop, U-turn, reverse turn, strand inversion, or hairpin loop.

[0072] When the ComP glycosylation fragment is located within the fusion protein, it has been discovered that the cysteine ​​residues on either side are not required for glycosylation, so the ComP glycosylation fragment disclosed herein may be shorter than previously thought. In certain embodiments, the ComP glycosylation fragment is a ComP glycosylation fragment. 110264 (SEQ ID NO: 1) , so long as it contains a serine residue corresponding to the conserved serine residue at position 82 of (SEQ ID NO: 1), it may be less than 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 amino acids in length. In certain embodiments, the ComP glycosylation fragment has a length of any one of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 to any one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids in length. In certain embodiments, the fragment has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues from the N-terminus of the ComP protein to a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO:1, e.g., [ka] wherein n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues of the ComP protein. In certain embodiments, the fragment has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues from the C-terminus of the ComP protein to a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO:1, e.g., [ka] wherein n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues of the ComP protein. Further, in certain embodiments, the amino acid sequence of the ComP glycosylation fragment is 110264 does not extend N-terminally beyond the amino acid residue corresponding to position 72 of (SEQ ID NO:1), and / or110264 It does not extend C-terminally beyond the corresponding amino acid residue at position 92 of (SEQ ID NO:1).

[0073] Consistent with the ComP protein of the present disclosure, in certain embodiments, the ComP protein from which the ComP glycosylation fragment is derived is SEQ ID NO: 9 (ComPΔ28 110264 ), SEQ ID NO: 10 (ComPΔ28 ADP1 ), SEQ ID NO: 11 (ComPΔ28 GFJ-2 ), SEQ ID NO: 12 (ComPΔ28 P50v1 ), SEQ ID NO: 13 (ComPΔ28 4466 ), SEQ ID NO: 14 (ComPΔ28 SFC ), SEQ ID NO: 15 (ComPΔ28 P5312 ), or SEQ ID NO: 16 (ComPΔ29 ANT_H59 In certain embodiments, the ComP protein from which the ComP glycosylation fragment is derived comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 (ComPΔ28). 110264 ), SEQ ID NO: 10 (ComPΔ28 ADP1 ), SEQ ID NO: 11 (ComPΔ28 GFJ-2 ), SEQ ID NO: 12 (ComPΔ28 P50v1 ), SEQ ID NO: 13 (ComPΔ28 4466 ), SEQ ID NO: 14 (ComPΔ28 SFC ), SEQ ID NO: 15 (ComPΔ28P 5312 ), or SEQ ID NO: 16 (ComPΔ29 ANT_H59 ).

[0074] In certain embodiments of the glycoconjugates of the present disclosure, the ComP glycosylation fragment has the following amino acid consensus sequence: [ka] where X1 is V, T, A, or I; X4 is Q, T, E, A, or S; X5 is E, Q, T, or L; X6 is I or V; X7 is S, N, A, or G; X8 is S or no amino acid; X9 is G, D, or no amino acid; X 12 is N, S, or A, X 13 is A, S, or K, X 15 is T, S, or K, X 18 is A, E, Q, or L, X 19 is T, S, or K, X 20 is A or S, X 21 is T, Q, A, or V, or a serine corresponding to position 11 of SEQ ID NO:17

number

number

number

[0075] Certain embodiments provide a ComP glycosylation fragment that is a variant of the amino acid consensus sequence of SEQ ID NO:17, SEQ ID NO:196, or SEQ ID NO:197, or a fragment thereof, having 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, additions, and / or deletions, the variant comprising a serine residue corresponding to position 11 of SEQ ID NO:17.

number

[0076] Whether a ComP glycosylated fragment (including subfragments and variants of the fragments disclosed herein, collectively referred to as ComP glycosylated fragments) can be glycosylated and the efficiency of glycosylation can be determined by methods such as those described herein. In certain embodiments, the ComP glycosylated fragment can be glycosylated when located within a fusion protein and / or within a carrier protein sequence described elsewhere herein. Furthermore, in certain embodiments, the ComP glycosylated fragment or variant is not glycosylated when located at the N-terminus and / or C-terminus of a fusion protein, or is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% less glycosylated when located at the N-terminus and / or C-terminus of a fusion protein compared to when located within the fusion protein.

[0077] In certain embodiments, the fusion protein is Pseudomonas aeruginosa exotoxin A (EPA), CRM 197 , cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae protein D, and fragment(s) thereof. For example, in certain embodiments, the Pseudomonas aeruginosa exotoxin A (EPA) carrier protein comprises the amino acid sequence of SEQ ID NO: 18, or a fragment(s) thereof. For example, in certain embodiments, the CRM 197 The carrier protein comprises the amino acid sequence of SEQ ID NO:24, or a fragment or fragments thereof.

[0078] As can be understood from this disclosure as a whole, internal to the fusion protein means that the ComP fusion protein is not located at the C-terminus or N-terminus of the fusion protein, apart from a C-terminal leader sequence or an N-terminal tag (His tag), etc. For example: N-terminal, not internal Leader sequence-ComP glycosylation fragment-carrier protein C-terminal, not internal Carrier protein-ComP glycosylation fragment-His tag internal Leader sequence-carrier protein-ComP glycosylation fragment-carrier protein-His tag In certain embodiments, the ComP glycosylation fragment may be attached to the carrier protein sequence via an amino acid linker.

[0079] Furthermore, in certain embodiments, the ComP glycosylation fragment may be inserted within the sequence of a carrier protein, rather than between carrier proteins. For example, in certain embodiments: (i) the ComP glycosylation fragment is inserted between Ala489 and Arg490 of Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (SEQ ID NO: 19); (ii) the ComP glycosylation fragment is inserted between Glu548 and Gly549 relative to the PDB molecule 1IKQ of Pseudomonas aeruginosa exotoxin A (EPA) (SEQ ID NO: 20); (iii) the ComP glycosylation fragment is inserted between Ala122 and Gly123 of Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (SEQ ID NO: 21); or (iv) the ComP glycosylation fragment is inserted between Thr355 and Gly356 of Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (SEQ ID NO: 22); or (v) The ComP glycosylation fragment is inserted between Lys20 and Asp21 relative to the PDB molecule 1IKQ of Pseudomonas aeruginosa exotoxin A (EPA) (SEQ ID NO: 23).

[0080] Furthermore, in certain embodiments, the ComP glycosylation fragment may be inserted within the sequence of a carrier protein, rather than between carrier proteins. For example, in certain embodiments: (i) the ComP glycosylated fragment is a CRM 197 The PDB molecule is inserted between Asn481 and Gly482 of 4AE0 (SEQ ID NO: 25), (ii) the ComP glycosylated fragment is a CRM 197 The PDB molecule is inserted between Asp392 and Gly393 of 4AE0 (SEQ ID NO: 26), (iii) the ComP glycosylated fragment is a CRMP 197 Inserted between Glu142 and Gly143 of the PDB molecule 4AE0 (SEQ ID NO: 27) (iv) the ComP glycosylated fragment is a CRM 197 or (v) the ComP glycosylated fragment is a CRM 197 The PDB molecule is inserted between Asn69 and Glu70 of 4AE0 (SEQ ID NO: 29).

[0081] In certain embodiments, the ComP glycosylation fragments can be located between carrier proteins and can also be inserted into the sequence of the carrier protein(s) within a fusion protein. In certain embodiments, a ComP glycosylation fragment can be located internally and one or more ComP glycosylation fragments can be located at the C-terminus and / or N-terminus, which are sufficient for glycosylation in such locations.

[0082] In aspects of the present disclosure, a fusion protein may be designed to contain multiple ComP glycosylation fragments, for example, to enhance the immunogenicity of the glycosylated fusion protein / glycoconjugate. In certain embodiments, the fusion protein contains 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more, or 20 or more ComP glycosylation fragments. In certain embodiments, the fusion protein does not contain more than 3, 5, 10, 15, 20, or 25 ComP glycosylation fragments. The identity of the ComP glycosylation fragments may also be controlled. For example, in certain embodiments, multiple ComP glycosylation fragments of a fusion protein are identical. In certain embodiments, the ComP glycosylation fragments of a fusion protein are different from each other. For example, in certain embodiments, at least three, at least four, or at least five of the ComP glycosylation fragments of a fusion protein are all different from each other. For example, in certain embodiments, none of the ComP glycosylation fragments of the fusion protein are the same.

[0083] In certain embodiments, the oligosaccharide or polysaccharide is derived from a saccharide produced by a bacterium of the genus Streptococcus. For example, in certain embodiments, the saccharide is a capsular polysaccharide of S.pneumoniae, S.agalactiae, or S.suis, in certain embodiments, the saccharide is a serotype 8 capsular polysaccharide from S.pneumoniae, and in certain embodiments, the saccharide is a type Ia, Ib, II, III, IV, V, VI, VII, VIII, or X capsular polysaccharide from S.agalactiae.

[0084] In certain embodiments, the oligosaccharide or polysaccharide is derived from a saccharide produced by a bacterium of the genus Klebsiella. For example, in certain embodiments, the saccharide is a capsular polysaccharide of K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca, and in certain embodiments, the saccharide is an O-antigen polysaccharide of K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

[0085] In certain embodiments, the glycoconjugate is produced in vivo, e.g., in a bacterial cell, e.g., in Escherichia coli, in a bacterium of the genus Klebsiella, and / or the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

[0086] Provided herein are glycoconjugates as described above (e.g., the ComP glycosylated fragments are ComP 110264 and / or the ComP glycosylated fragment does not contain a cysteine ​​(C) residue corresponding to the conserved cysteine ​​(C) residue at position 71 of (SEQ ID NO: 1). 110264 (e.g., the ComP glycosylated fragment does not contain a cysteine ​​(C) residue corresponding to the conserved cysteine ​​(C) residue at position 93 of SEQ ID NO:1), and the ComP glycosylated fragment comprises or consists of an amino acid sequence of SEQ ID NO:32-163, or 164. Provided herein are glycoconjugates as described above (e.g., the ComP glycosylated fragment does not contain a cysteine ​​(C) residue corresponding to the conserved cysteine ​​(C) residue at position 93 of SEQ ID NO:1), and the ComP glycosylated fragment comprises or consists of an amino acid sequence of SEQ ID NO:32-163, or 164. 110264 and / or the ComP glycosylated fragment does not contain a cysteine ​​(C) residue corresponding to the conserved cysteine ​​(C) residue at position 71 of (SEQ ID NO: 1). 110264 (not including the conserved cysteine ​​(C) residue corresponding to position 93 of SEQ ID NO:1), the ComP glycosylated fragment comprises or consists of the following amino acid sequence: [ka]

[0087] Also provided herein is a ComP glycosylation fragment that is a variant of any of the ComP glycosylation fragments disclosed above, which has 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, additions, and / or deletions, and which variants maintain a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO:1, and which variants are selected from the group consisting of ComP glycosylation fragments, ... 110264 and / or the variant does not contain a cysteine ​​(C) residue corresponding to the conserved cysteine ​​(C) residue at position 71 of (SEQ ID NO: 1); 110264 (SEQ ID NO: 1), does not contain a cysteine ​​(C) residue corresponding to the conserved cysteine ​​(C) residue at position 93 of SEQ ID NO: 1.

[0088] Whether a ComP glycosylated fragment (including subfragments and variants of the fragments disclosed herein, collectively referred to as ComP glycosylated fragments) is glycosylated and the efficiency of glycosylation can be determined by methods such as those described herein. In certain embodiments, the ComP glycosylated fragment can be glycosylated when located within a fusion protein and / or within a carrier protein sequence described elsewhere herein. Furthermore, in certain embodiments, the ComP glycosylated fragment is not glycosylated when located at the N-terminus and / or C-terminus of a fusion protein, or is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% less glycosylated when located at the N-terminus and / or C-terminus of a fusion protein compared to when located within the fusion protein.

[0089] In certain embodiments, the glycoconjugate is a conjugate vaccine. Thus, the present disclosure is directed to and provides, in certain embodiments, a conjugate vaccine. In certain embodiments, the conjugate vaccine is a vaccine against Streptococcus pneumoniae serotype 8. In certain embodiments, the conjugate vaccine induces an immune response when administered to a subject. In certain embodiments, the immune response induces long-term memory (memory B and T cells) and is an antibody response, optionally a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG or IgM response. In certain embodiments, the antibody response is an IgG response, optionally an IgG1 response. Also, in certain embodiments, the conjugate vaccine generates immune memory in a subject administered the vaccine.

[0090] Although the above describes glycoconjugates comprising ComP glycosylation fragments, including fragments of isolated ComP proteins, it is understood that the disclosure also expressly provides ComP glycosylation fragments consistent with any and all descriptions of ComP glycosylation fragments provided anywhere in this specification, including the claims appended below, e.g., the ComP glycosylation fragments include ComP 110264 (SEQ ID NO: 1), and / or 110264 (SEQ ID NO: 1), and the ComP glycosylated fragment does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 93 of ComP. 110264 (SEQ ID NO:1) contains a serine residue corresponding to the conserved serine residue at position 82 of (SEQ ID NO:1).

[0091] Provided herein is a fusion protein comprising the ComP glycosylation fragment of the present disclosure. In certain embodiments, the fusion protein comprises SEQ ID NO: 1 (ComP 110264) is glycosylated with an oligosaccharide or polysaccharide at a serine residue of the glycosylation fragment corresponding to the serine residue of the ComP glycosylation fragment at position 82 of ComP glycosylation fragment ... 197 , cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae protein D, and fragment(s) thereof.

[0092] Also provided herein is a method for in vivo conjugation of an oligosaccharide or polysaccharide to a receptor polypeptide. In certain embodiments, the method comprises culturing a host cell that contains the components necessary for conjugation of the oligosaccharide or polysaccharide to the polypeptide. Generally, these components are an oligosaccharyltransferase, a receptor polypeptide to be glycosylated, and an oligosaccharide or polysaccharide. The method comprises covalently attaching an oligosaccharide or polysaccharide to the receptor polypeptide (a fusion protein of the present disclosure) by a PglS oligosaccharyltransferase (OTase), the receptor polypeptide comprising a ComP glycosylation fragment as described herein. In certain embodiments, the PglS OTase is a PglS OTase. 110264 (SEQ ID NO: 165), PglS ADP1 (SEQ ID NO: 166), PglS GFJ-2 (SEQ ID NO: 167), PglS 50v1 (SEQ ID NO: 168), PglS 4466 (SEQ ID NO: 169), PglS SFC (SEQ ID NO: 170), Pgl SP5312 (SEQ ID NO: 171), or PglS ANT_H59(SEQ ID NO: 172). In certain embodiments, the oligosaccharide or polysaccharide is SEQ ID NO: 1 (ComP 110264 Serine corresponding to the serine residue at position 82 of

number

[0093] In certain embodiments, the glycoconjugates are produced in a cell-free system. Examples of the use of cell-free systems utilizing OTases other than PglS can be found in WO2013 / 067523A1, which is incorporated herein by reference.

[0094] Also provided is a host cell comprising (a) a gene cluster encoding proteins required for oligosaccharide or polysaccharide synthesis, (b) a PglS OTase, and (3) a receptor polypeptide comprising a ComP glycosylation fragment of the disclosure. In certain embodiments, the receptor polypeptide is a fusion protein. In certain embodiments, the host cell comprises a nucleic acid encoding the PglS OTase. In certain embodiments, the host cell comprises a nucleic acid encoding the receptor polypeptide.

[0095] Also provided herein is an isolated nucleic acid encoding the ComP glycosylation fragment and / or fusion protein of the present disclosure. In certain embodiments, the nucleic acid is a vector. In certain embodiments, the host cell comprises the isolated nucleic acid.

[0096] The glycoconjugates of the present invention may have one of many uses, including but not limited to use as conjugate vaccines.Thus, in certain methods, conjugate vaccines are produced.In certain embodiments, compositions comprising the conjugate vaccines or fusion proteins of the present disclosure and adjuvants. For example, in certain embodiments, the conjugate vaccine is selected from the group consisting of Streptococcus pneumoniae serotype 8, Streptococcus pneumoniae serotype 1, Streptococcus pneumoniae serotype 2, Streptococcus pneumoniae serotype 4, Streptococcus pneumoniae serotype 5, Streptococcus pneumoniae serotype 6A, Streptococcus pneumoniae serotype 6B, Streptococcus pneumoniae serotype 7F, Streptococcus pneumoniae serotype 9N, Streptococcus pneumoniae serotype 9V, Streptococcus pneumoniae serotype 10A, Streptococcus pneumoniae serotype 11A, Streptococcus pneumoniae serotype 12F, Streptococcus pneumoniae serotype 14, Streptococcus pneumoniae serotype 15B, Streptococcus pneumoniae serotype 17F, Streptococcus pneumoniae serotype 18C, Streptococcus pneumoniae serotype 19F, Streptococcus pneumoniae serotype 19A, Streptococcus pneumoniae serotype 20, Streptococcus pneumoniae serotype 22F, Streptococcus pneumoniae serotype 23F, Streptococcus pneumoniae serotype 33F, Klebsiella pneumoniae serotype K1, Klebsiella Klebsiella pneumoniae serotype K2, Klebsiella pneumoniae serotype K5, Klebsiella pneumoniae serotype K16, Klebsiellapneumoniae serotype K20, Klebsiella pneumoniae serotype K54, Klebsiella pneumoniae serotype K57, Streptococcus agalactiae serotype Ia, Streptococcus agalactiae serotype Ib, Streptococcus agalactiae serotype II, Streptococcus agalactiae serotype III, Streptococcus agalactiae serotype IV, Streptococcus agalactiae serotype V, Streptococcus agalactiae serotype VI, Streptococcus agalactiae serotype VII, Streptococcus agalactiae serotype VIII, Streptococcus agalactiae serotype IX, Streptococcus pyogenes group A carbohydrate, Enterococcus faecalis serotype A, Enterococcus faecalis serotype B, Enterococcus faecalis serotype C, Enterococcus The conjugate vaccine is a vaccine against Enterococcus faecalis serotype D, Enterococcus faecium capsular polysaccharide and lipoteichoic acid, Moraxella catarrhalis lipo-oligosaccharide A, Moraxella catarrhalis lipo-oligosaccharide B, Moraxella catarrhalis lipo-oligosaccharide C, and Staphylococcus aureus lipo-oligosaccharide. In certain embodiments, the conjugate vaccine is useful because it induces an immune response when administered to a subject. In certain embodiments, the immune response induces long-term memory (memory B and T cells) and is an antibody response, optionally a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG or IgM response. For example, in certain embodiments, the antibody response can be an IgG response, and in certain embodiments, an IgG1 response. In certain embodiments, the conjugate vaccine generates immune memory in the subject administered the vaccine.

[0097] Disclosed herein is a pneumococcal glycoconjugate vaccine comprising a conventional vaccine carrier, which can be produced by isolating a glycoconjugate or glycosylated fusion protein of the present disclosure comprising a ComP glycosylated fragment of the present disclosure, and combining the isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant. In certain embodiments, the ComP glycosylated fragment can be added to a conventional carrier protein, Pseudomonas aeruginosa exotoxin A (EPA). In certain embodiments, it has been demonstrated that the glycosylated fragment / carrier fusion protein can be combined with the use of CPS8 polysaccharide and PglS to generate a carrier protein-CPS8 bioconjugate, a first of its kind pneumococcal bioconjugate vaccine. For example, in certain embodiments, an EPA fusion can be combined with the use of the CPS8 polysaccharide and PglS to generate an EPA-CPS8 bioconjugate. The EPA-CPS8 bioconjugate vaccine was demonstrated to induce high serotype 8-specific IgG titers that were protective as judged by bactericidal killing. Importantly, vaccination with as little as 100 ng of polysaccharide contained in the EPA-CPS8 bioconjugate was able to provide protection. Thus, certain embodiments provide a CPS8 pneumococcal bioconjugate vaccine.

[0098] It is contemplated that the conjugate vaccine (e.g., the EPA vaccine construct) may contain additional / multiple glycosylation sites to increase the glycan-to-protein ratio and expand the number of serotypes to develop a comprehensive pneumococcal bioconjugate vaccine.

[0099] In certain embodiments, the glycoconjugates or glycosylated fusion proteins disclosed herein are conjugate vaccines that can be administered to a subject for the prevention and / or treatment of infection and / or disease. In certain embodiments, the conjugate vaccine is a prophylactic, which can be used, for example, to immunize a subject against infection and / or disease. In certain embodiments, the glycoconjugate is associated with an adjuvant (e.g., in a therapeutic composition) and / or administered with an adjuvant. Certain embodiments provide compositions (e.g., therapeutic compositions) that include a conjugate vaccine and an adjuvant described herein. In certain embodiments, the conjugate vaccine induces an immune response when administered to a subject. In certain embodiments, the immune response induces long-term memory (memory B and T cells). In certain embodiments, the immunity is an antibody response. In certain embodiments, the antibody response is a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG or IgM response. In certain embodiments, where the antibody response is an IgG response, the IgG response is an IgG1 response.Furthermore, in certain embodiments, the conjugate vaccine generates immune memory in a subject administered the vaccine.

[0100] Certain embodiments also provide for producing a vaccine against an infectious disease and / or disease. In certain embodiments, a method comprises isolating a glycoconjugate or fusion protein (conjugate vaccine) disclosed herein and combining the conjugate vaccine with an adjuvant. In certain embodiments, the infectious disease is a local or systemic infection of the skin, soft tissue, blood, or organs, or is autoimmune in nature. In certain embodiments, the vaccine is a conjugate vaccine against pneumococcal infection. In certain embodiments, the disease is pneumonia. In certain embodiments, the infectious disease is a systemic infection and / or a blood infection. In certain embodiments, the subject is a mammal. For example, in certain embodiments, a pig or a human.

[0101] Importantly, the embodiments disclosed herein are not limited to pneumococcal polysaccharides, but indeed have broad applicability for generating bioconjugate vaccines against many important human and animal pathogens that are incompatible with PglB and PglL. Notable examples include the human pathogens Klebsiella pneumoniae and group B Streptococcus, as well as the swine pathogen S. suis, all highly relevant pathogens for which no licensed vaccines are available.

[0102] Provided herein is a method for inducing a host immune response against a pathogen. In certain embodiments, the pathogen is a bacterial pathogen. In certain embodiments, the host is immunized against the pathogen. In certain embodiments, the method comprises administering an effective amount of a ComP conjugate vaccine, a glycosylated fusion protein, or any other therapeutic / immunogenic composition disclosed herein to a subject in need of the immune response. Certain embodiments provide a conjugate vaccine, a glycosylated fusion protein, or any other therapeutic / immunogenic composition disclosed herein for use in inducing a host immune response against a bacterial pathogen and immunizing against the bacterial pathogen. Examples of immune responses include, but are not limited to, innate responses, adaptive responses, humoral responses, antibody responses, cell-mediated responses, B cell responses, T cell responses, upregulation or downregulation of cytokines, immune system crosstalk, and combinations of two or more of these immune responses. In certain embodiments, the immune response is an antibody response. In certain embodiments, the immune response is an innate response, a humoral response, an antibody response, a T cell response, or a combination of two or more of these immune responses.

[0103] Also disclosed herein is a method of preventing or treating bacterial and / or infectious diseases in a subject, comprising administering to a subject in need thereof a conjugate vaccine, fusion protein, or composition disclosed herein. In certain embodiments, the infectious disease is a localized or systemic infection of the skin, soft tissue, blood, or organs, or is autoimmune in nature. In certain embodiments, the disease is pneumonia. In certain embodiments, the infectious disease is a systemic infection and / or an infection of the blood. In certain aspects disclosed herein, the subject is a vertebrate. In certain embodiments, the subject is a mammal, such as a dog, cat, cow, horse, pig, mouse, rat, rabbit, sheep, goat, guinea pig, monkey, ape, etc. Also, for example, in certain embodiments, the mammal is a human.

[0104] In any of the administration embodiments disclosed herein, the compositions are administered via intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.

[0105] In certain embodiments, the glycoconjugate, glycosylated fusion protein, or conjugate vaccine of any of the above claims for use in inducing a host immune response against a bacterial pathogen and / or for preventing or treating a bacterial disease and / or infection in a subject.

[0106] Immunization with glycosylated ComP bioconjugates elicits an immune response. T cell-dependent immune responses to conjugate vaccines are characterized by the secretion of high affinity IgG1 antibodies (Avci, FY, Li, X., Tsuji, M. & Kasper, DL Nat Med 17, 1602-1609 (2011)). The immunogenicity of the CPS14-ComP bioconjugate in a mouse vaccination model was evaluated (WO / 2020 / 131236, incorporated herein by reference in its entirety). Serum collected from mice vaccinated with the CPS14-ComP bioconjugate had a significant increase in CPS14-specific IgG titers, but not IgM titers. Furthermore, a secondary HRP-tagged anti-IgG subtype antibody was used to identify IgG subtypes with elevated titers. IgG1 titers appeared to be higher than other subtypes.

[0107] A second vaccination study was then conducted to compare the immunogenicity of trivalent CPS8-, CPS9V-, and CPS14-ComP bioconjugates with the current standard of care, PREVNAR 13®. Serotypes 9V and 14 are included in PREVNAR 13®, and elevated IgG titers were observed in PREVNAR 13®-immunized mice against these two serotypes. Monovalent immunization against serotype 14 also showed a significant induction of serotype-specific IgG titers similar to the preimmunization. Mice administered the trivalent bioconjugates, as expected, all had elevated serotype-specific IgG titers when compared to controls, with day 49 sera showing an even higher elevation of IgG titers against serotypes 8 and 14 compared to serotype 9V. Nevertheless, IgG titers against 9V were significantly higher than placebo. *****

[0108] Certain embodiments of the present disclosure may be defined in any of the following numbered paragraphs:

[0109] 1. A glycoconjugate comprising an oligosaccharide or polysaccharide covalently attached to a fusion protein, said fusion protein comprising a ComP protein (ComP) glycosylated fragment, said ComP glycosylated fragment being a glycoconjugate of ComP. 110264 (SEQ ID NO: 1), and / or 110264 (SEQ ID NO: 1), and the ComP glycosylation fragment is located within the fusion protein, and the fusion protein does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 93 of ComP (SEQ ID NO: 1), and the ComP glycosylation fragment is located within the fusion protein, and the fusion protein comprises a ComP glycosylation fragment having a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 93 of ComP (SEQ ID NO: 1), 110264 (SEQ ID NO:1), optionally wherein the glycoconjugate is immunogenic; optionally wherein the ComP glycosylated fragment is solvent (or surface) exposed; and optionally wherein the ComP glycosylated fragment is a C-terminal fragment of the fusion protein. 10 The glycoconjugate is incorporated into a β-turn, a β-turn, a β-twist, a β-loop, a U-turn, a reverse turn, a strand reversal, or a hairpin loop.

[0110] 2. The glycoconjugate of paragraph 1, wherein the ComP glycosylated fragment is 5-22 amino acids in length, 10-22 amino acids in length, 11-22 amino acids in length, 5-21 amino acids in length, 10-21 amino acids in length, or 11-21 amino acids in length, and optionally the fragment has at least 1, 2, 3, 4, or 5 amino acid residues N-terminal to the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO:1, and / or the fragment has at least 1, 2, 3, 4, or 5 amino acid residues C-terminal to the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO:1.

[0111] 3. The amino acid sequence of the ComP glycosylation fragment is 110264 does not extend N-terminally beyond the amino acid residue corresponding to position 72 of (SEQ ID NO:1), and / or110264 3. The glycoconjugate of paragraph 1 or 2, which does not extend C-terminally beyond the corresponding amino acid residue at position 92 of (SEQ ID NO:1).

[0112] 4. The ComP protein is selected from the group consisting of SEQ ID NO: 9 (ComPΔ28 110264 ), SEQ ID NO: 10 (ComPΔ28 ADP1 ), SEQ ID NO: 11 (ComPΔ28 GFJ-2 ), SEQ ID NO: 12 (ComPΔ28 P50v1 ), SEQ ID NO: 13 (ComPΔ28 4466 ), SEQ ID NO: 14 (ComPΔ28 SFC ), SEQ ID NO: 15 (ComPΔ28 P5312 ), or SEQ ID NO: 16 (ComPΔ29 ANT_H59 9 (ComPΔ28), and optionally the ComP protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 (ComPΔ28), 110264 ), SEQ ID NO: 10 (ComPΔ28 ADP1 ), SEQ ID NO: 11 (ComPΔ28 GFJ-2 ), SEQ ID NO: 12 (ComPΔ28 P50v1 ), SEQ ID NO: 13 (ComPΔ28 4466 ), SEQ ID NO: 14 (ComPΔ28 SFC ), SEQ ID NO: 15 (ComPΔ28 P5312 ), or SEQ ID NO: 16 (ComPΔ29 ANT_H59 4. The glycoconjugate of any one of paragraphs 1 to 3, comprising:

[0113] 5. The glycoconjugate according to any one of paragraphs 1 to 4, wherein the ComP glycosylation fragment has the following amino acid consensus sequence: [ka] with the proviso that X1 is V, T, A, or I, X4 is Q, T, E, A, or S, X5 is E, Q, T, or L, X6 is I or V, X7 is S, N, A, or G, X8 is S or no amino acid, X9 is G, D, or no amino acid, and X 12 is N, S, or A, and X 13 is A, S, or K, and X 15 is T, S, or K, and X 18 is A, E, Q, or L, and X 19 is T, S, or K, and X 20 is A or S, and X 21 is T, Q, A, or V, or the serine at position 11 of SEQ ID NO: 17.

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[0114] 6. The glycoconjugate according to any one of paragraphs 1 to 4, wherein the ComP glycosylation fragment has the following amino acid consensus sequence: [ka] with the proviso that X1 is V, T, A, or I, X4 is Q, T, E, A, or S, X5 is E, Q, T, or L, X6 is I or V, X7 is S, N, A, or G, X8 is S or no amino acid, X9 is G, D, or no amino acid, and X 12 is N, S, or A, and X 13 is A, S, or K, and X 15 is T, S, or K, and X 18 is A, E, Q, or L, and X 19 is T, S, or K, and X 20 is A or S, and X 21is T, Q, A, or V, or the serine at position 11 of SEQ ID NO: 17.

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[0115] 7. The fusion protein is selected from the group consisting of Pseudomonas aeruginosa exotoxin A (EPA), CRM 197 , cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae protein D, and fragment(s) thereof, optionally wherein the Pseudomonas aeruginosa exotoxin A (EPA) carrier protein comprises the amino acid sequence of SEQ ID NO: 18, or fragment(s) thereof, and optionally wherein the CRM 1977. The glycoconjugate of any one of paragraphs 1 to 6, wherein the carrier protein comprises the amino acid sequence of SEQ ID NO: 24, or a fragment or fragments thereof.

[0116] 8. (i) the ComP glycosylation fragment is inserted between Ala489 and Arg490 for Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (SEQ ID NO: 19); (ii) the ComP glycosylation fragment is inserted between Glu548 and Gly549 for Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (SEQ ID NO: 20); (iii) the ComP glycosylation fragment is inserted between Ala122 and Gly123 for Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (SEQ ID NO: 21); or (iv) the ComP glycosylation fragment is inserted between Pseudomonas aeruginosa 8. The glycoconjugate of paragraph 7, wherein (i) the ComP glycosylation fragment is inserted between Thr355 and Gly356 of Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (SEQ ID NO: 22); or (ii) the ComP glycosylation fragment is inserted between Lys20 and Asp21 of Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (SEQ ID NO: 23).

[0117] 9. (i) the ComP glycosylated fragment is a CRM 197 (ii) the ComP glycosylation fragment is inserted between Asn481 and Gly482 of the PDB molecule 4AE0 (SEQ ID NO: 25); or 197 (iii) the ComP glycosylation fragment is inserted between Asp392 and Gly393 of the PDB molecule 4AE0 (SEQ ID NO: 26); or 197 (iv) the ComP glycosylation fragment is inserted between Glu142 and Gly143 of the PDB molecule 4AE0 (SEQ ID NO: 27); or 197or (v) the ComP glycosylation fragment is inserted between Asp129 and Gly130 of the PDB molecule 4AE0 (SEQ ID NO: 28); or 197 8. The glycoconjugate of paragraph 7, wherein the glycoconjugate is inserted between Asn69 and Glu70 of PDB molecule 4AE0 (SEQ ID NO: 29).

[0118] 10. The glycoconjugate of any one of paragraphs 1 to 9, wherein the fusion protein comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more, or 20 or more ComP glycosylated fragments, optionally wherein the fusion protein does not comprise more than 3, more than 5, more than 10, more than 15, more than 20, or more than 25 ComP glycosylated fragments.

[0119] 11. The glycoconjugate according to any one of paragraphs 1 to 10, wherein the ComP glycosylation fragments are identical.

[0120] 12. The glycoconjugate of any one of paragraphs 1 to 10, wherein the ComP glycosylation fragments are different from each other, and optionally at least three, at least four, or at least five of the ComP glycosylation fragments are all different from each other, and optionally none of the ComP glycosylation fragments are the same.

[0121] 13. The glycoconjugate according to any one of paragraphs 1 to 12, wherein the oligosaccharide or polysaccharide is derived from a saccharide produced by a bacterium of the genus Streptococcus, optionally the saccharide is a capsular polysaccharide of S. pneumoniae, S. agalactiae, or S. suis, optionally the saccharide is a serotype 8 capsular polysaccharide from S. pneumoniae, optionally the saccharide is a type Ia, Ib, II, III, IV, V, VI, VII, VIII, or X capsular polysaccharide from S. agalactiae.

[0122] 14. The glycoconjugate of any one of paragraphs 1 to 12, wherein the oligosaccharide or polysaccharide is derived from a saccharide produced by a bacterium of the genus Klebsiella, and optionally the saccharide is a capsular polysaccharide of K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca, and optionally the saccharide is an O-antigen polysaccharide of K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

[0123] 15. The glycoconjugate according to any one of paragraphs 1 to 14, wherein the oligosaccharide or polysaccharide comprises glucose at its reducing end.

[0124] 16. The glycoconjugate according to any one of paragraphs 1 to 15, produced in vivo, optionally in a bacterial cell, optionally in Escherichia coli, optionally in a bacterium of the genus Klebsiella, optionally wherein the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

[0125] 17. The glycoconjugate according to any one of paragraphs 1 to 16, wherein the ComP glycosylated fragment comprises or consists of an amino acid sequence of SEQ ID NO: 32 to 163, or 164, or a variant thereof having one, two or three amino acid substitutions, additions and / or deletions, said variant including a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 1, wherein optionally the ComP glycosylated fragment is capable of being glycosylated when located internally in a fusion protein, and optionally the ComP glycosylated fragment is not glycosylated when located at the N-terminus and / or C-terminus of a fusion protein, or is at least 50%, 60%, 70%, 80%, 90%, 95% or 99% less glycosylated when located at the N-terminus and / or C-terminus of a fusion protein compared to when located internally in the fusion protein.

[0126] 18. The ComP glycosylation fragment has the following amino acid sequence: [ka] 18. The glycoconjugate according to paragraph 17, comprising or consisting of, or a variant thereof.

[0127] 19. The glycoconjugate according to paragraph 17, wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of SEQ ID NO: 32-163, or 164, and optionally the ComP glycosylation fragment is capable of being glycosylated when located internally in a fusion protein, and optionally the ComP glycosylation fragment is not glycosylated when located at the N-terminus and / or C-terminus of a fusion protein, or is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% less glycosylated when located at the N-terminus and / or C-terminus of a fusion protein compared to when located internally in the fusion protein.

[0128] 20. The ComP glycosylation fragment has the following amino acid sequence: [ka] 20. The glycoconjugate according to paragraph 19, comprising or consisting of:

[0129] 21. The glycoconjugate according to any one of paragraphs 1 to 20, wherein the bioconjugate is a conjugate vaccine, optionally wherein the conjugate vaccine is a vaccine against Streptococcus pneumoniae serotype 8.

[0130] 22. The glycoconjugate according to paragraph 21, wherein said conjugate vaccine induces an immune response when administered to a subject.

[0131] 23. The glycoconjugate according to paragraph 22, wherein said immune response induces long-term memory (memory B and T cells) and is an antibody response, optionally a serotype-specific antibody response.

[0132] 24. The glycoconjugate according to paragraph 23, wherein the antibody response is an IgG or IgM response.

[0133] 25. The glycoconjugate according to paragraph 24, wherein the antibody response is an IgG response, optionally an IgG1 response.

[0134] 26. The glycoconjugate according to any one of paragraphs 21 to 25, wherein the conjugate vaccine generates immune memory in a subject administered the vaccine.

[0135] 27. An isolated ComP glycosylation fragment comprising or consisting of a fragment of the ComP protein, comprising: 110264 (SEQ ID NO: 1), and / or 110264 (SEQ ID NO: 1), does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 93 of ComP 110264 The ComP glycosylated fragment comprises a serine residue corresponding to the conserved serine residue at position 82 of (SEQ ID NO:1), and is optionally immunogenic.

[0136] 28. A ComP glycosylation fragment according to paragraph 27 having a length of 5 to 22 amino acids, or having a length of 10 to 22 amino acids, or having a length of 11 to 22 amino acids, or having a length of 5 to 21 amino acids, or having a length of 10 to 21 amino acids, or having a length of 11 to 21 amino acids, and optionally having at least 1, 2, 3, 4, 5, or 6 amino acid residues N-terminal to the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO:1 and / or having at least 1, 2, 3, 4, 5, or 6 amino acid residues C-terminal to the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO:1.

[0137] 29. The amino acid sequence of the ComP glycosylation fragment is 110264 does not extend N-terminally beyond the amino acid residue corresponding to position 72 of (SEQ ID NO:1), and / or 11026429. A ComP glycosylation fragment according to paragraph 27 or 28, which does not extend C-terminally beyond the corresponding amino acid residue at position 92 of (SEQ ID NO:1).

[0138] 30. The ComP protein is selected from the group consisting of SEQ ID NO: 9 (ComPΔ28 110264 ), SEQ ID NO: 10 (ComPΔ28 ADP1 ), SEQ ID NO: 11 (ComPΔ28 GFJ-2 ), SEQ ID NO: 12 (ComPΔ28 P50v1 ), SEQ ID NO: 13 (ComPΔ28 4466 ), SEQ ID NO: 14 (ComPΔ28 SFC ), SEQ ID NO: 15 (ComPΔ28 P5312 ), or SEQ ID NO: 16 (ComPΔ29 ANT_H59 9 (ComPΔ28), and optionally the ComP protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9 (ComPΔ28), 110264 ), SEQ ID NO: 10 (ComPΔ28 ADP1 ), SEQ ID NO: 11 (ComPΔ28 GFJ-2 ), SEQ ID NO: 12 (ComPΔ28 P50v1 ), SEQ ID NO: 13 (ComPΔ28 4466 ), SEQ ID NO: 14 (ComPΔ28 SFC ), SEQ ID NO: 15 (ComPΔ28 P5312 ), or SEQ ID NO: 16 (ComPΔ29 ANT_H59 30. The ComP glycosylation fragment of any one of paragraphs 27 to 29, comprising:

[0139] 31. A ComP glycosylation fragment according to any one of paragraphs 27 to 30, comprising the following amino acid consensus sequence: [ka] with the proviso that X1 is V, T, A, or I, X4 is Q, T, E, A, or S, X5 is E, Q, T, or L, X6 is I or V, X7 is S, N, A, or G, X8 is S or no amino acid, X9 is G, D, or no amino acid, and X 12 is N, S, or A, and X 13 is A, S, or K, and X 15 is T, S, or K, and X 18 is A, E, Q, or L, and X 19 is T, S, or K, and X 20 is A or S, and X 21 is T, Q, A, or V, or the serine at position 11 of SEQ ID NO: 17.

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[0140] 32. A ComP glycosylation fragment according to any one of paragraphs 27 to 30, comprising the following amino acid consensus sequence: [ka] with the proviso that X1 is V, T, A, or I, X4 is Q, T, E, A, or S, X5 is E, Q, T, or L, X6 is I or V, X7 is S, N, A, or G, X8 is S or no amino acid, X9 is G, D, or no amino acid, and X 12 is N, S, or A, and X 13 is A, S, or K, and X 15 is T, S, or K, and X 18 is A, E, Q, or L, and X 19 is T, S, or K, and X 20 is A or S, and X 21 is T, Q, A, or V, or the serine at position 11 of SEQ ID NO: 17.

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[0141] 33. A ComP glycosylation fragment according to paragraph 27, having an amino acid sequence of SEQ ID NO: 32 to 163, or 164, or a variant thereof having one, two or three amino acid substitutions, additions and / or deletions, comprising the conserved serine at position 82 of SEQ ID NO: 1.

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[0142] 34. The amino acid sequence: [ka] 34. A ComP glycosylation fragment according to paragraph 33, comprising or consisting of, or a variant thereof.

[0143] 35. A ComP glycosylated fragment according to paragraph 33, comprising or consisting of an amino acid sequence of SEQ ID NO: 32-163, or 164, which is optionally capable of being glycosylated when placed within a fusion protein, and optionally is not glycosylated when located at the N-terminus or C-terminus of a fusion protein, or is at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less glycosylated when located at the N-terminus or C-terminus of a fusion protein compared to when located within the fusion protein.

[0144] 36. The amino acid sequence: [ka] 36. A ComP glycosylation fragment according to paragraph 35, comprising or consisting of:

[0145] 37. A fusion protein comprising a ComP glycosylation fragment according to any one of paragraphs 27 to 36, wherein the ComP glycosylation fragment is located internally in the fusion protein and optionally comprises SEQ ID NO: 1 (ComP 110264 The fusion protein is glycosylated with an oligosaccharide or polysaccharide at a serine residue of the glycosylation fragment corresponding to the serine residue of the ComP glycosylation fragment at position 82 of the glycosylation fragment.

[0146] 38. The fusion protein of paragraph 37, wherein the oligosaccharide or polysaccharide is derived from a saccharide produced by a bacterium of the genus Streptococcus, optionally the saccharide is a capsular polysaccharide of S. pneumoniae, S. agalactiae, or S. suis, optionally the saccharide is a serotype 8 capsular polysaccharide from S. pneumoniae, optionally the saccharide is a type Ia, Ib, II, III, IV, V, VI, VII, VIII, or X capsular polysaccharide from S. agalactiae.

[0147] 39. The fusion protein of paragraph 37, wherein the oligosaccharide or polysaccharide is derived from a saccharide produced by a bacterium of the genus Klebsiella, and optionally the saccharide is a capsular polysaccharide of K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca, and optionally the saccharide is an O-antigen polysaccharide of K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

[0148] 40. The fusion protein according to any one of paragraphs 37 to 39, wherein the oligosaccharide or polysaccharide comprises glucose at its reducing end.

[0149] 41. The fusion protein of any one of paragraphs 37 to 40, wherein the glycosylated fusion protein is produced in vivo, optionally in a bacterial cell, optionally in Escherichia coli, optionally in a bacterium of the genus Klebsiella, and optionally wherein the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

[0150] 42.Pseudomonas aeruginosa exotoxin A (EPA), CRM 197 42. The fusion protein of any one of paragraphs 37 to 41, comprising a carrier protein selected from the group consisting of: cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae protein D, and fragment(s) thereof, optionally wherein the Pseudomonas aeruginosa exotoxin A (EPA) carrier protein comprises the amino acid sequence of SEQ ID NO: 18, or fragment(s) thereof, and optionally wherein the CRM 197 The fusion protein, wherein the carrier protein comprises the amino acid sequence of SEQ ID NO: 24, or a fragment or fragments thereof.

[0151] 43. (i) the ComP glycosylation fragment is inserted between Ala489 and Arg490 for Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (SEQ ID NO: 19); (ii) the ComP glycosylation fragment is inserted between Glu548 and Gly549 for Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (SEQ ID NO: 20); (iii) the ComP glycosylation fragment is inserted between Ala122 and Gly123 for Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (SEQ ID NO: 21); or (iv) the ComP glycosylation fragment is inserted between Pseudomonas aeruginosa 43. The fusion protein of paragraph 42, wherein (i) the ComP glycosylation fragment is inserted between Thr355 and Gly356 of Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (sequence number 22); or (ii) the ComP glycosylation fragment is inserted between Lys20 and Asp21 of Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ (sequence number 23).

[0152] 44. (i) the ComP glycosylated fragment is a CRM 197 (ii) the ComP glycosylation fragment is inserted between Asn481 and Gly482 of the PDB molecule 4AE0 (SEQ ID NO: 25); or 197 (iii) the ComP glycosylation fragment is inserted between Asp392 and Gly393 of the PDB molecule 4AE0 (SEQ ID NO: 26); or 197 (iv) the ComP glycosylation fragment is inserted between Glu142 and Gly143 of the PDB molecule 4AE0 (SEQ ID NO: 27); or 197 or (v) the ComP glycosylation fragment is inserted between Asp129 and Gly130 of the PDB molecule 4AE0 (SEQ ID NO: 28); or 197 43. The fusion protein of paragraph 42, wherein the fusion protein is inserted between Asn69 and Glu70 of PDB molecule 4AE0 (SEQ ID NO: 29).

[0153] 45. The fusion protein of any one of paragraphs 37 to 44, comprising 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more, or 20 or more ComP glycosylation fragments, optionally not comprising more than 3, more than 5, more than 10, more than 15, more than 20, or more than 25 ComP glycosylation fragments.

[0154] 46. ​​The fusion protein according to any one of paragraphs 37 to 45, wherein the ComP glycosylation fragments are identical.

[0155] 47. The fusion protein of any one of paragraphs 37 to 45, wherein the ComP glycosylation fragments are different from each other, and optionally at least three, at least four, or at least five of the ComP glycosylation fragments are all different from each other, and optionally none of the ComP glycosylation fragments are the same.

[0156] 48. A method for in vivo conjugation of an oligosaccharide or polysaccharide to a receptor polypeptide, comprising covalently attaching the oligosaccharide or polysaccharide to the receptor polypeptide by PglS oligosaccharyltransferase (OTase), wherein the receptor polypeptide comprises a ComP glycosylated fragment described in any one of paragraphs 27 to 36.

[0157] 49. The PglS OTase is PglS 110264 (SEQ ID NO: 165), PglS ADP1 (SEQ ID NO: 166), PglS GFJ-2 (SEQ ID NO: 167), PglS 50v1 (SEQ ID NO: 168), PglS 4466 (SEQ ID NO: 169), PglS SFC (SEQ ID NO: 170), Pgl SP5312 (SEQ ID NO: 171), or PglS ANT_H59 49. The method of paragraph 48, wherein said amino acid sequence is (SEQ ID NO: 172).

[0158] 50. The oligosaccharide or polysaccharide is provided by the ComP glycosylation fragment with the sequence SEQ ID NO: 1 (ComP 110264 Serine corresponding to the serine residue at position 82 of

number

[0159] 51. The method of any one of paragraphs 48 to 50, wherein the in vivo conjugation is carried out within a host cell.

[0160] 52. The method of paragraph 51, wherein the host cell is a bacterial cell, optionally within Escherichia coli, optionally within the genus Klebsiella, and optionally the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

[0161] 53. The method of paragraph 51 or 52, comprising culturing a host cell containing (a) a gene cluster encoding proteins required for the synthesis of said oligosaccharide or polysaccharide, (b) PglS OTase, and (3) said receptor polypeptide.

[0162] 54. The method of any one of paragraphs 48 to 53, wherein production of the oligosaccharide or polysaccharide is enhanced by the transcriptional activator rmpA of K. pneumoniae (K. pneumoniae NTUH K-2044) or a homologue of the transcriptional activator rmpA of K. pneumoniae (K. pneumoniae NTUH K-2044).

[0163] 55. The method of any one of paragraphs 48 to 54 for producing a conjugate vaccine.

[0164] 56. A host cell comprising (a) a gene cluster encoding proteins required for the synthesis of said oligosaccharide or polysaccharide, (b) a PglS OTase, and (3) an acceptor polypeptide comprising a ComP glycosylation fragment according to any one of paragraphs 27 to 36.

[0165] 57. The host cell according to paragraph 56, wherein the receptor polypeptide is a fusion protein.

[0166] 58. A host cell according to paragraph 56 or 57, comprising a nucleic acid encoding the PglS OTase.

[0167] 59. A host cell according to any one of paragraphs 56 to 58, comprising a nucleic acid encoding the receptor polypeptide.

[0168] 60. An isolated nucleic acid encoding a ComP glycosylation fragment according to any one of paragraphs 27 to 36 and / or a fusion protein according to any one of paragraphs 37 to 47.

[0169] 61. The isolated nucleic acid of paragraph 60, which is a vector.

[0170] 62. A host cell comprising an isolated nucleic acid according to paragraph 60 or 61.

[0171] 63. A composition comprising a conjugate vaccine according to any one of paragraphs 21 to 26 or a fusion protein according to any one of paragraphs 37 to 47, and an adjuvant.

[0172] 64. A method for inducing a host immune response against a bacterial pathogen, comprising administering to a subject in need of said immune response an effective amount of a conjugate vaccine described in any one of paragraphs 21 to 26, a fusion protein described in any one of paragraphs 37 to 47, or a composition described in paragraph 63.

[0173] 65. The method of paragraph 64, wherein the immune response is an antibody response.

[0174] 66. The method of paragraph 64, wherein the immune response is selected from the group consisting of an innate response, an adaptive response, a humoral response, an antibody response, a cell-mediated response, a B cell response, a T cell response, upregulation or downregulation of cytokines, immune system crosstalk, and a combination of two or more of the immune responses.

[0175] 67. The method of paragraph 64, wherein the immune response is selected from the group consisting of an innate response, a humoral response, an antibody response, a T cell response, and a combination of two or more of the immune responses.

[0176] 68. A method for preventing or treating a bacterial disease and / or infectious disease in a subject, comprising administering to a subject in need thereof a conjugate vaccine described in any one of paragraphs 21 to 26, a fusion protein described in any one of paragraphs 37 to 47, or a composition described in paragraph 63.

[0177] 69. The method of paragraph 68, wherein the infection is a localized or systemic infection of the skin, soft tissue, blood, or organs, or is autoimmune in nature.

[0178] 70. The method of paragraph 69, wherein the disease is pneumonia.

[0179] 71. The method of paragraph 69, wherein the infection is a systemic infection and / or a blood infection.

[0180] 72. The method of any one of paragraphs 68 to 71, wherein the subject is a human.

[0181] 73. The method of any one of paragraphs 68-72, wherein the composition is administered via intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.

[0182] 74. A method for producing a pneumococcal conjugate vaccine against pneumococcal infection, the method comprising: (a) isolating a glycoconjugate according to any one of paragraphs 1 to 26 or a glycosylated fusion protein according to any one of paragraphs 37 to 47, and (b) combining the isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant.

[0183] 75. A glycoconjugate, glycosylated fusion protein, or conjugate vaccine according to any of the above paragraphs for use in inducing a host immune response against a bacterial pathogen and / or for preventing or treating a bacterial disease and / or infection in a subject.

[0184] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0185] array >Pseudomonas aeruginosa exotoxin A (EPA) PDB molecule 1IKQ

[0186] SEQ ID NO:18 AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFV RAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWN QVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARS QDLDAIWRGFYIAGDPALAYGYAQDQEPDARGIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK

[0187] >ssDsbA-EPA-iGT-SITE1-6xHis -DsbA signal sequence and hexahistidine tag are underlined, and the insertion site for the ComP glycosylation fragment is located between the underlined and bold amino acid residues.

[0188] [ka]

[0189] >ssDsbA-EPA-iGT-SITE2-6xHis -DsbA signal sequence and hexahistidine tag are underlined, and the insertion site for the ComP glycosylation fragment is located between the underlined and bold amino acid residues.

[0190] [ka]

[0191] >ssDsbA-EPA-iGT-SITE3-6xHis -The DsbA signal sequence and hexahistidine tag are underlined, and the insertion site for the ComP glycosylation fragment is located between the underlined and bold amino acid residues.

[0192] [ka]

[0193] >ssDsbA-EPA-iGT-SITE4-6xHis -The DsbA signal sequence and hexahistidine tag are underlined, and the insertion site for the ComP glycosylation fragment is located between the underlined and bold amino acid residues.

[0194] [ka]

[0195] >ssDsbA-EPA-iGT-SITE5-6xHis -The DsbA signal sequence and hexahistidine tag are underlined, and the insertion site for the ComP glycosylation fragment is located between the underlined and bold amino acid residues.

[0196] [ka]

[0197] >PDB molecule CRM197 4AE0

[0198] SEQ ID NO:24 GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASR VVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVT GTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTV EDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS

[0199] >ssFlgI-CRM197-iGT-SITE1-6xHis -FlgI signal sequence and hexahistidine tag are underlined, and the insertion site for the ComP glycosylation fragment is located between the underlined and bold amino acid residues.

[0200] [ka]

[0201] >ssFlgI-CRM197-iGT-SITE2-6xHis -FlgI signal sequence and hexahistidine tag are underlined, and the insertion site for the ComP glycosylation fragment is located between the underlined and bold amino acid residues.

[0202] [ka]

[0203] >ssFlgI-CRM197-iGT-SITE3-6xHis -FlgI signal sequence and hexahistidine tag are underlined, and the insertion site for the ComP glycosylation fragment is located between the underlined and bold amino acid residues.

[0204] [ka]

[0205] >ssFlgI-CRM197-iGT-SITE4-6xHis -FlgI signal sequence and hexahistidine tag are underlined, and the insertion site for the ComP glycosylation fragment is located between the underlined and bold amino acid residues.

[0206] [ka]

[0207] >ssFlgI-CRM197-iGT-SITE5-6xHis -FlgI signal sequence and hexahistidine tag are underlined, and the insertion site for the ComP glycosylation fragment is located between the underlined and bold amino acid residues.

[0208] [ka]

[0209] >PglS 110264

[0210] SEQ ID NO:165 MNFLISKLKFYVLFIGIVCFCLTFILPNTSYFSSSLFKEIVVVLGFLILLTNQILSLKEIILPKKAPLLFILFLFLFLFLFFQYLFKLIISFQDLFFNLIYISVFFLSIIFGLNSKKYNQIILIHWILFSLIFSA LISFLIGLNQKIRIIESPYLFGVSYNGRATANLGQPNQLSTLTLMAFFSLFYLKKYYKINKLFFYSIIISLIFCNVLTQSRSAWLSVILISIFFITKFPDKKNVLSVFCLNLVFWLSTILIPFFFNYFYPIGNSYT TLDRMKLSSSRFDIWPQLFLATFDKPFLGYGAGQVGLAQIESISNVSTRGEWFTYSHNIFLDFVIWYGWIVGSLVSFFIISLLIKISKSDLNRNETYLFVIILVFFFHCLLEYPYSYFYFLIPIGIISGFLLKLKS DDIFVLKKMYLCIVVFLSWLLFTLFTYQLIELGEKKESYSLQYLFKSSVKPIQSNLFILDGYSEKLDIEYLDYCYLIKNKDKEFFRRVAYRYPSTVSVSKYYSTQSDNLKNAENIVQAYQVISNRVYQPHIKKCNN

[0211] >PglS ADP1

[0212] SEQ ID NO:166 MNSIFKKIKNYTIVSGVFFLGSAFIIPNTSNLSSTLYKELIAVLGLLILLTVKSFDYKKILIPKNFYWFLFVIFIIFIQLIVGEIYFFQDFFFSISFLVILFLSFLLGFNERLNGDDLIVKKIAWIFIIVVQISFLI AINQKIEIVQNFFLFSSSYNGRSTANLGQPNQFSTLILITLFLLCYLREKNSLNNMVFNILSFCLIFANVMTQSRSAWISVILISLLYLLKFQKKIELRRVIFFNIVFWTLVYCVPLLFNLIFFQKNSYSTFDRLTM GSSRFEIWPQLLKAVFHKPFIGYGWGQTGVAQLETINKSSTKGEWFTYSHNLFLDLMLWNGFFIGLIISILILCFLIELYSSIKNKSDLFLFFCVVAFFVHCLLEYPFAYTYFLIPVGFLCGYISTQNIKNSISYFN LSKRKLTLFLGCCWLGYVAFWVEVLDISKKNEIYARQFLFSNHVKFYNIENYILDGFSKQLDFQYLDYCELKDKYQLLDFKKVAYRYPNASIVYKYYSISAEMKMDQKSANQIIRAYSVIKNQKIIKPKLKFCSIEY

[0213] >PglS GFJ-2

[0214] SEQ ID NO:167 MINILNKFKDCLIIIGLGCLCLAFFLPNTSNFSSSLFKEFFAVLGFLFILTVQFFFLKKIVVPSKLFILFILFILFIQYVFNLIINFQDLFFNLIYISIFFLSIIFGLNSKKYNNSVLIHWILFSLIFSALVS FLISLNQKIRIIESPYLFCVSYNGRATANLGQPNQLSTLTLMAFFSLFYLKKYYKINKLFFYTIIISLIFCNVLTQSRSAWLSVILISTFFITKFPDKKNVLSVFCLNLVFWLSTILIPFFFNYFYPIGNSYTTL DRMKLSSSRFDIWPQLFLATFDKPFLGYGAGQVGLAQIESISNASTRGEWFTYSHNIFLDFVIWYGWIVGSLVSFFIISLLIKISKSDLNRNKTYLFIIILVFFFHCLLEYPYSYFYFLIPIGIISGFLLKLKSD GVFVLKKIYLCIVIFLSWLLFALFTYQLIELDEKKESYSLQYLFKSSVKPIQSNLFILDGYSEKLDIEYLDYCYLIKNRDKEFFRRVAYRYPSTVSVSKYYSTQSDNLKNAENIVQAYQVISNRVYQPNIKKCNN

[0215] >PglS 50v1

[0216] SEQ ID NO:168 MRLYLSFLLLGLSYLSPNSSLLWPNSLQDFFAILSLILLLLTFNLNNFLINKYLFLVFLLLISIPVIQYNLKIIYFKQELFLSCLYITIFFSSIFLGSSIHNSQKVFIKFSIFFLVIGVLCVLIQIFQWIA VYSSIFINDLNSSRLSANIGQPNQLASLLSISLISCLILYKNKKIKVLIFSTCSVLIIFGIVLTQSRTSWLIFILIILFSYFKKNLKLTKYVTIFSTIFYGLLITYPFFYNSIHKKDISIIQRLNSDYSRL DIWQQMLFAIIERPWFGYGWNQTSVAQTEISLYHTTSIWIEYSHNLFLDFLIWNGIPLGIILITIIIFWFIYMYVNIKDLNSFMILIIISSFFIHCLLEFPFAYAYFIFPIGLYIGIINKRYLKYNYFNFN NWNYIFGLIIIFLLFFIVKDYIKITEKHKEYSLKYFSDNSILPNKLDIYLLDSLNVKEDIQYLDICYLIKIYNSEEIRNNFLRYPTNKSAVSLYYISLYNKNVSLETISFMKWKFQNLDLNTLKINKRCNTL

[0217] >PglS 4466

[0218] SEQ ID NO:169 MSFYCYKYLNLFGIFLMGVAYFSTITLFFSTTFYKEIFAVAGLLFFLTALCFQYKIVSTQLLLFNLALLLIPMIQYAFGIIFFLQDALLSTVYLCIFLCSILVGVNFKANHQTNILNIFLAMLVFVGCISVLMA FNQRFMWFNSYLLFSSSYGNRATANLAQPNQLSTLLIMSLFSLFYLYQAQKIKKIIMYGITFILLIGIVMTQSRSAWASCIVLSALYYYHQKQDIINVIKLNVVFIGLTLCIPFLLNVLTYSQASTAIDRLQG GSTRFKIWPQLLHAVMEQPWTGYGWGQVDVAQLSTMTPTSTKKELFTYSHNLFLDLLLWNGLVLGTLLSLLIIYILYRCYMNLQYKQDLLLFLGFMAFFVHSCLEYPYAYTYFLIPAGMFLGYVSYQQNIKEVLI QINKKLYVIFLILLCIIFGCFLIEVNHLNEKSDLYARQNLFHEKVDFNDQKFYFLDGYSTSLDFQTIPYCNLVQYYPLITFKQIAYRYPSALTIAKYWLFSQKQQQMVRDAEQLRQAYVLLTKSGQHTFNNKVCN

[0219] >PglS SFC

[0220] SEQ ID NO:170 MQLILIILGLSYLNPNSFLPWPNAMQDFCAMVALILLTATQFIKKNIQINKNTFYLFLFILSIPIIQFLFNILFFKQELFLSILYISIFFLSIIYGINQKEASNRIIKVSFFFVSVGIVCVFIQIIQWT NIYYSPFILESNYLRPSANLGQPNNLATLLFICLFSNLYIFKNKKINTSFYISINIFIIFGIALTQSRTSWIVFIALLILSHFKKELKLFKTIMINSILFFILVLITPYITLFYHGKGLTIIERINSDYS RLSIWKQIIIAITNKPLTGYGWNQTSVAQTQISLKYPIKVWLEYSHNMFLDILVWTGIPIGLLIITLINKWLFKTYQNIKNTNQLIIFFIIISFFIHCMFEFPFAYAYFLIPVGIYIGFLNKQDYNIITI NIFTILLFLLISTLLTIITIDYMVLSEKRNNYSTKYLFSKKISPLESNIKILDALDLHNDILFLNDCYILKNKSIKNIKHIFYRYPTNKNIVIYYRFSLYYKNSSKEVIEYMKLKYPNFDSNQSKYNMCN

[0221] >PglS P5312

[0222] SEQ ID NO:171 MPIFYFILGLSYLSPIFMQPWVSAFQDLCAIIAIILLMSIQSYRKNIEIDRRVLYVFGFIVCIPLVQYLFGILFFTQELVLSLIYISVFFLSIISGANFNRSYKNEEKLSFFFVFIGLSCVFIQLIQWSG LYHSALILDSSSRRPFANIGQPNNLATLLFIGFFSNILLFKNNRLKAKFYFLISAVLMTGIVLTQSRTSWLVFVSVLLLAFFKSKLELFSIMLKSSVLFFCLVLILPYITLFFHDQGLTVTERISSDSSR LYIWKQMLIAIMDKPWFGYGWNQTSVAQTSVTLKYPLDIWLEYSHNLFLDLIVWTGIPIGLSIIGIIIIWFLQTFKKINTLNQLLYFFIIAAFLIHCMLEYPFAYAYFLVPIGLYVGMLHQQLYETKNLK FKSLVITLVSILIITIIIISRDYFVLSDKRTIYTSESLFSEQVKPAFSKVLVLDALDVNNDILFLNRCYVLKKNTIENFKSNFYRYPTRMNLVMYYKSTIYYEKNSRDAERYMTAWYPDYKQNLSQYDICS

[0223] >ComP P5312

[0224] SEQ ID NO:7 MNAQKGFTLIELMIVIAIIGILAAIALPAYTDYTTRARVSEALTTASAMKATVSENIISKGGTSIDEDSACIGVATVGSDASAATKNVQKSVCDKGVITVTTTPDAKSVPLILTPSYSGDGVEWTCTTTADKKYVPAECR

[0225] >ComPΔ28 P5312

[0226] SEQ ID NO:15 MNAQKGFTLIELMIVIAIIGILAAIALPAYTDYTTRARVSEALTTASAMKATVSENIISKGGTSIDEDSACIGVATVGSDASAATKNVQKSVCDKGVITVTTTPDAKSVPLILTPSYSGDGVEWTCTTTADKKYVPAECR

[0227] >PglS ANT_H59

[0228] SEQ ID NO:172 MLIFYIMLGLSYLSPNIFLPWLNALQDLFAIFALIILVSKQSYRKDIEIDERVIYVFGLIALIPLVQYLFGLLFFTQELVLSLIYISAFFLSIISGINLTKSFKEIEKISFSFIFISLSCVLLQLIQWSN IYHSALLLDSSSRRPFANIGQPNNLATLLFIGFFSNILLFKNNKIKIYLYLLVSATLMTGIVLTQSRTSWLVFIAVLFITFLKKKLNLFSTMLKSSIAFLFLVLTLPYITLFFHDQGLTVIERISSDSSRL YIWKQMLIAIIDKPWFGYGWNQTSVAQTSVTLKYPLNIWLEYSHNLFLDIIVWTGIPIGISIITIIIIWFLQTFKKINTPNQLIYFLIITAFFIHCMLEFPFAYAYFLLPVGLYVGILHQQVYETKNSKVK GLVMTIVTVLIVAVIIISRDYFLFNNKRTIYASKNLFSQQIQPISSKILLLNALDINNDILFLDECYVLKNNKFKVLRNSFYRYPTNKNLITYYKSAIYNNQNTQYPEKYMQKEYSNFKSSPAIYNNCSKL

[0229] >ComP ANT_H59

[0230] SEQ ID NO:8 MNTAQKGFTLIELMIVIAIIGILAAIAIPAYSDYTARARVTEAVTTASSMKATVSENIISKGGTTIGAGSCAGVSLIGASNKTKNVLSSTCTDTTGVILVTTTADAKSVPLTLTPTYTGDAVTWKCTTTSDFTKYVPAECRPH

[0231] >ComPΔ29 ANT_H59

[0232]

Chem.

[0233]

Chem.

Chem.

Chem.

Chem.

[0234] References 1.Szymanski,C.M.,Yao,R.,Ewing,C.P.,Trust,T.J.& Guerry,P.Evidence for a system of general protein glycosylation in Campylobacter jejuni.Mol Microbiol 32,1022-1030,doi:10.1046 / j.1365-2958.1999.01415.x(1999). 2.Wacker,M.et al.N-linked glycosylation in Campylobacter jejuni and its functional transfer into E.coli.Science 298,1790-1793,doi:10.1126 / science.298.5599.1790(2002). 3.Nothaft,H.& Szymanski,C.M.Protein glycosylation in bacteria:sweeter than ever.Nat Rev Microbiol 8,765-778,doi:10.1038 / nrmicro2383(2010). 4.Schaffer,C.& Messner,P.Emerging facets of prokaryotic glycosylation.FEMS Microbiol Rev 41,49-91,doi:10.1093 / femsre / fuw036(2017). 5.Harding,C.M.& Feldman,M.F.Glycoengineering bioconjugate vaccines,therapeutics,and diagnostics in E.coli.Glycobiology 29,519-529,doi:10.1093 / glycob / cwz031(2019). 6.Valvano,M.A.Export of O-specific lipopolysaccharide.Front Biosci 8,s452-471,doi:10.2741 / 1079(2003). 7.Hug,I.& Feldman,M.F.Analogies and homologies in lipopolysaccharide and glycoprotein biosynthesis in bacteria.Glycobiology 21,138-151,doi:10.1093 / glycob / cwq148(2011). 8.Raetz,C.R.& Whitfield,C.Lipopolysaccharide endotoxins.Annu Rev Biochem 71,635-700,doi:10.1146 / annurev.biochem.71.110601.135414(2002). 9.Wacker,M.et al.Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems.Proc Natl Acad Sci U S A 103,7088-7093,doi:10.1073 / pnas.0509207103(2006). 10.Faridmoayer,A.et al.Extreme substrate promiscuity of the Neisseria oligosaccharyl transferase involved in protein O-glycosylation.J Biol Chem 283,34596-34604,doi:10.1074 / jbc.M807113200(2008). 11.Feldman,M.F.et al.Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli.Proc Natl Acad Sci U S A 102,3016-3021,doi:10.1073 / pnas.0500044102(2005). 12.Kowarik,M.et al.Definition of the bacterial N-glycosylation site consensus sequence.EMBO J 25,1957-1966,doi:10.1038 / sj.emboj.7601087(2006). 13.Faridmoayer,A.,Fentabil,M.A.,Mills,D.C.,Klassen,J.S.& Feldman,M.F.Functional characterization of bacterial oligosaccharyltransferases involved in O-linked protein glycosylation.J Bacteriol 189,8088-8098,doi:10.1128 / JB.01318-07(2007). 14.Vik,A.et al.Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae.Proc Natl Acad Sci U S A 106,4447-4452,doi:10.1073 / pnas.0809504106(2009). 15.Pan,C.et al.Biosynthesis of Conjugate Vaccines Using an O-Linked Glycosylation System.MBio 7,e00443-00416,doi:10.1128 / mBio.00443-16(2016). 16.Harding,C.M.et al.Acinetobacter strains carry two functional oligosaccharyltransferases,one devoted exclusively to type IV pilin,and the other one dedicated to O-glycosylation of multiple proteins.Mol Microbiol 96,1023-1041,doi:10.1111 / mmi.12986(2015). 17.Harding,C.M.et al.A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E.coli as a host.Nat Commun 10,891,doi:10.1038 / s41467-019-08869-9(2019). 18.Wacker,M.et al.Prevention of Staphylococcus aureus infections by glycoprotein vaccines synthesized in Escherichia coli.J Infect Dis 209,1551-1561,doi:10.1093 / infdis / jit800(2014). 19.Hatz,C.F.et al.Safety and immunogenicity of a candidate bioconjugate vaccine against Shigella dysenteriae type 1 administered to healthy adults:A single blind,partially randomized Phase I study.Vaccine 33,4594-4601,doi:10.1016 / j.vaccine.2015.06.102(2015). 20.Huttner,A.et al.Safety,immunogenicity,and preliminary clinical efficacy of a vaccine against extraintestinal pathogenic Escherichia coli in women with a history of recurrent urinary tract infection:a randomised,single-blind,placebo-controlled phase 1b trial.Lancet Infect Dis 17,528-537,doi:10.1016 / S1473-3099(17)30108-1(2017). 21.Sun,P.et al.Design and production of conjugate vaccines against S.Paratyphi A using an O-linked glycosylation system in vivo.NPJ Vaccines 3,4,doi:10.1038 / s41541-017-0037-1(2018). 22.van den Dobbelsteen,G.et al.Immunogenicity and safety of a tetravalent E.coli O-antigen bioconjugate vaccine in animal models.Vaccine 34,4152-4160,doi:10.1016 / j.vaccine.2016.06.067(2016). 23.Geno,K.A.et al.Pneumococcal Capsules and Their Types:Past,Present,and Future.Clin Microbiol Rev 28,871-899,doi:10.1128 / CMR.00024-15(2015). 24.Pan,Y.J.et al.Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp.Sci Rep 5,15573,doi:10.1038 / srep15573(2015). 25.Berti,F.et al.Structure of the type IX group B Streptococcus capsular polysaccharide and its evolutionary relationship with types V and VII.J Biol Chem 289,23437-23448,doi:10.1074 / jbc.M114.567974(2014). 26.Feldman,M.F.et al.A promising bioconjugate vaccine against hypervirulent Klebsiella pneumoniae.Proc Natl Acad Sci U S A 116,18655-18663,doi:10.1073 / pnas.1907833116(2019). 27.Ihssen,J.et al.Production of glycoprotein vaccines in Escherichia coli.Microb Cell Fact 9,61,doi:10.1186 / 1475-2859-9-61(2010). 28.Gerber,S.et al.Mechanism of bacterial oligosaccharyltransferase:in vitro quantification of sequon binding and catalysis.J Biol Chem 288,8849-8861,doi:10.1074 / jbc.M112.445940(2013). 29.Kay,E.J.,Yates,L.E.,Terra,V.S.,Cuccui,J.& Wren,B.W.Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli.Open Biol 6,150243,doi:10.1098 / rsob.150243(2016). 30.Garcia-Quintanilla,F.,Iwashkiw,J.A.,Price,N.L.,Stratilo,C.& Feldman,M.F.Production of a recombinant vaccine candidate against Burkholderia pseudomallei exploiting the bacterial N-glycosylation machinery.Front Microbiol 5,381,doi:10.3389 / fmicb.2014.00381(2014). 31.Berti,F.& Adamo,R.Antimicrobial glycoconjugate vaccines:an overview of classic and modern approaches for protein modification.Chem Soc Rev 47,9015-9025,doi:10.1039 / c8cs00495a(2018). 32.Goffin,P.,Dewerchin,M.,De Rop,P.,Blais,N.& Dehottay,P.High-yield production of recombinant CRM197,a non-toxic mutant of diphtheria toxin,in the periplasm of Escherichia coli.Biotechnol J 12,doi:10.1002 / biot.201700168(2017). 33.Wedekind,J.E.et al.Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity.J Mol Biol 314,823-837,doi:10.1006 / jmbi.2001.5195(2001).

Claims

1. 1. A glycoconjugate comprising an oligo- or polysaccharide covalently attached to a fusion protein, The fusion protein comprises a ComP protein (ComP) glycosylated fragment, comprising an isolated fragment of a ComP protein (ComP), The ComP glycosylated fragment is １１０２６４ and / or does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 71 of (SEQ ID NO: 1). １１０２６４ does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 93 of (SEQ ID NO:1); said ComP glycosylated fragment comprises a serine residue corresponding to the conserved serine residue at position 82 of ComP 110264 (SEQ ID NO: 1); the ComP glycosylation fragment is located internally in the fusion protein; The fusion protein comprises a ComP glycosylation fragment. １１０２６４ (SEQ ID NO:1), Optionally, the glycoconjugate is immunogenic. The glycoconjugate.

2. the ComP glycosylated fragment has a length of 5 to 22 amino acids; the fragment has at least two amino acid residues N-terminal to a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO:1; and 2. The glycoconjugate of claim 1, wherein the fragment has at least two amino acid residues C-terminal to a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO:

1.

3. The ComP protein is represented by SEQ ID NO: 9 (ComPΔ28 １１０２６４ ) or SEQ ID NO: 10 (ComPΔ28 ＡＤＰ１ or the ComP protein comprises an amino acid sequence at least 70% identical to SEQ ID NO: 9 (ComPΔ28 １１０２６４ ) or SEQ ID NO: 10 (ComPΔ28 ＡＤＰ１ 2. The glycoconjugate of claim 1 , comprising:

4. 2. The glycoconjugate of claim 1, wherein the ComP glycosylation fragment has the following amino acid consensus sequence: 【number】 however, X １ is V, T, A, or I; X ４ is Q, T, E, A, or S; X ５ is E, Q, T, or L; X ６ is I or V, X ７ is S, N, A, or G; X ８ is S or no amino acid, X ９ is G, D, or the absence of an amino acid, X １２ is N, S, or A; X １３ is A, S, or K; X １５ is T, S, or K; X １８ is A, E, Q, or L; X １９ is T, S, or K; X ２０ is A or S, X ２１ is T, Q, A, or V; or serine at position 11 of SEQ ID NO:17 [0010] a fragment thereof of at least 5 amino acids in length, comprising the residues Serine at position 11 of SEQ ID NO:17 [0025] and / or has at least two amino acid residues N-terminal to the serine at position 11 of SEQ ID NO:

17. [0030] the fragment having at least two amino acid residues on the C-terminal side of the residue, Alternatively, a variant or fragment thereof having one, two or three amino acid substitutions, additions and / or deletions of the amino acid consensus sequence of SEQ ID NO: 17, comprising the serine at position 11 of SEQ ID NO:

17. [0045] The residue is maintained at position 11 of SEQ ID NO:

17. [0050] and / or has at least two amino acid residues N-terminal to the serine at position 11 of SEQ ID NO:

17. [006] the variant or fragment thereof having at least two amino acid residues C-terminal to said residue, Optionally, the ComP glycosylation fragment is capable of being glycosylated when placed inside a fusion protein.

5. The fusion protein is selected from the group consisting of Pseudomonas aeruginosa exotoxin A (EPA), CRM １９７ cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae protein D, and fragments thereof; Optionally, the Pseudomonas aeruginosa exotoxin A (EPA) carrier protein comprises the amino acid sequence of SEQ ID NO: 18, or a fragment thereof; Optionally, the CRM １９７ The glycoconjugate of claim 1 , wherein the carrier protein comprises the amino acid sequence of SEQ ID NO: 24, or a fragment thereof.

6. (i) the ComP glycosylation fragment is inserted between Ala489 and Arg490 relative to Pseudomonas aeruginosa exotoxin A (EPA) PDB species 1IKQ (SEQ ID NO: 19); or (ii) the ComP glycosylation fragment is inserted between Glu548 and Gly549 for Pseudomonas aeruginosa exotoxin A (EPA) PDB species 1IKQ (SEQ ID NO: 20); or (iii) the ComP glycosylation fragment is inserted between Ala122 and Gly123 relative to Pseudomonas aeruginosa exotoxin A (EPA) PDB species 1IKQ (SEQ ID NO: 21); or (iv) the ComP glycosylation fragment is inserted between Thr355 and Gly356 of Pseudomonas aeruginosa exotoxin A (EPA) PDB species 1IKQ (SEQ ID NO: 22); or (v) The glycoconjugate of claim 5, wherein the ComP glycosylation fragment is inserted between Lys20 and Asp21 relative to Pseudomonas aeruginosa exotoxin A (EPA) PDB species 1IKQ (SEQ ID NO: 23).

7. (i) the ComP glycosylated fragment is a CRM １９７ or, (ii) the ComP glycosylated fragment is a CRM １９７ or, (iii) the ComP glycosylated fragment is a CRM １９７ or, (iv) the ComP glycosylated fragment is a CRM １９７ or (v) the ComP glycosylated fragment is a CRM １９７ 6. The glycoconjugate of claim 5 , wherein the glycoconjugate is inserted between Asn69 and Glu70 of PDB species 4AE0 of SEQ ID NO:

29.

8. The glycoconjugate of claim 1 , wherein the fusion protein comprises two or more ComP glycosylation fragments.

9. the oligosaccharide or polysaccharide is derived from a sugar produced by a bacterium of the genus Streptococcus; Optionally, the saccharide is a capsular polysaccharide of S. pneumoniae, S. agalactiae, or S. suis; Optionally, the saccharide is a serotype 8 capsular polysaccharide from S. pneumoniae; Optionally, the saccharide is a type Ia, Ib, II, III, IV, V, VI, VII, VIII, or X capsular polysaccharide from S. agalactiae.

10. the oligosaccharide or polysaccharide is derived from a sugar produced by a bacterium of the genus Klebsiella; Optionally, the saccharide is a capsular polysaccharide of K. pneumoniae, K. varricola, K. michinganensis, or K. oxytoca; Optionally, the saccharide is an O-antigen polysaccharide of K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

11. The glycoconjugate of claim 1 , wherein the oligosaccharide or polysaccharide comprises glucose at its reducing end.

12. 2. The glycoconjugate according to claim 1, wherein the ComP glycosylated fragment consists of the amino acid sequence of SEQ ID NO: 32-163, or 164; or a variant thereof having one, two or three amino acid substitutions, additions and / or deletions, the variant comprises a variant comprising a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO:1; The glycoconjugate, wherein the ComP glycosylation fragment is capable of being glycosylated when placed inside a fusion protein.

13. The ComP glycosylated fragment has the following amino acid sequence: 【Chemistry 2】 13. The glycoconjugate of claim 12, consisting of:

14. the glycoconjugate is a conjugate vaccine; Optionally, the conjugate vaccine is a vaccine against Streptococcus pneumoniae serotype 8.

15. A fusion protein comprising a ComP glycosylated fragment, the fusion protein comprising an isolated fragment of a ComP protein (ComP), said ComP glycosylated fragment does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 71 of ComP110264 (SEQ ID NO:1) and / or does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 93 of ComP110264 (SEQ ID NO:1); said ComP glycosylated fragment comprises a serine residue corresponding to the conserved serine residue at position 82 of ComP 110264 (SEQ ID NO: 1); and the ComP glycosylation fragment is located internally in the fusion protein; The fusion protein.

16. the oligosaccharide or polysaccharide is derived from a sugar produced by a bacterium of the genus Streptococcus; Optionally, the saccharide is a capsular polysaccharide of S. pneumoniae, S. agalactiae, or S. suis; Optionally, the saccharide is a serotype 8 capsular polysaccharide from S. pneumoniae; Optionally, the saccharide is a type Ia, Ib, II, III, IV, V, VI, VII, VIII, or X capsular polysaccharide from S. agalactiae.

17. the oligosaccharide or polysaccharide is derived from a sugar produced by a bacterium of the genus Klebsiella; Optionally, the saccharide is a capsular polysaccharide of K. pneumoniae, K. varricola, K. michinganensis, or K. oxytoca; Optionally, the saccharide is an O-antigen polysaccharide of K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

18. The fusion protein of claim 15 , wherein the oligosaccharide or polysaccharide contains glucose at its reducing end.

19. Pseudomonas aeruginosa exotoxin A (EPA), CRM １９７ 16. The fusion protein of claim 15, comprising a carrier protein selected from the group consisting of cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae protein D, and fragments thereof, Optionally, the Pseudomonas aeruginosa exotoxin A (EPA) carrier protein comprises the amino acid sequence of SEQ ID NO: 18, or a fragment thereof; Optionally, the CRM １９７ The fusion protein, wherein the carrier protein comprises the amino acid sequence of SEQ ID NO:24, or a fragment thereof.

20. (i) the ComP glycosylation fragment is inserted between Ala489 and Arg490 relative to Pseudomonas aeruginosa exotoxin A (EPA) PDB species 1IKQ (SEQ ID NO: 19); or (ii) the ComP glycosylation fragment is inserted between Glu548 and Gly549 for Pseudomonas aeruginosa exotoxin A (EPA) PDB species 1IKQ (SEQ ID NO: 20); or (iii) the ComP glycosylation fragment is inserted between Ala122 and Gly123 relative to Pseudomonas aeruginosa exotoxin A (EPA) PDB species 1IKQ (SEQ ID NO: 21); or (iv) the ComP glycosylation fragment is inserted between Thr355 and Gly356 of Pseudomonas aeruginosa exotoxin A (EPA) PDB species 1IKQ (SEQ ID NO: 22); or (v) the ComP glycosylation fragment is inserted between Lys20 and Asp21 of Pseudomonas aeruginosa exotoxin A (EPA) PDB species 1IKQ (SEQ ID NO: 23), the fusion protein of claim 19.

21. (i) the ComP glycosylated fragment is a CRM １９７ or, (ii) the ComP glycosylated fragment is a CRM １９７ or, (iii) the ComP glycosylated fragment is a CRM １９７ or, (iv) the ComP glycosylated fragment is a CRM １９７ or (v) the ComP glycosylated fragment is a CRM １９７ The fusion protein of claim 19, wherein the fusion protein is inserted between Asn69 and Glu70 of PDB species 4AE0 of (SEQ ID NO: 29).

22. The fusion protein of claim 15, comprising two or more ComP glycosylation fragments.

23. 1. A method of in vivo conjugation of an oligosaccharide or polysaccharide to a receptor polypeptide, the method comprising covalently attaching the oligosaccharide or polysaccharide to the receptor polypeptide in a host cell by PglS oligosaccharyltransferase (OTase), wherein the receptor polypeptide comprises a ComP glycosylated fragment, the ComP glycosylated fragment comprising an isolated fragment of a ComP protein (ComP); said ComP glycosylated fragment does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 71 of ComP110264 (SEQ ID NO:1) and / or does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 93 of ComP110264 (SEQ ID NO:1), and said ComP glycosylated fragment contains a serine residue corresponding to the conserved serine residue at position 82 of ComP110264 (SEQ ID NO:1); The method.

24. The PglS OTase is １１０２６４ (SEQ ID NO: 165), PglS ＡＤＰ１ (SEQ ID NO: 166), PglS ＧＦＪ－２ (SEQ ID NO: 167), PglS ５０ｖ１ (SEQ ID NO: 168), PglS ４４６６ (SEQ ID NO: 169), PglS ＳＦＣ (SEQ ID NO: 170), Pgl ＳＰ５３１２ (SEQ ID NO: 171), or PglS ＡＮＴ＿Ｈ５９ The method of claim 23, wherein the sequence is (SEQ ID NO: 172).

25. the host cell is a bacterial cell, Optionally, in Escherichia coli, Optionally, in a bacterium of the genus Klebsiella, 24. The method of claim 23, wherein optionally the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.

26. 24. The method of claim 23, comprising culturing a host cell containing (a) a gene cluster encoding proteins required for synthesis of the oligosaccharide or polysaccharide, (b) PglS OTase, and (3) the receptor polypeptide.

27. A host cell comprising (a) a gene cluster encoding proteins required for the synthesis of said oligosaccharides or polysaccharides, (b) a PglS OTase, and (3) a receptor polypeptide that is a fusion protein comprising an isolated fragment of a ComP protein (ComP), said receptor polypeptide being a fusion protein comprising a ComP glycosylation fragment, said ComP glycosylated fragment does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 71 of ComP110264 (SEQ ID NO:1) and / or does not contain a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 93 of ComP110264 (SEQ ID NO:1); and The host cell, wherein the ComP glycosylated fragment comprises a serine residue corresponding to the conserved serine residue at position 82 of ComP 110264 (SEQ ID NO:1).

28. 16. An isolated nucleic acid encoding the fusion protein of claim 15, optionally wherein the nucleic acid is a vector.

29. 29. A host cell comprising the isolated nucleic acid of claim 28.

30. A composition comprising the sugar conjugate of claim 14 and an adjuvant as a conjugate vaccine.

31. The composition of claim 30 for inducing a host immune response against a bacterial pathogen.

32. The composition of claim 30 for preventing or treating bacterial disease and / or infectious disease in a subject.

33. 1. A method for producing a pneumococcal conjugate vaccine against pneumococcal infection, the method comprising: (a) isolating the glycoconjugate of claim 1; and (b) combining said isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant.

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