O-linked glycosylation recognition motif

Abstract

Provided herein are glycoproteins containing O-linked glycosylation recognition motifs and methods for making them, eg, for use in the production of conjugate vaccines.

Description

Technical Field

[0001] Cross - Reference to Related Applications This PCT application claims the priority of U.S. Provisional Application No. 62 / 783,971, filed on December 21, 2018.

[0002] This application is related to U.S. Application No. 15 / 553,733, filed on August 25, 2017. This related application is the national stage application of PCT / CA2016 / 050208, filed on February 26, 2016, and claims the priority of U.S. Provisional Application No. 62 / 121,439, filed on February 24, 2015.

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

[0004] Statement of Government Funding This invention was made with government support under grant number R41 AI142928 - 01 awarded by the National Institute of Allergy and Infectious Diseases (NIAID). The government has certain rights in this invention.

Background Art

[0005] First, as a general protein glycosylation pathway in bacteria, the N-linked glycosylation system of Campylobacter jejuni was discovered 20 years ago (Szymanski CM, et al. (1999) Evidence for a system of general protein glycosylation in Campylobacter jejuni. Mol Microbiol 32(5):1022-1030). Since then, numerous prokaryotic glycosylation systems have been characterized, including O-linked glycosylation systems, for which there are no eukaryotic counterparts (Iwashkiw JA, et al. (2013) Pour some sugar on it: the expanding world of bacterial protein O-linked glycosylation. Mol Microbiol 89(1):14-28). Shortly after these discoveries, glycosylation pathways were recombinantly introduced into E. coli, creating the field of bacterial glycoengineering (Wacker M, et al. (2002) N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298(5599):1790-1793). Bacterial glycoengineering is a new biotechnology tool that uses E. coli or other Gram-negative organisms as hosts and prokaryotic glycosylation systems for the production of recombinantly glycosylated proteins. Currently, glycoengineering utilizes two major approaches for recombinantly glycosylating proteins, the oligosaccharyltransferase (OTase)-dependent and OTase-independent approaches, both of which can generate N-linked or O-linked linkages.

[0006] Protein glycosylation, i.e., the covalent attachment of carbohydrates to proteins, is a ubiquitous post-translational modification. In most cases, protein glycosylation is specified as either N-linked, in which the glycan is attached to an asparagine residue, or O-linked, in which the glycan is attached to a serine or threonine residue. Although the importance of eukaryotic glycosylation has been and continues to be the subject of intensive research, prokaryotic glycosylation has only recently begun to attract the attention of the scientific community with the discovery of a general N-linked protein glycosylation system in the ε-proteobacterium Campylobacter jejuni (Szymanski CM, et al. (1999) Evidence for a system of general protein glycosylation in Campylobacter jejuni. Mol Microbiol 32(5):1022-1030). Because it was first discovered in C. jejuni, prokaryotic glycosylation systems have been described across a wide range of Gram-negative and Gram-positive bacteria and have been shown to contribute not only to pathogenesis but also to normal bacterial physiology (Iwashkiw JA, et al. (2013) Pour some sugar on it: the expanding world of bacterial protein O-linked glycosylation. Mol Microbiol 89(1):14-28; Nothaft H & Szymanski CM (2010) Protein glycosylation in bacteria: sweeter than ever. Nat Rev Microbiol 8(11):765-778); Schaffer C & Messner P (2017) Emerging facets of prokaryotic glycosylation. FEMS Microbiol Rev 41(1):49-91).Given the simple and straightforward nature of prokaryotic genes, it was only a matter of time before protein glycosylation systems were manipulated and utilized to produce designer glycoproteins in a process called "bacterial glycan engineering."

[0007] Bacteria have developed an N-linked OTase pathway that is nearly identical to eukaryotic glycosylation, but they also employ a prokaryote-specific O-linked OTase system. OTase-independent glycosylation occurs in the cytoplasm and relies on glycosyltransferases to transfer monosaccharides from nucleotide-activated precursors for the sequential assembly of glycoproteins. Both OTase-dependent and -independent pathways are utilized for the bioconjugation of carbohydrates and proteins.

[0008] Bacterial surface polysaccharides are part of the first and most abundant microbial components encountered by the immune system during infection (Comstock LE & Kasper DL (2006) Bacterial glycans: key mediators of diverse host immune responses. Cell 126(5):847-850). These polysaccharides usually take the form of O antigens attached to the capsule or lipid A and serve a number of purposes, including protecting the microorganism from external threats and immune clearance. Given their abundance on invading organisms and their biochemical differences from eukaryotic carbohydrates, some microbial surface polysaccharides have been used as antigens for vaccine development. However, when polysaccharides are used alone in vaccine formulations, they usually act as T cell-independent antigens and thus do not stimulate immunoglobulin class switching and long-term B cell memory. Moreover, polysaccharide vaccines alone do not induce protection in vulnerable populations such as infants under 2 years of age. This poor immune response may be overcome by covalently attaching the polysaccharide to a protein carrier in a process known as conjugation (De Gregorio E & Rappuoli R (2014) From empiricism to rational design: a personal perspective of the evolution of vaccine development. Nat Rev Immunol 14(7):505-514).

[0009] Conventionally, conjugate polysaccharide vaccines are synthesized using a semi-synthetic approach. In this approach, the polysaccharide is extracted from the target microorganism, purified, chemically modified, and covalently linked to a carrier protein. This approach has led to the commercial approval of multiple conjugate polysaccharide vaccines for preventing colonization and infections caused by Haemophilus influenzae type B, as well as multiple serotypes of Streptococcus pneumoniae and Neisseria meningitidis. For a detailed review on the production of semi-synthetic or synthetic conjugate polysaccharide vaccines, see the following excellent review article (Berti F & Adamo R (2018) Antimicrobial glycoconjugate vaccines: an overview of classic and modern approaches for protein modification. Chem Soc Rev 47(24):9015-9025). Although chemically produced conjugate vaccines have shown great commercial success (the conjugate polysaccharide vaccine PREVNAR 13 was the best-selling Pfizer product from 2015 to 2018, with sales exceeding $24 billion), the manufacturing process is not without drawbacks; these include batch-to-batch variability, non-uniform product formation, large-scale production of pathogenic organisms, and high manufacturing costs (Frasch CE (2009) Preparation of bacterial polysaccharide-protein conjugates: analytical and manufacturing challenges. Vaccine 27(46):6468-6470).

[0010] Over the past 20 years, alternative strategies for manufacturing conjugate vaccines have emerged. These techniques are broad in approach, and some lead to vaccines that are closer to commercially approved vaccines than others. Specifically, the emergence of in vivo bacterial conjugation for manufacturing conjugate vaccines has led to some of the most clinically advanced products currently. The in vivo conjugation of polysaccharides and proteins for conjugate vaccine manufacture, commonly referred to as bioconjugation or protein-glycan coupling technology (PGCT), relies on OTase (Frasch CE (2009) Preparation of bacterial polysaccharide-protein conjugates: analytical and manufacturing challenges. Vaccine 27(46):6468-6470). Bioconjugation is generally considered to represent the simplification of the conjugate vaccine generation and manufacturing process (Rappuoli R, De Gregorio E, & Costantino P (2019) On the mechanisms of conjugate vaccines. Proc Natl Acad Sci U S A 116(1):14-16).

[0011] Both N-linked OTase and O-linked OTase have been used for the biological conjugation of polysaccharides and carrier proteins for conjugate vaccine manufacture. Regardless of which OTase is used, biological conjugation in any Gram-negative bacterium relies on three components: the locus (singular or plural) encoding the polysaccharide biosynthesis protein, the carrier protein to be glycosylated, and the OTase that transfers the desired carbohydrate to the carrier protein. These three components, although necessary, do not have to be on three separate plasmids.

[0012] Recently, a class 3 O-linked OTase has been used in the production of bioconjugate vaccines (Harding CM, et al. (2019) A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host. Nat Commun 10(1):891). This class 3 OTase, named PglS, glycosylates the pilin-like protein, ComP, much like the only other known O-linked OTases, PilO and PglL (Schulz BL, et al. (2013) Identification of bacterial protein O-oligosaccharyltransferases and their glycoprotein substrates. PLoS One 8(5):e62768). Follow-up studies have demonstrated that PglS is in fact a pilin-specific OTase and is likely to glycosylate only ComP, as no other glycoproteins were identified using a comprehensive glycoprotein screening approach (Harding CM, et al. (2015) 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(5):1023-1041). Initially characterized as a PglL homolog from the ADP1 strain of the environmental bacterium Acinetobacter baylyi, PglS is in fact phylogenetically distinct from the PglL protein.Strains of the genus Acinetobacter that encode the PglS protein also encode the PglL protein, which has been shown to act as a general OTase that glycosylates at least seven membrane-bound proteins in a manner similar to that of species of the genus Neisseria (Iwashkiw JA, et al. (2012) Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation. PLoS Pathog 8(6):e1002758). Some strains of the genus Acinetobacter also encode the PilO OTase, and thus the genus Acinetobacter is the only genus known to carry the genes for all three O-OTase families (PilO, PglL, and PglS) (Harding CM, et al. (2015) 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(5):1023-1041;Iwashkiw JA, et al. (2012) Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation. PLoS Pathog 8(6):e1002758).

[0013] Apart from the phylogenetic differences, PglS glycosylates its cognate pilin at a unique serine site that is not conserved when compared to the glycosylation sites of PilE (the pilin target of PglL) or PilA (the pilin target of PilO) and is not contained within the LCR (Harding CM, et al. (2019) A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host. Nat Commun 10(1):891). However, the most striking difference lies in the polysaccharide substrate that PglS transfers. PglS is the only OT-ase known to be able to transfer polysaccharides with glucose at the reducing end, whether N-linked or O-linked. Many pathogens produce capsules containing polysaccharides with glucose at the reducing end, such as Streptococcus pneumoniae (Geno KA, et al. (2015) Pneumococcal Capsules and Their Types: Past, Present, and Future. Clin Microbiol Rev 28(3):871-899), Streptococcus group B (Carboni F, et al. (2017) Structure of a protective epitope of group B Streptococcus type III capsular polysaccharide. Proc Natl Acad Sci U S A 114(19):5017-5022), and Klebsiella pneumoniae (Pan YJ, et al. (2015) Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp. Sci Rep 5:15573), and thus may be targets for PglS-dependent bioconjugate vaccine development.Indeed, PglS has been used in the production of a multivalent pneumococcal conjugate vaccine against serotypes 8, 9V, and 14 (all having glucose at the reducing end) using the native acceptor ComP as a carrier protein. Also, a ComP fragment lacking the first 28 amino acids was able to function as a glycotag when translationally fused to the C-terminus of Pseudomonas aeruginosa exotoxin A, paving the way for incorporating more conventional vaccine carriers into the PglS bioconjugation system (Harding CM, et al. (2019) A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host. Nat Commun 10(1):891).

[0014] Summary of the Invention The present disclosure provides a bioconjugate comprising an oligosaccharide or polysaccharide covalently bound to a fusion protein, provided that the fusion protein comprises a ComP protein (ComP) glycosylation tag, the ComP glycosylation tag comprising both a cysteine residue corresponding to the conserved cysteine residue at position 71 of SEQ ID NO: 2 (ComP110264: ENV58402.1) and a cysteine residue corresponding to the conserved cysteine residue at position 93 of SEQ ID NO: 2, or both a cysteine residue corresponding to the conserved cysteine residue at position 75 of SEQ ID NO: 1 (ComPADP1: AAC45886.1) and a cysteine residue corresponding to the conserved cysteine residue at position 95 of SEQ ID NO: 1, and that the fusion protein is glycosylated with an oligosaccharide or polysaccharide of the ComP glycosylation tag at a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 2 or position 84 of SEQ ID NO: 1. In certain embodiments, the ComP glycosylation tag does not include a methionine residue corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1). In certain embodiments, the fusion protein of the bioconjugate does not include a methionine residue corresponding to or nearly corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1) with respect to the ComP glycosylation tag. In certain embodiments, the bioconjugate is a conjugate vaccine.

[0015] In certain embodiments of the present disclosure, the ComP glycosylation tag comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 32 [C1], SEQ ID NO: 33 [D1], SEQ ID NO: 34 [E1], SEQ ID NO: 41 [E2], SEQ ID NO: 42 [F2], SEQ ID NO: 43 [G2], SEQ ID NO: 44 [H2], SEQ ID NO: 45 [A3], SEQ ID NO: 46 [B3], SEQ ID NO: 47 [C3], SEQ ID NO: 55 [D4], SEQ ID NO: 56 [E4], SEQ ID NO: 57 [F4], SEQ ID NO: 58 [G4], SEQ ID NO: 59 [A5], SEQ ID NO: 60 [B5], SEQ ID NO: 61 [D5], SEQ ID NO: 62 [E5], SEQ ID NO: 63 [F5], SEQ ID NO: 72 [H6], SEQ ID NO: 73 [B7], SEQ ID NO: 74 [C7], SEQ ID NO: 75 [D7], SEQ ID NO: 76 [E7], SEQ ID NO: 77 [F7], SEQ ID NO: 78 [A8], SEQ ID NO: 79 [B8], SEQ ID NO: 92 [A10], SEQ ID NO: 93 [B10], SEQ ID NO: 94 [C10], SEQ ID NO: 95 [D10], SEQ ID NO: 96 [F10], SEQ ID NO: 97 [G10], SEQ ID NO: 98 [H10], SEQ ID NO: 99 [A11], SEQ ID NO: 100 [B11], and SEQ ID NO: 101 [C11], or a variant thereof having one, two, three, four, five, six, or seven amino acid substitutions, additions, and / or deletions, provided that the variant maintains both the cysteine residue corresponding to the conserved cysteine residue at position 75 of SEQ ID NO: 1 (ComPADP1: AAC45886.1) and the cysteine residue corresponding to the conserved cysteine residue at position 95 of SEQ ID NO: 1, and further provided that the variant maintains the serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 1.

[0016] The present disclosure provides a ComP glycosylation tag comprising an isolated fragment of the ComP protein, provided that the fragment comprises a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 1 (ComPADP1: AAC45886.1), and both a cysteine residue corresponding to the conserved cysteine residue at position 71 of SEQ ID NO: 2 (ComP110264: ENV58402.1) and a cysteine residue corresponding to the conserved cysteine residue at position 93 of SEQ ID NO: 2, or both a cysteine residue corresponding to the conserved cysteine residue at position 75 of SEQ ID NO: 1 (ComPADP1: AAC45886.1) and a cysteine residue corresponding to the conserved cysteine residue at position 95 of SEQ ID NO: 1. In certain embodiments, the ComP glycosylation tag of claim 41, provided that the ComP glycosylation tag does not comprise a methionine residue corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP110264: ENV58402.1). In certain embodiments, the ComP glycosylation tag of claim 42, provided that the amino acid sequence of the ComP glycosylation tag does not continue beyond the amino acid residue corresponding to position 103 of SEQ ID NO: 2 (ComP110264: ENV58402.1) in the C-terminal direction.

[0017] Provided herein is a fusion protein comprising a ComP glycosylation tag of the present disclosure.

[0018] Also provided herein is an in vivo conjugation method of an oligosaccharide or polysaccharide with an acceptor polypeptide, the method comprising covalently bonding the oligosaccharide or polysaccharide with the acceptor polypeptide using PglS oligosaccharyltransferase (OTase), provided that the acceptor polypeptide comprises a ComP glycosylation tag of the present disclosure, and optionally, the ComP glycosylation tag is linked to a heterologous carrier protein.

[0019] Also provided herein is a host cell comprising (a) a gene cluster encoding a protein necessary for the synthesis of an oligosaccharide or polysaccharide, (b) an OT-ase of PglS, and (3) an acceptor polypeptide comprising the ComP glycosylation tag of the present disclosure.

[0020] Also provided herein is an isolated nucleic acid encoding a ComP glycosylation tag and / or a fusion protein of the present disclosure, and a host cell comprising the isolated nucleic acid.

[0021] Also provided herein is a composition comprising a conjugate vaccine or fusion protein of the present disclosure and an adjuvant.

[0022] A method of inducing a host immune response against a pathogenic microorganism, the method comprising administering to a subject in need of an immune response an effective amount of a conjugate vaccine, fusion protein, or composition of the present disclosure.

[0023] Also provided herein is a method for preventing or treating a bacterial disease and / or an infectious disease in a subject, the method comprising administering to a subject in need of prevention or treatment an effective amount of a conjugate vaccine, fusion protein, or composition of the present disclosure.

[0024] Also provided herein is a method for producing a pneumococcal conjugate vaccine against pneumococcal infection, the method comprising isolating a bioconjugate or glycosylated fusion protein of the present disclosure and combining the isolated bioconjugate or glycosylated fusion protein with an adjuvant. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

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Mode for Carrying Out the Invention

[0026] To the extent necessary to provide explanatory support, the subject matter and / or documents of the appended claims are hereby incorporated by reference in their entirety into this specification.

[0027] As will be apparent to all readers of this written description, the exemplary aspects and embodiments described and claimed herein are appropriately practicable whether or not any of the recited features, elements, or steps are present, regardless of whether or not they are specifically disclosed herein.

[0028] Definitions It should be noted that the entity of the term "a" or "an" refers to one or more of that entity. For example, "polysaccharide" is understood to represent one or more polysaccharides. In such cases, the terms "a" (or "an"), "one or more", and "at least one" may be used interchangeably herein.

[0029] Furthermore, "and / or" as used herein should be taken as specific disclosure of each of the particular features or components, with or without the others. Thus, when used in phrases such as "A and / or B" herein, the term "and / or" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, when used in phrases such as "A, B, and / or C", the term "and / or" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0030] When an aspect is described in this specification using the word "comprising" or "comprises", it should always be understood that similar aspects described using terms such as "consisting of", "consists of", "consisting essentially of" and / or "consists essentially of" are also provided.

[0031] Unless otherwise defined, technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the relevant technical fields of this disclosure.

[0032] Numeric ranges include the numbers defining the range. Unless explicitly identified by phrases such as "and any range therebetween" or, for example, when a listing of values such as 1, 2, 3 or 4 is described, the disclosure specifically includes any range between the values, such as 1 - 3, 1 - 4, 2 - 4, etc., unless otherwise specified.

[0033] The headings provided in this specification are for reference convenience only and are not limitations of the various aspects or aspects of the disclosure that can be obtained by referring to the entire specification.

[0034] As used herein, the term "non - naturally occurring" substance, composition, entity, and / or any combination of substances, compositions, or entities, or any grammatical variant thereof, is a conditional term that explicitly excludes substances, compositions, entities, and / or any combination of substances, compositions, or entities that are well understood by one of ordinary skill in the art as "naturally occurring" or that may at any time be determined or interpreted as "naturally occurring" by a judge or administrative or judicial body, but only to the extent of such exclusion.

[0035] As used herein, the term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptides", and this term refers 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 or chains of two or more amino acids and does not refer to a product of a specific length. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" can be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modification of polypeptides, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting groups / blocking groups, proteolytic cleavage, or modification by non-standard amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant techniques, but is not necessarily translated from a specified nucleic acid sequence. It can be produced by any method including chemical synthesis.

[0036] As used herein, the term "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 that are joined, for example, by disulfide bonds, hydrogen bonds, or hydrophobic interactions to produce a multimeric protein.

[0037] An "isolated" polypeptide or fragment, variant, or derivative thereof is intended to mean a polypeptide that is not in its natural environment. A specific level of purification is not required. For example, an isolated polypeptide can be removed from its native or natural environment. By any suitable technique, recombinant-produced polypeptides and proteins expressed in host cells, as well as separated, fractionated, or partially or substantially purified native polypeptides or recombinant polypeptides, are considered to be isolated for the purposes of the present invention. Isolated polypeptides or fragments, variants, or derivatives thereof can associate, bind, etc. with cofactors. Similarly, purified or purified and isolated polypeptides or fragments, variants, or derivatives thereof can associate, bind, etc. with cofactors.

[0038] As used herein, the term "non-naturally occurring" polypeptide, or any grammatical variation thereof, clearly excludes forms of polypeptides that are well understood by those of skill in the art to be "naturally occurring" or that may at any time be determined or construed to be "naturally occurring" by a judge or administrative or judicial body, but is a term that excludes only on that condition.

[0039] Disclosed herein are certain binding molecules, or antigen-binding fragments, variants, or derivatives thereof. Unless specifically indicated as full-length antibodies such as naturally occurring antibodies, the term "binding molecule" includes full-length antibodies as well as antigen-binding fragments, variants, analogs, or derivatives of such antibodies, e.g., modified antibody molecules or fragments that bind antigens in a manner similar to naturally occurring antibodies or immunoglobulin molecules or antibody molecules.

[0040] 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 can include one of a plurality of "binding domains". As used herein, a "binding domain" is a two - or three - dimensional polypeptide structure that can specifically bind to a given antigenic determinant, i.e., an epitope. By way of example and not limitation of a binding molecule, there are antibodies or fragments thereof that include a binding domain that specifically binds to an antigenic determinant or epitope. Another example of a binding molecule is a bispecific antibody that includes a first binding domain that binds to a first epitope and a second binding domain that binds to a second epitope.

[0041] The terms "antibody" and "immunoglobulin" can be used interchangeably herein. An antibody (or fragment, variant, or derivative thereof as disclosed herein) includes at least the variable domain of the heavy chain and at least the variable domains of the heavy and light chains. The basic immunoglobulin structure of the vertebrate system is relatively well - understood. See, for example, Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988).

[0042] Binding molecules, such as antibodies, or antigen-binding fragments, variants, or derivatives thereof, include polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, or chimeric antibodies, single-chain antibodies, epitope-binding fragments, such as Fab, Fab’, and F(ab’)2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, disulfide-bonded Fv (sdFv), fragments containing either a VL or VH domain, fragments generated by a Fab expression library, but are not limited thereto. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019. The immunoglobulin or antibody molecules encompassed by the present disclosure can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or isotype of immunoglobulin molecule.

[0043] "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 some complementarity between the antigen-binding domain and the epitope. According to this definition, a binding molecule "specifically binds" to an epitope when it binds to that epitope more readily than it binds randomly to an unrelated epitope via its antigen-binding domain. The term "specifically" is used herein to specify the relative affinity of a particular binding molecule for a particular epitope. For example, binding molecule "A" can be considered to have a higher specificity for a given epitope than binding molecule "B", or it can be said that binding molecule "A" binds to epitope "C" with a higher specificity than it binds to related epitope "D".

[0044] As used herein, the term "bispecific antibody" refers to an antibody having binding sites for two different antigens within a single antibody molecule. It will be understood that in addition to the classical antibody structure, other molecules can be constructed to have two binding specificities. Furthermore, it will be understood that antigen binding by a bispecific antibody can be simultaneous or sequential. Trioma and hybrid hybridoma are two examples of cell lines capable of secreting bispecific antibodies. Bispecific antibodies can also be constructed by recombinant techniques. (Strohlein and Heiss, Future Oncol. 6:1387-94 (2010); Mabry and Snavely, IDrugs. 13:543-9(2010)). A bispecific antibody can also be a diabody.

[0045] The term "polynucleotide" is intended to encompass single nucleic acids and plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). Polynucleotides can include conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds as found in peptide nucleic acid (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments present in a polynucleotide, e.g., a DNA or RNA fragment. An "isolated" nucleic acid or polynucleotide is intended to mean a nucleic acid molecule, DNA or RNA, recovered 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 in solution. Isolated RNA molecules include RNA transcripts of polynucleotides in vivo or in vitro. Isolated polynucleotides or nucleic acids further include such molecules generated synthetically. Further, a polynucleotide or nucleic acid can be, or can include, regulatory elements such as a promoter, ribosome binding site, or transcription terminator.

[0046] As used herein, the term "non-naturally occurring" polynucleotide, or any grammatical variation thereof, clearly excludes a form of polynucleotide that is well understood by one of ordinary skill in the art as "naturally occurring", or that may at any time be determined or construed as "naturally occurring" by a judge or administrative or judicial body, but is a conditional term that only excludes.

[0047] In certain embodiments, the polynucleotide or nucleic acid is DNA. In other embodiments, the polynucleotide can be RNA.

[0048] A "vector" is a nucleic acid molecule that is introduced into a host cell, thereby generating a transformed host cell. A vector can contain nucleic acid sequences that enable replication within 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.

[0049] A "transformed" cell, or "host" cell, is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformation encompasses techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection by viral vectors, transformation by plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. A transformed cell or host cell can be a bacterial cell or a eukaryotic cell.

[0050] The term "expression" as used herein refers to the process by which a gene produces a biochemical substance, such as a polypeptide. This process includes any expression of the functional presence of a gene within a cell, including, but not limited to, gene knockdown, as well as both transient and stable expression. This includes, but is not limited to, transcription of the gene into messenger RNA (mRNA), and translation of such mRNA into polypeptide(s). When the final desired product is a biochemical substance, expression includes the production of that biochemical substance and any precursors. Expression of a gene produces a "gene product". As used herein, a gene product can be either a nucleic acid, such as messenger RNA produced by transcription of the gene, or a polypeptide translated from the transcript. Gene products described herein further include nucleic acids having post-transcriptional modifications, such as polyadenylation, or polypeptides having post-translational modifications, such as methylation, glycosylation, lipid addition, association with other protein subunits, proteolytic cleavage, and the like.

[0051] As used herein, the terms "treating," "treatment," or "therapy of" (e.g., in the phrase "treating a subject") mean, for example, reducing the likelihood of a disease pathology, reducing the occurrence of disease symptoms, to the extent that the subject has a longer survival period or reduced discomfort. For example, treatment can indicate that a therapy has the ability to reduce the symptoms, signs, or causes of a disease when administered to a subject. Treatment also indicates reducing or decreasing at least one clinical symptom, and / or inhibiting or delaying the progression of symptoms, and / or preventing or delaying the onset of a disease or disorder.

[0052] The term "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, livestock animals, sport animals, and zoo animals, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, etc.

[0053] The term "pharmaceutical composition" refers to a formulation that enables 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 a composition can be sterile.

[0054] An "effective amount" of an antibody, as disclosed herein, is an amount sufficient to perform the specifically stated purpose. The "effective amount" can be determined empirically and in a conventional manner with respect to the stated purpose.

[0055] Summary. Conjugate vaccines consisting of polysaccharides linked to proteins are life-saving prophylactic agents. Traditionally, conjugate vaccines have been manufactured using chemical methodologies. However, in vivo bacterial conjugation has emerged as an alternative means of production. In vivo conjugation (bioconjugation) relies on oligosaccharyltransferase to bind polysaccharides to proteins. Currently, the oligosaccharyltransferases used in bioconjugation are not suitable for the production of conjugate vaccines when the polysaccharide contains glucose at the reducing end. Since approximately 75% of the Streptococcus pneumoniae capsule contains glucose as the reducing end sugar, this limitation has a significant impact. Disclosed herein is the use of O-linked oligosaccharide transferase to produce the first multivalent pneumococcal bioconjugate vaccine having polysaccharides that contain glucose at their reducing ends. The pneumococcal bioconjugates are immunogenic, protective, and are rapidly produced by recombinant techniques. Specific embodiments disclosed herein provide the engineering, characterization, and immunological responses of multivalent pneumococcal bioconjugate vaccines using the native acceptor protein ComP as a vaccine carrier, as well as monovalent pneumococcal bioconjugate vaccines using conventional vaccine carriers, e.g., those containing the Pseudomonas aeruginosa exotoxin A protein in certain embodiments. This establishes a platform for overcoming the limitations of other conjugate enzymes and enables the development of bioconjugate vaccines against many important human and animal pathogens.

[0056] Even though pneumococcal conjugate vaccines have been introduced and implemented over the past 20 years, approximately 15 million deaths per year are caused by S. pneumoniae. This is partly due to over 90 serotypes of S. pneumoniae and the complex manufacturing methods required for the synthesis of pneumococcal conjugate vaccines. These factors combined have hindered the global distribution and development of more extensive and protective variations of the vaccine. To facilitate development and reduce manufacturing costs, disclosed herein is a platform for developing conjugate vaccines, such as pneumococcal conjugate vaccines, using in vivo conjugation. This streamlined process has the potential to complement existing manufacturing pipelines or completely avoid dependence on chemical conjugation methodologies, enabling the production of more comprehensive conjugate vaccines.

[0057] Conventional chemical conjugate vaccine synthesis is considered complex, costly, and cumbersome (Frasch, C.E. Vaccine 27, 6468 - 6470 (2009)). However, in vivo conjugation has advanced significantly as a viable alternative to biosynthesis (Huttner, A. et al. Lancet Infect Dis 17, 528 - 537 (2017)). These advancements are most notably highlighted by the success of GlycoVaxyn (now LimmaTech Biologics AG, an independent company with direct ties to GlaxoSmithKline). This is a clinical-stage biopharmaceutical company that uses multiple bioconjugate vaccines in various phases of clinical trials, one of which (Flexyn2a) completed a phase 2b challenge study. GlycoVaxyn is at the forefront of the in vivo conjugation revolution, but its ability to glycosylate carrier / acceptor proteins with polysaccharides containing glucose (Glc) as the reducing-end sugar has been remarkable and, predictably, has hindered the development of pneumococcal bioconjugate vaccines.

[0058] The oligosaccharyltransferase previously called PglL by Schulz et al. (PMID23658772) and PglL by Harding et al. 2015 (PMID 26727908) has recently been characterized as a functional OT-ase (Schulz, B.L. 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 OT-ase and glycosylates multiple periplasmic and outer membrane proteins (Harding, C.M. et al. Mol Microbiol 96, 1023-1041 (2015)). Indeed, the genome of A. baylyi ADP1 encodes two OT-ases, PglL-like orthologs (UniProtKB / Swiss-Prot:Q6FFS6.1), which act as general OT-ases and PglS (UniProtKB / Swiss-Prot: Q6F7F9.1) and glycosylate the single protein ComP (Harding, C.M. et al. Mol Microbiol 96, 1023-1041 (2015)). ComP

[0059] ​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, P.M. et al. Mol Microbiol 49, 833-847 (2003)), respectively. TfpO and PglL also glycosylate cognate pilins at serine residues, but the glycosylation sites differ between the respective systems. TfpO glycosylates cognate pilins at a C-terminal serine residue that is not present in ComP (Comer, J.E., Marshall, M.A., Blanch, V.J., Deal, C.D. & 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 relative to PilE from N. meningiditis. However, comprehensive glycopeptide analysis has revealed that this serine and the surrounding residues are not the glycosylation site of ComP. Here, PglS is disclosed to glycosylate ComP at a single serine residue located at a position corresponding to the conserved serine at position 84 of AAC4588631 (SEQ ID NO: 1) (ComP ADP1 : AAC4588631 (SEQ ID NO: 1) (ComP 110264 : corresponding also to the conserved serine at position 82 of ENV58402.1 (SEQ ID NO: 2)), which is a novel glycosylation site not previously seen within the type IV pilin superfamily. In combination with the identification of a novel site of glycosylation within the pilin superfamily, the ability of PglS to transfer a polysaccharide containing glucose as the reducing-end sugar demonstrates that PglS is a functionally distinct OTase from PglL and TfpO.

[0060] PglS transferred polysaccharides containing glucose at the reducing end to the acceptor protein ComP, but neither PglB nor PglL did so. Two classes of OTases, PglB and PglL, have previously been used for in vivo conjugation (Feldman, M.F. et al. Proc Natl Acad Sci USA 102, 3016 - 3021 (2005); Faridmoayer, A., Fentabil, M.A., Mills, D.C., Klassen, J.S. & Feldman, M.F. J Bacteriol 189, 8088 - 8098 (2007)). PglB, the first OTase described, preferentially transfers glycans containing an acetamido group at the C-2 position of the reducing end (i.e., N-acetylglucosamine) because it is thought to play a role in substrate recognition (Wacker, M. et al. Proc Natl Acad Sci USA 103, 7088 - 7093 (2006)). However, polysaccharides containing galactose (Gal) at the reducing end, such as the S. enterica Typhimurium O antigen, can be transferred by engineered PglB variants (Ihssen, J. et al. Open Biol 5, 140227 (2015)). PglL from N. meningiditis, the second OTase described, has a more relaxed substrate specificity than PglB and naturally transfers polysaccharides with an acetamido group at the C-2 position and polysaccharides containing galactose (Gal) at the reducing end (Faridmoayer, A., Fentabil, M.A., Mills, D.C., Klassen, J.S. & Feldman, M.F. J Bacteriol 189, 8088 - 8098 (2007); Pan, C. et al. MBio 7 (2016)). However, there is no available evidence for the transfer of polysaccharides containing glucose (Glc) at the reducing end via PglB or PglL, which is particularly interesting considering that most of the pneumococcal CPS contains glucose at the reducing end (Geno, K.A. et al. Clin Microbiol Rev 28, 871 - 899 (2015)).The ability of PglB and PglL to transfer pneumococcal serotype 14 capsular polysaccharide (CPS14) to their cognate glycosylation targets AcrA (Wacker, M. et al. Science 298, 1790-1793 (2002)) and DsbA (Vik, A. et al. Proc Natl Acad Sci USA 106, 4447-4452 (2009)) was tested respectively. As seen in FIGS. 14A and 14B, both acceptor proteins were expressed, but no evidence of CPS14 glycosylation to either acceptor protein was observed.

[0061] Acinetobacter species are described to contain three O - ligases; a general PglL OT - ligase involved in the glycosylation of multiple proteins, and two pilin - specific OT - ligases (Harding, C.M. Mol Microbiol 96, 1023-1041 (2015)). The first pilin - specific OT - ligase is an ortholog of TfpO (also known as PilO) and cannot transfer polysaccharides with more than one repeating unit and thus is not used in the in vivo conjugation system (Faridmoayer, A., Fentabil, M.A., Mills, D.C., Klassen J.S. & Feldman, M.F. J Bacteriol 189, 8088-8098(2007)). The second pilin - specific OT - ligase, PglS, is a single protein, type IV pilin ComP 28Glycosylate. Bioinformatics analysis indicated that PglS is the prototype of a different family of OTases. Considering that PglS is a new class of O-OTase, its ability to transfer pneumococcal CPS14 to its cognate acceptor protein ComP (Harding, C. M. et al. Mol Microbiol 96, 1023-1041 (2015)) was tested. As seen in Figure 14C, co-expression of the CPS14 biosynthetic locus combined with hexahistag-tagged variants of PglS and ComP resulted in a typical ladder-like band pattern compatible with protein glycosylation when analyzed by Western blotting (Figure 14B). The high molecular weight modal distribution of the signal indicates protein glycosylation by the repetition of glycan subunits with increasing molecular weight. Collectively, these results show that, unlike previously characterized OTases, PglS can transfer polysaccharides containing glucose at the reducing end.

[0062] There are over 90 serotypes of S. pneumoniae (Geno, K. A. et al. Clin Microbiol Rev 28, 871-899 (2015)). Many serotypes that are becoming increasingly prevalent, such as serotypes 8, 22F, and 33F, are not included in currently approved vaccines. Therefore, the versatility of PglS was tested to generate a multivalent pneumococcal bioconjugate vaccine against two serotypes (serotypes 9V and 14) included in Prevnar 13 and one serotype (serotype 8) not included (Attachment - Prevnar 13 - FDA, World Wide Web (fda.gov / downloads / BiologicsBloodVaccines / Vaccines / ApprovedProducts / UCM201669.pdf)). Importantly, all three of these capsular polysaccharides contain glucose as the reducing - end sugar (Geno, K.A. et al. Clin Microbiol Rev 28, 871-899 (2015)). As seen in Figure 15, Western blot analysis of affinity - purified proteins from whole cells co - expressing PglS, a hexahistidine - tagged ComP variant, and either CPS8, CPS9V, or CPS14 generated CPS - specific bioconjugates. Furthermore, antisera specific for any of the CPS8, CPS9V, or CPS14 antigens also reacted with the anti - His - reactive bands, indicating that ComP - His was glycosylated with the correct polysaccharide. To confirm that the purified material was not contaminated with lipopolysaccharide, samples were treated with proteinase K and analyzed by Western blotting, and loss of signal was observed, confirming that the bioconjugate was proteinaceous.

[0063] Therefore, it has been demonstrated that PglS can transfer the polysaccharide of S. pneumoniae to ComP, while PglB and PglL cannot. Specifically, PglS is the only OT-ase known in the world that can transfer a polysaccharide containing glucose at the reducing end. In certain embodiments, PglS can be used to transfer a lipid-linked oligosaccharide or polysaccharide containing glucose at the reducing end (collectively referred to herein as "oligosaccharide or polysaccharide") to ComP or a fusion protein containing a fragment of ComP.

[0064] PglS can transfer Klebsiella capsular polysaccharides to ComP. Klebsiella pneumonia (K. pneumoniae), a Gram-negative opportunistic human pathogen, produces capsular polysaccharides that are known to be important for pathogenicity. To date, at least 79 antigenically distinct capsular polysaccharides have been reported for Klebsiella species (Pan, Y.J. et al. Sci Rep 5, 15573 (2015)). Furthermore, K. pneumoniae is known to produce at least 59 of the 77 capsular polysaccharides, more than half of which contain glucose as the reducing end sugar (Pan, Y.J. et al. Sci Rep 5, 15573 (2015)). To determine whether PglS can transfer K. pneumoniae capsular polysaccharides to ComP, genes encoding proteins required for the synthesis of K1 or K2 capsular polysaccharides were cloned into the IPTG-inducible pBBR1MCS-2 vector (Kovach, M.E. et al. Gene 166, 175-176 (1995)). The K1 capsular locus was cloned from K. pneumoniae NTUHK-2044, a previously characterized K1 capsular-producing strain (Wu, K.M. et al. J Bacteriol 191, 4492-4501 (2009)). The K2 capsular locus was cloned from K. pneumoniae 52.145, a previously characterized K2 capsular-producing strain (Lery, L.M. et al. BMC Biol 12, 41 (2014)). The K1 or K2 capsular polysaccharide expression plasmids were then individually introduced into E. coli that co-expresses PglS OT ase and the acceptor protein ComP from a separate plasmid vector.To enhance the expression of K1 and K2 specific polysaccharides, the Klebsiella pneumoniae transcriptional activator rmpA from K. pneumoniae NTUHK-2044 was subsequently cloned into the pACT3 (Dykxhoorn, D.M., St Pierre, R. & Linn, T. Gene 177, 133-136(1996)) low-copy, IPTG-inducible vector because it had previously been characterized as a regulator of the Klebsiella pneumoniae capsule (Arakawa, Y. et al. Infect Immun 59, 2043-2050(1991)); Yeh, K.M. et al. J Clin Microbiol 45, 466-471(2007)). Introduction of the rmpA gene into an E. coli strain co-expressing PglS and a hexahistidine-tagged ComP variant, and either K1 or K2 capsular polysaccharide from K. pneumoniae resulted in strong expression and detection of high molecular weight ComP bioconjugates, as shown by a typical ladder-like pattern of bands compatible with protein glycosylation when analyzed by Western blotting (Figure 16B). The modal distribution of the signals indicates protein glycosylation by repeating glycan subunits of increasing molecular weight. Thus, in summary, PglS was able to glycosylate ComP with K1 and K2 capsular polysaccharides from K. pneumonia. Co-expression of the transcriptional activator rmpA from K. pneumoniae resulted in an increase in conjugation efficiency.

[0065] PglS can transfer K. pneumoniae polysaccharides to ComP. Considering that most K. pneumoniae capsular polysaccharides contain glucose as the reducing terminal sugar, only the other commercially approved OT-ases (PglB and PglL) should not be able to use these polysaccharides to generate conjugate vaccines. Furthermore, co-expression of the transcriptional activator RmpA with the capsular gene cluster enhanced capsular expression to a detectable level. In certain embodiments, a pan-Klebsiella conjugate vaccine can be generated that includes all serotypes, including other species such as K. varricola, K. michiganensis, and K. oxytoca, using the method for producing Klebsiella conjugates.

[0066] Mass spectrometry and site-directed mutagenesis confirmed that PglS is an O-linked OT-ase and ComP is ComP ADP1It was confirmed that it was glycosylated at the serine residue corresponding to the 84th position. Bacterial N-glycosylation generally occurs within the sequence D-X-N-S-T (SEQ ID NO: 21), where X is any amino acid other than proline (Kowarik, M. et al. EMBO J 25, 1957-1966 (2006)). In contrast, O-glycosylation does not seem to follow a defined sequence. Most O-glycosylation events in bacterial proteins occur in regions of low complexity (LCR) rich in serine, alanine, and proline (Vik, A. et al. Proc Natl Acad Sci USA 106, 4447-4452 (2009)). Alternatively, some pyrins are O-glycosylated at the C-terminal serine residue (Comer, J.E., Marshall, M.A., Blanch, V.J., Deal, C.D. & Castric, P. Infect Immun 70, 2837-2845 (2002)). Since ComP does not appear to have an obvious LCR or C-terminal serine residue homologous to those found in other pyrin-like proteins, mass spectrometry was used to determine the site(s) of glycosylation. The purified CPS14-ComP bioconjugate was subjected to proteolytic digestion, ZIC-HILIC glycopeptide enrichment, and multiple MS analyses. As seen in FIGS. 17A and 17B, it was identified that a single glycopeptide consisting of the peptide ISASNATTNVATAT (SEQ ID NO: 22) was bound to a glycan consistent with the published CPS14 composition (Geno, K.A. et al. Clin Microbiol Rev 28, 871-899 (2015)). To enable confirmation of both the peptide and the bound glycan sequence, multiple collision energy regimes were run to HexNA C2 Glycosylation of the semi-GluC-derived peptide ISASNATTNVATAT (SEQ ID NO: 22) by a 1378.47 Da glycan corresponding to Hexose6 was confirmed (FIG. 17B). Additional glycopeptides decorated with extended glycans corresponding to up to four tetrasaccharide repeat units were also observed (FIG. 29).

[0067] Acinetobacter species have previously been shown to glycosylate proteins mainly at serine residues, and thus it was hypothesized that either serine (S) 82 or 84 numbered in SEQ ID NO: 1 was the site of glycosylation (Scott, N.E. et al. Mol Cell Proteomics 13, 2354-2370 (2014)). To determine which serine residue was the site of glycosylation, these serine residues were individually mutated to alanine (A), and the glycosylation status of both mutant proteins was analyzed. In this experiment, the C. jejuni heptasaccharide biosynthetic locus was used as the donor glycan. This is because glycosylation can be easily detected with hR6 antiglycan antiserum and also by an increase in electrophoretic mobility (Schwarz, F. et al. Nat Chem Biol 6, 264-266 (2010)). As shown in Fig. 18, wild-type hexahistidine-tagged ComP was glycosylated with C. jejuni heptasaccharide, as indicated by an increase in electrophoretic mobility when co-expressed with PglS and co-localization with the hR6 antiserum signal. MS analysis also confirmed the presence of C. jejuni heptasaccharide in the same semi-GluC-derived peptide ISASNATTNVATAT (SEQ ID NO: 22) modified with CPS14 (Figs. 30 and 31). As a negative control, a catalytically inactive PglS mutant (H324A) was generated, which was unable to glycosylate wild-type ComP when co-expressed with C. jejuni heptasaccharide glycan. Site-directed mutagenesis was performed, and it was observed that glycosylation of ComP by C. jejuni heptasaccharide was abolished in the ComP[S84A] mutant, whereas ComP[S82A] was glycosylated at wild-type levels. Collectively, these results indicate that ComP is glycosylated by PglS alone at serine 84 (numbered in SEQ ID NO: 1), which is a unique site different from other previously characterized pirin-like proteins. This corresponds to serine 82 numbered in SEQ ID NO: 2.

[0068] Bioinformatics features of the ComP pilin ortholog. 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)). In subsequent studies, ComP from A. baylyi ADP1 (referred to herein as ComP ADP1 was shown to be glycosylated by a new OT-ase, PglS, immediately downstream of ComP, rather than by the general OT-ase PglL found elsewhere on the chromosome (Harding, C.M. et al. Mol Microbiol 96, 1023-1041(2015)). ComP ADP1 protein (NCBI identifier AAC45886.1) belongs to a protein family called type IV pilins. Specifically, ComP shares homology with the major pilin of type IVa (Giltner, C.L., Nguyen, Y. & Burrows, L.L. Microbiol Mol Biol Rev 76, 740-772(2012)). Type IVa pilins share high sequence homology at the N-terminus encoding a highly conserved leader sequence and an N-terminal alpha helix, but the C-terminus shows significant differences even between genera and within species (Giltner, C.L., Nguyen, Y. & Burrows, L.L. Microbiol Mol Biol Rev 76, 740-772(2012)). To distinguish ComP orthologs 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)), a BLASTp analysis was performed to compare the primary amino acid sequence of ComP ADP1 with all proteins from bacteria of the genus Acinetobacter. As expected, ComP ADP1Many Acinetobacter type IVa pilin orthologs, including, share high homology at the N-terminus. However, few proteins show high sequence conservation across the entire amino acid sequence of ComP. At least six ComP orthologs (Figure 19) were identified based on the presence of a conserved serine at position 84 relative to ComP and a conserved disulfide bond adjacent to a predicted glycosylation site that connects a predicted alpha-beta loop to a beta-strand region (Giltner, C.L., Nguyen, Y. & Burrows, L.L. Microbiol Mol Biol Rev 76, 740-772 (2012)). Furthermore, all six ComP orthologs have both a pglS homolog immediately downstream of the comp gene and a pglL homolog somewhere on the chromosome. In summary, the presence of at least a conserved serine at position 84, a disulfide loop adjacent to the glycosylation site, the presence of the pglS gene immediately downstream of comP, and the presence of a pglL homolog somewhere on the chromosome distinguish ComP pilin variants from other type IVa pilin variants. ADP1 Based on the presence of a conserved serine at position 84 relative to ComP and a conserved disulfide bond adjacent to a predicted glycosylation site that connects a predicted alpha-beta loop to a beta-strand region (Giltner, C.L., Nguyen, Y. & Burrows, L.L. Microbiol Mol Biol Rev 76, 740-772 (2012)). Furthermore, all six ComP orthologs have both a pglS homolog immediately downstream of the comp gene and a pglL homolog somewhere on the chromosome. In summary, the presence of at least a conserved serine at position 84, a disulfide loop adjacent to the glycosylation site, the presence of the pglS gene immediately downstream of comP, and the presence of a pglL homolog somewhere on the chromosome distinguish ComP pilin variants from other type IVa pilin variants.

[0069] Thus, features common to ComP proteins that identify ComP orthologs of different Acinetobacter species are disclosed herein. ComP proteins can be distinguished from other pilins by the presence of a conserved glycosylated serine at position 84 relative to the ADP1 ComP protein and the presence of a disulfide loop adjacent to the glycosylation site. Furthermore, the presence of a pglS homolog immediately downstream of ComP is an indicator of ComP. Additionally, the OT-ase downstream of ComP needs to show higher sequence conservation with PglS (ACIAD3337) compared to PglL (ACIAD0103) of A. baylyi ADP1 in order to be classified as a PglS OT-ase protein rather than a PglL OT-ase protein. Also apparent to one of ordinary skill in the art is that in any of the embodiments disclosed herein, the ComP protein has the sequence of SEQ ID NO: 1 (ComP ADP1: It contains a serine residue corresponding to the conserved serine residue at position 84 of AAC45886.1, and this serine residue can be glycosylated.

[0070] ComP from A. soli CIP 110264 is glycosylated by PglS from A. baylyi ADP1. Considering the existence of multiple ComP orthologs, it was investigated whether PglS from A. baylyi ADP1 could glycosylate various ComP proteins. The ComP protein from A. soli CIP 110264 (ComP 110264 ) is 71% identical at the amino acid level when compared with ComP ADP1 . However, consistent with the above-mentioned characteristics, ComP 110264 contains a predicted disulfide bridge between the predicted alpha-beta loop and the second beta strand, and a conserved serine at position 84 relative to ComP ADP1 . Furthermore, the PglS ortholog may be immediately downstream of ComP 110264 . To determine whether PglS from A. baylyi ADP1 (PglS ADP1 ) could glycosylate ComP 110264 , PglS ADP1 was cloned into pACT3 and ComP 110264 was cloned into pEXT20 (Dykxhoorn, D.M., St Pierre, R. & Linn, T. Gene 177, 133-136 (1996)), and these plasmids were introduced into E. coli expressing the serotype 8 capsular polysaccharide (CPS8) from S. pneumoniae. Furthermore, the reverse experiment was carried out by cloning and expressing PglS from A. soli CIP 110264 (PglS 110264 ) together with ComP ADP1 . As shown in Figure 20, PglS 110264 , when compared with the whole cell lysate lacking PglS 110264 , as shown by the high molecular weight ComP pilin variant, the cognate acceptor pilin ComP 110264was minimally glycosylated. Based on Western blot analysis, PglS 110264 did not appear to glycosylate ComP ADP1 . On the other hand, PglS ADP1 efficiently glycosylated both ComP ADP1 and ComP 110264 , as indicated by a strong increase in His-reactive signal due to an increase in electrophoretic mobility. Collectively, PglS ADP1 appears to be the optimal OTase for heterologous glycosylation in E. coli, with the unique ability to cross-glycosylate multiple ComP substrates. Thus, it was demonstrated that PglS proteins from different Acinetobacter species can glycosylate diverse non-native ComP sequences.

[0071] Generation of Soluble Periplasmic Fusion Proteins Glycosylatable by PglS. All members of the type IVa pilin family are considered membrane proteins because a portion of their N-terminal alpha helix is embedded in the inner membrane (Giltner, C.L., Nguyen, Y. & Burrows, L.L. Microbiol Mol Biol Rev 76, 740-772 (2012)). Therefore, to generate a soluble variant of ComP that can be glycosylated by PglS, translational fusions of truncated ComP fragment proteins were constructed on three different carrier proteins. The carrier proteins DsbA and MalE (also known as maltose-binding protein - MBP) from E. coli were selected as appropriate carriers because they have previously been shown to promote periplasmic localization and solubility of the acceptor proteins fused to their C-termini (Malik, A. Biotech 6, 44 (2016)). Exotoxin A from Pseudomonas aeruginosa (EPA) was also selected because it has previously been shown to act as an immunogenic carrier protein in other conjugate vaccine formulations (Ravenscroft, N. et al. Glycobiology 26, 51-62 (2016)). The fusion proteins consisted of a leader sequence, a carrier protein, a short linker peptide, a ComP variant lacking the first 28 amino acids, and a hexahistidine tag. ComP ADP1 and ComP 110264 's first 28 amino acids were deleted because they contain a leader sequence and a hydrophobic region of the N-terminal alpha helix predicted to be embedded in the inner membrane. The fusion constructs were then introduced into E. coli expressing either pneumococcal serotype 8 capsular polysaccharide (CPS8) and pACT3 alone, or pglS 110264 or pglS ADP1 -carrying pACT3. As shown in Figure 21, either DsbA-AAA-ComPΔ28 110264 or DsbA-GGGS-ComPΔ28 110264 by PglS ADP1E. coli cells expressing in combination showed detectable levels of glycosylation as indicated by the modal distribution of the reactive signal of increased electrophoretic mobility. ComPΔ28 ADP1 E. coli cells expressing the fusion containing ADP1 did not show detectable glycosylation. The same glycosylation pattern was observed in E. coli cells expressing maltose-binding protein (MBP) fusions. In particular, as shown in Figure 22, MBP-AAA-ComPΔ28 110264 or MBP-GGGS-ComPΔ28 110264 expressed in combination with PglS ADP1 showed detectable levels of glycosylation as indicated by the modal distribution of the anti-His reactive signal; while the fusion with ComPΔ28 ADP1 was only minimally glycosylated. Finally, to demonstrate that a previously established carrier protein used in conjugate vaccine formulations could be glycosylated by PglS with pneumococcal CPS8, a fusion protein containing the DsbA signal peptide sequence fused to EPA was modified. Then, the ComPΔ28 110264 peptide was fused to a glycine-glycine-glycine-serine (GGGS; SEQ ID NO: 23) linker at the C-terminus of EPA, and glycosylation in the presence and absence of PglS ADP1 was tested in both whole cell extracts and periplasmic extracts. As shown in Figure 23, the EPA-GGGS-ComPΔ28 110264 construct was found to be glycosylated in both whole cell extracts and periplasmic extracts of cells co-expressing CPS8 glycan and PglS ADP1 as indicated by the modal distribution of the anti-His reactive signal. No detectable glycosylation was observed in samples lacking the PglS ortholog or samples expressing PglS 110264 . In summary, PglS ADP1 is the optimal OT-ase for transferring polysaccharides containing glucose at the reducing end to truncated ComP fusion proteins. The specific amino acid sequences of each fusion construct are shown in Figure 24.

[0072] Immunization with the glycosylated ComP bioconjugate induces an immune response. The T cell-dependent immune response to the conjugate vaccine is characterized by the secretion of high-affinity IgG1 antibodies (Avci, F.Y., Li, X., Tsuji, M. & Kasper, D.L. Nat Med 17, 1602-1609 (2011)). The immunogenicity of the CPS14-ComP bioconjugate in a mouse vaccination model was evaluated. As seen in Figure 25A, the sera collected from mice vaccinated with the CPS14-ComP bioconjugate showed a significant increase in CPS14-specific IgG titers, but no increase in IgM titers. Furthermore, a secondary HRP-tagged anti-IgG subtype antibody was used to determine which IgG subtype increased the titer. As seen in Figure 25B, the IgG1 titer appeared to be higher than the other subtypes.

[0073] Next, a second vaccination study was conducted to compare the immunogenicity of the trivalent CPS8-, CPS9V-, and CPS14-ComP bioconjugates with PREVNAR 13®, the current standard of care. Serotypes 9V and 14 are included in PREVNAR 13®, and an increase in IgG titers was seen in PREVNAR 13®-immunized mice against these two serotypes (Figure 26). Monovalent immunization against serotype 14 also showed significant induction of serotype-specific IgG titers similar to the prime immunization (Figures 25 and 26). Mice administered the trivalent bioconjugate, as expected, had increased serotype-specific IgG titers when compared to the control, and the sera on day 49 showed increased IgG titers for serotypes 8 and 14 compared to serotype 9V. Nevertheless, the IgG titer against 9V was significantly higher than that of the placebo (Figure 26).

[0074] Provided herein are bioconjugates comprising an oligosaccharide or polysaccharide linked to a fusion protein. In certain embodiments, the oligosaccharide or polysaccharide is covalently bound to the fusion protein. In certain embodiments, the fusion protein comprises a ComP protein (ComP). In certain other embodiments, the fusion protein comprises a glycosylation tag of the ComP protein (as described in detail anywhere herein).

[0075] As disclosed herein, it has been discovered that ComP is glycosylated at a serine (S) residue. This serine residue is conserved in the ComP protein and corresponds to position 84 of SEQ ID NO: 1 (ComP ADP1 : AAC45886.1). This serine residue also corresponds to position 82 of SEQ ID NO: 2 (ComP110264: ENV58402.1) (FIGS. 39A, B, and C). Thus, in certain embodiments, the fusion protein (and thus the bioconjugate) is glycosylated with an oligosaccharide or polysaccharide at a serine residue corresponding to position 84 of SEQ ID NO: 1 (ComP ADP1 : AAC45886.1) on its ComP glycosylation tag or at a serine residue corresponding to position 82 of SEQ ID NO: 2. FIG. 28 shows an alignment of a region of the ComP sequence containing a serine (S) residue (boxed) corresponding to position 84 of SEQ ID NO: 1 (ComPADP1: AAC45886.1) that is conserved among ComP sequences. In certain embodiments, for the purpose of enabling glycosylation, the ComP glycosylation tag comprises both a cysteine residue corresponding to the conserved cysteine residue at position 75 of SEQ ID NO: 1 (ComP ADP1 : AAC45886.1) and a cysteine residue corresponding to the conserved cysteine residue at position 95 of SEQ ID NO: 1. Alternatively, and by way of similar description, in certain embodiments, for the purpose of enabling glycosylation, the ComP glycosylation tag comprises SEQ ID NO: 2 (ComP ADP1:It contains both the cysteine residue corresponding to the conserved cysteine residue at position 71 of AAC45886.1) and the cysteine residue corresponding to the conserved cysteine residue at position 93 of SEQ ID NO: 2.

[0076] In certain embodiments of the bioconjugates of the present disclosure, the oligosaccharide or polysaccharide contains glucose at its reducing end.

[0077] As will be appreciated by those skilled in the art, by aligning the ComP sequence with SEQ ID NO: 1 (e.g., the full-length sequence or a partial sequence), the conserved residues of the ComP protein other than SEQ ID NO: 1 disclosed herein corresponding to the serine residue at position 84 of SEQ ID NO: 1 can be identified. Furthermore, as will be appreciated by those skilled in the art, by aligning the ComP sequence with SEQ ID NO: 1, other residues, regions, and / or features corresponding to the residues, regions, and / or features of SEQ ID NO: 1 referred to herein can be identified in the ComP sequence other than SEQ ID NO: 1 and referred to with respect to SEQ ID NO: 1. And although SEQ ID NO: 1 is generally referred to herein, by analogy, any residue, region, feature, etc. of any ComP sequence disclosed herein can be similarly referred to, for example, with respect to SEQ ID NO: 2.

[0078] The ComP protein is a protein identified as a ComP protein consistent with the description provided herein. For example, representative examples of the ComP protein 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]; and OAL75955.1 competence protein [Acinetobacter sp. SFC]. In certain embodiments, 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: 1 (ComP ADP1 :AAC45886.1) and comprises a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 1 (ComP ADP1 :AAC45886.1). SEQ ID NO: 1 includes a 28-amino acid leader sequence. In certain embodiments, the ComP protein does not include the 28-amino acid leader sequence, but comprises a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 1 (ComP ADP1 :AAC45886.1), SEQ ID NO: 7 (ComPΔ28 ADP1 ), SEQ ID NO: 8 (ComPΔ28 110264 ), SEQ ID NO: 9 (ComPΔ28 GFJ-2 ), SEQ ID NO: 10 (ComPΔ28 P50v1 ), SEQ ID NO: 11 (ComPΔ28 4466 ), or SEQ ID NO: 12 (ComPΔ28 SFC) contains an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical thereto. In certain embodiments, the ComP protein does not contain a 28 - amino - acid leader sequence, but contains a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 1 (ComP ADP1 : AAC45886.1), and contains 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: 7 (ComPΔ28 ADP1 ). In certain embodiments, the ComP protein contains SEQ ID NO: 7 (ComPΔ28 ADP1 ), SEQ ID NO: 8 (ComPΔ28 110264 ), SEQ ID NO: 9 (ComPΔ28 GFJ-2 ), SEQ ID NO: 10 (ComPΔ28 P50v1 ), SEQ ID NO: 11 (ComPΔ28 4466 ), or SEQ ID NO: 12 (ComPΔ28 SFC ). In certain embodiments, the ComP protein is SEQ ID NO: 1 (ComP ADP1 : AAC45886.1), SEQ ID NO: 2 (ComP 110264 : ENV58402.1), SEQ ID NO: 3 (ComP GFJ-2 : APV36638.1), SEQ ID NO: 4 (ComP 50v1 : PKD82822.1), SEQ ID NO: 5 (ComP 4466 : SNX44537.1), or SEQ ID NO: 6 (ComP SFC : OAL75955.1).

[0079] In certain embodiments, the bioconjugate is produced in vivo in a host cell, such as by any of the production methods disclosed herein. In certain embodiments, the bioconjugate is produced in bacterial cells, fungal cells, yeast cells, avian cells, algal cells, insect cells, or mammalian cells. In certain embodiments, the bioconjugate is produced in a cell-free system. Examples of the use of cell-free systems that utilize an OTase other than PglS can be found in WO2013 / 067523A1, which is incorporated herein by reference.

[0080] The methionine residue corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP 110264 :ENV58402.1), when present in the ComP glycosylation tag, has been found to potentially have an inhibitory effect on glycosylation, even if the full-length ComP protein containing this methionine residue is glycosylated. Thus, in certain embodiments, the ComP glycosylation tag of the present disclosure does not contain the methionine residue corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP 110264 :ENV58402.1). For example, in certain embodiments, the methionine residue in the ComP amino acid sequence is replaced with another amino acid that does not exhibit an inhibitory effect or is deleted from the ComP glycosylation tag amino acid sequence. In certain embodiments, the amino acid sequence of the ComP glycosylation tag does not continue beyond the amino acid residue corresponding to position 103 of SEQ ID NO: 2 (ComP 110264 :ENV58402.1) in the C-terminal direction. For example, in certain embodiments, the amino acid sequence of the ComP glycosylation tag ends with a residue corresponding to position 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, or 103 of SEQ ID NO: 2 (ComP 110264 :ENV58402.1). As will be appreciated by those skilled in the art, fusion proteins containing the ComP glycosylation tag are similarly related to the ComP glycosylation tag as SEQ ID NO: 2 (ComP 110264:ENV58402.1), does not contain a methionine residue corresponding to or approximating the 104th conserved methionine residue, even if the methionine residue results from the sequence of a fusion protein that does not belong to the ComP glycosylation tag sequence. For example, in certain embodiments, the fusion protein of the bioconjugate, with respect to the ComP glycosylation tag, has SEQ ID NO: 2 (ComP 110264 :ENV58402.1), does not contain a methionine residue corresponding to or approximating the 104th conserved methionine residue. In certain embodiments, the fusion protein of the bioconjugate, with respect to the ComP glycosylation tag, has SEQ ID NO: 2 (ComP 110264 :ENV58402.1), does not contain a methionine residue that appears to correspond to the 104th conserved methionine residue.

[0081] The ComP glycosylation tags of the present disclosure are generally not full-length ComP proteins. In certain embodiments of any of the ComP glycosylation tags described herein, the ComP glycosylation tag is 18 to 50 amino acids in length, e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In certain embodiments, the glycosylation tag is 21 to 45 amino acids in length. In certain embodiments, the glycosylation tag is 23 to 45 amino acids in length.

[0082] The ComP glycosylation tags of the present disclosure can be fragments, variants, or variant fragments of the ComP protein as described anywhere herein. In certain embodiments, the ComP protein has SEQ ID NO: 7 (ComPΔ28 ADP1 ), SEQ ID NO: 8 (ComPΔ28 110264 ), SEQ ID NO: 9 (ComPΔ28 GFJ-2 ), SEQ ID NO: 10 (ComPΔ28 P50v1 ), SEQ ID NO: 11 (ComPΔ284466 ) or 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: 12 (ComPΔ28 SFC ) and includes 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: 7 (ComPΔ28 ADP1 ) or SEQ ID NO: 8 (ComPΔ28 110264 ). In certain embodiments, the ComP protein includes 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: 7 (ComPΔ28 ADP1 ), SEQ ID NO: 8 (ComPΔ28 110264 ), SEQ ID NO: 9 (ComPΔ28 GFJ-2 ), SEQ ID NO: 10 (ComPΔ28 P50v1 ), SEQ ID NO: 11 (ComPΔ28 4466 ), or SEQ ID NO: 12 (ComPΔ28 SFC ). Further, in certain embodiments, the ComP protein is SEQ ID NO: 1 (ComP ADP1 : AAC45886.1), SEQ ID NO: 2 (ComP 110264 : ENV58402.1), SEQ ID NO: 3 (ComP GFJ-2 : APV36638.1), SEQ ID NO: 4 (ComP 50v1 : PKD82822.1), SEQ ID NO: 5 (ComP 4466 : SNX44537.1), or SEQ ID NO: 6 (ComP SFC : OAL75955.1) and includes 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: 1 (ComP ADP1 : AAC45886.1) or SEQ ID NO: 2 (ComP 110264: It contains 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: ENV58402.1). Further, in certain embodiments, the ComP protein is SEQ ID NO: 1 (ComP ADP1 : AAC45886.1), SEQ ID NO: 2 (ComP 110264 : ENV58402.1), SEQ ID NO: 3 (ComP GFJ-2 : APV36638.1), SEQ ID NO: 4 (ComP 50v1 : PKD82822.1), SEQ ID NO: 5 (ComP 4466 : SNX44537.1), or SEQ ID NO: 6 (ComP SFC : OAL75955.1).

[0083] In certain embodiments, the ComP glycosylation tag of the present disclosure can be defined as comprising or consisting of the amino acid consensus sequence of SEQ ID NO: 27:

Number

[0084] In certain embodiments, the ComP glycosylation tag comprises or consists of a fragment of the amino acid consensus sequence of SEQ ID NO: 27, provided that the fragment retains the cysteine residue at position 13 of SEQ ID NO: 27, the cysteine residue at position 35 of SEQ ID NO: 27, and the serine residue at position 24 of SEQ ID NO: 27. In certain embodiments, the ComP glycosylation tag comprises or consists of a variant of the amino acid consensus sequence of SEQ ID NO: 27 or a fragment thereof, the variant having one, two, three, four, five, six, or seven amino acid substitutions, additions, and / or deletions, provided that the variant retains the cysteine residue at position 13 of SEQ ID NO: 27, the cysteine residue at position 35 of SEQ ID NO: 27, and the serine residue at position 24 of SEQ ID NO: 27. In certain embodiments, the amino acid substitutions are conservative amino acid substitutions. As disclosed herein, in certain embodiments, the ComP glycosylation tag comprising SEQ ID NO: 27 does not contain a methionine residue at a position corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP 110264 :ENV58402.1). Further, in certain embodiments, the amino acid sequence of the ComP glycosylation tag comprising SEQ ID NO: 27 does not continue beyond the amino acid residue corresponding to position 44 of SEQ ID NO: 27 in the C-terminal direction. In certain embodiments, the ComP glycosylation tag comprising or consisting of the amino acid consensus sequence of SEQ ID NO: 27 or a fragment and / or variant thereof is 25, 30, 40, 45, or 50 amino acids or less in length.

[0085] In certain embodiments, the ComP glycosylation tag of the present disclosure can be defined as comprising or consisting of the amino acid consensus sequence of SEQ ID NO: 28:

Number

[0086] In certain embodiments, the ComP glycosylation tag comprises or consists of a variant of the amino acid consensus sequence of SEQ ID NO: 28, the variant having 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, additions, and / or deletions, provided that the variant maintains the cysteine residue at position 1 of SEQ ID NO: 28, the cysteine residue at position 23 of SEQ ID NO: 28, and the serine residue at position 12 of SEQ ID NO: 28. In certain embodiments, the amino acid substitutions are conservative amino acid substitutions.

[0087] In certain embodiments, the ComP glycosylation tag comprising SEQ ID NO: 28 does not contain a methionine residue at the position corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP 110264 :ENV58402.1). Further, in certain embodiments, the amino acid sequence of the ComP glycosylation tag comprising SEQ ID NO: 28, in the C-terminal direction, is SEQ ID NO: 2 (ComP 110264: It does not follow after the amino acid residue corresponding to position 103 of SEQ ID NO: ENV58402.1). In certain embodiments, the ComP glycosylation tag comprising the amino acid consensus sequence of SEQ ID NO: 28 or a variant thereof has a length of 25, 30, 40, 45, or 50 amino acids or less.

[0088] In certain embodiments, the ComP glycosylation tag comprises or consists of a variant having one, two, three, four, five, six, or seven amino acid substitutions, additions, and / or deletions to an amino acid sequence selected from the group consisting of SEQ ID NO: 32 [C1], SEQ ID NO: 33 [D1], SEQ ID NO: 34 [E1], SEQ ID NO: 41 [E2], SEQ ID NO: 42 [F2], SEQ ID NO: 43 [G2], SEQ ID NO: 44 [H2], SEQ ID NO: 45 [A3], SEQ ID NO: 46 [B3], SEQ ID NO: 47 [C3], SEQ ID NO: 55 [D4], SEQ ID NO: 56 [E4], SEQ ID NO: 57 [F4], SEQ ID NO: 58 [G4], SEQ ID NO: 59 [A5], SEQ ID NO: 60 [B5], SEQ ID NO: 61 [D5], SEQ ID NO: 62 [E5], SEQ ID NO: 63 [F5], SEQ ID NO: 72 [H6], SEQ ID NO: 73 [B7], SEQ ID NO: 74 [C7], SEQ ID NO: 75 [D7], SEQ ID NO: 76 [E7], SEQ ID NO: 77 [F7], SEQ ID NO: 78 [A8], SEQ ID NO: 79 [B8], SEQ ID NO: 92 [A10], SEQ ID NO: 93 [B10], SEQ ID NO: 94 [C10], SEQ ID NO: 95 [D10], SEQ ID NO: 96 [F10], SEQ ID NO: 97 [G10], SEQ ID NO: 98 [H10], SEQ ID NO: 99 [A11], SEQ ID NO: 100 [B11], and SEQ ID NO: 101 [C11], provided that the variant is SEQ ID NO: 1 (ComP ADP1: It maintains both the cysteine residue corresponding to the 75th conserved cysteine residue of AAC45886.1) and the cysteine residue corresponding to the 95th conserved cysteine residue of SEQ ID NO: 1, and the variant maintains the serine residue corresponding to the 84th conserved serine residue of SEQ ID NO: 1. In certain embodiments, the amino acid substitution is a conservative amino acid substitution. Further, in certain embodiments, the ComP glycosylation tag comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 32 [C1], SEQ ID NO: 33 [D1], SEQ ID NO: 34 [E1], SEQ ID NO: 41 [E2], SEQ ID NO: 42 [F2], SEQ ID NO: 43 [G2], SEQ ID NO: 44 [H2], SEQ ID NO: 45 [A3], SEQ ID NO: 46 [B3], SEQ ID NO: 47 [C3], SEQ ID NO: 55 [D4], SEQ ID NO: 56 [E4], SEQ ID NO: 57 [F4], SEQ ID NO: 58 [G4], SEQ ID NO: 59 [A5], SEQ ID NO: 60 [B5], SEQ ID NO: 61 [D5], SEQ ID NO: 62 [E5], SEQ ID NO: 63 [F5], SEQ ID NO: 72 [H6], SEQ ID NO: 73 [B7], SEQ ID NO: 74 [C7], SEQ ID NO: 75 [D7], SEQ ID NO: 76 [E7], SEQ ID NO: 77 [F7], SEQ ID NO: 78 [A8], SEQ ID NO: 79 [B8], SEQ ID NO: 92 [A10], SEQ ID NO: 93 [B10], SEQ ID NO: 94 [C10], SEQ ID NO: 95 [D10], SEQ ID NO: 96 [F10], SEQ ID NO: 97 [G10], SEQ ID NO: 98 [H10], SEQ ID NO: 99 [A11], SEQ ID NO: 100 [B11], and SEQ ID NO: 101 [C11]. In certain embodiments, the ComP glycosylation tag comprising one of the above sequences or variants thereof is SEQ ID NO: 2 (ComP 110264 : does not contain a methionine residue at the position corresponding to the 104th conserved methionine residue of ENV58402.1). Further, in certain embodiments, the amino acid sequence of the ComP glycosylation tag comprising one of the above sequences or variants thereof, in the C-terminal direction, is SEQ ID NO: 2 (ComP 110264: It does not follow after the amino acid residue corresponding to the 103rd position of ENV58402.1. In certain embodiments, the ComP glycosylation tag comprising the amino acid sequences listed above and / or their variants has a length of 25, 30, 40, 45, or 50 amino acids or less. In certain embodiments, the ComP glycosylation tag consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 32 [C1], SEQ ID NO: 33 [D1], SEQ ID NO: 34 [E1], SEQ ID NO: 41 [E2], SEQ ID NO: 42 [F2], SEQ ID NO: 43 [G2], SEQ ID NO: 44 [H2], SEQ ID NO: 45 [A3], SEQ ID NO: 46 [B3], SEQ ID NO: 47 [C3], SEQ ID NO: 55 [D4], SEQ ID NO: 56 [E4], SEQ ID NO: 57 [F4], SEQ ID NO: 58 [G4], SEQ ID NO: 59 [A5], SEQ ID NO: 60 [B5], SEQ ID NO: 61 [D5], SEQ ID NO: 62 [E5], SEQ ID NO: 63 [F5], SEQ ID NO: 72 [H6], SEQ ID NO: 73 [B7], SEQ ID NO: 74 [C7], SEQ ID NO: 75 [D7], SEQ ID NO: 76 [E7], SEQ ID NO: 77 [F7], SEQ ID NO: 78 [A8], SEQ ID NO: 79 [B8], SEQ ID NO: 92 [A10], SEQ ID NO: 93 [B10], SEQ ID NO: 94 [C10], SEQ ID NO: 95 [D10], SEQ ID NO: 96 [F10], SEQ ID NO: 97 [G10], SEQ ID NO: 98 [H10], SEQ ID NO: 99 [A11], SEQ ID NO: 100 [B11], and SEQ ID NO: 101 [C11].

[0089] In certain embodiments, the oligosaccharides or polysaccharides for conjugating to a glycosylation tag, fusion protein, and / or bioconjugate are produced by bacteria of the genus Streptococcus. For example, in certain embodiments, the polysaccharide is a capsular polysaccharide of S. pneumoniae, S. agalactiae, or S. suis. Further, in certain embodiments, the capsular polysaccharide is CPS14, CPS8, CPS9V, or CPS15b. In certain other embodiments, the oligosaccharide or polysaccharide is produced by bacteria of the genus Klebsiella. For example, in certain embodiments, the polysaccharide is a capsular polysaccharide of Klebsiella pneumoniae, Klebsiella varricola, Klebsiella michinganenis, or Klebsiella oxytoca. In certain embodiments, the polysaccharide is a capsular polysaccharide of Klebsiella pneumoniae. Further, in certain embodiments, the polysaccharide is a serotype K1 or serotype K2 capsular polysaccharide of Klebsiella pneumoniae.

[0090] In certain embodiments, the bioconjugate is produced in vivo. For example, in certain embodiments, the bioconjugate is produced in bacterial cells.

[0091] Since the bioconjugate contains an oligosaccharide or polysaccharide covalently linked to a fusion protein, in certain applications, it may be advantageous to form a fusion protein with a carrier protein or a fragment thereof. In certain embodiments, the carrier protein is one recognized in the art as useful for the production of conjugate vaccines. In certain embodiments, when the ComP glycosylated fragment is fused to a carrier protein or a fragment thereof, the glycosylated fragment, and thus the fusion protein, can be glycosylated at a conserved serine residue described anywhere herein. In certain embodiments, the fusion protein comprises a carrier protein selected from the group consisting of diphtheria toxoid CRM197, tetanus toxoid, Pseudomonas aeruginosa exotoxin A (EPA), tetanus toxin C fragment, cholera toxin B subunit, Haemophilus influenza protein D, or fragments thereof. In certain embodiments, the carrier protein or a fragment thereof is an amino acid linker, for example, (GGGS) n(SEQ ID NO: 23, wherein n is at least 1), or via AAA (SEQ ID NO: 24), is linked to the ComP protein or its glycosylation. For the purpose of increasing the potential immunogenicity of the ComP fusion protein, it may be advantageous to include more than one glycosylation tag. Thus, in certain embodiments, 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 glycosylation tag fragments of the ComP protein. In certain embodiments, the fusion protein comprises from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 glycosylation tag fragments of the ComP protein. In certain embodiments, multiple glycosylations are arranged in series with each other in the fusion protein. In certain embodiments, multiple glycosylation tag fragments are arranged apart from each other in the fusion protein, for example, separated by the sequence of the carrier protein. In certain embodiments, the glycosylation tag(s) can be located, for example, at the N-terminus of the carrier protein and / or the fusion protein. In certain embodiments, the glycosylation tag(s) can be located, for example, at the C-terminus of the carrier protein and / or the fusion protein. In certain embodiments, the glycosylation tag(s) can be located, for example, internally within the carrier protein and / or the fusion protein, and the glycosylation tag fragment is located between multiple carrier proteins in the fusion protein. In certain embodiments, the multiple carrier proteins can be of the same type or of different types. In certain embodiments, the glycosylation tags can be of the same type or of different types. In certain embodiments, these ComP glycosylation tags are identical. In certain embodiments, at least two of the ComP glycosylation tags are different from each other.In certain embodiments, at least three, at least four, or at least five of the ComP glycosylation tags are all different from each other. Further, in certain embodiments, none are identical as any one of the ComP glycosylation tags.

[0092] The bioconjugate of the present invention can be used for one of a number of applications, such as, but not limited to, use as a conjugate vaccine. For example, in certain embodiments, the conjugate vaccine is 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 pneumoniae serotype K2, Klebsiella pneumoniae serotype K5, Klebsiella pneumoniae serotype K16, Klebsiella pneumoniae serotype K20, Klebsiella pneumoniae serotype K54, KlebsiellaA vaccine against serotype K57 of Streptococcus pneumoniae, serotype Ia of Streptococcus agalactiae, serotype Ib of Streptococcus agalactiae, serotype II of Streptococcus agalactiae, serotype III of Streptococcus agalactiae, serotype IV of Streptococcus agalactiae, serotype V of Streptococcus agalactiae, serotype VI of Streptococcus agalactiae, serotype VII of Streptococcus agalactiae, serotype VIII of Streptococcus agalactiae, serotype IX of Streptococcus agalactiae, group A carbohydrate of Streptococcus pyogenes, serotype A of Enterococcus faecalis, serotype B of Enterococcus faecalis, serotype C of Enterococcus faecalis, serotype D of Enterococcus faecalis, capsular polysaccharide and lipoteichoic acid of Enterococcus faecium, lipooligosaccharide A of Moraxella catarrhalis, lipooligosaccharide B of Moraxella catarrhalis, lipooligosaccharide C of Moraxella catarrhalis, and lipoteichoic acid of Staphylococcus aureus. 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 cells and memory T cells), is an antibody response, and optionally, is a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG response or an IgM response. For example, in certain embodiments, the antibody response can be an IgG response, and in certain embodiments, it can be an IgG1 response. In certain embodiments, the conjugate vaccine generates immune memory in the subject to whom the vaccine is administered.

[0093] Provided herein is a fusion protein as further disclosed in detail elsewhere herein, which fusion protein contains a ComP glycosylation tag as detailed elsewhere herein. In certain embodiments, the fusion protein is glycosylated at a serine residue corresponding to the serine residue at position 84 of SEQ ID NO: 1 (ComP ADP1 : AAC45886.1). In certain embodiments, the fusion protein is glycosylated with an oligosaccharide or a polysaccharide. In certain embodiments, the oligosaccharide or polysaccharide is produced by a bacterium of the genus Streptococcus, such as the capsular polysaccharide of S. pneumoniae, S. agalactiae, or S. suis. In certain embodiments, the capsular polysaccharide is CPS14, CPS8, CPS9V, or CPS15b. In certain embodiments, the oligosaccharide or polysaccharide is produced by a bacterium of the genus Klebsiella, such as the capsular polysaccharide of Klebsiella pneumoniae, Klebsiella varricola, Klebsiella michinganenis, or Klebsiella oxytoca. In certain embodiments, the polysaccharide is the capsular polysaccharide of Klebsiella pneumoniae. In certain embodiments, the polysaccharide is the serotype K1 or serotype K2 capsular polysaccharide of Klebsiella pneumoniae. In certain ones of any of the embodiments disclosed herein, the oligosaccharide or polysaccharide contains glucose at its reducing end. Certain embodiments relate to a fusion protein, which fusion protein is produced in vivo. For example, in certain embodiments, the fusion protein is produced in mammalian cells, fungal cells, yeast cells, insect cells, avian cells, algal cells, or bacterial cells. In certain embodiments, the fusion protein is produced in bacterial cells, such as E. coli.

[0094] Disclosed herein is a method for in vivo conjugation (in vivo glycosylation) of an oligosaccharide or polysaccharide with a polypeptide. In certain embodiments, the method involves covalently attaching an oligosaccharide or polysaccharide to a polypeptide using a PglS oligosaccharyltransferase (OTase) (described anywhere herein). In certain embodiments, the polypeptide comprises a ComP protein or a glycosylation tag thereof. In certain embodiments, the polypeptide comprises a ComP protein or a glycosylation tag thereof linked to a heterologous polypeptide such as a carrier protein. Examples of PglS OTases include, but are not limited to, PglS 110264 , PglS ADP1 , PglS GFJ-2 , PglS 50v1 , PglS 4466 , and PglS SFC . The ComP protein is described in detail anywhere, and representative examples include ComP 110264 , ComP ADP1 , ComP GFJ-2 , ComP 50v1 , ComP 4466 , and ComP SFC . Of course, a PglS OTase from a certain organism may naturally glycosylate a ComP protein from that organism (e.g., PglS 110264 glycosylates ComP 110264 ), but in certain embodiments, a PglS from a certain organism glycosylates a ComP from a different organism (e.g., PglS ADP1 glycosylates ComP 110264 ). For example, in certain aspects, the PglS OTase is PglS ADP1 . In a particular embodiment where the PglS OTase is PglS ADP1 , the glycosylated ComP protein is not ComP ADP1 . For example, where the PglS OTase is PglS ADP1In certain embodiments, the ComP protein is ComP 110264 Of course, if the ComP protein or its glycosylated tag fragment is linked to a heterologous carrier protein, the PglSOT enzyme will not glycosylate them naturally, even if the ComP protein or its glycosylated tag fragment is from the same organism as the PglSOT enzyme.

[0095] In certain embodiments regarding any combination of PglS and ComP, the ComP protein or its glycosylated tag fragment is glycosylated at a serine residue corresponding to the serine residue at position 84 of SEQ ID NO: 1 (ComP ADP1 : AAC45886.1).

[0096] In certain embodiments disclosed herein, in vivo glycosylation occurs in host cells. In certain embodiments, for example, the host cell can be a mammalian cell, a fungal cell, a yeast cell, an insect cell, a bird cell, an algal cell, or a bacterial cell. In certain embodiments, the host cell is a bacterial cell, such as E. coli.

[0097] In certain embodiments, the method includes culturing a host cell that contains the components necessary to attach an oligosaccharide or polysaccharide to a polypeptide. Generally, such components are oligosaccharyltransferase, a glycosylated acceptor polypeptide, and an oligosaccharide or polysaccharide. In certain embodiments, the method includes culturing a host cell that includes: (a) a gene cluster encoding proteins necessary for the synthesis of an oligosaccharide or polysaccharide, (b) PglSOTase, and (c) an acceptor polypeptide. Further, it has been discovered that the production of oligosaccharides or polysaccharides can be improved by a transcriptional activator. In certain embodiments, the production of oligosaccharides or polysaccharides is improved by the Klebsiella pneumoniae transcriptional activator rmpA (Klebsiella pneumoniae NTUH K-2044) or a homolog of the Klebsiella pneumoniae transcriptional activator rmpA (Klebsiella pneumoniae NTUH K-2044). In certain embodiments, the method further includes expressing and / or providing such a transcriptional activator to the host cell, in combination with other components.

[0098] In certain embodiments, the carrier protein linked to the ComP glycosylation tag is, for example, diphtheria toxoid CRM197, tetanus toxoid, Pseudomonas aeruginosa exotoxin A (EPA), tetanus toxin C fragment, cholera toxin B subunit, Haemophilus influenza protein D, or a fragment thereof.

[0099] Certain embodiments relate to a conjugate vaccine comprising the bioconjugate of the present disclosure, or a method of manufacturing such a conjugate vaccine.

[0100] Certain embodiments also provide a host cell comprising components for in vivo glycosylation of an acceptor ComP protein or a glycosylated tag fragment thereof. In certain embodiments, the host cell comprises: (a) a gene cluster encoding proteins necessary for the synthesis of oligosaccharides or polysaccharides, (b) PglSOTase, and (c) an acceptor polypeptide comprising a ComP protein or a glycosylated tag fragment thereof. In certain embodiments, the acceptor polypeptide is a fusion protein. In certain embodiments, the host cell further comprises a transcriptional activator, such as those described above, in combination with other components.

[0101] In certain embodiments, the host cell comprises an isolated nucleic acid encoding PglSOTase. In certain embodiments, the host cell comprises an isolated nucleic acid encoding a ComP acceptor polypeptide. In certain embodiments, the host cell comprises a gene cluster encoding proteins necessary for the synthesis of oligosaccharides or polysaccharides. In certain embodiments, the host cell comprises at least two of an isolated nucleic acid encoding PglSOTase, an isolated nucleic acid encoding a ComP acceptor polypeptide, and a gene cluster encoding proteins necessary for the synthesis of oligosaccharides or polysaccharides. In an aspect of the embodiment, the host cell comprises a nucleic acid encoding a PglSOTase of one organism and a nucleic acid encoding a ComP acceptor polypeptide from a different organism.

[0102] Certain embodiments also provide an isolated nucleic acid encoding a ComP protein, a ComP glycosylated tag fragment, and / or a ComP fusion protein described anywhere herein. In certain embodiments, the isolated nucleic acid referred to herein is a vector or is contained within a vector. In certain embodiments, the isolated nucleic acid referred to herein is inserted into and / or integrated into a heterologous genome or a heterologous region of a genome.

[0103] Disclosed herein is a pneumococcal conjugate vaccine comprising a conventional vaccine carrier. Certain embodiments include the use of a ComP fragment as a glycosylation tag (also known as a "glycotag"). In certain embodiments, the glycosylation tag can be added to the C-terminus and / or N-terminus of the carrier protein. For example, in certain embodiments, a glycotag is added to the C-terminus of Pseudomonas aeruginosa exotoxin A (EPA), a conventional carrier protein. In certain embodiments, it has been demonstrated that the glycosylation tag / carrier fusion protein, in combination with the use of CPS8 polysaccharide and PglS, can generate a carrier protein-CPS8 conjugate, which is the first of its kind as a pneumococcal conjugate vaccine. For example, in certain embodiments, an EPA fusion can be combined with the use of CPS8 polysaccharide and PglS to generate an EPA-CPS8 conjugate. The EPA-CPS8 conjugate vaccine has been demonstrated to induce high IgG titers specific for serotype 8, which is protective as determined by bactericidal killing. Importantly, vaccination with 100 ng of polysaccharide in the EPA-CPS8 conjugate was able to provide protection. Thus, certain embodiments provide a CPS8 pneumococcal conjugate vaccine.

[0104] Conjugate vaccines (such as EPA vaccine constructs) can be thought to include additional / multiple sites of glycosylation not only to increase the glycan-to-protein ratio but also to expand the number of serotypes in order to develop a comprehensive pneumococcal conjugate vaccine.

[0105] In certain embodiments, the bioconjugates or glycosylated fusion proteins disclosed herein are conjugate vaccines that can be administered to a subject for the prevention and / or treatment of infectious diseases and / or disorders. In certain embodiments, the conjugate vaccine is, for example, a prophylactic that can be used to immunize a subject against an infectious disease and / or disorder. In certain embodiments, the bioconjugate is associated with an adjuvant and / or is administered with an adjuvant (such as in a therapeutic composition). Certain embodiments provide compositions (such as therapeutic compositions) comprising the conjugate vaccines and adjuvants described herein. In certain embodiments, when the conjugate vaccine is administered to a subject, it induces an immune response. In certain embodiments, the immune response induces long-term memory (memory B cells and memory 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. Further, in certain embodiments, the conjugate vaccine generates immune memory in a subject to whom the vaccine has been administered.

[0106] Certain embodiments provide for the manufacture of vaccines against infectious diseases and / or disorders. In certain embodiments, the method includes isolating a bioconjugate 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 disorder is pneumonia. In certain embodiments, the infectious disease is a systemic and / or bloodstream infection. In certain embodiments, the subject is a mammal. For example, in certain embodiments, the subject is a pig or a human.

[0107] Importantly, the aspects disclosed herein are not limited to pneumococcal polysaccharides and, in fact, have broad applicability for generating conjugate vaccines against many important human and animal pathogens that are not compatible with PglB and PglL. Notable examples include the human pathogens Klebsiella pneumoniae and group B streptococcus, and the porcine pathogen S. suis, and all highly relevant pathogens for which no approved vaccines are available.

[0108] Provided herein is a method of inducing a host immune response against a pathogen. In certain embodiments, the pathogen is a pathogenic microorganism. In certain embodiments, the host is immunized against the pathogen. In certain embodiments, the method comprises administering to a subject in need of an immune response an effective amount of a ComP conjugate vaccine, a glycosylated fusion protein, or any other therapeutic / immunogenic composition disclosed herein. Certain embodiments provide a conjugate vaccine, a glycosylated fusion protein, or other therapeutic / immunogenic composition disclosed herein for use in inducing a host immune response against a pathogenic microorganism and immunizing against a pathogenic microorganism. 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 the above 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 the above immune responses.

[0109] Also provided herein is a method of preventing or treating a bacterial disease and / or an infectious disease in a subject, comprising administering to the subject in need thereof a conjugate vaccine, a fusion protein, or a composition disclosed herein. In certain embodiments, the infectious disease is a local or systemic infection of the skin, soft tissue, blood, or organs, or is essentially autoimmune. In certain embodiments, the disease is pneumonia. In certain embodiments, the infectious disease is a systemic infection and / or a blood infection. In certain specific embodiments 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. And, for example, in certain embodiments, the mammal is a human.

[0110] In any of the administration embodiments disclosed herein, the composition is administered via intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.

Example

[0111] Example 1. Determination of whether the cysteine residues adjacent to the glycosylation sites in ComP contribute to ComP stability and glycosylation. ComP protein from Acinetobacter baylyi ADP1 (ComP ADP1 ) and ComP protein from strain 110264 of A. soli (ComP 110264 ) have been previously demonstrated to be glycosylated at homologous serine residues located at positions 84 and 82, respectively, by the O-linked OT-ase PglS (Harding CM, et al. (2019) A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host. Nat Commun 10(1):891). Specifically, the S84A point mutant of ComP ADP1 and the S82A point mutant of ComP 110264 were shown to be unable to be glycosylated with serotype 8 pneumococcal capsular polysaccharide by PglS. To further analyze the role of other amino acids important for PglS-dependent glycosylation, a series of point mutants were generated and the conserved cysteine residues adjacent to the glycosylation sites located at positions 75 and 95 of ComP ADP1 were modified (Figure 1A).

[0112] A series of point mutants were first generated by replacing cysteine 75 with either alanine or glycine. This is because these mutants are thought to block the formation of disulfide bonds that may form between cysteine 75 and cysteine 95. The point mutants were then used in combination with the C. jejuni heptasaccharide biosynthesis gene cluster and PglS ADP1were introduced into E. coli SDB1 co-expressing them. As seen in Figure 1B, the mutation at cysteine 75 significantly reduced the expression of the ComP protein variant compared to the wild-type (WT) ComP. Specifically, the C75A and C75G variants exhibited very low levels of protein expression and appeared to exist only as the low molecular weight non-glycosylated form. Next, a second series of point mutants were generated by replacing cysteine 95 with either alanine, glycine, or serine, and then these mutant ComP constructs were again introduced into E. coli SDB1 co-expressing the C. jejuni heptasaccharide biosynthetic gene cluster and PglS ADP1 were introduced into E. coli SDB1 co-expressing them. As seen in Figure 1B, ComPj variants having either alanine, glycine, or serine instead of cysteine 95 were undetectable. Finally, a series of double point mutants consisting of all possible combinations of changing cysteine 75 to alanine or glycine and cysteine 95 to alanine, glycine, or serine were generated. As seen in Figure 1B, the ComP double mutants were undetectable. Based on the lack of detectable expression for the various ComP point mutant variants, the cysteine residues located at positions 75 and 95 are likely to form a disulfide bond adjacent to the glycosylation site of serine 84. Blocking the formation of this disulfide bond appears to be detrimental to protein stability and protein glycosylation.

[0113] Example 2. Determination of a short PglS-dependent O-linked recognition motif by a reductive cloning strategy. ComP lacking the first 28 amino acids 110264 (ComPΔ28 herein 110264A translational fusion containing it (referred to as) fused to the C-terminus of a gene-inactivated variant of Pseudomonas aeruginosa-derived exotoxin A protein (EPA) was demonstrated to be efficiently glycosylated by PglS with multiple pneumococcal and K. pneumoniae capsular polysaccharides (Harding CM, et al. (2019) A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host. Nat Commun 10(1):891; Feldman MF, et al. (2019) A promising bioconjugate vaccine against hypervirulent Klebsiella pneumoniae. Proc Natl Acad Sci U S A). For the purpose of shortening and defining the minimal recognition site required for PglS-dependent glycosylation, ComP 110264 A shortened cloning strategy was pursued to translationally fuse the fragment to the C-terminus of the EPA protein between a glycine-glycine-glycine-serine (GGGS) linker and a hexahistidine tag (Figure 2). Specifically, ComP 110264 Multiple constructs containing either 25, 30, 35, 40, or 45 amino acid fragments of ComP 110264 were generated. Each fragment was shifted one amino acid at a time towards the stop codon of ComP 110264 regardless of size. As an example, the first construct contained a 25 amino acid fragment of ComP 110264 spanning residues 67 to 91, the second construct contained a 25 amino acid fragment of ComP 110264 spanning residues 68 to 92, and the third construct contained a 25 amino acid fragment of ComP ADP1 spanning residues 69 to 93, etc. All fragments contained serine 82, which is the PglS glycosylation site. The EPA fusion constructs were then introduced into E. coli SDB1 that co-expresses PglS

[0114] As can be seen in Figure 3, for three constructs containing a short fragment of 25 amino acids of ComP 110264 when analyzed by Western blot, as identified by a decrease in electrophoretic mobility and the presence of multiple glycoforms (observed as a ladder-like distribution above the non-glycosylated protein), it was found that they were glycosylated with pneumococcal CPS8 by PglS ADP1 . As a positive control, an EPA fusion containing the ComPΔ 28110264 fragment was included. This is because this protein has already been established to be glycosylated with CPS8 by PglS APD1 . Notably, it was found that only three 25-amino acid constructs were glycosylated: C1 (SEQ ID NO: 32), D1 (SEQ ID NO: 33), and E1 (SEQ ID NO: 34). All of these contained conserved cysteine residues predicted to form disulfide bonds adjacent to the ComP 110264 glycosylation site (serine 82).

[0115] As can be seen in Figures 3 and 4, for seven constructs containing a 30-amino acid fragment of ComP 110264 , it was found that they were glycosylated with pneumococcal CPS8 by PglS ADP1 : E2 (SEQ ID NO: 41), F2 (SEQ ID NO: 42), G2 (SEQ ID NO: 43), H2 (SEQ ID NO: 44), A3 (SEQ ID NO: 45), B3 (SEQ ID NO: 46), C3 (SEQ ID NO: 47). All seven constructs contained conserved cysteine residues predicted to form disulfide bonds adjacent to the ComP 110264 glycosylation site.

[0116] As can be seen in Figures 5 and 6, for nine constructs containing a 35-amino acid fragment of ComP 110264 , PglS ADP1It was found to be glycosylated with pneumococcal CPS8 at: D4 (SEQ ID NO: 55), E4 (SEQ ID NO: 56), F4 (SEQ ID NO: 57), G4 (SEQ ID NO: 58), A5 (SEQ ID NO: 59), B5 (SEQ ID NO: 60), D5 (SEQ ID NO: 61), E5 (SEQ ID NO: 62), F5 (SEQ ID NO: 63). All nine constructs were ComP 110264 were predicted to contain conserved cysteine residues that form disulfide bonds adjacent to the glycosylation site.

[0117] As can be seen in Figures 6 and 7, ComP 110264 For eight constructs containing the 40-amino acid fragment of, PglS ADP1 It was found to be glycosylated with pneumococcal CPS8 at: H6 (SEQ ID NO: 72), B7 (SEQ ID NO: 73), C7 (SEQ ID NO: 74), D7 (SEQ ID NO: 75), E7 (SEQ ID NO: 76), F7 (SEQ ID NO: 77), A8 (SEQ ID NO: 78), B8 (SEQ ID NO: 79). All eight constructs were ComP 110264 were predicted to contain conserved cysteine residues that form disulfide bonds adjacent to the glycosylation site.

[0118] As can be seen in Figures 8, 9, and 10, ComP 110264 For ten constructs containing the 45-amino acid fragment of, PglS ADP1 It was found to be glycosylated with pneumococcal CPS8 at: A10 (SEQ ID NO: 92), B10 (SEQ ID NO: 93), C10 (SEQ ID NO: 94), D10 (SEQ ID NO: 95), F10 (SEQ ID NO: 96), G10 (SEQ ID NO: 97), H10 (SEQ ID NO: 98), A11 (SEQ ID NO: 99), B11 (SEQ ID NO: 100), C11 (SEQ ID NO: 101). Again, all ten constructs were ComP 110264 were predicted to contain conserved cysteine residues that form disulfide bonds adjacent to the glycosylation site.

[0119] Based on the data shown in FIGS. 3, 4, 5, 6, 7, 8, 9, and 10, when translationally fused to the C-terminus of the EPA carrier protein, the cysteine residues located at positions 71 and 93 are essential for glycosylation by PglS ADP1 . Also, the methionine residue located at position 104 appears to block glycosylation by PglS ADP1 when it is part of the C-terminal glycosylation tag. This is particularly supported by the fact that constructs G5 (SEQ ID NO: 64), C8 (SEQ ID NO: 80), and D11 (SEQ ID NO: 102), which each contain the essential cysteines at positions 71 and 93, did not show any signs of glycosylation. G5 (SEQ ID NO: 64), C8 (SEQ ID NO: 80), and D11 (SEQ ID NO: 102) each contain ComP 110264 fragments that are 35, 40, and 45 amino acids in length, respectively, but each fragment has a methionine at the terminus at position 104, and when this residue is included in the C-terminal glyco-tag, this is demonstrated to be sufficient to block glycosylation. Moreover, all constructs containing methionine 104 in addition to the cysteines at positions 71 and 93 did not show any signs of glycosylation (G5 (SEQ ID NO: 64), H5 (SEQ ID NO: 65), C8 (SEQ ID NO: 80), D8 (SEQ ID NO: 81), E8 (SEQ ID NO: 82), F8 (SEQ ID NO: 83), G8 (SEQ ID NO: 84), H8 (SEQ ID NO: 85), A9 (SEQ ID NO: 86), D11 (SEQ ID NO: 102), E11 (SEQ ID NO: 103), F11 (SEQ ID NO: 104), H11 (SEQ ID NO: 105), A12 (SEQ ID NO: 106), B12 (SEQ ID NO: 107), C12 (SEQ ID NO: 108), D12 (SEQ ID NO: 109), E12 (SEQ ID NO: 110), F12 (SEQ ID NO: 111), G12 (SEQ ID NO: 112). Table 1 presents a list of all ComP ADP1 fragments tested for their ability to function as an O-linked glycosylation recognition motif by PglS 110264 .

Table 1-1

Table 1-2

[0120] The O-linked glycosylation recognition motif can be glycosylated by PglS when translationally fused at the N-terminus, either in series at the N- or C-terminus, or simultaneously at the N- and C-termini. Based on the data presented above, the D5 (SEQ ID NO: 61) fragment of ComP ADP1 was selected for the tracking experiment. As outlined in FIGS. 12A and 12B, in the tracking experiment, the D5 (SEQ ID NO: 61) fragment or its derivative (D5’) was translationally fused at the N- and C-termini in various combinations. As a positive control, an EPA fusion containing the ComPΔ28 110264 fragment was included. This is because this protein has already been established to be glycosylated with CPS8 by PglS 110264 . Subsequently, the EPA fusion constructs were introduced into E. coli SDB1 that co-expresses pneumococcal CPS8 in the presence or absence of PglS APD1 . As seen in FIG. 12C, all of the EPA-ComP110264 fusion constructs were glycosylated with pneumococcal CPS8. This indicates that the O-linked glycosylation recognition motif of ComP110264 can be translationally fused to the N- or C-terminus in multiple combinations and still be glycosylated by PglS ADP1 . ADP1

[0121] Example 3. Double ComPΔ28 fused in series at the C-terminus 110264 The glycosylation tag is glycosylated by PglS ADP1 . An EPA fusion construct containing a double ComPΔ28 110264 glycosylation tag fused in series at the C-terminus was constructed. ComPΔ28 110264 ​The glycosylation tag was separated by either a glycine-glycine-glycine-glycine-serine (GGGS) linker (SEQ ID NO: 23) or a proline-alanine-proline-alanine-proline (PAPAP) linker (SEQ ID NO: 25). Both constructs contained a hexahistidine tag to assist in downstream purification. As a positive control, ComPΔ28 110264 fragments-containing EPA fusions were included. This is because this protein has already been established to be glycosylated by PglS APD1 at CPS8. The dual-tagged EPA fusion constructs were then introduced into E. coli SDB1, which co-expresses pneumococcal CPS8, in the presence or absence of PglS ADP1 . As seen in Figure 13, both EPA variant 7 and EPA variant 8 were glycosylated with pneumococcal CPS8 when PglS ADP1 was present. Moreover, the glycosylation appeared to be of very high molecular weight and the immunoreactivity was close to the 250 kDa marker.

Number

[0122] The present disclosure is not intended to be limited by the specific embodiments or prior examples described, which are intended as single illustrations of the individual aspects of the present disclosure, and any composition or method that is functionally equivalent is within the scope of the present disclosure. Indeed, various modifications of the present disclosure will become apparent to those skilled in the art from the foregoing description and the accompanying drawings in addition to those shown and described herein. Such modifications are intended to fall within the scope of the appended claims.

[0123] All publications and patent applications mentioned herein are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference herein.

[0124] References

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Claims

1. A bioconjugate comprising an oligosaccharide or polysaccharide covalently bound to a fusion protein, wherein the fusion protein comprises a ComP protein (ComP) glycosylation tag, wherein the ComP glycosylation tag has a length of 18 to 50 amino acids, The ComP glycosylation tag includes both a cysteine residue corresponding to the conserved cysteine residue at position 71 of SEQ ID NO: 2 (ComP １１０２６４ : ENV58402.1) and a cysteine residue corresponding to the conserved cysteine residue at position 93 of SEQ ID NO:

2. The fusion protein is glycosylated with an oligosaccharide or a polysaccharide at a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 2 of the ComP glycosylation tag, and the ComP glycosylation tag does not contain a methionine residue corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP １１０２６４ : ENV58402.1), and does not contain a methionine residue corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP The fusion protein of the bioconjugate does not contain a methionine residue at a position that may correspond to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP １１０２６４ : ENV58402.1) in relation to the ComP glycosylation tag. wherein the ComP glycosylation tag consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 32 [C1], SEQ ID NO: 33 [D1], SEQ ID NO: 34 [E1], SEQ ID NO: 41 [E2], SEQ ID NO: 42 [F2], SEQ ID NO: 43 [G2], SEQ ID NO: 44 [H2], SEQ ID NO: 45 [A3], SEQ ID NO: 46 [B3], SEQ ID NO: 47 [C3], SEQ ID NO: 55 [D4], SEQ ID NO: 56 [E4], SEQ ID NO: 57 [F4], SEQ ID NO: 58 [G4], SEQ ID NO: 59 [A5], SEQ ID NO: 60 [B5], SEQ ID NO: 61 [D5], SEQ ID NO: 62 [E5], SEQ ID NO: 63 [F5], SEQ ID NO: 72 [H6], SEQ ID NO: 73 [B7], SEQ ID NO: 74 [C7], SEQ ID NO: 75 [D7], SEQ ID NO: 76 [E7], SEQ ID NO: 77 [F7], SEQ ID NO: 78 [A8], SEQ ID NO: 79 [B8], SEQ ID NO: 92 [A10], SEQ ID NO: 93 [B10], SEQ ID NO: 94 [C10], SEQ ID NO: 95 [D10], SEQ ID NO: 96 [F10], SEQ ID NO: 97 [G10], SEQ ID NO: 98 [H10], SEQ ID NO: 99 [A11], SEQ ID NO: 100 [B11], and SEQ ID NO: 101 [C11], or consists of these variants containing one or two amino acid substitutions, said bioconjugate.

2. The bioconjugate according to claim 1, wherein the oligosaccharide or polysaccharide is produced by bacteria of the genus Streptococcus.

3. The bioconjugate according to claim 2, wherein the polysaccharide is a capsular polysaccharide, and the capsular polysaccharide is CPS14, CPS8, CPS9V, or CPS15b.

4. The bioconjugate according to claim 1, wherein the oligosaccharide or polysaccharide is produced by bacteria of the genus Klebsiella.

5. The bioconjugate according to claim 4, wherein the polysaccharide is the capsular polysaccharide of Klebsiella pneumoniae serotype K1 or serotype K2.

6. The bioconjugate according to claim 1, wherein the oligosaccharide or polysaccharide contains glucose at its reducing end.

7. The bioconjugate according to claim 1, for use as a conjugate vaccine.

8. The bioconjugate according to claim 7, wherein the conjugate vaccine is a vaccine against Streptococcus pneumoniae serotype 8.

9. A fusion protein comprising a ComP glycosylation tag and an isolated fragment of the ComP protein, wherein the fragment comprises a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 2 (ComP110264: ENV58402.1), and both a cysteine residue corresponding to the conserved cysteine residue at position 71 of SEQ ID NO: 2 and a cysteine residue corresponding to the conserved cysteine residue at position 93 of SEQ ID NO: 2, wherein the ComP glycosylation tag is 18 to 50 amino acids in length, The ComP glycosylation tag does not contain a methionine residue corresponding to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP １１０２６４ : ENV58402.1); and, In relation to the ComP glycosylation tag, the fusion protein does not contain a methionine residue at a position that may correspond to the conserved methionine residue at position 104 of SEQ ID NO: 2 (ComP １１０２６４ : ENV58402.1), wherein the ComP glycosylation tag consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 32 [C1], SEQ ID NO: 33 [D1], SEQ ID NO: 34 [E1], SEQ ID NO: 41 [E2], SEQ ID NO: 42 [F2], SEQ ID NO: 43 [G2], SEQ ID NO: 44 [H2], SEQ ID NO: 45 [A3], SEQ ID NO: 46 [B3], SEQ ID NO: 47 [C3], SEQ ID NO: 55 [D4], SEQ ID NO: 56 [E4], SEQ ID NO: 57 [F4], SEQ ID NO: 58 [G4], SEQ ID NO: 59 [A5], SEQ ID NO: 60 [B5], SEQ ID NO: 61 [D5], SEQ ID NO: 62 [E5], SEQ ID NO: 63 [F5], SEQ ID NO: 72 [H6], SEQ ID NO: 73 [B7], SEQ ID NO: 74 [C7], SEQ ID NO: 75 [D7], SEQ ID NO: 76 [E7], SEQ ID NO: 77 [F7], SEQ ID NO: 78 [A8], SEQ ID NO: 79 [B8], SEQ ID NO: 92 [A10], SEQ ID NO: 93 [B10], SEQ ID NO: 94 [C10], SEQ ID NO: 95 [D10], SEQ ID NO: 96 [F10], SEQ ID NO: 97 [G10], SEQ ID NO: 98 [H10], SEQ ID NO: 99 [A11], SEQ ID NO: 100 [B11], and SEQ ID NO: 101 [C11], or consists of variants thereof comprising one or two amino acid substitutions. The fusion protein.

10. The fusion protein according to claim 9, comprising a carrier protein or a fragment thereof selected from the group consisting of diphtheria toxoid CRM197, tetanus toxoid, Pseudomonas aeruginosa exotoxin A (EPA), tetanus toxin C fragment, cholera toxin B subunit, and Haemophilus influenza protein D.

11. The fusion protein according to claim 9, wherein the ComP glycosylation tag is located at the N-terminus of the fusion protein, the C-terminus of the fusion protein, and / or inside the fusion protein.

12. The fusion protein according to claim 9, comprising two or more ComP glycosylation tags.

13. The fusion protein according to claim 12, comprising 2 to 20 ComP glycosylation tags.

14. The fusion protein according to claim 12, wherein the ComP glycosylation tags are identical.

15. The fusion protein according to claim 12, wherein at least two of the ComP glycosylation tags are different from each other.

16. An isolated nucleic acid encoding the fusion protein according to claim 9.

17. The isolated nucleic acid according to claim 16, which is a vector.

18. A host cell comprising the isolated nucleic acid according to claim 17.

19. A conjugate vaccine comprising the bioconjugate according to claim 1 and an adjuvant.

20. A conjugate vaccine used for inducing a host immune response against a pathogenic microorganism, administered in an effective amount to a subject in need of said immune response, the conjugate vaccine according to claim 19.

21. A conjugate vaccine used for preventing or treating a bacterial disease and / or an infectious disease in a subject, administered to a subject in need of prevention or treatment, the conjugate vaccine according to claim 19.

22. A method for producing a pneumococcal conjugate vaccine against pneumococcal infection, comprising: (a) isolating the bioconjugate according to claim 1; and (b) combining the isolated bioconjugate with an adjuvant. A method comprising the above steps.

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