O-linked glycosylation recognition motifs

Abstract

To provide a bioconjugate comprising an oligo- or polysaccharide covalently linked to a fusion protein.SOLUTION: The present invention provides a bioconjugate comprising an oligo- or polysaccharide covalently linked to a fusion protein, wherein the fusion protein comprises a ComP protein (ComP) glycosylation tag, wherein the ComP glycosylation tag comprises a cysteine residue conserved in a specific amino acid sequence, and the fusion protein is glycosylated with an oligo- or polysaccharide at a serine residue conserved in a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims priority to U.S. Provisional Application No. 62 / 783,971, filed December 21, 2018.

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

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

[0004] Government Funding Statement This invention was made with Government support under Grant No. R41 AI142928-01 awarded by the National Institute of Allergy and Infectious Diseases (NIAID). The Government has certain rights in this invention. [Background technology]

[0005] The first general protein glycosylation pathway in bacteria, the Campylobacter jejuni N-linked glycosylation system, 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, many diverse prokaryotic glycosylation systems have been characterized, including O-linked glycosylation systems that have no homologous counterparts in eukaryotes (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 engineered 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 an emerging biotechnological tool that uses E. coli or other Gram-negative organisms as hosts and exploits prokaryotic glycosylation systems for recombinant production of glycosylated proteins. Currently, glycoengineering utilizes two major approaches to recombinantly glycosylate proteins: oligosaccharyltransferase (OTase)-dependent and OTase-independent approaches, both of which can generate N- or O-linked glycosylation.

[0006] Protein glycosylation, the covalent attachment of carbohydrates to proteins, is a ubiquitous post-translational modification. In most cases, protein glycosylation is specified as either N-linked, where the glycan is attached to an asparagine residue, or O-linked, where the glycan is attached to a serine or threonine residue. Although the importance of eukaryotic glycosylation has been and continues to be the reason for intensive research, prokaryotic glycosylation has only recently attracted 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). Since first being discovered in C. jejuni, prokaryotic glycosylation systems have been described across a large number of Gram-negative and Gram-positive bacteria and have demonstrated contributions 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 straightforward nature of prokaryotic genes, it was only a matter of time before protein glycosylation systems were engineered and harnessed to generate designer glycoproteins in a process called "bacterial glycoengineering."

[0007] Bacteria have developed an N-linked OTase pathway similar to eukaryotic glycosylation, but also employ a prokaryotic unique 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 to proteins.

[0008] Bacterial surface polysaccharides are some 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 in the form of capsules or O antigens bound to lipid A, serve multiple purposes including protecting the microorganism from external threats and immune elimination. Given their abundance on invading organisms and their biochemical differences from eukaryotic carbohydrates, some of the 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 therefore 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 the age of 2 years. This poor immune response could potentially be overcome by covalently linking polysaccharides to protein carriers 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 RevI mmunol 14(7):505-514).

[0009] Traditionally, conjugate vaccines are synthesized using a semisynthetic approach, in which polysaccharides are extracted from the target microorganism, purified, chemically modified, and covalently conjugated to a carrier protein. This approach has led to the commercial approval of several conjugate vaccines to prevent colonization and infection by Haemophilus influenzae type B, as well as several serotypes of Streptococcus pneumoniae and Neisseria meningiditis. For a detailed review on the production of semisynthetic or synthetic conjugate 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 Pfizer's top selling product from 2015 to 2018, with sales exceeding USD 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 last two decades, alternative strategies to produce conjugate vaccines have emerged. These techniques vary in approach, with some resulting in vaccines closer to commercially licensed vaccines than others. Specifically, the emergence of in vivo bacterial conjugation to produce conjugate vaccines has resulted in some of the most clinically advanced products to date. In vivo conjugation of polysaccharides and proteins for the production of conjugate vaccines, 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 a simplification of the generation and production process of conjugate polysaccharide vaccines (Rappuoli R, De Gregorio E, & Costantino P (2019) On the mechanisms of conjugate vaccines. Proc Natl Acad Sci USA 116(1):14-16).

[0011] Both N-linked and O-linked OTases have been used in the biological conjugation of polysaccharides with carrier proteins for the production of conjugated polysaccharide vaccines. Regardless of which OTase is used, biological conjugation in any Gram-negative bacterium relies on three components: the locus or loci encoding the polysaccharide biosynthetic 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 third class of O-linked OTase was used to produce a bioconjugate vaccine (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 third class of OTase, named PglS, naturally glycosylates the pilin-like protein, ComP, in a manner similar to 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 demonstrated that PglS is in fact a pilin-specific OTase, likely glycosylating only ComP, since 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). Although initially characterized as a PglL orthologue from the environmental bacterium Acinetobacter baylyi strain ADP1, PglS is in fact phylogenetically distinct from the PglL protein.Acinetobacter strains 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 Neisseria species (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 Acinetobacter also encode the PilO OTase, making Acinetobacter the only known genus of bacteria that possesses 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 (pilin target of PglL) or PilA (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 substrates transferred by PglS. PglS is the only OTase known that can transfer polysaccharides with glucose at the reducing end, either N- or O-linked. Many pathogens are 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 USA 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), produce a capsule that contains a polysaccharide with glucose at the reducing end and therefore may be a target for the development of PglS-dependent bioconjugate vaccines.Indeed, PglS was used to generate a polyvalent pneumococcal bioconjugate vaccine against serotypes 8, 9V, and 14 (all with glucose at the reducing end) using the natural acceptor ComP as a carrier protein. Also, a ComP fragment lacking the first 28 amino acids could function as a glycotag when translationally fused to the C-terminus of P. 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 oligo- or polysaccharide covalently attached to a fusion protein, wherein the fusion protein comprises a ComP protein (ComP) glycosylation tag, wherein the ComP glycosylation tag comprises 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 wherein the fusion protein is glycosylated with the oligo- or polysaccharide of the ComP glycosylation tag at a serine residue corresponding to position 82 of SEQ ID NO:2 or a serine residue corresponding to position 84 of SEQ ID NO:1. In certain embodiments, the ComP glycosylation tag does not include a methionine residue that corresponds 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 that corresponds or most likely corresponds 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 disclosure, the ComP glycosylation tag is 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[B 10], 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 the variant maintains the serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO:1.

[0016] The disclosure provides a ComP glycosylation tag comprising an isolated fragment of a ComP protein, wherein 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, wherein 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 described in claim 42, wherein the amino acid sequence of the ComP glycosylation tag does not continue C-terminally beyond the amino acid residue corresponding to position 103 of SEQ ID NO: 2 (ComP110264:ENV58402.1).

[0017] Provided herein are fusion proteins that include the ComP glycosylation tags of the disclosure.

[0018] Also provided herein is an in vivo method for conjugating an oligosaccharide or polysaccharide to an acceptor polypeptide, the method comprising using a PglS oligosaccharyltransferase (OTase) to covalently conjugate an oligosaccharide or polysaccharide to an acceptor polypeptide, wherein the acceptor polypeptide comprises a ComP glycosylation tag of the 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 proteins required for oligosaccharide or polysaccharide synthesis, (b) a PglS OTase, and (3) an acceptor polypeptide comprising the ComP glycosylation tag of the disclosure.

[0020] Also provided herein are isolated nucleic acids encoding ComP glycosylation tags and / or fusion proteins of the disclosure, as well as host cells containing the isolated nucleic acids.

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

[0022] A method for 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 bacterial disease and / or 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] [Figure 1]We show that the cysteine ​​residue immediately adjacent to serine 84 in ComP derived from ADP1 of Acinetobacter baylyi (ComPADP1) contributes to PglS-dependent glycosylation and ComP stability. (A) The amino acid sequence of ComPADP1 from amino acid residues 75 to 95, in which two cysteine ​​residues are adjacent to serine 84, the site of PglS-dependent glycosylation, is shown. (B) Point mutations replacing either cysteine ​​75, cysteine ​​95, or both cysteines 75 and 95 with alanine, glycine, or serine negatively affect ComP stability and block glycosylation of serine 84 with the Campylobacter jejuni heptasaccharide by PglS. Western blot analysis of E. coli whole cell lysates co-expressing PglS, the C. jejuni heptasaccharide, and variants of ComPADP1. E. coli strains expressing the single mutations C95A, C95G, and C95S, and the double mutations C75A / C95A, C75A / C95G, C75A / C95S, C75G / C95A, C75G / C95G, and C75G / C95S all had ComP levels below the lower limit of detection, indicating the intrinsic instability of these mutant proteins and the importance of cysteines 75 and 95. [Diagram 2] 1 shows a schematic diagram of a recombinant fusion protein containing a C-terminal fragment of ComP from Acinetobacter soli strain 110264 (referred to herein as ComP110264). [Diagram 3]Figure 1 shows glycosylation by PglSADP1 of a recombinant fusion protein composed of a ComP110264 fragment containing cysteine ​​residues at positions 71 and 93, adjacent to the previously established glycosylation site at serine 82. (A) Western blot analysis of E. coli whole cell lysates co-expressing fusion proteins containing PglSADP1, pneumococcal serotype 8 capsular polysaccharide, and a ComP110264 fragment. PglSADP1 was only able to glycosylate the recombinant fusion protein containing the fragment of ComP110264 containing cysteine ​​71, serine 82, and cysteine ​​93. Specifically, fusion proteins C1, D1, and E1 were found to be glycosylated as indicated by immunoreactive bands running at higher molecular weights. The "+" sample (SEQ ID NO: 29) serves as a positive control, as it has been previously shown that this fusion protein containing the ComP110264 fragment consisting of amino acids 29-145 is efficiently glycosylated by PglSADP1. (B) Diagrammatic format summarizing the definition of the ComP110264 fragment used in the recombinant fusion glycosylation experiments and the Western blot observations regarding the presence or absence of glycosylation. For illustrative purposes, serine 82, a known PglS-dependent glycosylation site, is in bold underlined type. [Figure 4]Glycosylation of recombinant fusion proteins composed of ComP110264 fragments containing cysteine ​​residues at positions 71 and 93 by PglSADP1, adjacent to the previously established glycosylation site at serine 82. (A) Western blot analysis of E. coli whole cell lysates co-expressing fusion proteins containing PglSADP1, pneumococcal serotype 8 capsular polysaccharide, and ComP110264 fragments. PglSADP1 was only able to glycosylate recombinant fusion proteins containing the fragment of ComP110264 containing cysteine ​​71, serine 82, and cysteine ​​93. Specifically, fusion proteins E2, F2, G2, H2, A3, B3, and C3 were found to be glycosylated as indicated by immunoreactive bands running at higher molecular weights. The "+" sample (SEQ ID NO: 29) serves as a positive control, as it has been previously shown that this fusion protein containing the ComP110264 fragment consisting of amino acids 29-145 is efficiently glycosylated by PglSADP1. (B) Diagrammatic format summarizing the definition of the ComP110264 fragment used in the recombinant fusion glycosylation experiments and the Western blot observations regarding the presence or absence of glycosylation. For illustrative purposes, serine 82, a known PglS-dependent glycosylation site, is in bold underlined type. [Diagram 5]Figure 1 shows glycosylation by PglSADP1 of a recombinant fusion protein composed of a ComP110264 fragment containing cysteine ​​residues at positions 71 and 93, adjacent to the previously established glycosylation site at serine 82. (A) Western blot analysis of E. coli whole cell lysates co-expressing PglSADP1, pneumococcal serotype 8 capsular polysaccharide, and a fusion protein containing a ComP110264 fragment. PglSADP1 was only able to glycosylate the recombinant fusion protein containing the fragment of ComP110264 containing cysteine ​​71, serine 82, and cysteine ​​93. Specifically, fusion proteins D4, E4, F4, G4, A5, B5, D5, and E5 were found to be glycosylated as indicated by immunoreactive bands running at higher molecular weights. The "+" sample (SEQ ID NO: 29) serves as a positive control, as it has been previously shown that this fusion protein containing the ComP110264 fragment consisting of amino acids 29-145 is efficiently glycosylated by PglSADP1. (B) Diagrammatic format summarizing the definition of the ComP110264 fragment used in the recombinant fusion glycosylation experiments and the Western blot observations regarding the presence or absence of glycosylation. For illustrative purposes, serine 82, a known PglS-dependent glycosylation site, is in bold underlined type. [Figure 6]Figure 2 shows glycosylation of recombinant fusion proteins composed of ComP110264 fragments containing cysteine ​​residues at positions 71 and 93 by PglSADP1. (A) Western blot analysis of E. coli whole cell lysates co-expressing PglSADP1, pneumococcal serotype 8 capsular polysaccharide, and fusion proteins containing ComP110264 fragments. PglSADP1 was only able to glycosylate recombinant fusion proteins containing the fragment of ComP110264 containing cysteine ​​71, serine 82, and cysteine ​​93. Specifically, fusion proteins F5 and H6 were found to be glycosylated as indicated by immunoreactive bands running at higher molecular weights. The "+" sample (SEQ ID NO: 29) serves as a positive control, as it has been previously shown that this fusion protein containing the ComP110264 fragment consisting of amino acids 29-145 is efficiently glycosylated by PglSADP1. (B) Diagrammatic format summarizing the definition of ComP110264 fragments used in recombinant fusion glycosylation experiments and the Western blot observations for the presence or absence of glycosylation. For illustration, serine 82, a known PglS-dependent glycosylation site, is in bold underlined type. [Figure 7]Figure 2 shows that PglSADP1 glycosylation is blocked by methionine at position 104, even in the presence of cysteine ​​71, serine 82, and cysteine ​​93. (A) Western blot analysis of E. coli whole cell lysates co-expressing fusion proteins containing PglSADP1, pneumococcal serotype 8 capsular polysaccharide, and a fragment of ComP110264. PglSADP1 was only able to glycosylate the recombinant fusion protein containing the fragment of ComP110264 that contained cysteine ​​71, serine 82, and cysteine ​​93, and lacked methionine 104. Specifically, fusion proteins B7, C7, D7, E7, F7, A8, and B8 were found to be glycosylated, as indicated by immunoreactive bands running at higher molecular weights. The "+" sample (SEQ ID NO: 29) serves as a positive control, as it has been previously shown that this fusion protein containing the ComP110264 fragment consisting of amino acids 29-145 is efficiently glycosylated by PglSADP1. (B) Diagrammatic format summarizing the definition of the ComP110264 fragment used in the recombinant fusion glycosylation experiments and the Western blot observations regarding the presence or absence of glycosylation. For illustrative purposes, serine 82, a known PglS-dependent glycosylation site, is in bold underlined type. [Figure 8]We show that glycosylation of serine 82 by PglSADP1 is not blocked by the presence of multiple methionine residues 5' of cysteine ​​71 and cysteine ​​93. (A) Western blot analysis of E. coli whole cell lysates co-expressing fusion proteins containing PglSADP1, pneumococcal serotype 8 capsular polysaccharide, and a fragment of ComP110264. PglSADP1 was only able to glycosylate the recombinant fusion protein containing the fragment of ComP110264 that contained cysteine ​​71, serine 82, and cysteine ​​93, and lacked methionine 104. Specifically, fusion proteins A10 and B10 were found to be glycosylated, as indicated by the immunoreactive bands running at higher molecular weights. The "+" sample (SEQ ID NO: 29) serves as a positive control, as it has been previously shown that this fusion protein containing the ComP110264 fragment consisting of amino acids 29-145 is efficiently glycosylated by PglSADP1. (B) Diagrammatic format summarizing the definition of the ComP110264 fragment used in the recombinant fusion glycosylation experiments and the Western blot observations regarding the presence or absence of glycosylation. For illustrative purposes, serine 82, a known PglS-dependent glycosylation site, is in bold underlined type. [Figure 9]We show that glycosylation of serine 82 by PglSADP1 is not blocked by the presence of multiple methionine residues 5' of cysteine ​​71 and cysteine ​​93. (A) Western blot analysis of E. coli whole cell lysates co-expressing fusion proteins containing PglSADP1, pneumococcal serotype 8 capsular polysaccharide, and a fragment of ComP110264. PglSADP1 was only able to glycosylate the recombinant fusion protein containing the fragment of ComP110264 that contained cysteine ​​71, serine 82, and cysteine ​​93 and lacked methionine 104. Specifically, fusion proteins C10, D10, F10, G10, H10, A11, B11, and C11 were found to be glycosylated as indicated by immunoreactive bands running at higher molecular weights. The "+" sample (SEQ ID NO: 29) serves as a positive control, as it has been previously shown that this fusion protein containing the ComP110264 fragment consisting of amino acids 29-145 is efficiently glycosylated by PglSADP1. (B) Diagrammatic format summarizing the definition of the ComP110264 fragment used in the recombinant fusion glycosylation experiments and the Western blot observations regarding the presence or absence of glycosylation. For illustrative purposes, serine 82, a known PglS-dependent glycosylation site, is in bold underlined type. [Figure 10]It shows that glycosylation of serine 82 by PglSADP1 is blocked by the presence of methionine at position 104. (A) Western blot analysis of E. coli whole cell lysates co-expressing PglSADP1, pneumococcal serotype 8 capsular polysaccharide, and fusion proteins containing a fragment of ComP110264. PglSADP1 was only able to glycosylate the recombinant fusion protein containing the fragment of ComP110264 that contains cysteine ​​71, serine 82, and cysteine ​​93, and lacks methionine 104. Specifically, none of the fusion proteins was found to be glycosylated by PglSADP1. The "+" sample (SEQ ID NO: 29) serves as a positive control, as it has already been shown that this fusion protein containing the fragment of ComP110264 consisting of amino acids 29-145 is efficiently glycosylated by PglSADP1. (B) Diagrammatic format summarizing the definition of ComP110264 fragments used in recombinant fusion glycosylation experiments and the Western blot observations for the presence or absence of glycosylation. For illustration, serine 82, a known PglS-dependent glycosylation site, is in bold underlined type. [Figure 11A] Figure 1 shows that ComP110264 fragments exhibit efficient glycosylation with serotype 8 pneumococcal capsular polysaccharide by PglSADP1. Western blot analysis of E. coli whole cell lysates co-expressing fusion proteins containing PglSADP1, pneumococcal serotype 8 capsular polysaccharide, and fragments of ComP110264. Western blots were performed in duplicate and probed with either anti-exotoxin A antiserum (A) or anti-His antiserum (B). All of the different ComP110264 fragments showed similar levels of glycosylation as indicated by immunoreactive bands running at higher molecular weights. All fragments contained cysteine ​​71, serine 82, and cysteine ​​93. [Figure 11B]Figure 1 shows that ComP110264 fragments exhibit efficient glycosylation with serotype 8 pneumococcal capsular polysaccharide by PglSADP1. Western blot analysis of E. coli whole cell lysates co-expressing fusion proteins containing PglSADP1, pneumococcal serotype 8 capsular polysaccharide, and fragments of ComP110264. Western blots were performed in duplicate and probed with either anti-exotoxin A antiserum (A) or anti-His antiserum (B). All of the different ComP110264 fragments showed similar levels of glycosylation as indicated by immunoreactive bands running at higher molecular weights. All fragments contained cysteine ​​71, serine 82, and cysteine ​​93. [Figure 11C] The ComP110264 fragments used in the recombinant fusion glycosylation experiments and the Western blot observations regarding the presence or absence of glycosylation are summarized in tabular form. For illustrative purposes, serine 82, a known PglS-dependent glycosylation site, is in bold underlined type. [Figure 12] N- or C-terminal O-linked glycosylation motifs translationally fused to EPA carrier proteins are glycosylated in the presence of PglSADP1. (A) Legend defining the properties of each EPA carrier fusion protein used in this experiment. Six different fusion proteins were used as represented by the presence of a single O-linked glycosylation tag or dual glycosylation tags. (B) D5 and D5' ComP110264 amino acid fragment sequences. (C) Western blot analysis of E. coli whole cell lysates co-expressing pneumococcal CPS8 and fusion carrier proteins in the presence or absence of PglSADP1. The D5 and D5' glycosylation motifs, whether N-terminal, C-terminally in tandem, or at both the N- and C-termini, were all glycosylated only in the presence of PglSADP1. [Figure 13]Two ComP110264 fragments translationally fused in tandem to the C-terminus of a carrier protein are glycosylated with high molecular weight polysaccharides. Fusion proteins were purified from E. coli co-expressing pneumococcal serotype 8 capsular polysaccharide in the presence or absence of PglSADP1. (A) Western blot analysis of nickel affinity purified EPA fusion proteins probed with anti-His antibody shows both the nonglycosylated EPA carrier protein and the higher molecular weight EPA carrier protein glycosylated with pneumococcal CPS8. (B) Western blot analysis of nickel affinity purified EPA fusion proteins probed with anti-CPS8 antibody shows the presence of CPS8 polysaccharide only in samples co-expressing PglSADP1. We also show that the EPA carrier protein, which contains two ComP110264 fragments lacking the first 28 amino acids (ComPΔ28110264) separated by either glycine-glycine-glycine-serine (GGGS; SEQ ID NO: 23) or proline-alanine-proline-alanine-proline (PAPAP; SEQ ID NO: 25) linkers, is glycosylated with high molecular weight pneumococcal CPS8. (C) Merged Western blot image of 13A and 13B shows both anti-His (red channel) and anti-CPS8 (green channel). [Figure 14] Figure 1 shows that PglS (C), but not PglB (B) or PglL (A), can bind pneumococcal CPS14 to its cognate acceptor / carrier protein. Western blot analysis of E. coli whole cell lysates probing hexahistidine-tagged acceptor protein variants. [Figure 15]Figure 1 shows that PglS from A. baylyi ADP1 (PglSADP1) can transfer multiple pneumococcal capsular polysaccharides from A. baylyi ADP1 (ComPADP1) to ComP. Western blot analysis of hexahistidine-tagged ComPADP1 variants and purified ComPADP1 variants probing either pneumococcal CPS8 (left), CPS9V (middle), or CPS14 (right). Colocalization of anti-His and anti-glycan signals indicates that ComPADP1 was glycosylated with the correct pneumococcal polysaccharides. Asterisks indicate samples treated with proteinase K for 2 h. [Figure 16] Figure 1 shows that PglSADP1 can transfer K. pneumoniae K1 and K2 capsular polysaccharides to ComPADP1. Western blot analysis of E. coli whole cell lysates probing hexahistidine-tagged ComPADP1 variants and RNA polymerase. RNA polymerase was used as a loading control. [Figure 17A] 1 shows mass spectrometry of CPS14-ComPADP1 in which a single glycosylated peptide was identified: ISASNATTNVATAT (SEQ ID NO: 22). [Figure 17B] Mass spectrometry analysis of CPS14-ComPADP1 in which a single glycosylated peptide was identified. [Figure 18] Serine 84 of ComPADP1 is shown to be a PglS-dependent glycosylation site. Western blot analysis of E. coli whole cell lysates probing hexahistidine-tagged ComPADP1 variants and the Campylobacter jejuni heptasaccharide. The ComP[S84A]ADP1 variant was expressed but not glycosylated, as shown by the absence of a reactive band probing with anti-hR6 heptasaccharide antiserum. [Figure 19] The amino acid sequences of ComP orthologues are listed, with predicted glycosylation sites shown in bold, flanked by predicted disulfide bonds (underlined) linking the predicted alpha-beta loop to the beta strand region. [Figure 20]We show that PglSADP1, but not PglS110264, efficiently glycosylates both the cognate ComPADP1 and ComP110264 from A. soli CIP110264. Western blot analysis of E. coli whole cell lysates probing hexahistidine-tagged ComP variants and RNA polymerase. RNA polymerase was used as a loading control. [Figure 21] We show that PglSADP1 efficiently glycosylates the DsbA-ComPΔ28110264 fusion but not the DsbA-ComPΔ28ADP1 fusion. All fusions had either a triple alanine peptide (AAA; SEQ ID NO:24) or a glycine-glycine-glycine-serine peptide (GGGS; SEQ ID NO:23) linking DsbA to either hexahistidine-tagged ComPΔ28110264 or ComPΔ28ADP1. Western blot analysis of E. coli whole cell lysates probing hexahistidine-tagged ComP variants and RNA polymerase. RNA polymerase was used as a loading control. [Figure 22] We show that PglSADP1 efficiently glycosylates the MBP-ComPΔ28110264 fusion but not the MBP-ComPΔ28ADP1 fusion. All fusions had either a triple alanine peptide (AAA; SEQ ID NO:24) or a glycine-glycine-glycine-serine peptide (GGGS; SEQ ID NO:23) linking maltose binding protein (MBP) to either hexahistidine-tagged ComPΔ28110264 or ComPΔ28ADP1. Western blot analysis of E. coli whole cell lysates probing hexahistidine-tagged ComP variants and RNA polymerase. RNA polymerase was used as a loading control. [Figure 23]PglSADP1, but not PglS110264, is efficient against the EPA-GGGS-ComPΔ28110264 fusion. Western blot analysis of E. coli whole cell lysates or periplasmic extracts probing hexahistidine-tagged ComP variants. Exotoxin A with glycine-glycine-glycine-serine peptide (GGGS; SEQ ID NO: 23) linking EPA-GGGS-hexahistidine-tagged ComPΔ28110264 variants. [Figure 24] The amino acid sequence of a representative ComPΔ28110264 fusion protein is shown. [Figure 25A] 1 shows that monovalent CPS14-ComPADP1 bioconjugate vaccine induces serotype-specific IgG antibodies. [Figure 25B] 1 shows that monovalent CPS14-ComPADP1 bioconjugate vaccine induces serotype-specific IgG antibodies. [Figure 25C] 1 shows that monovalent CPS14-ComPADP1 bioconjugate vaccine induces serotype-specific IgG antibodies. [Figure 26] 1 shows that trivalent bioconjugate vaccines against serotypes 8, 9V, and 14 induce serotype-specific IgG titers at levels comparable to PREVNAR 13. [Figure 27] The ComP Δ28 ortholog amino acid sequence is listed, with amino acids corresponding to the 28 N-terminal amino acids of SEQ ID NO:1 (ComPADP1:AAC45886.1) removed. Predicted glycosylation sites are shown in bold, flanked by predicted disulfide bonds (underlined) linking the predicted alpha-beta loop to the beta strand region. [Figure 28] An alignment of the region of the ComP sequence including the serine (S) residue (boxed) corresponding to the serine residue at position 84 of SEQ ID NO:1 (ComPADP1:AAC45886.1) is shown. [Figure 29]High-energy collision dissociation (HCD) fragmentation spectrum of the CPS14-ComP bioconjugate digested with GluC. CPS14-ComP digested with GluC was subjected to HCD fragmentation, allowing the confirmation of a single peptide derived from semi-GluC bound to a glycan at the CPS14 repeating subunit. Additional glycopeptides decorated with extended glycans corresponding to up to four tetrasaccharide repeating units were also observed. [Diagram 30] Figure 1 shows a high-energy collision dissociation (HCD) fragmentation spectrum of GluC-digested ComP glycosylated with a heptasaccharide (ComP-glycan Cj) from C. jejuni. GluC-digested ComP-glycan Cj was subjected to HCD fragmentation, allowing the confirmation of a single peptide attached to the glycan at the CPS14 repeating subunit. A low collision energy regime was implemented to confirm the glycosylation of peptide ISASNATTNVATAT (SEQ ID NO: 22) with a 1380.53 Da glycan corresponding to 6*HexNAc, 1*Hexose. [Diagram 31] Figure 1 shows a high-energy collision dissociation (HCD) fragmentation spectrum of GluC-digested ComP glycosylated with a heptasaccharide (ComP-glycan Cj) from C. jejuni. GluC-digested ComP-glycan Cj was subjected to HCD fragmentation, allowing the confirmation of a single peptide attached to the glycan at the CPS14 repeating subunit. A high collision energy regime was implemented to confirm the glycosylation of peptide ISASNATTNVATAT (SEQ ID NO: 22) with a 1380.53 Da glycan corresponding to 6*HexNAc, 1*Hexose. [Diagram 32]We show that the oligosaccharyltransferase PglS can glycosylate the acceptor protein ComP with the pneumococcal CPS14 polysaccharide. E. coli SDB1 cells co-expressing the acceptor protein (DsbA, AcrA, or ComP), OTase (PglL, PglB, or PglS), and CPS14 polysaccharide were analyzed for protein glycosylation by Western blot analysis of affinity purified acceptor proteins. (A-C): DsbA purified from SDB1 cells in the presence or absence of PglL. (A): Hexahistidine-tagged DsbA probed with the anti-His channel. (B): Probing with the anti-glycan channel of CPS14. (C): Merged image of panels A and B. (D-F): AcrA purified from SDB1 cells in the presence or absence of PglB. (D): Probing of hexahistidine-tagged AcrA with the anti-His channel. (E): Probing of CPS14 with the anti-glycan channel. (F): Merged image of panels D and E. (GI): ComP purified from SDB1 cells in the presence or absence of PglS. (G): Probing of hexahistidine-tagged ComP with the anti-His channel. (H): Probing of CPS14 with the anti-glycan channel. (I): Merged image of panels G and H. Asterisks indicate samples treated with proteinase K for 1 h at 55°C. [Figure 33A] High-energy collision dissociation (HCD) fragmentation spectra of the CPS14-ComP bioconjugate digested with GluC. CPS14-ComP digested with GluC was subjected to HCD fragmentation, allowing the identification of a single peptide attached to a glycan at the CPS14 repeating subunit. A high collision energy regime (A) and a low collision energy regime (B) were performed to confirm the glycosylation of the peptide ISASNATTNVATAT (SEQ ID NO: 22) with a 1378.47 Da glycan corresponding to HexNAc2Hexose6. [Figure 33B]High-energy collision dissociation (HCD) fragmentation spectra of the CPS14-ComP bioconjugate digested with GluC. CPS14-ComP digested with GluC was subjected to HCD fragmentation, allowing the identification of a single peptide attached to a glycan at the CPS14 repeating subunit. A high collision energy regime (A) and a low collision energy regime (B) were performed to confirm the glycosylation of the peptide ISASNATTNVATAT (SEQ ID NO: 22) with a 1378.47 Da glycan corresponding to HexNAc2Hexose6. [Diagram 34] Western blot analysis of CPS8-ComP and CPS9V-ComP glycoproteins. SDB1 cells of E. coli were prepared co-expressing ComP, PglS, and either pneumococcal CPS8 or CPS9V. Affinity purified glycosylated ComP from each strain was analyzed for protein glycosylation by Western blot analysis. (AC): Western blot analysis of CPS8-ComP bioconjugates compared to ComP alone. (A): Hexahistidine-tagged ComP purified from SDB1 expressing CPS8 in the presence or absence of PglS was probed with the anti-His channel. (B): CPS8 was probed with the anti-glycan channel. (C): Merged image of panels A and B. (D-F): Western blot analysis of CPS9V-ComP bioconjugates compared to ComP alone. (D): Hexahistidine-tagged ComP purified from SDB1 expressing CPS9V in the presence or absence of PglS was probed with the anti-His channel. (E): CPS9V was probed with the anti-glycan channel. (F): Merged image of panels D and E. Asterisks indicate samples treated with proteinase K for 1 h at 55 °C. [Figure 35A]IgG responses of mice vaccinated with ComP, PREVNAR 13®, monovalent CPS14-ComP bioconjugate, and trivalent CPS8- / CPS9V- / CPS14-ComP biconjugate. Groups of mice were vaccinated with ComP alone, PREVNAR 13®, monovalent CPS14-ComP bioconjugate vaccine, or CPS8- / CPS9V- / CPS14-ComP biconjugate vaccine. Serum was collected on day 49 and analyzed for serotype-specific IgG responses by ELISA compared to sera collected on day 0. (A-C): No detectable increase in IgG responses was detected in placebo-vaccinated mice with serotypes 8 (A), 9V (B), or 14 (C). An unpaired t-test (Mann-Whitney) was performed to statistically analyze pre-immune sera from day 49 sera. P value in each case tested was ****p=0.0001. Each point represents a single vaccinated mouse. Error bars indicate standard deviation of the mean. [Figure 35B] IgG responses of mice vaccinated with ComP, PREVNAR 13®, monovalent CPS14-ComP bioconjugate, and trivalent CPS8- / CPS9V- / CPS14-ComP biconjugate. Groups of mice were vaccinated with ComP alone, PREVNAR 13®, monovalent CPS14-ComP bioconjugate vaccine, or CPS8- / CPS9V- / CPS14-ComP biconjugate vaccine. Serum was collected on day 49 and analyzed for serotype-specific IgG responses by ELISA compared to sera collected on day 0. (A-C): No detectable increase in IgG responses was detected in placebo-vaccinated mice with serotypes 8 (A), 9V (B), or 14 (C). An unpaired t-test (Mann-Whitney) was performed to statistically analyze pre-immune sera from day 49 sera. P value in each case tested was ****p=0.0001. Each point represents a single vaccinated mouse. Error bars indicate standard deviation of the mean. [Figure 35C]IgG responses of mice vaccinated with ComP, PREVNAR 13®, monovalent CPS14-ComP bioconjugate, and trivalent CPS8- / CPS9V- / CPS14-ComP biconjugate. Groups of mice were vaccinated with ComP alone, PREVNAR 13®, monovalent CPS14-ComP bioconjugate vaccine, or CPS8- / CPS9V- / CPS14-ComP biconjugate vaccine. Serum was collected on day 49 and analyzed for serotype-specific IgG responses by ELISA compared to sera collected on day 0. (A-C): No detectable increase in IgG responses was detected in placebo-vaccinated mice with serotypes 8 (A), 9V (B), or 14 (C). An unpaired t-test (Mann-Whitney) was performed to statistically analyze pre-immune sera from day 49 sera. P value in each case tested was ****p=0.0001. Each point represents a single vaccinated mouse. Error bars indicate standard deviation of the mean. [Figure 35D] IgG responses of mice vaccinated with ComP, PREVNAR 13®, monovalent CPS14-ComP bioconjugate, and trivalent CPS8- / CPS9V- / CPS14-ComP biconjugate. Groups of mice were vaccinated with ComP alone, PREVNAR 13®, monovalent CPS14-ComP bioconjugate vaccine, or CPS8- / CPS9V- / CPS14-ComP biconjugate vaccine. Serum was collected on day 49 and analyzed for serotype-specific IgG responses by ELISA compared to sera collected on day 0. (D-F): In PREVNAR 13® vaccinated mice, no increase in IgG response titers against serotype 8 (D) was detected, but there was an increase in IgG responses specific to serotypes 9V (E) and 14 (F). An unpaired t-test (Mann-Whitney) was performed to statistically analyze pre-immune sera from day 49 sera. P value in each case tested was ****p=0.0001. Each point represents a single vaccinated mouse. Error bars indicate standard deviation of the mean. [Figure 35E] IgG responses of mice vaccinated with ComP, PREVNAR 13®, monovalent CPS14-ComP bioconjugate, and trivalent CPS8- / CPS9V- / CPS14-ComP biconjugate. Groups of mice were vaccinated with ComP alone, PREVNAR 13®, monovalent CPS14-ComP bioconjugate vaccine, or CPS8- / CPS9V- / CPS14-ComP biconjugate vaccine. Serum was collected on day 49 and analyzed for serotype-specific IgG responses by ELISA compared to sera collected on day 0. (D-F): In PREVNAR 13® vaccinated mice, no increase in IgG response titers against serotype 8 (D) was detected, but there was an increase in IgG responses specific to serotypes 9V (E) and 14 (F). An unpaired t-test (Mann-Whitney) was performed to statistically analyze pre-immune sera from day 49 sera. P value in each case tested was ****p=0.0001. Each point represents a single vaccinated mouse. Error bars indicate standard deviation of the mean. [Fig. 35F]IgG responses of mice vaccinated with ComP, PREVNAR 13®, monovalent CPS14-ComP bioconjugate, and trivalent CPS8- / CPS9V- / CPS14-ComP biconjugate. Groups of mice were vaccinated with ComP alone, PREVNAR 13®, monovalent CPS14-ComP bioconjugate vaccine, or CPS8- / CPS9V- / CPS14-ComP biconjugate vaccine. Serum was collected on day 49 and analyzed for serotype-specific IgG responses by ELISA compared to sera collected on day 0. (D-F): In PREVNAR 13® vaccinated mice, no increase in IgG response titers against serotype 8 (D) was detected, but there was an increase in IgG responses specific to serotypes 9V (E) and 14 (F). An unpaired t-test (Mann-Whitney) was performed to statistically analyze pre-immune sera from day 49 sera. P value in each case tested was ****p=0.0001. Each point represents a single vaccinated mouse. Error bars indicate standard deviation of the mean. [Figure 35G]IgG responses of mice vaccinated with ComP, PREVNAR 13®, monovalent CPS14-ComP bioconjugate, and trivalent CPS8- / CPS9V- / CPS14-ComP bioconjugate. Groups of mice were vaccinated with ComP alone, PREVNAR 13®, monovalent CPS14-ComP bioconjugate vaccine, or CPS8- / CPS9V- / CPS14-ComP bioconjugate vaccine and serum was collected on day 49 and analyzed for serotype-specific IgG responses by ELISA compared to serum collected on day 0. (GI): In mice vaccinated with the CPS14-ComP bioconjugate vaccine, there was no detectable increase in IgG titers specific for serotype 8 (G) or 9V (H), but there was a statistically significant increase in IgG responses to serotype 14 (I). An unpaired t-test (Mann-Whitney) was performed to statistically analyze pre-immune sera from day 49 sera. The P value in each case tested was ****p=0.0001. Each point represents a single vaccinated mouse. Error bars indicate the standard deviation of the mean. [Fig. 35H]IgG responses of mice vaccinated with ComP, PREVNAR 13®, monovalent CPS14-ComP bioconjugate, and trivalent CPS8- / CPS9V- / CPS14-ComP bioconjugate. Groups of mice were vaccinated with ComP alone, PREVNAR 13®, monovalent CPS14-ComP bioconjugate vaccine, or CPS8- / CPS9V- / CPS14-ComP bioconjugate vaccine and serum was collected on day 49 and analyzed for serotype-specific IgG responses by ELISA compared to serum collected on day 0. (GI): In mice vaccinated with the CPS14-ComP bioconjugate vaccine, there was no detectable increase in IgG titers specific for serotype 8 (G) or 9V (H), but there was a statistically significant increase in IgG responses to serotype 14 (I). An unpaired t-test (Mann-Whitney) was performed to statistically analyze pre-immune sera from day 49 sera. The P value in each case tested was ****p=0.0001. Each point represents a single vaccinated mouse. Error bars indicate the standard deviation of the mean. [Fig. 35I]IgG responses of mice vaccinated with ComP, PREVNAR 13®, monovalent CPS14-ComP bioconjugate, and trivalent CPS8- / CPS9V- / CPS14-ComP bioconjugate. Groups of mice were vaccinated with ComP alone, PREVNAR 13®, monovalent CPS14-ComP bioconjugate vaccine, or CPS8- / CPS9V- / CPS14-ComP bioconjugate vaccine and serum was collected on day 49 and analyzed for serotype-specific IgG responses by ELISA compared to serum collected on day 0. (GI): In mice vaccinated with the CPS14-ComP bioconjugate vaccine, there was no detectable increase in IgG titers specific for serotype 8 (G) or 9V (H), but there was a statistically significant increase in IgG responses to serotype 14 (I). An unpaired t-test (Mann-Whitney) was performed to statistically analyze pre-immune sera from day 49 sera. The P value in each case tested was ****p=0.0001. Each point represents a single vaccinated mouse. Error bars indicate the standard deviation of the mean. [Fig. 35J]IgG responses of mice vaccinated with ComP, PREVNAR 13®, monovalent CPS14-ComP bioconjugate, and trivalent CPS8- / CPS9V- / CPS14-ComP bioconjugate. Groups of mice were vaccinated with ComP alone, PREVNAR 13®, monovalent CPS14-ComP bioconjugate vaccine, or CPS8- / CPS9V- / CPS14-ComP bioconjugate vaccine and serum was collected on day 49 and analyzed for serotype-specific IgG responses by ELISA compared to serum collected on day 0. (J-L): Mice vaccinated with trivalent CPS8- / CPS9V- / CPS14-ComP bioconjugate all showed statistically significant increases in IgG responses in IgG titers to serotypes 8 (J), 9V (K), and 14 (L). An unpaired t-test (Mann-Whitney) was performed to statistically analyze pre-immune sera from day 49 sera. The P value in each case tested was ****p=0.0001. Each point represents a single vaccinated mouse. Error bars indicate the standard deviation of the mean. [Figure 35K]IgG responses of mice vaccinated with ComP, PREVNAR 13®, monovalent CPS14-ComP bioconjugate, and trivalent CPS8- / CPS9V- / CPS14-ComP bioconjugate. Groups of mice were vaccinated with ComP alone, PREVNAR 13®, monovalent CPS14-ComP bioconjugate vaccine, or CPS8- / CPS9V- / CPS14-ComP bioconjugate vaccine and serum was collected on day 49 and analyzed for serotype-specific IgG responses by ELISA compared to serum collected on day 0. (J-L): Mice vaccinated with trivalent CPS8- / CPS9V- / CPS14-ComP bioconjugate all showed statistically significant increases in IgG responses in IgG titers to serotypes 8 (J), 9V (K), and 14 (L). An unpaired t-test (Mann-Whitney) was performed to statistically analyze pre-immune sera from day 49 sera. The P value in each case tested was ****p=0.0001. Each point represents a single vaccinated mouse. Error bars indicate the standard deviation of the mean. [Figure 35L]IgG responses of mice vaccinated with ComP, PREVNAR 13®, monovalent CPS14-ComP bioconjugate, and trivalent CPS8- / CPS9V- / CPS14-ComP bioconjugate. Groups of mice were vaccinated with ComP alone, PREVNAR 13®, monovalent CPS14-ComP bioconjugate vaccine, or CPS8- / CPS9V- / CPS14-ComP bioconjugate vaccine and serum was collected on day 49 and analyzed for serotype-specific IgG responses by ELISA compared to serum collected on day 0. (J-L): Mice vaccinated with trivalent CPS8- / CPS9V- / CPS14-ComP bioconjugate all showed statistically significant increases in IgG responses in IgG titers to serotypes 8 (J), 9V (K), and 14 (L). An unpaired t-test (Mann-Whitney) was performed to statistically analyze pre-immune sera from day 49 sera. The P value in each case tested was ****p=0.0001. Each point represents a single vaccinated mouse. Error bars indicate the standard deviation of the mean. [Diagram 36] Bactericidal activity of sera from mice vaccinated against S. pneumoniae serotypes 8 and 14. Opsonophagocytosis assay (OPA) of sera from mice vaccinated with either buffer control, PREVNAR 13®, or bioconjugate vaccine against both S. pneumoniae serotypes 8 (A) and 14 (B). Serotype-specific commercial rabbit anti-S. pneumoniae serum was used as a positive control. Assays were performed with fresh whole blood of naive mice spiked with 5% (v / v) sample serum and a bacterial MOI of 0.01. Viable counts were performed after 4 hours of incubation. To determine bacterial killing, viable counts from tubes incubated with sample serum were compared to those incubated with control naive mouse serum. Results are expressed as percent killing for individual mice, with bars representing standard deviation of the mean. [Figure 37A]Analysis of EPA glycosylation by CPS8 capsular polysaccharide. Western blot analysis of EPA-CPS8 bioconjugate compared to EPA alone. (A-left panel) Hexahistidine-tagged EPA purified from SDB1 expressing CPS8 in the presence or absence of PglS was probed with the anti-His channel. (A-middle panel) CPS8 was probed with the anti-glycan channel. (A-right) Merged image of left and middle panels. [Figure 37B] Analysis of EPA glycosylation by CPS8 capsular polysaccharide is shown. (B): EPA-CPS8 separated on a Coomassie-stained SDS-polyacrylamide gel. [Figure 37C] Intact protein mass spectrometry showing the MS1 mass spectrum of purified EPA-CPS8. The theoretical mass of the EPA fusion protein is 79,526.15 Daltons and can be observed as a peak at 79,514.76. The EPA fusion protein was also observed in multiple states of mass increase corresponding to the CPS8 repeating subunits with a theoretical mass of 662 Daltons. Various glycoforms of EPA-CPS8 were observed and are represented by "g number", where "g" represents the glycoform and "number" corresponds to the number of repeating CPS8 subunits. The EPA fusion protein was modified with up to 11 repeating subunits of the CPS8 glycan. Panel D presents a zoomed-in view of the various EPA-CPS8 glycoforms. [Figure 37D] A close-up view of the various EPA-CPS8 glycoforms in FIG. 37C is presented. [Figure 38]Analysis of immune responses to ComP-CPS8 and EPA-CPS8 bioconjugates in mice. (A): CPS8 IgG antibody titers in mice immunized with CPS8 bioconjugate vaccine. Mice groups were as follows: EPA (n=9, mice vaccinated with 5 μg total protein), ComP-CPS8 (n=10, mice vaccinated with 5 μg total polysaccharide), and EPA-CPS8 (n=10, mice vaccinated with 100 ng total polysaccharide). On days 1, 14, and 28, all mice were immunized with 100 μL of vaccine diluted 1:1 in Imject Alum Adjuvant. Serum was collected on day 4. For titration, ELISA plates were coated with whole cell serotype 8 pneumococci and incubated with serial two-fold dilutions of sera. Titers of individual mice are shown, horizontal bars represent standard error of the mean. Statistically significant titers compared to the EPA placebo group are indicated with an asterisk and were determined using Kruskal-Wallis one-way analysis of variance. **, P=0.0223 and ****, P<0.0001. For analytical and presentation purposes, negative titer values ​​(<100) were given an arbitrary value of 10. (B): Opsonophagocytic killing of S. pneumoniae serotype 8 by day 42 sera from mice immunized with ComP-CPS8 and EPA-CPS8 bioconjugate vaccines. The same groups of mice described for IgG titers were used for OPA. Assays were performed by adding 40% (vol / vol) samples of serum and a bacterial MOI of 0.01 to fresh whole blood from naive mice. Results are expressed as percent killing of individual mice, with horizontal bars representing the standard deviation of the mean. Statistically significant kills compared to the EPA placebo group are indicated with an asterisk and were determined using Kruskal-Wallis one-way ANOVA. **, P=0.0015. [Figure 39A]We show that the conserved homologous serines are the likely glycosylation sites for ComP proteins from ADP1 of A. baylyi and 110264 of A. soli. Serines 82 and 84 in ComPADP1 and the homologous serines 79 and 82 in ComP110264 were mutated to alanine and probed for glycosylation in the presence of PglS and serotype 8 capsular polysaccharide. SDB1 cells expressing ComP variants in the presence of PglS and CPS8 were probed by western blotting for protein glycosylation. ComP expression and probed with anti-His channel for glycosylation. [Figure 39B] We show that the conserved homologous serines are the likely glycosylation sites for ComP proteins from ADP1 of A. baylyi and 110264 of A. soli. Serines 82 and 84 in ComPADP1 and the homologous serines 79 and 82 in ComP110264 were mutated to alanine and probed for glycosylation in the presence of PglS and serotype 8 capsular polysaccharide. SDB1 cells expressing ComP variants in the presence of PglS and CPS8 were probed by western blotting for protein glycosylation. ComP expression and probed with anti-His channel for glycosylation. [Figure 39C] We show that the conserved homologous serines are the likely glycosylation sites for ComP proteins from ADP1 of A. baylyi and 110264 of A. soli. Serines 82 and 84 in ComPADP1 and the homologous serines 79 and 82 in ComP110264 were mutated to alanine and probed for glycosylation in the presence of PglS and serotype 8 capsular polysaccharide. SDB1 cells expressing ComP variants in the presence of PglS and CPS8 were probed by western blotting for protein glycosylation. ComP expression and probed with anti-His channel for glycosylation. [Diagram 40]Analysis of EPA glycosylation by Klebsiella pneumoniae K1 and K2 capsular polysaccharides. Western blot analysis of purified (A) unglycosylated EPA, (B) EPA glycosylated by K. pneumoniae K1 capsular polysaccharide, or (C) EPA glycosylated by K. pneumoniae K2 capsular polysaccharide. "g0" indicates unglycosylated EPA fusion, and "gn" indicates EPA fusion glycosylated with the different sizes of K1 or K2 repeating subunits, as shown in panels B or C, respectively. [Figure 41A] Figure 1 shows an intact protein mass analysis showing the MS1 mass spectrum of purified EPA-K2. The theoretical mass of the EPA fusion protein is 79,526.15 Daltons and can be observed as a peak at 79,518.73. The EPA fusion protein was also observed in multiple states of mass increase corresponding to the repeating subunits of the K2 capsular polysaccharide of K. pneumoniae with a theoretical mass of 662 Daltons. Various glycoforms of EPA-K2 were observed and are represented by "g number", where "g" represents the glycoform and "number" corresponds to the number of repeating K2 subunits. The EPA fusion protein was modified with up to 11 repeating subunits of the K2 capsule. [Figure 41B] Figure 1 shows an intact protein mass analysis showing the MS1 mass spectrum of purified EPA-K2. The theoretical mass of the EPA fusion protein is 79,526.15 Daltons and can be observed as a peak at 79,518.73. The EPA fusion protein was also observed in multiple states of mass increase corresponding to the repeating subunits of the K2 capsular polysaccharide of K. pneumoniae with a theoretical mass of 662 Daltons. Various glycoforms of EPA-K2 were observed and are represented by "g number", where "g" represents the glycoform and "number" corresponds to the number of repeating K2 subunits. The EPA fusion protein was modified with up to 11 repeating subunits of the K2 capsule. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0027] It will be understood by any reader of this written description that the exemplary aspects and embodiments described and claimed herein may be suitably practiced in the absence of any of the recited features, elements, or steps, whether or not specifically disclosed herein.

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

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

[0030] Whenever an embodiment is described herein with the words "comprising" or "comprises," it is understood that analogous embodiments described with 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 herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0032] Numeric ranges are inclusive of the numbers defining the range. Unless expressly identified by "and any range therebetween," or a list of values ​​is set forth, e.g., 1, 2, 3, or 4, the disclosure specifically includes any range between the values, e.g., 1 to 3, 1 to 4, 2 to 4, etc., unless otherwise stated.

[0033] The headings provided herein are for ease of reference only and are not limitations of the various aspects or aspects of the disclosure that can be had by reference to the specification as a whole.

[0034] As used herein, the term "non-naturally occurring" material, composition, entity, and / or any combination of materials, compositions, or entities, or any grammatical variations thereof, is a qualified term that expressly excludes, but only excludes, materials, compositions, entities, and / or any combination of materials, compositions, or entities in a form that is well understood by one of ordinary skill in the art as being "naturally occurring" or that may be determined or construed at any time by a judge or administrative or judicial body as being "naturally occurring."

[0035] As used herein, the term "polypeptide" is intended to encompass the singular "polypeptide" and the plural "polypeptides," which 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 specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," 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" may be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the product of post-expression modifications of the polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-standard amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It may be produced in any manner, including by chemical synthesis.

[0036] As used herein, "protein" can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides linked together, for example, by disulfide bonds, hydrogen bonds, or hydrophobic interactions to produce a multimeric protein.

[0037] By "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide may be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of the present invention, as are naturally occurring or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique. An isolated polypeptide or a fragment, variant, or derivative thereof, etc., may be associated, bound, etc. with a cofactor. Similarly, a purified or purified and isolated polypeptide or a fragment, variant, or derivative thereof, etc., may be associated, bound, etc. with a cofactor.

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

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

[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 contain one of a number of "binding domains". As used herein, a "binding domain" is a two-dimensional or three-dimensional polypeptide structure that can specifically bind to a given antigenic determinant, i.e., epitope. A non-limiting example of a binding molecule is an antibody or a fragment thereof that contains a binding domain that specifically binds to an antigenic determinant or epitope. Another example of a binding molecule is a bispecific antibody that contains a first binding domain that binds to a first epitope and a second binding domain that binds to a second epitope.

[0041] The terms "antibody" and "immunoglobulin" can be used interchangeably herein. An antibody (or a fragment, variant, or derivative thereof as disclosed herein) contains at least a variable domain of a heavy chain and at least variable domains of heavy and light chains. Basic immunoglobulin structures in vertebrate systems are relatively well characterized. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988).

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

[0043] "Specifically binds" means that a binding molecule, e.g., an antibody, or a fragment, variant, or derivative thereof, binds to an epitope through 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 is said to "specifically bind" to an epitope when it binds to the epitope through its antigen-binding domain more readily than it would randomly bind to an unrelated epitope. The term "specifically" is used herein to specify the relative affinity with which a particular binding molecule binds to 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 binding molecule "A" can be said to bind to epitope "C" with a higher specificity than it has to the related epitope "D."

[0044] The term "bispecific antibody" as used herein refers to an antibody that has binding sites for two different antigens within a single antibody molecule. In addition to classical antibody structures, it will be understood that other molecules can be constructed with two binding specificities. It will be further understood that antigen binding by bispecific antibodies can be simultaneous or sequential. Triomas and hybrid hybridomas 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)). Bispecific antibodies can also be diabodies.

[0045] The term "polynucleotide" is intended to encompass single and multiple nucleic acids and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds as found in peptide nucleic acids (PNAs)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. An "isolated" nucleic acid or polynucleotide contemplates a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding a polypeptide subunit contained in a vector is considered isolated as disclosed herein. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. Isolated RNA molecules include RNA transcripts of polynucleotides in vivo or in vitro. Isolated polynucleotides or nucleic acids further include such molecules produced synthetically. In addition, a polynucleotide or nucleic acid may be or may 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, is a qualified term that expressly excludes, but only excludes, polynucleotides in a form that is well understood by those of skill in the art as being "naturally occurring" or that may be determined or construed at any time by a judge or administrative or judicial body as being "naturally occurring."

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

[0048] A "vector" is a nucleic acid molecule that is introduced into a host cell, thereby producing a transformed host cell. A vector can contain nucleic acid sequences that allow it to replicate in the host cell, such as an origin of replication. A vector can also contain one or more selectable marker genes and other genetic elements known in the art.

[0049] A "transformed" cell, or a "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 with a viral vector, transformation with a plasmid vector, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. A transformed or host cell can be a bacterial cell or a eukaryotic cell.

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

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

[0052] "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, farm animals, sport animals, and zoo animals, including, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, and the like.

[0053] The term "pharmaceutical composition" refers to a formulation that is in a form that permits the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the composition is administered. Such compositions may be sterile.

[0054] An "effective amount" of an antibody, as disclosed herein, is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and routinely for the stated purpose.

[0055] Overview. Conjugate vaccines consisting of polysaccharides linked to proteins are life-saving prophylactics. Traditionally, conjugate vaccines are produced using chemical methodologies. However, in vivo bacterial conjugation has emerged as an alternative means of production. In vivo conjugation (bioconjugation) relies on oligosaccharyl transferases to attach polysaccharides to proteins. Currently, oligosaccharyl transferases used for bioconjugation are not suitable for the production of conjugate vaccines when the polysaccharide contains glucose at the reducing end. This limitation has major implications, as approximately 75% of Streptococcus pneumoniae capsules contain glucose as the reducing end sugar. Disclosed herein is the use of O-linked oligosaccharide transferases to generate the first multivalent pneumococcal bioconjugate vaccines with polysaccharides containing glucose at their reducing ends. The pneumococcal bioconjugates are immunogenic, protective, and rapidly generated with recombinant techniques. Certain 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 traditional vaccine carriers, such as, in certain embodiments, those that include Pseudomonas aeruginosa exotoxin A protein, establishing a platform to overcome the limitations of other conjugation enzymes and enabling the development of bioconjugate vaccines against many important human and animal pathogens.

[0056] Even with the introduction and implementation of pneumococcal conjugate vaccines over the past two decades, approximately 1.5 million deaths are attributable to S. pneumoniae each year. This is due in part to the more than 90 serotypes of S. pneumoniae and the complex manufacturing methods required to synthesize pneumococcal conjugate vaccines. These factors combine to impede global distribution and development of broader, more protective variants of the vaccine. To expedite development and lower production 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 reliance on chemical conjugation methodologies, allowing for the production of more comprehensive conjugate vaccines.

[0057] Traditional chemical conjugate vaccine synthesis is considered complex, costly, and laborious (Frasch, CEVaccine 27, 6468-6470 (2009)). However, in vivo conjugation has been thoroughly advanced as a viable biosynthetic alternative (Huttner, A. et al. Lancet Infect Dis 17, 528-537 (2017)). These advances are best highlighted by the success of GlycoVaxyn (now LimmaTech Biologics AG, an independent company with direct ties to GlaxoSmithKline). This is a clinical-stage biopharmaceutical company with multiple bioconjugate vaccines in various phases of clinical trials, one of which (Flexyn2a) has completed a phase 2b challenge study. Although GlycoVaxyn is at the forefront of the in vivo conjugation revolution, the ability to glycosylate carrier / acceptor proteins with polysaccharides containing glucose (Glc) as the reducing end sugar has been elusive and, as expected, has hindered the development of pneumococcal bioconjugate vaccines.

[0058] Schulz et al. (PMID 23658772) and Harding et al. 2015 (PMID 26727908) ComP Oligosaccharyltransferase, formerly called PglS, was recently characterized as a functional OTase (Schulz, BL et al. PLoS One 8, e62768 (2013)). Subsequent mass spectrometry studies on total glycopeptides showed that PglS does not act as a general PglL-like OTase, but glycosylates multiple periplasmic and outer membrane proteins (Harding, CM et al. Mol Microbiol 96, 1023-1041 (2015)). Indeed, the genome of A. baylyi ADP1 encodes two OTases, a PglL-like orthologue (UniProtKB / Swiss-Prot:Q6FFS6.1), which acts as a general OTase and PglS (UniProtKB / Swiss-Prot: Q6F7F9.1), which glycosylates the single protein ComP (Harding, CM et al. Mol Microbiol 96, 1023-1041 (2015)).

[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, PM et al. Mol Microbiol 49, 833-847 (2003)), respectively. TfpO and PglL also glycosylate the cognate pilin at serine residues, but the sites of glycosylation differ between each system. TfpO glycosylates the cognate pilin at a C-terminal serine residue that is not present in ComP (Comer, JE, Marshall, MA, Blanch, VJ, Deal, CD & Castric, P. Infect Immun 70, 2837-2845 (2002)). PglL glycosylates PilE at an internal serine at position 63 (Stimson, E. et al. Mol Microbiol 17, 1201-1214 (1995)). ComP also contains a serine residue near position 63, and the surrounding residues show moderate conservation with PilE from N. meningiditis. However, comprehensive glycopeptide analysis revealed that this serine and the surrounding residues are not the glycosylation site of ComP. Here, PglS is a glycosylation site for ComP. ADP1 : AAC4588631 (SEQ ID NO: 1) (ComP 110264 It has been disclosed that PglS glycosylates CoMP at a single serine residue located at a position corresponding to the conserved serine at position 84 of ENV58402.1 (which also corresponds to the conserved serine at position 82 of SEQ ID NO:2), a novel glycosylation site not previously seen within the type IV pilin superfamily. The ability of PglS to transfer polysaccharides containing glucose as the reducing end sugar, combined with the identification of a novel site of glycosylation within the pilin superfamily, demonstrates that PglS is a functionally distinct OTase from PglL and TfpO.

[0060] PglS, but not PglB or PglL, transferred polysaccharides containing glucose at the reducing end to the acceptor protein ComP. Two classes of OTases, PglB and PglL, have been used previously for in vivo conjugation (Feldman, MF et al. Proc Natl Acad Sci USA 102, 3016-3021 (2005); Faridmoayer, A., Fentabil, MA, Mills, DC, Klassen, JS & Feldman, MF J Bacteriol 189, 8088-8098(2007)). PglB, the first OTase described, preferentially transfers glycans containing an acetamide group at the C-2 position of the reducing end (i.e., N-acetylglucosamine), which 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 S. enterica Typhimurium O antigen, can be transferred by engineered PglB variants (Ihssen, J. et al. Open Biol 5, 140227(2015)). The second described OTase, PglL from N. meningiditis, has a more relaxed substrate specificity than PglB and naturally transfers polysaccharides with an acetamide group at C-2 and polysaccharides containing galactose (Gal) at the reducing end (Faridmoayer, A., Fentabil, MA, Mills, DC, Klassen, JS & Feldman, MF J Bacteriol 189, 8088-8098 (2007); Pan, C. et al. MBio 7 (2016)). However, no evidence is available for PglB- or PglL-mediated transfer of polysaccharides containing glucose (Glc) at the reducing end, which is particularly interesting considering that the majority of pneumococcal CPS contain glucose at the reducing end (Geno, KA et al. Clin Microbiol Rev 28, 871-899 (2015)).The abilities 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)), respectively, were tested. As can be seen in Figures 14A and 14B, although both acceptor proteins were expressed, no evidence of CPS14 glycosylation onto either acceptor protein was observed.

[0061] Acinetobacter species have been described to contain three O-linked OTases; a general PglL OTase involved in the glycosylation of multiple proteins, and two pilin-specific OTases (Harding, CM Mol Microbiol 96, 1023-1041 (2015)). The first pilin-specific OTase is an orthologue of TfpO (also known as PilO) and is not used in in vivo conjugation systems, as it cannot transfer polysaccharides with more than one repeating unit (Faridmoayer, A., Fentabil, MA, Mills, DC, Klassen JS & Feldman, MFJ Bacteriol 189, 8088-8098(2007)). The second pilin-specific OTase, PglS, is linked to a single protein, the type IV pilin ComP. 28Bioinformatics analysis showed that PglS is the prototype of a distinct family of OTases. Given that PglS is a new class of O-OTase, its ability to transfer pneumococcal CPS14 to its cognate acceptor protein, ComP (Harding, CM et al. Mol Microbiol 96, 1023-1041 (2015)) was tested. As seen in Figure 14C, co-expression of the CPS14 biosynthetic locus in combination with hexahistyl-tagged variants of PglS and ComP resulted in a typical ladder-like banding pattern compatible with protein glycosylation when analyzed by Western blotting (Figure 14B). The high molecular weight modal distribution of the signals indicates protein glycosylation with repeating glycan subunits of increasing molecular weight. Taken together, these results indicate that PglS, unlike previously characterized OTases, can transfer polysaccharides containing glucose at the reducing end.

[0062] There are more than 90 serotypes of S. pneumoniae (Geno, KA et al. Clin Microbiol Rev 28, 871-899 (2015)). Many increasingly prevalent serotypes, such as serotypes 8, 22F, and 33F, are not currently included in licensed vaccines. Therefore, we tested the versatility of PglS to generate a multivalent pneumococcal bioconjugate vaccine against two serotypes included in Prevnar 13 (serotypes 9V and 14) and one serotype not included (serotype 8) (Package Insert - Prevnar 13 - FDA, available on the World Wide Web at (fda.gov / downloads / BiologicsBloodVaccines / Vaccines / ApprovedProducts / UCM201669.pdf). Importantly, all three of these capsular polysaccharides contain glucose as the reducing end sugar (Geno, KA 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, hexahis-tagged ComP variants, and either CPS8, CPS9V, or CPS14 produced CPS-specific bioconjugates. Furthermore, antisera specific for either CPS8, CPS9V, or CPS14 antigens also reacted with the anti-His reactive band, indicating that ComP-His was glycosylated with the correct polysaccharide. To confirm that the purified material was not contaminated with lipid-linked polysaccharides, samples were treated with proteinase K and a loss of signal was observed when analyzed by Western blotting, confirming that the bioconjugates were proteinaceous.

[0063] Thus, it was demonstrated that PglS, but not PglB and PglL, can transfer polysaccharides of S. pneumoniae to ComP. Specifically, PglS is the only OTase known in the world that can transfer polysaccharides containing glucose at the reducing end. In certain embodiments, PglS can be used to transfer lipid-linked oligosaccharides or polysaccharides containing glucose at the reducing end (collectively referred to herein as "oligosaccharides or polysaccharides") to ComP or a fusion protein containing a fragment of ComP.

[0064] PglS can transfer Klebsiella capsular polysaccharides to ComP. Klebsiella pneumoniae, a Gram-negative opportunistic human pathogen, produces capsular polysaccharides known to be important for virulence. To date, at least 79 antigenically distinct capsular polysaccharides have been reported for Klebsiella species (Pan, YJ 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, YJ et al. Sci Rep 5, 15573 (2015)). To determine whether PglS can transfer K. pneumoniae capsular polysaccharide to ComP, genes encoding proteins required for synthesis of K1 or K2 capsular polysaccharide were cloned into the IPTG-inducible pBBR1MCS-2 vector (Kovach, ME et al. Gene 166, 175-176(1995)). The K1 capsule locus was cloned from K. pneumoniae NTUHK-2044, a previously characterized K1 capsule producer (Wu, KM et al. J Bacteriol 191, 4492-4501(2009)). The K2 capsule locus was cloned from K. pneumoniae 52.145, a previously characterized K2 capsule producer (Lery, LM et al. BMC Biol 12, 41(2014)). The K1 or K2 capsular polysaccharide expression plasmids were then individually introduced into E. coli coexpressing the PglS OTase and the acceptor protein ComP from separate plasmid vectors.To enhance expression of the K1- and K2-specific polysaccharides, the K. pneumoniae transcriptional activator rmpA from K. pneumoniae NTUHK-2044 was subsequently cloned into the pACT3 (Dykxhoorn, DM, St Pierre, R. & Linn, T. Gene 177, 133-136(1996)) low-copy, IPTG-inducible vector, since it had previously been characterized as a regulator of the K. pneumoniae capsule (Arakawa, Y. et al. Infect Immun 59, 2043-2050(1991)); Yeh, KM et al. J Clin Microbiol 45, 466-471(2007)). Introduction of the rmpA gene into E. coli strains co-expressing PglS and hexahistyl-tagged ComP variants and either the K1 or K2 capsular polysaccharide from K. pneumoniae resulted in strong expression and detection of macromolecular ComP bioconjugates, as shown by the typical ladder-like pattern of bands, compatible with protein glycosylation, when analyzed by Western blotting (Figure 16B). The modal distribution of signals indicates protein glycosylation with repeating glycan subunits of increasing molecular weight. Thus, taken together, PglS was able to glycosylate ComP with the K1 and K2 capsular polysaccharides from K. pneumoniae. An increase in conjugation efficiency was observed upon co-expression of the transcriptional activator rmpA from K. pneumoniae.

[0065] PglS can transfer K. pneumoniae polysaccharides to ComP. Given that most K. pneumoniae capsular polysaccharides contain glucose as the reducing end sugar, other commercially approved OTases (PglB and PglL) alone should not be able to use these polysaccharides to generate conjugate vaccines. Furthermore, co-expression of the transcriptional activator RmpA with the capsule gene cluster enhanced capsule expression to detectable levels. In certain embodiments, the method for producing Klebsiella conjugates can be used to generate pan-Klebsiella conjugate vaccines encompassing all serotypes, including other species such as K. varricola, K. michiganensis, and K. oxytoca.

[0066] Mass spectrometry and site-directed mutagenesis confirmed that PglS is an O-linked OTase, and ComP is a ComP ADP1It was confirmed that N-glycosylation of ribozyme A is glycosylated at the serine residue corresponding to position 84 of the ribozyme A. Bacterial N-glycosylation generally occurs within the sequon DXNST (SEQ ID NO: 21), where X is any amino acid except proline (Kowarik, M. et al. EMBO J 25, 1957-1966 (2006)). In contrast, O-glycosylation does not appear to follow a defined sequon. Most O-glycosylation events in bacterial proteins occur in low-complexity (LCR) regions that are rich in serine, alanine, and proline (Vik, A. et al. Proc Natl Acad Sci USA 106, 4447-4452 (2009)). Alternatively, some pilins are O-glycosylated at a C-terminal serine residue (Comer, JE, Marshall, MA, Blanch, VJ, Deal, CD & Castric, P. Infect Immun 70, 2837-2845(2002)). Because ComP does not appear to have obvious LCRs or C-terminal serine residues homologous to those found in other pilin-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 Figure 17A and Figure 17B, a single glycopeptide consisting of the peptide ISASNATTNVATAT (SEQ ID NO: 22) was identified attached to a glycan matching the published CPS14 composition (Geno, KA et al. Clin Microbiol Rev 28, 871-899(2015)). To be able to ascertain both the peptide and the attached glycan sequence, multiple collision energy regimes were performed to characterize the HexNA C2 Glycosylation of the semi-GluC-derived peptide ISASNATTNVATAT (SEQ ID NO: 22) with a 1378.47 Da glycan corresponding to Hexose6 was confirmed (Figure 17B). Additional glycopeptides decorated with extended glycans corresponding to up to four tetrasaccharide repeating units were also observed (Figure 29).

[0067] It has been previously shown that Acinetobacter species glycosylate proteins primarily at serine residues, and therefore it was hypothesized that either serine (S) 82 or 84, numbered in SEQ ID NO:1, was the site of glycosylation (Scott, NE 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, since glycosylation can be easily detected with the hR6 anti-glycan antiserum and by increased electrophoretic mobility (Schwarz, F. et al. Nat Chem Biol 6, 264-266 (2010)). As shown in Figure 18, wild-type hexaHis-tagged ComP was glycosylated with the C. jejuni heptasaccharide, as indicated by increased electrophoretic mobility when co-expressed with PglS and co-localization with the hR6 antiserum signal. MS analysis also confirmed the presence of the C. jejuni heptasaccharide in the same semi-GluC-derived peptide ISASNATTNVATAT (SEQ ID NO: 22) modified with CPS14 (Figures 30 and 31). As a negative control, a catalytically inactive PglS mutant (H324A) was generated that was unable to glycosylate wild-type ComP when co-expressed with the C. jejuni heptasaccharide glycan. Site-directed mutagenesis was performed and it was observed that glycosylation of ComP with the C. jejuni heptasaccharide was abolished in the ComP[S84A] mutant, whereas ComP[S82A] was glycosylated at wild-type levels. Taken together, these results indicate that ComP is singly glycosylated by PglS at serine 84 (numbered in SEQ ID NO:1), a unique site distinct from other previously characterized pilin-like proteins, which corresponds to serine 82 numbered in SEQ ID NO:2.

[0068] Bioinformatics characterization of ComP pilin orthologues. ComP was first described as a factor required for natural transformation of Acinetobacter baylyi ADP1 (Porstendorfer, D., Drotschmann, U. & Averhoff, B. Appl Environ Microbiol 63, 4150-4157(1997)). Subsequent studies have demonstrated that A. baylyi ADP1 (referred to herein as ComP ADP1 It was shown that ComP from the chromosome 10 (hereafter referred to as ComP) is glycosylated by a novel OTase, PglS, located immediately downstream of ComP, rather than the general OTase PglL located elsewhere on the chromosome (Harding, CM et al. Mol Microbiol 96, 1023-1041 (2015)). ADP1 The protein (NCBI identifier AAC45886.1) belongs to a family of proteins called type IV pilins. Specifically, ComP shares homology with the major pilins of type IVa (Giltner, CL, Nguyen, Y. & Burrows, LLMicrobiol Mol Biol Rev 76, 740-772(2012)). Type IVa pilins share high sequence homology at the N-terminus, which encodes a highly conserved leader sequence and an N-terminal alpha helix, but the C-terminus shows significant divergence between genera and even within species (Giltner, CL, Nguyen, Y. & Burrows, LLMicrobiol Mol Biol Rev 76, 740-772(2012)). To distinguish the ComP orthologues from other type IVa pilin proteins, such as PilA from A. baumannii, P. aeruginosa, and Haemophilus influenzae and PilE from Neisseria species (Pelicic, V. Mol Microbiol 68, 827-837(2008)), BLASTp analysis was performed to identify the ComP orthologues. ADP1 The primary amino acid sequence of ComP was compared with all proteins from bacteria of the genus Acinetobacter. ADP1Many Acinetobacter type IVa pilin orthologues, including those from ComP, 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 orthologues (Figure 19) share high homology with ComP. ADP1 ComP pilin variants were identified based on the presence of a conserved serine at position 84 relative to the comP pilin variant and a conserved disulfide bond adjacent to a predicted site of glycosylation connecting the predicted alpha-beta loop to the beta strand region (Giltner, CL, Nguyen, Y. & Burrows, LLMicrobiol Mol Biol Rev 76, 740-772(2012)). In addition, all six ComP orthologs have both a pglS homolog immediately downstream of the comP gene and a pglL homolog somewhere on the chromosome. Taken together, the presence of at least a conserved serine at position 84, a disulfide loop adjacent to a site of glycosylation, the presence of a pglS gene immediately downstream of comP, and the presence of a pglL homolog somewhere on the chromosome distinguish ComP pilin variants from other type IVa pilin variants.

[0069] Thus, disclosed herein are common features in ComP proteins that identify ComP orthologues of different Acinetobacter species. ComP proteins can be distinguished from other pilins by the presence of a conserved glycosylated serine located at position 84 relative to the ADP1 ComP protein and the presence of a disulfide loop adjacent to the glycosylation site. Furthermore, the presence of a pglS homolog immediately downstream of ComP is indicative of ComP. Furthermore, to be classified as a PglS OTase protein rather than a PglL OTase protein, the OTase downstream of ComP must show higher sequence conservation with PglS (ACIAD3337) when compared to PglL (ACIAD0103) of A. baylyi ADP1. It is also clear to one of skill in the art that in any of the embodiments disclosed herein, the ComP protein may be a nucleotide sequence similar to that of SEQ ID NO: 1 (ComP ADP1: AAC45886.1) and is capable of glycosylation at this serine residue.

[0070] ComP from A. soli CIP 110264 is glycosylated by PglS from A. baylyi ADP1. Considering the presence of multiple ComP orthologs, we investigated whether PglS from A. baylyi ADP1 could glycosylate diverse ComP proteins. 110264 ) is ComP ADP1 However, consistent with the above characteristics, ComP 110264 contains a predicted disulfide bridge between the predicted alpha-beta loop and the second beta strand, and a predicted disulfide bridge between the predicted alpha-beta loop and the second beta strand, ADP1 Furthermore, the PglS orthologue contains a conserved serine at position 84 relative to ComP. 110264 It is possible that PglS from A. baylyi ADP1 (PglS ADP1 ) is ComP 110264 To determine whether PglS can be glycosylated, ADP1 to pACT3, ComP 110264 were cloned into pEXT20 (Dykxhoorn, DM, St Pierre, R. & Linn, T. Gene 177, 133-136 (1996)) and these plasmids were introduced into E. coli expressing serotype 8 capsular polysaccharide (CPS8) from S. pneumoniae. In addition, PglS from A. soli CIP 110264 (PglS 110264 ) to ComP ADP1 The reverse experiment was performed by cloning and expressing PglS together with 110264 , PglS 110264 The cognate acceptor pilin ComP, as indicated by the higher molecular weight ComP pilin mutants when compared to whole cell lysates lacking 110264Based on Western blot analysis, PglS 110264 is ComP ADP1 On the other hand, PglS did not appear to glycosylate. ADP1 , as indicated by a strong increase in His-reactive signal with increased electrophoretic mobility. ADP1 and ComP 110264 In summary, PglS efficiently glycosylated both ADP1 appears to be an 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 glycosylated by PglS. All members of the type IVa pilin family are considered membrane proteins because part of their N-terminal alpha helices is embedded in the inner membrane (Giltner, CL, Nguyen, Y. & Burrows, LLMicrobiol Mol Biol Rev 76, 740-772(2012)). Therefore, to generate soluble variants of ComP that can be glycosylated by PglS, translational fusions of truncated ComP fragment proteins onto three different carrier proteins were constructed. The carrier proteins DsbA and MalE (also known as maltose binding protein-MBP) from E. coli were selected as suitable carriers because they have previously been shown to promote periplasmic localization and solubility of acceptor proteins fused at their C-terminus (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 protein consisted of a leader sequence, a carrier protein, a short linker peptide, a ComP variant that does not contain the first 28 amino acids, and a hexahistidine tag. ComP ADP1 and ComP 110264 The first 28 amino acids of pglS were deleted, as they contain the leader sequence and the hydrophobic region of the N-terminal alpha helix predicted to be embedded in the inner membrane. The fusion construct was then co-transfected with pneumococcal serotype 8 capsular polysaccharide (CPS8) and pACT3 alone or with pglS. 110264 Or pglS ADP1 As shown in FIG. 21, pACT3 carrying either DsbA-AAA-ComPΔ28 was introduced into E. coli expressing DsbA-AAA-ComPΔ28. 110264 or DsbA-GGGS-ComPΔ28 110264 Either PglS ADP1E. coli cells expressing ComPΔ28 in combination with ComPΔ28 showed detectable levels of glycosylation as indicated by a modal distribution of reactive signals with increased electrophoretic mobility. ADP1 E. coli cells expressing a fusion containing ComPΔ28 did not show detectable glycosylation. The same glycosylation pattern was observed in E. coli cells expressing a maltose binding protein (MBP) fusion. In particular, as shown in FIG. 22, the MBP-AAA-ComPΔ28 110264 or MBP-GGGS-ComPΔ28 110264 Either PglS ADP1 E. coli cells expressing ComPΔ28 in combination with ComPΔ28 showed detectable levels of glycosylation, as indicated by the modal distribution of anti-His reactive signals; ADP1 Finally, to demonstrate that a previously established carrier protein used in conjugate vaccine formulations could be glycosylated by PglS in pneumococcal CPS8, we engineered a fusion protein containing the DsbA signal peptide sequence fused to EPA. ComPΔ28 was then glycosylated with 110264 The peptide was fused with a glycine-glycine-glycine-serine (GGGS; SEQ ID NO: 23) linker to the C-terminus of EPA and expressed in PglS in both whole cell and periplasmic extracts. ADP1 Glycosylation was tested in the presence and absence of EPA-GGGS-ComPΔ28 as shown in FIG. 110264 The constructs expressed CPS8 glycans and PglS as indicated by the modal distribution of anti-His reactive signals. ADP1 We found that PglS was glycosylated in both whole-cell and periplasmic extracts of cells co-expressing PglS. 110264 No detectable glycosylation was observed in samples expressing PglS. ADP1 is the optimal OTase for transferring polysaccharides containing glucose at the reducing end to the truncated ComP fusion protein. The specific amino acid sequences of each fusion construct are shown in FIG.

[0072] Immunization with glycosylated ComP bioconjugates induces immune responses. T cell-dependent immune responses to conjugate vaccines are characterized by the secretion of high affinity IgG1 antibodies (Avci, FY, Li, X., Tsuji, M. & Kasper, DLNat Med 17, 1602-1609(2011)). The immunogenicity of CPS14-ComP bioconjugates in a mouse vaccination model was evaluated. As seen in Figure 25A, sera collected from mice vaccinated with CPS14-ComP bioconjugates showed a significant increase in CPS14-specific IgG titers, but not IgM titers. Furthermore, a secondary HRP-tagged anti-IgG subtype antibody was used to determine which IgG subtypes raised the titers. As seen in Figure 25B, IgG1 titers appeared to be higher than other subtypes.

[0073] Next, a second vaccination study was conducted comparing the immunogenicity of trivalent CPS8-, CPS9V-, and CPS14-ComP bioconjugates with the current standard of care, PREVNAR 13®. Serotypes 9V and 14 are included in PREVNAR 13®, and elevated IgG titers were observed in PREVNAR 13®-immunized mice against these two serotypes (Figure 26). Monovalent immunization against serotype 14 also showed significant induction of serotype-specific IgG titers similar to preimmunization (Figures 25 and 26). Mice administered the trivalent bioconjugates, as expected, all showed elevated serotype-specific IgG titers when compared to controls, with day 49 sera showing elevated IgG titers for serotypes 8 and 14 compared to serotype 9V. Nevertheless, IgG titers against 9V were significantly higher than placebo (Figure 26).

[0074] Provided herein is a bioconjugate 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 other embodiments, the fusion protein comprises a ComP protein (ComP). In certain other embodiments, the fusion protein comprises a glycosylation tag of a ComP protein (as described in detail elsewhere 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 is shown in SEQ ID NO:1 (ComP ADP1 :AAC45886.1). This serine residue also corresponds to position 82 of SEQ ID NO:2 (ComP110264:ENV58402.1) (Figures 39A, B, and C). Thus, in certain embodiments, the fusion protein (and thus the bioconjugate) contains SEQ ID NO:1 (ComP ADP1 2. The ComP glycosylation tag is glycosylated with an oligosaccharide or polysaccharide at a serine residue corresponding to the serine residue at position 84 of SEQ ID NO:1 (ComPADP1:AAC45886.1) or corresponding to the serine residue at position 82 of SEQ ID NO:2. Figure 28 shows an alignment of regions of ComP sequences that include a serine (S) residue (boxed) that corresponds to the serine residue at position 84 of SEQ ID NO:1 (ComPADP1:AAC45886.1), which is conserved among ComP sequences. In certain embodiments, the ComP glycosylation tag is glycosylated with an oligosaccharide or polysaccharide at a serine residue corresponding to the serine residue at position 84 of SEQ ID NO:1 (ComPADP1:AAC45886.1) or corresponding to the serine residue at position 82 of SEQ ID NO:2. ADP1 :AAC45886.1) and a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 95 of SEQ ID NO: 1. Or, similarly described, but in certain embodiments, for purposes of being capable of being glycosylated, the ComP glycosylation tag comprises both a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 75 of SEQ ID NO:AAC45886.1 and a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 95 of SEQ ID NO: 1. ADP1:AAC45886.1) and a 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 one of skill in the art, by aligning a ComP sequence with SEQ ID NO:1 (e.g., with a full-length sequence or with a partial sequence), one can identify conserved residues in non-SEQ ID NO:1 ComP proteins disclosed herein that correspond to the serine residue at position 84 of SEQ ID NO:1. Furthermore, as will be appreciated by one of skill in the art, by aligning a ComP sequence with SEQ ID NO:1, other residues, regions, and / or features that correspond to residues, regions, and / or features of SEQ ID NO:1 referred to herein can be identified in non-SEQ ID NO:1 ComP sequences and referenced in relation to SEQ ID NO:1. And, although reference is made generally herein to SEQ ID NO:1, by analogy, any residue, region, feature, etc. of any ComP sequence disclosed herein can similarly be referenced in relation to, for example, SEQ ID NO:2.

[0078] ComP protein is a protein that is identified as a ComP protein consistent with the description provided herein.For example, representative examples of 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 is represented by SEQ ID NO:1 (ComP ADP1 : AAC45886.1), comprising 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 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 includes a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO:1 (ComP ADP1 SEQ ID NO: 7 (ComPΔ28) containing a serine residue corresponding to the conserved serine residue at position 84 of AAC45886.1 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 SFCIn certain embodiments, the ComP protein does not include the 28 amino acid leader sequence, but comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1 (ComP ADP1 SEQ ID NO: 7 (ComPΔ28) containing a serine residue corresponding to the conserved serine residue at position 84 of AAC45886.1 ADP1 In certain embodiments, the ComP protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: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 comprises SEQ ID NO:1 (ComP ADP1 :AAC45886.1), SEQ ID NO:2 (ComP 110264 :ENV58402.1), sequence number 3 (ComP GFJ-2 :APV36638.1), SEQ ID NO:4 (ComP 50v1 :PKD82822.1), SEQ ID NO:5 (ComP 4466 :SNX44537.1), 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 a bacterial cell, a fungal cell, a yeast cell, an avian cell, an algae cell, an insect cell, or a mammalian cell.In certain embodiments, the bioconjugate is produced in a cell-free system.An example of the use of a cell-free system that utilizes OTase other than PglS can be found in WO2013 / 067523A1, which is incorporated herein by reference.

[0080] Sequence number 2 (ComP 110264 It has been discovered that a methionine residue corresponding to the conserved methionine residue at position 104 of ComP Glycosylation Tag (SEQ ID NO: ... 110264 :ENV58402.1) does not contain a methionine residue corresponding to the conserved methionine residue at position 104. 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 is, toward the C-terminus, SEQ ID NO:2 (ComP 110264 :ENV58402.1)。 For example, in certain embodiments, the amino acid sequence of the ComP glycosylation tag is SEQ ID NO: 2 (ComP 110264 As will be appreciated by those skilled in the art, fusion proteins containing a ComP glycosylation tag may also be prepared by integrating the ComP glycosylation tag with the ComP glycosylation tag at a residue corresponding to positions 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, or 103 of SEQ ID NO:2 (ComP 110264:ENV58402.1), even if that methionine residue results from a sequence of the fusion protein that is not part of the ComP glycosylation tag sequence. For example, in certain embodiments, the fusion protein of the bioconjugate does not contain a methionine residue that corresponds to or is approximately equivalent to the conserved methionine residue at position 104 of SEQ ID NO:2 (ComP 110264 In certain embodiments, the fusion protein of the bioconjugate does not contain a methionine residue that corresponds to or is approximately equivalent to the conserved methionine residue at position 104 of SEQ ID NO:2 (ComP 110264 :ENV58402.1), which does not contain a methionine residue that would correspond to the conserved methionine residue at position 104.

[0081] The ComP glycosylation tag of the present disclosure is generally not the full-length ComP protein. In certain embodiments of any of the ComP glycosylation tags described herein, the ComP glycosylation tag is 18-50 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 21-45 amino acids in length. In certain embodiments, the glycosylation tag is 23-45 amino acids in length.

[0082] The ComP glycosylation tag of the present disclosure can be a fragment, variant, or variant fragment of a ComP protein as described anywhere herein. In certain embodiments, the ComP protein is 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 SEQ ID NO: 12 (ComPΔ28 SFC ), for example, 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: 7 (ComPΔ28 ADP1 ) or SEQ ID NO: 8 (ComPΔ28 110264 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: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 Additionally, in certain embodiments, the ComP protein comprises SEQ ID NO:1 (ComP ADP1 :AAC45886.1), SEQ ID NO:2 (ComP 110264 :ENV58402.1), sequence number 3 (ComP GFJ-2 :APV36638.1), SEQ ID NO:4 (ComP 50v1 :PKD82822.1), SEQ ID NO:5 (ComP 4466 :SNX44537.1), or SEQ ID NO: 6 (ComP SFC :OAL75955.1). For example, 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) or SEQ ID NO:2 (ComP 110264:ENV58402.1). Further, 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), SEQ ID NO:2 (ComP 110264 :ENV58402.1), sequence number 3 (ComP GFJ-2 :APV36638.1), SEQ ID NO:4 (ComP 50v1 :PKD82822.1), SEQ ID NO:5 (ComP 4466 :SNX44537.1), or SEQ ID NO: 6 (ComP SFC :OAL75955.1).

[0083] In certain embodiments, a 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, where 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, where the variant has one, two, three, four, five, six, or seven amino acid substitutions, additions, and / or deletions, where 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 comprises the ComP glycosylation tag of SEQ ID NO:27, where 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. 110264 :ENV58402.1). Moreover, in certain embodiments, the amino acid sequence of a ComP glycosylation tag comprising SEQ ID NO:27 does not continue C-terminally beyond the amino acid residue corresponding to position 44 of SEQ ID NO:27. In certain embodiments, a 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 no more than 25, 30, 40, 45, or 50 amino acids in length.

[0085] In certain embodiments, a 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, which has one, two, three, four, five, six, or seven 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 is SEQ ID NO:2 (ComP 110264 :ENV58402.1). Furthermore, in certain embodiments, the amino acid sequence of the ComP glycosylation tag comprising SEQ ID NO:28 does not include a methionine residue at a position corresponding to the conserved methionine residue at position 104 of SEQ ID NO:28. 110264:ENV58402.1) beyond the amino acid residue corresponding to position 103. In certain embodiments, a ComP glycosylation tag comprising the amino acid consensus sequence of SEQ ID NO:28, or a variant thereof, is no more than 25, 30, 40, 45, or 50 amino acids in length.

[0088] In certain embodiments, the ComP glycosylation tag is 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[D 7], 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], with the proviso that the variant comprises or consists of a variant having one, two, three, four, five, six, or seven amino acid substitutions, additions, and / or deletions in an amino acid sequence selected from the group consisting of SEQ ID NO:1 (ComP ADP11), and variants that maintain the conserved cysteine ​​residue at position 75 of SEQ ID NO:AAC45886.1) and the conserved cysteine ​​residue at position 95 of SEQ ID NO:1, and variants that maintain the conserved serine residue at position 84 of SEQ ID NO:1. In certain embodiments, the amino acid substitutions are conservative amino acid substitutions. Further, in certain embodiments, the ComP glycosylation tag is 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:64[G6], SEQ ID NO:65[G7], SEQ ID NO:66[G8], SEQ ID NO:67[G9], SEQ ID NO:68[H10], SEQ ID NO:69[H11], SEQ ID NO:60[H12], SEQ ID NO:61[H13], SEQ ID NO:62[H14], SEQ ID NO:63[H15], SEQ ID NO:64[H16], SEQ ID NO:65[H17], SEQ ID NO:66[H18], SEQ ID NO:67[H19], SEQ ID NO:70[H20], SEQ ID NO:71[H21], SEQ ID NO:72[H22], SEQ ID NO:73[H23], SEQ ID NO:74[H24], SEQ ID NO:75[H25], SEQ ID NO:76 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 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 (ComP 110264 In certain embodiments, the amino acid sequence of the ComP glycosylation tag comprising one of the above sequences or variants thereof does not include a methionine residue at a position corresponding to the conserved methionine residue at position 104 of ComP Glycosylation Tag SEQ ID NO:2 (ComP 110264:ENV58402.1). In certain embodiments, the ComP glycosylation tag comprising the above-listed amino acid sequences and / or variants thereof is 25, 30, 40, 45, or 50 amino acids or less in length. In certain embodiments, the ComP glycosylation tag is 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 oligosaccharide or polysaccharide for attachment to the glycosylation tag, fusion protein, and / or bioconjugate is 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. Furthermore, 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. Furthermore, in certain embodiments, the polysaccharide is a serotype K1 or serotype K2 capsular polysaccharide of Klebsiella pneumoniae.

[0090] In certain embodiments, the bioconjugates are produced in vivo, for example, in certain embodiments, the bioconjugates are produced in bacterial cells.

[0091] Since the bioconjugate comprises an oligosaccharide or polysaccharide covalently linked to the 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 that is art-recognized as useful for the production of conjugate vaccines. In certain embodiments, when a ComP glycosylation fragment is fused to a carrier protein or a fragment thereof, the glycosylation fragment, and thus the fusion protein, may be glycosylated at a conserved serine residue as described elsewhere 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 a fragment thereof. In certain embodiments, the carrier protein or a fragment thereof is linked to an amino acid linker, such as (GGGS) n(SEQ ID NO:23, where n is at least 1), or AAA (SEQ ID NO:24) to the ComP protein or its glycosylation. In order to increase 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 any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 to any of 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, the multiple glycosylations are arranged in tandem with each other in the fusion protein. In certain embodiments, the glycosylation tag fragments are spaced apart from one another in the fusion protein, e.g., separated by the sequence of the carrier protein. In certain embodiments, the glycosylation tag(s) can be located, e.g., at the N-terminus of the carrier protein and / or fusion protein. In certain embodiments, the glycosylation tag(s) can be located, e.g., at the C-terminus of the carrier protein and / or fusion protein. In certain embodiments, the glycosylation tag(s) can be located, e.g., internally within the carrier protein and / or fusion protein, and the glycosylation tag fragments are located between the carrier proteins in the fusion protein. In certain embodiments, the carrier proteins can be of the same type or different types. In certain embodiments, the glycosylation tags can be of the same type or different types. In certain embodiments, the ComP glycosylation tags are identical. In certain embodiments, at least two of the ComP glycosylation tags are different from one another.In certain embodiments, at least three, at least four, or at least five of the ComP glycosylation tags are all different from one another. Moreover, in certain embodiments, no one of the ComP glycosylation tags is identical.

[0092] The bioconjugates of the present invention can be used in one of a number of applications, including, but not limited to, as conjugate vaccines. For example, in certain embodiments, the conjugate vaccine is selected from the group consisting of Streptococcus pneumoniae serotype 8, Streptococcus pneumoniae serotype 1, Streptococcus pneumoniae serotype 2, Streptococcus pneumoniae serotype 4, Streptococcus pneumoniae serotype 5, Streptococcus pneumoniae serotype 6A, Streptococcus pneumoniae serotype 6B, Streptococcus pneumoniae serotype 7F, Streptococcus pneumoniae serotype 9N, Streptococcus pneumoniae serotype 9V, Streptococcus pneumoniae serotype 10A, Streptococcus pneumoniae serotype 11A, Streptococcus pneumoniae serotype 12F, Streptococcus pneumoniae serotype 14, Streptococcus pneumoniae serotype 15B, Streptococcus pneumoniae serotype 17F, Streptococcus pneumoniae serotype 18C, Streptococcus pneumoniae serotype 19F, Streptococcus pneumoniae serotype 19A, Streptococcus pneumoniae serotype 20, Streptococcus pneumoniae serotype 22F, Streptococcus pneumoniae serotype 23F, Streptococcus pneumoniae serotype 33F, Klebsiella pneumoniae serotype K1, Klebsiella Klebsiella pneumoniae serotype K2, Klebsiella pneumoniae serotype K5, Klebsiella pneumoniae serotype K16, Klebsiella pneumoniae serotype K20, Klebsiella pneumoniae serotype K54, Klebsiella pneumoniae serotype K54pneumoniae serotype K57, Streptococcus agalactiae serotype Ia, Streptococcus agalactiae serotype Ib, Streptococcus agalactiae serotype II, Streptococcus agalactiae serotype III, Streptococcus agalactiae serotype IV, Streptococcus agalactiae serotype V, Streptococcus agalactiae serotype VI, Streptococcus agalactiae serotype VII, Streptococcus agalactiae serotype VIII, Streptococcus agalactiae serotype IX, Streptococcus pyogenes group A carbohydrate, Enterococcus faecalis serotype A, Enterococcus faecalis serotype B, Enterococcus faecalis serotype C, Enterococcus faecalis serotype D, Enterococcus faecium capsular polysaccharide and lipoteichoic acid, Moraxella The conjugate vaccine is a vaccine against Moraxella catarrhalis lipooligosaccharide A, Moraxella catarrhalis lipooligosaccharide B, Moraxella catarrhalis lipooligosaccharide C, and Staphylococcus aureus lipoteichoic acid. 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, an IgG1 response. In certain embodiments, the conjugate vaccine generates immune memory in a subject administered the vaccine.

[0093] Provided herein is a fusion protein as disclosed in further detail elsewhere herein, which comprises a ComP glycosylation tag as disclosed in further detail elsewhere herein. In certain embodiments, the fusion protein comprises SEQ ID NO:1 (ComP ADP1 :AAC45886.1). In certain embodiments, the fusion protein is glycosylated with an oligosaccharide or polysaccharide. In certain embodiments, the oligosaccharide or polysaccharide is produced by a bacterium of the genus Streptococcus, such as the capsular polysaccharide of S. neumoniae, 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 a serotype K1 or serotype K2 capsular polysaccharide of Klebsiella pneumoniae. In certain of any of the embodiments disclosed herein, the oligosaccharide or polysaccharide comprises glucose at its reducing end. Certain embodiments relate to fusion proteins, which are produced in vivo. For example, in certain embodiments, the fusion protein is produced in a mammalian cell, a fungal cell, a yeast cell, an insect cell, an avian cell, an algal cell, or a bacterial cell. In certain embodiments, the fusion protein is produced in a bacterial cell, such as E. coli.

[0094] Disclosed herein is a method for in vivo conjugation (in vivo glycosylation) of an oligosaccharide or polysaccharide to a polypeptide. In certain embodiments, the method comprises covalently attaching an oligosaccharide or polysaccharide to a polypeptide using a PglS oligosaccharyltransferase (OTase) (described elsewhere 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. An example of a PglSOTase is PglS. 110264 , PglS ADP1 , PglS GFJ-2 , PglS 50v1 , PglS 4466 , and PglS SFC The ComP protein is described in detail elsewhere, and a representative example is ComP 110264 , ComP ADP1 , ComP GFJ-2 , ComP 50v1 , ComP 4466 , and ComP SFC Of course, a PglSOTase from a given organism naturally glycosylates the ComP protein from that organism (e.g., PglS 110264 But, ComP 110264 Although it is possible that PglS from one organism glycosylates ComP from a different organism, in certain embodiments, PglS from one organism glycosylates ComP from a different organism (e.g., PglS ADP1 But, ComP 110264 For example, in certain embodiments, PglSOTase glycosylates PglS ADP1 PglSOTase is PglS ADP1 In certain embodiments, the glycosylated ComP protein is ComP ADP1 For example, PglSOTase is not PglS ADP1In certain embodiments, the ComP protein is 110264 Of course, it will be appreciated that when the ComP protein or its glycosylation tag fragment is linked to a heterologous carrier protein, PglSOTase will not naturally glycosylate it, even if the ComP protein or its glycosylation tag fragment is from the same organism as the PglSOTase.

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

[0096] In certain embodiments disclosed herein, in vivo glycosylation occurs in a host cell. In certain embodiments, for example, the host cell can be a mammalian cell, a fungal cell, a yeast cell, an insect cell, an avian 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 comprises culturing a host cell that comprises the components necessary to conjugate an oligosaccharide or polysaccharide to a polypeptide. Typically, such components are an oligosaccharyltransferase, an acceptor polypeptide to be glycosylated, and an oligosaccharide or polysaccharide. In certain embodiments, the method comprises culturing a host cell that comprises: (a) a gene cluster encoding proteins necessary for the synthesis of an oligosaccharide or polysaccharide, (b) a PglSOTase, and (3) an acceptor polypeptide. Additionally, it has been discovered that the production of oligosaccharides or polysaccharides can be enhanced by transcriptional activators. In certain embodiments, the production of oligosaccharides or polysaccharides is enhanced by K. pneumoniae transcriptional activator rmpA (K. pneumoniae NTUH K-2044) or a homologue of K. pneumoniae transcriptional activator rmpA (K. pneumoniae NTUH K-2044). In certain embodiments, the methods further comprise expressing and / or providing such a transcriptional activator, together with other components, to the host cell.

[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 conjugate vaccines comprising a bioconjugate of the present disclosure or methods of making such conjugate vaccines.

[0100] Certain embodiments also provide a host cell comprising components for in vivo glycosylation of an acceptor ComP protein or a glycosylation-tagged fragment thereof. In certain embodiments, the host cell comprises: (a) a gene cluster encoding proteins required for the synthesis of an oligosaccharide or polysaccharide, (b) a PglSOTase, and (3) an acceptor polypeptide comprising a ComP protein or a glycosylation-tagged 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, together with other components.

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

[0102] Certain embodiments also provide isolated nucleic acids encoding ComP proteins, ComP glycosylation tag fragments, and / or ComP fusion proteins described anywhere herein. In certain embodiments, the isolated nucleic acids referred to herein are vectors or are contained within vectors. In certain embodiments, the isolated nucleic acids referred to herein are inserted and / or integrated into a heterologous genome or heterologous region of a genome.

[0103] Disclosed herein are pneumococcal bioconjugate vaccines that include conventional vaccine carriers. Certain embodiments include the use of ComP fragments as glycosylation tags (also known as "glycotags"). In certain embodiments, glycosylation tags 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 the conventional carrier protein, Pseudomonas aeruginosa exotoxin A (EPA). In certain embodiments, it has been demonstrated that the glycosylation tag / carrier fusion protein can be combined with the use of CPS8 polysaccharide and PglS to generate a carrier protein-CPS8 bioconjugate, a first of its kind pneumococcal bioconjugate vaccine. For example, in certain embodiments, the EPA fusion can be combined with the use of CPS8 polysaccharide and PglS to generate an EPA-CPS8 bioconjugate. The EPA-CPS8 bioconjugate vaccine has been demonstrated to elicit high serotype 8 specific IgG titers that are protective as determined by bactericidal killing. Importantly, vaccination with 100 ng of polysaccharide in the EPA-CPS8 bioconjugate was able to provide protection. Thus, certain embodiments provide a CPS8 pneumococcal bioconjugate vaccine.

[0104] Conjugate vaccines (such as EPA vaccine constructs) could include additional / multiple sites of glycosylation to expand the number of serotypes as well as increase the glycan-to-protein ratio to develop a comprehensive pneumococcal bioconjugate 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 infection and / or disease. In certain embodiments, the conjugate vaccine is a prophylactic that can be used, for example, to immunize a subject against infection and / or disease. In certain embodiments, the bioconjugate is associated with and / or administered with an adjuvant (such as in a therapeutic composition). Certain embodiments provide a composition (such as a therapeutic composition) that includes the conjugate vaccine described herein and an adjuvant. 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, the antibody response is an IgG response, the IgG response is an IgG1 response. Furthermore, in certain embodiments, the conjugate vaccine generates immune memory in a subject administered the vaccine.

[0106] Certain embodiments provide for the manufacture of a vaccine against an infectious disease and / or disease. In certain embodiments, the method comprises 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 disease is pneumonia. In certain embodiments, the infectious disease is a systemic infection and / or a blood infection. In certain embodiments, the subject is a mammal. For example, in certain embodiments, a pig or a human.

[0107] Importantly, the embodiments disclosed herein are not limited to pneumococcal polysaccharides, and indeed have broad applicability for generating bioconjugate vaccines against many important human and animal pathogens that are incompatible with PglB and PglL. Notable examples include the human pathogens Klebsiella pneumoniae and group B Streptococcus, and the swine pathogen S. suis, all highly relevant pathogens for which there are no licensed vaccines 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 any 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, an innate response, an adaptive response, a humoral response, an antibody response, a cell-mediated response, a B cell response, a T cell response, upregulation or downregulation of cytokines, immune system crosstalk, and a combination of two or more of the 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 bacterial and / or infectious diseases in a subject, comprising administering to a subject in need thereof a conjugate vaccine, fusion protein, or composition disclosed herein. In certain embodiments, the infectious disease is a localized or systemic infection of the skin, soft tissue, blood, or organs, or is autoimmune in nature. In certain embodiments, the disease is pneumonia. In certain embodiments, the infectious disease is a systemic infection and / or an infection of the blood. In certain 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 compositions are administered via intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration. EXAMPLES

[0111] Example 1. Determining whether cysteine ​​residues adjacent to glycosylation sites in ComP contribute to ComP stability and glycosylation. The ComP protein from Acinetobacter baylyi ADP1 (ComP ADP1 ) and the ComP protein from A. soli strain 110264 (ComP 110264 ) was previously demonstrated to be glycosylated by the O-linked OTase PglS at the homologous serine residues located at positions 84 and 82, respectively (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, ComP ADP1 S84A point mutant and ComP 110264 It was shown that the S82A point mutant of ComP is unable to be glycosylated by PglS on the serotype 8 pneumococcal capsular polysaccharide. To further analyze the role of other amino acids important for PglS-dependent glycosylation, a series of point mutants were generated and the ComP ADP1 The conserved cysteine ​​residues adjacent to the glycosylation sites at positions 75 and 95 of were modified (Figure 1A).

[0112] A series of point mutants was first generated replacing cysteine ​​75 with either alanine or glycine, as these mutants would block the formation of a potential disulfide bond between cysteine ​​75 and cysteine ​​95. The point mutants were then transformed into the C. jejuni heptasaccharide biosynthetic gene cluster and PglS. ADP1As can be seen in Figure 1B, mutation of cysteine ​​75 significantly reduced the expression of the ComP protein mutants compared to wild-type (WT) ComP. In particular, the C75A and C75G mutants exhibited very low levels of protein expression and appeared to exist only as low molecular weight unglycosylated forms. A second series of point mutants was then generated replacing cysteine ​​95 with either alanine, glycine, or serine, and these mutant ComP constructs were then again transfected with the C. jejuni heptasaccharide biosynthetic gene cluster and PglS. ADP1 ComPj mutants with either alanine, glycine, or serine instead of cysteine ​​95 were undetectable, as seen in Figure 1B. Finally, a series of double point mutants were generated consisting of all combinations of cysteine ​​75 to alanine or glycine and cysteine ​​95 to alanine, glycine, or serine. ComP double mutants were undetectable, as seen in Figure 1B. Based on the lack of detectable expression for the various ComP point mutant variants, the cysteine ​​residues located at positions 75 and 95 likely form disulfide bonds 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 short PglS-dependent O-linked recognition motifs by a reductive cloning strategy. ComP lacking the first 28 amino acids 110264 (In this specification, ComPΔ28 110264A translational fusion containing a polyvalent pneumococcal bioconjugate vaccine (referred to as ComP) fused to the C-terminus of a genetically inactivated variant of the Pseudomonas aeruginosa 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 USA). In order to shorten and define the minimal recognition site required for PglS-dependent glycosylation, ComP 110264 A truncated cloning strategy was pursued in which the fragment was translationally fused at the C-terminus of the EPA protein between a glycine-glycine-glycine-serine (GGGS) linker and a hexahistidine tag (Figure 2). 110264 Multiple constructs were generated containing either 25, 30, 35, 40, or 45 amino acid fragments of ComP. 110264 As an example, the first construct was a ComP spanning residues 67-91. 110264 The second construct contains a 25 amino acid fragment of ComP spanning residues 68 to 92. 110264 and the third construct contains a 25 amino acid fragment of ComP spanning residues 69 to 93. 110264 All fragments contained serine 82, the PglS glycosylation site. The EPA fusion constructs were then transfected with PglS ADP1 and was introduced into E. coli SDB1 co-expressing pneumococcal CPS8.

[0114] As can be seen in Figure 3, ComP 110264 When analyzed by Western blot, three constructs containing short 25 amino acid fragments of PglS were identified as having reduced electrophoretic mobility and the presence of multiple glycoforms (observed as a ladder-like distribution above the non-glycosylated protein). ADP1 As a positive control, ComPΔ 28110264 EPA fusions containing the fragment were included because this protein already has a PglS APD1 Notably, only three 25 amino acid constructs were found to be glycosylated: C1 (SEQ ID NO: 32), D1 (SEQ ID NO: 33), and E1 (SEQ ID NO: 34). 110264 It contained a conserved cysteine ​​residue predicted to form a disulfide bond adjacent to the glycosylation site (serine 82).

[0115] As can be seen in Figures 3 and 4, ComP 110264 Seven constructs containing the 30 amino acid fragment of PglS ADP1 The following constructs were found to be glycosylated in pneumococcal CPS8 by ComP: 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), and C3 (SEQ ID NO: 47). All seven constructs were glycosylated by ComP 110264 It contained a conserved cysteine ​​residue predicted to form a disulfide bond adjacent to the glycosylation site.

[0116] As can be seen in Figures 5 and 6, ComP 110264 Nine constructs containing the 35 amino acid fragment of PglS ADP1The following constructs were found to be glycosylated in pneumococcal CPS8 by ComP: 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), and F5 (SEQ ID NO:63). 110264 It contained a conserved cysteine ​​residue predicted to form a disulfide bond adjacent to the glycosylation site.

[0117] As can be seen in Figures 6 and 7, ComP 110264 Eight constructs containing a 40 amino acid fragment of PglS ADP1 The following constructs were found to be glycosylated by ComP in pneumococcal CPS8: 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), and B8 (SEQ ID NO: 79). 110264 It contained a conserved cysteine ​​residue predicted to form a disulfide bond adjacent to the glycosylation site.

[0118] As can be seen in Figs. 8, 9, and 10, ComP 110264 Ten constructs containing the 45 amino acid fragment of PglS ADP1 The following constructs were found to be glycosylated in pneumococcal CPS8 by ComP: 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), and C11 (SEQ ID NO: 101). Again, all ten constructs were glycosylated by ComP. 110264 It contained a conserved cysteine ​​residue predicted to form a disulfide bond adjacent to the glycosylation site.

[0119] Based on the data presented in Figures 3, 4, 5, 6, 7, 8, 9, and 10, the cysteine ​​residues located at positions 71 and 93 act to mediate the binding of EPA to the C-terminus of the PglS carrier protein. ADP1 In addition, the methionine residue at position 104 is essential for glycosylation by PglS when it is part of a C-terminal glycosylation tag. ADP1 This is especially supported by the fact that constructs G5 (SEQ ID NO:64), C8 (SEQ ID NO:80), and D11 (SEQ ID NO:102) did not show any indication of glycosylation, even though they contain essential cysteines at positions 71 and 93, respectively. G5 (SEQ ID NO:64), C8 (SEQ ID NO:80), and D11 (SEQ ID NO:102) are ComPs that are 35, 40, and 45 amino acids in length, respectively. 110264 Although containing fragments, each fragment is terminal to a methionine at position 104, demonstrating that this residue is sufficient to block glycosylation when included in a C-terminal glycotag. Moreover, all constructs containing methionine 104 in addition to cysteines at positions 71 and 93 did not show any indication 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 shows the PglS ADP1 ComP was tested for its ability to function as an O-linked glycosylation recognition motif by 110264 Provides a list of all fragments. [Table 1-1] [Table 1-2]

[0120] The O-linked glycosylation recognition motifs bind to PglS when translationally fused to the N-terminus, in tandem at the N- or C-terminus, or simultaneously at the N- and C-terminus. ADP1 Based on the data presented above, ComP 110264 The D5 (SEQ ID NO: 61) fragment of ComPΔ28 was selected for follow-up experiments. As outlined in Figures 12A and 12B, the D5 (SEQ ID NO: 61) fragment or its derivative (D5') was translationally fused to the N-terminus and C-terminus in various combinations in the follow-up experiments. 110264 EPA fusions containing the fragment were included because this protein already has a PglS APD1 The EPA fusion construct was then transformed with PglS, as it has been established that PglS is glycosylated at CPS8. ADP1 The EPA-ComP110264 fusion constructs were introduced into E. coli SDB1 co-expressing pneumococcal CPS8 in the presence or absence of PglS. As seen in Fig. 12C, all EPA-ComP110264 fusion constructs were glycosylated with pneumococcal CPS8, indicating 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 This indicates that the glycosylation can be performed by

[0121] Example 3. Dual ComPΔ28 fused in tandem to the C-terminus 110264 The glycosylation tag is PglS ADP1 Double ComPΔ28 fused in tandem to the C-terminus 110264 An EPA fusion construct containing a glycosylation tag was constructed. ComPΔ28 110264The glycosylation tags were 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 aid in downstream purification. As a positive control, ComPΔ28 110264 EPA fusions containing the fragment were included because this protein already has a PglS APD1 The dual tag EPA fusion construct was then transformed with PglS ADP1 EPA variant 7 and EPA variant 8 were introduced into E. coli SDB1 co-expressing pneumococcal CPS8 in the presence or absence of PglS. As can be seen in FIG. 13, both EPA variant 7 and EPA variant 8 ADP1 When present, it was glycosylated in pneumococcal CPS8, and the glycosylation appeared to be of very high molecular weight, with immunoreactivity approaching the 250 kDa marker.

number

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

[0123] All publications and patent applications mentioned in this specification are herein 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

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Claims

1. 1. A bioconjugate comprising an oligo- or polysaccharide covalently attached to a fusion protein, the fusion protein comprises a ComP protein (ComP) glycosylation tag; The ComP glycosylation tag is SEQ ID NO:2 (ComP １１０２６４ :ENV58402.1) and the cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 93 of SEQ ID NO:2, or ＡＤＰ１ 1, comprising both a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 75 of SEQ ID NO:AAC45886.1) and a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 95 of SEQ ID NO:1; and the fusion protein is glycosylated with an oligosaccharide or polysaccharide 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 of the ComP glycosylation tag; The bioconjugate.

2. The ComP glycosylation tag is SEQ ID NO:2 (ComP １１０２６４ :ENV58402.1), Optionally, the fusion protein of the bioconjugate has, with respect to the ComP glycosylation tag, SEQ ID NO:2 ( １１０２６４ : ENV58402.1), The bioconjugate of claim 1.

3. The amino acid sequence of the ComP glycosylation tag is, from the C-terminal direction, SEQ ID NO: 2 (ComP １１０２６４ 3. The bioconjugate of claim 2, wherein the amino acid residue does not extend beyond the amino acid residue corresponding to position 103 of ENV58402.

1.

4. 4. The bioconjugate of any one of claims 1 to 3, wherein the ComP glycosylation tag is 18-50 amino acids in length, 21-45 amino acids in length, or 23-45 amino acids in length.

5. The ComP protein is represented by 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 ), Optionally, the ComP protein is SEQ ID NO:7 (ComPΔ28 ＡＤＰ１ ) or SEQ ID NO: 8 (ComPΔ28 １１０２６４ ), Optionally, the ComP protein is 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 ), A bioconjugate according to any one of claims 1 to 4.

6. The ComP protein is represented by 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 5, wherein the amino acid sequence is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of ... Optionally, the ComP protein is SEQ ID NO:1 (ComP ＡＤＰ１ : AAC45886.1) or SEQ ID NO: 2 (ComP １１０２６４ :ENV58402.1), Optionally, 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), A bioconjugate according to any one of claims 1 to 4.

7. The ComP glycosylation tag has the following amino acid consensus sequence: [0010] During the ceremony, X １ is V, A or the absence of an amino acid, X ２ is A, G, T or the absence of an amino acid, X ５ is P, S, or Q; X ６ is S, M, or I; X ７ is T, P, or V; X ８ is A, S, or T; X ９ is G, N, S, or T; X １０ is N or the absence of an amino acid, X １１ is S, G, or A; X １２ is S or N, X １４ is V, T, or A; X １７ is Q, T, or E; X １８ is E, Q, or T; X ２０ is S, N, A, or G; X ２１ is S or the absence of an amino acid, X ２２ is G or the absence of an amino acid, X ２５ is N, S, or A; X ２６ is A, S, or K; X ２８ is T, S, or K; X ３１ is A or E, X ３２ is T or S, X ３４ is T, Q, or A; X ３６ is G, S, or T; X ３７ is A, G, or D; X ３８ is S, L, or A; X ３９ is S, G, D, or T; X ４０ is A, V, or G; X ４１ is G, I, or V; X ４２ is Q, T, or I; X ４３ is I, V, T, or L; and X ４４ is I, T, or V; or a fragment thereof, wherein said fragment of said ComP glycosylation tag comprises a cysteine ​​residue at position 13 of SEQ ID NO:27, a cysteine ​​residue at position 35 of SEQ ID NO:27, and a serine residue at position 24 of SEQ ID NO:27; or a variant of the amino acid consensus sequence of SEQ ID NO: 27, or a fragment thereof, having one, two, three, four, five, six, or seven amino acid substitutions, additions, and / or deletions; provided that said variant maintains a cysteine ​​residue at position 13 of SEQ ID NO:27, a cysteine ​​residue at position 35 of SEQ ID NO:27, and a serine residue at position 24 of SEQ ID NO:27; 2. The bioconjugate of claim 1, comprising or consisting of:

8. The ComP glycosylation tag has the following amino acid consensus sequence: [0025] During the ceremony, X １ is V, A or the absence of an amino acid, X ２ is A, G, T or the absence of an amino acid, X ５ is P, S, or Q; X ６ is S, M, or I; X ７ is T, P, or V; X ８ is A, S, or T; X ９ is G, N, S, or T; X １０ is N or the absence of an amino acid, X １１ is S, G, or A; X １２ is S or N, X １４ is V, T, or A; X １７ is Q, T, or E; X １８ is E, Q, or T; X ２０ is S, N, A, or G; X ２１ is S or the absence of an amino acid, X ２２ is G or the absence of an amino acid, X ２５ is N, S, or A; X ２６ is A, S, or K; X ２８ is T, S, or K; X ３１ is A or E, X ３２ is T or S, X ３４ is T, Q, or A; X ３６ is G, S, or T; X ３７ is A, G, or D; X ３８ is S, L, or A; X ３９ is S, G, D, or T; X ４０ is A, V, or G; X ４１ is G, I, or V; X ４２ is Q, T, or I; X ４３ is I, V, T, or L; and X ４４ is I, T, or V; or a fragment thereof, wherein said fragment of said ComP glycosylation tag comprises a cysteine ​​residue at position 13 of SEQ ID NO:27, a cysteine ​​residue at position 35 of SEQ ID NO:27, and a serine residue at position 24 of SEQ ID NO:27; 8. The bioconjugate of claim 7, comprising or consisting of:

9. The ComP glycosylation tag is SEQ ID NO:2 (ComP １１０２６４ 9. The bioconjugate of claim 8, which does not contain a methionine residue at a position corresponding to the conserved methionine residue at position 104 of SEQ ID NO: ENV58402.

1.

10. 10. The bioconjugate of claim 9, wherein the amino acid sequence of the ComP glycosylation tag does not continue C-terminally beyond the amino acid residue corresponding to position 44 of SEQ ID NO:

27.

11. 11. The bioconjugate of claim 8, wherein the ComP glycosylation tag is no more than 25, 30, 40, 45, or 50 amino acids in length.

12. The ComP glycosylation tag has the following amino acid consensus sequence: [0030] During the ceremony, X ２ is V, T, or A, or optionally V; X ５ is Q, T, or E, or optionally Q; X ６ is E, Q, or T; X ８ is S, N, A, or G; X ９ is S or the absence of an amino acid, X １０ is G or the absence of an amino acid, X １３ is N, S, or A, or optionally N; X １４ is A, S, or K, or optionally A; X １６ is T, S, or K; X １９ is A or E, or optionally A; X ２０ is T or S, or optionally is T, or X ２２ is T, Q, or A, or optionally T; or variants thereof having 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, additions, and / or deletions; provided that said variant maintains a cysteine ​​residue at position 1 of SEQ ID NO:28, a cysteine ​​residue at position 23 of SEQ ID NO:28, and a serine residue at position 12 of SEQ ID NO:28; 2. The bioconjugate of claim 1, comprising or consisting of:

13. The ComP glycosylation tag is SEQ ID NO:2 (ComP １１０２６４ 13. The bioconjugate of claim 12, which does not contain a methionine residue at a position corresponding to the conserved methionine residue at position 104 of GAL V2R (ENV58402.1).

14. The amino acid sequence of the ComP glycosylation tag is, from the C-terminal direction, SEQ ID NO: 2 (ComP １１０２６４ 14. The bioconjugate of claim 13, wherein the amino acid sequence does not extend beyond the amino acid residue corresponding to position 103 of ENV58402.

1.

15. 15. The bioconjugate of any one of claims 12 to 14, wherein the ComP glycosylation tag is no more than 25, 30, 40, 45, or 50 amino acids in length.

16. The ComP glycosylation tag is 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 [H2], SEQ ID NO:63 [H2], SEQ ID NO:64 [H3], SEQ ID NO:65 [H4], SEQ ID NO:66 [H4], SEQ ID NO:67 [H4], SEQ ID NO:68 [H4], SEQ ID NO:69 [H5], SEQ ID NO:69 [H5], SEQ ID NO:60 [H5], SEQ ID NO:61 [H5], SEQ ID NO:62 [H6], SEQ ID NO:63 [H6], SEQ ID NO:64 [H6], SEQ ID NO:65 [H7], SEQ ID NO:66 [H7], SEQ ID NO:67 [H8], SEQ ID NO:68 [H9], SEQ ID NO:69 [H10], SEQ ID NO:69 [H11], SEQ ID NO:69 [H12], SEQ ID NO:69 [H13], SEQ ID NO:69 [H14], SEQ ID NO:69 2 [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 variants thereof having 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, additions, and / or deletions; However, the variant is SEQ ID NO: 1 (ComP ＡＤＰ１ 1) and a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 95 of SEQ ID NO:1; and The variant maintains a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO:1; 2. The bioconjugate of claim 1, comprising or consisting of:

17. The ComP glycosylation tags are 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 [ 17. The bioconjugate of claim 16, comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:73 [A10], 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].

18. The ComP glycosylation tag is SEQ ID NO:2 (ComP １１０２６４ 18. The bioconjugate of claim 16 or 17, which does not contain a methionine residue at a position corresponding to the conserved methionine residue at position 104 of GAL V2R (ENV58402.1).

19. The amino acid sequence of the ComP glycosylation tag is, from the C-terminal end, SEQ ID NO: 2 (ComP １１０２６４ 20. The bioconjugate of claim 18, wherein the amino acid sequence does not extend beyond the amino acid residue corresponding to position 103 of ENV58402.1).

20. 20. The bioconjugate of any one of claims 16 to 19, wherein the ComP glycosylation tag is no more than 25, 30, 40, 45, or 50 amino acids in length.

21. The ComP glycosylation tags are 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:

2. The bioconjugate of claim 1, comprising an amino acid sequence selected from the group consisting of 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].

22. The oligosaccharide or polysaccharide is produced by a bacterium of the genus Streptococcus, Optionally, the polysaccharide is a capsular polysaccharide of S. pneumoniae, S. agalactiae, or S. suis; 22. The bioconjugate of any one of claims 1 to 21.

23. 23. The bioconjugate of claim 22, wherein the capsular polysaccharide is CPS14, CPS8, CPS9V, or CPS15b.

24. The oligosaccharide or polysaccharide is produced by a bacterium of the genus Klebsiella, Optionally, the polysaccharide is a capsular polysaccharide of Klebsiella pneumoniae, Klebsiella varricola, Klebsiella michinganenis, or Klebsiella oxytoca; 22. The bioconjugate of any one of claims 1 to 21.

25. 22. The bioconjugate of any one of claims 1 to 21, wherein the polysaccharide is a capsular polysaccharide of Klebsiella pneumoniae.

26. 26. The bioconjugate of claim 25, wherein the polysaccharide is a serotype K1 or serotype K2 capsular polysaccharide of Klebsiella pneumoniae.

27. 27. The bioconjugate of any one of claims 1 to 26, wherein the oligosaccharide or polysaccharide comprises glucose at its reducing end.

28. The bioconjugate is produced in vivo; 28. The bioconjugate of any one of claims 1 to 27, optionally produced in a bacterial cell.

29. 29. The bioconjugate of any one of claims 1 to 28, wherein the fusion protein comprises 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.

30. the ComP glycosylation tag is located at the N-terminus of the fusion protein, the C-terminus of the fusion protein, and / or internally within the fusion protein; 30. The bioconjugate of claim 29, wherein optionally, the carrier protein or fragment thereof is linked to the glycosylation tag via an amino acid linker.

31. 31. The bioconjugate of any one of claims 1 to 30, wherein the fusion protein comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 8 or more, 10 or more, 15 or more, or 20 or more ComP glycosylation tags.

32. 31. The bioconjugate of any one of claims 1 to 30, wherein the fusion protein comprises any number of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 to any number of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 ComP glycosylation tags.

33. The bioconjugate of claim 31 or 32, wherein the ComP glycosylation tags are identical.

34. at least two of the ComP glycosylation tags are different from each other; Optionally, at least three, at least four, or at least five of said ComP glycosylation tags are all different from one another; Optionally, none of said ComP glycosylation tags are the same.

33. The bioconjugate of claim 31 or 32.

35. the bioconjugate is a conjugate vaccine; Optionally, the conjugate vaccine is a vaccine against Streptococcus pneumoniae serotype 8.

36. 36. The bioconjugate of claim 35, wherein the conjugate vaccine induces an immune response when administered to a subject.

37. 37. The bioconjugate of claim 35 or 36, wherein 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.

38. 38. The bioconjugate of claim 37, wherein the antibody response is an IgG response or an IgM response.

39. the antibody response is an IgG response; Optionally, the bioconjugate of claim 38 is IgG1 responsive.

40. 40. The bioconjugate of any one of claims 35 to 39, wherein the conjugate vaccine generates immune memory in a subject to which the vaccine is administered.

41. A ComP glycosylation tag comprising an isolated fragment of a ComP protein, said fragment being represented by SEQ ID NO: 1 (ComP ＡＤＰ１ A serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO:2 (ComP １１０２６４ :ENV58402.1) and the cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 93 of SEQ ID NO:2, or ＡＤＰ１ 1, comprising both a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 75 of SEQ ID NO:AAC45886.1) and a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 95 of SEQ ID NO:1, The ComP glycosylation tag.

42. The ComP glycosylation tag is SEQ ID NO:2 (ComP １１０２６４ 42. The ComP glycosylation tag of claim 41, which does not contain a methionine residue corresponding to the conserved methionine residue at position 104 of GAL58402.

1.

43. The amino acid sequence of the ComP glycosylation tag is, from the C-terminal direction, SEQ ID NO: 2 (ComP １１０２６４ 43. The ComP glycosylation tag of claim 42, which does not extend beyond the amino acid residue corresponding to position 103 of ENV58402.1).

44. 44. The ComP glycosylation tag of any one of claims 41 to 43, wherein the ComP glycosylation tag is 18 to 50 amino acids in length, 21 to 45 amino acids in length, or 23 to 45 amino acids in length.

45. The ComP protein is represented by 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 ), Optionally, the ComP protein is SEQ ID NO:7 (ComPΔ28 ＡＤＰ１ ) or SEQ ID NO: 8 (ComPΔ28 １１０２６４ ), Optionally, the ComP protein is 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 ), 45. A ComP glycosylation tag according to any one of claims 41 to 44.

46. The ComP protein is represented by 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 5, wherein the amino acid sequence is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of ... Optionally, the ComP protein is SEQ ID NO:1 (ComP ＡＤＰ１ : AAC45886.1) or SEQ ID NO: 2 (ComP １１０２６４ :ENV58402.1), Optionally, 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), 45. A ComP glycosylation tag according to any one of claims 41 to 44.

47. The ComP glycosylation tag has the following amino acid consensus sequence: [0045] During the ceremony, X １ is V, A or the absence of an amino acid, X ２ is A, G, T or the absence of an amino acid, X ５ is P, S, or Q; X ６ is S, M, or I; X ７ is T, P, or V; X ８ is A, S, or T; X ９ is G, N, S, or T; X １０ is N or the absence of an amino acid, X １１ is S, G, or A; X １２ is S or N, X １４ is V, T, or A; X １７ is Q, T, or E; X １８ is E, Q, or T; X ２０ is S, N, A, or G; X ２１ is S or the absence of an amino acid, X ２２ is G or the absence of an amino acid, X ２５ is N, S, or A; X ２６ is A, S, or K; X ２８ is T, S, or K; X ３１ is A or E, X ３２ is T or S, X ３４ is T, Q, or A; X ３６ is G, S, or T; X ３７ is A, G, or D; X ３８ is S, L, or A; X ３９ is S, G, D, or T; X ４０ is A, V, or G; X ４１ is G, I, or V; X ４２ is Q, T, or I; X ４３ is I, V, T, or L; and X ４４ is I, T, or V; or a fragment thereof, wherein said fragment of said ComP glycosylation tag comprises a cysteine ​​residue at position 13 of SEQ ID NO:27, a cysteine ​​residue at position 35 of SEQ ID NO:27, and a serine residue at position 24 of SEQ ID NO:27; or a variant of the amino acid consensus sequence of SEQ ID NO: 27, or a fragment thereof, having one, two, three, four, five, six, or seven amino acid substitutions, additions, and / or deletions; provided that said variant maintains a cysteine ​​residue at position 13 of SEQ ID NO:27, a cysteine ​​residue at position 35 of SEQ ID NO:27, and a serine residue at position 24 of SEQ ID NO:27; 42. The ComP glycosylation tag of claim 41, comprising or consisting of:

48. The ComP glycosylation tag has the following amino acid consensus sequence: [0050] During the ceremony, X １ is V, A or the absence of an amino acid, X ２ is A, G, T or the absence of an amino acid, X ５ is P, S, or Q; X ６ is S, M, or I; X ７ is T, P, or V; X ８ is A, S, or T; X ９ is G, N, S, or T; X １０ is N or the absence of an amino acid, X １１ is S, G, or A; X １２ is S or N, X １４ is V, T, or A; X １７ is Q, T, or E; X １８ is E, Q, or T; X ２０ is S, N, A, or G; X ２１ is S or the absence of an amino acid, X ２２ is G or the absence of an amino acid, X ２５ is N, S, or A; X ２６ is A, S, or K; X ２８ is T, S, or K; X ３１ is A or E, X ３２ is T or S, X ３４ is T, Q, or A; X ３６ is G, S, or T; X ３７ is A, G, or D; X ３８ is S, L, or A; X ３９ is S, G, D, or T; X ４０ is A, V, or G; X ４１ is G, I, or V; X ４２ is Q, T, or I; X ４３ is I, V, T, or L; and X ４４ is I, T, or V; or a fragment thereof, wherein said fragment of said ComP glycosylation tag comprises a cysteine ​​residue at position 13 of SEQ ID NO:27, a cysteine ​​residue at position 35 of SEQ ID NO:27, and a serine residue at position 24 of SEQ ID NO:27; 48. The ComP glycosylation tag of claim 47, comprising or consisting of:

49. The ComP glycosylation tag is SEQ ID NO:2 (ComP １１０２６４ 49. The ComP glycosylation tag of claim 48, which does not contain a methionine residue at a position corresponding to the conserved methionine residue at position 104 of ENV58402.

1.

50. The amino acid sequence of the ComP glycosylation tag is, from the C-terminal direction, SEQ ID NO: 2 (ComP １１０２６４ 50. The ComP glycosylation tag of claim 49, which does not extend beyond the amino acid residue corresponding to position 103 of ENV58402.1).

51. 51. The ComP glycosylation tag of any one of claims 48 to 50, wherein the ComP glycosylation tag is no more than 25, 30, 40, 45, or 50 amino acids in length.

52. The ComP glycosylation tag has the following amino acid consensus sequence: [006] During the ceremony, X ２ is V, T, or A, or optionally V; X ５ is Q, T, or E, or optionally Q; X ６ is E, Q, or T; X ８ is S, N, A, or G; X ９ is S or the absence of an amino acid, X １０ is G or the absence of an amino acid, X １３ is N, S, or A, or optionally N; X １４ is A, S, or K, or optionally A; X １６ is T, S, or K; X １９ is A or E, or optionally A; X ２０ is T or S, or optionally is T, or X ２２ is T, Q, or A, or optionally T; or variants thereof having 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, additions, and / or deletions; provided that said variant maintains a cysteine ​​residue at position 13 of SEQ ID NO:28, a cysteine ​​residue at position 35 of SEQ ID NO:28, and a serine residue at position 14 of SEQ ID NO:28; 42. The ComP glycosylation tag of claim 41, comprising or consisting of:

53. The ComP glycosylation tag is SEQ ID NO:2 (ComP １１０２６４ 53. The ComP glycosylation tag of claim 52, which does not contain a methionine residue at a position corresponding to the conserved methionine residue at position 104 of ENV58402.

1.

54. The amino acid sequence of the ComP glycosylation tag is, from the C-terminal direction, SEQ ID NO: 2 (ComP １１０２６４ 54. The ComP glycosylation tag of claim 53, which does not extend beyond the amino acid residue corresponding to position 103 of ENV58402.1).

55. 55. The ComP glycosylation tag of any one of claims 52 to 54, wherein the ComP glycosylation tag is no more than 25, 30, 40, 45, or 50 amino acids in length.

56. The ComP glycosylation tag is 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 [H2], SEQ ID NO:63 [H2], SEQ ID NO:64 [H3], SEQ ID NO:65 [H4], SEQ ID NO:66 [H4], SEQ ID NO:67 [H4], SEQ ID NO:68 [H4], SEQ ID NO:69 [H5], SEQ ID NO:69 [H5], SEQ ID NO:60 [H5], SEQ ID NO:61 [H5], SEQ ID NO:62 [H6], SEQ ID NO:63 [H6], SEQ ID NO:64 [H6], SEQ ID NO:65 [H7], SEQ ID NO:66 [H7], SEQ ID NO:67 [H8], SEQ ID NO:68 [H9], SEQ ID NO:69 [H10], SEQ ID NO:69 [H11], SEQ ID NO:69 [H12], SEQ ID NO:69 [H13], SEQ ID NO:69 [H14], SEQ ID NO:69 2 [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 variants thereof having 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, additions, and / or deletions; However, the variant is SEQ ID NO: 1 (ComP ＡＤＰ１ 1) and a cysteine ​​residue corresponding to the conserved cysteine ​​residue at position 95 of SEQ ID NO:1; and The variant maintains a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO:1; 42. The ComP glycosylation tag of claim 41, comprising or consisting of:

57. The ComP glycosylation tags are 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 [B 57], 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].

58. The ComP glycosylation tag is SEQ ID NO:2 (ComP １１０２６４ 58. The ComP glycosylation tag of claim 56 or 57, which does not contain a methionine residue at a position corresponding to the conserved methionine residue at position 104 of ComP glycosylation tag GAGGGGGGGGGGGGGGGGG.

59. The amino acid sequence of the ComP glycosylation tag is, from the C-terminal end, SEQ ID NO: 2 (ComP １１０２６４ 59. The ComP glycosylation tag of claim 58, which does not extend beyond the amino acid residue corresponding to position 103 of ENV58402.1).

60. 60. The ComP glycosylation tag of any one of claims 56 to 59, wherein the ComP glycosylation tag is no more than 25, 30, 40, 45, or 50 amino acids in length.

61. The ComP glycosylation tags are 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:

42. The ComP glycosylation tag of claim 41, comprising an amino acid sequence selected from the group consisting of 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].

62. 62. A fusion protein comprising a ComP glycosylation tag according to any one of claims 41 to 61, Optionally, the fusion protein comprises a sequence similar to SEQ ID NO:1 (ComP ＡＤＰ１ : AAC45886.1), The fusion protein.

63. the fusion protein is glycosylated with an oligosaccharide or polysaccharide, and the oligosaccharide or polysaccharide is produced by a bacterium of the genus Streptococcus; Optionally, the polysaccharide is a capsular polysaccharide of S. pneumoniae, S. agalactiae, or S. suis; 63. The fusion protein of claim 62.

64. 64. The fusion protein of claim 63, wherein the capsular polysaccharide is CPS14, CPS8, CPS9V, or CPS15b.

65. the fusion protein is glycosylated with an oligosaccharide or polysaccharide, and the oligosaccharide or polysaccharide is produced by a bacterium of the genus Klebsiella; Optionally, the polysaccharide is a capsular polysaccharide of Klebsiella pneumoniae, Klebsiella varricola, Klebsiella michinganenis, or Klebsiella oxytoca.

66. 66. The fusion protein of claim 65, wherein the polysaccharide is a capsular polysaccharide of Klebsiella pneumoniae.

67. 67. The fusion protein of claim 66, wherein the polysaccharide is a serotype K1 or serotype K2 capsular polysaccharide of Klebsiella pneumoniae.

68. 68. The fusion protein of any one of claims 62 to 67, wherein the oligosaccharide or polysaccharide comprises glucose at its reducing end.

69. The fusion protein is produced in vivo; Optionally, produced in a bacterial cell.

69. A fusion protein according to any one of claims 62 to 68.

70. 70. The fusion protein of any one of claims 62 to 69, 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.

71. the ComP glycosylation tag is located at the N-terminus of the fusion protein, the C-terminus of the fusion protein, and / or internally within the fusion protein; Optionally, the carrier protein or fragment thereof is linked to the glycosylation tag via an amino acid linker.

72. 72. The fusion protein of any one of claims 62 to 71, wherein the fusion protein comprises two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, fifteen or more, or twenty or more ComP glycosylation tags.

73. 72. The fusion protein of any one of claims 62 to 71, comprising from any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 to any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 ComP glycosylation tags.

74. 74. The fusion protein of claim 72 or 73, wherein the ComP glycosylation tags are identical.

75. at least two of the ComP glycosylation tags are different from each other; Optionally, at least three, at least four, or at least five of said ComP glycosylation tags are all different from one another; Optionally, none of said ComP glycosylation tags are the same.

74. A fusion protein according to claim 72 or 73.

76. 62. A method for in vivo conjugation of an oligosaccharide or polysaccharide to an acceptor polypeptide, the method comprising covalently attaching the oligosaccharide or polysaccharide to the acceptor polypeptide using a PglS oligosaccharyltransferase (OTase), wherein the acceptor polypeptide comprises a ComP glycosylation tag according to any one of claims 41 to 61; Optionally, the ComP glycosylation tag is linked to a heterologous carrier protein. The method.

77. The PglSOTase is １１０２６４ , Pgls ＡＤＰ１ , Pgls ＧＦＪ－２ , Pgls ５０ｖ１ , Pgls ４４６６ , or Pgls ＳＦＣ 77. The method of claim 76, wherein:

78. The oligosaccharide or polysaccharide is represented by SEQ ID NO: 1 (ComP ＡＤＰ１ 78. The method of claim 76 or 77, wherein the ComP glycosylation tag is linked to the ComP glycosylation tag at a serine residue corresponding to the serine residue at position 84 of GlcNAcAsp (AAC4588631).

79. 79. The method of any one of claims 76 to 78, wherein the in vivo binding occurs in a host cell.

80. 80. The method of claim 79, wherein the host cell is a bacterial cell.

81. 81. The method of claim 80, wherein the bacterial host cell is E. coli.

82. 82. The method of any one of claims 79 to 81, comprising culturing a host cell comprising: (a) a gene cluster encoding proteins necessary to synthesize the oligo- or polysaccharide; (b) a PglSOTase; and (3) the acceptor polypeptide.

83. 83. The method of any one of claims 76 to 82, wherein the production of the oligosaccharide or polysaccharide is enhanced by the K. pneumoniae transcriptional activator rmpA (K. pneumoniae NTUH K-2044) or a homologue of the K. pneumoniae transcriptional activator rmpA (K. pneumoniae NTUH K-2044).

84. 84. The method of any one of claims 76 to 83, wherein the method produces a conjugate vaccine.

85. 62. A host cell comprising: (a) a gene cluster encoding proteins necessary to synthesize an oligosaccharide or polysaccharide, (b) a PglSOTase, and (3) an acceptor polypeptide comprising a ComP glycosylation tag described in any one of claims 41 to 61.

86. 86. The method of claim 85, wherein the acceptor polypeptide is a fusion protein.

87. 87. The method of claim 85 or claim 86, wherein the host cell comprises a nucleic acid encoding the PglSOTase.

88. 88. The method of any one of claims 85 to 87, wherein the host cell comprises a nucleic acid encoding the acceptor polypeptide.

89. 8. An isolated nucleic acid encoding a ComP glycosylation tag according to any one of claims 41 to 61 and / or a fusion protein according to any one of claims 62 to 75.

90. 79. The isolated nucleic acid of claim 78, wherein the nucleic acid is a vector.

91. 80. A host cell comprising the isolated nucleic acid of claim 78 or 79.

92. 80. A composition comprising a conjugate vaccine according to any one of claims 35 to 40 or a fusion protein according to any one of claims 62 to 75, and an adjuvant.

93. A method for inducing a host immune response against a pathogenic microorganism, the method comprising administering to a subject in need of said immune response an effective amount of a conjugate vaccine described in any one of claims 35 to 40, a fusion protein described in any one of claims 62 to 75, or a composition described in claim 92.

94. 94. The method of claim 93, wherein the immune response is an antibody response.

95. 95. The method of claim 94, wherein the immune response is selected from the group consisting of an innate response, an adaptive response, a humoral response, an antibody response, a cell-mediated response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system crosstalk, and a combination of two or more of the immune responses.

96. 96. The method of claim 95, wherein the immune response is selected from the group consisting of an innate response, a humoral response, an antibody response, a T cell response, and a combination of two or more of the immune responses.

97. A method for preventing or treating a bacterial disease and / or infectious disease in a subject, the method comprising administering to a subject in need of prevention or treatment a conjugate vaccine described in any one of claims 35 to 40, a fusion protein described in any one of claims 62 to 75, or a composition described in claim 92.

98. 98. The method of claim 97, wherein the infection is a local or systemic infection of the skin, soft tissue, blood, or organs, or is autoimmune in nature.

99. 98. The method of claim 97, wherein the disease is pneumonia.

100. 99. The method of claim 98, wherein the infection is a systemic infection and / or a blood infection.

101. 101. The method of any one of claims 97 to 100, wherein the subject is a human.

102. 102. The method of any one of claims 97 to 101, wherein the composition is administered via intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.

103. 1. A method for producing a pneumococcal conjugate vaccine against pneumococcal infection, comprising: (a) isolating the bioconjugate of any one of claims 1 to 40 or the glycosylated fusion protein of any one of claims 62 to 75, and (b) combining the isolated conjugate vaccine or the isolated glycosylated fusion protein with an adjuvant; The method comprising:

Images