WO2024182291A2 - Compositions and methods for producing glycoconjugate polypeptides having isopeptide bonds with a second polypeptide partner and uses thereof - Google Patents

Compositions and methods for producing glycoconjugate polypeptides having isopeptide bonds with a second polypeptide partner and uses thereof Download PDF

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WO2024182291A2
WO2024182291A2 PCT/US2024/017287 US2024017287W WO2024182291A2 WO 2024182291 A2 WO2024182291 A2 WO 2024182291A2 US 2024017287 W US2024017287 W US 2024017287W WO 2024182291 A2 WO2024182291 A2 WO 2024182291A2
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seq
glycosylation
polypeptide
fragment
certain embodiments
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Cory James Knoot
Christian Michael Harding
Lloyd Sherwood ROBINSON
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Vaxnewmo Llc
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  • glycoprotein conjugate vaccines can stimulate protective immune responses against the O antigen or capsule present on the surface of gram-positive or gram-negative pathogens.
  • glycoprotein conjugate vaccines can stimulate robust immune memory by inducing T-cell recruitment, memory cell formation, and B-cell IgM-to-IgG antibody class switching leading to immune memory establishment (Rappuoli, et al. (2019) PNAS, 116 (1) 14-16; Avci, F., et al. (2011) Nat Med 17, 1602–1609).
  • Glycoprotein conjugate vaccines can be generated via a number of different methods.
  • the glycan substrate of the OTase is linked to a membrane-bound lipid carrier such as undecaprenyl pyrophosphate (UNDPP).
  • UNDPP undecaprenyl pyrophosphate
  • O-linking OTases catalyze the transfer of UNDPP-linked oligo- or polysaccharides to serine or threonine sidechain hydroxyls in conserved protein motifs termed sequons (Knoot, C., et al. (2021) Glycobiology, Volume 31, Issue 9, September 2021, Pages 1192–1203; Knoot, C., et al. (2023) Glycobiology, Volume 33, Issue 1, January 2023, Pages 57–74).
  • bioconjugate vaccines can be generated in a ‘one-pot’ biological reaction followed by downstream purification (Harding, C. and Feldman, M. (2019) Glycobiology, Volume 29, Issue 7, July 2019, Pages 519–529).
  • bioconjugate vaccines are currently in clinical trials and each is composed of a bacterial glycan linked to a periplasmic protein, primarily Pseudomonas aeruginosa exotoxin A (EPA), Haemophilus Protein D, or CRM197 (a modified diphtheria toxin) (Sorieul, C., et al. (2023) Expert Review of Vaccines, 22:1, 1055-1078).
  • carrier proteins are protein nanoparticles (NPs) or virus-like particles (VLPs): symmetric, self- assembling protein ‘cages’ that are either derivatives of natural virus capsids or rationally engineered protein assemblies (Nguyen, B.
  • NP/VLP-based therapeutics have been shown to improve immune response in part through increased antibody avidity arising from the larger immunogen particle size (Nguyen, B. and Tolia, N.
  • VLPs/NPs Two examples of VLPs/NPs are AP205 and mi3.AP205, is derived from the CP3 coat protein of the RNA bacteriophage AP205 (Brune, K., et al. (2016) Sci Rep 6, 19234). AP205 VLPs assemble into a 120-mer with a diameter of roughly 20 nm (Cohen, et al. (2021) PLoS ONE 16(3): e0247963). mi3 is a porous dodecahedral 60-mer derived from a computationally designed NP 32080280 - 2 - Atty. Dkt.
  • the SpyTag/SpyCatcher system was derived from an immunoglobulin-like collagen adhesin domain (CnaB2) from the fibronectin binding protein, FbaB2, of Streptococcus pyogenes (Zakeri, B. et al. (2012)).
  • the CnaB2 domain naturally forms an intra- protein isopeptide bond between lysine at position 31 and aspartic acid at position 117; specifically, the unprotonated amine of Lys31 acts as a nucleophile attacking the carbonyl carbon of Asp117, which is catalyzed by glutamic acid located at position 77 (Zakeri, B. et al. (2012)).
  • This isopeptide reaction happens spontaneously and seems to be a feature of some members of the prealbumin-like fold domain bacterial proteins to which CnaB2 belongs.
  • the CnaB2 domain was split, separating CnaB2 into (1) a peptide containing the C-terminal ⁇ - strand that contains the reactive Asp117 defined as the SpyTag and (2) a protein binding partner derived from the remaining CnaB2 polypeptide defined as the SpyCatcher (Zakeri, B. et al. (2012)).
  • the system was dubbed SpyTag and SpyCatcher to denote the bacterial source (S. pyogenes) of the CnaB2 fragments.
  • SpyTag003 and SpyCatcher003 Later forms of the SpyTag and SpyCatcher system, designated SpyTag003 and SpyCatcher003 were created through a phage display and subsequent rational engineering approach resulting in reaction rate of 5.5 x 105 M-1 s-1.
  • SpyTag003/SpyCatcher003 reacts approximately 400-fold faster than the original SpyTag/SpyCatcher system (Keeble, A. H. et al. (2019)).
  • the SpyTag/SpyCatcher system(s) have been broadly applied to enable covalently attachment of two, separate polypeptides, one containing a SpyTag and the other containing the SpyCatcher, through the formation of the isopeptide bond.
  • the SpyTag/SpyCatcher system has been applied across a range of biological applications all seeking to covalently attach a polypeptide of interest containing a SpyTag to a different protein or material containing a SpyCatcher, including but not limited, to anchor polypeptides to the surface different solid organic and inorganic materials, to attach polypeptides to different multimerization architectures like nanoparticles, virus-like particles, or Adenoviral vectors, as well as to directly attach polypeptides to the surface of intact cells (Keeble, A. H. & Howarth, M. (2020); Brune, K. D. et al. (2016); Bruun, T. U. J., Andersson, A. C., Draper, S. J.
  • a fusion protein comprising: (i) a glycosylation fragment and (ii) a first polypeptide tag, wherein the first polypeptide tag can spontaneously form an isopeptide bond with a second polypeptide tag binding partner.
  • the fusion protein is a glycoconjugate comprising a saccharide covalently attached to the fusion protein via the glycosylation fragment, and optionally is immunogenic.
  • the first polypeptide tag include a SpyTag (SEQ ID NO: 416), SpyTag002 (SEQ ID NO: 417), SpyTag003 (SEQ ID NO: 418), or a DogTag (SEQ ID NO: 419).
  • the glycosylation fragment is a ComP glycosylation fragment or a variant thereof as described herein.
  • the glycosylation fragment is a TfpM-associated pilin glycosylation fragment or a variant thereof as described herein.
  • the glycosylation fragment is a PilE glycosylation fragment or a variant thereof as described herein.
  • the glycosylation fragment is a PglB glycosylation fragment or a variant thereof as described herein.
  • the glycosylation fragment is a PilA glycosylation fragment or a variant thereof as described herein.
  • the glycosylation fragment is a STT3 glycosylation fragment or a variant thereof as described herein.
  • the glycosylation fragment is a N-linking glycosyltransferase glycosylation fragment.
  • the glycosylation fragment is an O-linking glycosyltransferase glycosylation fragment.
  • the glycosylation fragment is a PilA_Pa5196-associated pilin glycosylation fragment or a variant thereof as described herein.
  • the fusion protein comprises a carrier protein, optionally, wherein the carrier protein is selected from the group consisting of Escherichia coli maltose binding protein, Pseudomonas aeruginosa Exotoxin A (EPA), Pseudomonas aeruginosa PcrV, CRM197, Haemophilus influenzae Protein D, cholera toxin B subunit, or tetanus toxin, and a fragment of any thereof.
  • the carrier protein is selected from the group consisting of Escherichia coli maltose binding protein, Pseudomonas aeruginosa Exotoxin A (EPA), Pseudomonas aeruginosa PcrV, CRM197, Haemophilus influenzae Protein D, cholera toxin B subunit,
  • composition comprising a polypeptide pair that comprises a first polypeptide and a second polypeptide, wherein the first polypeptide is a fusion 32080280 - 4 - Atty. Dkt. No.: 64100-234947 protein of this disclosure, wherein the second polypeptide comprises a second polypeptide tag binding partner to the first polypeptide tag of the first polypeptide, and wherein the first polypeptide is attached to the second polypeptide via an isopeptide bond between the first polypeptide tag and the second polypeptide tag.
  • the second polypeptide comprises a monomeric polypeptide that can spontaneously multimerize/self- assemble into a higher-order, multimeric structure; and optionally, said higher-order, multimeric structure is an icosahedron or dodecahedron particle (e.g,. resembling nanocages), virus-like particle, or Adenoviral vector.
  • the second polypeptide tag is a SpyCatcher (SEQ ID NO: 420), SpyCatcher002 (SEQ ID NO: 421), SpyCatcher003 (SEQ ID NO: 422), or a DogCatcher (SEQ ID NO: 423).
  • the first polypeptide is a bioconjugate comprising a saccharide covalently attached to the glycosylation fragment of the first polypeptide; optionally, wherein said composition is immunogenic.
  • a complex comprising two or more of the polypeptide pairs of this disclosure.
  • the complex is a self-assembled, multimeric higher- order structure.
  • the self-assembled, multimeric higher-order structure is an icosahedron or dodecahedron particle (e.g,. resembling nanocages), virus-like particle, or Adenoviral vector.
  • Also provided for is a method of making the polypeptide pair of this disclosure, the method comprising contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag binding partner.
  • Also provided for is a method of making the complex of this disclosure, the method comprising: (i) forming a self-assembled, multimeric higher-order structure of the second polypeptide and then contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag; or (ii) contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag and then forming of a self-assembled, multimeric higher-order structure of the second polypeptide.
  • a ComP glycosylation fragment is glycosylated by a PglS OTase; a TfpM-associated pilin glycosylation fragment is glycosylated by a TfpM OTase, optionally, wherein a ComP glycosylation fragment is 32080280 - 5 - Atty. Dkt.
  • FIG. 1 Schematic overview of glycoprotein polypeptide production using enzymes that form glycosidic linkages and the subsequent isopeptide bond formation with a second protein containing a polypeptide tag.
  • a first polypeptide contains both (i) a SpyTag, which will spontaneously form an isopeptide bond with a polypeptide tag (SpyCatcher) of a second partner polypeptide (PROTEIN 2), as well as (ii) a glycosylation fragment (Sequon), which is recognized by a specific enzyme that forms glycosidic linkages by covalently transferring a saccharide to the glycosylation fragment (Sequon).
  • PROTEIN 2 containing the polypeptide tag also can self-assemble into higher- order structures like icosahedron or dodecahedron nanoparticles resembling nanocages, virus- like particles (VLPs), or Adenoviral vectors.
  • Figure 2 Coomassie-stained SDS-PAGE denaturing gel of purified MBP-SpyTag- v1 E. coli O16 O-antigen bioconjugate generated using the TfpM oligosaccharyltransferase system, mi3-SpyCatcher, and the isopeptide bonded MBP-SpyTag-v1 E. coli O16:mi3- SpyCatcher.
  • the boxed diagrams show the schematic structure of the SpyTag protein (corresponding to PROTEIN 1 in Figure 1) and SpyCatcher protein (corresponding to PROTEIN 2 in Figure 1) designed for compatibility with enzymes that form glycosidic linkages.
  • MBP maltose binding protein
  • MBPsp E. coli maltose binding 32080280 - 6 - Atty. Dkt. No.: 64100-234947 protein secretory (Sec) signal peptide
  • Pil20 20 amino acid pilin sequon that is glycosylated by the TfpM oligosaccharyltransferase
  • mi3, mi3 nanoparticle monomer 6xHis, hexahistidine tag.
  • Lane E 2:1 reaction mixture of mi3- SpyCatcher and MBP-SpyTag-v1-O16.
  • Figure 3 Western blot of purified MBP-SpyTag-v1 E. coli O16 O-antigen bioconjugate generated using the TfpM oligosaccharyltransferase system, mi3-SpyCatcher, and the isopeptide bonded MBP-SpyTag-v1 E. coli O16:mi3-SpyCatcher.
  • SpyTag and SpyCatcher proteins alone or at a 1:1 or 2:1 ratio (based on protein concentration) were reacted for 2 hours in Tris buffered saline prior western blot analysis.
  • Figure 4 Size-exclusion chromatography of mi3-SpyCatcher and isopeptide bonded MBP-SpyTag-v1-O16:mi3-SpyCatcher separated using a Sephacryl S-400 HR 16/600 column. Shown are UV absorbance traces of 250 ⁇ g mi3-SpyCatcher (solid line) and 250 ⁇ g mi3-SpyCatcher after reaction with 250 ⁇ g MBP-SpyTag-v1-O16 (dashed line) in Tris buffered saline for 2 hours at room temperature. [0029] Figure 5. Coomassie-stained SDS-PAGE denaturing gel of purified MBP-SpyTag- v2 E.
  • Lane A Protein ladder with standards masses in kDa marked to the left.
  • Lane B Purified MBP-SpyTag-v2- O16 bioconjugate.
  • Lane C Purified mi3-SpyCatcher.
  • Lane D 1:1 reaction mixture of mi3- SpyCatcher and MBP-SpyTag-v2-O16.
  • Lane E 2:1 reaction mixture of mi3-SpyCatcher and MBP-SpyTag-v2-O16.
  • Figure 6. Western blot of purified MBP-SpyTag-v2 E.
  • Lane A Protein ladder with standards masses in kDa marked to the left.
  • Lane B Purified MBP-SpyTag-v2-O16 bioconjugate.
  • Lane C Purified mi3-SpyCatcher bioconjugate.
  • Lane D 1:1 reaction mixture of mi3-SpyCatcher and MBP-SpyTag-v2-O16.
  • Lane E 2:1 reaction mixture of mi3-SpyCatcher and MBP-SpyTag- v2-O16.
  • Figure 7. Coomassie-stained SDS-PAGE denaturing gel of purified MBP-SpyTag- v1 E.
  • coli maltose binding protein secretory (Sec) signal peptide Pil20, 20 amino acid pilin sequon that is glycosylated by the TfpM oligosaccharyltransferase; AP205, AP205 virus-like protein coat protein monomer; 6xHis, hexahistidine tag.
  • SpyCatcher and SpyTag fusion proteins alone or at a 1:1 or 2:1 ratio (based on protein concentration) were reacted for 2 hours in Tris buffered saline at room temperature before gel loading. Protein masses associated with proteins and glycoproteins are indicated above each lane. Lane A) Protein 32080280 - 8 - Atty. Dkt.
  • Lane B Purified MBP-SpyTag-v1- O16 bioconjugate.
  • Lane C Purified AP205-SpyCatcher.
  • Lane D 1:1 reaction mixture of AP205-SpyCatcher and MBP-SpyTag-v1-O16.
  • Lane E 2:1 reaction mixture of AP205- SpyCatcher and MBP-SpyTag-v1-O16.
  • Figure 8. Coomassie-stained SDS-PAGE denaturing gel of purified MBP-SpyTag- v2 E.
  • coli O16 O-antigen bioconjugate generated using the PglS oligosaccharyltransferase system, AP205-SpyCatcher, and the isopeptide bonded MBP-SpyTag-v2 E. coli O16:AP205- SpyCatcher.
  • the boxed diagrams show the schematic structure of the SpytTag and SpyCatcher proteins in this experiment.
  • MBP maltose binding protein
  • MBPsp E.
  • coli maltose binding protein secretory (Sec) signal peptide ; ComP sequon, 23 amino acid ComP- derived sequon that is glycosylated by the PglS OTase; AP205, AP205 Virus-like protein coat protein monomer; 6xHis, hexahistidine tag.
  • SpyTag and SpyCatcher fusion proteins alone or at a 1:1 or 2:1 ratio (based on protein concentration) were reacted for 2 hours in Tris buffered saline at room. Isopeptide bond reaction formation was stopped by the addition of Laemmli buffer and subsequent heating of the sample for 10 mins at 100 ⁇ C prior to gel loading.
  • coli O16 O-antigen bioconjugates generated using the PglS oligosaccharyltransferase system, mi3-SpyCatcher, and isopeptide-bonded EPA-Spytag E. coli O16:mi3-SpyCatcher.
  • the boxed diagrams show the schematic structure of the EPA SpyTag proteins designed for compatibility with enzymes that form glycosidic linkages.
  • coli maltose binding protein secretory (Sec) signal peptide ; ComP Sequon, 23 amino acid pilin sequon that is glycosylated by the PglS oligosaccharyltransferase; mi3, mi3 nanoparticle monomer; 6xHis, hexahistidine tag.
  • the isopeptide bond formation reactions were performed using purified EPA-Spycatcher-O16 bioconjugates and purified mi3-Spycatcher. Lane A) Protein ladder with standards masses in kDa marked to the left. Lane B) Purified mi3-Spycatcher. Lane C) Purified EPA-Spytag-v1- O16.
  • Lane D 1:1 reaction mixture of mi3-Spycatcher and EPA-SpyTag-v1-O16.
  • Lane E 1:2 32080280 - 9 - Atty. Dkt. No.: 64100-234947 reaction mixture of mi3-Spycatcher and EPA-SpyTag-v1-O16.
  • Lane F Purified EPA-Spytag- v2-O16.
  • Lane G 1:1 reaction mixture of mi3-Spycatcher and EPA-SpyTag-v2-O16.
  • Lane H 1:2 reaction mixture of mi3-Spycatcher and EPA-SpyTag-v2-O16.
  • Lane I Purified EPA- Spytag-v3-O16.
  • Lane J 1:1 reaction mixture of mi3-Spycatcher and EPA-SpyTag-v3-O16.
  • Lane K 1:2 reaction mixture of mi3-Spycatcher and EPA-SpyTag-v3-O16. Isopeptide-bonded proteins were not observed using EPA-Spytag-v2-O16 or EPA-Spytag-v3-O16 and mi3- Spycatcher.
  • Figure 10 Western blot of purified, unglycosylated EPA-SpyTag-v1 protein linker variants, mi3-SpyCatcher, and the isopeptide bonded EPA-SpyTag E. coli:mi3-SpyCatcher.
  • Each protein variant has a different amino acid linker between Spytag003 and EPA.
  • the isopeptide bond formation reactions were performed using EPA-Spytag proteins from E. coli periplasmic extracts and purified mi3-Spycatcher. The western blot was probed with anti-His tag antibody.
  • Lane A Protein ladder with standards masses in kDa marked to the left.
  • Lane B Purified EPA-Spytag-v1 with linker L1 (SGG).
  • Lane C 1:1 reaction mixture of mi3- Spycatcher and EPA-Spytag-v1 with linker L1.
  • Lane D Purified EPA-Spytag-v1 with linker L2 (SEQ ID NO: 430).
  • Lane E 1:1 reaction mixture of mi3-Spycatcher and EPA-Spytag-v1 with linker L2.
  • Lane F Purified EPA-Spytag-v1 with linker L3 (SEQ ID NO: 431).
  • Lane G 1:1 reaction mixture of mi3-Spycatcher and EPA-Spytag-v1 with linker L3.
  • Lane H Purified EPA-Spytag-v1 with linker L4 (SEQ ID NO: 432).
  • Lane I 1:1 reaction mixture of mi3- Spycatcher and EPA-Spytag-v1 with linker L4.
  • Lane J Purified EPA-Spytag-v1 with linker L5 (SEQ ID NO: 433).
  • Lane Q 1:1 reaction mixture of mi3-Spycatcher and EPA-Spytag-v1 with linker L8.
  • Lane R Purified EPA-Spytag-v1 with linker L9 (SEQ ID NO: 437).
  • Lane S 1:1 reaction mixture of mi3-Spycatcher and EPA-Spytag-v1 with linker L9.
  • Lane T Purified EPA-Spytag-v1 with linker L10 (SEQ ID NO: 438).
  • Lane U 1:1 reaction mixture of mi3- Spycatcher and EPA-Spytag-v1 with linker L10.
  • Figure A Protein ladder with standards masses in kDa marked to the left. Lane B) mi3-Spycatcher only. Lane C) EPA-Spytag-v1 with linker L7 only. Lane D) Isopeptide bond reaction between mi3-Spycatcher and EPA-Spytag-v1 with linker L7 after 0.5 hours. Lane E) The reaction after 1 hour. Lane F) The reaction after 2 hours. Lane G) The reaction after 5 hours. Lane H) The reaction after 24 hours.
  • Figure 12A-E Figure 12A shows a schematic of EPA-ComP110264 fusion proteins where the ComP glycosylation fragment is fused at the C-terminus of the fusion protein.
  • “ssDsbA” corresponds to the DsbA Sec secretion signal.
  • GGGS SEQ ID NO: 382
  • Figure 12B shows different amino acid sequences for ComP glycosylation fragments fused to C-terminus of the EPA fusion protein.
  • the bold, underlined serine residue in each sequence corresponds to the conserved serine 82 of ComP110264 and is the site of glycosylation.
  • the bold, underlined cysteine residues corresponding to Cys71 and Cys93 are also highlighted.
  • Figure 12C, Figure 12D, and Figure 12E show Western blot analysis of periplasmic extracts from E. coli SDB1 expressing PglS, the CPS8 glycan and an EPA-ComP 110264 variant.
  • Each lane of the Western blot panel corresponds to a strain of SDB1 expressing a different EPA-ComP variant with the ComP glycosylation fragment corresponding to the sequence shown in Figure 12B.
  • Figure 12C shows proteins reacting with the anti-EPA antisera.
  • Figure 12D shows proteins reacting with the anti-His antisera.
  • Figure 12E shows the merged western blot images of Figure 12C and Figure 12D. Equivalent amounts of periplasmic extract based on OD600 were loaded per lane.
  • g 0 denotes unglycosylated EPA- ComP110264 and gn denotes EPA-ComP110264 glycosylated with different numbers of CPS8 repeat units.
  • FIG. 13A shows a schematic of the CRM 197 -ComP C1 fusion protein.
  • “ssFlgI” corresponds to the FlgI SRP secretion signal.
  • GGGS SEQ ID NO: 382 is a flexible linker between CRM197 and ComPC1.
  • Figure 13B, Figure 13C, and Figure 13D show 32080280 - 11 - Atty. Dkt. No.: 64100-234947 Western blot analysis of the purified CRM 197 -ComP C1 -CPS8 glycoconjugate.
  • Figure 13B shows the proteins reacting with the anti-CPS8 antisera.
  • Figure 13C shows the proteins reacting with the anti-CRM197 antisera.
  • Figure 2D shows the merged western blot images of Figure 13B and Figure 13C.
  • Loss of CRM197 and CPS8 signals in the proteinase K (PK)- treated samples demonstrate that the pneumococcal serotype 8 signal is CRM 197 -linked and not the result of contamination from free polysaccharide or lipid-linked polysaccharide precursors.
  • Protein mass markers (in kDa) are indicated to the left of panels Figure 13B-D.
  • Figure 14A,B Figure 14A shows schematic diagrams of the C- and N-terminal CRM 197 variants containing the C1 ComP glycosylation fragment.
  • Figure 14B shows Western blot analysis of periplasmic extracts of E. coli SDB1 expressing CRM197-ComPC1 or ComPC1- CRM197 and the CPS8 glycan in the presence (+) or absence (-) of PglS. Equivalent amounts of periplasmic extracts based on OD 600 were loaded per lane. Protein mass markers (in kDa) are indicated to the left. GGGS (SEQ ID NO: 382). [0039] Figure 15A-E. Figure 15A shows a schematic diagram of EPA fusion proteins containing ComP glycosylation fragments integrated internal of the EPA amino acid sequence.
  • Figure 15B shows amino acid sequences of the two iGT ComP glycosylation fragments inserted between EPA residues Ala489 and Arg489. These have either two terminal cysteines (“iG CC ”; SEQ ID NO: 230) or serines (“iG SS ”; SEQ ID NO: 231).
  • Figure 15C and Figure 15D show Western blots on periplasmic extracts of E. coli SDB1 expressing the CPS8 glycan, EPAiGTcc or EPAiGTss, with (+) or without (-) PglS.
  • Figure 15C shows proteins reacting with the anti-EPA antisera.
  • Figure 15D shows proteins reacting with the anti-His antisera.
  • Figure 15E shows the merged Western blot images of Figure 15C and Figure 15D. Equivalent amounts of periplasmic extracts based on OD600 were loaded per lane. Protein mass markers (in kDa) are indicated to the left of panels.
  • Figure 16A-D Figure 16A show a schematic Diagram of EPA constructs containing ComP glycosylation fragments used for these experiments (from top to bottom, SEQ ID NOs: 6-28). Twenty-two to five amino acid-truncated variants of the iGTCC ComP glycosylation fragment were inserted into the EPA coding sequence between Ala489 and Arg489.
  • Figure 16B shows the amino acid sequences of the 22 truncated iGT ComP glycosylation fragments with name designations assigned to the left.
  • the underlined, bolded serine is the glycosylation site.
  • Each lane of the Western blot panel corresponds to a strain of SDB1 expressing a different EPAiGT fusion protein containing a truncated ComP glycosylation fragment with the ComP glycosylation fragment corresponding to the sequence shown in Figure 16B.
  • Figure 16C shows proteins reacting with the anti-EPA antisera probing with an anti-EPA antibody.
  • EPAiGTcc is shown for comparison.
  • the “EPA” lane corresponds to EPA lacking any ComP-derived sequences and serves as a negative control.
  • Figure 16D shows the same Western blot as above with an increase anti-EPA signal brightness in order to show low- level glycosylation for the smallest ComP glycosylation fragments.
  • Figure 17A,B,C shows Western blot analysis of Ni affinity chromatography purified EPA fusion proteins containing the iGT ⁇ 6-6 ComP glycosylation fragment integrated between residues Ala489 and Arg490 of EPA. The fusion protein was purified from SDB1 cells expressing the CPS8 glycan in the presence (+) or absence (-) of PglS.
  • Figure 17A shows proteins reacting with anti-His antisera.
  • Figure 17B shows proteins reacting with anti-CPS8 antisera.
  • Figure 17C shows a merge of Figure 17A and Figure 17B. Protein mass markers (in kDa) are indicated to the left of panels Figure 17A-C.
  • Figure 18A and 18B show a schematic diagram of the EPA fusion protein containing the iGT ⁇ 3-4 ComP glycosylation fragment integrated between residues Glu548 and Gly549 of EPA. The iGT ⁇ 3-4 amino acid sequence is listed below the schematic (SEQ ID NO: 271).
  • Figure 18B shows Western blot analysis on periplasmic extracts of E.
  • Figure 19A,B,C shows Western blot analysis of Ni affinity chromatography purified EPA fusion proteins containing the iGT ⁇ 3-4 ComP glycosylation fragment integrated between residues Glu548 and Gly549 of EPA.
  • the fusion protein was purified from SDB cells expressing the CPS8 glycan in the presence (+) or absence (-) of PglS.
  • Figure 19A shows proteins reacting with anti-His antisera.
  • Figure 19B shows proteins reacting 32080280 - 13 - Atty. Dkt. No.: 64100-234947 with anti-CPS8 antisera.
  • Figure 19C shows a merge of Figure 19A and Figure 19B. Protein mass markers (in kDa) are indicated to the left of panels Figure 19A-C.
  • Figure 20 lists ComP ortholog amino acid sequences. The site of predicted glycosylation is bolded.
  • Figure 21 lists ComP ⁇ 28 ortholog amino acid sequences in which the amino acids corresponding to the 28 N-terminal amino acids of ComPADP1: AAC45886.1 have been removed.
  • Figure 22 shows an alignment of a region ComP sequences including the serine (S) residue (boxed) corresponding to the serine residue at position 82 of ComP 110264 (SEQ ID NO: 201) also corresponding to the serine residue at position 84 of ComPADP1 (SEQ ID NO: 202).
  • Figure 23A-D Figure 23 shows the characterization of 13 TfpM orthologs from species in the Moraxellaceae family.
  • FIG. 23B Diagram showing the EPA-pilin fusion protein and TfpM construct design. Colored arrows indicate genes. Gene expression was driven from an IPTG-inducible tac promoter with a lacO operator (tac1O). The rrnB T2 terminator is marked by a black hairpin structure.
  • H286A indicates the M. osloensis TfpM site directed OTase active site mutant.
  • “g0” indicates unglycosylated EPA-pilin and “gn” indicates CPS8-glycosylated EPA-pilin protein. Reference protein masses are marked left of the western blot in kDa.
  • Figure 24 shows a phylogram of TfpM, PilO, PglL, and PglS orthologs with relative distances shown.
  • the phylogenetic tree was generated using the phylogeny.fr server (on the world wide web at phylogeny.fr/) which uses MUSCLE, PhyML, and TreeDyn for sequence alignment, tree calculation, and image generation, respectively.
  • Figure 25 Figure 25 Phylogram of TfpM-associated pilin-like proteins, select PilA proteins from Neisseria and Pseudomonas, and ComP from A. soli CIP 110264 with relative distances shown. The red numbers indicate branch confidence.
  • the phylogenetic tree was generated using the phylogeny.fr server (on the world wide web at phylogeny.fr/) which 32080280 - 14 - Atty. Dkt.
  • FIG. 64100-234947 uses MUSCLE, PhyML, and TreeDyn for sequence alignment, tree calculation, and image generation, respectively.
  • Figure 26 shows multiple sequence alignment of select bacterial O- linking oligosaccharyltransferase. The alignment was generated using Clustal Omega with default settings found on the world wide web at ebi.ac.uk/Tools/msa/clustalo/. N_menigitidis_MC58_PglL (SEQ ID NO: 105).
  • Figure 27 shows an anti-EPA whole-cell western blot examining the glycosylation status of the EPA-PilMo ⁇ 28 fusions as well as EPA-PilMo ⁇ 28 C-terminal Thr 167 mutants. All lanes were normalized to the same OD 600 .
  • Figure 28A and 28B Figure 28 shows Targeted MS/MS analysis of the HexHexA-modified C-terminal EPA-PilMo ⁇ 28 peptide 762 FLPANCRGT 770 (SEQ ID NO: 61).
  • Figure 28B) HCD fragmentation enables the confirmation of the peptide sequence as well as the linkage of the disaccharide HexHexA through the Hex monosaccharide by the observation of multiple y-ions linked solely to a Hex residue.
  • Figure 29A-G Figure 29A-G.
  • Figure 29 shows TfpM Mo can transfer diverse bacterial glycans to the EPA-PilMo ⁇ 28 fusion protein.
  • Figure 29A Structures of the repeat units of the five bacterial glycans tested with TfpMMo. The linkages between sugar monomers are indicated in rounded brackets.
  • Figure 29D Anti-LT2.
  • Figure 29E Anti-O2a.
  • Figure 29F Anti-GBSIII.
  • Figure 29G Anti-EPA.
  • +/- labels indicate whether the samples were incubated with (+) or without (-) proteinase K prior to SDS-PAGE separation. Reference protein masses are marked next to the western blots in kDa. 32080280 - 15 - Atty. Dkt. No.: 64100-234947 [0054]
  • Figure 30A and 30B Figure 30 shows TfpM Mo glycosylates truncated EPA-fused pilin variants as small as three amino acids.
  • FIG 30A Sequences of the EPA-fused Pil Mo fragments tested for bioconjugation with TfpMMo. Blue letters mark the C-terminal residues of EPA (i.e., EDLK; SEQ ID NO: 132). Underlined residues indicate the glycine linker placed between EPA and the pilin sequence.
  • Figure 30B Anti-EPA western blot of whole cell extracts expressing the truncated pilin variants, CPS8, and TfpMMo. The calculated EPA-PilMo ⁇ 28 mass is 80.3 kDa and that of the truncated variants ranges from 67.1 to 69.0 kDa. All lanes were normalized to the same OD 600 .
  • Pil20 (SEQ ID NO: 60).
  • Pil 15 (SEQ ID NO: 109).
  • Pil 13 (SEQ ID NO: 110).
  • GGGG plus Pil 10 is Pil 10L (SEQ ID NO: 111).
  • Pil 10 (SEQ ID NO: 112).
  • Pil 7 (SEQ ID NO: 113).
  • Pil 6 (SEQ ID NO: 114).
  • Pil 5 (SEQ ID NO: 115).
  • Pil4 (SEQ ID NO: 116).
  • Figure 31 shows multiple sequence alignment of select pilin proteins. Accession numbers for these proteins are given in the main text. The alignment was generated using Clustal Omega with default settings found on the world wide web at ebi.ac.uk/Tools/msa/clustalo/. P_aeruginosa_1244_PilA (SEQ ID NO: 119). N_menigitidis_M2_PilA (SEQ ID NO: 120). A_junii_65_pilin (SEQ ID NO: 97). A_CIP102143_pilin (SEQ ID NO: 88). A_CIP102637_pilin (SEQ ID NO: 100).
  • FIG. 32 shows purified TfpM Mo -derived GBSIII bioconjugate elicits a robust IgG immune response in mice.
  • Figure 32A Western blot of purified GBSIII- 291 bioconjugate, anti-EPA channel.
  • Figure 32B Anti-GBSIII.
  • Figure 32F GBSIII- specific IgG kinetics over the course of immunization as measured by ELISA and converted to ng/mL IgG using a standard IgG curve. **P ⁇ 0.01.
  • Figure 33A and 33B Figure 33 shows glycosylation of EPA constructs containing sequons from different O-linking oligosaccharyltransferase systems.
  • Figure 33A Diagrams of the plasmid-based operons expressing EPA with PglS- or TfpM-specific sequons.
  • iGT Internal glycotag
  • SDB1 periplasmic extracts expressing one of the four constructs and the E. coli O16 O-antigen. Load amounts per lane were normalized to OD 600 .
  • g 0 indicates unglycosylated EPA carrier protein and the singly or doubly glycosylated EPA proteins are indicated. Reference protein masses are marked left of the western blot in kDa.
  • a or “an” entity refers to one or more of that entity; for example, "a polysaccharide,” is understood to represent one or more polysaccharides.
  • the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.
  • polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
  • peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms.
  • polypeptide is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-standard amino acids.
  • a polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.
  • a “protein” as used herein can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides that are associated, e.g., by disulfide bonds, hydrogen bonds, or hydrophobic interactions, to produce a multimeric protein.
  • an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For 32080280 - 18 - Atty. Dkt. No.: 64100-234947 example, an isolated polypeptide can be removed from its native or natural environment.
  • a "vector” (also use interchangeably herein with “plasmid”) is nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell.
  • a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector can encode and express a protein.
  • a vector can also include one or more selectable marker gene and other genetic elements known in the art.
  • a "transformed” cell, or a "host” cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques.
  • transformation encompasses those techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.
  • a transformed cell or a host cell can be a bacterial cell or a eukaryotic cell.
  • expression refers to a process by which a gene produces a biochemical, for example, a polypeptide.
  • the process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), and the translation of such mRNA into 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 that is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like.
  • the terms "treat,” “treatment,” or “treatment of” refers to reducing the potential for disease pathology, reducing the occurrence of disease symptoms, e.g., to an extent that the subject has a longer survival rate or reduced discomfort.
  • treating can refer to the ability of a therapy when administered to a subject, to reduce disease symptoms, signs, or causes. Treating also refers to 32080280 - 19 - Atty. Dkt. No.: 64100-234947 mitigating or decreasing at least one clinical symptom and/or inhibition or delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness.
  • subject or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals, including, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, llamas, bears, and so on.
  • pharmaceutical composition or “therapeutic composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile.
  • a “saccharide” is a general term used to refer to any sized carbohydrate molecule; including but not limited to, monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, oligosaccharide, or polysaccharide.
  • a “glycosidic linkage” is a covalent bond between a saccharide and another organic molecule, including but not limited to, another saccharide, a protein, a lipid, or a nucleic acid.
  • a “glycosylation fragment” or “sequon” is a sequence of consecutive amino acids in a protein that serves as the recognition and attachment site for a saccharide, which is covalently transferred to the protein by a glycosyltransferase or oligosaccharyltransferase.
  • the term “translationally fused” can mean directly attached to (e.g., a carrier protein, N-terminal leader sequence, C-terminal tag, or the like of the fusion protein), or it can mean indirectly attached via an amino acid linker.
  • polypeptide pair comprising of a polypeptide tag and a binding partner (e.g., another polypeptide tag), wherein the polypeptide tag and the binding partner can bind to one another via the spontaneous formation of an isopeptide bond between one reactive residue comprised within said binding partner and another reactive residue comprised within said polypeptide tag.
  • a binding partner e.g., another polypeptide tag
  • the other can be referred to as its partner. 32080280 - 20 - Atty. Dkt.
  • One component of a polypeptide pair of this disclosure can be a fusion protein.
  • a fusion protein comprising: (i) a glycosylation fragment and (ii) a first polypeptide tag, wherein the first polypeptide tag can spontaneously form an isopeptide bond with a second polypeptide tag binding partner.
  • a fusion protein comprising a glycosylation fragment can comprise a fragment containing the glycosylation site (also referred to herein as a “sequon”) of a protein that is glycosylated or, in other embodiments, can comprise the full-length protein that is glycosylated, which in turn comprises the glycosylation fragment.
  • the fusion protein comprises a carrier protein.
  • the carrier protein can be Escherichia coli maltose binding protein (MPB), Pseudomonas aeruginosa Exotoxin A (EPA), Pseudomonas aeruginosa PcrV, CRM197, Haemophilus influenzae Protein D, cholera toxin B subunit, tetanus toxin, or a fragment of any thereof.
  • the glycosylation fragment can be as little as 3 amino acids in length.
  • TfpM OTase can recognize a three-amino acid sequon.
  • the glycosylation fragment can be longer, including a full-length or nearly full-length protein.
  • the glycosylation fragment is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, or 40 amino acids in length.
  • the glycosylation is not a full-length glycosylated protein, but a shorter fragment thereof, and thus is not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, 40, 50, 60, 80, or 100 amino acids in length. Therefore, in certain embodiments, the glycosylation fragment is from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 24 amino acids in length to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, or 25 amino acids in length.
  • the glycosylation fragment is from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, or 40 amino acids in length to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, 40, or 50 amino acids in length. In certain embodiments, the glycosylation fragment is from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, 40, 50, 60, or 80 amino acids in length to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, 40, 50, 60, 80, or 100 amino acids in length. 32080280 - 21 - Atty. Dkt.
  • the fusion protein is a glycoconjugate comprising a saccharide covalently attached to the fusion protein via the glycosylation site/residue of the glycosylation fragment (sequon).
  • the glycoconjugate is immunogenic.
  • the saccharide can be covalently attached to the glycosylation fragment, for example, through an N-linkage, O-linkage, or C-linkage.
  • the first polypeptide tag is translationally fused at the N- terminal end of the fusion protein.
  • the first polypeptide tag is translationally fused at the C-terminal end of the fusion protein. In certain embodiments, the first polypeptide tag is translationally fused internally within the fusion protein. In certain embodiments, the first polypeptide tag is translationally fused internally within the sequence of a carrier protein. [0082] In certain embodiments, the glycosylation fragment is translationally fused at the N-terminal end of the fusion protein. In certain embodiments, the glycosylation fragment is translationally fused at the C-terminal end of the fusion protein. In certain embodiments, the glycosylation fragment is translationally fused internally within the fusion protein. In certain embodiments, the glycosylation fragment is translationally fused internally within the sequence of a carrier protein.
  • glycosylation fragment As can be understood from this disclosure as a whole, by fused internally within the fusion protein, it is meant that the glycosylation fragment, polypeptide tag, or the like is not located at the C-terminal end or the N-terminal end of the fusion protein, not including any signal/leader sequence, purification tag (e.g., His-Tag), or the like.
  • N-terminal Glycosylation fragment Carrier protein N-terminal, not internal Leader sequence
  • Glycosylation fragment Carrier protein C-terminal Carrier protein—Glycosylation fragment C-terminal, not internal Carrier protein—Glycosylation fragment—His-Tag 32080280 - 22 - Atty. Dkt.
  • the glycosylation fragment, polypeptide tag, or the like can be placed (translationally fused) between separate carrier proteins (even if the same type of carrier protein). In certain embodiments of an internal placement, the glycosylation fragment, polypeptide tag, or the like can be placed (translationally fused) internally within the sequence of a single carrier protein.
  • representative examples of one polypeptide tag partner of a pair include SpyTag (SEQ ID NO: 416), SpyTag002 (SEQ ID NO: 417), SpyTag003 (SEQ ID NO: 418), or a DogTag (SEQ ID NO: 419).
  • the SpyTag, Spytag002, or Spytag003 is translationally fused at the N-terminal end of the fusion protein (e.g., Figure 2).
  • the SpyTag, Spytag002, or Spytag003 is translationally fused at the C-terminal end of the fusion protein (e.g., Figure 5).
  • the DogTag is translationally fused internally within the fusion protein.
  • Representative examples of fusion proteins of this disclosure are shown in Figure 2, Figure 5, Figure 7, Figure 8, and Figure 9.
  • EPA-Spytag-v1 SEQ ID NO: 427
  • EPA-Spytag-v2 SEQ ID NO: 428
  • EPA-Spytag-v3 SEQ ID NO: 429
  • an amino acid linker sequence is translationally inserted between components of the fusion protein (e.g., a signal peptide, a polypeptide tag sequence, a glycosylation fragment, a carrier protein, a histidine tag, etc.).
  • the amino acid linker sequence is GGS, GGGGGG (SEQ ID NO: 430), GGGGGGGG (SEQ ID NO: 431), GGGGS (SEQ ID NO: 432), EAAAK (SEQ ID NO: 433), PAPAPPAPAP (SEQ ID NO: 434), EAAAKEAAAK (SEQ ID NO: 435), GGGGSPAPAP (SEQ ID NO: 436), GGGGSGGGGS (SEQ ID NO: 437), or EAAAKGGGGS (SEQ ID NO: 438).
  • certain embodiments comprise an EPA-Spytag-v1 of SEQ ID NO: 427, an EPA-Spytag-v2 of SEQ ID NO: 428, or an EPA-Spytag-v3 of SEQ ID NO: 429, having one or more amino acid linkers, such as above, translationally inserted between components of the fusion protein.
  • an EPA- Spytag-v1 of SEQ ID NO: 427 having the amino acid linker SSG translationally inserted after the polypeptide tag.
  • an EPA-Spytag-v1 of SEQ ID NO: 440 having the amino 32080280 - 23 - Atty. Dkt.
  • a glycosylation fragment is a ComP glycosylation fragment.
  • a sequence comprising a glycosylation fragment of ComP could be a full-length ComP protein.
  • the ComP glycosylation fragment comprises or consists of the amino acid sequence CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 412) or a fragment thereof comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412. In certain embodiments, the ComP glycosylation fragment comprises or consists of the amino acid sequence CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 412) or a fragment thereof at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids in length comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412.
  • the ComP glycosylation fragment comprises or consists of the amino acid sequence CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 412) or a fragment thereof at least 10 amino acids in length comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412. In certain embodiments, the ComP glycosylation fragment comprises or consists of the amino acid sequence CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 412) or a fragment thereof at least 11 amino acids in length comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412.
  • the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions.
  • SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions.
  • substitutions can be conservative amino acid substitutions.
  • the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having cumulatively one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. 32080280 - 24 - Atty. Dkt. No.: 64100-234947 [0089]
  • the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions and/or additions.
  • the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or additions. [0090] In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions and/or deletions.
  • the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or deletions.
  • the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid additions and/or deletions.
  • the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having cumulatively one, two, three, four, five, or six amino acid additions and/or deletions. [0092] In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions.
  • the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid additions. In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid deletions. [0093] In certain embodiments, the ComP glycosylation fragment comprises or consists of any of the further ComP glycosylation fragment sequences described elsewhere herein. 32080280 - 25 - Atty.
  • the glycosylation fragment is a TfpM-associated pilin glycosylation fragment.
  • a sequence comprising a glycosylation fragment of a TfpM-associated pilin could be a full- length TfpM-associated pilin protein.
  • the TfpM-associated pilin glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (SEQ ID NO: 413) or a fragment thereof comprising at least the last three amino acids from the TfpM- associated pilin C-terminus (i.e., RGT).
  • the TfpM-associated pilin glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (SEQ ID NO: 413) or a fragment thereof at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids in length comprising at least the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT).
  • the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO:) 413 comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions.
  • SEQ ID NO: 413 comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions.
  • the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having cumulatively one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions.
  • SEQ ID NO: 413 the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having cumulatively one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions.
  • the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid substitutions and/or additions.
  • SEQ ID NO: 413 the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid substitutions and/or additions.
  • the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or additions.
  • SEQ ID NO: 413 the TfpM-associated pilin C-terminus
  • the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid substitutions and/or deletions.
  • SEQ ID NO: 413 the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid substitutions and/or deletions.
  • the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or deletions.
  • SEQ ID NO: 413 the TfpM-associated pilin C-terminus
  • the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid additions and/or deletions.
  • SEQ ID NO: 413 the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid additions and/or deletions.
  • the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having cumulatively one, two, three, four, five, or six amino acid additions and/or deletions.
  • SEQ ID NO: 413 the TfpM-associated pilin C-terminus
  • the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid substitutions.
  • SEQ ID NO: 413 the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid substitutions.
  • the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid additions.
  • SEQ ID NO: 413 the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid additions.
  • the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid deletions.
  • the TfpM-associated pilin glycosylation fragment comprises or consists of any of the further TfpM-associated pilin glycosylation fragment sequences described elsewhere herein.
  • the glycosylation fragment is a PilE glycosylation fragment.
  • a sequence comprising a glycosylation fragment of PilE could be a full-length PilE protein.
  • the PilE glycosylation fragment comprises or consists of the amino acid SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) or a fragment thereof comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 32080280 - 27 - Atty. Dkt. No.: 64100-234947 of SEQ ID NO: 414.
  • the PilE glycosylation fragment comprises or consists of the amino acid SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) or a fragment thereof at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acids in length comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414.
  • the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions.
  • the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having cumulatively one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions.
  • the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid substitutions and/or additions.
  • the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or additions.
  • the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid substitutions and/or deletions.
  • the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or deletions. 32080280 - 28 - Atty. Dkt.
  • the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid additions and/or deletions.
  • the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having cumulatively one, two, three, four, five, or six amino acid additions and/or deletions.
  • the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid substitutions.
  • the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid deletions.
  • PglB glycosylation fragments [0107]
  • the glycosylation fragment is a PglB glycosylation fragment.
  • a sequence comprising a glycosylation fragment of PglB could be a full-length PglB protein.
  • the PglB glycosylation fragment comprises or consists of the consensus motif amino acid sequence X1 X2 N X3 X4, wherein X1 is D or E, X2 is any amino acid except proline, X 3 is any amino acid except proline, and X 4 is S or T.
  • PilA glycosylation fragments [0108] Without being limited to any particular glycosylation sequence, in certain embodiments the glycosylation fragment is a PilA glycosylation fragment. A sequence comprising a glycosylation fragment of PilA could be a full-length PilA protein. In certain embodiments, the PilA glycosylation fragment comprises or consists of the PilA pilin disulfide 32080280 - 29 - Atty.
  • the PilA glycosylation fragment comprises or consists of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) or a fragment thereof at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids in length comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS).
  • the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having cumulatively one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions.
  • the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having one, two, three, four, five, or six amino acid substitutions and/or additions.
  • the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or additions.
  • the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having one, two, three, four, five, or six amino acid substitutions and/or deletions.
  • the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or deletions.
  • the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID 32080280 - 30 - Atty. Dkt.
  • the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having cumulatively one, two, three, four, five, or six amino acid additions and/or deletions.
  • the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having one, two, three, four, five, or six amino acid substitutions.
  • the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having one, two, three, four, five, or six amino acid additions.
  • the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having one, two, three, four, five, or six amino acid deletions.
  • PilA Pa5196 glycosylation fragments [0114]
  • the glycosylation fragment is a PilA_Pa5196-associated pilin glycosylation fragment.
  • a sequence comprising a glycosylation fragment of PilA_Pa5196-associated pilin could be a full-length PilA_Pa5196-associated pilin protein.
  • the PilA_Pa5196-associated pilin glycosylation fragment comprises or consists of the strands 1 and 2 of the antiparallel beta-sheet domain of PilA_Pa5196 GKYSSVDSTIASGYPNGQITVTMTQG (SEQ ID NO: 426) or a fragment thereof.
  • the PilA_Pa5196-associated pilin glycosylation fragment comprises or consists of the strands 1 and 2 of the antiparallel beta-sheet domain of PilA_Pa5196 GKYSSVDSTIASGYPNGQITVTMTQG (SEQ ID NO: 426) or a fragment thereof at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
  • the glycosylation fragment is a variant of PilA_Pa5196- associated pilin glycosylation fragment consistent with the variants of other glycosylation 32080280 - 31 - Atty. Dkt.
  • STT3 glycosylation fragments [0116] Without being limited to any particular glycosylation sequence, in certain embodiments the glycosylation fragment is a STT3 glycosylation fragment. A sequence comprising a glycosylation fragment of STT3 could be a full-length STT3 protein. In certain embodiments, the STT3 glycosylation fragment comprises or consists of the consensus motif amino acid sequence N-X-S/T, wherein X is any amino acids except proline and S/T is serine (S) or threonine (T).
  • the glycosylation fragment is a N-linking glycosyltransferase glycosylation fragment.
  • the N-linking glycosyltransferase glycosylation fragment comprises or consists of the consensus motif amino acid sequence N-X-S/T, wherein X is any amino acids except proline and S/T is serine (S) or threonine (T).
  • the glycosylation fragment is an O-linking glycosyltransferase glycosylation fragment.
  • the O-linking glycosyltransferase glycosylation fragment comprises or consists of a fragment of the serine or threonine rich repeats from the serine-rich repeats (SRR) adhesins of streptococci or staphylococci bacteria.
  • the O-linking glycosyltransferase glycosylation fragment comprises or consists of serine (S) or threonine (T) rich repeats from the adhesin GspB from Streptococcus gordonii.
  • Multiple glycosylation fragments [0119] Certain aspects of this disclosure are drawn to fusion proteins comprising two or more glycosylation fragments. For example, wherein the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 glycosylation fragments.
  • the fusion protein comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 glycosylation fragments to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 glycosylation fragments.
  • at least one glycosylation fragment is located at the N-terminal end of the fusion protein and at least one glycosylation fragment is located internally within the fusion protein.
  • at least one glycosylation fragment is located at the C-terminal end of 32080280 - 32 - Atty. Dkt. No.: 64100-234947 the fusion protein and at least one glycosylation fragment is located internally within the fusion protein.
  • At least two glycosylation fragments are located internally within the fusion protein. Further, in certain embodiments, one glycosylation fragment is located at the N-terminal end of the fusion protein and one glycosylation fragment is located at the C-terminal end of the fusion protein. In certain embodiments, the two or more glycosylation fragments are the same. For example, a fusion protein having multiple ComP glycosylation fragments. In certain embodiments, at least one of the two or more glycosylation fragments is different or each of the glycosylation fragments is different.
  • fusion protein wherein one glycosylation fragment is a ComP glycosylation fragment and one glycosylation fragment is a TfpM-associated pilin glycosylation fragment.
  • the fusion protein is a glycoconjugate comprising two or more saccharide covalently attached to the fusion protein via the two or more glycosylation fragments.
  • the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 covalently attached saccharides.
  • the fusion protein comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 covalently attached saccharides to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 covalently attached saccharides.
  • the two or more saccharides are the same. In certain embodiments, at least one of the two or more saccharides is different or each of the saccharides is different.
  • the ability to create fusion proteins with multiple glycosylation fragments, and further that can be recognized by different glycosylating enzymes and/or attaching different saccharides, in addition to combining different fusion proteins within the same complex allows for the mixing and matching and amplification of various immunogenic components.
  • the fusion protein comprises a carrier protein.
  • carrier proteins have been used in glycoconjugate vaccines and all are contemplated herein.
  • the carrier protein is selected from the group consisting of Escherichia coli maltose binding protein, Pseudomonas aeruginosa Exotoxin A (EPA), Pseudomonas aeruginosa PcrV, CRM197, Haemophilus influenzae Protein D, cholera toxin B subunit, or tetanus toxin, and a fragment of any thereof.
  • Polypeptide pair [0123] Certain aspects of this disclosure are drawn to a composition comprising a polypeptide pair that comprises a first polypeptide and a second polypeptide. The first 32080280 - 33 - Atty. Dkt.
  • polypeptide comprises a first polypeptide tag that is a binding partner to the second polypeptide tag of the second polypeptide.
  • the first polypeptide is a fusion protein of this disclosure comprising a glycosylation fragment as described in detail elsewhere herein.
  • the second polypeptide comprises a second polypeptide tag binding partner to the first polypeptide tag of the first polypeptide.
  • the first polypeptide can be attached to the second polypeptide via an isopeptide bond between the first polypeptide tag and the second polypeptide tag.
  • the second polypeptide comprises a monomeric polypeptide that can spontaneously multimerize/self-assemble into a higher-order, multimeric structure.
  • an icosahedron or dodecahedron particle e.g,. resembling nanocages
  • virus-like particle VLP
  • Adenoviral vector e.g. resembling nanocages
  • the second polypeptide comprises an Adenoviral capsid structural protein.
  • the second polypeptide comprises a coat protein of the bacteriophage AP205.
  • the second polypeptide comprises a fragment of the 2-keto-3-deoxy- phosphogluconate aldolase (i301).
  • the second polypeptide comprises a fragment of a mutated 2-keto-3-deoxy-phosphogluconate aldolase (mi3).
  • the polypeptide tag of the second polypeptide is a SpyCatcher (SEQ ID NO: 420). In certain embodiments, the second polypeptide tag is a SpyCatcher002 (SEQ ID NO: 421). In certain embodiments, the second polypeptide tag is a SpyCatcher003 (SEQ ID NO: 422). In certain embodiments, the second polypeptide tag a DogCatcher (SEQ ID NO: 423).
  • the polypeptide tag of the second polypeptide can be located on the second polypeptide at an end (N-terminal or C-terminal end) or internally.
  • the second polypeptide tag is translationally fused at the N-terminal end of the second polypeptide (e.g., Figure 2). In certain embodiments, the second polypeptide tag is translationally fused at the C-terminal end of the second polypeptide. In certain embodiments, the second polypeptide tag is translationally fused internally within the second polypeptide.
  • the first polypeptide e.g., a fusion protein of this disclosure
  • is a bioconjugate comprising a saccharide covalently attached to the glycosylation fragment of the first polypeptide.
  • the composition comprising a polypeptide pair is immunogenic, e.g., wherein the first polypeptide is a bioconjugate comprising a saccharide covalently attached to the glycosylation fragment of the first polypeptide.
  • the polypeptide pair composition further comprises an adjuvant and/or an 32080280 - 34 - Atty. Dkt. No.: 64100-234947 excipient.
  • Example adjuvants could include, but are not limited to: alum (aluminum hydroxide gel or aluminum phosphate gel), squalene emulsions (e.g., MF59, AddaS03, or AddaVax), lipid A derivatives such as monophosphoryl lipid A (MPLA), or saponins (e.g., Quil-A).
  • the polypeptide pair composition is a pharmaceutical and/or therapeutic composition.
  • the polypeptide pair composition is a conjugate vaccine.
  • first polypeptide e.g., a fusion protein of this disclosure
  • second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with its corresponding second polypeptide tag binding partner.
  • the method further comprises glycosylating the first polypeptide with a saccharide before contact and isopeptide bond formation with the second polypeptide (e.g., Figure 1).
  • the first polypeptide is glycosylated in vivo (such as in a host cell, e.g., in bacteria) before contact and isopeptide bond formation with the second polypeptide.
  • the method comprises isolating/purifying the in vivo glycosylated first polypeptide before contact and isopeptide bond formation with the second polypeptide.
  • the first polypeptide is glycosylated after contact and isopeptide bond formation with the second polypeptide.
  • Complexes [0128] Provided for in this disclosure is a complex comprising two or more of the polypeptide pairs disclosed herein.
  • an individual complex comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250 or more complexed polypeptide pairs of this disclosure.
  • an individual complex comprises from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, or 250 complexed polypeptide pairs of this disclosure to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, or 500 complexed polypeptide pairs of this disclosure.
  • such a complex is a self-assembled, multimeric higher- order structure.
  • such a self-assembled, multimeric higher-order structure is an icosahedron or dodecahedron particle (e.g,. resembling nanocages), virus-like particle, or Adenoviral vector.
  • the second polypeptide is not limited to partnering with just on type of first polypeptide (and vice versa).
  • all of the first polypeptides of the complex comprise the same fusion protein.
  • the first polypeptides can comprises different fusion proteins. In certain embodiments, at least two, three, four, five, or more of the first polypeptides of the complex comprise different fusion proteins. In certain embodiments, at least two of the first polypeptides of the complex comprise different fusion proteins. In certain embodiments, two, three, four, five, or six of the first polypeptides of the complex comprise different fusion proteins. In certain embodiments, all of the first polypeptides of the complex are different fusion proteins. [0130] In certain embodiments, at least one first polypeptide of the complex is a bioconjugate comprising a saccharide covalently attached to the glycosylation fragment of the first polypeptide.
  • At least about 5%, 10%, 25%, 50%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% of the first polypeptides of the complex are bioconjugates. In certain embodiments, from any of about 5%, 10%, 25%, 50%, 75%, 80%, 90%, 95%, 97%, or 98% of the first polypeptides of the complex to about any of about 10%, 25%, 50%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% of the first polypeptides of the complex are bioconjugates. In certain embodiments, about 100% or 100% of the first polypeptides of the complex are bioconjugates.
  • two or more of the first polypeptides of the complex are bioconjugates comprising a covalently attached saccharide.
  • the number of covalently attached saccharides can be numerous and will depend on the number of first polypeptide/second polypeptide pairs in the complex and the number of saccharides attached to each first polypeptide.
  • the AP205 VLP comprises about 180 first polypeptide binding partners (e.g., SpyCatcher) per VLP. In theory if 100% are isopeptide-bonded to a first polypeptide that is a bioconjugate, that would allow 180 bioconjugates per VLP.
  • each bioconjugate can be covalently attached to multiple saccharides.
  • Mi3 is lower, about 60 first polypeptide binding partners per NP.
  • the complex comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 2,000, 2,500, or 5,000 covalently attached saccharides.
  • the complex comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 2,000, or 2,500 covalently attached saccharides to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 32080280 - 36 - Atty. Dkt. No.: 64100-234947 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 2,000, 2,500, or 5,000 covalently attached saccharides.
  • the complex comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, or 750 covalently attached saccharides.
  • the complex comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, or 500 covalently attached saccharides to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, or 750 covalently attached saccharides.
  • all of the saccharides attached to the complex are the same.
  • at least two, three, four, five, or more saccharides attached to the complex are different.
  • the complex is immunogenic.
  • the complex is immunogenic.
  • Certain embodiments provide for a pharmaceutical and/or therapeutic composition comprising a complex of this invention and an adjuvant and/or an excipient.
  • the complex is a conjugate vaccine.
  • Certain aspects provide for a method of making the complex of this invention.
  • a second polypeptide of this disclosure e.g., an icosahedron or dodecahe
  • the first polypeptide can be glycosylated before the isopeptide bond is formed between the first polypeptide and the second polypeptide (e.g., 32080280 - 37 - Atty. Dkt. No.: 64100-234947 Figure 1).
  • the first polypeptide can be glycosylated after the isopeptide bond is formed between the first polypeptide and the second polypeptide.
  • the first polypeptide can be glycosylated before it is incorporated into a multimeric, higher-order structure (e.g., Figure 1).
  • the first polypeptide can be glycosylated after is has been incorporated into a multimeric, higher-order structure.
  • Glycosylation [0135]
  • the glycosylated residue, glycosylation site, glycosylation fragment, sequon, first polypeptide fusion protein, complex, or the like of this disclosure can be covalently linked to a saccharide by any of numerous method of glycosylation including, but not limited to, the following illustrative examples.
  • a saccharide is transferred to a fusion protein comprising a glycosylation fragment by the action of an N-linking oligosaccharyltransferase (N-OTase), an O-linking oligosaccharyltransferase (O-Otase), an N- linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C- mannosyltransferase (CMT).
  • N-OTase N-linking oligosaccharyltransferase
  • O-Otase O-linking oligosaccharyltransferase
  • NTT N- linking glycosyltransferase
  • OHT O-linking glycosyltransferase
  • CMT C- mannosyltransferase
  • a saccharide is transferred to a fusion protein comprising a glycosylatin fragment by the action of a PglS OTase, a TfpM OTase, a PglL OTase, a PglB OTase, a TfpO/PilO OTase, a STT3 OTase, a TfpW glycosyltransferase, and/or an AlgB OTase.
  • the glycosylation fragment is a ComP glycosylation fragment glycosylated by a PglS OTase.
  • the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a PglS OTase.
  • the glycosylation fragment is a TfpM-associated pilin glycosylation fragment glycosylated by a TfpM OTase.
  • the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpM OTase.
  • the glycosylation fragment is a PilE glycosylation fragment glycosylated by a PglL OTase.
  • the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a PglL OTase.
  • the glycosylation fragment is a PglB glycosylation fragment glycosylated by a PglB OTase.
  • the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using a PglB OTase.
  • the glycosylation fragment is a PilA glycosylation fragment glycosylated by a TfpO or PilO OTase.
  • the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpO or PilO OTase.
  • the glycosylation fragment is a STT3 glycosylation fragment glycosylated by the STT3 catalytic subunit.
  • the saccharide is covalently linked to a nitrogen atom within a glycosylation 32080280 - 38 - Atty. Dkt. No.: 64100-234947 fragment using a STT3 OTase.
  • the glycosylation fragment is a PilA_Pa5196-associated pilin glycosylation fragment glycosylated by a TfpW glycosyltransferase.
  • the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpW glycosyltransferase.
  • the glycosylation fragment is an Archaeal AlgB glycosylation fragment glycosylated by an AlgB OTase.
  • the saccharide is covalently linked to a nitrogen atom within the Archaeal AlgB glycosylation fragment glycosylation fragment using an AlgB OTase.
  • the glycosylation fragment is an N-linking glycosyltransferase glycosylation fragment glycosylated by an N-linking glycosyltransferase, for example from Actinobacillus pleuropneumoniae, from Haemophilus influenzae, or from Yersinia enterocolitica.
  • the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using an N-linking glycosyltransferase.
  • the glycosylation fragment is an O-linking glycosyltransferase glycosylation fragment glycosylated by an O-linking glycosyltransferase, for example GtfA/GtfB glycosyltransferases.
  • the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using an O-linking glycosyltransferase. Further in certain embodiments, the saccharide is covalently linked to a carbon atom within a glycosylation fragment using a C-mannosyltransferase.
  • the saccharide is covalently linked to an oxygen atom within a ComP glycosylation fragment (e.g., SEQ ID NO: 412 or a variant thereof) using a PglS OTase (e.g., SEQ ID NO: 400).
  • the saccharide is covalently linked to an oxygen atom within a TfpM glycosylation fragment (e.g., SEQ ID NO: 413 or a variant thereof) using a TfpM OTase (e.g., SEQ ID NO: 402).
  • the saccharide is covalently linked to an oxygen atom within a PilE glycosylation fragment (e.g., SEQ ID NO: 414 or a variant thereof, e.g., SEQ ID NO: 439) using a PglL OTase (e.g., SEQ ID NO: 404).
  • the saccharide is covalently linked to an oxygen atom within a PilE glycosylation fragment (e.g., SEQ ID NO: 414 or a variant thereof) using a PglL OTase (e.g., SEQ ID NO: 404).
  • the saccharide is covalently linked to a nitrogen atom within a PglB glycosylation fragment using a PglB Otase (e.g., SEQ ID NO: 405).
  • the saccharide is covalently linked to an oxygen atom within a PilA glycosylation fragment (e.g., SEQ ID NO: 415 or a variant thereof) using a TfpO/PilO Otase (e.g., SEQ ID NO: 407).
  • the saccharide is covalently linked to a nitrogen atom within a STT3 32080280 - 39 - Atty. Dkt.
  • the saccharide is covalently linked to a nitrogen atom within an Archaeal AlgB glycosylation fragment using an AlgB Otase (e.g., SEQ ID NO: 409).
  • the saccharide is covalently linked to an oxygen atom within PilA_Pa5196-associated pilin glycosylation fragment (e.g., SEQ ID NO: 426 or a variant thereof) using a TfpW glycosyltransferase (e.g., SEQ ID NO: 424).
  • the saccharide is covalently linked to a nitrogen atom within a N-linking glycosyltransferase sequon using an N-linking glycosyltransferase (e.g., SEQ ID NO: 410).
  • the saccharide is covalently linked to an oxygen atom within an O-linking glycosyltransferase sequon using an O-linking glycosyltransferase (e.g., SEQ ID NO: 411).
  • the saccharide is covalently linked to a carbon atom within a C-mannosyltransferase glycosylation fragment using a C-mannosyltransferase.
  • the method is a method of producing a conjugate vaccine. This can involve adding to a partner pair and/or complex of this disclosure an adjuvant and/or an excipient.
  • a further aspect provides for a system comprising the first polypeptide and the second polypeptide of the composition of this disclosure.
  • the first polypeptide is a glycosylated bioconjugate.
  • the system comprises a multimeric, higher-order structure assembled of the second polypeptide.
  • the system comprises a saccharide and an N-linking oligosaccharyltransferase (N-Otase), an O-linking oligosaccharyltransferase (O-OTase), an N-linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C- mannosyltransferase (CMT) as disclosed herein.
  • N-Otase N-linking oligosaccharyltransferase
  • O-OTase O-linking oligosaccharyltransferase
  • NTT N-linking glycosyltransferase
  • OHT O-linking glycosyltransferase
  • CMT C- mannosyltransferase
  • kits comprising two or more components comprising the fusion protein, the first polypeptide, the second polypeptide, a saccharide, an N-linking oligosaccharyltransferase (N-OTase), an O-linking oligosaccharyltransferase (O-OTase), an N-linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C- mannosyltransferase (CMT), the bioconjugate, the multimeric, higher-order structure 32080280 - 40 - Atty.
  • N-OTase N-linking oligosaccharyltransferase
  • O-OTase O-linking oligosaccharyltransferase
  • NTT N-linking glycosyltransferase
  • OHT O-linking glycosyltransferase
  • CMT C- man
  • Dkt. No.: 64100-234947 assembled from the second polypeptide, the isolated nucleic acid, the vector, and the host cell of this disclosure.
  • Another aspect provides for a method of eliciting an immune response in a subject by administering to said subject an effective amount of any composition, complex, and/or conjugate vaccine of this disclosure. Further provided for is a composition, complex, and/or conjugate vaccine of this disclosure for use in eliciting an immune response in a subject.
  • a composition or complex disclosed herein is a conjugate vaccine that can be administered to a subject for the prevention and/or treatment of an infection and/or disease.
  • the conjugate vaccine is a prophylaxis that can be used, e.g., to immunize a subject against an infection and/or disease.
  • the glycoconjugate is associated with (such as in a therapeutic composition) and/or administered with an adjuvant.
  • a composition such as a therapeutic composition
  • the conjugate vaccine when administered to a subject, it induces an immune response.
  • the immune response elicits long term memory (memory B and T cells).
  • the immune is an antibody response.
  • the antibody response is a serotype-specific antibody response.
  • the antibody response is an IgG or IgM response. In certain embodiments where the antibody response is an IgG response, the IgG response is an IgG1 response. Further, in certain embodiments, the conjugate vaccine generates immunological memory in a subject administered the vaccine. [0143] Certain embodiments also provide for producing a vaccine against an infection and/or disease. In certain embodiments a method comprises isolating a glycoconjugate or fusion protein disclosed herein (conjugate vaccine) and combining the conjugate vaccine with an adjuvant. In certain embodiments, the infection is a localized or systemic infection of skin, soft tissue, blood, or an organ, or is auto-immune in nature.
  • the vaccine is a conjugate vaccine against pneumococcal infection.
  • the disease is pneumonia.
  • the infection is a systemic infection and/or an infection of the blood.
  • the subject is a mammal.
  • a pig or a human is a mammal.
  • the aspects disclosed herein are not limited to pneumococcal polysaccharides, but in fact, have vast applicability for generating bioconjugate vaccines for many important human and animal pathogens that are incompatible with PglB and PglL. 32080280 - 41 - Atty. Dkt.
  • the pathogen is a bacterial pathogen.
  • the host is immunized against the pathogen.
  • the method comprises administering to a subject in need of the immune response an effective amount of a ComP conjugate vaccine, glycosylated fusion protein, or any other therapeutic/immunogenic composition disclosed herein.
  • Certain embodiments provide a conjugate vaccine, glycosylated fusion protein, or other therapeutic/immunogenic composition disclosed herein for use in inducing a host immune response against a bacterial pathogen and immunization against the bacterial pathogen.
  • immune responses include but are not limited to an innate response, an adaptive response, a humoral response, an antibody response, cell mediated response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system cross-talk, and a combination of two or more of said immune responses.
  • the immune response is an antibody response.
  • 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 said immune responses.
  • Also provided herein are methods of preventing or treating a bacterial disease and/or infection in a subject comprising administering to a subject in need thereof a conjugate vaccine, a fusion protein, or a composition disclosed herein.
  • the infection is a localized or systemic infection of skin, soft tissue, blood, or an organ, or is auto- immune in nature.
  • the disease is pneumonia.
  • the infection is a systemic infection and/or an infection of the blood.
  • the subject is a vertebrate.
  • the subject is a mammal such as a dog, cat, cow, horse, pig, mouse, rat, rabbit, sheep, goat, guinea pig, monkey, ape, llama, etc. And, for example, in certain embodiments the mammal is a human.
  • the composition is administered via intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.
  • the glycoconjugate, glycosylated fusion protein, or conjugate vaccine of any of the above claims for use in inducing a host immune response 32080280 - 42 - Atty. Dkt. No.: 64100-234947 against a bacterial pathogen and/or preventing or treating a bacterial disease and/or infection in a subject.
  • Minimal Sequons Sufficient for O-Linking Glycosylation [0149] Traditional, chemical conjugate vaccine synthesis is considered complex, costly, and laborious (Frasch, C.E. Vaccine 27, 6468-6470 (2009)) however, in vivo conjugation has been thoroughly progressing as a viable biosynthetic alternative (Huttner, A.
  • baylyi ADP1 encodes for two OTase, a PglL-like ortholog (UniProtKB/Swiss-Prot: Q6FFS6.1), which acts as the general OTase and PglS (UniProtKB/Swiss-Prot: Q6F7F9.1), which glycosylates a single protein, ComP (Harding, C.M. et al. Mol Microbiol 96, 1023-1041 (2015)).
  • ComP is orthologous to type IV pilin proteins, like PilA from Pseudomonas aeruginosa and PilE from Neisseria meningiditis, both of which are glycosylated by the OTases TfpO (Castric, P. Microbiology 141 ( Pt 5), 1247-1254 (1995)) and PglL (Power, P.M. et al. Mol Microbiol 49, 833-847 (2003)), respectively. Although TfpO and PglL also glycosylate their cognate pilins at serine residues, the sites of glycosylation differ between each system.
  • TfpO glycosylate has cognate pilin at a C-terminal serine residue (Comer, J.E., Marshall, M.A., Blanch, V.J., Deal, C.D. & Castric, P. Infect Immun 70, 2837-2845 (2002)), which is not present in ComP.
  • ComP also contains serine residues near position 63 and 32080280 - 43 - Atty. Dkt. No.: 64100-234947 the surrounding residues show moderate conservation to PilE from N. meningiditis.
  • ComP ADP1 baylyi ADP1
  • the ComP ADP1 protein belongs to a family of proteins called type IV pilins. Specifically, ComP shares homology to type IVa major pilins (Giltner, C.L., Nguyen, Y. & Burrows, L.L. Microbiol Mol Biol Rev 76, 740-772 (2012)).
  • Type IVa pilins share high sequence homology at their N-terminus, which encode for the highly conserved leader sequence and N-terminal alpha helix; however, the C-terminus display remarkable divergences across genera and even within species (Giltner, C.L., Nguyen, Y. & Burrows, L.L. Microbiol Mol Biol Rev 76, 740-772 (2012)).
  • ComP orthologs from other type IVa pilin proteins such as, PilA from A. baumannii, P. aeruginosa, and Haemophilus influenzae as well as PilE from Neisseria species (Pelicic, V.
  • ComP proteins can be differentiated from other pilins by the presence of the conserved glycosylated serine located at position 84 relative to the ADP1 ComP protein and the presence of a disulfide loop flanking the site of glycosylation.
  • a ComP protein comprises and is capable of being glycosylated on a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201 (ComP 110264 : ENV58402.1).
  • the ComP glycosylation fragment can be, or can be from or derived from, any of the ComP proteins disclosed herein.
  • the PglS OTase can be any of the following.
  • PglS was engineered to functionally glycosylate heterologous proteins by translationally fusing a large fragment (117 amino acids) of ComP to the C-terminus of a known carrier protein.
  • the 117 amino acid ComP110264 fragment was fused at the C-terminus of a genetically deactivated exotoxin A from Pseudomonas aeruginosa (EPA) between a flexible GGGS linker (SEQ ID NO: 382).
  • This chimeric carrier protein also had an N-terminal DsbA signal sequence (ssDsbA) for translocation to the periplasm via the Sec-pathway as well as a C-terminal hexahistidine tag for detection.
  • Figure 12A and Figure 12B show ComP 110264 fragments that were designed to shift one amino acid N- to C-terminal relative to serine 82, which is the site of PglS glycosylation when the ComP glycosylation fragment was fused to the extreme C-terminus of the EPA carrier protein.
  • the ComP glycosylation fragments were PCR amplified, cloned onto the C-terminus of EPA, and tested for bioconjugation by PglS.
  • the serotype 8 pneumococcal capsular polysaccharide (CPS8) expressed from the pB-8 plasmid as the glycan source was used.
  • the CPS8 glycan was selected as it contains glucose as the reducing end sugar and was previously demonstrated to be efficiently transferred to ComP by PglS (Harding, C. M. et al., 2019).
  • bioconjugation was performed in the E. coli strain, SDB1.
  • SDB1 has deletions of WecA, which initiates biosynthesis of the enterobacterial common antigen and the O-antigen polysaccharides, and WaaL, which transfers undecaprenyl-pyrophosphate linked glycan precursors to the outer core of lipid-A (Garcia-Quintanilla, F., et al., 2014).
  • Figure 12C, Figure 12D, and Figure 12E reaffirm that the presence of Cys71 and Cys93 residues flanking Ser82 in ComP 110264 are essential for EPA-ComP 110264 glycosylation when the ComP glycosylation fragment is fused at the C-terminus.
  • Cross-reactive material 197 is a genetically deactivated form of the diphtheria toxin that has been used extensively as the carrier protein in multiple conjugate vaccines for pneumococcus, Neisseria meningitidis, and Haemophilus influenza type b (Berti, F. & Adamo, R., 2018). Given the frequent use of CRM197 in conjugate vaccine formulations the PglS bioconjugation system was extended to function with CRM 197 . For these experiments, the 25-amino acid “C1” ComP glycosylation fragment (ComP C1 ) previously identified was translationally fused to the C-terminus of CRM197 linked by a GGGS sequence (SEQ ID NO: 382).
  • glycoconjugates contain a C-terminal hexahistidine tag. Fractions containing glycoconjugates were pooled and enriched for glycosylated glycoconjugates using a MonoQ column and eluted with a linear salt gradient. A final polishing step to remove large aggregates was performed on a Superdex 200 Increase column. As seen in Figure 13B, Figure 13C, and 32080280 - 47 - Atty. Dkt.
  • the N-linking sequon recognized by PglB has been engineered into multiple sites on surface loops of EPA and used as an “internal” glycotag (Ihssen, J. et al., 2010).
  • the entire 23 amino acid ComP 110264 glycosylation fragment spanning Cys71 to Cys93 ⁇ referred to herein as the iGT CC for internal GlycoTag – cysteine-cysteine ⁇ was integrated internal of the EPA amino acid sequence.
  • the ComP 110264 iGT CC was inserted between residues Ala489 and Arg490 of EPA, which is in a ⁇ -turn structure on the surface of the catalytic domain ( Figure 15A).
  • a variant of the iGT CC ComP glycosylation fragment containing serine residues instead of cysteine residues at positions 71 and 93 of ComP termed iGTss (“serine-serine”) was also integrated.
  • This iGTSS ComP glycosylation fragment was also integrated between residues Ala489 and Arg490 of EPA.
  • Serine residues are hypothesized to contribute a similar steric bulk as the cysteine residues, but are unable to oxidize and form a disulfide bond (Figure 15B).
  • the 32080280 - 48 - Atty. Dkt. No.: 64100-234947 ability of PglS to transfer CPS8 to the EPA iGTcc or EPA iGTss was assessed in a three-plasmid system as described above.
  • both the cysteine-cysteine and serine-serine variants of EPAiGT were glycosylated, demonstrating that Cys71 and Cys93 (and the putative disulfide bond formed between them) are not required for glycosylation by PglS when the ComP fragment is introduced internal of the EPA protein.
  • the cysteine residues are not necessary for PglS dependent glycosylation only when the ComP glycosylation fragment is integrated internal of the fusion protein, it was contemplated that a shorter ComP glycosylation fragment representing the minimal O-linking ComP sequon could be found within the 23-amino acid ComP glycosylation fragment spanning Cys71 to Cys93.
  • the glycosylation ratio was comparable to the 23 amino acid iGTCC ComP glycosylation fragment, suggesting modest truncations on either side of Ser82 do not have a significant impact on the glycosylation efficiency by PglS. Although these fusion proteins were glycosylated, a mild decrease in glycosylation efficiency was observed as the iGT ComP glycosylation fragment amino acid sequence was shortened.
  • the shortest internal ComP glycosylation fragment that was efficiently glycosylated was iGT ⁇ 6-6 having the sequence IASGASAATTN (SEQ ID NO: 309); Figure 16C).
  • the CPS8 glycosylated EPA fusion protein containing the iGT ⁇ 6-6 ComP glycosylation fragment located between residues Ala489-Arg490 was purified from whole-cell lysates using a Ni-affinity chromatography and performed western blot analysis on the eluate using antisera specific to either the EPA protein or the CPS8 glycan.
  • the results of these experiments clearly show that the EPA fusion protein containing the iGT ⁇ 6-6 ComP glycosylation fragment located between residues Ala489-Arg490 was being glycosylated with CPS8 by PglS ( Figure 17A, Figure 17B, and Figure 17C).
  • the CPS8 glycosylated EPA fusion protein containing the iGT ⁇ 3-4 ComP glycosylation fragment located between residues Glu548 and Gly549 was then purified from whole-cell lysates using a Ni-affinity chromatography and performed Western blot analysis on the eluate using antisera specific to either the EPA protein or the CPS8 glycan.
  • the results of these experiments again show that the EPA fusion protein containing the iGT ⁇ 3-4 ComP glycosylation fragment located between residues Glu548 and Gly549 was being glycosylated 32080280 - 50 - Atty. Dkt. No.: 64100-234947 with CPS8 by PglS.
  • glycoconjugates comprising an oligo- or polysaccharide linked to a fusion protein.
  • the oligo- or polysaccharide is covalently linked to the fusion protein.
  • the fusion protein comprises a glycosylation fragment of a ComP protein (as described in detail elsewhere herein).
  • the oligo- or polysaccharide comprises a glucose at its reducing end.
  • ComP is glycosylated on a serine (S) residue. This serine residue corresponds to position 82 of SEQ ID NO: 201 (ComP110264: ENV58402.1). This serine residue is conserved in ComP proteins and, for example, corresponds to position 84 of SEQ ID NO: 202 (ComPADP1: AAC45886.1).
  • a fusion protein (and thus the glycoconjugate) is glycosylated with an oligo- or polysaccharide on a ComP glycosylation fragment at a serine residue corresponding to the serine residue at position 84 of SEQ ID NO: 202 (ComPADP1: AAC45886.1) or corresponding to the serine residue at position 82 of SEQ ID NO: 201 (ComP 110264 : ENV58402.1).
  • Figure 22 shows an alignment of a region of ComP sequences including the serine (S) residue (boxed) corresponding to the serine residue at position 82 of SEQ ID NO: 201 (ComP 110264 : ENV58402.1), which is conserved across the ComP sequences.
  • a ComP protein is a protein that has been identified as a ComP protein consistent with the description provided herein.
  • representative examples of ComP proteins 32080280 - 51 - Atty. Dkt. No.: 64100-234947 include, but are not limited to: AAC45886.1 ComP [Acinetobacter sp.
  • a 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: 201 (ComP ADP1 ) or SEQ ID NO: 201 (ComP 110264 ) and contains a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 202 or at position 82 of SEQ ID NO: 201.
  • SEQ ID NO: 202 comprises a leader sequence of 28 amino acids.
  • a 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: 210 (ComP ⁇ 28ADP1), SEQ ID NO: 209 (ComP ⁇ 28110264), SEQ ID NO: 211 (ComP ⁇ 28GFJ-2), SEQ ID NO: 212 (ComP ⁇ 28P50v1), SEQ ID NO: 213 (ComP ⁇ 28 4466 ), SEQ ID NO: 214 (ComP ⁇ 28 SFC ), SEQ ID NO: 215 (ComP ⁇ 28 P5312 ), or SEQ ID NO: 216 (ComP ⁇ 29ANT_H59) that do not include the amino acid leader sequence but do contain a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201 (ComP 110264 : AAC45886.1).
  • a 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: 209 (ComP ⁇ 28110264) that does not include the 28 amino acid leader sequence but does contain a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201 (ComP 110264 ).
  • the ComP protein comprises SEQ ID NO: 210 (ComP ⁇ 28ADP1), SEQ ID NO: 209 (ComP ⁇ 28110264), SEQ ID NO: 211 (ComP ⁇ 28GFJ-2), SEQ ID NO: 212 (ComP ⁇ 28P50v1), SEQ ID NO: 213 (ComP ⁇ 28 4466 ), SEQ ID NO: 214 (ComP ⁇ 28 SFC ), SEQ ID NO: 215 (ComP ⁇ 28 P5312 ), or SEQ ID NO: 216 (ComP ⁇ 29ANT_H59).
  • the ComP protein is SEQ ID NO: 202 (ComPADP1: AAC45886.1), SEQ ID NO: 201 (ComP110264: ENV58402.1), SEQ ID NO: 203 (ComP GFJ-2 : APV36638.1), SEQ ID NO: 204 (ComP 50v1 : PKD82822.1), SEQ ID NO: 205 (ComP4466: SNX44537.1), SEQ ID NO: 206 (ComPSFC: OAL75955.1), SEQ ID NO: 207 (ComPP5312), or SEQ ID NO: 208 (ComPANT_H59).
  • SEQ ID NO: 202 ComPADP1: AAC45886.1
  • SEQ ID NO: 201 ComP110264: ENV58402.1
  • SEQ ID NO: 203 ComP GFJ-2 : APV36638.1
  • SEQ ID NO: 204 ComP 50v1 : PKD82822.1
  • SEQ ID NO: 205 ComP4466: SN
  • a glycoconjugate comprising an oligo- or polysaccharide covalently linked to a fusion protein wherein the fusion protein comprises a ComP protein (ComP) glycosylation fragment.
  • the ComP glycosylation fragment 32080280 - 52 - Atty. Dkt. No.: 64100-234947 does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 71 of ComP 110264 (SEQ ID NO: 201).
  • the ComP glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 93 of ComP110264 (SEQ ID NO: 201).
  • the fusion protein is glycosylated with the oligo- or polysaccharide on the ComP glycosylation fragment at serine residue corresponding to the conserved serine residue at position 82 of ComP110264 (SEQ ID NO: 201).
  • the ComP glycosylation fragment is located internally within the fusion protein.
  • the ComP glycosylation fragment portion of the fusion protein is solvent (or surface)-exposed and/or is integrated into a C10 ⁇ -turn, ⁇ -turn, ⁇ -twist, ⁇ -loop, U turn, reverse turn, chain reversal, or a hairpin loop of the fusion protein.
  • the ComP glycosylation fragment can be shorter than 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 amino acids long, as long as it comprises a serine residue corresponding to the conserved serine residue at position 82 of ComP110264 (SEQ ID NO: 201).
  • the ComP glycosylation fragment has a length of from any one of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 to any one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids in length.
  • the fragment has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues of the ComP protein N-terminal to the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201, e.g., X n S[Y], wherein n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues of the ComP protein.
  • the fragment has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues of the ComP protein C-terminal to the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201, e.g., [X]SYn, wherein n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues of the ComP protein.
  • the amino acid sequence of the ComP glycosylation fragment does not extend in the N-terminus direction beyond the amino acid residue corresponding to position 72 of ComP 110264 (SEQ ID NO: 201) and/or does not extend in the C-terminus beyond the amino acid residue corresponding to position 92 of ComP110264 (SEQ ID NO: 201). 32080280 - 53 - Atty. Dkt.
  • a ComP protein from which the ComP glycosylation fragment is derived comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 209 (ComP ⁇ 28110264) SEQ ID NO: 210 (ComP ⁇ 28ADP1), SEQ ID NO: 211 (ComP ⁇ 28 GFJ-2 ), SEQ ID NO: 212 (ComP ⁇ 28 P50v1 ), SEQ ID NO: 213 (ComP ⁇ 284466), SEQ ID NO: 214 (ComP ⁇ 28SFC); SEQ ID NO: 215 (ComP ⁇ 28P5312), or SEQ ID NO: 216 (ComP ⁇ 29ANT_H59).
  • the ComP protein from which the ComP glycosylation fragment is derived comprises SEQ ID NO: 209 (ComP ⁇ 28 110264 ), SEQ ID NO: 210 (ComP ⁇ 28 ADP1 ), SEQ ID NO: 211 (ComP ⁇ 28 GFJ-2 ), SEQ ID NO: 212 (ComP ⁇ 28P50v1), SEQ ID NO: 213 (ComP ⁇ 284466), SEQ ID NO: 214 (ComP ⁇ 28SFC); SEQ ID NO: 215 (ComP ⁇ 28 P5312 ), or SEQ ID NO: 216 (ComP ⁇ 29 ANT_H59 ).
  • X4
  • the fragment has at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues N-terminal to the serine (S) residue corresponding to position 11 of SEQ ID NO: 217. In certain embodiments, the fragment has at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues C-terminal to the serine (S) residue corresponding to position 11 of SEQ ID NO: 217. But, the ComP 32080280 - 54 - Atty. Dkt.
  • glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 71 of ComP 110264 (SEQ ID NO: 201) and/or the ComP glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 93 of ComP110264 (SEQ ID NO: 201).
  • Certain embodiments provide for a ComP glycosylation fragment that is a variant of the amino acid consensus sequence of SEQ ID NO: 217, SEQ ID NO: 396, or SEQ ID NO: 397, or the fragment thereof, having 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions, additions, and/or deletions, wherein the variant maintains the serine (S) residue corresponding to position 11 of SEQ ID NO: 217 and wherein the variant does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 71 of ComP110264 (SEQ ID NO: 201) and/or the variant does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 93 of ComP 110264 (SEQ ID NO: 201).
  • ComP glycosylation fragment can be glycosylated (including subfragments of a fragment and variants as disclosed herein and collectively referred to as ComP glycosylation fragments), and the efficiency of glycosylation, can be determined such as by methods described herein.
  • the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein and/or internally in a carrier protein sequence as described elsewhere herein. Further, in certain embodiments, the ComP glycosylation fragment or variant is not glycosylated when located at the N-terminal and/or C- terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal and/or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein.
  • the fusion protein comprises a carrier protein selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM 197 , cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae Protein D, and a fragment or fragments thereof.
  • the Pseudomonas aeruginosa Exotoxin A (EPA) carrier protein comprises the amino acid sequence of SEQ ID NO: 218, or a fragment or fragments thereof.
  • No.: 64100-234947 protein comprises the amino acid sequence of SEQ ID NO: 224, or a fragment or fragments thereof.
  • the ComP fusion protein is not located at the C-terminal end or the N- terminal end of the fusion protein, not including any C-terminal leader sequence or N-terminal tag (e.g., His-Tag), or the like.
  • the ComP glycosylation fragment can be attached to the carrier protein sequence via an amino acid linker.
  • the ComP glycosylation fragment can be inserted into the sequence of a carrier protein rather than between carrier proteins.
  • the ComP glycosylation fragment is inserted between Ala489 and Arg490 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 219); (ii) the ComP glycosylation fragment is inserted between Glu548 and Gly549 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 220); (iii) the ComP glycosylation fragment is inserted between Ala122 and Gly123 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 221); (iv) the ComP glycosylation fragment is inserted between Thr355 and Gly356 relative to the PDB entity 1IKQ of Pseudomonas
  • the ComP glycosylation fragment can be inserted into the sequence of a carrier protein rather than between carrier proteins.
  • the ComP glycosylation fragment is inserted between Asn481 and Gly482 relative to the PDB entity 4AE0 of CRM 197 (SEQ ID NO: 225);
  • the ComP glycosylation fragment is inserted between Asp392 and Gly393 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 226); 32080280 - 56 - Atty. Dkt.
  • ComP glycosylation fragments can be located between carrier proteins and also inserted into the sequence of a carrier protein(s) within one fusion protein.
  • a ComP glycosylation fragment can be located internally and one or more ComP glycosylation fragments can be located at the C-terminal and/or N-terminal end that are sufficient for glycosylation at such location.
  • An aspect of this disclosure is that a fusion protein can be designed to comprise multiple ComP glycosylation fragments such as to increase the immunogenicity of the glycosylated fusion protein/glycoconjugate.
  • 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 fragments.
  • the fusion protein does not comprise more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five ComP glycosylation fragments.
  • the identity of the ComP glycosylation fragments can also be controlled.
  • a plurality of ComP glycosylation fragments of a fusion protein are identical.
  • ComP glycosylation fragments of a fusion protein differ from each other.
  • at least three, at least four, or at least five of the ComP glycosylation fragments of a fusion protein all differ from each other.
  • none of the ComP glycosylation fragments of a fusion protein are the same.
  • the oligo- or polysaccharide is derived from a saccharide produced by bacteria from the genus Streptococcus.
  • the saccharide is a S. pneumoniae, S. agalactiae, or S. suis capsular polysaccharide; in certain embodiments, the saccharide is the serotype 8 capsular polysaccharide from S. pneumoniae; and in certain embodiments, the saccharide is the type Ia, Ib, II, III, IV, V, VI, VII, VIII, or X capsular polysaccharide from S. agalactiae.
  • the oligo- or polysaccharide is derived from a saccharide produced by the bacteria from the genus Klebsiella.
  • the 32080280 - 57 - Atty. Dkt. No.: 64100-234947 saccharide is a K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca capsular polysaccharide; and in certain embodiments, the saccharide is a K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca O-antigen polysaccharide.
  • the glycoconjugate is produced in vivo, for example: in a bacterial cell; in Escherichia coli; in a bacterium from the genus Klebsiella; and/or wherein the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.
  • a glycoconjugate as described above (e.g., the ComP glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 71 of ComP 110264 (SEQ ID NO: 201) and/or the ComP glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 93 of ComP 110264 (SEQ ID NO: 201)), wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of SEQ ID NOs: 232- 363, or 364.
  • the ComP glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 71 of ComP 110264 (SEQ ID NO: 201) and/or the ComP glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 93 of ComP110264 (SEQ ID NO: 201)
  • the ComP glycosylation fragment comprises or consists of an amino acid sequence of: 32080280 - 58 - Atty. Dkt.
  • ComP glycosylation fragment that is a variant of any of the above disclosed ComP glycosylation fragments having 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, additions, and/or deletions, wherein the variant maintains the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201 and wherein the variant does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 71 of ComP110264 (SEQ ID NO: 201) and/or the variant does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 93 of ComP 110264 (SEQ ID NO: 201).
  • ComP glycosylation fragment can be glycosylated (including subfragments of a fragment and variants as disclosed herein and collectively referred to as ComP glycosylation fragments), and the efficiency of glycosylation, can be determined such as by methods described herein.
  • the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein and/or internally in a carrier protein sequence as described elsewhere herein.
  • the ComP glycosylation fragment is not glycosylated when located at the N-terminal and/or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal and/or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein.
  • the glycoconjugate is a conjugate vaccine.
  • this disclosure in certain embodiments is directed to and provides for a conjugate vaccine.
  • the conjugate vaccine is a vaccine against Streptococcus pneumoniae serotype 8.
  • the conjugate vaccine induces an immune response when administered to a subject.
  • the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response.
  • the antibody response is an IgG or IgM response.
  • the antibody response is an IgG response; optionally an IgG1 response.
  • the conjugate vaccine generates immunological memory in a subject administered the vaccine.
  • the fusion protein is glycosylated by an oligo- or polysaccharide at a serine residue on the glycosylation fragment corresponding to the serine ComP glycosylation fragment residue at position 82 of SEQ ID NO: 201 (ComP 110264 ).
  • a glycoconjugate comprising a ComP glycosylation fragment that comprises a fusion protein
  • this disclosure also explicitly provides for a fusion protein consistent with any and all description of a fusion protein provided anywhere herein, including in the appended Claims below.
  • the fusion protein comprises a carrier protein selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM 197 , cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae Protein D, and a fragment or fragments thereof.
  • EPA Pseudomonas aeruginosa Exotoxin A
  • CRM 197 cholera toxin B subunit
  • tetanus toxin C fragment Haemophilus influenzae Protein D
  • a fragment or fragments thereof also provided for herein is a method of in vivo conjugation of an oligo- or polysaccharide to an acceptor polypeptide.
  • the method comprises culturing a host cell comprising the components necessary for the conjugation of the oligo- or polysaccharide to the polypeptide.
  • these components are the oligosaccharyltransferase, the acceptor polypeptide to be glycosylated, and the oligo- or polysaccharide.
  • the method comprises covalently linking an oligo- or polysaccharide to the acceptor polypeptide (fusion protein of this disclosure) with a PglS oligosaccharyltransferase (OTase), wherein the acceptor polypeptide comprises a ComP glycosylation fragment as described herein.
  • the PglS OTase is PglS 110264 (SEQ ID NO: 365), PglSADP1 (SEQ ID NO: 366), PglSGFJ-2 (SEQ ID NO: 367), PglS50v1 (SEQ ID NO: 368), PglS4466 (SEQ ID NO: 369), PglSSFC (SEQ ID NO: 370), PglSP5312 (SEQ ID NO: 371), or PglS ANT_H59 (SEQ ID NO: 372).
  • the oligo- or polysaccharide is linked to the ComP glycosylation fragment at a serine (S) residue corresponding to the serine residue at position 82 of SEQ ID NO: 201 (ComP110264).
  • the in vivo conjugation occurs in a host cell.
  • the glycoconjugate is produced in a bacterial cell, a fungal cell, a yeast cell, an avian cell, an algal cell, an insect cell, or a mammalian cell.
  • the host cell is a bacterial cell, e.g.: in Escherichia 32080280 - 60 - Atty. Dkt.
  • Certain embodiments comprise culturing a host cell that comprises: (a) a genetic cluster encoding for the proteins required to synthesize the oligo- or polysaccharide; (b) a PglS OTase; and (3) the acceptor polypeptide.
  • the production of the oligo- or polysaccharide is enhanced by the K. pneumoniae transcriptional activator rmpA (K. pneumoniae NTUH K-2044) or a homolog of the K.
  • the method further comprises expressing and/or providing such a transcriptional activator in the host cell along with the other components.
  • the glycoconjugate is produced in a cell free system. Examples of the use of a cell free system utilizing OTases other than PglS can be found in WO2013/067523A1, which in incorporated herein by reference.
  • a host cell comprising (a) a genetic cluster encoding for the proteins required to synthesize an oligo- or polysaccharide; (b) a PglS OTase; and (3) an acceptor polypeptide comprising a ComP glycosylation fragment of this disclosure.
  • the acceptor polypeptide is a fusion protein.
  • the host cell comprises a nucleic acid encoding the PglS OTase.
  • the host cell comprises a nucleic acid encoding the acceptor polypeptide.
  • nucleic acid encoding a ComP glycosylation fragment and/or a fusion protein of this disclosure.
  • the nucleic acid is a vector.
  • a host cell comprises the isolated nucleic acid.
  • a glycoconjugate of this invention may have one of numerous uses including, but not limited to, use as a conjugate vaccine. Thus in certain methods, a conjugate vaccine is produced. In certain embodiments, a composition comprising the conjugate vaccine or the fusion protein of this disclosure and an adjuvant.
  • the conjugate vaccine is a vaccine against 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, 32080280 - 61 - Att
  • Streptococcus pneumoniae serotype 19F Streptococcus pneumoniae serotype 19A, Streptococcus pneumoniae serotype 20, Streptococcus pneumoniae serotype 22F, Streptococcus pneumoniae serotype 23F, Streptococcus pneumoniae serotype 33F, Klebsiella pneumoniae serotype K1, Klebsiella pneumoniae serotype K2, Klebsiella pneumoniae serotype K5, Klebsiella pneumoniae serotype K16, Klebsiella pneumoniae serotype K20, Klebsiella pneumoniae serotype K54, Klebsiella pneumoniae serotype K57, Streptococcus agalactiae serotype Ia, Streptococcus agalactiae serotype Ib, Streptococcus agalactiae serotype II, Streptococcus agalactiae serotype
  • the conjugate vaccine is useful because it induces an immune response when administered to a subject.
  • the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response.
  • the antibody response is an IgG or IgM response.
  • the antibody response can be an IgG response, and in certain embodiments, an IgG1 response.
  • the conjugate vaccine generates immunological memory in a subject administered the vaccine.
  • a pneumococcal glyconjugate vaccine containing a conventional vaccine carrier that can be produced by isolating a glycoconjugate or a glycosylated fusion protein of this disclosure comprising a ComP glycosylation fragment of this disclosure and combining the isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant.
  • the ComP glycosylation fragment can be added to a conventional carrier protein Pseudomonas aeruginosa Exotoxin A (EPA).
  • the glycosylation fragment/carrier fusion protein can be paired with the CPS8 polysaccharide and use of PglS, generating a carrier protein-CPS8 bioconjugate, a first of its kind pneumococcal bioconjugate vaccine.
  • an EPA fusion can be paired with the CPS8 polysaccharide and use of PglS, generating an EPA-CPS8 bioconjugate. It has been demonstrated that the EPA-CPS8 bioconjugate vaccine elicited high 32080280 - 62 - Atty. Dkt.
  • a conjugate vaccine (such as the EPA vaccine construct) can comprise additional/multiple sites of glycosylation to increase the glycan to protein ratio as well as expand upon the number of serotypes in order to develop a comprehensive pneumococcal bioconjugate vaccine.
  • TfpM bacterial O-linking oligosaccharyltransferases
  • Certain embodiments of this disclosure comprise any of the TfpM-associated pilin glycosylation fragments and/or TfpM OTases that follow.
  • TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins.
  • Phylogenetic analyses demonstrate that TfpM proteins cluster in different clades than known bacterial oligosaccharyltransferases.
  • TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and the minimal sequon required for glycosylation was identified. It was demonstrated that TfpM has a broad substrate tolerance and can transfer diverse polysaccharides including those with reducing-end glucose, galactose, or 2-N-acetyl sugars. It was also shown that a TfpM- derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice.
  • TfpM proteins (‘M’ for Moraxellaceae). Given the similar size of TfpM proteins to known TfpO proteins, it was initially hypothesized that these genes encoded variants of TfpO-PilA pairs like those found in Acinetobacter and Pseudomonas (Harding, C. M., et al. (2015) Molecular Microbiology 96, 1023-1041).
  • TfpM proteins were particularly interested in determining if TfpM proteins could transfer only short oligosaccharides to acceptor proteins, like TfpO proteins.
  • TfpM oligosaccharyltransferases listed in Table 1, the inventors selected 13 representatives from different clades to test for glycosylation activity in a glycoengineered E. coli strain (Harding, C. M., and Feldman, M. F. (2019) Glycobiology 29, 519-529; Feldman, M. F., et al. (2005) Proceedings of the National Academy of Sciences of the United States of America 102, 3016).
  • a chimeric acceptor protein strategy consisting of the exotoxin A protein from Pseudomonas aeruginosa (EPA) fused to different-sized, soluble fragments of ComP (the natural substrate of PglS) (Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203). All type IV pilin-like proteins contain a conserved, N-terminal pilin signal sequence and membrane-anchoring domain that is not required for glycosylation but is essential for pilin stability.
  • the fusion protein approach allows for the removal of the conserved N-terminal pilin signal sequence and membrane-anchoring domain and was used to determine the minimum sequon that PglSADP1 could recognize and still efficiently glycosylate (Harding, C. M., et al. (2019) Nature Communications 10, 891; Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203).
  • the inventors adapted this approach and designed 13 synthetic double- stranded DNA blocks encoding an N-terminally truncated fragment of the upstream pilin gene and the downstream tfpM gene.
  • the synthetic DNA blocks 32080280 - 66 - Atty. Dkt. No.: 64100-234947 were designed such that, when cloned into an EPA expression vector using Gibson assembly, it placed the pilin coding region in frame with the C-terminus of EPA, creating a gene fusion that translated as a single protein with the tfpM gene immediately downstream ( Figure 23B).
  • the truncated pilin fragments ranged from 113 to 140 amino acids in size.
  • the fusion protein was secreted into the periplasm using a DsbA signal sequence at the N-terminus of EPA. Oligos and primers used for the assemblies are listed in Table 2. Table 2. Primers and Oligos 32080280 - 67 - Atty. Dkt. No.: 64100-234947 32080280 - 68 - Atty. Dkt. No.: 64100-234947 [0203] Using this design, the inventors assessed the ability of the 13 TfpM proteins to transfer the Streptococcus pneumoniae capsular polysaccharide 8 (CPS8) glycan to their cognate pilin domain on the EPA-pilin fusions.
  • CPS8 Streptococcus pneumoniae capsular polysaccharide 8
  • the CPS8 repeat unit is a tetrasaccharide with a glucose at the reducing end.
  • PglS is thus far the only known oligosaccharyltransferase able to naturally transfer this glycan to acceptor proteins (Harding, C. M., et al. (2019) Nature Communications 10, 891).
  • the 13 EPA-pilin fusions/TfpM expression vectors were transformed individually into E. coli SDB1 strains (Feldman, M. F., et al. (2005) Proceedings of the National Academy of Sciences of the United States of America 102, 3016) expressing the CPS8 glycan and assessed for protein glycosylation.
  • the unglycosylated fusion proteins had expected masses ranging from 78.3 to 80.5 kDa.
  • TfpM proteins were found to glycosylate their cognate EPA-pilin fusion with glycosylation appearing as higher molecular-weight laddering (g n ) above the unglycosylated band (g 0 ) ( Figure 23C).
  • Each higher-weight band represents the attachment of a glycan with one additional CPS8 repeat unit to the EPA-pilin protein.
  • Glycosylation was readily observed in seven TfpM orthologs tested: Acinetobacter sp. YZSX-1-1, Acinetobacter sp. CIP102637, Acinetobacter sp. YH01026, A.
  • wzy_C family pfam04932 is an “O-antigen ligase” domain present in membrane-bound enzymes that catalyze the transfer and covalent attached of lipid-linked oligosaccharide (liposaccharide) to lipid A or protein substrates.
  • TfpMMo is an O-linking oligosaccharyltransferase that glycosylates the C-terminal threonine of its pilin substrate
  • osloensis 1202 and Acinetobacter sp. YH01026 exhibited the most efficient transfer of glycans 32080280 - 69 - Atty. Dkt. No.: 64100-234947 of diverse sizes. Due to the slightly higher apparent stability of the pilin from M. osloensis FDAARGOS_1202 (1202 hereafter), the inventors chose the oligosaccharyltransferase from this organism as a representative for further characterization and have termed the enzyme TfpMMo (SEQ ID NO: 56). For clarity, reference is made to the intact, native M.
  • osloensis 1202 pilin protein as Pil Mo SEQ ID NO: 57
  • the N-terminally truncated fusion domain as PilMo ⁇ 28
  • TfpMMo next was to identify the glycosylation site of PilMo ⁇ 28 and thereby determine whether the enzyme acted like a TfpO protein, glycosylating the C-terminal amino acid of its cognate pilin acceptor, or if it was more like a PglL or PglS protein, glycosylating an internal residue.
  • TfpO proteins transfer short oligosaccharides typically containing 3-6 sugars to the sidechain of the C-terminal serine residue of their cognate pilin. All except one of the cognate pilin proteins located immediately upstream a tfpM open reading frame ended in a C-terminal threonine residue, namely that from Psychrobacter sp.72-O-c which ends in a serine. Based on this observation, as well as the size similarity of TfpM and TfpO proteins, the inventors hypothesized that TfpM enzymes also transfer glycans to a C-terminal residue and thus designed point mutants of the pilin to test this.
  • TfpM Mo transfers polysaccharides containing glucose, galactose, or 2-N-acetyl monosaccharides at the reducing end [0207]
  • the inventors selected polysaccharides containing different reducing-end sugars, varying disaccharide sugar linkages near the reducing end, and/or were polymers composed of linear or branched repeat units.
  • glycoproteins were split into two equal fractions, one half of which were digested with proteinase K prior to SDS-PAGE separation and western blotting.
  • the western blots were probed using antisera specific to each polysaccharide and, separately, with anti-EPA antibody as all antibodies used in this experiment were from rabbits.
  • TfpM Mo was found to efficiently transfer all five different polysaccharides to the EPA-Pil Mo ⁇ 28 protein.
  • the inventors fused a seven-amino acid variant, termed Pil7, of PilMo consisting of a similar motif (modified to ‘P-A-N-A-R-G-T’, where the cysteine is mutated to an alanine – bolded residue) to EPA and assessed glycosylation (Figure 30A).
  • the inventors also built stepwise single-amino acid truncations of this ‘P-A-N-A-R-G-T’ sequence from seven to two amino acids and assessed the ability of TfpM to glycosylate these fragments with CPS8. The results showed that all variants except for Pil 2 were glycosylated by TfpM Mo at similar levels as Pil 10 (Figure 30B).
  • pVNM291 was introduced into SDB1 cells expressing the GBSIII glycan and resulting bioconjugates purified using Nickel immobilized metal affinity chromatography (IMAC) followed by anion-exchange and size-exclusion chromatography on FPLC.
  • IMAC Nickel immobilized metal affinity chromatography
  • Western blot and Coomassie staining of the SDS-PAGE resolved GBSIII-291 bioconjugate confirmed high molecular weight glycosylation of the EPA-Pil 20 protein with the GBSIII glycan ( Figure 32A, Figure 32B, Figure 32C, and Figure 32D).
  • Intact protein MS of the purified EPA-Pil20-GBSIII (“GBSIII-291”) conjugate supported a glycan:protein ratio of 20% ( Figure 32E).
  • Each dose was formulated to contain 1 ⁇ g of GBSIII polysaccharide.
  • unglycosylated pVNM291-derived carrier protein (“291”) was purified from SDB1 cells without glycan plasmid and dosed at the same protein concentration as for the GBSIII bioconjugates. 32080280 - 73 - Atty. Dkt. No.: 64100-234947 [0211]
  • TfpM Mo -generated GBSIII-291 bioconjugate was immunogenic.
  • mice received either placebo (unglycosylated 291 carrier protein) or the GBSIII-291 bioconjugate at two-week intervals starting with a priming dose followed by two booster doses. All vaccines were formulated with a 1:9 ratio of Alhydrogel ® 2% as an adjuvant. Serum was collected prior to each immunization and two weeks after the final booster. To determine the level of GBSIII-specific antibodies elicited, the inventors used an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • mice immunized with the bioconjugate GBSIII-291 were observed to express high levels of anti- GBSIII IgG antibodies, excluding a single mouse that exhibited a low anti-GBSIII IgG response, that was able to be boosted over the course of the immunization (Figure 32F).
  • the GBSIII bioconjugate-vaccinated mice had increased GBSIII-specific IgG titers compared with the mock-vaccinated mice (291 alone, Figure 32F).
  • TfpM Mo and PglS ADP1 glycosylate a single protein engineered to contain sequons specific to each oligosaccharyltransferase
  • PglL ComP PglS ADP1 glycosylate a single protein engineered to contain sequons specific to each oligosaccharyltransferase
  • the inventors engineered an EPA fusion protein containing a PglS sequon (CTGVTQIASGASAATTNVASAQC) (SEQ ID NO: 59) integrated between residues Ala 489 and Arg 490 as previously described (Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203) as well as the Pil20 sequon (CGGTGTTVAAKFLPANCRGT) (SEQ ID NO: 60; same as Pil DSL) at the C-terminus ( Figure 33A).
  • the construct was designed such that the open reading frame of the gene encoding for the EPA fusion and the start codon of tfpM overlapped by a single nucleotide.
  • the open reading frame encoding for pglS from A. baylyi ADP1 was cloned 100 bp downstream of the tfpM open reading frame stop codon.
  • This vector (pVNM337) was the introduced into E. coli SDB1 expressing the E. coli O16 antigen and tested for glycosylation via western blotting.
  • the inventors individually introduced the following constructs into E. coli SDB1 expressing the O16 antigen: (i) EPA containing only the TfpM-associated Pil 20 sequon (pVNM297) or (ii) EPA containing the PglS sequon integrated between residues Ala 489 and Arg 490 (pVNM167).
  • the EPA construct containing two PglS sequons exhibited a predominantly mono-glycosylated profile around 100 kDa but also displayed a di-glycosylated population migrating around 150 kDa.
  • Western blot analysis of the EPA fusion containing a sequon from TfpM Mo and PglS ADP1 displayed both mono- and di-glycosylated populations like that seen with construct pVNM245. It was concluded from these results that an acceptor protein can be glycosylated by two different OTase classes in one expression system.
  • Glycoconjugates [0213] This disclosure provides for a glycoconjugate comprising an oligo- or polysaccharide covalently linked to an acceptor protein.
  • the acceptor protein comprises or consists of a TfpM-associated pilin-like protein or glycosylation fragment thereof of this disclosure.
  • the oligo- or polysaccharide is covalently linked to the pilin-like protein or glycosylation fragment thereof.
  • the TfpM-associated pilin-like protein or glycosylation fragment thereof comprises a C- terminus serine or threonine residue and the oligo- or polysaccharide is covalently linked to the C-terminus serine or threonine.
  • the acceptor protein is a fusion protein comprising the TfpM-associated pilin-like protein or glycosylation fragment thereof translationally fused/linked to a heterologous amino acid sequence (e.g., a carrier protein) and the TfpM-associated pilin-like protein or glycosylation fragment thereof is the C-terminus- most sequence of the acceptor protein such that the acceptor protein comprises a C-terminus serine or threonine residue and the oligo- or polysaccharide is covalently linked to the C- terminus serine or threonine.
  • a heterologous amino acid sequence e.g., a carrier protein
  • carrier proteins include but are not limited to Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, or a fragment of any thereof.
  • the TfpM-associated pilin-like protein or glycosylation fragment thereof is translationally fused/linked to a heterologous amino acid sequence/carrier protein via an amino acid linker.
  • the oligo- or polysaccharide comprises a glucose at its reducing end.
  • the glycoconjugate is immunogenic. 32080280 - 75 - Atty. Dkt.
  • the acceptor protein comprises or consists of a full-length TfpM-associated pilin-like protein. In certain embodiments, the acceptor protein comprises or consists of a glycosylation fragment of a TfpM-associated pilin-like-protein that is less than a full-length TfpM-associated pilin-like protein.
  • the pilin-like-protein glycosylation fragment has a length of from 3 to 138 amino acids in length, has a length of from 10 to 138 amino acids in length, has a length of from 20 to 138 amino acids in length, 50 to 138 amino acids in length, has a length of from 100 to 138 amino acids in length, or has a length of from 116 to 138 amino acids in length.
  • the pilin-like-protein glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 10 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, 50 to 139 amino acids in length, has a length of from 100 to 139 amino acids in length, or has a length of from 116 to 139 amino acids in length.
  • the glycosylation fragment has a length of from 3 to 140 amino acids in length, has a length of from 10 to 140 amino acids in length, has a length of from 20 to 140 amino acids in length, 50 to 140 amino acids in length, has a length of from 100 to 140 amino acids in length, or has a length of from 116 to 140 amino acids in length.
  • the glycosylation fragment has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 3 to 21 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length.
  • the glycosylation fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
  • the glycosylation fragment has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
  • the TfpM- associated pilin-like-protein or glycosylation fragment thereof is PilMo (SEQ ID NO: 57) or Pil Mo lacking amino acids corresponding to residues 1–28 (Pil Mo ⁇ 28, SEQ ID NO: 58) or a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, for example, wherein the C-terminus threonine is substituted with serine.
  • the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ 32080280 - 76 - Atty. Dkt.
  • the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil jun
  • the pilin-like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (PilMo_DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148).
  • PilMo_DSL also referred to as Pil20
  • SEQ ID NO: 60 the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine
  • the pilin-like-protein glycosylation fragment consists of Pil20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil 16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil 12 (SEQ ID NO: 138), Pil 11 (SEQ ID NO: 139), Pil 10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine.
  • the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S (SEQ ID NO: 153), Pil14S (SEQ ID NO: 154), Pil13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil11S (SEQ ID NO: 157), Pil10S (SEQ ID NO: 158), Pil9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil 6S (SEQ ID NO: 162), Pil 5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acids, or a variant
  • the acceptor protein can be glycosylated at two or more different positions. In certain embodiments, the acceptor protein can be glycosylated by at least two different OTase classes in one expression system.
  • the acceptor protein is a fusion protein and the fusion protein further comprises an additional glycosylation sequence (e.g., glycosylation fragment) of an OTase other than TfpM oligosaccharyltransferase (OTase) in addition to the TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus.
  • the other OTase can be PglB, PglL, or PglS.
  • the additional glycosylation sequence is a sequence internal to the fusion protein (i.e., not the C-terminal or N-terminal most sequence).
  • the additional glycosylation sequence is a sequence internal within the sequence of a carrier protein (e.g., Figure 33A). In certain embodiments, the additional glycosylation sequence is also covalently linked to an oligo- or polysaccharide. In certain embodiments, 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 additional glycosylation sequences. In certain embodiments, the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five additional glycosylation sequences. In certain embodiments, the additional glycosylation sequences are identical.
  • the acceptor protein is a fusion protein and the fusion protein further comprises an internal glycosylation fragment of ComP in addition to the TfpM-associated pilin-like-protein glycosylation fragment located at its C- terminus.
  • the ComP glycosylation fragment is internal within the sequence of a carrier protein.
  • the ComP glycosylation fragment is also covalently linked to an oligo- or polysaccharide.
  • the ComP glycosylation fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least the amino acids ASA in positions 11-13.
  • 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 fragments.
  • the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, 32080280 - 78 - Atty. Dkt.
  • the ComP glycosylation fragments are identical. In certain embodiments, the ComP glycosylation fragments differ from each other. In certain embodiments, at least three, at least four, or at least five of the ComP glycosylation fragments all differ from each other. And, in certain embodiments, none of the ComP glycosylation fragments are the same. [0217] In certain embodiments of the glycoconjugate of this disclosure, the oligo- or polysaccharide covalently linked to the pilin-like protein or glycosylation fragment thereof has a size of at least three repeating units of oligo- or polysaccharide structure.
  • the oligo- or polysaccharide covalently linked to the pilin-like protein or glycosylation fragment thereof has a size of at least ten monosaccharides.
  • the oligo- or polysaccharide is produced by bacteria of the genus Streptococcus (for example, S. pneumoniae or S. agalactiae) and the polysaccharide is capsular polysaccharide such as Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX.
  • the oligo- or polysaccharide is produced by bacteria of the genus Klebsiella (for example, K. pneumoniae) and the polysaccharide is a capsular polysaccharide or O-antigen polysaccharide.
  • the oligo- or polysaccharide is produced by bacteria of the genus Salmonella and the polysaccharide is O- antigen polysaccharide.
  • the bacteria is S. enterica and the S. enterica polysaccharide is a Group B O-antigen.
  • the glycoconjugate is produced in vivo, such as in a bacterial cell.
  • the bacteria is Escherichia coli.
  • the bacterium is from the genus Klebsiella.
  • the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.
  • the glycoconjugate is produced in a cell free system.
  • the bioconjugate is a conjugate vaccine that induces an immune response when administered to a subject.
  • the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response.
  • the antibody response is an IgG or IgM response.
  • the antibody response is an IgG response, for example, an IgG1 response.
  • the conjugate vaccine generates immunological memory in a subject administered the vaccine.
  • TfpM-associated pilin-like protein or pilin-like-protein glycosylation fragment comprises or consists of PilMo (SEQ ID NO: 57) or PilMo lacking amino acids corresponding to residues 1–28 (Pil Mo ⁇ 28, SEQ ID NO: 58) or a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, for example, wherein the C-terminus threonine is substituted with serine.
  • the TfpM-associated pilin-like-protein is selected from the group consisting of Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO:
  • the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii
  • the pilin-like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (Pil Mo _DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148).
  • PilMo pilin disulfide loop region also referred to as Pil20; SEQ ID NO: 60
  • truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine
  • the pilin-like-protein glycosylation fragment consists of Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ 32080280 - 80 - Atty. Dkt.
  • the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil 15S (SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acids, or a variant
  • the isolated fragment of a TfpM-associated pilin-like protein of this disclosure has a length of from 3 to 138 amino acids in length, has a length of from 10 to 138 amino acids in length, has a length of from 20 to 138 amino acids in length, 50 to 138 amino acids in length, has a length of from 100 to 138 amino acids in length, or has a length of from 116 to 138 amino acids in length.
  • the glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 10 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, 50 to 139 amino acids in length, has a length of from 100 to 139 amino acids in length, or has a length of from 116 to 139 amino acids in length.
  • the glycosylation fragment has a length of from 3 to 140 amino acids in length, has a length of from 10 to 140 amino acids in length, has a length of from 20 to 140 amino acids in length, 50 to 140 amino acids in length, has a length of from 100 to 140 amino acids in length, or has a length of from 116 to 140 amino acids in length.
  • the glycosylation fragment has a length of has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length.
  • the glycosylation fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
  • the 32080280 - 81 - Atty. Dkt. No.: 64100-234947 glycosylation fragment has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
  • Fusion protein [0225] Provided for herein is a fusion protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof of this disclosure translationally fused/linked to a heterologous carrier protein such as, but not limited to, Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, or a fragment of any thereof.
  • the TfpM-associated pilin-like protein or glycosylation fragment thereof is translationally fused/linked to a heterologous carrier protein via an amino acid linker.
  • the pilin-like protein or glycosylation fragment comprises a C-terminus serine or threonine residue. In certain embodiments, the pilin-like protein or glycosylation fragment is the C-terminus-most sequence of the fusion protein. And, in certain embodiments, the fusion protein comprises a C-terminus serine or threonine residue. Further, in certain embodiments, the fusion protein is glycosylated by an oligo- or polysaccharide covalently linked to the C-terminus serine or threonine.
  • the fusion protein is glycosylated by an oligo- or polysaccharide comprising glucose at its reducing end covalently linked to the C-terminus serine or threonine.
  • the glycosylated fusion protein is immunogenic.
  • the glycosylated fusion protein is a conjugate vaccine. [0226]
  • the fusion protein comprises a full-length TfpM-associated pilin-like protein.
  • the fusion protein comprises or consists of a glycosylation fragment of a TfpM-associated pilin-like- protein that is less than a full-length TfpM-associated pilin-like protein.
  • the pilin-like-protein glycosylation fragment has a length of from 3 to 138 amino acids in length, has a length of from 10 to 138 amino acids in length, has a length of from 20 to 138 amino acids in length, 50 to 138 amino acids in length, has a length of from 100 to 138 amino acids in length, or has a length of from 116 to 138 amino acids in length.
  • the glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 10 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, 50 to 139 amino acids in length, has a length of from 100 to 139 amino acids in length, or has a length of from 116 to 139 amino acids in length.
  • the glycosylation fragment has a length of from 3 to 140 amino acids in length, has a length of from 10 to 140 amino acids in length, has a length of from 20 to 140 amino acids in length, 50 32080280 - 82 - Atty. Dkt.
  • the glycosylation fragment has a length of has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length.
  • the glycosylation fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, the glycosylation fragment has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
  • the pilin-like-protein glycosylation fragment comprises or consists of PilMo (SEQ ID NO: 57) or PilMo lacking amino acids corresponding to residues 1–28 (PilMo ⁇ 28, SEQ ID NO: 58) or a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, for example, wherein the C-terminus threonine is substituted with serine.
  • the TfpM-associated pilin-like-protein is selected from the group consisting of Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K91 (SEQ ID NO: 95), Pil CIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO:
  • the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65
  • the pilin-like-protein glycosylation 32080280 - 83 - Atty. Dkt. No.: 64100-234947 fragment comprises or consists of the PilMo pilin disulfide loop region (Pil Mo _DSL, also referred to as Pil 20 ; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148).
  • PilMo pilin disulfide loop region also referred to as Pil 20 ; SEQ ID NO: 60
  • truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine
  • the pilin-like-protein glycosylation fragment consists of Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil 10 (SEQ ID NO: 112), Pil 9 (SEQ ID NO: 140), Pil 8 (SEQ ID NO: 141), Pil 7 (SEQ ID NO: 113), Pil 6 (SEQ ID NO: 114), Pil 5 (SEQ ID NO: 115), Pil 4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine.
  • the pilin-like-protein glycosylation fragment consists of Pil 20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil15S (SEQ ID NO: 153), Pil14S (SEQ ID NO: 154), Pil13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil 8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil4S (SEQ ID NO: 164), or Pil3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acids, or a variant
  • the fusion protein can be glycosylated at two or more different positions.
  • the fusion protein can be glycosylated by at least two different OTase classes in one expression system.
  • the fusion protein further comprises a glycosylation sequence (e.g., glycosylation fragment) of an OTase other than TfpM oligosaccharyltransferase (OTase) in addition to the TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus.
  • the other OTase can be PglB, PglL, or PglS.
  • the additional glycosylation sequence is a sequence internal to the fusion protein (i.e., not the C-terminal or N-terminal most sequence). In certain embodiments, the additional glycosylation sequence is a sequence internal within the sequence of a carrier protein (e.g., Figure 11A). In certain embodiments, the additional glycosylation sequence is also covalently linked to an oligo- or polysaccharide. In certain embodiments, 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 additional glycosylation sequences.
  • the fusion protein does not comprise more than two, more than three, more than five, more than 32080280 - 84 - Atty. Dkt. No.: 64100-234947 ten, more than fifteen, more than twenty, or more than twenty five additional glycosylation sequences.
  • the additional glycosylation sequences are identical.
  • at least one additional glycosylation sequence differs from another.
  • at least three, at least four, or at least five of the additional glycosylation sequences all differ from each other. And, in certain embodiments, none of the additional glycosylation sequences are the same.
  • the fusion protein further comprises an internal glycosylation fragment of ComP in addition to the TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus.
  • the ComP glycosylation fragment is also covalently linked to an oligo- or polysaccharide.
  • the ComP glycosylation fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least the amino acids ASA in positions 11-13.
  • 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 fragments. In certain embodiments, the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five ComP glycosylation fragments. In certain embodiments, the ComP glycosylation fragments are identical. In certain embodiments, the ComP glycosylation fragments differ from each other. In certain embodiments, at least three, at least four, or at least five of the ComP glycosylation fragments all differ from each other.
  • the oligo- or polysaccharide covalently linked to the pilin-like protein or glycosylation fragment thereof has a size of at least three repeating units of oligo- or polysaccharide structure. In certain embodiments of the fusion protein of this disclosure, the oligo- or polysaccharide covalently linked to the pilin-like protein or glycosylation fragment thereof has a size of at least ten monosaccharides.
  • the oligo- or polysaccharide is produced by bacteria of the genus Streptococcus (for example, S. pneumoniae or S. agalactiae) and the polysaccharide is capsular polysaccharide such as Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX. 32080280 - 85 - Atty. Dkt. No.: 64100-234947 [0231] In certain embodiments of the fusion protein of this disclosure, the oligo- or polysaccharide is produced by bacteria of the genus Klebsiella (for example, K.
  • the polysaccharide is a capsular polysaccharide or O-antigen polysaccharide.
  • the oligo- or polysaccharide is produced by bacteria of the genus Salmonella and the polysaccharide is O- antigen polysaccharide.
  • the bacteria is S. enterica and the S. enterica polysaccharide is a Group B O-antigen.
  • the glycosylated fusion protein is produced in vivo, such as in a bacterial cell.
  • the bacteria is Escherichia coli.
  • the bacterium is from the genus Klebsiella.
  • the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.
  • the fusion protein is a vaccine that induces an immune response when administered to a subject.
  • the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response.
  • the antibody response is an IgG or IgM response.
  • the antibody response is an IgG response, for example, an IgG1 response.
  • the fusion protein generates immunological memory in a subject administered the fusion protein.
  • Method of producing a glycoconjugate [0235] Provided for herein is a method of producing a glycoconjugate. In certain embodiments, the method occurs in vivo. In certain aspects, the glycoconjugate is produced in a cell free system. Examples of the use of a cell free system utilizing OTases other than TfpM can be found in WO2013/067523A1, which is in incorporated herein by reference.
  • the method comprises covalently linking (conjugating) an oligo- or polysaccharide to an acceptor protein comprising or consisting of a TfpM-associated pilin-like protein or glycosylation fragment thereof using a TfpM oligosaccharyltransferase (OTase) of this disclosure.
  • the pilin-like protein or glycosylation fragment comprises a C-terminus serine or threonine residue
  • the acceptor protein comprises a C- terminus serine or threonine residue
  • the oligo- or polysaccharide is covalently linked to the C-terminus serine or threonine residue of the acceptor protein.
  • the oligo- or polysaccharide comprises a glucose at its reducing end.
  • the 32080280 - 86 - Atty. Dkt. No.: 64100-234947 acceptor protein is a fusion protein of this disclosure as described in detail elsewhere herein.
  • the glycoconjugate is immunogenic.
  • the TfpM OTase contains a wzy_C superfamily domain that is defined by the .National Library of Science (NCBI) conserved protein domain family cl04850, an O-antigen ligase domain, and/or the TfpM OTase contains a wzy_C domain defined by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI, EMBL-EBI) protein family (pfam) conserved protein domain family motif pfam04932, an O-antigen ligase domain, wherein pfam04932 is a protein domain family in the cl04850 superfamily protein domain.
  • NCBI National Library of Science
  • the TfpM OTase comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to TfpM Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpMZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpMYH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpM AI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpMANC4282 (SEQ ID NO: 73), TfpMCIP102159 (SEQ ID NO: 74), TfpMjuni
  • the TfpM OTase is TfpM Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpMZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpM VE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpM CIP102143 (SEQ ID NO: 69), TfpM AI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpMANC4282 (SEQ ID NO: 73), TfpMCIP102159 (SEQ ID NO: 74), TfpMjunii-65 (SEQ ID NO: 75), TfpMYZS-X (SEQ ID NO: 76
  • the TfpM OTase is TfpMMo (SEQ ID NO: 56).
  • the acceptor protein comprises or consists of a full-length TfpM-associated pilin- like protein.
  • the acceptor protein comprises or consists of a glycosylation fragment of a TfpM-associated pilin-like-protein that is less than a full-length TfpM-associated pilin-like protein.
  • the pilin-like-protein glycosylation fragment has a length of from 3 to 138 amino acids in length, has a length of from 10 to 138 amino acids in length, has a length of from 20 to 138 amino acids in length, 50 32080280 - 87 - Atty. Dkt. No.: 64100-234947 to 138 amino acids in length, has a length of from 100 to 138 amino acids in length, or has a length of from 116 to 138 amino acids in length.
  • the pilin-like-protein glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 10 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, 50 to 139 amino acids in length, has a length of from 100 to 139 amino acids in length, or has a length of from 116 to 139 amino acids in length.
  • the glycosylation fragment has a length of from 3 to 140 amino acids in length, has a length of from 10 to 140 amino acids in length, has a length of from 20 to 140 amino acids in length, 50 to 140 amino acids in length, has a length of from 100 to 140 amino acids in length, or has a length of from 116 to 140 amino acids in length.
  • the glycosylation fragment has a length of has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length.
  • the glycosylation fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, the glycosylation fragment has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
  • the pilin-like-protein glycosylation fragment comprises or consists of Pil Mo (SEQ ID NO: 57) or Pil Mo lacking amino acids corresponding to residues 1–28 (Pil Mo ⁇ 28, SEQ ID NO: 58) or a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, for example, wherein the C-terminus threonine is substituted with serine.
  • the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), Pil CIP102143 (SEQ ID NO: 88), Pil AI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Pil junii-65 (SEQ ID NO: 97), Pil YZS-X (SEQ
  • the TfpM-associated pilin- like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino 32080280 - 88 - Atty. Dkt. No.: 64100-234947 acid sequence selected from the group consisting of Pil DSM16617 (SEQ ID NO: 82), Pil ZZC3-9 (SEQ ID NO: 83), Pil TUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), Pil BI730 (SEQ ID NO: 94), Pil A3K
  • the pilin- like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (PilMo_DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148).
  • PilMo_DSL also referred to as Pil20
  • SEQ ID NO: 60 the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine
  • the pilin-like-protein glycosylation fragment consists of Pil20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil 15 (SEQ ID NO: 109), Pil 14 (SEQ ID NO: 137), Pil 13 (SEQ ID NO: 110), Pil 12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil 4 (SEQ ID NO: 116), or Pil 3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine.
  • the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil 19S (SEQ ID NO: 149), Pil 18S (SEQ ID NO: 150), Pil 17S (SEQ ID NO: 151), Pil 16S (SEQ ID NO: 152), Pil 15S (SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil11S (SEQ ID NO: 157), Pil10S (SEQ ID NO: 158), Pil9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil 4S (SEQ ID NO: 164), or Pil 3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acids, or a variant
  • the acceptor protein is a fusion protein and the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and a fragment of any thereof.
  • the TfpM-associated pilin-like protein or glycosylation fragment thereof is translationally fused/linked to a heterologous carrier protein via an amino acid linker. 32080280 - 89 - Atty. Dkt.
  • the acceptor protein is a fusion protein and the method comprises glycosylating the acceptor protein at two or more different positions.
  • the method comprises glycosylating the acceptor protein with at least two different OTase classes in one expression system.
  • the fusion protein comprises two or more glycosylation sequences (e.g., glycosylation fragment) associated with at least two different OTases.
  • Representative examples of OTases that may be used in combination include PglB, PglL, PglS, TfpO, and TfpM.
  • the acceptor protein comprises a TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus in addition to an additional glycosylation sequence of an OTase other than TfpM oligosaccharyltransferase (OTase).
  • the other OTase is PglB, PglL, and/or PglS.
  • one or more glycosylation sequence is a sequence internal to the fusion protein (i.e., not the C-terminal or N-terminal most sequence).
  • the one or more glycosylation sequence is a sequence internal within the sequence of a carrier protein (e.g., Figure 33A).
  • the additional glycosylation sequence is a sequence internal to the fusion protein (i.e., not the C-terminal or N-terminal most sequence).
  • the additional glycosylation sequence is a sequence internal within the sequence of a carrier protein (e.g., Figure 33A).
  • At least two different glycosylation sequences of two different OTase systems are covalently linked to an oligo- or polysaccharide.
  • the TfpM-associated pilin-like-protein glycosylation fragment located at the fusion protein C-terminus and the additional glycosylation sequence are covalently linked to an oligo- or polysaccharide.
  • 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 additional glycosylation sequences.
  • the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five additional glycosylation sequences.
  • the additional glycosylation sequences are identical.
  • at least one additional glycosylation sequence differs from another.
  • at least three, at least four, or at least five of the additional 32080280 - 90 - Atty. Dkt. No.: 64100-234947 glycosylation sequences all differ from each other. And, in certain embodiments, none of the additional glycosylation sequences are the same.
  • the method comprises further glycosylating an internal glycosylation fragment of ComP using a PglS OTase in addition to glycosylating the TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus.
  • the ComP glycosylation fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least the amino acids ASA in positions 11-13.
  • 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 fragments.
  • the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five ComP glycosylation fragments.
  • the ComP glycosylation fragments are identical.
  • the ComP glycosylation fragments differ from each other.
  • at least three, at least four, or at least five of the ComP glycosylation fragments all differ from each other.
  • none of the ComP glycosylation fragments are the same.
  • the host cell is a bacterial cell.
  • the conjugation occurs in in Escherichia coli.
  • the conjugation occurs in a bacterium from the genus Klebsiella.
  • the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.
  • the method comprises culturing a host cell that comprises: (a) a genetic cluster encoding for the proteins required to synthesize the oligo- or polysaccharide; (b) a TfpM OTase; and (3) the acceptor protein.
  • the method produces a conjugate vaccine.
  • a host cell comprising (a) a genetic cluster encoding for the proteins required to synthesize an oligo- or polysaccharide; (b) a TfpM OTase of this disclosure; and (3) an acceptor protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof of this disclosure.
  • the acceptor protein 32080280 - 91 - Atty. Dkt. No.: 64100-234947 is a fusion protein.
  • the host cell comprises a nucleic acid encoding the TfpM OTase. In certain embodiments, the host cell comprises a nucleic acid encoding the acceptor protein. And, in certain embodiments, the TfpM OTase and the acceptor protein are encoded by the same nucleic acid.
  • an isolated nucleic acid encoding the pilin-like-protein glycosylation fragment and/or the fusion protein of this disclosure. In certain embodiments, the nucleic acid is a vector. Also provided for is a host cell comprising such isolated nucleic acid of this disclosure. In certain embodiments, the host cell is a bacterial cell.
  • the host cell is Escherichia coli. In certain embodiments, the host cell is from the genus Klebsiella. And, in certain embodiments, the host cell is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.
  • a composition comprising the conjugate vaccine or the fusion protein of this disclosure and an adjuvant and/or carrier. In certain embodiments, the composition is a pharmaceutical or therapeutic composition suitable for administration to a subject/patient.
  • a method of inducing a host immune response against a bacterial pathogen comprising administering to a subject in need of the immune response an effective amount of the conjugate vaccine, the fusion protein, or the composition comprising the conjugate vaccine or the fusion protein and an adjuvant and/or carrier of this disclosure.
  • Treatment with pharmaceutical compositions comprising an immunogenic composition can occur separately or in conjunction with other treatments, as appropriate.
  • an amount adequate to accomplish this is defined as “effective amount,” “effective dose,” or “unit dose.” Amounts effective for this use will depend on, e.g., the glycoconjugate composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician. In some aspects, a priming dose is followed by a boosting dose over a period of time. In certain embodiments, the immune response is an antibody response.
  • the immune response is selected from the group consisting of an innate response, an adaptive response, a humoral response, an antibody response, cell mediated response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system cross-talk, and a combination of two or more of said immune responses.
  • the immune response is selected from the group consisting of an innate response, a humoral response, an 32080280 - 92 - Atty. Dkt. No.: 64100-234947 antibody response, a T cell response, and a combination of two or more of said immune responses.
  • the subject is a mammal.
  • the subject is a human.
  • the subject is a companion animal.
  • the subject is livestock.
  • the infection is a localized or systemic infection of skin, soft tissue, blood, or an organ, or is auto-immune in nature.
  • the disease is pneumonia.
  • the infection is a systemic infection and/or an infection of the blood.
  • the conjugate vaccine, the fusion protein, or the composition is administered via intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration.
  • a method of producing a pneumococcal conjugate vaccine against pneumococcal infection comprising: (a) isolating the glycoconjugate or a glycosylated fusion protein of this disclosure; and (b) combining the isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant and/or carrier.
  • a glycoconjugate, glycosylated fusion protein, or conjugate vaccine or a composition of any thereof of this disclosure for use in inducing a host immune response against a bacterial pathogen and/or preventing or treating a bacterial disease and/or infection in a subject.
  • a recombinant nucleic acid construct comprising a nucleotide sequence encoding a TfpM oligosaccharyltransferase (OTase) operably linked to at least one heterologous transcriptional regulatory sequence.
  • the TfpM OTase comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to TfpM Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpMYH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpMAI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74), TfpM
  • the TfpM OTase is TfpM Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpMYH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpMAI40 (SEQ ID NO: 70), TfpM F78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), Tf
  • the TfpM OTase is TfpM Mo (SEQ ID NO: 56).
  • the heterologous transcriptional regulatory sequence is a promotor sequence.
  • the recombinant construct further comprises a nucleotide sequence encoding a TfpM-associated pilin-like protein or glycosylation fragment thereof of this disclosure or a fusion protein of this disclosure comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof operably linked to the nucleotide sequence encoding the a TfpM OTase.
  • the recombinant construct further comprises a nucleotide sequence encoding a TfpM-associated pilin-like protein or glycosylation fragment thereof of this disclosure or a fusion protein of this disclosure comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof 5’ of and operably linked to the nucleotide sequence encoding the a TfpM OTase.
  • the fusion protein of the construct also comprises a glycosylation sequence of an OTase other than TfpM, such as for PglB, PglL, PglS (e.g., ComP or a glycosylation fragment thereof).
  • the coding sequence of the TfpM-associated pilin-like protein or glycosylation fragment thereof or a fusion protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof is within 2, 5, 10, 20, 30, 40, or 50 nucleotides of the sequence encoding the TfpM OTase. In certain embodiments, the coding sequence of the TfpM-associated pilin-like protein or glycosylation fragment thereof or a fusion protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof overlaps the operably linked nucleotide sequence encoding the a TfpM OTase.
  • the TfpM-associated pilin-like protein comprises or consists of a full-length TfpM-associated pilin-like protein. In certain embodiments, the TfpM- associated pilin-like protein comprises or consists of a glycosylation fragment of a TfpM- associated pilin-like-protein that is less than a full-length TfpM-associated pilin-like protein. In certain embodiments, the pilin-like-protein glycosylation fragment has a length of from 3 to 138 amino acids in length, has a length of from 10 to 138 amino acids in length, has a length 32080280 - 94 - Atty. Dkt.
  • the pilin-like-protein glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 10 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, 50 to 139 amino acids in length, has a length of from 100 to 139 amino acids in length, or has a length of from 116 to 139 amino acids in length.
  • the glycosylation fragment has a length of from 3 to 140 amino acids in length, has a length of from 10 to 140 amino acids in length, has a length of from 20 to 140 amino acids in length, 50 to 140 amino acids in length, has a length of from 100 to 140 amino acids in length, or has a length of from 116 to 140 amino acids in length.
  • the glycosylation fragment has a length of has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length.
  • the glycosylation fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, the glycosylation fragment has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
  • the pilin-like-protein glycosylation fragment comprises or consists of PilMo (SEQ ID NO: 57) or Pil Mo lacking amino acids corresponding to residues 1–28 (Pil Mo ⁇ 28, SEQ ID NO: 58) or a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, for example, wherein the C-terminus threonine is substituted with serine.
  • PilMo SEQ ID NO: 57
  • Pil Mo lacking amino acids corresponding to residues 1–28
  • a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, for example, wherein the C-terminus threonine is substituted with serine.
  • the TfpM- associated pilin-like-protein is selected from the group consisting of Pil DSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), Pil F78 (SEQ ID NO: 90), Pil S71 (SEQ ID NO: 91), Pil ANC4282 (SEQ ID NO: 92), Pil 72- O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO:
  • the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID 32080280 - 95 - Atty. Dkt.
  • the pilin- like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (Pil Mo _DSL, also referred to as Pil 20 ; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148).
  • PilMo pilin disulfide loop region also referred to as Pil 20 ; SEQ ID NO: 60
  • truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine
  • the pilin-like-protein glycosylation fragment consists of Pil 20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil 11 (SEQ ID NO: 139), Pil 10 (SEQ ID NO: 112), Pil 9 (SEQ ID NO: 140), Pil 8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine.
  • the pilin-like-protein glycosylation fragment consists of Pil 20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil 15S (SEQ ID NO: 153), Pil 14S (SEQ ID NO: 154), Pil 13S (SEQ ID NO: 155), Pil 12S (SEQ ID NO: 156), Pil 11S (SEQ ID NO: 157), Pil 10S (SEQ ID NO: 158), Pil 9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil4S (SEQ ID NO: 164), or Pil3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acids, or a variant
  • the fusion protein is a fusion protein of this disclosure.
  • the recombinant construct further comprises a nucleotide sequence encoding an additional OTase as described elsewhere herein operably linked to the TpfM OTase.
  • the recombinant construct further comprises a nucleotide sequence encoding the additional OTase 3’ of and operably linked to the TpfM OTase.
  • the recombinant construct further comprises a nucleotide sequence encoding the additional OTase 5’ of and operably linked to the TpfM OTase.
  • the coding sequence of the additional OTase is within 10, 20, 30, 40, 50, 75, or 32080280 - 96 - Atty. Dkt. No.: 64100-234947 100 nucleotides of the sequence encoding the TfpM OTase.
  • the recombinant construct further comprises a nucleotide sequence encoding a PglS OTase 3’ operably linked to the TpfM OTase.
  • the recombinant construct further comprises a nucleotide sequence encoding a PglS OTase 3’ of and operably linked to the TpfM OTase.
  • the recombinant construct further comprises a nucleotide sequence encoding a PglS OTase 5’ of and operably linked to the TpfM OTase.
  • the coding sequence of the PglS OTase is within 10, 20, 30, 40, 50, 75, or 100 nucleotides of the sequence encoding the TfpM OTase.
  • a vector comprising the recombinant nucleic acid construct.
  • a host cell comprising the recombinant nucleic acid construct or the vector. In certain embodiments, the host cell is a bacterial cell.
  • the host cell is Escherichia coli. In certain embodiments, the host cell is from the genus Klebsiella. And, in certain embodiments, the host cell is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca.
  • a method for producing a TfpM OTase comprising culturing the host cell, wherein said vector is an expression vector, and recovering the TfpM OTase.
  • bioconjugate-mi3 and bioconjugate-AP205 assemblies were generated using two different bacterial O-linking OTases: Acinetobacter baylyi PglS and Moraxella osloensis TfpM. PglS and TfpM belong to recently characterized O-linking OTase families that have the broadest saccharide substrate range of known OTases (Harding, C.M., et al. (2019) Nat Commun 10, 891; Knoot, C.J., et al. (2023) Glycobiology, Volume 33, Pages 57-74).
  • PglS and TfpM are both able to transfer glycans with glucose at the reducing end, allowing these enzymes to be used to generate bioconjugate vaccines against a variety of pathogens whose native polysaccharides have this sugar at the reducing end (Harding, C.M., et al. (2019) Nat Commun 10, 891; Feldman, et al. (2019) PNAS, 116 (37) 18655-18663).
  • E. coli maltose binding protein (MBP) and P. aeruginosa EPA were engineered to contain PglS- or TfpM-specific sequons (Knoot, C.J., et al.
  • the SpyTagged MBP-O16 bioconjugates (“first polypeptide”; e.g., Figure 1: “PROTEIN 1”) were produced in CLM24 glycoengineered E. coli strains.
  • the MBP-SpyTag fusion proteins were separately expressed from a pEXT20 expression plasmid and WbbL was expressed from plasmid pMF19, a pEXT21 derivative. Expression of WbbL restores production of O16 O-antigen in most laboratory strains of E. coli.
  • the mi3- and AP205- SpyCatcher fusion proteins (“second polypeptide”; e.g., Figure 1: “PROTEIN 2”) were separately produced in C41(DE3) E. coli.
  • SpyTagged O16-bioconjugates were purified from periplasmic cell extracts by using immobilized metal-affinity (Ni) chromatography (IMAC). The IMAC eluate was concentrated and buffer-exchanged prior to loading on an Akta FPLC instrument for further purification using anion-exchange chromatography. Anion-exchange was used to separate unglycosylated SpyTagged MBP from glycosylated SpyTagged MBP-O16. Fractions containing glycosylated SpyTagged MBP-O16 were pooled, concentrated and quantified using a BCA assay kit.
  • IMAC immobilized metal-affinity
  • NPs nanoparticles
  • TfpM-derived Spytagged bioconjugates more completely reacted with Spycatcher-mi3 or AP205 to form isopeptide-bonded assemblies based on the intensity of the cross-linked protein band in Coomassie-stained gels or western blotting and the observation that less of the either the NP/VLP monomer or bioconjugate band remained after the isopeptide bond reaction.
  • Size-exclusion chromatography showed the bioconjugate-NP/VLP assemblies were larger in mass than those before the isopeptide reaction (Figure 3).
  • Working antibiotic concentrations used for liquid culture and in LB-agar plates were as follows: ampicillin (Amp), 100 ⁇ g/mL, kanamycin (Kan), 20 ⁇ g/mL, tetracycline (Tet), 10 ⁇ g/mL, spectinomycin (Sp), 50 ⁇ g/mL.
  • ampicillin Amp
  • Kan kanamycin
  • Tet tetracycline
  • Sp spectinomycin
  • HiFi gblocks were ordered (Integrated DNA Technologies, IDT) designed with terminal 25 base pair overlaps for Gibson assembly with PCR-linearized plasmid.
  • the plasmid backbone for these fragments was amplified from a pEXT20 plasmid (Dykxhoorn, D.
  • coli Stellar cells (Takara Bio) by heat shock, out-grown for an hour at 37oC, and plated on LB-agar supplemented with Amp. Individual colonies were picked and grown in LB media with appropriate antibiotic and plasmids isolated using a GeneJet Plasmid Miniprep Kit (Thermo Fisher). All plasmids were sequence-verified by Sanger sequencing (Genewiz). The plasmid expressing the M. osloensis 1202 EPA-Pil ⁇ 28 fusion and TfpMMo was named pVNM227.
  • the inventors designed overlapping PCR primers that introduced the necessary codon changes in the pilin gene and amplified each fragment from pVNM227 plasmid.
  • the resulting PCR product was DpnI-digested (NEB) for 30 min at 37oC and gel- 32080280 - 100 - Atty. Dkt. No.: 64100-234947 purified from agarose gel using a Pure-Link Gel Extraction Kit (Thermo Fisher).
  • NEB DpnI-digested
  • gel- 32080280 - 100 - Atty. Dkt. No.: 64100-234947 purified from agarose gel using a Pure-Link Gel Extraction Kit (Thermo Fisher).
  • the oligos were re-suspended in purified water, mixed, and annealed together in thermocycler by heating to 98oC for 5 min followed by a slow cooling to 4oC at 0.1oC/minute.
  • the annealed oligos were diluted 1 to 5 in water and assembled with PCR-linearized pVNM227 using an NEBuilder HiFi DNA Assembly Kit (NEB).
  • the resulting DNA was transformed into Stellar cells and plasmids isolated and verified as described above.
  • the plasmid comprising the construct encoding EPA-Pil 20 and TfpM was termed pVNM297.
  • the N-terminally His-tagged EPA-Pil 20 variant was constructed by linearizing pVNM297 using PCR and using this fragment in Gibson assembly with complementary annealed oligos containing the 6xHis coding region and terminal homologous regions, resulting in pVNM291.
  • pVNM167 was generated by digesting the previously described EPAiGTcc plasmid (Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203) with SalI.
  • the purified SalI fragment was Gibson assembled with the pglS gene with its native 100 bp 5’ UTR amplified from A. baylyi ADP1 gDNA.
  • pVNM245 was generated from pVNM167 template by separate PCR reactions to amplify products with overhangs for Gibson assembly: (i) the vector backbone with PglS and EPA with one iGT, (ii) the second iGT for integration between E548 and G549 and (iii) the C-terminus of EPA downstream of the iGT.
  • the plasmid pVNM337 was created by amplifying tfpM from pVNM291 with the primers EPA 3’ F1 and pglS-tfpM R1 and cloning the product into PCR-linearized pVNM167, which was amplified with pglS 5’ F1 and EPA 3’ R1.
  • Phylogenetic trees for the TfpM and pilin proteins were generated using the phylogeny.fr server (on the world wide web at phylogeny.fr/) which uses MUSCLE, PhyML, and TreeDyn for sequence alignment, tree calculation, and image generation, respectively.
  • Example 5 Expression of glycans and cloning of K. pneumoniae O2a glycan genes [0265]
  • the S. pneumonia CPS8 glycan was expressed from plasmid pB8 (TetR) (Kay, E. J., et al. (2016) Open Biology 6, 150243), the Salmonella enterica LT2 glycan from plasmid pPR1347 (Kan R ) (Neal, B.
  • the plasmid backbone for the O2a cluster was amplified from plasmid pBBR1MCS2 (Kan R ) (Kovach, M. E., et al. (1995) Gene 166, 175-176). Primers for these reactions are listed in Table 2 The PCR products from these reactions were assembled using Gibson assembly with a NEBuilder HiFi DNA Assembly Kit (NEB). Stellar cells were transformed and plasmid isolated and verified as described in the previous section. Example 6. Bioconjugation and western blots [0266] E. coli strains used the bioconjugation experiments were either SDB1 or CLM24 (Feldman, M. F., et al.
  • SDB1 is a W3110 E. coli derivative with mutations in the genes encoding for WecA, the glycosyltransferase that initiates synthesis of the endogenous E. coli O16 antigen and WaaL, the enzyme transferring Und-PP linked glycans to Lipid A-core saccharide to produce LPS.
  • CLM24 is a W3110 derivative with only a deletion of waaL. Elimination of these genes prevents crosstalk of the heterologous bioconjugation system and endogenous E. coli glycosylation pathways.
  • coli strains for bioconjugation the inventors electroporated plasmids using competent cells prepared as previously described (Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203) followed by out-growth at 37oC in SOB media.
  • the cells were plated on LB-agar with appropriate antibiotics.
  • 8 – 10 colonies were picked and inoculated into LB or TB with antibiotics and grown overnight while shaking at 30oC.
  • starter cultures were inoculated into either 30 mL media in a 125 mL Erlenmeyer flask or 1 L media in a 2 L flask to a starting optical density at 600 nm (OD 600 ) of 0.05.
  • the boiled samples were briefly centrifuged at 10,000 rcf and equivalent amounts, normalized to the same OD 600 per lane, loaded for SDS-PAGE separation on a 7.5% Mini-Protean TGX gel (Biorad). Proteins were transferred to a nitrocellulose membrane using 32080280 - 102 - Atty. Dkt. No.: 64100-234947 a semi-dry electrode system and blocked with Intercept Blocking Buffer (Li-Cor) for one hour. The membrane was incubated with primary antibodies in 1:1 blocking and TBST for 45 min. For protein detection, commercial rabbit anti-EPA and mouse anti-6xHis antibodies (Millipore- Sigma) were used.
  • Rabbit glycan antibodies for CPS8, GBSIII, and O16 were purchased from SSI Diagnostica.
  • K. pneumoniae rabbit O2a antibodies were a generous gift from Prof. Chris Whitfield (University of Guelph) (Clarke, B. R., et al. (2016) Journal of Biological Chemistry 293, 4666-4679).
  • Salmonella Group B rabbit antibodies were purchased from BD. Primary incubation was followed by three washes with TBST buffer totaling 15 min The membranes were then incubated with secondary antibodies IRDye 680RD goat anti-mouse and/or IRDye 800CW goat anti-rabbit (Li-Cor) in 1:1 blocking buffer and TBST for 30 min.
  • the destained band was then dehydrated with 100% ethanol for 10 min and dried by vacuum- centrifugation for 10 min before being rehydrated in 10 mM DTT in 50 mM NH 4 HCO 3 . Reduction was carried out for 60 min at 56°C after which the gel band was dehydrated twice with 100% ethanol for 10 min to remove the remaining reduction buffer. The reduced sample was then sequentially alkylated with 55 mM iodoacetamide in 50 mM NH 4 HCO 3 for 45 min at RT in the dark.
  • the alkylated sample was then washed four times for 10 min with 50 mM NH4HCO3 followed by 100% ethanol, followed by 50 mM NH4HCO3 followed by 100% ethanol before being dried by vacuum-centrifugation.
  • the dried alkylated sample was then rehydrated with 20 ng/ ⁇ l Lys-C endoprotease (Wako Chemicals) in 40 mM NH 4 HCO 3 at 4°C for 1hr. Excess Lys-C was removed, gel pieces were covered in 40mM NH4HCO3 and incubated overnight at 37°C.
  • Peptides were concentrated and desalted using C18 stage tips (Ishihama, Y., et al.
  • the sample was concentrated onto the trap column at 5 ⁇ l/minute using 0.1% formic acid (FA) for 5 min and infused into an Orbitrap FusionTM LumosTM TribridTM Mass Spectrometer equipped with a FAIMS Pro interface (Thermo Fisher Scientific) at 300 nl/min via the analytical column using a Dionex Ultimate 3000 UPLC (Thermo Fisher Scientific) by altering the concentration of buffer A (2% DMSO, 0.1% FA) and buffer B (78% ACN, 2% DMSO and 0.1% FA). Identification of potential glycopeptides utilized a 140-minute analytical run while targeted analysis utilized a 60-minute run.
  • FA formic acid
  • the buffer composition was altered from 3% buffer B to 28% buffer B over 120 min, 28% buffer B to 40% buffer B over 9 min, 40% buffer B to 100% buffer B over 3 min, then the composition was held at 100% buffer B for 2 min, and then dropped to 3% buffer B over 2 min and held at 3% buffer B for another 8 min
  • the LumosTM Mass Spectrometer was operated in a stepped FAIMS data-dependent mode at three different FAIMS CVs, -25, -45 and -65 as previously described (Ahmad Izaham, A. R., et al.
  • the washed cell pellets were suspended in 200 mM Tris-HCl pH 8.5, 100 mM EDTA, 25% sucrose and incubated while rolling for 30 min at 4oC. Cells were pelleted by centrifugation at 4,700 rcf for 30 min and the resulting pellet suspended in 20 mM Tris-HCl pH 8.5 and incubated while rolling for 45 min at 4oC. The suspension was centrifuged for 30 min at 18,000 rcf. The supernatant containing the periplasmic fraction was concentrated and either loaded directly on an FPLC anion-exchange column or, for His-tagged EPA-Pil ⁇ 28 bioconjugates, purified using Nickel IMAC as previously described (Knoot, C.
  • bioconjugates were eluted at 2 mL/min using a stepwise gradient with buffer A (20 mM Tris pH 8) and buffer B (20 mM Tris pH 8, 1 M NaCl) from 0% B to 25% in 5% increments at 10 column volumes for each concentration.
  • Bioconjugates for immunization were further purified using a Superdex 200 Increase 10/300 GL column.
  • the concentrated bioconjugates pooled from the anion-exchange column were loaded on a pre-equilibrated Superdex 200 column in PBS buffer and eluted at a flow rate of 0.75 mL/min Fractions containing the purified bioconjugates were pooled, concentrated, and frozen at -80oC for storage.
  • mice Five-week-old female CD-1 outbred mice (Charles River Laboratories) were subcutaneously injected with 100 ⁇ L of a vaccine formulation on days 0, 14, and 28.
  • the vaccination groups were 291 alone (5 ⁇ g protein) and GBSIII-291 (5 ⁇ g protein, 1 ⁇ g polysaccharide). Mice had sera collected on days 0, 14, 28, and 42. All vaccines were formulated with Alhydrogel ® 2% aluminum hydroxide gel (InvivoGen) at a 1:9 ratio (50 ⁇ L vaccine to 5.5 ⁇ L alum in 44.5 ⁇ L 1x sterile phosphate buffered saline).
  • Example 12 Example 12
  • Enzyme-linked immunosorbent assay [0273] IgG kinetic titers were determined using enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates (TRP Immunomaxi plates) were coated in triplicate overnight with approximately 10 6 CFU/100 ⁇ L of glycoengineered E. coli expressing the GBSIII capsular polysaccharide in sodium carbonate buffer. The coating E. coli strain was grown the same as referenced above and after overnight induction to induce GBSIII expression was washed and diluted to coat plates. Wells were blocked with 1% BSA in PBS and washed with 0.05% PBS-Tween (PBST), all subsequent washes were the same.
  • PBST PBS-Tween
  • Serum from mice was 32080280 - 106 - Atty. Dkt. No.: 64100-234947 diluted to 1:100 and added to wells for 1 hr at room temperature then washed.
  • Total IgG titers were detected by HRP conjugated anti-mouse IgG (GE Lifesciences, 1:5000 dilution) added to wells for 1 hour at room temperature. After washing, plates were developed using 3,3′,5,5′ tetramethyl benzidine (TMB) substrate (Biolegend) and stopped with 2 N H2SO4. The optical densities were determined at 450 nm using a microplate reader (Bio-Tek). Total IgG product was determined using IgG standards to generate a standard curve for data fitting.
  • a fusion protein comprising: (i) a glycosylation fragment and (ii) a first polypeptide tag, wherein the first polypeptide tag can spontaneously form an isopeptide bond with a second polypeptide tag binding partner; optionally, wherein the glycosylation fragment is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, or 40 amino acids in length; optionally, wherein the glycosylation fragment is not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, 40, 50, 60, 80, or 100 amino acids in length; optionally, wherein the glycosylation fragment is from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, or 30 amino acids in length to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, or 40 amino acids in length; and optionally, wherein the fusion protein comprises a carrier protein.
  • the fusion protein of Paragraph 1 wherein the fusion protein is a glycoconjugate comprising a saccharide covalently attached to the fusion protein via the glycosylation fragment; optionally, wherein the saccharide is covalently attached to the glycosylation fragment through an N-linkage, O-linkage, or C-linkage; and optionally, wherein said glycoconjugate is immunogenic. 32080280 - 107 - Atty. Dkt. No.: 64100-234947 [0278] 3. The fusion protein of Paragraph 1 or 2, wherein the first polypeptide tag is translationally fused at the N-terminal or C-terminal end of the fusion protein. [0279] 4.
  • the fusion protein of Paragraph 1 or 2 wherein the first polypeptide tag is translationally fused internally within the fusion protein; optionally, wherein the first polypeptide tag is translationally fused internally within the sequence of a carrier protein.
  • the fusion protein of any one of Paragraphs 1 to 4 wherein the glycosylation fragment is translationally fused at the N-terminal or C-terminal end of the fusion protein.
  • 6. The fusion protein of any one of Paragraphs 1 to 5, wherein the glycosylation fragment is translationally fused internally within the fusion protein; [0282] optionally, wherein the glycosylation fragment is translationally fused internally within the sequence of a carrier protein. [0283] 7.
  • the first polypeptide tag is a SpyTag (SEQ ID NO: 416), SpyTag002 (SEQ ID NO: 417), SpyTag003 (SEQ ID NO: 418), or a DogTag (SEQ ID NO: 419); optionally, wherein the SpyTag, Spytag002, or Spytag003 is translationally fused at the N-terminal or C-terminal end of the fusion
  • the glycosylation fragment is a ComP glycosylation fragment; optionally, wherein the ComP glycosylation fragment comprises or consists of the amino acid sequence CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 412) or a fragment thereof comprising the amino acids ASA, or a variant thereof comprising the amino acids ASA in positions 11-13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions; optionally, wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of: iGTcc ⁇ 0-1 CTGVTQIASGASAATTNVASAQ (SEQ ID NO: 232); iGTcc ⁇ 1-0 TGVTQIASGASAATTNVASAQC (SEQ ID NO: 243); iGTcc ⁇ 1-1 TGVTQIASGASAATTNVASAQ (SEQ ID NO: 244); iGTcc
  • the glycosylation fragment is a TfpM-associated pilin glycosylation fragment; [0286] optionally, wherein the TfpM-associated pilin glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (SEQ ID NO: 413) or a fragment thereof comprising at least the last three amino acids from the TfpM-associated pilin C-terminus; [0287] or a variant thereof comprising the last three amino acids from the TfpM-associated pilin C-terminus and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. [0288] 10.
  • the TfpM-associated pilin glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (SEQ ID NO: 413) or a fragment thereof comprising at least the last three amino acids from the TfpM-associated pilin C-terminus; [0287] or a variant thereof comprising the last
  • the glycosylation fragment is a PilE glycosylation fragment; optionally, wherein the PilE glycosylation fragment comprises or consists of the amino acid SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) or a fragment thereof comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414; or a variant thereof comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. [0289] 11.
  • glycosylation fragment is a PglB glycosylation fragment; optionally, wherein the PglB glycosylation fragment comprises or consists of the consensus motif amino acid sequence X1 X2 N X3 X4, wherein X1 is D or E, X2 is any amino acid except proline, X3 is any amino acid except proline, and X4 is S or T. 32080280 - 109 - Atty. Dkt. No.: 64100-234947 [0290] 12.
  • the glycosylation fragment is a PilA glycosylation fragment; optionally, wherein the PilA glycosylation fragment comprises or consists of the PilA pilin disulfide loop region (SEQ ID NO: 415) or a fragment thereof comprising at least the last three amino acids from the PilA C-terminus; or a variant thereof comprising at least the last three amino acids from the PilA terminus and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. [0291] 13.
  • glycosylation fragment is a STT3 glycosylation fragment; optionally, wherein the STT3 glycosylation fragment comprises or consists of the consensus motif amino acid sequence N X 1 X 2 , wherein X 1 is any amino acid except proline and X2 is S or T. [0292] 14.
  • glycosylation fragment is a N-linking glycosyltransferase glycosylation fragment; optionally, wherein the N-linking glycosyltransferase glycosylation fragment comprises or consists of the consensus motif amino acid sequence N X1 X2, wherein X1 is any amino acid and X 2 is S or T [0293] 15.
  • glycosylation fragment is an O-linking glycosyltransferase glycosylation fragment; optionally, wherein the O-linking glycosyltransferase glycosylation fragment comprises or consists of a fragment of the serine or threonine rich repeats from the serine-rich repeats (SRR) adhesins of streptococci or staphylococci bacteria; optionally, wherein the O-linking glycosyltransferase glycosylation fragment comprises or consists of serine or threonine rich repeats from the adhesin GspB from Streptococcus gordonii. [0294] 16.
  • SRR serine-rich repeats
  • No.: 64100-234947 optionally, wherein at least one glycosylation fragment is located at the N-terminal or C-terminal end of the fusion protein and at least one glycosylation fragment is located internally within the fusion protein; optionally, wherein at least two glycosylation fragments are located internally within the fusion protein; and/or optionally, wherein one glycosylation fragment is located at the N-terminal end of the fusion protein and where glycosylation fragment is located at the C-terminal end of the fusion protein. [0295] 17.
  • the fusion protein of Paragraph 16 wherein the two or more glycosylation fragments are the same; wherein at least one of the two or more glycosylation fragments is different; or wherein each of the glycosylation fragments is different; optionally, wherein one glycosylation fragment is a ComP glycosylation fragment and wherein one glycosylation fragment is a TfpM-associated pilin glycosylation fragment; optionally, wherein the fusion protein is a glycoconjugate comprising two or more saccharide covalently attached to the fusion protein via the two or more glycosylation fragments, optionally, wherein the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 covalently attached saccharides; optionally, wherein the fusion protein comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 covalently attached saccharides to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
  • EPA Pseudomonas aeruginosa Exotoxin A
  • CRM197 Haemophilus influenzae Protein D
  • cholera toxin B subunit cholera toxin B subunit
  • tetanus toxin a fragment of any thereof.
  • a composition comprising a polypeptide pair that comprises a first polypeptide and a second polypeptide, wherein the first polypeptide is the fusion protein of any one of Paragraphs 1 to 18, wherein the second polypeptide comprises a second polypeptide tag binding partner to the first polypeptide tag of the first polypeptide, and wherein the first polypeptide is attached to the second polypeptide via an isopeptide bond between the first polypeptide tag and the second polypeptide tag; optionally, where the second polypeptide comprises a monomeric polypeptide that can spontaneously multimerize/self-assemble into a higher-order, multimeric structure; and/or further optionally, wherein said higher-order, multimeric structure is an icosahedron or dodecahedron particle (e.g,.
  • the second polypeptide comprises a fragment of a mutated 2-keto-3-deoxy- phosphogluconate aldolase (mi3).
  • the first polypeptide tag is a SpyCatcher (SEQ ID NO: 420), SpyCatcher002 (SEQ ID NO: 421), SpyCatcher003 (SEQ
  • a complex comprising two or more of the polypeptide pairs of any one of Paragraphs 19 to 26; optionally, wherein the complex comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, or more complexed polypeptide pairs of any one of Paragraphs 19 to 26; optionally, wherein the complex comprises from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, or 400 complexed polypeptide pairs of any one of Paragraphs 19 to 26 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, or 500 complexed polypeptide pairs of any one of Paragraphs 19 to 26;
  • a pharmaceutical/therapeutic composition comprising the complex of any one of Paragraphs 27 to 32 and adjuvant and/or an excipient. 32080280 - 114 - Atty. Dkt. No.: 64100-234947 [0312] 34. The complex of any one of Paragraphs 27 to 32 or the pharmaceutical/therapeutic composition of Paragraph 33, wherein the complex or composition is a conjugate vaccine. [0313] 35. A method of making the polypeptide pair of any one of Paragraphs 19 to 26, the method comprising contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag binding partner. [0314] 36.
  • the method of Paragraph 35 further comprising glycosylating the first polypeptide with a saccharide before contact and isopeptide bond formation with the second polypeptide; optionally, wherein the first polypeptide is glycosylated in vivo before contact and isopeptide bond formation with the second polypeptide; and optionally, wherein the method comprises isolating/purifying the in vivo glycosylated first polypeptide before contact and isopeptide bond formation with the second polypeptide. [0315] 37. The method of Paragraph 35, wherein the first polypeptide is glycosylated after contact and isopeptide bond formation with the second polypeptide. [0316] 38.
  • a method of making the complex of any one of Paragraphs 27 to 32 comprising: (i) forming a self-assembled, multimeric higher-order structure of the second polypeptide and then contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag; or (ii) contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag and then forming of a self-assembled, multimeric higher-order structure of the second polypeptide. [0317] 39.
  • the method of Paragraph 38 further comprising glycosylating the first polypeptide with a saccharide, optionally: wherein the first polypeptide is glycosylated before the isopeptide bond is formed between the first polypeptide and the second polypeptide; wherein the first polypeptide is glycosylated after the isopeptide bond is formed between the first polypeptide and the second polypeptide; 32080280 - 115 - Atty. Dkt. No.: 64100-234947 wherein the first polypeptide is glycosylated before it is incorporated into a multimeric, higher-order structure; and/or wherein the first polypeptide is glycosylated after is has been incorporated into a multimeric, higher-order structure.
  • N- OTase N-linking oligosaccharyltransferase
  • O-OTase O-linking oligosaccharyltransferase
  • NTT N-linking glycosyltransferase
  • OHT O-linking glycosyltransferase
  • CMT C-mannosyltransferase
  • a ComP glycosylation fragment is glycosylated by a PglS OTase; wherein a TfpM-associated pilin glycosylation fragment is glycosylated by a TfpM OTase, optionally, wherein a ComP glycosylation fragment is glycosylated by a PglS OTase and a TfpM-associated pilin glycosylation fragment is glycosylated by a TfpM OTase; wherein a PilE glycosylation fragment is glycosylated by a PglL OTase; wherein a PglB glycosylation fragment is glycosylated by a PglB OTase; wherein a PilA glycosylation fragment is glycosylated by a TfpO or PilO OTase; wherein a STT3
  • Dkt. No.: 64100-234947 wherein the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using a PglB OTase; wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpO or PilO OTase; wherein the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using a STT3 OTase; wherein the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using an AlgB OTase; wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpW glycosyltransferase; wherein the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using an N-linking glycosyltransferase; wherein the saccharide is co
  • Dkt. No.: 64100-234947 wherein the saccharide is covalently linked to a nitrogen atom within a STT3 glycosylation fragment (e.g., N X 1 X 2 , wherein X 1 is any amino acid except proline and X 2 is S or T) using a STT3 OTase (e.g., SEQ ID NO: 408); wherein the saccharide is covalently linked to a nitrogen atom within an Archaeal AlgB glycosylation fragment (e.g., N X 1 X 2 , wherein X 1 is any amino acid except proline and X 2 is S or T) using an AlgB OTase (e.g., SEQ ID NO: 409); wherein the saccharide is covalently linked to an oxygen atom within PilA_Pa5196- associated pilin glycosylation fragment (e.g., SEQ ID NO: 426 or a variant thereof) using a TfpW glyco
  • a system comprising the first polypeptide and the second polypeptide of the composition of any one of Paragraphs 1 to 26; optionally, wherein the first polypeptide is a glycosylated bioconjugate; optionally, wherein the system comprises a multimeric, higher-order structure assembled of the second polypeptide; optionally, wherein the system comprises a saccharide and an N-linking oligosaccharyltransferase (N-OTase), an O-linking oligosaccharyltransferase (O-Otase), an N- linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C- mannosyltransferase (CMT).
  • N-OTase N-linking oligosaccharyltransferase
  • O-Otase O-linking oligosaccharyltransferase
  • NTT N- linking glyco
  • a kit comprising two or more components comprising the fusion protein, the first polypeptide, the second polypeptide, a saccharide, an N-linking oligosaccharyltransferase (N-Otase), an O-linking oligosaccharyltransferase (O-Otase), an N-linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C-mannosyltransferase (CMT), the bioconjugate, the multimeric, higher-order structure assembled from the second polypeptide, the isolated nucleic acid, the vector, and the host cell of any of the Paragraphs above. [0328] 50.
  • N-Otase N-linking oligosaccharyltransferase
  • O-Otase O-linking oligosaccharyltransferase
  • NTT N-linking glycosyl
  • a method of eliciting an immune response in a subject by administering to said subject an effective amount of any composition, complex, and/or conjugate vaccine of any of the Paragraphs above or a composition, complex, and/or conjugate vaccine of any of the Paragraphs above for use in eliciting an immune response in a subject.
  • glycosylation fragment comprises means for having a saccharide attached to the glycosylation fragment by a PglS OTase, a TfpM OTase, a PglL OTase, a PglB OTase, a TfpO/PilO OTase, a STT3 OTase, a AlgB OTase, a N-Linking Glycosyltransferase, and/or an O-Linking Glycosyltransferase.
  • PglS OTase a TfpM OTase
  • PglL OTase a PglB OTase
  • TfpO/PilO OTase a STT3 OTase
  • AlgB OTase a N-Linking Glycosyltransferase
  • O-Linking Glycosyltransferase [03
  • the fusions protein in anyone of the above Paragraphs comprising an amino acid linker, optionally, wherein the amino acid linker is selected from the group consisting of SGG, SEQ ID NO: 430, SEQ ID NO: 431, SEQ ID NO: 432, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 437, and SEQ ID NO: 438, optionally, wherein the amino acid linker in translationally fused immediately following a leader sequence, a polypeptide tag, a glycosylation fragment, a carrier protein, and or a polyhistidine tag, and/or optionally, wherein the amino acid linker in translationally fused immediately preceding a leader sequence, a polypeptide tag, a glycosylation fragment, a carrier protein, and or a polyhistidine tag.
  • a glycoconjugate comprising an oligo- or polysaccharide covalently linked to a fusion protein: wherein the fusion protein comprises a ComP protein (ComP) glycosylation fragment; wherein the ComP glycosylation fragment does not contain a cysteine residue corresponding to the conserved cysteine residue at position 71 of ComP110264 (SEQ ID NO: 201) and/or does not contain a cysteine residue corresponding to the conserved cysteine residue at position 93 of ComP 110264 (SEQ ID NO: 201); wherein the ComP glycosylation fragment is located internally within the fusion protein; and wherein the fusion protein is glycosylated with the oligo- or polysaccharide on the ComP glycosylation fragment at serine residue corresponding to the conserved serine residue at position 82 of ComP 110264 (SEQ ID NO: 201); optionally, wherein the glycoconjugate is immunogenic; optionally, wherein the ComP glycosylation fragment is solvent (or surface)
  • SEQ ID NO: 213 ComP ⁇ 28 4466
  • SEQ ID NO: 214 ComP ⁇ 28 SFC
  • SEQ ID NO: 215 ComP ⁇ 28 P5312
  • SEQ ID NO: 216 ComP ⁇ 29 ANT_H59
  • the ComP protein comprises SEQ ID NO: 209 (ComP ⁇ 28110264), SEQ ID NO: 210 (ComP ⁇ 28ADP1), SEQ ID NO: 211 (ComP ⁇ 28GFJ-2), SEQ ID NO: 212 (ComP ⁇ 28P50v1), SEQ ID NO: 213 (ComP ⁇ 28 4466 ), SEQ ID NO: 214 (ComP ⁇ 28 SFC ); SEQ ID NO: 215 (ComP ⁇ 28 P5312 ), or SEQ ID NO: 216 (ComP ⁇ 29ANT_H59).
  • X 9 is G, D, or no amino acid
  • X 12 is N, S, or A
  • X 13 is A, S, or K
  • X 15 is T, S, or K
  • X 18 is A, E, Q, or L
  • X 19 is T, S, or K
  • X 20 is A or S
  • X 21 is T, Q, A, or V; or a fragment of thereof of at least 5, 6, 7, 8, 9, 10, or 11 amino acids in length comprising the serine (S) residue at position 11 of SEQ ID NO: 217, optionally, wherein the fragment has at least 1, 2, 3, 4, 5, or 6 amino acid residues N-terminal to the serine (S) residue at position 11 of SEQ ID NO: 217 and/or wherein the fragment has at least 1, 2, 3, 4, 5, or 6 amino acid residues C- terminal to the serine (S) residue at position 11 of SEQ ID NO: 217; optionally, wherein the ComP glycosylation fragment can be glycosyl
  • the fusion protein comprises a carrier protein selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae Protein D, and a fragment or fragments thereof; optionally, wherein the Pseudomonas aeruginosa Exotoxin A (EPA) carrier protein comprises the amino acid sequence of SEQ ID NO: 218, or a fragment or fragments thereof; optionally, wherein the CRM 197 carrier protein comprises the amino acid sequence of SEQ ID NO: 224, or a fragment or fragments thereof.
  • EPA Pseudomonas aeruginosa Exotoxin A
  • CRM197 cholera toxin B subunit
  • tetanus toxin C fragment Haemophilus influenzae Protein D
  • the oligo- or polysaccharide is derived from a saccharide produced by bacteria from the genus Streptococcus; optionally, wherein the saccharide is a S. pneumoniae, S. agalactiae, or S.
  • oligo- or polysaccharide comprises glucose at its reducing end. 32080280 - 123 - Atty. Dkt. No.: 64100-234947 [0348] 16.
  • the ComP glycosylation fragment comprises or consists of an amino acid sequence of SEQ ID NO: 232- 363, or 364, or a variant thereof having one, two, or three amino acid substitutions, additions, and/or deletions, wherein the variant comprises the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201; optionally, wherein the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein; and optionally, wherein the ComP glycosylation fragment is not glycosylated when located at the N-terminal and/or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal and/or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein.
  • the ComP glycosylation fragment comprises or consists of an amino acid sequence of: iGTcc ⁇ 0-1 CTGVTQIASGASAATTNVASAQ (SEQ ID NO: 232); iGTcc ⁇ 1-0 TGVTQIASGASAATTNVASAQC (SEQ ID NO: 243); iGTcc ⁇ 1-1 TGVTQIASGASAATTNVASAQ (SEQ ID NO: 244); iGTcc ⁇ 1-2TGVTQIASGASAATTNVASA (SEQ ID NO: 245); iGTcc ⁇ 2-1 GVTQIASGASAATTNVASAQ (SEQ ID NO: 256); iGTcc ⁇ 2- 2 GVTQIASGASAATTNVASA (SEQ ID NO: 257); iGTcc ⁇ 2-3 GVTQIASGASAATTNVAS (SEQ ID NO: 258); iGTcc ⁇ 3-2 VTQIASGASAATTNVASA (SEQ ID NO: 269);
  • the ComP glycosylation fragment is not glycosylated when located at the N-terminal and/or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal and/or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein. [0352] 20.
  • a ComP glycosylation fragment comprising or consisting of an isolated fragment of a ComP protein, wherein the ComP glycosylation fragment does not contain a cysteine residue corresponding to the conserved cysteine residue at position 71 of ComP 110264 (SEQ ID NO: 201) and/or does not contain a cysteine residue corresponding to the conserved cysteine residue at position 93 of ComP110264 (SEQ ID NO: 201); and wherein the ComP glycosylation fragment comprises the serine residue corresponding to the conserved serine residue at position 82 of ComP 110264 (SEQ ID NO: 201); optionally, wherein the ComP glycosylation fragment is immunogenic. [0360] 28.
  • ComP glycosylation fragment of Paragraph 33 wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of: iGTcc ⁇ 0-1 CTGVTQIASGASAATTNVASAQ (SEQ ID NO: 232); iGTcc ⁇ 1-0 TGVTQIASGASAATTNVASAQC (SEQ ID NO: 243); iGTcc ⁇ 1-1 TGVTQIASGASAATTNVASAQ (SEQ ID NO: 244); iGTcc ⁇ 1-2 TGVTQIASGASAATTNVASA (SEQ ID NO: 245); iGTcc ⁇ 2-1 GVTQIASGASAATTNVASAQ (SEQ ID NO: 256); iGTcc ⁇ 2-2 GVTQIASGASAATTNVASA (SEQ ID NO: 257); iGTcc ⁇ 2-3 GVTQIASGASAATTNVAS (SEQ ID NO: 258); iGTcc ⁇ 3-2 VTQIASGASAATTNVASA (SEQ ID NO: 232); i
  • the ComP glycosylation fragment of Paragraph 33 wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of SEQ ID NO: 232- 363, or 364, optionally, wherein the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein; and optionally, wherein the ComP glycosylation fragment is not glycosylated when located at the N-terminal or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein. [0368] 36.
  • the ComP glycosylation fragment of Paragraph 35 wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of: iGTcc ⁇ 0-1 CTGVTQIASGASAATTNVASAQ (SEQ ID NO: 232); iGTcc ⁇ 1-0 TGVTQIASGASAATTNVASAQC (SEQ ID NO: 243); iGTcc ⁇ 1-1 TGVTQIASGASAATTNVASAQ (SEQ ID NO: 244); iGTcc ⁇ 1-2 TGVTQIASGASAATTNVASA (SEQ ID NO: 245); iGTcc ⁇ 2-1 GVTQIASGASAATTNVASAQ (SEQ ID NO: 256); iGTcc ⁇ 2-2 GVTQIASGASAATTNVASA (SEQ ID NO: 257); iGTcc ⁇ 2-3 GVTQIASGASAATTNVAS (SEQ ID NO: 258); iGTcc ⁇ 3-2 VTQIASGASAATTNVASA (SEQ ID NO: 269); i
  • a fusion protein comprising the ComP glycosylation fragment of any of Paragraphs 27 to 36, wherein the ComP glycosylation fragment is located internally within the fusion protein; optionally, wherein the fusion protein is glycosylated by an oligo- or polysaccharide at a serine residue on the glycosylation fragment corresponding to the serine ComP glycosylation fragment residue at position 82 of SEQ ID NO: 201 (ComP110264).
  • the fusion protein of Paragraph 37 wherein the oligo- or polysaccharide is derived from a saccharide produced by the bacteria from the genus Klebsiella; optionally, wherein the saccharide is a K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca capsular polysaccharide; optionally, wherein the saccharide is a K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca O-antigen polysaccharide. [0372] 40. The fusion protein of any one of Paragraphs 37 to 39, wherein oligo- or polysaccharide comprises glucose at its reducing end. [0373] 41.
  • the fusion protein of any one of Paragraphs 37 to 41 wherein the fusion protein comprises a carrier protein selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM 197 , cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae Protein D, and a fragment or fragments thereof; optionally, wherein the Pseudomonas aeruginosa Exotoxin A (EPA) carrier protein comprises the amino acid sequence of SEQ ID NO: 218, or a fragment or fragments thereof; optionally, wherein the CRM 197 carrier protein comprises the amino acid sequence of SEQ ID NO: 224, or a fragment or fragments thereof.
  • EPA Pseudomonas aeruginosa Exotoxin A
  • CRM 197 carrier protein comprises the amino acid sequence of SEQ ID NO: 224, or a fragment or fragments thereof.
  • the fusion protein of Paragraph 42 wherein: (i) the ComP glycosylation fragment is inserted between Asn481 and Gly482 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 225); (ii) the ComP glycosylation fragment is inserted between Asp392 and Gly393 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 226); (iii) the ComP glycosylation fragment is inserted between Glu142 and Gly143 relative to the PDB entity 4AE0 of CRM 197 (SEQ ID NO: 227); (iv) the ComP glycosylation fragment is inserted between Asp129 and Gly130 relative to the PDB entity 4AE0 of CRM 197 (SEQ ID NO: 228); or (v) the ComP glycosylation fragment is inserted between Asn69 and Glu70 relative to the PDB entity 4AE0 of CRM 197 (SEQ ID NO: 229).
  • [0377] 45 The fusion protein of any one of Paragraph s 37 to 44, 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 fragments; optionally, wherein the fusion protein does not comprise more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five ComP glycosylation fragments. [0378] 46. The fusion protein of any one of Paragraphs 37 to 45, wherein the ComP glycosylation fragments are identical. [0379] 47.
  • a method of in vivo conjugation of an oligo- or polysaccharide to an acceptor polypeptide comprising covalently linking the oligo- or polysaccharide to the acceptor polypeptide with a PglS oligosaccharyltransferase (OTase), wherein the acceptor polypeptide comprises the ComP glycosylation fragment of any one of Paragraphs 27 to 36.
  • OTase PglS oligosaccharyltransferase
  • PglS OTase is PglS 110264 (SEQ ID NO: 365), PglSADP1 (SEQ ID NO: 366), PglSGFJ-2 (SEQ ID NO: 367), PglS50v1 (SEQ ID NO: 368), PglS4466 (SEQ ID NO: 369), PglSSFC (SEQ ID NO: 370), PglSP5312 (SEQ ID NO: 371), or PglS ANT_H59 (SEQ ID NO: 372).
  • a host cell comprising (a) a genetic cluster encoding for the proteins required to synthesize an oligo- or polysaccharide; (b) a PglS OTase; and (3) an acceptor polypeptide comprising the ComP glycosylation fragment of any one of Paragraphs 27 to 36. [0389] 57. The host cell of Paragraph 56, wherein the acceptor polypeptide is a fusion protein. [0390] 58. The host cell of Paragraph 56 or 57, wherein the host cell comprises a nucleic acid encoding the PglS OTase. [0391] 59.
  • 62. A host cell comprising the isolated nucleic acid of Paragraph 60 or 61.
  • a composition comprising the conjugate vaccine of any one of Paragraphs 21 to 26 or the fusion protein of any one of Paragraphs 37 to 47, and an adjuvant.
  • a method of inducing a host immune response against a bacterial pathogen comprising administering to a subject in need of the immune response an effective amount of the conjugate vaccine of any one of Paragraphs 21 to 26, the fusion protein of any one of Paragraphs 37 to 47, or the composition of Paragraph 63.
  • the method of Paragraph 64 wherein the immune response is selected from the group consisting of an innate response, an adaptive response, a humoral response, an antibody response, cell mediated response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system cross-talk, and a combination of two or more of said immune responses.
  • 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 said immune responses.
  • a method of preventing or treating a bacterial disease and/or infection in a subject comprising administering to a subject in need thereof the conjugate vaccine of any one of Paragraphs 21 to 26, the fusion protein of any one of Paragraphs 37 to 47, or the composition of Paragraph 63.
  • the method of Paragraph 68, wherein the infection is a localized or systemic infection of skin, soft tissue, blood, or an organ, or is auto-immune in nature.
  • 70. The method of Paragraph 69, wherein the disease is pneumonia.
  • a method of producing a pneumococcal conjugate vaccine against pneumococcal infection comprising:(a) isolating the glycoconjugate of any one of Paragraphs 1 to 26 or a glycosylated fusion protein of any one of Paragraphs 37 to 47; and (b) combining the isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant. 32080280 - 133 - Atty. Dkt. No.: 64100-234947 [0407] 75.
  • glycoconjugate, glycosylated fusion protein, or conjugate vaccine of any of the above paragraphs for use in inducing a host immune response against a bacterial pathogen and/or preventing or treating a bacterial disease and/or infection in a subject.
  • ***** [0408] In addition to all of the above, certain embodiments in the numbered paragraphs above can be further defined in any of the following numbered paragraphs: [0409] 1.
  • a glycoconjugate comprising an oligo- or polysaccharide covalently linked to an acceptor protein, wherein the acceptor protein comprises or consists of a TfpM-associated pilin-like protein or glycosylation fragment thereof and the oligo- or polysaccharide is covalently linked to the TfpM-associated pilin-like protein or glycosylation fragment thereof, and wherein the TfpM-associated pilin-like protein or glycosylation fragment thereof comprises a C-terminus serine or threonine residue and the oligo- or polysaccharide is covalently linked to the C-terminus serine or threonine; optionally, wherein the TfpM-associated pilin-like protein glycosylation fragment comprises at least the last three amino acids from the pilin-like protein C-terminal end; optionally, wherein the acceptor protein is a fusion protein comprising the TfpM- associated pilin-like protein or glycosylation fragment thereof translationally
  • TfpM-associated pilin-like protein glycosylation fragment comprises a C- terminus serine or threonine residue.
  • TfpM-associated pilin-like protein or glycosylation fragment thereof is: (i) Pil Mo (SEQ ID NO: 57), (ii) Pil Mo lacking amino acids corresponding to residues 1–28 (PilMo ⁇ 28, SEQ ID NO: 58), or (iii) a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, wherein the TfpM-associated pilin-like protein comprises a C-terminus serine or threonine residue, optionally, wherein the C-terminus threonine is substituted with serine; (b) wherein the TfpM-associated pilin-like protein comprises a C-terminus serine or threonine residue, optional
  • the pilin-like-protein glycosylation fragment consists of Pil 20 (SEQ ID NO: 60), Pil 19 (SEQ ID NO: 133), Pil 18 (SEQ ID NO: 134), Pil 17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil 8 (SEQ ID NO: 141), Pil 7 (SEQ ID NO: 113), Pil 6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining
  • the acceptor protein is a fusion protein comprising a heterologous carrier protein and the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and a fragment of any thereof.
  • EPA Pseudomonas aeruginosa Exotoxin A
  • CRM197 cholera toxin B subunit
  • tetanus toxin C fragment and a fragment of any thereof.
  • OTase TfpM oligosaccharyltransferase
  • 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 additional glycosylation sequences; optionally, wherein the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five additional glycosylation sequences; optionally, wherein the additional glycosylation sequences are identical; optionally, wherein the additional glycosylation sequences differ from each other; and/or optionally, wherein at least three, at least four, or at least five of the additional glycosylation sequences all differ from each other; and/or optionally, wherein none of the additional glycosylation sequences are the same.
  • agalactiae capsular polysaccharide is Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX; (ii) wherein the oligo- or polysaccharide is produced by bacteria of the genus Klebsiella and the polysaccharide is a capsular polysaccharide or O-antigen polysaccharide, optionally wherein the bacteria is K. pneumoniae; or (iii) wherein the oligo- or polysaccharide is produced by bacteria of the genus Salmonella and the polysaccharide is O-antigen polysaccharide; optionally wherein the bacteria is S. enterica and the S. enterica polysaccharide is a Group B O-antigen.
  • the bioconjugate is a conjugate vaccine that induces an immune response when administered to a subject; optionally, wherein the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response; optionally, wherein the antibody response is an IgG or IgM response; optionally, wherein the antibody response is an IgG response; optionally an IgG1 response; and/or optionally, wherein the conjugate vaccine generates immunological memory in a subject administered the vaccine. [0419] 11.
  • a pilin-like-protein glycosylation fragment comprising or consisting of an isolated fragment of a TfpM-associated pilin-like protein, (a) wherein the TfpM-associated pilin-like-protein or glycosylation fragment thereof is: (i) PilMo (SEQ ID NO: 57), (ii) PilMo lacking amino acids corresponding to residues 1–28 32080280 - 138 - Atty. Dkt.
  • TfpM-associated pilin-like protein contains a C-terminus serine or threonine residue, optionally, wherein the C-terminus threonine is substituted with serine, (b) wherein the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), Pil CIP
  • No.: 64100-234947 has a length of from 20 to 139 amino acids in length, has a length of from 116 to 139 amino acids in length, has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length, wherein the TfpM-associated pilin-like protein glycosylation fragment comprises a C- terminus serine or threonine residue. [0421] 13.
  • a fusion protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof translationally fused to a heterologous carrier protein, wherein the TfpM-associated pilin-like protein or glycosylation fragment comprises a C-terminus serine or threonine residue, wherein the TfpM-associated pilin-like protein or glycosylation fragment is the C- terminus-most sequence of the fusion protein, and wherein the fusion protein comprises a C-terminus serine or threonine residue; optionally, wherein the fusion protein is glycosylated by an olio- or polysaccharide covalently linked to the C-terminus serine or threonine; optionally, wherein the fusion protein is glycosylated by an oligo- or polysaccharide comprising glucose at its reducing end covalently linked to the C-terminus serine or threonine.
  • the fusion protein of Paragraph 13 wherein the pilin-like-protein glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, has a length of from 116 to 139 amino acids in length, has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length, wherein the TfpM-associated pilin-like protein glycosylation fragment comprises a C- terminus serine or threonine residue.
  • TfpM-associated pilin-like-protein or glycosylation fragment thereof is: (i) Pil Mo (SEQ ID NO: 57), (ii) Pil Mo lacking amino acids corresponding to residues 1–28 (Pil Mo ⁇ 28, SEQ ID NO: 58), or (ii) a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or 32080280 - 141 - Atty. Dkt.
  • TfpM-associated pilin-like protein contains a C-terminus serine or threonine residue, optionally, wherein the C-terminus threonine is substituted with serine; (b) wherein the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), Pil AI7 (SEQ ID NO: 85), Pil VE-C3 (SEQ ID NO: 86), Pil YH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 58, wherein the TfpM-associated pilin-like
  • the glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end, further optionally, wherein the pilin- like-protein glycosylation fragment consists of Pil20[A] (SEQ ID NO: 166), Pil19[A] (SEQ ID NO: 167), Pil18[A] (SEQ ID NO: 168), Pil17[A] (SEQ ID NO: 169), Pil16[A] (SEQ ID NO: 170), Pil 15[A] (SEQ ID NO: 171), Pil 14[A] (SEQ ID NO: 172), Pil 13[A] (SEQ ID NO: 173), Pil 12[A] (SEQ ID NO: 174), Pil11[A] (SEQ ID NO: 175), Pil10[A] (SEQ ID NO: 176), Pil9[A] (SEQ ID NO: 177), Pil8[A] (SEQ ID NO: 178
  • the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and a fragment of any thereof. 32080280 - 143 - Atty. Dkt. No.: 64100-234947 [0425] 17.
  • OTase TfpM oligosaccharyltransferase
  • the fusion protein of Paragraph 17 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 additional glycosylation sequence; optionally, wherein the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five additional glycosylation sequence; optionally, wherein the additional glycosylation sequence are identical; optionally, wherein the additional glycosylation sequence differ from each other; and/or optionally, wherein at least three, at least four, or at least five of the additional glycosylation sequences all differ from each other; and/or optionally, wherein none of the additional glycosylation sequence are the same.
  • oligo- or polysaccharide covalently linked to the pilin-like protein or glycosylation fragment thereof has a size of at least three repeating units of oligo- or polysaccharide structure and/or has a size of at least ten monosaccharides.
  • agalactiae capsular polysaccharide is Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX; 32080280 - 144 - Atty. Dkt. No.: 64100-234947 (ii) wherein the oligo- or polysaccharide is produced by bacteria of the genus Klebsiella and the polysaccharide is a capsular polysaccharide or O-antigen polysaccharide, optionally wherein the bacteria is K.
  • oligo- or polysaccharide is produced by bacteria of the genus Salmonella and the polysaccharide is O-antigen polysaccharide; optionally wherein the bacteria is S. enterica and the S. enterica polysaccharide is a Group B O-antigen.
  • 21. The fusion protein of any one of Paragraphs 13 to 20, wherein the glycosylated fusion protein is produced in vivo; optionally, in a bacterial cell; optionally, in Escherichia coli; optionally, in a bacterium from the genus Klebsiella; and/or optionally, wherein the bacterial species is K. pneumoniae, K. varricola, K.
  • fusion protein of any one of Paragraphs 13 to 21, wherein when the fusion protein is a vaccine that induces an immune response when administered to a subject; optionally, wherein the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response; optionally, wherein the antibody response is an IgG or IgM response; optionally, wherein the antibody response is an IgG response; optionally an IgG1 response; and/or optionally, wherein the fusion protein generates immunological memory in a subject administered the vaccine.
  • the fusion protein generates immunological memory in a subject administered the vaccine.
  • a method of producing a glycoconjugate comprising covalently linking an oligo- or polysaccharide to an acceptor protein comprising or consisting of a TfpM- associated pilin-like protein or glycosylation fragment thereof using a TfpM oligosaccharyltransferase (OTase); wherein the pilin-like protein or glycosylation fragment comprises a C-terminus serine or threonine residue, the acceptor protein comprises a C-terminus serine or threonine residue, and the oligo- or polysaccharide is covalently linked to the C-terminus serine or threonine residue of the acceptor protein; optionally, wherein the oligo- or polysaccharide comprises a glucose at its reducing end; 32080280 - 145 - Atty.
  • Dkt. No.: 64100-234947 optionally, wherein the acceptor protein is a fusion protein of any one of Paragraphs 13 to 22; optionally, wherein the method is a method of in vivo conjugation of an oligo- or polysaccharide to an acceptor protein; and/or optionally, wherein the glycoconjugate is immunogenic. [0432] 24.
  • TfpM OTase comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to TfpMMo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpM TUM15069 (SEQ ID NO: 65), TfpM AI7 (SEQ ID NO: 66), TfpM VE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpMAI40 (SEQ ID NO: 70), TfpMF78 (SEQ ID NO: 71), TfpM S71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74
  • TfpM-associated pilin-like-protein or glycosylation fragment thereof is: (i) Pil Mo (SEQ ID NO: 57), (ii) Pil Mo lacking amino acids corresponding to residues 1–28 (PilMo ⁇ 28, SEQ ID NO: 58), or (ii) a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, wherein the TfpM-associated pilin-like protein contains a C-terminus serine or threonine residue, optionally, wherein the C-terminus threonine is substituted with serine; (b) wherein the TfpM-associated pilin-like-protein
  • the acceptor protein is a fusion protein comprising an additional glycosylation sequence of an OTase other than TfpM oligosaccharyltransferase (OTase) in addition to the TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus
  • the method further comprises covalently linking an oligo- or polysaccharide to the additional glycosylation sequence with an OTase other than TfpM OTase
  • the acceptor protein is a fusion protein comprising a ComP glycosylation fragment
  • the method further comprises covalently linking an oligo- or polysaccharide to the ComP glycosylation fragment using a PglS OTase, optionally, wherein the ComP glycosylation fragment is an internal glycosylation fragment of ComP, further optionally, wherein the ComP glycosylation fragment comprises or consists of C
  • the method of Paragraph 29 or 30, comprising culturing a host cell that comprises: (a) a genetic cluster encoding for the proteins required to synthesize the oligo- or polysaccharide; (b) a TfpM OTase; and (3) the acceptor protein.
  • 32 The method of any one of Paragraphs 23 to 31, wherein the method produces a conjugate vaccine.
  • 33 A host cell comprising (a) a genetic cluster encoding for the proteins required to synthesize an oligo- or polysaccharide; (b) a TfpM OTase; and (3) an acceptor protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof.
  • a composition comprising the conjugate vaccine of Paragraph 10 or the fusion protein of Paragraph 22, and an adjuvant and/or carrier.
  • 40. A method of inducing a host immune response against a bacterial pathogen, the method comprising administering to a subject in need of the immune response an effective amount of the conjugate vaccine of Paragraph 10, the fusion protein of Paragraph 22, or the composition of Paragraph 39.
  • the immune response is an antibody response; wherein the immune response is selected from the group consisting of an innate response, an adaptive response, a humoral response, an antibody response, cell mediated response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system cross-talk, and a combination of two or more of said immune responses; and/or 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 said immune responses.
  • 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 said immune responses.
  • a method of preventing or treating a bacterial disease and/or infection in a subject comprising administering to a subject in need thereof the conjugate vaccine of Paragraph 10, the fusion protein of Paragraph 22, or the composition of Paragraph 39; 32080280 - 150 - Atty. Dkt. No.: 64100-234947 optionally, wherein the subject is a human.
  • a method of producing a pneumococcal conjugate vaccine against pneumococcal infection comprising: (a) isolating the glycoconjugate of any one of Paragraphs 1 to 10 or a glycosylated fusion protein of any one of Paragraphs 13 to 22; and (b) combining the isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant and/or carrier.
  • glycoconjugate, glycosylated fusion protein, or conjugate vaccine of any of the above Paragraphs for use in inducing a host immune response against a bacterial pathogen and/or preventing or treating a bacterial disease and/or infection in a subject.
  • a recombinant nucleic acid construct comprising a nucleotide sequence encoding a TfpM oligosaccharyltransferase (OTase) operably linked to at least one heterologous transcriptional regulatory sequence.
  • OTase oligosaccharyltransferase
  • TfpM OTase comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to TfpMMo (SEQ ID NO: 56), TfpMDSM16617 (SEQ ID NO: 63), TfpMZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpM YH01026 (SEQ ID NO: 68), TfpM CIP102143 (SEQ ID NO: 69), TfpM AI40 (SEQ ID NO: 70), TfpMF78 (SEQ ID NO: 71), TfpMS71 (SEQ ID NO: 72), TfpMANC4282 (SEQ ID NO: 73), TfpMCIP102159 (SEQ ID NO: ).
  • TfpM OTase is TfpM Mo (SEQ ID NO: 56), TfpM DSM16617 (SEQ ID NO: 63), TfpM ZZC3 (SEQ ID NO: 64), TfpM TUM15069 (SEQ ID NO: 65), TfpM AI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpMYH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpMAI40 (SEQ ID NO: 70), TfpMF78 (SEQ ID NO: 71), TfpMS71 (SEQ ID NO: 72), TfpM ANC4282 (SEQ ID NO: 73), TfpM CIP102159 (SEQ ID NO: 74), TfpM junii-65 (SEQ ID NO: 75), TfpMY
  • TfpM-associated pilin-like-protein or glycosylation fragment thereof is: (i) PilMo (SEQ ID NO: 57), (ii) PilMo lacking amino acids corresponding to residues 1–28 (PilMo ⁇ 28, SEQ ID NO: 58), or (ii) a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, wherein the TfpM-associated pilin-like protein contains a C-terminus serine or threonine residue, optionally, wherein the C-terminus threonine is substituted with serine; (b) wherein the TfpM-associated pilin
  • Dkt. No.: 64100-234947 glycosylation fragment comprises at least the last four amino acids from the pilin C-terminal end.
  • 53. The recombinant construct of any one of Paragraphs 50 to 52, wherein the fusion protein is a fusion protein of any one of Paragraphs 13 to 22.
  • the recombinant construct of any one of Paragraphs 50 to 53 further comprising a nucleotide sequence encoding a PglS OTase operably linked to the TpfM OTase; optionally, wherein the coding sequence of the PglS OTase is within 10, 20, 30, 40, 50, 75, or 100 nucleotides of the sequence encoding the TfpM OTase.
  • 56 A vector comprising the recombinant nucleic acid construct of any one of Paragraphs 47 to 54.
  • a host cell comprising the recombinant nucleic acid construct of any one of Paragraphs 47 to 54, or the vector of Paragraph 55; optionally, wherein the host cell is a bacterial cell; and further: optionally, wherein the host cell is Escherichia coli; optionally, wherein the host cell is from the genus Klebsiella; or optionally, wherein the host cell is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0465] 57.
  • a method for producing a TfpM OTase comprising culturing the host cell of Paragraph 56, wherein said vector of Paragraph 54 is an expression vector, and recovering the TfpM OTase.
  • a glycoconjugate comprising an oligo- or polysaccharide covalently linked to an acceptor protein, wherein the acceptor protein comprises means for being covalently linked to the oligo- or polysaccharide by a TfpM OTase.
  • a fusion protein comprising means for being covalently linked to an oligo- or polysaccharide by a TfpM OTase, wherein said means is translationally fused to a heterologous carrier protein, wherein the means is located at the C-terminus-most sequence of the fusion protein.
  • 61. The fusion protein of Paragraph 60, wherein the fusion protein is the fusion protein of any one of Paragraphs 13 to 22. 32080280 - 155 - Atty. Dkt. No.: 64100-234947
  • 62. The glycoconjugate of Paragraph 58 or 59 or the fusion protein of Paragraph 60 or 61, for use in any of the methods above.
  • This “bare” version has a non-native N-terminal DsbA secretion signal sequence and has a deletion of residue E553 which inactivates the toxin)
  • Type IV pilin is glycosylated in Pseudomonas syringae pv. tabaci 6605 and is required for surface motility and virulence. Molecular Plant Pathology 13, 764-774.
  • Acinetobacter strains carry two functional oligosaccharyltransferases, one devoted exclusively to type IV pilin, and the other one dedicated to O-glycosylation of multiple proteins.
  • Burkholderia PglL enzymes are Serine preferring oligosaccharyltransferases which target conserved proteins across the Burkholderia genus. Communications Biology 4, 1045. Porstendörfer, D., Gohl, O., Mayer, F., and Averhoff, B. (2000) ComP, a Pilin-Like Protein Essential for Natural Competence in Acinetobacter sp.

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Abstract

This disclosure provides descriptions of compositions and methods used to produce a glycoconjugate polypeptide using enzymes that form glycosidic linkages that subsequently form an isopeptide bond with a second polypeptide containing a polypeptide tag and uses thereof.

Description

COMPOSITIONS AND METHODS FOR PRODUCING GLYCOCONJUGATE POLYPEPTIDES HAVING ISOPEPTIDE BONDS WITH A SECOND POLYPEPTIDE PARTNER AND USES THEREOF Inventors: Cory James Knoot Christian Michael Harding Lloyd Sherwood Robinson CROSS REFERENCE TO RELATED APPLICATIONS [0001] This PCT application claims the benefit of U.S. Provisional Appl. No.63/448,382, filed on February 27, 2023, U.S. Provisional Appl. No.63/448,386, filed on February 27, 2023, and U.S. Provisional Appl. No. 63/448,408, filed on February 27, 2023, each of which is incorporated herein in their entireties. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY [0002] A sequence listing in the file named “64100_234947_SL.xml”, which is 441,619 bytes (measured in MS-Windows®), contains 440 sequences, and was created on February 26, 2024, is provided herewith via the USPTO's Patent Center, and is incorporated herein by reference in its entirety. BACKGROUND [0003] Glycan-protein or glycoprotein conjugate vaccines are effective therapeutics against a variety of bacterial pathogens and are, in essence, a covalent linkage of bacterial surface oligo- or polysaccharides to a carrier protein. These vaccines can induce protective immune responses against the O antigen or capsule present on the surface of gram-positive or gram-negative pathogens. Unlike pure polysaccharide vaccines, glycoprotein conjugate vaccines can stimulate robust immune memory by inducing T-cell recruitment, memory cell formation, and B-cell IgM-to-IgG antibody class switching leading to immune memory establishment (Rappuoli, et al. (2019) PNAS, 116 (1) 14-16; Avci, F., et al. (2011) Nat Med 17, 1602–1609). Glycoprotein conjugate vaccines can be generated via a number of different methods. Historically, those in routine medical use have been synthesized conjugates made by chemically cross-linking purified oligo- or polysaccharides to amino acid side-chains on purified proteins using different linker molecules and chemistries (Berti, F. and Adamo, R. (2018) Chem. Soc. Rev., 2018, 47, 9015-9025). Bioconjugation is an alternative method to 32080280 - 1 - Atty. Dkt. No.: 64100-234947 chemical conjugation and relies on an oligosaccharyltransferase (OTase) enzyme that catalyzes the covalent attachment of lipid-linked oligo- or polysaccharides to specific amino acid residues on substrate proteins (Harding, C. and Feldman, M. (2019) Glycobiology, Volume 29, Issue 7, July 2019, Pages 519–529; Feldman, M. (2005) PNAS, 102 (8) 3016-3021). These bioconjugates are often produced in engineered strains of E. coli and conjugation occurs in the bacterial periplasm. The glycan substrate of the OTase is linked to a membrane-bound lipid carrier such as undecaprenyl pyrophosphate (UNDPP). O-linking OTases catalyze the transfer of UNDPP-linked oligo- or polysaccharides to serine or threonine sidechain hydroxyls in conserved protein motifs termed sequons (Knoot, C., et al. (2021) Glycobiology, Volume 31, Issue 9, September 2021, Pages 1192–1203; Knoot, C., et al. (2023) Glycobiology, Volume 33, Issue 1, January 2023, Pages 57–74). By chromosomally and/or episomally co-expressing desired glycan biosynthetic gene clusters, carrier protein(s), and OTase(s) in a bacterial host, bioconjugate vaccines can be generated in a ‘one-pot’ biological reaction followed by downstream purification (Harding, C. and Feldman, M. (2019) Glycobiology, Volume 29, Issue 7, July 2019, Pages 519–529). [0004] Several bioconjugate vaccines are currently in clinical trials and each is composed of a bacterial glycan linked to a periplasmic protein, primarily Pseudomonas aeruginosa exotoxin A (EPA), Haemophilus Protein D, or CRM197 (a modified diphtheria toxin) (Sorieul, C., et al. (2023) Expert Review of Vaccines, 22:1, 1055-1078). An alternative to using such carrier proteins are protein nanoparticles (NPs) or virus-like particles (VLPs): symmetric, self- assembling protein ‘cages’ that are either derivatives of natural virus capsids or rationally engineered protein assemblies (Nguyen, B. and Tolia, N. (2021) npj Vaccines 6, 70; Bruun, T., et al. (2018) ACS Nano 2018, 12, 9, 8855–8866; Cohen, et al. (2021) PLoS ONE 16(3): e0247963). Expression of NP/VLP monomer proteins in the bacterial cytoplasm results in the spontaneous assembly of megadalton-sized particles that can be purified from cell biomass (Cohen, et al. (2021) PLoS ONE 16(3): e0247963). NP/VLP-based therapeutics have been shown to improve immune response in part through increased antibody avidity arising from the larger immunogen particle size (Nguyen, B. and Tolia, N. (2021) npj Vaccines 6, 70). Two examples of VLPs/NPs are AP205 and mi3.AP205, is derived from the CP3 coat protein of the RNA bacteriophage AP205 (Brune, K., et al. (2016) Sci Rep 6, 19234). AP205 VLPs assemble into a 120-mer with a diameter of roughly 20 nm (Cohen, et al. (2021) PLoS ONE 16(3): e0247963). mi3 is a porous dodecahedral 60-mer derived from a computationally designed NP 32080280 - 2 - Atty. Dkt. No.: 64100-234947 with a diameter of 20 – 30 nm (Bruun, T., et al. (2018) ACS Nano 2018, 12, 9, 8855–8866) (Hsia, Y., et al. (2016) Nature volume 535, pages 136–139). [0005] The SpyTag/SpyCatcher system was derived from an immunoglobulin-like collagen adhesin domain (CnaB2) from the fibronectin binding protein, FbaB2, of Streptococcus pyogenes (Zakeri, B. et al. (2012)). The CnaB2 domain naturally forms an intra- protein isopeptide bond between lysine at position 31 and aspartic acid at position 117; specifically, the unprotonated amine of Lys31 acts as a nucleophile attacking the carbonyl carbon of Asp117, which is catalyzed by glutamic acid located at position 77 (Zakeri, B. et al. (2012)). This isopeptide reaction happens spontaneously and seems to be a feature of some members of the prealbumin-like fold domain bacterial proteins to which CnaB2 belongs. To adapt this into a tool for creating covalent fusions between two, separate polypeptides, the CnaB2 domain was split, separating CnaB2 into (1) a peptide containing the C-terminal β- strand that contains the reactive Asp117 defined as the SpyTag and (2) a protein binding partner derived from the remaining CnaB2 polypeptide defined as the SpyCatcher (Zakeri, B. et al. (2012)). The system was dubbed SpyTag and SpyCatcher to denote the bacterial source (S. pyogenes) of the CnaB2 fragments. Later forms of the SpyTag and SpyCatcher system, designated SpyTag003 and SpyCatcher003 were created through a phage display and subsequent rational engineering approach resulting in reaction rate of 5.5 x 105 M-1 s-1. SpyTag003/SpyCatcher003 reacts approximately 400-fold faster than the original SpyTag/SpyCatcher system (Keeble, A. H. et al. (2019)). The SpyTag/SpyCatcher system(s) have been broadly applied to enable covalently attachment of two, separate polypeptides, one containing a SpyTag and the other containing the SpyCatcher, through the formation of the isopeptide bond. The SpyTag/SpyCatcher system has been applied across a range of biological applications all seeking to covalently attach a polypeptide of interest containing a SpyTag to a different protein or material containing a SpyCatcher, including but not limited, to anchor polypeptides to the surface different solid organic and inorganic materials, to attach polypeptides to different multimerization architectures like nanoparticles, virus-like particles, or Adenoviral vectors, as well as to directly attach polypeptides to the surface of intact cells (Keeble, A. H. & Howarth, M. (2020); Brune, K. D. et al. (2016); Bruun, T. U. J., Andersson, A. C., Draper, S. J. & Howarth, M. (2018)). [0006] There remains a need to create new and more effective bioconjugate vaccines including flexible methods of generating diverse immunogenic compositions. 32080280 - 3 - Atty. Dkt. No.: 64100-234947 SUMMARY [0007] Provided for herein is a fusion protein comprising: (i) a glycosylation fragment and (ii) a first polypeptide tag, wherein the first polypeptide tag can spontaneously form an isopeptide bond with a second polypeptide tag binding partner. In certain embodiments, the fusion protein is a glycoconjugate comprising a saccharide covalently attached to the fusion protein via the glycosylation fragment, and optionally is immunogenic. Representative examples of the first polypeptide tag include a SpyTag (SEQ ID NO: 416), SpyTag002 (SEQ ID NO: 417), SpyTag003 (SEQ ID NO: 418), or a DogTag (SEQ ID NO: 419). [0008] In certain embodiments, the glycosylation fragment is a ComP glycosylation fragment or a variant thereof as described herein. [0009] In certain embodiments, the glycosylation fragment is a TfpM-associated pilin glycosylation fragment or a variant thereof as described herein. [0010] In certain embodiment, the glycosylation fragment is a PilE glycosylation fragment or a variant thereof as described herein. [0011] In certain embodiments, the glycosylation fragment is a PglB glycosylation fragment or a variant thereof as described herein. [0012] In certain embodiments, the glycosylation fragment is a PilA glycosylation fragment or a variant thereof as described herein. [0013] In certain embodiments, the glycosylation fragment is a STT3 glycosylation fragment or a variant thereof as described herein. [0014] In certain embodiments, the glycosylation fragment is a N-linking glycosyltransferase glycosylation fragment. [0015] In certain embodiments, the glycosylation fragment is an O-linking glycosyltransferase glycosylation fragment. [0016] In certain embodiments, the glycosylation fragment is a PilA_Pa5196-associated pilin glycosylation fragment or a variant thereof as described herein. [0017] In certain embodiments, the fusion protein comprises a carrier protein, optionally, wherein the carrier protein is selected from the group consisting of Escherichia coli maltose binding protein, Pseudomonas aeruginosa Exotoxin A (EPA), Pseudomonas aeruginosa PcrV, CRM197, Haemophilus influenzae Protein D, cholera toxin B subunit, or tetanus toxin, and a fragment of any thereof. [0018] Also provided for herein is a composition comprising a polypeptide pair that comprises a first polypeptide and a second polypeptide, wherein the first polypeptide is a fusion 32080280 - 4 - Atty. Dkt. No.: 64100-234947 protein of this disclosure, wherein the second polypeptide comprises a second polypeptide tag binding partner to the first polypeptide tag of the first polypeptide, and wherein the first polypeptide is attached to the second polypeptide via an isopeptide bond between the first polypeptide tag and the second polypeptide tag. In certain embodiments, the second polypeptide comprises a monomeric polypeptide that can spontaneously multimerize/self- assemble into a higher-order, multimeric structure; and optionally, said higher-order, multimeric structure is an icosahedron or dodecahedron particle (e.g,. resembling nanocages), virus-like particle, or Adenoviral vector. [0019] In certain embodiments, the second polypeptide tag is a SpyCatcher (SEQ ID NO: 420), SpyCatcher002 (SEQ ID NO: 421), SpyCatcher003 (SEQ ID NO: 422), or a DogCatcher (SEQ ID NO: 423). [0020] In certain embodiments, the first polypeptide is a bioconjugate comprising a saccharide covalently attached to the glycosylation fragment of the first polypeptide; optionally, wherein said composition is immunogenic. [0021] Also provided for is a complex comprising two or more of the polypeptide pairs of this disclosure. In certain embodiments, the complex is a self-assembled, multimeric higher- order structure. In certain embodiments, the self-assembled, multimeric higher-order structure is an icosahedron or dodecahedron particle (e.g,. resembling nanocages), virus-like particle, or Adenoviral vector. [0022] Also provided for is a method of making the polypeptide pair of this disclosure, the method comprising contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag binding partner. [0023] Also provided for is a method of making the complex of this disclosure, the method comprising: (i) forming a self-assembled, multimeric higher-order structure of the second polypeptide and then contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag; or (ii) contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag and then forming of a self-assembled, multimeric higher-order structure of the second polypeptide. In certain embodiments, a ComP glycosylation fragment is glycosylated by a PglS OTase; a TfpM-associated pilin glycosylation fragment is glycosylated by a TfpM OTase, optionally, wherein a ComP glycosylation fragment is 32080280 - 5 - Atty. Dkt. No.: 64100-234947 glycosylated by a PglS OTase and a TfpM-associated pilin glycosylation fragment is glycosylated by a TfpM OTase; a PilE glycosylation fragment is glycosylated by a PglL OTase; a PglB glycosylation fragment is glycosylated by a PglB OTase; a PilA glycosylation fragment is glycosylated by a TfpO or PilO OTase; a STT3 glycosylation fragment is glycosylated by the STT3 catalytic subunit; a PilA_Pa5196-associated pilin glycosylation fragment is glycosylated by a TfpW glycosyltransferase; an N-linking glycosyltransferase glycosylation fragment is glycosylated by an N-linking glycosyltransferase from Actinobacillus pleuropneumoniae; from Haemophilus influenzae; or from Yersinia enterocolitica; and/or an O-linking glycosyltransferase glycosylation fragment is glycosylated by GtfA/GtfB glycosyltransferases. [0024] Also provided for herein is a method of eliciting an immune response in a subject by administering to said subject an effective amount of any composition, complex, and/or conjugate vaccine of any of this disclosure or a composition, complex, and/or conjugate vaccine of this disclosure for use in eliciting an immune response in a subject. BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES [0025] Figure 1. Schematic overview of glycoprotein polypeptide production using enzymes that form glycosidic linkages and the subsequent isopeptide bond formation with a second protein containing a polypeptide tag. A first polypeptide (PROTEIN 1) contains both (i) a SpyTag, which will spontaneously form an isopeptide bond with a polypeptide tag (SpyCatcher) of a second partner polypeptide (PROTEIN 2), as well as (ii) a glycosylation fragment (Sequon), which is recognized by a specific enzyme that forms glycosidic linkages by covalently transferring a saccharide to the glycosylation fragment (Sequon). In this example, PROTEIN 2 containing the polypeptide tag (SpyCatcher) also can self-assemble into higher- order structures like icosahedron or dodecahedron nanoparticles resembling nanocages, virus- like particles (VLPs), or Adenoviral vectors. [0026] Figure 2. Coomassie-stained SDS-PAGE denaturing gel of purified MBP-SpyTag- v1 E. coli O16 O-antigen bioconjugate generated using the TfpM oligosaccharyltransferase system, mi3-SpyCatcher, and the isopeptide bonded MBP-SpyTag-v1 E. coli O16:mi3- SpyCatcher. The boxed diagrams show the schematic structure of the SpyTag protein (corresponding to PROTEIN 1 in Figure 1) and SpyCatcher protein (corresponding to PROTEIN 2 in Figure 1) designed for compatibility with enzymes that form glycosidic linkages. Abbreviations: MBP, maltose binding protein; MBPsp, E. coli maltose binding 32080280 - 6 - Atty. Dkt. No.: 64100-234947 protein secretory (Sec) signal peptide; Pil20, 20 amino acid pilin sequon that is glycosylated by the TfpM oligosaccharyltransferase; mi3, mi3 nanoparticle monomer; 6xHis, hexahistidine tag. For SDS-PAGE analysis, SpyCatcher and SpyTag proteins alone or at a 1:1 or 2:1 ratio (based on protein concentration) were reacted for 2 hours in Tris buffered saline at room temperature. Isopeptide bond reaction formation was stopped by the addition of Laemmli buffer and subsequent heating of the sample for 10 mins at 100˚C prior to gel loading. Protein masses associated with proteins and glycoproteins are indicated above each lane. Lane A) Protein ladder with standards masses in kDa marked to the left. Lane B) Purified MBP- SpyTag-v1-O16 bioconjugate. Lane C) Purified mi3-Spycatcher. Lane D) 1:1 reaction mixture of mi3-SpyCatcher and MBP-SpyTag-v1-O16. Lane E) 2:1 reaction mixture of mi3- SpyCatcher and MBP-SpyTag-v1-O16. [0027] Figure 3. Western blot of purified MBP-SpyTag-v1 E. coli O16 O-antigen bioconjugate generated using the TfpM oligosaccharyltransferase system, mi3-SpyCatcher, and the isopeptide bonded MBP-SpyTag-v1 E. coli O16:mi3-SpyCatcher. SpyTag and SpyCatcher proteins alone or at a 1:1 or 2:1 ratio (based on protein concentration) were reacted for 2 hours in Tris buffered saline prior western blot analysis. Protein masses associated with proteins and glycoproteins are indicated above each lane. The western blot was probed with anti-His tag ( ^-His, top panel) and E. coli O16 O-antigen antisera ( ^-O16, middle panel). The merged image is shown in the bottom panel. Lane A) Protein ladder with standards masses in kDa marked to the left. Lane B) Purified MBP-SpyCatcher-v1-O16 bioconjugate. Lane C) Purified mi3-Spycatcher. Lane D) 1:1 reaction mixture of mi3-SpyTag and MBP-SpyTag-v1- O16. Lane E) 2:1 reaction mixture of mi3-SpyCatcher and MBP-SpyTag-v1-O16. [0028] Figure 4. Size-exclusion chromatography of mi3-SpyCatcher and isopeptide bonded MBP-SpyTag-v1-O16:mi3-SpyCatcher separated using a Sephacryl S-400 HR 16/600 column. Shown are UV absorbance traces of 250 ^g mi3-SpyCatcher (solid line) and 250 ^g mi3-SpyCatcher after reaction with 250 ^g MBP-SpyTag-v1-O16 (dashed line) in Tris buffered saline for 2 hours at room temperature. [0029] Figure 5. Coomassie-stained SDS-PAGE denaturing gel of purified MBP-SpyTag- v2 E. coli O16 O-antigen bioconjugate generated using the PglS oligosaccharyltransferase system, mi3-SpyCatcher, and isopeptide bonded MBP-SpyTag-v2 E. coli O16:mi3- SpyCatcher. The boxed diagrams show the schematic structure of the SpyTag and SpyCatcher proteins in this experiment. Abbreviations: MBP, maltose binding protein; MBPsp, E. coli maltose binding protein secretory (Sec) signal peptide; ComP sequon, 23 amino acid ComP- 32080280 - 7 - Atty. Dkt. No.: 64100-234947 derived sequon glycosylated by the PglS oligosaccharyltransferase; mi3, mi3 nanoparticle monomer; 6xHis, hexahistidine tag. For SDS-PAGE analysis, SpyTag and SpyCatcher proteins alone or at a 1:1 or 2:1 ratio (based on protein concentration) were reacted for 2 hours in Tris buffered saline at room temperature. Isopeptide bond reaction formation was stopped by the addition of Laemmli buffer and subsequent heating of the sample for 10 mins at 100˚C prior to gel loading. Samples were taken from each reaction for SDS-PAGE analysis. Protein masses associated with proteins and glycoproteins are indicated above each lane. Lane A) Protein ladder with standards masses in kDa marked to the left. Lane B) Purified MBP-SpyTag-v2- O16 bioconjugate. Lane C) Purified mi3-SpyCatcher. Lane D) 1:1 reaction mixture of mi3- SpyCatcher and MBP-SpyTag-v2-O16. Lane E) 2:1 reaction mixture of mi3-SpyCatcher and MBP-SpyTag-v2-O16. [0030] Figure 6. Western blot of purified MBP-SpyTag-v2 E. coli O16 O-antigen bioconjugate generated using the PglS oligosaccharyltransferase system, mi3-SpyCatcher, and isopeptide bonded MBP-SpyTag-v2 E. coli O16:mi3-SpyCatcher. SpyTag and SpyCatcher fusion proteins alone or at a 1:1 or 2:1 ratio (based on protein concentration) were reacted for 2 hours in Tris buffered saline prior to western blot analysis. The western blot was probed with anti-His tag ( ^-His, top panel) and E. coli O16 O-antigen antisera ( ^-O16, middle panel). The merged image is shown in the bottom panel. Lane A) Protein ladder with standards masses in kDa marked to the left. Lane B) Purified MBP-SpyTag-v2-O16 bioconjugate. Lane C) Purified mi3-SpyCatcher bioconjugate. Lane D) 1:1 reaction mixture of mi3-SpyCatcher and MBP-SpyTag-v2-O16. Lane E) 2:1 reaction mixture of mi3-SpyCatcher and MBP-SpyTag- v2-O16. [0031] Figure 7. Coomassie-stained SDS-PAGE denaturing gel of purified MBP-SpyTag- v1 E. coli O16 O-antigen bioconjugate generated using the TfpM oligosaccharyltransferase system, AP205-SpyCatcher, and the isopeptide bonded MBP-SpyTag-v1 E. coli O16:AP205- SpyCatcher. The boxed diagrams show the schematic structure of the SpyTag and SpyCatcher proteins in this experiment. Abbreviations: MBP, maltose binding protein; MBPsp, E. coli maltose binding protein secretory (Sec) signal peptide; Pil20, 20 amino acid pilin sequon that is glycosylated by the TfpM oligosaccharyltransferase; AP205, AP205 virus-like protein coat protein monomer; 6xHis, hexahistidine tag. For SDS-PAGE analysis, SpyCatcher and SpyTag fusion proteins alone or at a 1:1 or 2:1 ratio (based on protein concentration) were reacted for 2 hours in Tris buffered saline at room temperature before gel loading. Protein masses associated with proteins and glycoproteins are indicated above each lane. Lane A) Protein 32080280 - 8 - Atty. Dkt. No.: 64100-234947 ladder with standards masses in kDa marked to the left. Lane B) Purified MBP-SpyTag-v1- O16 bioconjugate. Lane C) Purified AP205-SpyCatcher. Lane D) 1:1 reaction mixture of AP205-SpyCatcher and MBP-SpyTag-v1-O16. Lane E) 2:1 reaction mixture of AP205- SpyCatcher and MBP-SpyTag-v1-O16. [0032] Figure 8. Coomassie-stained SDS-PAGE denaturing gel of purified MBP-SpyTag- v2 E. coli O16 O-antigen bioconjugate generated using the PglS oligosaccharyltransferase system, AP205-SpyCatcher, and the isopeptide bonded MBP-SpyTag-v2 E. coli O16:AP205- SpyCatcher. The boxed diagrams show the schematic structure of the SpytTag and SpyCatcher proteins in this experiment. Abbreviations: MBP, maltose binding protein; MBPsp, E. coli maltose binding protein secretory (Sec) signal peptide; ComP sequon, 23 amino acid ComP- derived sequon that is glycosylated by the PglS OTase; AP205, AP205 Virus-like protein coat protein monomer; 6xHis, hexahistidine tag. For SDS-PAGE analysis, SpyTag and SpyCatcher fusion proteins alone or at a 1:1 or 2:1 ratio (based on protein concentration) were reacted for 2 hours in Tris buffered saline at room. Isopeptide bond reaction formation was stopped by the addition of Laemmli buffer and subsequent heating of the sample for 10 mins at 100˚C prior to gel loading. Samples were taken from each reaction for SDS-PAGE analysis. Protein masses associated with proteins and glycoproteins are indicated above each lane. Lane A) Protein ladder with standards masses in kDa marked to the left. Lane B) Purified MBP-Spycatcher- v2-O16 bioconjugate. Lane C) Purified AP205-Spycatcher. Lane D) 1:1 reaction mixture of AP205-Spytag and MBP-Spytag-v2-O16. Lane E) 2:1 reaction mixture of AP205-Spycatcher and MBP-Spytag-v2-O16. [0033] Figure 9. Coomassie-stained SDS-PAGE denaturing gel of three purified EPA- SpyTag E. coli O16 O-antigen bioconjugates generated using the PglS oligosaccharyltransferase system, mi3-SpyCatcher, and isopeptide-bonded EPA-Spytag E. coli O16:mi3-SpyCatcher. The boxed diagrams show the schematic structure of the EPA SpyTag proteins designed for compatibility with enzymes that form glycosidic linkages. Abbreviations: EPA, Pseudomonas aeruginosa exotoxin A; MBPsp, E. coli maltose binding protein secretory (Sec) signal peptide; ComP Sequon, 23 amino acid pilin sequon that is glycosylated by the PglS oligosaccharyltransferase; mi3, mi3 nanoparticle monomer; 6xHis, hexahistidine tag. The isopeptide bond formation reactions were performed using purified EPA-Spycatcher-O16 bioconjugates and purified mi3-Spycatcher. Lane A) Protein ladder with standards masses in kDa marked to the left. Lane B) Purified mi3-Spycatcher. Lane C) Purified EPA-Spytag-v1- O16. Lane D) 1:1 reaction mixture of mi3-Spycatcher and EPA-SpyTag-v1-O16. Lane E) 1:2 32080280 - 9 - Atty. Dkt. No.: 64100-234947 reaction mixture of mi3-Spycatcher and EPA-SpyTag-v1-O16. Lane F) Purified EPA-Spytag- v2-O16. Lane G) 1:1 reaction mixture of mi3-Spycatcher and EPA-SpyTag-v2-O16. Lane H) 1:2 reaction mixture of mi3-Spycatcher and EPA-SpyTag-v2-O16. Lane I) Purified EPA- Spytag-v3-O16. Lane J) 1:1 reaction mixture of mi3-Spycatcher and EPA-SpyTag-v3-O16. Lane K) 1:2 reaction mixture of mi3-Spycatcher and EPA-SpyTag-v3-O16. Isopeptide-bonded proteins were not observed using EPA-Spytag-v2-O16 or EPA-Spytag-v3-O16 and mi3- Spycatcher. [0034] Figure 10. Western blot of purified, unglycosylated EPA-SpyTag-v1 protein linker variants, mi3-SpyCatcher, and the isopeptide bonded EPA-SpyTag E. coli:mi3-SpyCatcher. Each protein variant has a different amino acid linker between Spytag003 and EPA. The isopeptide bond formation reactions were performed using EPA-Spytag proteins from E. coli periplasmic extracts and purified mi3-Spycatcher. The western blot was probed with anti-His tag antibody. Lane A) Protein ladder with standards masses in kDa marked to the left. Lane B) Purified EPA-Spytag-v1 with linker L1 (SGG). Lane C) 1:1 reaction mixture of mi3- Spycatcher and EPA-Spytag-v1 with linker L1. Lane D) Purified EPA-Spytag-v1 with linker L2 (SEQ ID NO: 430). Lane E) 1:1 reaction mixture of mi3-Spycatcher and EPA-Spytag-v1 with linker L2. Lane F) Purified EPA-Spytag-v1 with linker L3 (SEQ ID NO: 431). Lane G) 1:1 reaction mixture of mi3-Spycatcher and EPA-Spytag-v1 with linker L3. Lane H) Purified EPA-Spytag-v1 with linker L4 (SEQ ID NO: 432). Lane I) 1:1 reaction mixture of mi3- Spycatcher and EPA-Spytag-v1 with linker L4. Lane J) Purified EPA-Spytag-v1 with linker L5 (SEQ ID NO: 433). Lane K) 1:1 reaction mixture of mi3-Spycatcher and EPA-Spytag-v1 with linker L5. Lane L) Purified EPA-Spytag-v1 with linker L6 (SEQ ID NO: 434). Lane M) 1:1 reaction mixture of mi3-Spycatcher and EPA-Spytag-v1 with linker L6. Lane N) Purified EPA-Spytag-v1 with linker L7 (SEQ ID NO: 435). Lane O) 1:1 reaction mixture of mi3- Spycatcher and EPA-Spytag-v1 with linker L7. Lane P) Purified EPA-Spytag-v1 with linker L8 (SEQ ID NO: 436). Lane Q) 1:1 reaction mixture of mi3-Spycatcher and EPA-Spytag-v1 with linker L8. Lane R) Purified EPA-Spytag-v1 with linker L9 (SEQ ID NO: 437). Lane S) 1:1 reaction mixture of mi3-Spycatcher and EPA-Spytag-v1 with linker L9. Lane T) Purified EPA-Spytag-v1 with linker L10 (SEQ ID NO: 438). Lane U) 1:1 reaction mixture of mi3- Spycatcher and EPA-Spytag-v1 with linker L10. Some variants are poorly expressed and/or unable to form isopeptide bonds with mi3-Spycatcher. [0035] Figure 11. Western blot of unglycosylated EPA-Spytag-v1 with linker L7 (EAAAKEAAAK; SEQ ID NO: 435) and mi3-Spycatcher isopeptide-formation reaction 32080280 - 10 - Atty. Dkt. No.: 64100-234947 timecourse. The isopeptide bond formation reactions were performed using EPA-Spytag-v1 with linker L7 from E. coli periplasmic extracts and purified mi3-Spycatcher at a 1:1 ratio. The left panel shows the western blot probed with anti-His tag antibody. The middle panel shows the western blot probed with anti-EPA antibody. The right panel is the merged image of the two channels. Lane A) Protein ladder with standards masses in kDa marked to the left. Lane B) mi3-Spycatcher only. Lane C) EPA-Spytag-v1 with linker L7 only. Lane D) Isopeptide bond reaction between mi3-Spycatcher and EPA-Spytag-v1 with linker L7 after 0.5 hours. Lane E) The reaction after 1 hour. Lane F) The reaction after 2 hours. Lane G) The reaction after 5 hours. Lane H) The reaction after 24 hours. [0036] Figure 12A-E. Figure 12A shows a schematic of EPA-ComP110264 fusion proteins where the ComP glycosylation fragment is fused at the C-terminus of the fusion protein. “ssDsbA” corresponds to the DsbA Sec secretion signal. GGGS (SEQ ID NO: 382) is a flexible linker between EPA and the ComP110264 fragment. Figure 12B shows different amino acid sequences for ComP glycosylation fragments fused to C-terminus of the EPA fusion protein. The bold, underlined serine residue in each sequence corresponds to the conserved serine 82 of ComP110264 and is the site of glycosylation. The bold, underlined cysteine residues corresponding to Cys71 and Cys93 are also highlighted. (C2, SEQ ID NO: 383; D2; SEQ ID NO: 384; E2, SEQ ID NO: 385; F2, SEQ ID NO: 386; G2, SEQ ID NO: 387; H2, SEQ ID NO: 388; A3, SEQ ID NO: 389; B3, SEQ ID NO: 390; C3, SEQ ID NO: 391; D3, SEQ ID NO: 392; E3, SEQ ID NO: 393; F3, SEQ ID NO: 394; and C1, SEQ ID NO: 395). Figure 12C, Figure 12D, and Figure 12E show Western blot analysis of periplasmic extracts from E. coli SDB1 expressing PglS, the CPS8 glycan and an EPA-ComP110264 variant. Each lane of the Western blot panel corresponds to a strain of SDB1 expressing a different EPA-ComP variant with the ComP glycosylation fragment corresponding to the sequence shown in Figure 12B. Figure 12C shows proteins reacting with the anti-EPA antisera. Figure 12D shows proteins reacting with the anti-His antisera. Figure 12E shows the merged western blot images of Figure 12C and Figure 12D. Equivalent amounts of periplasmic extract based on OD600 were loaded per lane. To the right of panels Figure 12C-E, g0 denotes unglycosylated EPA- ComP110264 and gn denotes EPA-ComP110264 glycosylated with different numbers of CPS8 repeat units. Protein mass markers (in kDa) are indicated to the left of panels Figure 12C-E. [0037] Figure 13A-D. Figure 13A shows a schematic of the CRM197-ComPC1 fusion protein. “ssFlgI” corresponds to the FlgI SRP secretion signal. GGGS (SEQ ID NO: 382) is a flexible linker between CRM197 and ComPC1. Figure 13B, Figure 13C, and Figure 13D show 32080280 - 11 - Atty. Dkt. No.: 64100-234947 Western blot analysis of the purified CRM197-ComPC1-CPS8 glycoconjugate. Figure 13B shows the proteins reacting with the anti-CPS8 antisera. Figure 13C shows the proteins reacting with the anti-CRM197 antisera. Figure 2D shows the merged western blot images of Figure 13B and Figure 13C. Loss of CRM197 and CPS8 signals in the proteinase K (PK)- treated samples demonstrate that the pneumococcal serotype 8 signal is CRM197-linked and not the result of contamination from free polysaccharide or lipid-linked polysaccharide precursors. Protein mass markers (in kDa) are indicated to the left of panels Figure 13B-D. [0038] Figure 14A,B. Figure 14A shows schematic diagrams of the C- and N-terminal CRM197 variants containing the C1 ComP glycosylation fragment. Figure 14B shows Western blot analysis of periplasmic extracts of E. coli SDB1 expressing CRM197-ComPC1 or ComPC1- CRM197 and the CPS8 glycan in the presence (+) or absence (-) of PglS. Equivalent amounts of periplasmic extracts based on OD600 were loaded per lane. Protein mass markers (in kDa) are indicated to the left. GGGS (SEQ ID NO: 382). [0039] Figure 15A-E. Figure 15A shows a schematic diagram of EPA fusion proteins containing ComP glycosylation fragments integrated internal of the EPA amino acid sequence. Figure 15B shows amino acid sequences of the two iGT ComP glycosylation fragments inserted between EPA residues Ala489 and Arg489. These have either two terminal cysteines (“iGCC”; SEQ ID NO: 230) or serines (“iGSS”; SEQ ID NO: 231). Figure 15C and Figure 15D show Western blots on periplasmic extracts of E. coli SDB1 expressing the CPS8 glycan, EPAiGTcc or EPAiGTss, with (+) or without (-) PglS. Figure 15C shows proteins reacting with the anti-EPA antisera. Figure 15D shows proteins reacting with the anti-His antisera. Figure 15E shows the merged Western blot images of Figure 15C and Figure 15D. Equivalent amounts of periplasmic extracts based on OD600 were loaded per lane. Protein mass markers (in kDa) are indicated to the left of panels. [0040] Figure 16A-D. Figure 16A show a schematic Diagram of EPA constructs containing ComP glycosylation fragments used for these experiments (from top to bottom, SEQ ID NOs: 6-28). Twenty-two to five amino acid-truncated variants of the iGTCC ComP glycosylation fragment were inserted into the EPA coding sequence between Ala489 and Arg489. Figure 16B shows the amino acid sequences of the 22 truncated iGT ComP glycosylation fragments with name designations assigned to the left. The underlined, bolded serine is the glycosylation site. (iGTcc SEQ ID NO: 230; Δ0-1 SEQ ID NO: 232; Δ1-0 SEQ ID NO: 243; Δ1-2 SEQ ID NO: 245; Δ2-1 SEQ ID NO: 256; Δ2-3 SEQ ID NO: 258; Δ3-2 SEQ ID NO: 269; Δ3-4 SEQ ID NO: 271; Δ4-3 SEQ ID NO: 282; Δ4-5 SEQ ID NO: 284; Δ5- 32080280 - 12 - Atty. Dkt. No.: 64100-234947 4 SEQ ID NO: 295; Δ5-6 SEQ ID NO: 297; Δ6-5 SEQ ID NO: 308; Δ6-6 SEQ ID NO: 309; Δ6-7 SEQ ID NO: 310; Δ7-6 SEQ ID NO: 321; Δ7-7 SEQ ID NO: 322; Δ7-8 SEQ ID NO: 323; Δ8-7 SEQ ID NO: 334; Δ8-8 SEQ ID NO: 335; Δ8-9 SEQ ID NO: 336; Δ9-8 SEQ ID NO: 346; Δ9-9 SEQ ID NO: 347). Figure 16C shows Western blot analysis on periplasmic extracts of E. coli SDB1 expressing PglS, CPS8 and an EPAiGT fusion protein containing a truncated ComP glycosylation fragment. Each lane of the Western blot panel corresponds to a strain of SDB1 expressing a different EPAiGT fusion protein containing a truncated ComP glycosylation fragment with the ComP glycosylation fragment corresponding to the sequence shown in Figure 16B. Figure 16C shows proteins reacting with the anti-EPA antisera probing with an anti-EPA antibody. EPAiGTcc is shown for comparison. The “EPA” lane corresponds to EPA lacking any ComP-derived sequences and serves as a negative control. Equivalent amounts of periplasmic extract based on OD600 were loaded per lane. Figure 16D shows the same Western blot as above with an increase anti-EPA signal brightness in order to show low- level glycosylation for the smallest ComP glycosylation fragments. [0041] Figure 17A,B,C. Figure 17 shows Western blot analysis of Ni affinity chromatography purified EPA fusion proteins containing the iGT ^6-6 ComP glycosylation fragment integrated between residues Ala489 and Arg490 of EPA. The fusion protein was purified from SDB1 cells expressing the CPS8 glycan in the presence (+) or absence (-) of PglS. Figure 17A shows proteins reacting with anti-His antisera. Figure 17B shows proteins reacting with anti-CPS8 antisera. Figure 17C shows a merge of Figure 17A and Figure 17B. Protein mass markers (in kDa) are indicated to the left of panels Figure 17A-C. [0042] Figure 18A and 18B. Figure 18A shows a schematic diagram of the EPA fusion protein containing the iGT∆3-4 ComP glycosylation fragment integrated between residues Glu548 and Gly549 of EPA. The iGT∆3-4 amino acid sequence is listed below the schematic (SEQ ID NO: 271). Figure 18B shows Western blot analysis on periplasmic extracts of E. coli SDB1 expressing PglS, CPS8 and the EPA fusion protein containing the iGT∆3-4 ComP glycosylation fragment integrated between residues Glu548 and Gly549. Protein reacting with the anti-EPA antisera probing with an anti-EPA antibody are shown. [0043] Figure 19A,B,C. Figure 19 shows Western blot analysis of Ni affinity chromatography purified EPA fusion proteins containing the iGT ^3-4 ComP glycosylation fragment integrated between residues Glu548 and Gly549 of EPA. The fusion protein was purified from SDB cells expressing the CPS8 glycan in the presence (+) or absence (-) of PglS. Figure 19A shows proteins reacting with anti-His antisera. Figure 19B shows proteins reacting 32080280 - 13 - Atty. Dkt. No.: 64100-234947 with anti-CPS8 antisera. Figure 19C shows a merge of Figure 19A and Figure 19B. Protein mass markers (in kDa) are indicated to the left of panels Figure 19A-C. [0044] Figure 20. Figure 20 lists ComP ortholog amino acid sequences. The site of predicted glycosylation is bolded. [0045] Figure 21. Figure 21 lists ComP Δ28 ortholog amino acid sequences in which the amino acids corresponding to the 28 N-terminal amino acids of ComPADP1: AAC45886.1 have been removed. The site of predicted glycosylation is bolded. [0046] Figure 22. Figure 22 shows an alignment of a region ComP sequences including the serine (S) residue (boxed) corresponding to the serine residue at position 82 of ComP110264 (SEQ ID NO: 201) also corresponding to the serine residue at position 84 of ComPADP1 (SEQ ID NO: 202). [0047] Figure 23A-D. Figure 23 shows the characterization of 13 TfpM orthologs from species in the Moraxellaceae family. Figure 23A) Cladogram of the 20 TfpM orthologs with archetypal TfpO, PglL, and PglS from Pseudomonas, Neisseria, and Acinetobacter, respectively. Branch confidence is indicated in red. Starred OTase/pilin pairs were cloned and tested in bioconjugation experiments. Figure 23B) Diagram showing the EPA-pilin fusion protein and TfpM construct design. Colored arrows indicate genes. Gene expression was driven from an IPTG-inducible tac promoter with a lacO operator (tac1O). The rrnB T2 terminator is marked by a black hairpin structure. Figure 23C) and Figure 23D) Anti-EPA western blot of whole-cell E. coli extracts expressing different EPA-pilin carrier open reading frames and tfpM genes. Panel D shows the same image as panel C but at higher exposure. Whole cell extracts loaded in each lane were normalized by OD600. H286A indicates the M. osloensis TfpM site directed OTase active site mutant. “g0” indicates unglycosylated EPA-pilin and “gn” indicates CPS8-glycosylated EPA-pilin protein. Reference protein masses are marked left of the western blot in kDa. [0048] Figure 24. Figure 24 shows a phylogram of TfpM, PilO, PglL, and PglS orthologs with relative distances shown. The phylogenetic tree was generated using the phylogeny.fr server (on the world wide web at phylogeny.fr/) which uses MUSCLE, PhyML, and TreeDyn for sequence alignment, tree calculation, and image generation, respectively. [0049] Figure 25. Figure 25 Phylogram of TfpM-associated pilin-like proteins, select PilA proteins from Neisseria and Pseudomonas, and ComP from A. soli CIP 110264 with relative distances shown. The red numbers indicate branch confidence. The phylogenetic tree was generated using the phylogeny.fr server (on the world wide web at phylogeny.fr/) which 32080280 - 14 - Atty. Dkt. No.: 64100-234947 uses MUSCLE, PhyML, and TreeDyn for sequence alignment, tree calculation, and image generation, respectively. [0050] Figure 26. Figure 26 shows multiple sequence alignment of select bacterial O- linking oligosaccharyltransferase. The alignment was generated using Clustal Omega with default settings found on the world wide web at ebi.ac.uk/Tools/msa/clustalo/. N_menigitidis_MC58_PglL (SEQ ID NO: 105). A_baylyi_ADP1_PglS (SEQ ID NO: 106); P_aeruginosa_1244_TfPO (SEQ ID NO: 107); M_osloensis_1202_TfpM (SEQ ID NO: 56); A_nosocomialis_M2_TfpO (SEQ ID NO: 108). [0051] Figure 27. Figure 27 shows an anti-EPA whole-cell western blot examining the glycosylation status of the EPA-PilMo∆28 fusions as well as EPA-PilMo∆28 C-terminal Thr167 mutants. All lanes were normalized to the same OD600. Reference protein masses are marked next to the western blot in kDa. [0052] Figure 28A and 28B. Figure 28 shows Targeted MS/MS analysis of the HexHexA-modified C-terminal EPA-PilMo∆28 peptide 762FLPANCRGT770 (SEQ ID NO: 61). Figure 28A) EThcD fragmentation enabled the localization of the HexHexA glycosylation event to the terminal residue Thr770. Figure 28B) HCD fragmentation enables the confirmation of the peptide sequence as well as the linkage of the disaccharide HexHexA through the Hex monosaccharide by the observation of multiple y-ions linked solely to a Hex residue. [0053] Figure 29A-G. Figure 29 shows TfpMMo can transfer diverse bacterial glycans to the EPA-PilMo ^28 fusion protein. Figure 29A) Structures of the repeat units of the five bacterial glycans tested with TfpMMo. The linkages between sugar monomers are indicated in rounded brackets. Glycan abbreviations used: CPS8, S. pneumoniae capsular polysaccharide Type 8; GBSIII, Group B Streptococcus capsular polysaccharide Type III; LT2, Salmonella enterica Group B serotype LT2 O-antigen; O16, E. coli serotype O16 O-antigen; O2a, Klebsiella pneumoniae serotype O2a O-antigen; All sugars are the pyranose form except where noted. Abbreviations used: Glc, glucose; Gal, galactose; Galf, galactofuranose; Rha, rhamnose; GlcNAc, N-acetylglucosamine; Abe, abequose; NeuNAc, N-acetylneuraminic acid, sialic acid. Figures 29B)-29F) Anti-glycan western blots with partially purified TfpMMo-derived bioconjugates. Figure 29B) Anti-CPS8. Figure 29C) Anti-O16. Figure 29D) Anti-LT2. Figure 29E) Anti-O2a. Figure 29F) Anti-GBSIII. Figure 29G) Anti-EPA. In panels B)-G), +/- labels indicate whether the samples were incubated with (+) or without (-) proteinase K prior to SDS-PAGE separation. Reference protein masses are marked next to the western blots in kDa. 32080280 - 15 - Atty. Dkt. No.: 64100-234947 [0054] Figure 30A and 30B. Figure 30 shows TfpMMo glycosylates truncated EPA-fused pilin variants as small as three amino acids. Figure 30A) Sequences of the EPA-fused PilMo fragments tested for bioconjugation with TfpMMo. Blue letters mark the C-terminal residues of EPA (i.e., EDLK; SEQ ID NO: 132). Underlined residues indicate the glycine linker placed between EPA and the pilin sequence. Figure 30B) Anti-EPA western blot of whole cell extracts expressing the truncated pilin variants, CPS8, and TfpMMo. The calculated EPA-PilMo∆28 mass is 80.3 kDa and that of the truncated variants ranges from 67.1 to 69.0 kDa. All lanes were normalized to the same OD600. Reference protein masses are marked left of the western blot in kDA. “g0” indicates the unglycosylated truncated EPA-pilins and “gn” indicates the glycosylated EPA-pilin protein. Unglycosylated EPA-PilMo∆28 runs near 75 kDa. Pil20 (SEQ ID NO: 60). Pil15 (SEQ ID NO: 109). Pil13 (SEQ ID NO: 110). GGGG plus Pil10 is Pil10L (SEQ ID NO: 111). Pil10 (SEQ ID NO: 112). Pil7 (SEQ ID NO: 113). Pil6 (SEQ ID NO: 114). Pil5 (SEQ ID NO: 115). Pil4 (SEQ ID NO: 116). Pil3 (SEQ ID NO: 117). EDLK plus Pil2 (SEQ ID NO: 118). EDLKGGGG plus Pil20 (SEQ ID NO: 122). EDLK plus Pil15 (SEQ ID NO: 123). EDLK plus Pil13 (SEQ ID NO: 124). EDLK plus Pil10L (SEQ ID NO: 125). EDLK plus Pil10 (SEQ ID NO: 126). EDLK plus Pil7 (SEQ ID NO: 127). EDLK plus Pil6 (SEQ ID NO: 128). EDLK plus Pil5 (SEQ ID NO: 129). EDLK plus Pil4 (SEQ ID NO: 130). EDLK plus Pil3 (SEQ ID NO: 131). [0055] Figure 31. Figure 31 shows multiple sequence alignment of select pilin proteins. Accession numbers for these proteins are given in the main text. The alignment was generated using Clustal Omega with default settings found on the world wide web at ebi.ac.uk/Tools/msa/clustalo/. P_aeruginosa_1244_PilA (SEQ ID NO: 119). N_menigitidis_M2_PilA (SEQ ID NO: 120). A_junii_65_pilin (SEQ ID NO: 97). A_CIP102143_pilin (SEQ ID NO: 88). A_CIP102637_pilin (SEQ ID NO: 100). A_YZS-X1- 1_pilin (SEQ ID NO: 98). A_soli_110264_ComP (SEQ ID NO: 121). A_YH01026_pilin (SEQ ID NO: 87). M_osloensis_1202_pilin (SEQ ID NO: 57). A_junii_TUM15069_pilin (SEQ ID NO: 84). [0056] Figure 32A-F. Figure 32 shows purified TfpMMo-derived GBSIII bioconjugate elicits a robust IgG immune response in mice. Figure 32A) Western blot of purified GBSIII- 291 bioconjugate, anti-EPA channel. Figure 32B) Anti-GBSIII. Figure 32C) Merged image of A and B. Figure 32D) Coomassie stain of purified GBSIII-291 bioconjugate. Figure 32E) MS1 spectrum of intact, purified GBSIII-291 bioconjugate. The 291 protein (EPA-Pil20) has a theoretical mass of 69,582.19 Da. The GBSIII-291 bioconjugate is observed in multiple states 32080280 - 16 - Atty. Dkt. No.: 64100-234947 of increasing mass separated by near 980 Da that corresponds to the calculated mass of a GBSIII glycan repeat unit. Figure 32F) GBSIII- specific IgG kinetics over the course of immunization as measured by ELISA and converted to ng/mL IgG using a standard IgG curve. **P<0.01. [0057] Figure 33A and 33B. Figure 33 shows glycosylation of EPA constructs containing sequons from different O-linking oligosaccharyltransferase systems. Figure 33A) Diagrams of the plasmid-based operons expressing EPA with PglS- or TfpM-specific sequons. Internal glycotag (“iGT”) is a 23 amino acid fragment derived from ComP110264 inserted between EPA Ala489 – Arg490 and/or Glu548 – Gly549. Figure 33B) Anti-EPA western blot of SDB1 periplasmic extracts expressing one of the four constructs and the E. coli O16 O-antigen. Load amounts per lane were normalized to OD600. “g0” indicates unglycosylated EPA carrier protein and the singly or doubly glycosylated EPA proteins are indicated. Reference protein masses are marked left of the western blot in kDa. DETAILED DESCRIPTION [0058] This disclosure provides descriptions of compositions of and methods used to produce a glycoconjugate polypeptide using enzymes that form glycosidic linkages, where said polypeptide can spontaneously form an isopeptide bond with a second partner polypeptide containing a polypeptide tag and uses thereof. Definitions [0059] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a polysaccharide," is understood to represent one or more polysaccharides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. [0060] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the specified features or components with or without the other. Thus, the term and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). [0061] It is understood that wherever aspects are described herein with the language "comprising" or “comprises” otherwise analogous aspects described in terms of "consisting 32080280 - 17 - Atty. Dkt. No.: 64100-234947 of," “consists of,” "consisting essentially of," and/or “consists essentially of,” and the like are also provided. [0062] Unless defined otherwise, 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 is related. [0063] Numeric ranges are inclusive of the numbers defining the range. Even when not explicitly identified by “and any range in between,” or the like, where a list of values is recited, e.g., 1, 2, 3, or 4, unless otherwise stated, the disclosure specifically includes any range in between the values, e.g., 1 to 3, 1 to 4, 2 to 4, etc. [0064] The headings provided herein are solely for ease of reference and are not limitations of the various aspects or aspects of the disclosure, which can be had by reference to the specification as a whole. [0065] As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed 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, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-standard amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis. [0066] A “protein” as used herein can refer to a single polypeptide, i.e., a single amino acid chain as defined above, but can also refer to two or more polypeptides that are associated, e.g., by disulfide bonds, hydrogen bonds, or hydrophobic interactions, to produce a multimeric protein. [0067] By an "isolated" polypeptide or a fragment, variant, or derivative thereof is intended a polypeptide that is not in its natural milieu. No particular level of purification is required. For 32080280 - 18 - Atty. Dkt. No.: 64100-234947 example, an isolated polypeptide can be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated as disclosed herein, as are recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique. [0068] A "vector" (also use interchangeably herein with “plasmid”) is nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can encode and express a protein. A vector can also include one or more selectable marker gene and other genetic elements known in the art. [0069] 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 those techniques by which a nucleic acid molecule can be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration. A transformed cell or a host cell can be a bacterial cell or a eukaryotic cell. [0070] The term “expression” as used herein refers to a process by which a gene produces a biochemical, for example, a polypeptide. The process includes any manifestation of the functional presence of the gene within the cell including, without limitation, gene knockdown as well as both transient expression and stable expression. It includes without limitation transcription of the gene into messenger RNA (mRNA), and the translation of such mRNA into 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 that is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, proteolytic cleavage, and the like. [0071] As used herein the terms "treat," "treatment," or "treatment of" (e.g., in the phrase "treating a subject") refers to reducing the potential for disease pathology, reducing the occurrence of disease symptoms, e.g., to an extent that the subject has a longer survival rate or reduced discomfort. For example, treating can refer to the ability of a therapy when administered to a subject, to reduce disease symptoms, signs, or causes. Treating also refers to 32080280 - 19 - Atty. Dkt. No.: 64100-234947 mitigating or decreasing at least one clinical symptom and/or inhibition or delay in the progression of the condition and/or prevention or delay of the onset of a disease or illness. [0072] By "subject" or "individual" or "animal" or "patient" or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals, including, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, llamas, bears, and so on. [0073] The term "pharmaceutical composition" or “therapeutic composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile. [0074] As used herein, a “saccharide” is a general term used to refer to any sized carbohydrate molecule; including but not limited to, monosaccharide, disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide, heptasaccharide, oligosaccharide, or polysaccharide. [0075] As used herein, a “glycosidic linkage” is a covalent bond between a saccharide and another organic molecule, including but not limited to, another saccharide, a protein, a lipid, or a nucleic acid. [0076] As used herein, a “glycosylation fragment” or “sequon” is a sequence of consecutive amino acids in a protein that serves as the recognition and attachment site for a saccharide, which is covalently transferred to the protein by a glycosyltransferase or oligosaccharyltransferase. [0077] As used herein, the term “translationally fused” can mean directly attached to (e.g., a carrier protein, N-terminal leader sequence, C-terminal tag, or the like of the fusion protein), or it can mean indirectly attached via an amino acid linker. Glycoconjugate Polypeptides Having Isopeptide Bonds With a Second Polypeptide Partner [0078] Provided for herein is a polypeptide pair comprising of a polypeptide tag and a binding partner (e.g., another polypeptide tag), wherein the polypeptide tag and the binding partner can bind to one another via the spontaneous formation of an isopeptide bond between one reactive residue comprised within said binding partner and another reactive residue comprised within said polypeptide tag. As used herein, where one component of the polypeptide pair is referred to, the other can be referred to as its partner. 32080280 - 20 - Atty. Dkt. No.: 64100-234947 Fusion Proteins [0079] One component of a polypeptide pair of this disclosure, generally comprising what is referred to as the first polypeptide tag, can be a fusion protein. Thus, certain embodiments of this disclosure provide for a fusion protein comprising: (i) a glycosylation fragment and (ii) a first polypeptide tag, wherein the first polypeptide tag can spontaneously form an isopeptide bond with a second polypeptide tag binding partner. For the avoidance of doubt, a fusion protein comprising a glycosylation fragment can comprise a fragment containing the glycosylation site (also referred to herein as a “sequon”) of a protein that is glycosylated or, in other embodiments, can comprise the full-length protein that is glycosylated, which in turn comprises the glycosylation fragment. Provided elsewhere herein in detail are numerous, representative examples of glycosylated proteins and sequons, as well as relevant enzymes, that can be used in the compositions and methods of this disclosure. In certain embodiments, the fusion protein comprises a carrier protein. For example, in certain embodiments the carrier protein can be Escherichia coli maltose binding protein (MPB), Pseudomonas aeruginosa Exotoxin A (EPA), Pseudomonas aeruginosa PcrV, CRM197, Haemophilus influenzae Protein D, cholera toxin B subunit, tetanus toxin, or a fragment of any thereof. In certain embodiments, the glycosylation fragment can be as little as 3 amino acids in length. For example, TfpM OTase can recognize a three-amino acid sequon. In certain embodiments, the glycosylation fragment can be longer, including a full-length or nearly full-length protein. Thus, in certain embodiments, the glycosylation fragment is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, or 40 amino acids in length. In certain embodiments, the glycosylation is not a full-length glycosylated protein, but a shorter fragment thereof, and thus is not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, 40, 50, 60, 80, or 100 amino acids in length. Therefore, in certain embodiments, the glycosylation fragment is from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 24 amino acids in length to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, or 25 amino acids in length. In certain embodiments, the glycosylation fragment is from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, or 40 amino acids in length to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, 40, or 50 amino acids in length. In certain embodiments, the glycosylation fragment is from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, 40, 50, 60, or 80 amino acids in length to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, 40, 50, 60, 80, or 100 amino acids in length. 32080280 - 21 - Atty. Dkt. No.: 64100-234947 [0080] In certain embodiments, the fusion protein is a glycoconjugate comprising a saccharide covalently attached to the fusion protein via the glycosylation site/residue of the glycosylation fragment (sequon). In certain embodiments, the glycoconjugate is immunogenic. One of ordinary skill in the art will recognize that the saccharide can be covalently attached to the glycosylation fragment, for example, through an N-linkage, O-linkage, or C-linkage. [0081] In certain embodiments, the first polypeptide tag is translationally fused at the N- terminal end of the fusion protein. In certain embodiments, the first polypeptide tag is translationally fused at the C-terminal end of the fusion protein. In certain embodiments, the first polypeptide tag is translationally fused internally within the fusion protein. In certain embodiments, the first polypeptide tag is translationally fused internally within the sequence of a carrier protein. [0082] In certain embodiments, the glycosylation fragment is translationally fused at the N-terminal end of the fusion protein. In certain embodiments, the glycosylation fragment is translationally fused at the C-terminal end of the fusion protein. In certain embodiments, the glycosylation fragment is translationally fused internally within the fusion protein. In certain embodiments, the glycosylation fragment is translationally fused internally within the sequence of a carrier protein. [0083] As can be understood from this disclosure as a whole, by fused internally within the fusion protein, it is meant that the glycosylation fragment, polypeptide tag, or the like is not located at the C-terminal end or the N-terminal end of the fusion protein, not including any signal/leader sequence, purification tag (e.g., His-Tag), or the like. For example: N-terminal Glycosylation fragment—Carrier protein N-terminal, not internal Leader sequence—Glycosylation fragment—Carrier protein C-terminal Carrier protein—Glycosylation fragment C-terminal, not internal Carrier protein—Glycosylation fragment—His-Tag 32080280 - 22 - Atty. Dkt. No.: 64100-234947 INTERNAL Leader Sequence—Carrier protein 1—Glycosylation fragment—Carrier protein 2—His-Tag INTERNAL Leader Sequence—Carrier protein 1—Glycosylation fragment—Carrier protein 1—His-Tag [0084] As shown above, in certain embodiments of internal placement, the glycosylation fragment, polypeptide tag, or the like can be placed (translationally fused) between separate carrier proteins (even if the same type of carrier protein). In certain embodiments of an internal placement, the glycosylation fragment, polypeptide tag, or the like can be placed (translationally fused) internally within the sequence of a single carrier protein. [0085] Without being limited to any particular sequence, representative examples of one polypeptide tag partner of a pair (generally referred to herein as the first polypeptide tag) include SpyTag (SEQ ID NO: 416), SpyTag002 (SEQ ID NO: 417), SpyTag003 (SEQ ID NO: 418), or a DogTag (SEQ ID NO: 419). In certain embodiments, the SpyTag, Spytag002, or Spytag003 is translationally fused at the N-terminal end of the fusion protein (e.g., Figure 2). In certain embodiments, the SpyTag, Spytag002, or Spytag003 is translationally fused at the C-terminal end of the fusion protein (e.g., Figure 5). In certain embodiments, the DogTag is translationally fused internally within the fusion protein. [0086] Representative examples of fusion proteins of this disclosure are shown in Figure 2, Figure 5, Figure 7, Figure 8, and Figure 9. For example EPA-Spytag-v1 (SEQ ID NO: 427), EPA-Spytag-v2 (SEQ ID NO: 428), and EPA-Spytag-v3 (SEQ ID NO: 429). In certain embodiments, an amino acid linker sequence is translationally inserted between components of the fusion protein (e.g., a signal peptide, a polypeptide tag sequence, a glycosylation fragment, a carrier protein, a histidine tag, etc.). In certain embodiments, the amino acid linker sequence is GGS, GGGGGG (SEQ ID NO: 430), GGGGGGGG (SEQ ID NO: 431), GGGGS (SEQ ID NO: 432), EAAAK (SEQ ID NO: 433), PAPAPPAPAP (SEQ ID NO: 434), EAAAKEAAAK (SEQ ID NO: 435), GGGGSPAPAP (SEQ ID NO: 436), GGGGSGGGGS (SEQ ID NO: 437), or EAAAKGGGGS (SEQ ID NO: 438). Thus, certain embodiments comprise an EPA-Spytag-v1 of SEQ ID NO: 427, an EPA-Spytag-v2 of SEQ ID NO: 428, or an EPA-Spytag-v3 of SEQ ID NO: 429, having one or more amino acid linkers, such as above, translationally inserted between components of the fusion protein. For example an EPA- Spytag-v1 of SEQ ID NO: 427, having the amino acid linker SSG translationally inserted after the polypeptide tag. For example an EPA-Spytag-v1 of SEQ ID NO: 440, having the amino 32080280 - 23 - Atty. Dkt. No.: 64100-234947 acid linker EAAAKEAAAK (SEQ ID NO: 435) translationally inserted after the polypeptide tag. It is contemplated that in certain embodiments, other amino acid linkers, such as but not limited to those disclosed herein, could be similarly situated. ComP glycosylation fragments [0087] Without being limited to any particular glycosylation sequence, in certain embodiments, a glycosylation fragment is a ComP glycosylation fragment. A sequence comprising a glycosylation fragment of ComP could be a full-length ComP protein. In certain embodiments, the ComP glycosylation fragment comprises or consists of the amino acid sequence CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 412) or a fragment thereof comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412. In certain embodiments, the ComP glycosylation fragment comprises or consists of the amino acid sequence CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 412) or a fragment thereof at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids in length comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412. In certain embodiments, the ComP glycosylation fragment comprises or consists of the amino acid sequence CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 412) or a fragment thereof at least 10 amino acids in length comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412. In certain embodiments, the ComP glycosylation fragment comprises or consists of the amino acid sequence CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 412) or a fragment thereof at least 11 amino acids in length comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412. [0088] In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. One of ordinary skill in the art would understand in any of the embodiments of a glycosylation fragment of this disclosure, for example, if an addition occurs at a certain position, and a deletion occurs at a different position, the net effect on the length of the sequence is zero, etc. Further, multiple additions or deletions can be consecutive and/or non-consecutive. And, in certain embodiments, substitutions can be conservative amino acid substitutions. In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having cumulatively one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. 32080280 - 24 - Atty. Dkt. No.: 64100-234947 [0089] In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions and/or additions. In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or additions. [0090] In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions and/or deletions. In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or deletions. [0091] In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid additions and/or deletions. In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having cumulatively one, two, three, four, five, or six amino acid additions and/or deletions. [0092] In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions. In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid additions. In certain embodiments, the ComP glycosylation fragment comprises or consists of a variant of SEQ ID NO: 412 comprising the amino acids ASA in positions 11 to 13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid deletions. [0093] In certain embodiments, the ComP glycosylation fragment comprises or consists of any of the further ComP glycosylation fragment sequences described elsewhere herein. 32080280 - 25 - Atty. Dkt. No.: 64100-234947 TfpM-associated pilin glycosylation fragments [0094] Without being limited to any particular glycosylation sequence, in certain embodiments, the glycosylation fragment is a TfpM-associated pilin glycosylation fragment. A sequence comprising a glycosylation fragment of a TfpM-associated pilin could be a full- length TfpM-associated pilin protein. In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (SEQ ID NO: 413) or a fragment thereof comprising at least the last three amino acids from the TfpM- associated pilin C-terminus (i.e., RGT). In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (SEQ ID NO: 413) or a fragment thereof at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids in length comprising at least the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT). [0095] In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO:) 413 comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having cumulatively one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. [0096] In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid substitutions and/or additions. In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or additions. [0097] In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid substitutions and/or deletions. In certain 32080280 - 26 - Atty. Dkt. No.: 64100-234947 embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or deletions. [0098] In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid additions and/or deletions. In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having cumulatively one, two, three, four, five, or six amino acid additions and/or deletions. [0099] In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid substitutions. In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid additions. In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of a variant of the PilMo pilin disulfide loop region (SEQ ID NO: 413) comprising the last three amino acids from the TfpM-associated pilin C-terminus (i.e., RGT) and having one, two, three, four, five, or six amino acid deletions. [0100] In certain embodiments, the TfpM-associated pilin glycosylation fragment comprises or consists of any of the further TfpM-associated pilin glycosylation fragment sequences described elsewhere herein. PilE glycosylation fragments [0101] Without being limited to any particular glycosylation sequence, in certain embodiments the glycosylation fragment is a PilE glycosylation fragment. A sequence comprising a glycosylation fragment of PilE could be a full-length PilE protein. In certain embodiments, the PilE glycosylation fragment comprises or consists of the amino acid SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) or a fragment thereof comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 32080280 - 27 - Atty. Dkt. No.: 64100-234947 of SEQ ID NO: 414. In certain embodiments, the PilE glycosylation fragment comprises or consists of the amino acid SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) or a fragment thereof at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 amino acids in length comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414. [0102] In certain embodiments, the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. In certain embodiments, the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having cumulatively one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. [0103] In certain embodiments, the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid substitutions and/or additions. In certain embodiments, the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or additions. [0104] In certain embodiments, the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid substitutions and/or deletions. In certain embodiments, the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or deletions. 32080280 - 28 - Atty. Dkt. No.: 64100-234947 [0105] In certain embodiments, the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid additions and/or deletions. In certain embodiments, the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having cumulatively one, two, three, four, five, or six amino acid additions and/or deletions. [0106] In certain embodiments, the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid substitutions. In certain embodiments, the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid additions. In certain embodiments, the PilE glycosylation fragment comprises or consists of a variant of SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid deletions. PglB glycosylation fragments [0107] Without being limited to any particular glycosylation sequence, in certain embodiments the glycosylation fragment is a PglB glycosylation fragment. A sequence comprising a glycosylation fragment of PglB could be a full-length PglB protein. In certain embodiments, the PglB glycosylation fragment comprises or consists of the consensus motif amino acid sequence X1 X2 N X3 X4, wherein X1 is D or E, X2 is any amino acid except proline, X3 is any amino acid except proline, and X4 is S or T. PilA glycosylation fragments [0108] Without being limited to any particular glycosylation sequence, in certain embodiments the glycosylation fragment is a PilA glycosylation fragment. A sequence comprising a glycosylation fragment of PilA could be a full-length PilA protein. In certain embodiments, the PilA glycosylation fragment comprises or consists of the PilA pilin disulfide 32080280 - 29 - Atty. Dkt. No.: 64100-234947 loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) or a fragment thereof comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS). In certain embodiments, the PilA glycosylation fragment comprises or consists of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) or a fragment thereof at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids in length comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS). [0109] In certain embodiments, the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. In certain embodiments, the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having cumulatively one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. [0110] In certain embodiments, the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having one, two, three, four, five, or six amino acid substitutions and/or additions. In certain embodiments, the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or additions. [0111] In certain embodiments, the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having one, two, three, four, five, or six amino acid substitutions and/or deletions. In certain embodiments, the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having cumulatively one, two, three, four, five, or six amino acid substitutions and/or deletions. [0112] In certain embodiments, the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID 32080280 - 30 - Atty. Dkt. No.: 64100-234947 NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having one, two, three, four, five, or six amino acid additions and/or deletions. In certain embodiments, the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having cumulatively one, two, three, four, five, or six amino acid additions and/or deletions. [0113] In certain embodiments, the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having one, two, three, four, five, or six amino acid substitutions. In certain embodiments, the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having one, two, three, four, five, or six amino acid additions. In certain embodiments, the PilA glycosylation fragment comprises or consists of a variant of the PilA pilin disulfide loop region CKITKTPTAWKPNYAPANCPKS (SEQ ID NO: 415) comprising at least the last three amino acids from the PilA C-terminus (i.e., PKS) and having one, two, three, four, five, or six amino acid deletions. PilA Pa5196 glycosylation fragments [0114] Without being limited to any particular glycosylation sequence, in certain embodiments the glycosylation fragment is a PilA_Pa5196-associated pilin glycosylation fragment. A sequence comprising a glycosylation fragment of PilA_Pa5196-associated pilin could be a full-length PilA_Pa5196-associated pilin protein. In certain embodiments, the PilA_Pa5196-associated pilin glycosylation fragment comprises or consists of the strands 1 and 2 of the antiparallel beta-sheet domain of PilA_Pa5196 GKYSSVDSTIASGYPNGQITVTMTQG (SEQ ID NO: 426) or a fragment thereof. In certain embodiments, the PilA_Pa5196-associated pilin glycosylation fragment comprises or consists of the strands 1 and 2 of the antiparallel beta-sheet domain of PilA_Pa5196 GKYSSVDSTIASGYPNGQITVTMTQG (SEQ ID NO: 426) or a fragment thereof at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. [0115] In certain embodiments, the glycosylation fragment is a variant of PilA_Pa5196- associated pilin glycosylation fragment consistent with the variants of other glycosylation 32080280 - 31 - Atty. Dkt. No.: 64100-234947 fragments disclosed herein having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. STT3 glycosylation fragments [0116] Without being limited to any particular glycosylation sequence, in certain embodiments the glycosylation fragment is a STT3 glycosylation fragment. A sequence comprising a glycosylation fragment of STT3 could be a full-length STT3 protein. In certain embodiments, the STT3 glycosylation fragment comprises or consists of the consensus motif amino acid sequence N-X-S/T, wherein X is any amino acids except proline and S/T is serine (S) or threonine (T). N-linking glycosyltransferase glycosylation fragments [0117] Without being limited to any particular glycosylation sequence, in certain embodiments the glycosylation fragment is a N-linking glycosyltransferase glycosylation fragment. In certain embodiments, the N-linking glycosyltransferase glycosylation fragment comprises or consists of the consensus motif amino acid sequence N-X-S/T, wherein X is any amino acids except proline and S/T is serine (S) or threonine (T). O-linking glycosyltransferase glycosylation fragments [0118] Without being limited to any particular glycosylation sequence, in certain embodiments the glycosylation fragment is an O-linking glycosyltransferase glycosylation fragment. In certain embodiments, the O-linking glycosyltransferase glycosylation fragment comprises or consists of a fragment of the serine or threonine rich repeats from the serine-rich repeats (SRR) adhesins of streptococci or staphylococci bacteria. In certain embodiments, the O-linking glycosyltransferase glycosylation fragment comprises or consists of serine (S) or threonine (T) rich repeats from the adhesin GspB from Streptococcus gordonii. Multiple glycosylation fragments [0119] Certain aspects of this disclosure are drawn to fusion proteins comprising two or more glycosylation fragments. For example, wherein the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 glycosylation fragments. In certain embodiments, the fusion protein comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 glycosylation fragments to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 glycosylation fragments. In certain embodiments, at least one glycosylation fragment is located at the N-terminal end of the fusion protein and at least one glycosylation fragment is located internally within the fusion protein. In certain embodiments, at least one glycosylation fragment is located at the C-terminal end of 32080280 - 32 - Atty. Dkt. No.: 64100-234947 the fusion protein and at least one glycosylation fragment is located internally within the fusion protein. In certain embodiments, at least two glycosylation fragments are located internally within the fusion protein. Further, in certain embodiments, one glycosylation fragment is located at the N-terminal end of the fusion protein and one glycosylation fragment is located at the C-terminal end of the fusion protein. In certain embodiments, the two or more glycosylation fragments are the same. For example, a fusion protein having multiple ComP glycosylation fragments. In certain embodiments, at least one of the two or more glycosylation fragments is different or each of the glycosylation fragments is different. For example, a fusion protein wherein one glycosylation fragment is a ComP glycosylation fragment and one glycosylation fragment is a TfpM-associated pilin glycosylation fragment. [0120] In certain embodiments, the fusion protein is a glycoconjugate comprising two or more saccharide covalently attached to the fusion protein via the two or more glycosylation fragments. In certain embodiments, the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 covalently attached saccharides. In certain embodiments, the fusion protein comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 covalently attached saccharides to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 covalently attached saccharides. In certain embodiments, the two or more saccharides are the same. In certain embodiments, at least one of the two or more saccharides is different or each of the saccharides is different. [0121] As can be appreciated, the ability to create fusion proteins with multiple glycosylation fragments, and further that can be recognized by different glycosylating enzymes and/or attaching different saccharides, in addition to combining different fusion proteins within the same complex, allows for the mixing and matching and amplification of various immunogenic components. [0122] In certain embodiments, the fusion protein comprises a carrier protein. Various carrier proteins have been used in glycoconjugate vaccines and all are contemplated herein. For example, in certain embodiments the carrier protein is selected from the group consisting of Escherichia coli maltose binding protein, Pseudomonas aeruginosa Exotoxin A (EPA), Pseudomonas aeruginosa PcrV, CRM197, Haemophilus influenzae Protein D, cholera toxin B subunit, or tetanus toxin, and a fragment of any thereof. Polypeptide pair [0123] Certain aspects of this disclosure are drawn to a composition comprising a polypeptide pair that comprises a first polypeptide and a second polypeptide. The first 32080280 - 33 - Atty. Dkt. No.: 64100-234947 polypeptide comprises a first polypeptide tag that is a binding partner to the second polypeptide tag of the second polypeptide. In certain embodiments, the first polypeptide is a fusion protein of this disclosure comprising a glycosylation fragment as described in detail elsewhere herein. The second polypeptide comprises a second polypeptide tag binding partner to the first polypeptide tag of the first polypeptide. The first polypeptide can be attached to the second polypeptide via an isopeptide bond between the first polypeptide tag and the second polypeptide tag. In certain embodiments, the second polypeptide comprises a monomeric polypeptide that can spontaneously multimerize/self-assemble into a higher-order, multimeric structure. For example, an icosahedron or dodecahedron particle (e.g,. resembling nanocages), virus-like particle (VLP), or Adenoviral vector. In certain embodiments, the second polypeptide comprises an Adenoviral capsid structural protein. In certain embodiments, the second polypeptide comprises a coat protein of the bacteriophage AP205. In certain embodiments, the second polypeptide comprises a fragment of the 2-keto-3-deoxy- phosphogluconate aldolase (i301). In certain embodiments, the second polypeptide comprises a fragment of a mutated 2-keto-3-deoxy-phosphogluconate aldolase (mi3). In certain embodiments, the polypeptide tag of the second polypeptide (second polypeptide tag) is a SpyCatcher (SEQ ID NO: 420). In certain embodiments, the second polypeptide tag is a SpyCatcher002 (SEQ ID NO: 421). In certain embodiments, the second polypeptide tag is a SpyCatcher003 (SEQ ID NO: 422). In certain embodiments, the second polypeptide tag a DogCatcher (SEQ ID NO: 423). [0124] The polypeptide tag of the second polypeptide can be located on the second polypeptide at an end (N-terminal or C-terminal end) or internally. In certain embodiments, the second polypeptide tag is translationally fused at the N-terminal end of the second polypeptide (e.g., Figure 2). In certain embodiments, the second polypeptide tag is translationally fused at the C-terminal end of the second polypeptide. In certain embodiments, the second polypeptide tag is translationally fused internally within the second polypeptide. [0125] In certain embodiments, the first polypeptide (e.g., a fusion protein of this disclosure) is a bioconjugate comprising a saccharide covalently attached to the glycosylation fragment of the first polypeptide. [0126] In certain embodiments, the composition comprising a polypeptide pair is immunogenic, e.g., wherein the first polypeptide is a bioconjugate comprising a saccharide covalently attached to the glycosylation fragment of the first polypeptide. In certain embodiments, the polypeptide pair composition further comprises an adjuvant and/or an 32080280 - 34 - Atty. Dkt. No.: 64100-234947 excipient. Example adjuvants could include, but are not limited to: alum (aluminum hydroxide gel or aluminum phosphate gel), squalene emulsions (e.g., MF59, AddaS03, or AddaVax), lipid A derivatives such as monophosphoryl lipid A (MPLA), or saponins (e.g., Quil-A). In certain embodiments, the polypeptide pair composition is a pharmaceutical and/or therapeutic composition. In certain embodiments, the polypeptide pair composition is a conjugate vaccine. [0127] Certain aspects provide for a method of making the polypeptide pair of this disclosure. This can be done by contacting a first polypeptide (e.g., a fusion protein of this disclosure) and a second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with its corresponding second polypeptide tag binding partner. In certain embodiments, the method further comprises glycosylating the first polypeptide with a saccharide before contact and isopeptide bond formation with the second polypeptide (e.g., Figure 1). In certain embodiments, the first polypeptide is glycosylated in vivo (such as in a host cell, e.g., in bacteria) before contact and isopeptide bond formation with the second polypeptide. In certaine embodiments, the method comprises isolating/purifying the in vivo glycosylated first polypeptide before contact and isopeptide bond formation with the second polypeptide. Alternatively, in certain embodiments, the first polypeptide is glycosylated after contact and isopeptide bond formation with the second polypeptide. Complexes [0128] Provided for in this disclosure is a complex comprising two or more of the polypeptide pairs disclosed herein. In certain embodiments, an individual complex comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250 or more complexed polypeptide pairs of this disclosure. In certain embodiments, an individual complex comprises from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, or 250 complexed polypeptide pairs of this disclosure to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, or 500 complexed polypeptide pairs of this disclosure. In certain embodiments, such a complex is a self-assembled, multimeric higher- order structure. In certain embodiments, such a self-assembled, multimeric higher-order structure is an icosahedron or dodecahedron particle (e.g,. resembling nanocages), virus-like particle, or Adenoviral vector. [0129] Because it is the polypeptide tag of the first polypeptide, and not the glycosylation fragment or the carrier protein, e.g., of a fusion protein, that determines it partner polypeptide, 32080280 - 35 - Atty. Dkt. No.: 64100-234947 the second polypeptide is not limited to partnering with just on type of first polypeptide (and vice versa). In certain embodiments, all of the first polypeptides of the complex comprise the same fusion protein. In certain embodiments, however, the first polypeptides can comprises different fusion proteins. In certain embodiments, at least two, three, four, five, or more of the first polypeptides of the complex comprise different fusion proteins. In certain embodiments, at least two of the first polypeptides of the complex comprise different fusion proteins. In certain embodiments, two, three, four, five, or six of the first polypeptides of the complex comprise different fusion proteins. In certain embodiments, all of the first polypeptides of the complex are different fusion proteins. [0130] In certain embodiments, at least one first polypeptide of the complex is a bioconjugate comprising a saccharide covalently attached to the glycosylation fragment of the first polypeptide. In certain embodiments, at least about 5%, 10%, 25%, 50%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% of the first polypeptides of the complex are bioconjugates. In certain embodiments, from any of about 5%, 10%, 25%, 50%, 75%, 80%, 90%, 95%, 97%, or 98% of the first polypeptides of the complex to about any of about 10%, 25%, 50%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% of the first polypeptides of the complex are bioconjugates. In certain embodiments, about 100% or 100% of the first polypeptides of the complex are bioconjugates. [0131] In certain embodiments, two or more of the first polypeptides of the complex are bioconjugates comprising a covalently attached saccharide. Because of the number of first polypeptides in the complex, the number of covalently attached saccharides can be numerous and will depend on the number of first polypeptide/second polypeptide pairs in the complex and the number of saccharides attached to each first polypeptide. For example, the AP205 VLP comprises about 180 first polypeptide binding partners (e.g., SpyCatcher) per VLP. In theory if 100% are isopeptide-bonded to a first polypeptide that is a bioconjugate, that would allow 180 bioconjugates per VLP. And, as described elsewhere herein, each bioconjugate can be covalently attached to multiple saccharides. Mi3 is lower, about 60 first polypeptide binding partners per NP. In certain embodiments, the complex comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 2,000, 2,500, or 5,000 covalently attached saccharides. In certain embodiments, the complex comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 2,000, or 2,500 covalently attached saccharides to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 32080280 - 36 - Atty. Dkt. No.: 64100-234947 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 2,000, 2,500, or 5,000 covalently attached saccharides. In certain embodiments, the complex comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, or 750 covalently attached saccharides. In certain embodiments, the complex comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, or 500 covalently attached saccharides to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, or 750 covalently attached saccharides. In certain embodiments, all of the saccharides attached to the complex are the same. In certain embodiments, at least two, three, four, five, or more saccharides attached to the complex are different. In certain embodiments, at least two saccharides attached to the complex are different. In certain embodiments, two, three, four, five, or six saccharides attached to the complex are different from each other. In certain embodiments, each of the saccharides attached to the complex is different. [0132] In certain embodiments of the complex, such as where it is attached to a saccharide, the complex is immunogenic. [0133] Certain embodiments provide for a pharmaceutical and/or therapeutic composition comprising a complex of this invention and an adjuvant and/or an excipient. In certain embodiments, the complex is a conjugate vaccine. [0134] Certain aspects provide for a method of making the complex of this invention. This can be done by forming a self-assembled, multimeric higher-order structure of a second polypeptide of this disclosure (e.g., an icosahedron or dodecahedron particle (e.g,. resembling nanocages), virus-like particle (VLP), or Adenoviral vector) and then contacting the first polypeptide of this disclosure (e.g., a fusion protein comprising a glycosylation fragment) and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag (e.g., Figure 1), This can also be done by contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag and then forming of a self-assembled, multimeric higher-order structure of the second polypeptide. It will be understood that such methods encompass numerous possibilities and combinations for glycosylating the first polypeptide of the complex with a saccharide, all of which are contemplated herein. For example, the first polypeptide can be glycosylated before the isopeptide bond is formed between the first polypeptide and the second polypeptide (e.g., 32080280 - 37 - Atty. Dkt. No.: 64100-234947 Figure 1). The first polypeptide can be glycosylated after the isopeptide bond is formed between the first polypeptide and the second polypeptide. The first polypeptide can be glycosylated before it is incorporated into a multimeric, higher-order structure (e.g., Figure 1). And, the first polypeptide can be glycosylated after is has been incorporated into a multimeric, higher-order structure. Glycosylation [0135] The glycosylated residue, glycosylation site, glycosylation fragment, sequon, first polypeptide fusion protein, complex, or the like of this disclosure can be covalently linked to a saccharide by any of numerous method of glycosylation including, but not limited to, the following illustrative examples. In certain embodiments, a saccharide is transferred to a fusion protein comprising a glycosylation fragment by the action of an N-linking oligosaccharyltransferase (N-OTase), an O-linking oligosaccharyltransferase (O-Otase), an N- linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C- mannosyltransferase (CMT). In certain embodiments, a saccharide is transferred to a fusion protein comprising a glycosylatin fragment by the action of a PglS OTase, a TfpM OTase, a PglL OTase, a PglB OTase, a TfpO/PilO OTase, a STT3 OTase, a TfpW glycosyltransferase, and/or an AlgB OTase. For example, in certain embodiments, the glycosylation fragment is a ComP glycosylation fragment glycosylated by a PglS OTase. In certain embodiments, the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a PglS OTase. In certain embodiments, the glycosylation fragment is a TfpM-associated pilin glycosylation fragment glycosylated by a TfpM OTase. In certain embodiments, the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpM OTase. In certain embodiments, the glycosylation fragment is a PilE glycosylation fragment glycosylated by a PglL OTase. In certain embodiments, the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a PglL OTase. In certain embodiments, the glycosylation fragment is a PglB glycosylation fragment glycosylated by a PglB OTase. In certain embodiments, the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using a PglB OTase. In certain embodiments, the glycosylation fragment is a PilA glycosylation fragment glycosylated by a TfpO or PilO OTase. In certain embodiments, the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpO or PilO OTase. In certain embodiments, the glycosylation fragment is a STT3 glycosylation fragment glycosylated by the STT3 catalytic subunit. In certain embodiments, the saccharide is covalently linked to a nitrogen atom within a glycosylation 32080280 - 38 - Atty. Dkt. No.: 64100-234947 fragment using a STT3 OTase. In certain embodiments, the glycosylation fragment is a PilA_Pa5196-associated pilin glycosylation fragment glycosylated by a TfpW glycosyltransferase. In certain embodiments, the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpW glycosyltransferase. In certain embodiments, the glycosylation fragment is an Archaeal AlgB glycosylation fragment glycosylated by an AlgB OTase. In certain embodiments, the saccharide is covalently linked to a nitrogen atom within the Archaeal AlgB glycosylation fragment glycosylation fragment using an AlgB OTase. In certain embodiments, the glycosylation fragment is an N-linking glycosyltransferase glycosylation fragment glycosylated by an N-linking glycosyltransferase, for example from Actinobacillus pleuropneumoniae, from Haemophilus influenzae, or from Yersinia enterocolitica. In certain embodiments, the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using an N-linking glycosyltransferase. In certain embodiments, the glycosylation fragment is an O-linking glycosyltransferase glycosylation fragment glycosylated by an O-linking glycosyltransferase, for example GtfA/GtfB glycosyltransferases. In certain embodiments, the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using an O-linking glycosyltransferase. Further in certain embodiments, the saccharide is covalently linked to a carbon atom within a glycosylation fragment using a C-mannosyltransferase. [0136] Further, illustratively, in certain embodiments, the saccharide is covalently linked to an oxygen atom within a ComP glycosylation fragment (e.g., SEQ ID NO: 412 or a variant thereof) using a PglS OTase (e.g., SEQ ID NO: 400). In certain embodiments, the saccharide is covalently linked to an oxygen atom within a TfpM glycosylation fragment (e.g., SEQ ID NO: 413 or a variant thereof) using a TfpM OTase (e.g., SEQ ID NO: 402). In certain embodiments, the saccharide is covalently linked to an oxygen atom within a PilE glycosylation fragment (e.g., SEQ ID NO: 414 or a variant thereof, e.g., SEQ ID NO: 439) using a PglL OTase (e.g., SEQ ID NO: 404). In certain embodiments, the saccharide is covalently linked to an oxygen atom within a PilE glycosylation fragment (e.g., SEQ ID NO: 414 or a variant thereof) using a PglL OTase (e.g., SEQ ID NO: 404). In certain embodiments, the saccharide is covalently linked to a nitrogen atom within a PglB glycosylation fragment using a PglB Otase (e.g., SEQ ID NO: 405). In certain embodiments, the saccharide is covalently linked to an oxygen atom within a PilA glycosylation fragment (e.g., SEQ ID NO: 415 or a variant thereof) using a TfpO/PilO Otase (e.g., SEQ ID NO: 407). In certain embodiments, the saccharide is covalently linked to a nitrogen atom within a STT3 32080280 - 39 - Atty. Dkt. No.: 64100-234947 glycosylation fragment using a STT3 Otase (e.g., SEQ ID NO: 408). In certain embodiments, the saccharide is covalently linked to a nitrogen atom within an Archaeal AlgB glycosylation fragment using an AlgB Otase (e.g., SEQ ID NO: 409). In certain embodiments, the saccharide is covalently linked to an oxygen atom within PilA_Pa5196-associated pilin glycosylation fragment (e.g., SEQ ID NO: 426 or a variant thereof) using a TfpW glycosyltransferase (e.g., SEQ ID NO: 424). In certain embodiments, the saccharide is covalently linked to a nitrogen atom within a N-linking glycosyltransferase sequon using an N-linking glycosyltransferase (e.g., SEQ ID NO: 410). In certain embodiments, the saccharide is covalently linked to an oxygen atom within an O-linking glycosyltransferase sequon using an O-linking glycosyltransferase (e.g., SEQ ID NO: 411). In certain embodiments, the saccharide is covalently linked to a carbon atom within a C-mannosyltransferase glycosylation fragment using a C-mannosyltransferase. [0137] In certain embodiments, the method is a method of producing a conjugate vaccine. This can involve adding to a partner pair and/or complex of this disclosure an adjuvant and/or an excipient. [0138] A further aspect provides for a system comprising the first polypeptide and the second polypeptide of the composition of this disclosure. In certain embodiments, the first polypeptide is a glycosylated bioconjugate. In certain embodiments, the system comprises a multimeric, higher-order structure assembled of the second polypeptide. In certain embodiments, the system comprises a saccharide and an N-linking oligosaccharyltransferase (N-Otase), an O-linking oligosaccharyltransferase (O-OTase), an N-linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C- mannosyltransferase (CMT) as disclosed herein. [0139] Another aspect provides for an isolated nucleic acid encoding the first polypeptide and/or the second polypeptide of the composition and/or the complex of this disclosure. Certain embodiments are directed to a vector comprising the isolated nucleic acid. Certain embodiments are directed to a host cell comprising the vector. [0140] Another aspect provides for a kit comprising two or more components comprising the fusion protein, the first polypeptide, the second polypeptide, a saccharide, an N-linking oligosaccharyltransferase (N-OTase), an O-linking oligosaccharyltransferase (O-OTase), an N-linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C- mannosyltransferase (CMT), the bioconjugate, the multimeric, higher-order structure 32080280 - 40 - Atty. Dkt. No.: 64100-234947 assembled from the second polypeptide, the isolated nucleic acid, the vector, and the host cell of this disclosure. [0141] Another aspect provides for a method of eliciting an immune response in a subject by administering to said subject an effective amount of any composition, complex, and/or conjugate vaccine of this disclosure. Further provided for is a composition, complex, and/or conjugate vaccine of this disclosure for use in eliciting an immune response in a subject. [0142] In certain embodiments, a composition or complex disclosed herein is a conjugate vaccine that can be administered to a subject for the prevention and/or treatment of an infection and/or disease. In certain embodiments, the conjugate vaccine is a prophylaxis that can be used, e.g., to immunize a subject against an infection and/or disease. In certain embodiments, the glycoconjugate is associated with (such as in a therapeutic composition) and/or administered with an adjuvant. Certain embodiments provide for a composition (such as a therapeutic composition) comprising a 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 elicits long term memory (memory B and T cells). In certain embodiments, the immune is an antibody response. In certain embodiments, the antibody response is a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG or IgM response. In certain embodiments where the antibody response is an IgG response, the IgG response is an IgG1 response. Further, in certain embodiments, the conjugate vaccine generates immunological memory in a subject administered the vaccine. [0143] Certain embodiments also provide for producing a vaccine against an infection and/or disease. In certain embodiments a method comprises isolating a glycoconjugate or fusion protein disclosed herein (conjugate vaccine) and combining the conjugate vaccine with an adjuvant. In certain embodiments, the infection is a localized or systemic infection of skin, soft tissue, blood, or an organ, or is auto-immune 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 infection is a systemic infection and/or an infection of the blood. In certain embodiments, the subject is a mammal. For example, in certain embodiments, a pig or a human. [0144] Importantly, the aspects disclosed herein are not limited to pneumococcal polysaccharides, but in fact, have vast applicability for generating bioconjugate vaccines for many important human and animal pathogens that are incompatible with PglB and PglL. 32080280 - 41 - Atty. Dkt. No.: 64100-234947 Notable examples include the human pathogens Klebsiella pneumoniae and Group B Streptococcus as well as the swine pathogen S. suis, all immensely relevant pathogens with no licensed vaccines available. [0145] Provided herein are methods of inducing a host immune response against a pathogen. In certain embodiments, the pathogen is a bacterial pathogen. In certain embodiments, the host is immunized against the pathogen. In certain embodiments, the method comprises administering to a subject in need of the immune response an effective amount of a ComP conjugate vaccine, glycosylated fusion protein, or any other therapeutic/immunogenic composition disclosed herein. Certain embodiments provide a conjugate vaccine, glycosylated fusion protein, or other therapeutic/immunogenic composition disclosed herein for use in inducing a host immune response against a bacterial pathogen and immunization against the bacterial pathogen. Examples of immune responses include but are not limited to an innate response, an adaptive response, a humoral response, an antibody response, cell mediated response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system cross-talk, and a combination of two or more of said 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 said immune responses. [0146] Also provided herein are methods of preventing or treating a bacterial disease and/or infection in a subject comprising administering to a subject in need thereof a conjugate vaccine, a fusion protein, or a composition disclosed herein. In certain embodiments, the infection is a localized or systemic infection of skin, soft tissue, blood, or an organ, or is auto- immune in nature. In certain embodiments, the disease is pneumonia. In certain embodiments, the infection 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, llama, etc. And, for example, in certain embodiments the mammal is a human. [0147] In any of the embodiments of administration disclose herein, the composition is administered via intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration. [0148] In certain embodiments, the glycoconjugate, glycosylated fusion protein, or conjugate vaccine of any of the above claims for use in inducing a host immune response 32080280 - 42 - Atty. Dkt. No.: 64100-234947 against a bacterial pathogen and/or preventing or treating a bacterial disease and/or infection in a subject. Minimal Sequons Sufficient for O-Linking Glycosylation [0149] Traditional, chemical conjugate vaccine synthesis is considered complex, costly, and laborious (Frasch, C.E. Vaccine 27, 6468-6470 (2009)) however, in vivo conjugation has been thoroughly progressing as a viable biosynthetic alternative (Huttner, A. et al. Lancet Infect Dis 17, 528-537 (2017)). These strides are best highlighted by the successes of GlycoVaxyn, (now LimmaTech Biologics AG an independent company with direct ties to GlaxoSmithKline), a clinical stage biopharmaceutical company with multiple bioconjugate vaccines in various phases of clinical trials, one of which (Flexyn2a) has just completed a Phase 2b challenge study. Although GlycoVaxyn has been 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, expectedly, has stymied the development of a pneumococcal bioconjugate vaccine. [0150] The oligosaccharyltransferase PglS – previously referred to as PglL by Schulz et al. (PMID 23658772) and PglLComP by Harding et al.2015 (PMID 26727908) – was only recently characterized as a functional OTase (Schulz, B.L. et al. PLoS One 8, e62768 (2013)). Subsequent mass spectrometry studies on total glycopeptides demonstrated that PglS does not act as a general PglL-like OTase, glycosylating multiple periplasmic and outer membrane proteins (Harding, C.M. et al. Mol Microbiol 96, 1023-1041 (2015)). In fact, the genome of A. baylyi ADP1 encodes for two OTase, a PglL-like ortholog (UniProtKB/Swiss-Prot: Q6FFS6.1), which acts as the general OTase and PglS (UniProtKB/Swiss-Prot: Q6F7F9.1), which glycosylates a single protein, ComP (Harding, C.M. et al. Mol Microbiol 96, 1023-1041 (2015)). [0151] ComP is orthologous to type IV pilin proteins, like PilA from Pseudomonas aeruginosa and PilE from Neisseria meningiditis, both of which are glycosylated by the OTases TfpO (Castric, P. Microbiology 141 ( Pt 5), 1247-1254 (1995)) and PglL (Power, P.M. et al. Mol Microbiol 49, 833-847 (2003)), respectively. Although TfpO and PglL also glycosylate their cognate pilins at serine residues, the sites of glycosylation differ between each system. TfpO glycosylates its cognate pilin at a C-terminal serine residue (Comer, J.E., Marshall, M.A., Blanch, V.J., Deal, C.D. & Castric, P. Infect Immun 70, 2837-2845 (2002)), which is not present in ComP. PglL glycosylates PilE at an internal serine located at position 63 (Stimson, E. et al. Mol Microbiol 17, 1201-1214 (1995)). ComP also contains serine residues near position 63 and 32080280 - 43 - Atty. Dkt. No.: 64100-234947 the surrounding residues show moderate conservation to PilE from N. meningiditis. Comprehensive glycopeptide analysis, however, revealed this serine and the surrounding residues were not the site of glycosylation in ComP. PglS glycosylates ComP at a single serine residue located at position corresponding to the conserved serine at position 82 of ComP110264: ENV58402.1 (SEQ ID NO: 201) (also corresponding to the conserved serine at position 84 of ComPADP1: AAC4588631 (SEQ ID NO: 202)), which is a novel glycosylation site not previously found within the type IV pilin superfamily. The ability of PglS to transfer polysaccharides containing glucose as the reducing end sugar coupled with the identification of a novel site of glycosylation within the pilin superfamilies demonstrates that PglS is a functionally distinct OTase from PglL and TfpO. Bioinformatic features of ComP pilin orthologs [0152] ComP was first described as a factor required for natural transformation in Acinetobacter baylyi ADP1 (Porstendorfer, D., Drotschmann, U. & Averhoff, B. Appl Environ Microbiol 63, 4150-4157 (1997)). In a subsequent study, it was demonstrated that ComP from A. baylyi ADP1 (herein referred to as ComPADP1) was glycosylated by a novel OTase, PglS, located immediately downstream of ComP, and not the general OTase PglL located elsewhere on the chromosome (Harding, C.M. et al. Mol Microbiol 96, 1023-1041 (2015)). The ComPADP1 protein (NCBI identifier AAC45886.1) belongs to a family of proteins called type IV pilins. Specifically, ComP shares homology to type IVa major pilins (Giltner, C.L., Nguyen, Y. & Burrows, L.L. Microbiol Mol Biol Rev 76, 740-772 (2012)). Type IVa pilins share high sequence homology at their N-terminus, which encode for the highly conserved leader sequence and N-terminal alpha helix; however, the C-terminus display remarkable divergences across genera and even within species (Giltner, C.L., Nguyen, Y. & Burrows, L.L. Microbiol Mol Biol Rev 76, 740-772 (2012)). To help differentiate ComP orthologs from other type IVa pilin proteins, such as, PilA from A. baumannii, P. aeruginosa, and Haemophilus influenzae as well as PilE from Neisseria species (Pelicic, V. Mol Microbiol 68, 827-837 (2008)), a BLASTp analysis was performed comparing the primary amino acid sequence of ComPADP1 against all proteins from bacteria in the Acinetobacter genus. Expectedly, many Acinetobacter type IVa pilin orthologs, including ComPADP1, share high homology at their N-termini; however, very few proteins display high sequence conservation across the entire amino acid sequence of ComP. At least six ComP orthologs (Figure 20) were identified based on the presence of the conserved serine at position 84 relative to ComPADP1 as well as a conserved disulfide bond flanking the site of predicted glycosylation connecting the predicted alpha beta loop to the beta 32080280 - 44 - Atty. Dkt. No.: 64100-234947 strand region (Giltner, C.L., Nguyen, Y. & Burrows, L.L. Microbiol Mol Biol Rev 76, 740-772 (2012)). Furthermore, all six ComP orthologs carry both a pglS homolog immediately downstream of the comP gene as well as a pglL homolog located elsewhere in the chromosome. Together, at least the presence of the conserved serine at position 84, the disulfide loop flanking the site of glycosylation, the presence of a pglS gene immediately downstream of comP, and the presence of a pglL homolog located elsewhere on the chromosome differentiate ComP pilin variants from other type IVa pilin variants. [0153] Therefore, features common to ComP proteins are disclosed herein that identify ComP orthologs in different Acinetobacter species. ComP proteins can be differentiated from other pilins by the presence of the conserved glycosylated serine located at position 84 relative to the ADP1 ComP protein and the presence of a disulfide loop flanking the site of glycosylation. In addition, the presence of a pglS homolog immediately downstream of ComP is an indicator of ComP. Further to be classified as a PglS OTase protein rather than a PglL OTase protein, the OTase downstream of ComP must display higher sequence conservation with PglS (ACIAD3337) when compared to PglL (ACIAD0103) in A. baylyi ADP1. It is also evident to one of ordinary skill in the art that in any embodiment disclosed herein, a ComP protein comprises and is capable of being glycosylated on a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201 (ComP110264: ENV58402.1). ComP protein glycosylation fragments [0154] In certain embodiments, the ComP glycosylation fragment can be, or can be from or derived from, any of the ComP proteins disclosed herein. Further, the PglS OTase can be any of the following. [0155] It was previously demonstrated that the PglS ortholog from Acinetobacter baylyi strain ADP1 glycosylates the ComP ortholog from A. soli strain CIP 110264 at a single serine residue located at position 82 (Harding, C. M. et al., 2019; WO/2019/241672, which is incorporated by reference herein in its entirety). PglS was engineered to functionally glycosylate heterologous proteins by translationally fusing a large fragment (117 amino acids) of ComP to the C-terminus of a known carrier protein. Specifically, the 117 amino acid ComP110264 fragment was fused at the C-terminus of a genetically deactivated exotoxin A from Pseudomonas aeruginosa (EPA) between a flexible GGGS linker (SEQ ID NO: 382). This chimeric carrier protein also had an N-terminal DsbA signal sequence (ssDsbA) for translocation to the periplasm via the Sec-pathway as well as a C-terminal hexahistidine tag for detection. 32080280 - 45 - Atty. Dkt. No.: 64100-234947 [0156] Even shorter ComP glycosylation fragments sufficient for glycosylation by PglS have been identified (WO/2020/131236, which is incorporated by reference herein in its entirety). It has been shown that ComP110264 glycosylation fragments fused to the C-terminus of the EPA carrier protein could also be glycosylated by PglS, but only if the ComP glycosylation fragments contained both cysteine residues corresponding to Cys71 and Cys93 relative to ComP110264. These observations were confirmed in a series of experiments aimed at identifying even shorter ComP glycosylation fragments. Figure 12A and Figure 12B show ComP110264 fragments that were designed to shift one amino acid N- to C-terminal relative to serine 82, which is the site of PglS glycosylation when the ComP glycosylation fragment was fused to the extreme C-terminus of the EPA carrier protein. The ComP glycosylation fragments were PCR amplified, cloned onto the C-terminus of EPA, and tested for bioconjugation by PglS. For these and all experiments described below, the serotype 8 pneumococcal capsular polysaccharide (CPS8) expressed from the pB-8 plasmid as the glycan source (Kay, E. J., et al., 2016) was used. The CPS8 glycan was selected as it contains glucose as the reducing end sugar and was previously demonstrated to be efficiently transferred to ComP by PglS (Harding, C. M. et al., 2019). In addition, for these and all experiments described below, bioconjugation was performed in the E. coli strain, SDB1. SDB1 has deletions of WecA, which initiates biosynthesis of the enterobacterial common antigen and the O-antigen polysaccharides, and WaaL, which transfers undecaprenyl-pyrophosphate linked glycan precursors to the outer core of lipid-A (Garcia-Quintanilla, F., et al., 2014). Collectively, these mutations facilitate the accumulation of heterologously expressed lipid-linked glycan precursors, like the CPS8 polysaccharide lipid-linked precursor, for exclusive use by PglS. SDB1 strains expressing the CPS8 glycan, PglS, and a fusion EPA-ComP110264 construct from IPTG inducible vectors were cultured in LB broth, induced at mid-log and grown overnight. Samples were harvested ~20 hours after induction for western blot analysis on periplasmic extracts to assess EPA- ComP110264 fusion protein expression and protein glycosylation. Western blots were probed using antibodies against EPA (anti-EPA) and the hexahistidine tag (anti-His). Probing with both antibodies allowed ascertainment whether the EPA protein and/or the C-terminal ComP fragment remained intact. [0157] Figure 12C, Figure 12D, and Figure 12E reaffirm that the presence of Cys71 and Cys93 residues flanking Ser82 in ComP110264 are essential for EPA-ComP110264 glycosylation when the ComP glycosylation fragment is fused at the C-terminus. As seen in Figure 12C, Figure 12D, and Figure 12E, fusion proteins containing ComP glycosylation fragments that 32080280 - 46 - Atty. Dkt. No.: 64100-234947 lacked either Cys71 or Cys93 were not glycosylated. Only in fusion proteins containing ComP glycosylation fragments with both cysteine residues was transfer of the CPS8 glycan observed. The glycosylation efficiency and average number of the CPS8 repeat units transferred by PglS were similar for all fusion proteins containing ComP glycosylation fragments containing both Cys71 and Cys93. Upon closer examination of the Western blots, it was observed that chimeric EPA-ComP110264 variants (listed as C2, D2, E3, and F3 in Figure 12C, Figure 12D, and Figure 12E) barely reacted with the anti-His antibody when compared to the anti-EPA signal (Figure 12D). Furthermore, the anti-EPA channel revealed that these variants migrated with a slightly lower molecular weight when compared to the unglycosylated EPA-ComP110264 variants containing both Cys71 and Cys93 (Figure 12C). Taken together, these observations indicated that the ComP fragments lacking both cysteine residues are unstable and likely prone to C- terminal degradation, thereby preventing glycosylation by PglS. Without being bound by theory, it is believed that Cys71 and Cys93 are able to stabilize ComP110264 by forming a covalent disulfide bridge. [0158] A variety of proteins from different organisms, typically inactivated bacterial toxins, have been used as carriers for conjugate and bioconjugate vaccines. Cross-reactive material 197 (CRM197) is a genetically deactivated form of the diphtheria toxin that has been used extensively as the carrier protein in multiple conjugate vaccines for pneumococcus, Neisseria meningitidis, and Haemophilus influenza type b (Berti, F. & Adamo, R., 2018). Given the frequent use of CRM197 in conjugate vaccine formulations the PglS bioconjugation system was extended to function with CRM197. For these experiments, the 25-amino acid “C1” ComP glycosylation fragment (ComPC1) previously identified was translationally fused to the C-terminus of CRM197 linked by a GGGS sequence (SEQ ID NO: 382). An SRP-dependent FlgI secretion sequence (ssFlgI) was added to the N-terminus for CRM197 for export to the periplasm (Goffin, P., et al., 2017). Finally, a C-terminal hexahistidine tag was added to aid purification (Figure 13A). E. coli SDB1 cells expressing the CPS8 glycan along with PglS and the CRM197-ComPC1 carrier (expected size of 61.8 kDa) were cultured in shake flasks and harvested after 24 hours. The CRM197-ComPC1-CPS8 glycoconjugate was purified with three successive rounds of chromatography. First, nickel-affinity chromatography was employed as the glycoconjugates contain a C-terminal hexahistidine tag. Fractions containing glycoconjugates were pooled and enriched for glycosylated glycoconjugates using a MonoQ column and eluted with a linear salt gradient. A final polishing step to remove large aggregates was performed on a Superdex 200 Increase column. As seen in Figure 13B, Figure 13C, and 32080280 - 47 - Atty. Dkt. No.: 64100-234947 Figure 13D, Western blotting on the purified samples using anti-CRM197 and pneumococcal CPS8 antisera demonstrated that the CRM197-ComPC1 carrier was glycosylated with CPS8. Digestion of the purified glycoconjugates with Proteinase K prior to separation on SDS-PAGE resulted in a complete loss of the CRM197 and polysaccharide specific signals, indicating that the CPS8 glycans were covalently attached to CRM197-ComPC1 protein. [0159] Next, whether the ComPC1 glycotag could be moved to another site in the CRM197 fusion was tested. As such, a new construct was designed placing ComPC1 N-terminal to the CRM197 coding region (Figure 14A). The FlgI secretion signal was placed immediately N- terminal to ComPC1 glycosylation fragment and CRM197 was C-terminally tagged with hexahistidine. E. coli SDB1 cells expressing the CPS8 glycan along with PglS and the ComPC1- CRM197 carrier were cultured in shake flasks and harvested after 24 hours. As seen in Figure 14B, Western blot analysis of periplasmic extracts probing with an anti-His antibody showed that the ComPC1-CRM197 was also glycosylated by PglS. The average number of CPS8 repeat units and glycosylation efficiency of both fusions was comparable, indicating that ComPC1 glycotag can be placed at the N- or C-terminus of a carrier protein. Identification of an 11 amino acid ComP110263 sequon sufficient for PglS glycosylation [0160] While prior reports indicated that Cys71 and Cys93 were required for glycosylation of fusion proteins containing a ComP110264 glycosylation fragment translationally fused at the C-terminus of EPA (e.g., Figure 12C, Figure 12D, and Figure 12E), these data do not ascertain whether the two cysteine residues and the putative disulfide bridge formed between them are absolutely required for glycosylation by PglS in all circumstances. The N-linking sequon recognized by PglB has been engineered into multiple sites on surface loops of EPA and used as an “internal” glycotag (Ihssen, J. et al., 2010). In order to determine whether Cys71 and Cys93 of ComP110264 are necessary for PglS glycosylation, the entire 23 amino acid ComP110264 glycosylation fragment spanning Cys71 to Cys93 ^referred to herein as the iGTCC for internal GlycoTag – cysteine-cysteine ^was integrated internal of the EPA amino acid sequence. The ComP110264 iGTCC was inserted between residues Ala489 and Arg490 of EPA, which is in a ^-turn structure on the surface of the catalytic domain (Figure 15A). As a control, a variant of the iGTCC ComP glycosylation fragment containing serine residues instead of cysteine residues at positions 71 and 93 of ComP termed iGTss (“serine-serine”) was also integrated. This iGTSS ComP glycosylation fragment was also integrated between residues Ala489 and Arg490 of EPA. Serine residues are hypothesized to contribute a similar steric bulk as the cysteine residues, but are unable to oxidize and form a disulfide bond (Figure 15B). The 32080280 - 48 - Atty. Dkt. No.: 64100-234947 ability of PglS to transfer CPS8 to the EPAiGTcc or EPAiGTss was assessed in a three-plasmid system as described above. As seen in Figure 15C and Figure 15D, both the cysteine-cysteine and serine-serine variants of EPAiGT were glycosylated, demonstrating that Cys71 and Cys93 (and the putative disulfide bond formed between them) are not required for glycosylation by PglS when the ComP fragment is introduced internal of the EPA protein. [0161] Since the cysteine residues are not necessary for PglS dependent glycosylation only when the ComP glycosylation fragment is integrated internal of the fusion protein, it was contemplated that a shorter ComP glycosylation fragment representing the minimal O-linking ComP sequon could be found within the 23-amino acid ComP glycosylation fragment spanning Cys71 to Cys93. To investigate this, shorter variants of the iGTCC ComP glycosylation fragment integrated between EPA residues Ala489-Arg490 were generated in order to identify which ComP residues were necessary for glycosylation. Alternate single amino acids were deleted from either side of the 23-amino acid iGTCC, generating 22 truncated variants that each contained Ser82, the site of PglS glycosylation (Figure 16A and Figure 16B). These variants were named after the number of deleted residues from either side of the iGTCC, e.g. ^3-4 corresponds to a deletion of three amino acids from the N-terminal side of iGTCC and a four amino acid deletion from the C-terminal side. The shortest variant generated was five amino acids long. These truncated EPA-iGTCC variants were tested for bioconjugation with CPS8 and PglS in shake flasks under the same conditions as the preceding experiments. As a negative control, we included a construct expressing only the EPA coding sequence along with DsbA secretion and hexahistidine tags. [0162] Figure 16C shows robust glycosylation for all EPA fusion proteins containing ComP glycosylation fragments that were at least 11 amino acids in length was observed. The glycosylation ratio was comparable to the 23 amino acid iGTCC ComP glycosylation fragment, suggesting modest truncations on either side of Ser82 do not have a significant impact on the glycosylation efficiency by PglS. Although these fusion proteins were glycosylated, a mild decrease in glycosylation efficiency was observed as the iGT ComP glycosylation fragment amino acid sequence was shortened. The shortest internal ComP glycosylation fragment that was efficiently glycosylated was iGT ^6-6 having the sequence IASGASAATTN (SEQ ID NO: 309); Figure 16C). Removal of either the N-terminal isoleucine residue (iGT ^7-6; SEQ ID NO: 321) or C-terminal asparagine residue (iGT ^6-7; SEQ ID NO: 310) dramatically reduced the glycosylation efficiency of the carrier protein, suggesting that these residues play an important role in PglS glycosylation. Variants smaller than iGT ^6-6 mostly showed minimal 32080280 - 49 - Atty. Dkt. No.: 64100-234947 glycosylation, the best of these being iGT ^7-6 with sequence ASGASAATTN (SEQ ID NO: 321). Interestingly, a small amount of higher molecular weight laddering was also observed in fusion proteins containing the smallest ComP glycosylation fragments, iGT ^9-8 (SEQ ID NO: 346) and iGT ^9-9 (SEQ ID NO: 347) (Figure 16D), suggesting that these six and five amino acid variants, respectively, were glycosylated by PglS at very low levels. This implies that the ComP110264 glycosylation sequon recognized by PglS can be as small as five amino acids in size. [0163] Next, the CPS8 glycosylated EPA fusion protein containing the iGT ^6-6 ComP glycosylation fragment located between residues Ala489-Arg490 was purified from whole-cell lysates using a Ni-affinity chromatography and performed western blot analysis on the eluate using antisera specific to either the EPA protein or the CPS8 glycan. The results of these experiments clearly show that the EPA fusion protein containing the iGT ^6-6 ComP glycosylation fragment located between residues Ala489-Arg490 was being glycosylated with CPS8 by PglS (Figure 17A, Figure 17B, and Figure 17C). Overall, these experiments show that ComP110264 glycosylation fragment can be shortened from the 117 amino acid ComP110264 to a sequon as short as or shorter than 11 amino acids while maintaining glycosylation. These results unexpectedly show that the cysteine residues corresponding to Cys71 and Cys93 of ComP110264, previously shown to be required when fused at the C-terminus, are not required for PglS dependent glycosylation when the ComP glycosylation fragment is integrated internal of the fusion protein. [0164] The preceding iGT truncation series was tested at one internal site on EPA between residues Ala489 and Arg490. Next, a second site between EPA residues Glu548 and Gly549 and incorporated the iGT ^3-4 ComP glycosylation fragment (SEQ ID NO: 271) was tested. Like the first site, the second site is found on a surface-exposed loop in the catalytic domain of EPA. This alternately tagged variant for bioconjugation with CPS8 and PglS was tested under the same conditions as the other truncations. It was observed that this construct was glycosylated with CPS8 at a similar efficiency as when iGT ^3-4 was placed in the first site on EPA. The CPS8 glycosylated EPA fusion protein containing the iGT ^3-4 ComP glycosylation fragment located between residues Glu548 and Gly549 was then purified from whole-cell lysates using a Ni-affinity chromatography and performed Western blot analysis on the eluate using antisera specific to either the EPA protein or the CPS8 glycan. The results of these experiments again show that the EPA fusion protein containing the iGT ^3-4 ComP glycosylation fragment located between residues Glu548 and Gly549 was being glycosylated 32080280 - 50 - Atty. Dkt. No.: 64100-234947 with CPS8 by PglS. Overall, these experiments show that ComP110264 glycosylation fragment can be shortened from the 117 amino acid ComP110264 to a sequon as short as 11 amino acids or shorter while maintaining glycosylation. These results unexpectedly show that the cysteine residues corresponding to Cys71 and Cys93 of ComP110264 are not required for PglS dependent glycosylation when the ComP glycosylation fragment is integrated internal of the fusion protein. [0165] Provided herein are glycoconjugates comprising an oligo- or polysaccharide linked to a fusion protein. In certain embodiments, the oligo- or polysaccharide is covalently linked to the fusion protein. The fusion protein comprises a glycosylation fragment of a ComP protein (as described in detail elsewhere herein). In certain embodiments of a glycoconjugate of this disclosure, the oligo- or polysaccharide comprises a glucose at its reducing end. [0166] ComP is glycosylated on a serine (S) residue. This serine residue corresponds to position 82 of SEQ ID NO: 201 (ComP110264: ENV58402.1). This serine residue is conserved in ComP proteins and, for example, corresponds to position 84 of SEQ ID NO: 202 (ComPADP1: AAC45886.1). Thus, in certain aspects, a fusion protein (and thus the glycoconjugate) is glycosylated with an oligo- or polysaccharide on a ComP glycosylation fragment at a serine residue corresponding to the serine residue at position 84 of SEQ ID NO: 202 (ComPADP1: AAC45886.1) or corresponding to the serine residue at position 82 of SEQ ID NO: 201 (ComP110264: ENV58402.1). Figure 22 shows an alignment of a region of ComP sequences including the serine (S) residue (boxed) corresponding to the serine residue at position 82 of SEQ ID NO: 201 (ComP110264: ENV58402.1), which is conserved across the ComP sequences. [0167] One of ordinary skill in the art would recognize that by aligning ComP sequences with SEQ ID NO: 201, (e.g., either full sequences or partial sequences) the conserved serine residue of a non-SEQ ID NO: 201 ComP protein, corresponding to the serine residue at position 82 of SEQ ID NO: 201, can be identified. Further, one of ordinary skill in the art would recognize that by aligning ComP sequences with SEQ ID NO: 201, other residues, regions, and/or features corresponding to residues, regions, and/or features of SEQ ID NO: 201 as referred to herein can be identified in the non-SEQ ID NO: 201 ComP sequence and referenced in relation to SEQ ID NO: 201. And, while reference is generally made herein to SEQ ID NO: 201, by analogy, reference can similarly be made to any residue, region, feature and the like of any ComP sequence disclosed herein, for example, in reference to SEQ ID NO: 202. [0168] A ComP protein is a protein that has been identified as a ComP protein consistent with the description provided herein. For example, representative examples of ComP proteins 32080280 - 51 - Atty. Dkt. No.: 64100-234947 include, but are not limited to: AAC45886.1 ComP [Acinetobacter sp. ADP1]; ENV58402.1 hypothetical protein F951_00736 [Acinetobacter soli CIP 110264]; APV36638.1 competence protein [Acinetobacter soli GFJ-2]; PKD82822.1 competence protein [Acinetobacter radioresistens 50v1]; SNX44537.1 type IV pilus assembly protein PilA [Acinetobacter puyangensis ANC 4466]; OAL75955.1 competence protein [Acinetobacter sp. SFC]; ComPP5312; and ComPANT_H59. In certain aspects, a 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: 201 (ComPADP1) or SEQ ID NO: 201 (ComP110264) and contains a serine residue corresponding to the conserved serine residue at position 84 of SEQ ID NO: 202 or at position 82 of SEQ ID NO: 201. SEQ ID NO: 202 comprises a leader sequence of 28 amino acids. In certain aspects, a 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: 210 (ComPΔ28ADP1), SEQ ID NO: 209 (ComPΔ28110264), SEQ ID NO: 211 (ComPΔ28GFJ-2), SEQ ID NO: 212 (ComPΔ28P50v1), SEQ ID NO: 213 (ComPΔ284466), SEQ ID NO: 214 (ComPΔ28SFC), SEQ ID NO: 215 (ComPΔ28P5312), or SEQ ID NO: 216 (ComPΔ29ANT_H59) that do not include the amino acid leader sequence but do contain a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201 (ComP110264: AAC45886.1). In certain aspects, a 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: 209 (ComPΔ28110264) that does not include the 28 amino acid leader sequence but does contain a serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201 (ComP110264). In certain aspects, the ComP protein comprises SEQ ID NO: 210 (ComPΔ28ADP1), SEQ ID NO: 209 (ComPΔ28110264), SEQ ID NO: 211 (ComPΔ28GFJ-2), SEQ ID NO: 212 (ComPΔ28P50v1), SEQ ID NO: 213 (ComPΔ284466), SEQ ID NO: 214 (ComPΔ28SFC), SEQ ID NO: 215 (ComPΔ28P5312), or SEQ ID NO: 216 (ComPΔ29ANT_H59). In certain aspects, the ComP protein is SEQ ID NO: 202 (ComPADP1: AAC45886.1), SEQ ID NO: 201 (ComP110264: ENV58402.1), SEQ ID NO: 203 (ComPGFJ-2: APV36638.1), SEQ ID NO: 204 (ComP50v1: PKD82822.1), SEQ ID NO: 205 (ComP4466: SNX44537.1), SEQ ID NO: 206 (ComPSFC: OAL75955.1), SEQ ID NO: 207 (ComPP5312), or SEQ ID NO: 208 (ComPANT_H59). [0169] Provided for herein is a glycoconjugate comprising an oligo- or polysaccharide covalently linked to a fusion protein wherein the fusion protein comprises a ComP protein (ComP) glycosylation fragment. In certain embodiments, the ComP glycosylation fragment 32080280 - 52 - Atty. Dkt. No.: 64100-234947 does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 71 of ComP110264 (SEQ ID NO: 201). In certain embodiments, the ComP glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 93 of ComP110264 (SEQ ID NO: 201). As described in greater detail herein, the fusion protein is glycosylated with the oligo- or polysaccharide on the ComP glycosylation fragment at serine residue corresponding to the conserved serine residue at position 82 of ComP110264 (SEQ ID NO: 201). In certain embodiments, the ComP glycosylation fragment is located internally within the fusion protein. Further, in certain embodiments, the ComP glycosylation fragment portion of the fusion protein is solvent (or surface)-exposed and/or is integrated into a C10 ^-turn, ^-turn, ^-twist, ^-loop, U turn, reverse turn, chain reversal, or a hairpin loop of the fusion protein. [0170] Because it has been discovered that when the ComP glycosylation fragment is located internally within the fusion protein, it does not require the flanking cysteine residues for glycosylation, the ComP glycosylation fragments disclosed herein can be shorter than previously believed. In certain embodiments, the ComP glycosylation fragment can be shorter than 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, or 6 amino acids long, as long as it comprises a serine residue corresponding to the conserved serine residue at position 82 of ComP110264 (SEQ ID NO: 201). In certain embodiment, the ComP glycosylation fragment has a length of from any one of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 to any one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids in length. In certain embodiments, the fragment has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues of the ComP protein N-terminal to the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201, e.g., XnS[Y], wherein n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues of the ComP protein. In certain embodiments, the fragment has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues of the ComP protein C-terminal to the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201, e.g., [X]SYn, wherein n is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues of the ComP protein. Further, in certain embodiments, the amino acid sequence of the ComP glycosylation fragment does not extend in the N-terminus direction beyond the amino acid residue corresponding to position 72 of ComP110264 (SEQ ID NO: 201) and/or does not extend in the C-terminus beyond the amino acid residue corresponding to position 92 of ComP110264 (SEQ ID NO: 201). 32080280 - 53 - Atty. Dkt. No.: 64100-234947 [0171] Consistent with a ComP protein of this disclosure, in certain embodiments, a ComP protein from which the ComP glycosylation fragment is derived comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 209 (ComPΔ28110264) SEQ ID NO: 210 (ComPΔ28ADP1), SEQ ID NO: 211 (ComPΔ28GFJ-2), SEQ ID NO: 212 (ComPΔ28P50v1), SEQ ID NO: 213 (ComPΔ284466), SEQ ID NO: 214 (ComPΔ28SFC); SEQ ID NO: 215 (ComPΔ28P5312), or SEQ ID NO: 216 (ComPΔ29ANT_H59). In certain embodiments, the ComP protein from which the ComP glycosylation fragment is derived comprises SEQ ID NO: 209 (ComPΔ28110264), SEQ ID NO: 210 (ComPΔ28ADP1), SEQ ID NO: 211 (ComPΔ28GFJ-2), SEQ ID NO: 212 (ComPΔ28P50v1), SEQ ID NO: 213 (ComPΔ284466), SEQ ID NO: 214 (ComPΔ28SFC); SEQ ID NO: 215 (ComPΔ28P5312), or SEQ ID NO: 216 (ComPΔ29ANT_H59). [0172] In certain embodiments of the glycoconjugate of this disclosure, the ComP glycosylation fragment comprises or consists of the amino acid consensus sequence of: X1GVX4X5X6X7X8X9ASX12X13TX15NVX18X19X20X21 (SEQ ID NO: 217); CX1GVX4X5X6X7X8X9ASX12X13TX15NVX18X19X20X21 (SEQ ID NO: 396); or X1GVX4X5X6X7X8X9ASX12X13TX15NVX18X19X20X21C (SEQ ID NO: 397); wherein: X1 is V, T, A, or I; X4 is Q, T, E, A, or S; X5 is E, Q, T, or L; X6 is I or V; X7 is S, N, A, or G; X8 is S or no amino acid; X9 is G, D, or no amino acid; X12 is N, S, or A; X13 is A, S, or K; X15 is T, S, or K; X18 is A, E, Q, or L; X19 is T, S, or K; X20 is A or S; and X21 is T, Q, A, or V; or a fragment of thereof of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length comprising the serine (S) residue corresponding to position 11 of SEQ ID NO: 217. In certain embodiments, the fragment has at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues N-terminal to the serine (S) residue corresponding to position 11 of SEQ ID NO: 217. In certain embodiments, the fragment has at least 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues C-terminal to the serine (S) residue corresponding to position 11 of SEQ ID NO: 217. But, the ComP 32080280 - 54 - Atty. Dkt. No.: 64100-234947 glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 71 of ComP110264 (SEQ ID NO: 201) and/or the ComP glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 93 of ComP110264 (SEQ ID NO: 201). [0173] Certain embodiments provide for a ComP glycosylation fragment that is a variant of the amino acid consensus sequence of SEQ ID NO: 217, SEQ ID NO: 396, or SEQ ID NO: 397, or the fragment thereof, having 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions, additions, and/or deletions, wherein the variant maintains the serine (S) residue corresponding to position 11 of SEQ ID NO: 217 and wherein the variant does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 71 of ComP110264 (SEQ ID NO: 201) and/or the variant does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 93 of ComP110264 (SEQ ID NO: 201). One of ordinary skill in the art will understand that the number of amino acid substitutions, additions, and/or deletions that can be tolerated within a sequence without abolishing its function (e.g., ability to function as a sequon) can depend on the length of the sequence. For example, a six amino acid long sequence will tolerate less changes than a 21 amino acid long sequence. [0174] Whether a ComP glycosylation fragment can be glycosylated (including subfragments of a fragment and variants as disclosed herein and collectively referred to as ComP glycosylation fragments), and the efficiency of glycosylation, can be determined such as by methods described herein. In certain embodiments, the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein and/or internally in a carrier protein sequence as described elsewhere herein. Further, in certain embodiments, the ComP glycosylation fragment or variant is not glycosylated when located at the N-terminal and/or C- terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal and/or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein. [0175] In certain embodiments, the fusion protein comprises a carrier protein selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae Protein D, and a fragment or fragments thereof. For example, in certain embodiments, the Pseudomonas aeruginosa Exotoxin A (EPA) carrier protein comprises the amino acid sequence of SEQ ID NO: 218, or a fragment or fragments thereof. For example, in certain embodiments, the CRM197 carrier 32080280 - 55 - Atty. Dkt. No.: 64100-234947 protein comprises the amino acid sequence of SEQ ID NO: 224, or a fragment or fragments thereof. [0176] As can be understood from this disclosure as a whole, by internally within the fusion protein, it is meant that the ComP fusion protein is not located at the C-terminal end or the N- terminal end of the fusion protein, not including any C-terminal leader sequence or N-terminal tag (e.g., His-Tag), or the like. [0177] In certain embodiments, the ComP glycosylation fragment can be attached to the carrier protein sequence via an amino acid linker. [0178] Further, in certain embodiments, the ComP glycosylation fragment can be inserted into the sequence of a carrier protein rather than between carrier proteins. For example, in certain embodiments: (i) the ComP glycosylation fragment is inserted between Ala489 and Arg490 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 219); (ii) the ComP glycosylation fragment is inserted between Glu548 and Gly549 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 220); (iii) the ComP glycosylation fragment is inserted between Ala122 and Gly123 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 221); (iv) the ComP glycosylation fragment is inserted between Thr355 and Gly356 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 222); or (v) the ComP glycosylation fragment is inserted between Lys20 and Asp21 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 223). [0179] Further, in certain embodiments, the ComP glycosylation fragment can be inserted into the sequence of a carrier protein rather than between carrier proteins. For example, in certain embodiments: (i) the ComP glycosylation fragment is inserted between Asn481 and Gly482 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 225); (ii) the ComP glycosylation fragment is inserted between Asp392 and Gly393 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 226); 32080280 - 56 - Atty. Dkt. No.: 64100-234947 (iii) the ComP glycosylation fragment is inserted between Glu142 and Gly143 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 227); (iv) the ComP glycosylation fragment is inserted between Asp129 and Gly130 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 228); or (v) the ComP glycosylation fragment is inserted between Asn69 and Glu70 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 229). [0180] In certain embodiments, ComP glycosylation fragments can be located between carrier proteins and also inserted into the sequence of a carrier protein(s) within one fusion protein. In certain embodiments, a ComP glycosylation fragment can be located internally and one or more ComP glycosylation fragments can be located at the C-terminal and/or N-terminal end that are sufficient for glycosylation at such location. [0181] An aspect of this disclosure is that a fusion protein can be designed to comprise multiple ComP glycosylation fragments such as to increase the immunogenicity of the glycosylated fusion protein/glycoconjugate. In certain embodiments, 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 fragments. In certain embodiments, the fusion protein does not comprise more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five ComP glycosylation fragments. The identity of the ComP glycosylation fragments can also be controlled. For example, in certain embodiments, a plurality of ComP glycosylation fragments of a fusion protein are identical. In certain embodiments, ComP glycosylation fragments of a fusion protein differ from each other. For example, in certain embodiments, at least three, at least four, or at least five of the ComP glycosylation fragments of a fusion protein all differ from each other. For example, in certain embodiments, none of the ComP glycosylation fragments of a fusion protein are the same. [0182] In certain embodiments, the oligo- or polysaccharide is derived from a saccharide produced by bacteria from the genus Streptococcus. For example, in certain embodiments, the saccharide is a S. pneumoniae, S. agalactiae, or S. suis capsular polysaccharide; in certain embodiments, the saccharide is the serotype 8 capsular polysaccharide from S. pneumoniae; and in certain embodiments, the saccharide is the type Ia, Ib, II, III, IV, V, VI, VII, VIII, or X capsular polysaccharide from S. agalactiae. [0183] In certain embodiments, the oligo- or polysaccharide is derived from a saccharide produced by the bacteria from the genus Klebsiella. For example, in certain embodiments, the 32080280 - 57 - Atty. Dkt. No.: 64100-234947 saccharide is a K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca capsular polysaccharide; and in certain embodiments, the saccharide is a K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca O-antigen polysaccharide. [0184] In certain embodiments, the glycoconjugate is produced in vivo, for example: in a bacterial cell; in Escherichia coli; in a bacterium from the genus Klebsiella; and/or wherein the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0185] Provided for herein is a glycoconjugate as described above (e.g., the ComP glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 71 of ComP110264 (SEQ ID NO: 201) and/or the ComP glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 93 of ComP110264 (SEQ ID NO: 201)), wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of SEQ ID NOs: 232- 363, or 364. Provided for herein is a glycoconjugate as described above (e.g., the ComP glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 71 of ComP110264 (SEQ ID NO: 201) and/or the ComP glycosylation fragment does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 93 of ComP110264 (SEQ ID NO: 201)), wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of:
Figure imgf000060_0001
32080280 - 58 - Atty. Dkt. No.: 64100-234947 [0186] Also provided for herein is a ComP glycosylation fragment that is a variant of any of the above disclosed ComP glycosylation fragments having 1, 2, 3, 4, 5, 6, or 7 amino acid substitutions, additions, and/or deletions, wherein the variant maintains the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201 and wherein the variant does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 71 of ComP110264 (SEQ ID NO: 201) and/or the variant does not contain a cysteine (C) residue corresponding to the conserved cysteine (C) residue at position 93 of ComP110264 (SEQ ID NO: 201). [0187] Whether a ComP glycosylation fragment can be glycosylated (including subfragments of a fragment and variants as disclosed herein and collectively referred to as ComP glycosylation fragments), and the efficiency of glycosylation, can be determined such as by methods described herein. In certain embodiments, the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein and/or internally in a carrier protein sequence as described elsewhere herein. Further, in certain embodiments, the ComP glycosylation fragment is not glycosylated when located at the N-terminal and/or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal and/or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein. [0188] In certain embodiments, the glycoconjugate is a conjugate vaccine. Thus, this disclosure in certain embodiments is directed to and provides for a conjugate vaccine. In certain embodiments the conjugate vaccine is a vaccine against Streptococcus pneumoniae serotype 8. In certain embodiments, the conjugate vaccine induces an immune response when administered to a subject. In certain embodiments, the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG or IgM response. In certain embodiments, the antibody response is an IgG response; optionally an IgG1 response. And, in certain embodiments, the conjugate vaccine generates immunological memory in a subject administered the vaccine. [0189] Whereas the above describes a glycoconjugate comprising a ComP glycosylation fragment that comprises an isolated fragment of a ComP protein, it is understood that this disclosure also explicitly provides for a ComP glycosylation fragment consistent with any and all description of a ComP glycosylation fragment provided anywhere herein, including in the appended Claims below, e.g., wherein the ComP glycosylation fragment does not contain a 32080280 - 59 - Atty. Dkt. No.: 64100-234947 cysteine residue corresponding to the conserved cysteine residue at position 71 of ComP110264 (SEQ ID NO: 201) and/or does not contain a cysteine residue corresponding to the conserved cysteine residue at position 93 of ComP110264 (SEQ ID NO: 201) and wherein the ComP glycosylation fragment comprises the serine residue corresponding to the conserved serine residue at position 82 of ComP110264 (SEQ ID NO: 201). [0190] Provided for herein is as fusion protein comprising a ComP glycosylation fragment of this disclosure. In certain embodiments, the fusion protein is glycosylated by an oligo- or polysaccharide at a serine residue on the glycosylation fragment corresponding to the serine ComP glycosylation fragment residue at position 82 of SEQ ID NO: 201 (ComP110264). Further, whereas the above describes a glycoconjugate comprising a ComP glycosylation fragment that comprises a fusion protein, it is understood that this disclosure also explicitly provides for a fusion protein consistent with any and all description of a fusion protein provided anywhere herein, including in the appended Claims below. In certain embodiments, the fusion protein comprises a carrier protein selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae Protein D, and a fragment or fragments thereof. [0191] Also provided for herein is a method of in vivo conjugation of an oligo- or polysaccharide to an acceptor polypeptide. In certain embodiments, the method comprises culturing a host cell comprising the components necessary for the conjugation of the oligo- or polysaccharide to the polypeptide. In general, these components are the oligosaccharyltransferase, the acceptor polypeptide to be glycosylated, and the oligo- or polysaccharide. The method comprises covalently linking an oligo- or polysaccharide to the acceptor polypeptide (fusion protein of this disclosure) with a PglS oligosaccharyltransferase (OTase), wherein the acceptor polypeptide comprises a ComP glycosylation fragment as described herein. In certain embodiments, the PglS OTase is PglS110264 (SEQ ID NO: 365), PglSADP1 (SEQ ID NO: 366), PglSGFJ-2 (SEQ ID NO: 367), PglS50v1 (SEQ ID NO: 368), PglS4466 (SEQ ID NO: 369), PglSSFC (SEQ ID NO: 370), PglSP5312 (SEQ ID NO: 371), or PglSANT_H59 (SEQ ID NO: 372). In certain embodiments, the oligo- or polysaccharide is linked to the ComP glycosylation fragment at a serine (S) residue corresponding to the serine residue at position 82 of SEQ ID NO: 201 (ComP110264). In certain embodiments, the in vivo conjugation occurs in a host cell. In certain aspects, the glycoconjugate is produced in a bacterial cell, a fungal cell, a yeast cell, an avian cell, an algal cell, an insect cell, or a mammalian cell. In certain embodiments, the host cell is a bacterial cell, e.g.: in Escherichia 32080280 - 60 - Atty. Dkt. No.: 64100-234947 coli; in a bacterium from the genus Klebsiella; the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. Certain embodiments comprise culturing a host cell that comprises: (a) a genetic cluster encoding for the proteins required to synthesize the oligo- or polysaccharide; (b) a PglS OTase; and (3) the acceptor polypeptide. In certain embodiments, the production of the oligo- or polysaccharide is enhanced by the K. pneumoniae transcriptional activator rmpA (K. pneumoniae NTUH K-2044) or a homolog of the K. pneumoniae transcriptional activator rmpA (K. pneumoniae NTUH K-2044). In certain embodiments, the method further comprises expressing and/or providing such a transcriptional activator in the host cell along with the other components. [0192] In certain aspects, the glycoconjugate is produced in a cell free system. Examples of the use of a cell free system utilizing OTases other than PglS can be found in WO2013/067523A1, which in incorporated herein by reference. [0193] Also provided for is a host cell comprising (a) a genetic cluster encoding for the proteins required to synthesize an oligo- or polysaccharide; (b) a PglS OTase; and (3) an acceptor polypeptide comprising a ComP glycosylation fragment of this disclosure. In certain embodiments, the acceptor polypeptide is a fusion protein. In certain embodiments, the host cell comprises a nucleic acid encoding the PglS OTase. In certain embodiments, the host cell comprises a nucleic acid encoding the acceptor polypeptide. [0194] Also provided for herein is an isolated nucleic acid encoding a ComP glycosylation fragment and/or a fusion protein of this disclosure. In certain embodiments, the nucleic acid is a vector. In certain embodiments, a host cell comprises the isolated nucleic acid. [0195] A glycoconjugate of this invention may have one of numerous uses including, but not limited to, use as a conjugate vaccine. Thus in certain methods, a conjugate vaccine is produced. In certain embodiments, a composition comprising the conjugate vaccine or the fusion protein of this disclosure and an adjuvant. For example, in certain embodiments, the conjugate vaccine is a vaccine against 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, 32080280 - 61 - Atty. Dkt. No.: 64100-234947 Streptococcus pneumoniae serotype 19F, Streptococcus pneumoniae serotype 19A, Streptococcus pneumoniae serotype 20, Streptococcus pneumoniae serotype 22F, Streptococcus pneumoniae serotype 23F, Streptococcus pneumoniae serotype 33F, Klebsiella pneumoniae serotype K1, Klebsiella pneumoniae serotype K2, Klebsiella pneumoniae serotype K5, Klebsiella pneumoniae serotype K16, Klebsiella pneumoniae serotype K20, Klebsiella pneumoniae serotype K54, Klebsiella pneumoniae serotype K57, Streptococcus agalactiae serotype Ia, Streptococcus agalactiae serotype Ib, Streptococcus agalactiae serotype II, Streptococcus agalactiae serotype III, Streptococcus agalactiae serotype IV, Streptococcus agalactiae serotype V, Streptococcus agalactiae serotype VI, Streptococcus agalactiae serotype VII, Streptococcus agalactiae serotype VIII, Streptococcus agalactiae serotype IX, Streptococcus pyogenes Group A Carbohydrate, Enterococcus faecalis serotype A, Enterococcus faecalis serotype B, Enterococcus faecalis serotype C, Enterococcus faecalis serotype D, Enterococcus faecium capsular polysaccharide and lipotechoic acid, Moraxella catarrhalis lipooligosaccharide A, Moraxella catarrhalis lipooligosaccharide B, Moraxella catarrhalis lipooligosaccharide C, and Staphylococcus aureus lipotechoic 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 elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG or IgM response. For example, in certain embodiments the antibody response can be an IgG response, and in certain embodiments, an IgG1 response. In certain embodiments, the conjugate vaccine generates immunological memory in a subject administered the vaccine. [0196] Disclosed herein is a pneumococcal glyconjugate vaccine containing a conventional vaccine carrier that can be produced by isolating a glycoconjugate or a glycosylated fusion protein of this disclosure comprising a ComP glycosylation fragment of this disclosure and combining the isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant. In certain embodiments, the ComP glycosylation fragment can be added to a conventional carrier protein Pseudomonas aeruginosa Exotoxin A (EPA). It has been demonstrated that in certain embodiments, the glycosylation fragment/carrier fusion protein can be paired with the CPS8 polysaccharide and use of PglS, generating a carrier protein-CPS8 bioconjugate, a first of its kind pneumococcal bioconjugate vaccine. For example, in certain embodiments, an EPA fusion can be paired with the CPS8 polysaccharide and use of PglS, generating an EPA-CPS8 bioconjugate. It has been demonstrated that the EPA-CPS8 bioconjugate vaccine elicited high 32080280 - 62 - Atty. Dkt. No.: 64100-234947 IgG titers specific to serotype 8 specific that were protective as determined via bactericidal killing. Importantly, vaccination with as little as 100 ng of polysaccharide in the EPA-CPS8 bioconjugate was able to provide protection. Thus, certain embodiments provide for a CPS8 pneumococcal bioconjugate vaccine. [0197] It is contemplated that a conjugate vaccine (such as the EPA vaccine construct) can comprise additional/multiple sites of glycosylation to increase the glycan to protein ratio as well as expand upon the number of serotypes in order to develop a comprehensive pneumococcal bioconjugate vaccine. Moraxellaceae O-Linking Oligosaccharyltransferases [0198] Also part of this disclosure is a novel family of bacterial O-linking oligosaccharyltransferases termed TfpM from Moraxellaceae bacteria. Certain embodiments of this disclosure comprise any of the TfpM-associated pilin glycosylation fragments and/or TfpM OTases that follow. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analyses demonstrate that TfpM proteins cluster in different clades than known bacterial oligosaccharyltransferases. Using a representative TfpM enzyme from Moraxella osloensis, it was determined that TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and the minimal sequon required for glycosylation was identified. It was demonstrated that TfpM has a broad substrate tolerance and can transfer diverse polysaccharides including those with reducing-end glucose, galactose, or 2-N-acetyl sugars. It was also shown that a TfpM- derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice. Thus the glycan substrate promiscuity of TfpM and identification of the minimal TfpM sequon renders this enzyme a valuable additional tool for expanding the glycoengineering toolbox. Bioinformatic identification of a new class of OTase carried by bacteria from the Moraxellaceae family. [0199] To identify genes encoding O-linking oligosaccharyltransferases, the inventors first searched the NCBI genome and whole-genome shotgun contig sequence databases using the Basic Local Alignment Search Tool (BLAST) and the PglSADP1 amino acid sequence (SEQ ID NO: 1) as the query. 32080280 - 63 - Atty. Dkt. No.: 64100-234947 SEQ ID NO: 1_ PglSADP1 amino acid sequence MNSIFKKIKNYTIVSGVFFLGSAFIIPNTSNLSSTLYKELIAVLGLLILLTVKSFDYKKILI PKNFYWFLFVIFIIFIQLIVGEIYFFQDFFFSISFLVILFLSFLLGFNERLNGDDLIVKKIA WIFIIVVQISFLIAINQKIEIVQNFFLFSSSYNGRSTANLGQPNQFSTLILITLFLLCYLRE KNSLNNMVFNILSFCLIFANVMTQSRSAWISVILISLLYLLKFQKKIELRRVIFFNIVFW TLVYCVPLLFNLIFFQKNSYSTFDRLTMGSSRFEIWPQLLKAVFHKPFIGYGWGQTGV AQLETINKSSTKGEWFTYSHNLFLDLMLWNGFFIGLIISILILCFLIELYSSIKNKSDLFL FFCVVAFFVHCLLEYPFAYTYFLIPVGFLCGYISTQNIKNSISYFNLSKRKLTLFLGCC WLGYVAFWVEVLDISKKNEIYARQFLFSNHVKFYNIENYILDGFSKQLDFQYLDYCE LKDKYQLLDFKKVAYRYPNASIVYKYYSISAEMKMDQKSANQIIRAYSVIKNQKIIKP KLKFCSIEY [0200] To shorten the list of hits and reduce the likelihood of identifying very similar orthologs of PglSADP1, the search was further refined to candidates that had less than 50% amino acid sequence identity to PglSADP1. Some of the top hits from this refined list were proteins that were much more similar in size to TfpO proteins, but the upstream cognate pilin protein contained both a ComP disulfide-flanked sequon as well as a PilA-like sequon at the C- terminus. The first identified pilin- oligosaccharyltransferase pairs were encoded in two Acinetobacter species: A. parvus DSM 16617 and A. townerii ZZC-3 (Table 1). Table 1. Organisms and accession numbers for the TfpM enzymes and their associated pilins
Figure imgf000066_0001
32080280 - 64 - Atty. Dkt. No.: 64100-234947
Figure imgf000067_0001
[0201] The two oligosaccharyltransferases from these strains were closely related, having >96% sequence identity. Intrigued by these findings, the inventors looked further and identified other strains in the Moraxellaceae family that carried genes encoding similar putative oligosaccharyltransferase/pilin pairs. While many genes like those in A. parvus DSM 16617 and A. townerii ZZC-3 were found, most of the associated pilin proteins encoded upstream of the oligosaccharyltransferase lacked a ComP-like sequon. The accession numbers and protein sizes for twenty of these putative oligosaccharyltransferases-pilin-like protein pairs are listed in Table 1. The inventors did not observe any homologues in species outside of the Moraxellaceae family and, to distinguish these different oligosaccharyltransferases from other known enzymes, have termed them TfpM proteins (‘M’ for Moraxellaceae). Given the similar size of TfpM proteins to known TfpO proteins, it was initially hypothesized that these genes encoded variants of TfpO-PilA pairs like those found in Acinetobacter and Pseudomonas (Harding, C. M., et al. (2015) Molecular Microbiology 96, 1023-1041). However, a multiple sequence alignment of the twenty TfpM proteins with known PglS, PglL, and TfpO proteins demonstrated that the former had less than 26% sequence identity to archetypal 32080280 - 65 - Atty. Dkt. No.: 64100-234947 oligosaccharyltransferases. Analysis of the phylogenetic tree produced by the multiple alignment showed that TfpM proteins cluster in different clades than TfpO, PglS and PglL proteins (Figure 23A and Figure 24). In contrast, the pilin genes located immediately upstream of tfpM did not cluster in discrete clades (Figure 25) and displayed overall higher identity to PilA proteins, namely between 37% and 60%. Based on the coding sequence, most of the associated pilin proteins belong to the type IV major pilin family with the exception of those from Acinetobacter sp. CIP102143 and Acinetobacter sp. CIP102637 which lack the characteristic type III signal sequence at the N-terminus (Giltner Carmen, L., et al. (2012) Microbiology and Molecular Biology Reviews 76, 740-772). TfpM orthologs glycosylate engineered pilin-fusion proteins with the pneumococcal serotype 8 capsular polysaccharide [0202] Although similar in size to TfpO proteins, TfpM proteins are sufficiently different in terms of their amino acid sequence warranting further investigation. The inventors were particularly interested in determining if TfpM proteins could transfer only short oligosaccharides to acceptor proteins, like TfpO proteins. Of the twenty TfpM oligosaccharyltransferases listed in Table 1, the inventors selected 13 representatives from different clades to test for glycosylation activity in a glycoengineered E. coli strain (Harding, C. M., and Feldman, M. F. (2019) Glycobiology 29, 519-529; Feldman, M. F., et al. (2005) Proceedings of the National Academy of Sciences of the United States of America 102, 3016). Previously, the inventors developed a chimeric acceptor protein strategy consisting of the exotoxin A protein from Pseudomonas aeruginosa (EPA) fused to different-sized, soluble fragments of ComP (the natural substrate of PglS) (Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203). All type IV pilin-like proteins contain a conserved, N-terminal pilin signal sequence and membrane-anchoring domain that is not required for glycosylation but is essential for pilin stability. The fusion protein approach allows for the removal of the conserved N-terminal pilin signal sequence and membrane-anchoring domain and was used to determine the minimum sequon that PglSADP1 could recognize and still efficiently glycosylate (Harding, C. M., et al. (2019) Nature Communications 10, 891; Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203). The inventors adapted this approach and designed 13 synthetic double- stranded DNA blocks encoding an N-terminally truncated fragment of the upstream pilin gene and the downstream tfpM gene. In most of the strains carrying tfpM, the protein coding regions of the upstream pilin gene and the start codon of tfpM overlap by a single nucleotide. This genetic architecture was left intact in the expression constructs. The synthetic DNA blocks 32080280 - 66 - Atty. Dkt. No.: 64100-234947 were designed such that, when cloned into an EPA expression vector using Gibson assembly, it placed the pilin coding region in frame with the C-terminus of EPA, creating a gene fusion that translated as a single protein with the tfpM gene immediately downstream (Figure 23B). The truncated pilin fragments ranged from 113 to 140 amino acids in size. No purification tag was added to the C-terminus of the EPA-pilin fusion as previous studies with TfpO from P. aeruginosa 1244 reported that adding additional C-terminal residues after the serine residue prevented glycosylation (Horzempa, J., et al. (2006) Journal of Biological Chemistry 281, 1128-1136). Expression of the EPA-pilin and TfpM proteins was driven from an IPTG- inducible tac promoter on a pEXT20 plasmid (Dykxhoorn, D. M., et al. (1996) Gene 177, 133- 136). The fusion protein was secreted into the periplasm using a DsbA signal sequence at the N-terminus of EPA. Oligos and primers used for the assemblies are listed in Table 2. Table 2. Primers and Oligos
Figure imgf000069_0001
32080280 - 67 - Atty. Dkt. No.: 64100-234947
Figure imgf000070_0001
32080280 - 68 - Atty. Dkt. No.: 64100-234947 [0203] Using this design, the inventors assessed the ability of the 13 TfpM proteins to transfer the Streptococcus pneumoniae capsular polysaccharide 8 (CPS8) glycan to their cognate pilin domain on the EPA-pilin fusions. The CPS8 repeat unit is a tetrasaccharide with a glucose at the reducing end. Notably, PglS is thus far the only known oligosaccharyltransferase able to naturally transfer this glycan to acceptor proteins (Harding, C. M., et al. (2019) Nature Communications 10, 891). The 13 EPA-pilin fusions/TfpM expression vectors were transformed individually into E. coli SDB1 strains (Feldman, M. F., et al. (2005) Proceedings of the National Academy of Sciences of the United States of America 102, 3016) expressing the CPS8 glycan and assessed for protein glycosylation. The unglycosylated fusion proteins had expected masses ranging from 78.3 to 80.5 kDa. Several TfpM proteins were found to glycosylate their cognate EPA-pilin fusion with glycosylation appearing as higher molecular-weight laddering (gn) above the unglycosylated band (g0) (Figure 23C). Each higher-weight band represents the attachment of a glycan with one additional CPS8 repeat unit to the EPA-pilin protein. Glycosylation was readily observed in seven TfpM orthologs tested: Acinetobacter sp. YZSX-1-1, Acinetobacter sp. CIP102637, Acinetobacter sp. YH01026, A. junii 65, Acinetobacter sp. CIP102143, Acinetobacter sp. TUM15069, and M. osloensis 1202 (Figures 23C). Some of these extracts required higher exposure of the western blot to observe the glycosylation pattern (Figure 23D). As a control, the inventors generated a mutant of the M. osloensis TfpM protein gene with a single-residue change in a conserved histidine (His286) previously shown to be required for activity of wzy_C domain-containing enzymes (Figure 26) The wzy_C family pfam04932 is an “O-antigen ligase” domain present in membrane-bound enzymes that catalyze the transfer and covalent attached of lipid-linked oligosaccharide (liposaccharide) to lipid A or protein substrates. (Ruan, X., et al. (2012) Glycobiology 22, 288-299; Musumeci, M. A., et al. (2014) Glycobiology 24, 39-50). [0204] Glycosylation in SDB1 cell extracts expressing this H286A mutant alongside the EPA-pilin fusion protein and CPS8 glycan was not observed (Figure 23C) indicating that the glycosylation is due to the activity of the tfpM gene product and that His286 is required for catalytic activity and/or stability of this oligosaccharyltransferase. TfpMMo is an O-linking oligosaccharyltransferase that glycosylates the C-terminal threonine of its pilin substrate [0205] Of the 13 TfpM-pilin pairs tested in the preceding experiment, those from M. osloensis 1202 and Acinetobacter sp. YH01026 exhibited the most efficient transfer of glycans 32080280 - 69 - Atty. Dkt. No.: 64100-234947 of diverse sizes. Due to the slightly higher apparent stability of the pilin from M. osloensis FDAARGOS_1202 (1202 hereafter), the inventors chose the oligosaccharyltransferase from this organism as a representative for further characterization and have termed the enzyme TfpMMo (SEQ ID NO: 56). For clarity, reference is made to the intact, native M. osloensis 1202 pilin protein as PilMo (SEQ ID NO: 57) and the N-terminally truncated fusion domain as PilMo ^28 (SEQ ID NO: 58) throughout the text. Using TfpMMo, next was to identify the glycosylation site of PilMo ^28 and thereby determine whether the enzyme acted like a TfpO protein, glycosylating the C-terminal amino acid of its cognate pilin acceptor, or if it was more like a PglL or PglS protein, glycosylating an internal residue. TfpO proteins transfer short oligosaccharides typically containing 3-6 sugars to the sidechain of the C-terminal serine residue of their cognate pilin. All except one of the cognate pilin proteins located immediately upstream a tfpM open reading frame ended in a C-terminal threonine residue, namely that from Psychrobacter sp.72-O-c which ends in a serine. Based on this observation, as well as the size similarity of TfpM and TfpO proteins, the inventors hypothesized that TfpM enzymes also transfer glycans to a C-terminal residue and thus designed point mutants of the pilin to test this. Two mutants of the C-terminal pilin threonine (Thr167) were generated, converting this residue to either serine or alanine and tested for glycosylation with CPS8. Whole cell E. coli extracts of the strains expressing the PilMo ^28 mutants were probed using anti-EPA antibody in western blots. As seen in Figure 27, TfpMMo was also able to glycosylate EPA- PilMo ^28 T167S, but no glycosylation was observed in the alanine mutant. These results indicate that the hydroxyl group on the sidechain of the Thr167 or Ser167 residue is likely the site of glycan attachment by TfpMMo. [0206] To confirm the site of pilin glycosylation by TfpMMo, CPS8-glycosylated EPA- PilMo ^28 was partially purified and separated via SDS-PAGE analysis and Coomassie-stained the resolved glycoproteins. Gel slices corresponding to EPA- PilMo ^28 glycosylated with 1 – 3 CPS8 repeat units were excised, digested with LysC, and analyzed for glycopeptides. Open searching-based analysis (Chick, J. M., et al. (2015) Nature Biotechnology 33, 743-749; Polasky, D. A., et al. (2020) Nature Methods 17, 1125-1132) of LysC-derived EPA-PilMo ^28 peptides resulted in the identification of Hexose (Hex)-Hexuronic Acid (HexA)-modified 762FLPANCRGT770 peptide consistent with an incomplete monomer of the CPS8 glycan (HexHexAHex2). Higher-energy C-trap dissociation (HCD) analysis supports linkage of this disaccharide through the hexose residue and targeted Electron-Transfer/Higher-Energy Collision Dissociation (EThcD) analysis confirmed the attachment of HexHexA to the C- 32080280 - 70 - Atty. Dkt. No.: 64100-234947 terminal threonine residue (Figure 28A and Figure 28B). While this MS analysis did not identify multimers of the CPS8 tetrasaccharide this is not surprising due to elongated glycoconjugates being extremely difficult to detect using peptide-centric LC-MS methods without the use of specialized chemical additives such as supercharging agents (Lin, C.-w., et al. (2016) Analytical Chemistry 88, 8484-8494). Nevertheless, the identification of a disaccharide consistent with the partially completed CPS8 tetrasaccharide still supports the identity of the high molecular weight laddering as polymerized CPS8 tetrasaccharides. TfpMMo transfers polysaccharides containing glucose, galactose, or 2-N-acetyl monosaccharides at the reducing end [0207] Next, it was sought to explore the range of polysaccharide substrates that TfpMMo can transfer in glycoengineered E. coli backgrounds. The inventors selected polysaccharides containing different reducing-end sugars, varying disaccharide sugar linkages near the reducing end, and/or were polymers composed of linear or branched repeat units. Four polysaccharides, E. coli O16 antigen, Salmonella enterica LT2 O-antigen, Klebsiella pneumoniae O2a antigen, and type III Group B Streptococcus capsular polysaccharide, in addition to the pneumococcal CPS8, were tested as glycan substrates for TfpMMo. The structures of all five repeat units tested are shown in Figure 29A (Liu, B., et al. (2020) FEMS Microbiology Reviews 44, 655-683; Curd, H., et al. (1998) Journal of Bacteriology 180, 1002-1007; Whitfield, C., et al. (1992) Journal of Bacteriology 174, 4913-4919; Pinto, V., and Berti, F. (2014) Journal of Pharmaceutical and Biomedical Analysis 98, 9-15; Geno, K. A., et al. (2015) Clinical Microbiology Reviews 28, 871-899). Each of the five polysaccharides were individually co- expressed with EPA-PilMo ^28 and TfpMMo in E. coli SDB1 cells, induced, and grown for subsequent glycoprotein purification. Periplasmic extractions of SDB1 cells were partially purified using anion-exchange chromatography to remove any contaminating undecaprenol- pyrophosphate-linked polysaccharides that would convolute interpretation of the western blots. To demonstrate that the glycan-specific antibody signal observed in the western was indeed from glycosylated protein and not due to contaminating lipid-linked polysaccharide carryover from whole cell lysates, purified glycoproteins were split into two equal fractions, one half of which were digested with proteinase K prior to SDS-PAGE separation and western blotting. The western blots were probed using antisera specific to each polysaccharide and, separately, with anti-EPA antibody as all antibodies used in this experiment were from rabbits. As seen in Figure 7, TfpMMo was found to efficiently transfer all five different polysaccharides to the EPA-PilMo ^28 protein. Digestion with proteinase-K eliminated both the anti-glycan (Figure 32080280 - 71 - Atty. Dkt. No.: 64100-234947 29B, Figure 29C, Figure 29D, Figure 29E, and Figure 29F) and anti-EPA (Figure 29G) signals in the western blots, confirming that the anti-glycan signals originated from protein- linked rather than contaminating lipid-linked polysaccharide. TfpMMo can glycosylate truncated PilMo ^28 variants [0208] The prior experiments all utilized an N-terminally truncated variant of PilAMo that was 139 amino acids in length. To gain insights into the minimal features required for C- terminal pilin glycosylation by TfpMMo, a series of further-truncated variants of PilMo ^28 and were generated and it was tested whether they could be glycosylated. The inventors first generated a 20 amino acid fragment of PilMo fused C-terminally to EPA via a flexible four- residue glycine linker, termed Pil20 (Figure 30A). This 20-amino acid fragment was chosen because it contains a disulfide loop (“DSL”) region that is conserved in many type IV pilins (Figure 31) (Horzempa, J., et al. (2006) Journal of Biological Chemistry 281, 1128-1136; Harvey, H., et al. (2009) Journal of Bacteriology 191, 6513-6524). It is noted that this DSL corresponds to a different motif than the disulfide-flanked sequon present in ComP proteins (Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203). Based on sequence alignments with P. aeruginosa 1244 PilA, the DSL in M. osloensis pilin is formed by residues Cys148 and Cys164. As such, the inventors designed the sequence downstream of the glycine linker to start with Cys148. The plasmid comprising the construct encoding EPA-Pil20 and TfpM was termed pVNM297. Glycosylation experiments with Pil20 revealed that it was able to be glycosylated by TfpMMo with CPS8 at similar levels to PilMo ^28 (Figure 29B). To test whether the DSL region was required for glycosylation, several shorter variants were generated that lacked part of this feature. In these smaller constructs, the inventors also mutated Cys164 to alanine to prevent the formation of non-natural disulfide linkages upon oxidation in the periplasm (Harvey, H., et al. (2009) Journal of Bacteriology 191, 6513-6524). 15-, 13-, and 10-amino acid PilMo variants were tested, with one variant of the 10-amino acid version containing an amino acid linker (e.g., GGGG linker) and one without. These constructs were termed Pil15, Pil13, Pil10L, and Pil10, respectively (Figure 30A). TfpMMo was able to glycosylate all four of these variants, albeit at lower levels than Pil20 and PilMo ^28 (Figure 27B). Both Pil10 and Pil10L were glycosylated comparably, indicating that the presence of the upstream glycine linker does not have a marked effect on TfpMMo activity. This linker was omitted from all subsequent constructs. Of the four proteins, the Pil13 was glycosylated notably worse than the others. [0209] From a sequence alignment, it was noted that each of the pilin proteins that were glycosylated by TfpM OTases have a conserved ‘P-A-N/E-C-R-G’ motif found near the C- 32080280 - 72 - Atty. Dkt. No.: 64100-234947 terminus, immediately upstream of the penultimate threonine residue (Figure 31). Given the presence of this feature in all glycosylated pilins, it was asked whether this motif was required for glycosylation by TfpMMo. The inventors fused a seven-amino acid variant, termed Pil7, of PilMo consisting of a similar motif (modified to ‘P-A-N-A-R-G-T’, where the cysteine is mutated to an alanine – bolded residue) to EPA and assessed glycosylation (Figure 30A). The inventors also built stepwise single-amino acid truncations of this ‘P-A-N-A-R-G-T’ sequence from seven to two amino acids and assessed the ability of TfpM to glycosylate these fragments with CPS8. The results showed that all variants except for Pil2 were glycosylated by TfpMMo at similar levels as Pil10 (Figure 30B). The overall glycosylation was again lower than for the Pil20 and PilMo ^28. Glycosylated Pil2 was barely detectable though some trace laddering was visible at higher exposure of the western blot. This indicates that TfpMMo can glycosylate this variant but at markedly reduced levels than the construct with even one additional amino acid. From these results, it was concluded that TfpMMo can recognize and glycosylate a three-amino acid PilMo fragment when fused to the C-terminus of the heterologous EPA protein. Immunogenicity of a TfpMMo-derived GBSIII bioconjugate [0210] Given that the EPA-Pil20 construct was efficiently glycosylated by TfpM, the inventors next assessed the ability of the EPA-Pil20 protein glycosylated with the type III capsular polysaccharide from Group B Streptococcus (GBSIII) for immunogenicity in a murine vaccination model. To aid in protein purification for these experiments, a plasmid expressing an N-terminally 6x-His tagged variant of the EPA-Pil20 carrier protein (pVNM291) was constructed. The His tag was added immediately downstream of the predicted N-terminal DsbA signal sequence cleavage site of EPA. pVNM291 was introduced into SDB1 cells expressing the GBSIII glycan and resulting bioconjugates purified using Nickel immobilized metal affinity chromatography (IMAC) followed by anion-exchange and size-exclusion chromatography on FPLC. Western blot and Coomassie staining of the SDS-PAGE resolved GBSIII-291 bioconjugate confirmed high molecular weight glycosylation of the EPA-Pil20 protein with the GBSIII glycan (Figure 32A, Figure 32B, Figure 32C, and Figure 32D). Intact protein MS of the purified EPA-Pil20-GBSIII (“GBSIII-291”) conjugate supported a glycan:protein ratio of 20% (Figure 32E). Each dose was formulated to contain 1 μg of GBSIII polysaccharide. As a control for these experiments, unglycosylated pVNM291-derived carrier protein (“291”) was purified from SDB1 cells without glycan plasmid and dosed at the same protein concentration as for the GBSIII bioconjugates. 32080280 - 73 - Atty. Dkt. No.: 64100-234947 [0211] To test whether the TfpMMo-generated GBSIII-291 bioconjugate was immunogenic, 5-week-old female CD-1 mice were immunized. Mice received either placebo (unglycosylated 291 carrier protein) or the GBSIII-291 bioconjugate at two-week intervals starting with a priming dose followed by two booster doses. All vaccines were formulated with a 1:9 ratio of Alhydrogel® 2% as an adjuvant. Serum was collected prior to each immunization and two weeks after the final booster. To determine the level of GBSIII-specific antibodies elicited, the inventors used an enzyme-linked immunosorbent assay (ELISA). All mice immunized with the bioconjugate GBSIII-291 were observed to express high levels of anti- GBSIII IgG antibodies, excluding a single mouse that exhibited a low anti-GBSIII IgG response, that was able to be boosted over the course of the immunization (Figure 32F). As expected, the GBSIII bioconjugate-vaccinated mice had increased GBSIII-specific IgG titers compared with the mock-vaccinated mice (291 alone, Figure 32F). Collectively, these data suggest that TfpMMo can produce bioconjugates capable of eliciting polysaccharide-specific IgG responses. TfpMMo and PglSADP1 (PglLComP) glycosylate a single protein engineered to contain sequons specific to each oligosaccharyltransferase [0212] Lastly, the inventors wanted to determine if a protein engineered to contain sequons from two different OTase systems could be glycosylated by both OTases at each site. As such, an EPA fusion protein containing a sequon associated with TfpM as well as a sequon associated with PglS was constructed. To this end, the inventors engineered an EPA fusion protein containing a PglS sequon (CTGVTQIASGASAATTNVASAQC) (SEQ ID NO: 59) integrated between residues Ala489 and Arg490 as previously described (Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203) as well as the Pil20 sequon (CGGTGTTVAAKFLPANCRGT) (SEQ ID NO: 60; same as Pil DSL) at the C-terminus (Figure 33A). As above, the construct was designed such that the open reading frame of the gene encoding for the EPA fusion and the start codon of tfpM overlapped by a single nucleotide. The open reading frame encoding for pglS from A. baylyi ADP1 was cloned 100 bp downstream of the tfpM open reading frame stop codon. This vector (pVNM337) was the introduced into E. coli SDB1 expressing the E. coli O16 antigen and tested for glycosylation via western blotting. To compare against proteins containing only a single sequon, the inventors individually introduced the following constructs into E. coli SDB1 expressing the O16 antigen: (i) EPA containing only the TfpM-associated Pil20 sequon (pVNM297) or (ii) EPA containing the PglS sequon integrated between residues Ala489 and Arg490 (pVNM167). To compare against a protein that has two sequons and can be 32080280 - 74 - Atty. Dkt. No.: 64100-234947 di-glycosylated, the inventors also introduced an EPA construct containing the sequon integrated between residues Ala489 and Arg490 as well as between residues Glu548 and Gly549 (pVNM245) (EPA_PglS sequon 2X). Diagrams of these constructs are shown in Figure 33A. As seen in Figure 33B, western blot analysis of EPA constructs containing only one sequon either from ComP or PilMo exhibited a glycoprofile around 100 kDa, indicative of a mono- glycosylation. The EPA construct containing two PglS sequons exhibited a predominantly mono-glycosylated profile around 100 kDa but also displayed a di-glycosylated population migrating around 150 kDa. Western blot analysis of the EPA fusion containing a sequon from TfpMMo and PglSADP1 displayed both mono- and di-glycosylated populations like that seen with construct pVNM245. It was concluded from these results that an acceptor protein can be glycosylated by two different OTase classes in one expression system. Glycoconjugates [0213] This disclosure provides for a glycoconjugate comprising an oligo- or polysaccharide covalently linked to an acceptor protein. In certain embodiments, the acceptor protein comprises or consists of a TfpM-associated pilin-like protein or glycosylation fragment thereof of this disclosure. In certain embodiments, the oligo- or polysaccharide is covalently linked to the pilin-like protein or glycosylation fragment thereof. And, in certain embodiments, the TfpM-associated pilin-like protein or glycosylation fragment thereof comprises a C- terminus serine or threonine residue and the oligo- or polysaccharide is covalently linked to the C-terminus serine or threonine. Further, in certain embodiments, the acceptor protein is a fusion protein comprising the TfpM-associated pilin-like protein or glycosylation fragment thereof translationally fused/linked to a heterologous amino acid sequence (e.g., a carrier protein) and the TfpM-associated pilin-like protein or glycosylation fragment thereof is the C-terminus- most sequence of the acceptor protein such that the acceptor protein comprises a C-terminus serine or threonine residue and the oligo- or polysaccharide is covalently linked to the C- terminus serine or threonine. Illustrative examples of carrier proteins include but are not limited to Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, or a fragment of any thereof. In certain embodiments, the TfpM-associated pilin-like protein or glycosylation fragment thereof is translationally fused/linked to a heterologous amino acid sequence/carrier protein via an amino acid linker. In certain embodiments, the oligo- or polysaccharide comprises a glucose at its reducing end. In certain embodiments, the glycoconjugate is immunogenic. 32080280 - 75 - Atty. Dkt. No.: 64100-234947 [0214] In certain embodiments of a glycoconjugate of this disclosure, the acceptor protein comprises or consists of a full-length TfpM-associated pilin-like protein. In certain embodiments, the acceptor protein comprises or consists of a glycosylation fragment of a TfpM-associated pilin-like-protein that is less than a full-length TfpM-associated pilin-like protein. In certain embodiments, the pilin-like-protein glycosylation fragment has a length of from 3 to 138 amino acids in length, has a length of from 10 to 138 amino acids in length, has a length of from 20 to 138 amino acids in length, 50 to 138 amino acids in length, has a length of from 100 to 138 amino acids in length, or has a length of from 116 to 138 amino acids in length. In certain embodiments, the pilin-like-protein glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 10 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, 50 to 139 amino acids in length, has a length of from 100 to 139 amino acids in length, or has a length of from 116 to 139 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from 3 to 140 amino acids in length, has a length of from 10 to 140 amino acids in length, has a length of from 20 to 140 amino acids in length, 50 to 140 amino acids in length, has a length of from 100 to 140 amino acids in length, or has a length of from 116 to 140 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 3 to 21 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, the glycosylation fragment has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. [0215] In certain embodiments of the glycoconjugate of this disclosure, the TfpM- associated pilin-like-protein or glycosylation fragment thereof is PilMo (SEQ ID NO: 57) or PilMo lacking amino acids corresponding to residues 1–28 (PilMoΔ28, SEQ ID NO: 58) or a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, for example, wherein the C-terminus threonine is substituted with serine. In certain embodiments of the glycoconjugate of this disclosure, the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ 32080280 - 76 - Atty. Dkt. No.: 64100-234947 ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), and PilCIP102637 (SEQ ID NO: 100). In certain embodiments, the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT- 3-2 (SEQ ID NO: 99), PilCIP102637 (SEQ ID NO: 100), and a fragment (e.g., C-terminus fragment) of any thereof and/or a variant wherein the C-terminus threonine is substituted with serine. And, in certain embodiments, the pilin-like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (PilMo_DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148). Further, in certain embodiments, the pilin-like-protein glycosylation fragment consists of Pil20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine. Further, in certain embodiments, the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil15S (SEQ ID NO: 153), Pil14S (SEQ ID NO: 154), Pil13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil11S (SEQ ID NO: 157), Pil10S (SEQ ID NO: 158), Pil9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil4S (SEQ ID NO: 164), or Pil3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine. 32080280 - 77 - Atty. Dkt. No.: 64100-234947 [0216] In certain embodiments of the glycoconjugate of this disclosure, the acceptor protein can be glycosylated at two or more different positions. In certain embodiments, the acceptor protein can be glycosylated by at least two different OTase classes in one expression system. For example, in certain embodiments, the acceptor protein is a fusion protein and the fusion protein further comprises an additional glycosylation sequence (e.g., glycosylation fragment) of an OTase other than TfpM oligosaccharyltransferase (OTase) in addition to the TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus. For example, the other OTase can be PglB, PglL, or PglS. In certain embodiments, the additional glycosylation sequence is a sequence internal to the fusion protein (i.e., not the C-terminal or N-terminal most sequence). In certain embodiments, the additional glycosylation sequence is a sequence internal within the sequence of a carrier protein (e.g., Figure 33A). In certain embodiments, the additional glycosylation sequence is also covalently linked to an oligo- or polysaccharide. In certain embodiments, 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 additional glycosylation sequences. In certain embodiments, the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five additional glycosylation sequences. In certain embodiments, the additional glycosylation sequences are identical. In certain embodiments, at least one additional glycosylation sequence differs from another. In certain embodiments, at least three, at least four, or at least five of the additional glycosylation sequences all differ from each other. And, in certain embodiments, none of the additional glycosylation sequences are the same. In certain embodiments, the acceptor protein is a fusion protein and the fusion protein further comprises an internal glycosylation fragment of ComP in addition to the TfpM-associated pilin-like-protein glycosylation fragment located at its C- terminus. In certain embodiments, the ComP glycosylation fragment is internal within the sequence of a carrier protein. In certain embodiments, the ComP glycosylation fragment is also covalently linked to an oligo- or polysaccharide. And, in certain embodiments, the ComP glycosylation fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least the amino acids ASA in positions 11-13. In certain embodiments, 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 fragments. In certain embodiments, the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, 32080280 - 78 - Atty. Dkt. No.: 64100-234947 or more than twenty five ComP glycosylation fragments. In certain embodiments, the ComP glycosylation fragments are identical. In certain embodiments, the ComP glycosylation fragments differ from each other. In certain embodiments, at least three, at least four, or at least five of the ComP glycosylation fragments all differ from each other. And, in certain embodiments, none of the ComP glycosylation fragments are the same. [0217] In certain embodiments of the glycoconjugate of this disclosure, the oligo- or polysaccharide covalently linked to the pilin-like protein or glycosylation fragment thereof has a size of at least three repeating units of oligo- or polysaccharide structure. In certain embodiments of the glycoconjugate of this disclosure, the oligo- or polysaccharide covalently linked to the pilin-like protein or glycosylation fragment thereof has a size of at least ten monosaccharides. [0218] In certain embodiments of the glycoconjugate of this disclosure, the oligo- or polysaccharide is produced by bacteria of the genus Streptococcus (for example, S. pneumoniae or S. agalactiae) and the polysaccharide is capsular polysaccharide such as Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX. [0219] In certain embodiments of the glycoconjugate of this disclosure, the oligo- or polysaccharide is produced by bacteria of the genus Klebsiella (for example, K. pneumoniae) and the polysaccharide is a capsular polysaccharide or O-antigen polysaccharide. [0220] In certain embodiments of the glycoconjugate of this disclosure, the oligo- or polysaccharide is produced by bacteria of the genus Salmonella and the polysaccharide is O- antigen polysaccharide. In certain embodiments, the bacteria is S. enterica and the S. enterica polysaccharide is a Group B O-antigen. [0221] In certain embodiments of the glycoconjugate of this disclosure, the glycoconjugate is produced in vivo, such as in a bacterial cell. In certain embodiments, the bacteria is Escherichia coli. In certain embodiments, the bacterium is from the genus Klebsiella. In certain embodiments, the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. In certain embodiments, the glycoconjugate is produced in a cell free system. [0222] In certain embodiments of the glycoconjugate of this disclosure, the bioconjugate is a conjugate vaccine that induces an immune response when administered to a subject. In certain embodiments, the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG or IgM response. In certain embodiments, the antibody response is an IgG response, for example, an IgG1 response. And, in certain 32080280 - 79 - Atty. Dkt. No.: 64100-234947 embodiments, the conjugate vaccine generates immunological memory in a subject administered the vaccine. Glycosylation fragments [0223] This disclosure provides for a pilin-like-protein glycosylation fragment comprising or consisting of an isolated fragment of a TfpM-associated pilin-like protein of this disclosure. In certain embodiments, the TfpM-associated pilin-like protein or pilin-like-protein glycosylation fragment comprises or consists of PilMo (SEQ ID NO: 57) or PilMo lacking amino acids corresponding to residues 1–28 (PilMoΔ28, SEQ ID NO: 58) or a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, for example, wherein the C-terminus threonine is substituted with serine. In certain embodiments, the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), and PilCIP102637 (SEQ ID NO: 100). In certain embodiments, the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), PilCIP102637 (SEQ ID NO: 100), and a fragment (e.g., C-terminus fragment) of any thereof and/or a variant wherein the C-terminus threonine is substituted with serine. And, in certain embodiments, the pilin-like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (PilMo_DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148). Further, in certain embodiments, the pilin-like-protein glycosylation fragment consists of Pil20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ 32080280 - 80 - Atty. Dkt. No.: 64100-234947 ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine. Further, in certain embodiments, the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil15S (SEQ ID NO: 153), Pil14S (SEQ ID NO: 154), Pil13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil11S (SEQ ID NO: 157), Pil10S (SEQ ID NO: 158), Pil9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil4S (SEQ ID NO: 164), or Pil3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine. [0224] In certain embodiments of the glycosylation fragment of this invention, the isolated fragment of a TfpM-associated pilin-like protein of this disclosure has a length of from 3 to 138 amino acids in length, has a length of from 10 to 138 amino acids in length, has a length of from 20 to 138 amino acids in length, 50 to 138 amino acids in length, has a length of from 100 to 138 amino acids in length, or has a length of from 116 to 138 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 10 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, 50 to 139 amino acids in length, has a length of from 100 to 139 amino acids in length, or has a length of from 116 to 139 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from 3 to 140 amino acids in length, has a length of from 10 to 140 amino acids in length, has a length of from 20 to 140 amino acids in length, 50 to 140 amino acids in length, has a length of from 100 to 140 amino acids in length, or has a length of from 116 to 140 amino acids in length. In certain embodiments, the glycosylation fragment has a length of has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, the 32080280 - 81 - Atty. Dkt. No.: 64100-234947 glycosylation fragment has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. Fusion protein [0225] Provided for herein is a fusion protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof of this disclosure translationally fused/linked to a heterologous carrier protein such as, but not limited to, Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, or a fragment of any thereof. In certain embodiments, the TfpM-associated pilin-like protein or glycosylation fragment thereof is translationally fused/linked to a heterologous carrier protein via an amino acid linker. In certain embodiments, the pilin-like protein or glycosylation fragment comprises a C-terminus serine or threonine residue. In certain embodiments, the pilin-like protein or glycosylation fragment is the C-terminus-most sequence of the fusion protein. And, in certain embodiments, the fusion protein comprises a C-terminus serine or threonine residue. Further, in certain embodiments, the fusion protein is glycosylated by an oligo- or polysaccharide covalently linked to the C-terminus serine or threonine. Further, in certain embodiments, the fusion protein is glycosylated by an oligo- or polysaccharide comprising glucose at its reducing end covalently linked to the C-terminus serine or threonine. In certain embodiments, the glycosylated fusion protein is immunogenic. In certain embodiments, the glycosylated fusion protein is a conjugate vaccine. [0226] In certain embodiments of the fusion protein of this disclosure, the fusion protein comprises a full-length TfpM-associated pilin-like protein. In certain embodiments, the fusion protein comprises or consists of a glycosylation fragment of a TfpM-associated pilin-like- protein that is less than a full-length TfpM-associated pilin-like protein. In certain embodiments, the pilin-like-protein glycosylation fragment has a length of from 3 to 138 amino acids in length, has a length of from 10 to 138 amino acids in length, has a length of from 20 to 138 amino acids in length, 50 to 138 amino acids in length, has a length of from 100 to 138 amino acids in length, or has a length of from 116 to 138 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 10 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, 50 to 139 amino acids in length, has a length of from 100 to 139 amino acids in length, or has a length of from 116 to 139 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from 3 to 140 amino acids in length, has a length of from 10 to 140 amino acids in length, has a length of from 20 to 140 amino acids in length, 50 32080280 - 82 - Atty. Dkt. No.: 64100-234947 to 140 amino acids in length, has a length of from 100 to 140 amino acids in length, or has a length of from 116 to 140 amino acids in length. In certain embodiments, the glycosylation fragment has a length of has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, the glycosylation fragment has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. [0227] In certain embodiments of the fusion protein of this disclosure, the pilin-like-protein glycosylation fragment comprises or consists of PilMo (SEQ ID NO: 57) or PilMo lacking amino acids corresponding to residues 1–28 (PilMoΔ28, SEQ ID NO: 58) or a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, for example, wherein the C-terminus threonine is substituted with serine. In certain embodiments, the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), and PilCIP102637 (SEQ ID NO: 100). In certain embodiments, the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), PilCIP102637 (SEQ ID NO: 100), and a fragment (e.g., C-terminus fragment) of any thereof and/or a variant wherein the C-terminus threonine is substituted with serine. And, in certain embodiments, the pilin-like-protein glycosylation 32080280 - 83 - Atty. Dkt. No.: 64100-234947 fragment comprises or consists of the PilMo pilin disulfide loop region (PilMo_DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148). Further, in certain embodiments, the pilin-like-protein glycosylation fragment consists of Pil20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine. Further, in certain embodiments, the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil15S (SEQ ID NO: 153), Pil14S (SEQ ID NO: 154), Pil13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil11S (SEQ ID NO: 157), Pil10S (SEQ ID NO: 158), Pil9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil4S (SEQ ID NO: 164), or Pil3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine. [0228] In certain embodiments of the fusion protein of this disclosure, the fusion protein can be glycosylated at two or more different positions. In certain embodiments, the fusion protein can be glycosylated by at least two different OTase classes in one expression system. For example, in certain embodiments, the fusion protein further comprises a glycosylation sequence (e.g., glycosylation fragment) of an OTase other than TfpM oligosaccharyltransferase (OTase) in addition to the TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus. For example, the other OTase can be PglB, PglL, or PglS. In certain embodiments, the additional glycosylation sequence is a sequence internal to the fusion protein (i.e., not the C-terminal or N-terminal most sequence). In certain embodiments, the additional glycosylation sequence is a sequence internal within the sequence of a carrier protein (e.g., Figure 11A). In certain embodiments, the additional glycosylation sequence is also covalently linked to an oligo- or polysaccharide. In certain embodiments, 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 additional glycosylation sequences. In certain embodiments, the fusion protein does not comprise more than two, more than three, more than five, more than 32080280 - 84 - Atty. Dkt. No.: 64100-234947 ten, more than fifteen, more than twenty, or more than twenty five additional glycosylation sequences. In certain embodiments, the additional glycosylation sequences are identical. In certain embodiments, at least one additional glycosylation sequence differs from another. In certain embodiments, at least three, at least four, or at least five of the additional glycosylation sequences all differ from each other. And, in certain embodiments, none of the additional glycosylation sequences are the same. In certain embodiments, the fusion protein further comprises an internal glycosylation fragment of ComP in addition to the TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus. In certain embodiments, the ComP glycosylation fragment is also covalently linked to an oligo- or polysaccharide. And, in certain embodiments, the ComP glycosylation fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least the amino acids ASA in positions 11-13. In certain embodiments, 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 fragments. In certain embodiments, the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five ComP glycosylation fragments. In certain embodiments, the ComP glycosylation fragments are identical. In certain embodiments, the ComP glycosylation fragments differ from each other. In certain embodiments, at least three, at least four, or at least five of the ComP glycosylation fragments all differ from each other. And, in certain embodiments, none of the ComP glycosylation fragments are the same. [0229] In certain embodiments of the fusion protein of this disclosure, the oligo- or polysaccharide covalently linked to the pilin-like protein or glycosylation fragment thereof has a size of at least three repeating units of oligo- or polysaccharide structure. In certain embodiments of the fusion protein of this disclosure, the oligo- or polysaccharide covalently linked to the pilin-like protein or glycosylation fragment thereof has a size of at least ten monosaccharides. [0230] In certain embodiments of the fusion protein of this disclosure, the oligo- or polysaccharide is produced by bacteria of the genus Streptococcus (for example, S. pneumoniae or S. agalactiae) and the polysaccharide is capsular polysaccharide such as Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX. 32080280 - 85 - Atty. Dkt. No.: 64100-234947 [0231] In certain embodiments of the fusion protein of this disclosure, the oligo- or polysaccharide is produced by bacteria of the genus Klebsiella (for example, K. pneumoniae) and the polysaccharide is a capsular polysaccharide or O-antigen polysaccharide. [0232] In certain embodiments of the fusion protein of this disclosure, the oligo- or polysaccharide is produced by bacteria of the genus Salmonella and the polysaccharide is O- antigen polysaccharide. In certain embodiments, the bacteria is S. enterica and the S. enterica polysaccharide is a Group B O-antigen. [0233] In certain embodiments of the fusion protein of this disclosure, the glycosylated fusion protein is produced in vivo, such as in a bacterial cell. In certain embodiments, the bacteria is Escherichia coli. In certain embodiments, the bacterium is from the genus Klebsiella. In certain embodiments, the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0234] In certain embodiments of the fusion protein of this disclosure, the fusion protein is a vaccine that induces an immune response when administered to a subject. In certain embodiments, the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response. In certain embodiments, the antibody response is an IgG or IgM response. In certain embodiments, the antibody response is an IgG response, for example, an IgG1 response. And, in certain embodiments, the fusion protein generates immunological memory in a subject administered the fusion protein. Method of producing a glycoconjugate [0235] Provided for herein is a method of producing a glycoconjugate. In certain embodiments, the method occurs in vivo. In certain aspects, the glycoconjugate is produced in a cell free system. Examples of the use of a cell free system utilizing OTases other than TfpM can be found in WO2013/067523A1, which is in incorporated herein by reference. In certain embodiments, the method comprises covalently linking (conjugating) an oligo- or polysaccharide to an acceptor protein comprising or consisting of a TfpM-associated pilin-like protein or glycosylation fragment thereof using a TfpM oligosaccharyltransferase (OTase) of this disclosure. In certain embodiments, the pilin-like protein or glycosylation fragment comprises a C-terminus serine or threonine residue, the acceptor protein comprises a C- terminus serine or threonine residue, and the oligo- or polysaccharide is covalently linked to the C-terminus serine or threonine residue of the acceptor protein. In certain embodiments, the oligo- or polysaccharide comprises a glucose at its reducing end. In certain embodiments, the 32080280 - 86 - Atty. Dkt. No.: 64100-234947 acceptor protein is a fusion protein of this disclosure as described in detail elsewhere herein. And, in certain embodiments, the glycoconjugate is immunogenic. [0236] In certain embodiments of a method of producing a glycoconjugate of this disclosure or with any other compositions or methods disclosed herein, the TfpM OTase contains a wzy_C superfamily domain that is defined by the .National Library of Science (NCBI) conserved protein domain family cl04850, an O-antigen ligase domain, and/or the TfpM OTase contains a wzy_C domain defined by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI, EMBL-EBI) protein family (pfam) conserved protein domain family motif pfam04932, an O-antigen ligase domain, wherein pfam04932 is a protein domain family in the cl04850 superfamily protein domain. In certain embodiments, the TfpM OTase comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to TfpMMo (SEQ ID NO: 56), TfpMDSM16617 (SEQ ID NO: 63), TfpMZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpMYH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpMAI40 (SEQ ID NO: 70), TfpMF78 (SEQ ID NO: 71), TfpMS71 (SEQ ID NO: 72), TfpMANC4282 (SEQ ID NO: 73), TfpMCIP102159 (SEQ ID NO: 74), TfpMjunii-65 (SEQ ID NO: 75), TfpMYZS-X (SEQ ID NO: 76), TfpMCIP102637 (SEQ ID NO: 77), TfpMT-3-2 (SEQ ID NO: 78), TfpMBI730 (SEQ ID NO: 79), TfpMA3K91 (SEQ ID NO: 80), and/or TfpM72-O-c (SEQ ID NO: 81). In certain embodiments, the TfpM OTase is TfpMMo (SEQ ID NO: 56), TfpMDSM16617 (SEQ ID NO: 63), TfpMZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpMYH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpMAI40 (SEQ ID NO: 70), TfpMF78 (SEQ ID NO: 71), TfpMS71 (SEQ ID NO: 72), TfpMANC4282 (SEQ ID NO: 73), TfpMCIP102159 (SEQ ID NO: 74), TfpMjunii-65 (SEQ ID NO: 75), TfpMYZS-X (SEQ ID NO: 76), TfpMCIP102637 (SEQ ID NO: 77), TfpMT-3-2 (SEQ ID NO: 78), TfpMBI730 (SEQ ID NO: 79), TfpMA3K91 (SEQ ID NO: 80), or TfpM72-O-c (SEQ ID NO: 81). In certain embodiments, the TfpM OTase is TfpMMo (SEQ ID NO: 56). [0237] In certain embodiments of a method of producing a glycoconjugate of this disclosure, the acceptor protein comprises or consists of a full-length TfpM-associated pilin- like protein. In certain embodiments, the acceptor protein comprises or consists of a glycosylation fragment of a TfpM-associated pilin-like-protein that is less than a full-length TfpM-associated pilin-like protein. In certain embodiments, the pilin-like-protein glycosylation fragment has a length of from 3 to 138 amino acids in length, has a length of from 10 to 138 amino acids in length, has a length of from 20 to 138 amino acids in length, 50 32080280 - 87 - Atty. Dkt. No.: 64100-234947 to 138 amino acids in length, has a length of from 100 to 138 amino acids in length, or has a length of from 116 to 138 amino acids in length. In certain embodiments, the pilin-like-protein glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 10 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, 50 to 139 amino acids in length, has a length of from 100 to 139 amino acids in length, or has a length of from 116 to 139 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from 3 to 140 amino acids in length, has a length of from 10 to 140 amino acids in length, has a length of from 20 to 140 amino acids in length, 50 to 140 amino acids in length, has a length of from 100 to 140 amino acids in length, or has a length of from 116 to 140 amino acids in length. In certain embodiments, the glycosylation fragment has a length of has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, the glycosylation fragment has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. [0238] In certain embodiments of a method of producing a glycoconjugate of this disclosure, the pilin-like-protein glycosylation fragment comprises or consists of PilMo (SEQ ID NO: 57) or PilMo lacking amino acids corresponding to residues 1–28 (PilMoΔ28, SEQ ID NO: 58) or a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, for example, wherein the C-terminus threonine is substituted with serine. In certain embodiments, the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), and PilCIP102637 (SEQ ID NO: 100). In certain embodiments, the TfpM-associated pilin- like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino 32080280 - 88 - Atty. Dkt. No.: 64100-234947 acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii- 65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), PilCIP102637 (SEQ ID NO: 100), and a fragment (e.g., C-terminus fragment) of any thereof and/or a variant wherein the C-terminus threonine is substituted with serine. And, in certain embodiments, the pilin- like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (PilMo_DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148). Further, in certain embodiments, the pilin-like-protein glycosylation fragment consists of Pil20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine. Further, in certain embodiments, the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil15S (SEQ ID NO: 153), Pil14S (SEQ ID NO: 154), Pil13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil11S (SEQ ID NO: 157), Pil10S (SEQ ID NO: 158), Pil9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil4S (SEQ ID NO: 164), or Pil3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine. [0239] In certain embodiments of a method of producing a glycoconjugate of this disclosure, the acceptor protein is a fusion protein and the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and a fragment of any thereof. In certain embodiments, the TfpM-associated pilin-like protein or glycosylation fragment thereof is translationally fused/linked to a heterologous carrier protein via an amino acid linker. 32080280 - 89 - Atty. Dkt. No.: 64100-234947 [0240] In certain embodiments of a method of producing a glycoconjugate of this disclosure, the acceptor protein is a fusion protein and the method comprises glycosylating the acceptor protein at two or more different positions. In certain embodiments, the method comprises glycosylating the acceptor protein with at least two different OTase classes in one expression system. In certain embodiments, the fusion protein comprises two or more glycosylation sequences (e.g., glycosylation fragment) associated with at least two different OTases. Representative examples of OTases that may be used in combination include PglB, PglL, PglS, TfpO, and TfpM. One of ordinary skill in the art would recognize that OTases cannot be used together if they both require a glycosylation sequence (sequon) in the same position, e.g., both at the N-terminus or both at the C-terminus. For example, generally, TfpO and TfpM cannot be used together if they both need a sequon at the extreme C-terminus. For example, in certain non-limiting illustrative embodiments, the acceptor protein comprises a TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus in addition to an additional glycosylation sequence of an OTase other than TfpM oligosaccharyltransferase (OTase). In certain embodiments, the other OTase is PglB, PglL, and/or PglS. In certain embodiments, one or more glycosylation sequence is a sequence internal to the fusion protein (i.e., not the C-terminal or N-terminal most sequence). In certain embodiments, the one or more glycosylation sequence is a sequence internal within the sequence of a carrier protein (e.g., Figure 33A). In certain embodiments, the additional glycosylation sequence is a sequence internal to the fusion protein (i.e., not the C-terminal or N-terminal most sequence). In certain embodiments, the additional glycosylation sequence is a sequence internal within the sequence of a carrier protein (e.g., Figure 33A). In certain embodiments, at least two different glycosylation sequences of two different OTase systems are covalently linked to an oligo- or polysaccharide. In certain embodiments, the TfpM-associated pilin-like-protein glycosylation fragment located at the fusion protein C-terminus and the additional glycosylation sequence are covalently linked to an oligo- or polysaccharide. In certain embodiments, 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 additional glycosylation sequences. In certain embodiments, the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five additional glycosylation sequences. In certain embodiments, the additional glycosylation sequences are identical. In certain embodiments, at least one additional glycosylation sequence differs from another. In certain embodiments, at least three, at least four, or at least five of the additional 32080280 - 90 - Atty. Dkt. No.: 64100-234947 glycosylation sequences all differ from each other. And, in certain embodiments, none of the additional glycosylation sequences are the same. For example, in certain embodiments, the method comprises further glycosylating an internal glycosylation fragment of ComP using a PglS OTase in addition to glycosylating the TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus. In certain embodiments, the ComP glycosylation fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least the amino acids ASA in positions 11-13. In certain embodiments, 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 fragments. In certain embodiments, the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five ComP glycosylation fragments. In certain embodiments, the ComP glycosylation fragments are identical. In certain embodiments, the ComP glycosylation fragments differ from each other. In certain embodiments, at least three, at least four, or at least five of the ComP glycosylation fragments all differ from each other. And, in certain embodiments, none of the ComP glycosylation fragments are the same. [0241] In certain embodiments of a method of producing a glycoconjugate of this disclosure (e.g., conjugation) occurs in vivo in a host cell. In certain embodiments, the host cell is a bacterial cell. In certain embodiments, the conjugation occurs in in Escherichia coli. In certain embodiments, the conjugation occurs in a bacterium from the genus Klebsiella. In certain embodiments, the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0242] In certain embodiments of a method of producing a glycoconjugate of this disclosure, the method comprises culturing a host cell that comprises: (a) a genetic cluster encoding for the proteins required to synthesize the oligo- or polysaccharide; (b) a TfpM OTase; and (3) the acceptor protein. [0243] In certain embodiments of a method of producing a glycoconjugate of this disclosure, the method produces a conjugate vaccine. Additional embodiments [0244] Provided for herein is a host cell comprising (a) a genetic cluster encoding for the proteins required to synthesize an oligo- or polysaccharide; (b) a TfpM OTase of this disclosure; and (3) an acceptor protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof of this disclosure. In certain embodiments, the acceptor protein 32080280 - 91 - Atty. Dkt. No.: 64100-234947 is a fusion protein. In certain embodiments, the host cell comprises a nucleic acid encoding the TfpM OTase. In certain embodiments, the host cell comprises a nucleic acid encoding the acceptor protein. And, in certain embodiments, the TfpM OTase and the acceptor protein are encoded by the same nucleic acid. [0245] Provided for herein is an isolated nucleic acid encoding the pilin-like-protein glycosylation fragment and/or the fusion protein of this disclosure. In certain embodiments, the nucleic acid is a vector. Also provided for is a host cell comprising such isolated nucleic acid of this disclosure. In certain embodiments, the host cell is a bacterial cell. In certain embodiments, the host cell is Escherichia coli. In certain embodiments, the host cell is from the genus Klebsiella. And, in certain embodiments, the host cell is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0246] Provided for herein is a composition comprising the conjugate vaccine or the fusion protein of this disclosure and an adjuvant and/or carrier. In certain embodiments, the composition is a pharmaceutical or therapeutic composition suitable for administration to a subject/patient. [0247] Provided for herein is a method of inducing a host immune response against a bacterial pathogen, the method comprising administering to a subject in need of the immune response an effective amount of the conjugate vaccine, the fusion protein, or the composition comprising the conjugate vaccine or the fusion protein and an adjuvant and/or carrier of this disclosure. Treatment with pharmaceutical compositions comprising an immunogenic composition can occur separately or in conjunction with other treatments, as appropriate. An amount adequate to accomplish this is defined as “effective amount,” “effective dose,” or “unit dose.” Amounts effective for this use will depend on, e.g., the glycoconjugate composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician. In some aspects, a priming dose is followed by a boosting dose over a period of time. In certain embodiments, the immune response is an antibody response. In certain embodiments, the immune response is selected from the group consisting of an innate response, an adaptive response, a humoral response, an antibody response, cell mediated response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system cross-talk, and a combination of two or more of said immune responses. In certain embodiments, the immune response is selected from the group consisting of an innate response, a humoral response, an 32080280 - 92 - Atty. Dkt. No.: 64100-234947 antibody response, a T cell response, and a combination of two or more of said immune responses. [0248] Provided for herein is a method of preventing or treating a bacterial disease and/or infection in a subject comprising administering an effective amount to a subject in need thereof the conjugate vaccine, the fusion protein, or the composition comprising the conjugate vaccine or the fusion protein and an adjuvant and/or carrier of this disclosure. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject is a companion animal. In certain embodiments, the subject is livestock. In certain embodiments, the infection is a localized or systemic infection of skin, soft tissue, blood, or an organ, or is auto-immune in nature. In certain embodiments, the disease is pneumonia. In certain embodiments, the infection is a systemic infection and/or an infection of the blood. In certain embodiments, the conjugate vaccine, the fusion protein, or the composition is administered via intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration. [0249] Provided for herein is a method of producing a pneumococcal conjugate vaccine against pneumococcal infection comprising: (a) isolating the glycoconjugate or a glycosylated fusion protein of this disclosure; and (b) combining the isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant and/or carrier. [0250] Provided for herein is a glycoconjugate, glycosylated fusion protein, or conjugate vaccine, or a composition of any thereof of this disclosure for use in inducing a host immune response against a bacterial pathogen and/or preventing or treating a bacterial disease and/or infection in a subject. [0251] Provided for herein is a recombinant nucleic acid construct comprising a nucleotide sequence encoding a TfpM oligosaccharyltransferase (OTase) operably linked to at least one heterologous transcriptional regulatory sequence. In certain embodiments, the TfpM OTase comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to TfpMMo (SEQ ID NO: 56), TfpMDSM16617 (SEQ ID NO: 63), TfpMZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpMYH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpMAI40 (SEQ ID NO: 70), TfpMF78 (SEQ ID NO: 71), TfpMS71 (SEQ ID NO: 72), TfpMANC4282 (SEQ ID NO: 73), TfpMCIP102159 (SEQ ID NO: 74), TfpMjunii-65 (SEQ ID NO: 75), TfpMYZS-X (SEQ ID NO: 76), TfpMCIP102637 (SEQ ID NO: 77), TfpMT-3-2 (SEQ ID NO: 78), TfpMBI730 (SEQ ID NO: 79), 32080280 - 93 - Atty. Dkt. No.: 64100-234947 TfpMA3K91 (SEQ ID NO: 80), and/or TfpM72-O-c (SEQ ID NO: 81). In certain embodiments, the TfpM OTase is TfpMMo (SEQ ID NO: 56), TfpMDSM16617 (SEQ ID NO: 63), TfpMZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpMYH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpMAI40 (SEQ ID NO: 70), TfpMF78 (SEQ ID NO: 71), TfpMS71 (SEQ ID NO: 72), TfpMANC4282 (SEQ ID NO: 73), TfpMCIP102159 (SEQ ID NO: 74), TfpMjunii-65 (SEQ ID NO: 75), TfpMYZS-X (SEQ ID NO: 76), TfpMCIP102637 (SEQ ID NO: 77), TfpMT-3-2 (SEQ ID NO: 78), TfpMBI730 (SEQ ID NO: 79), TfpMA3K91 (SEQ ID NO: 80), and/or TfpM72-O-c (SEQ ID NO: 81). In certain embodiments, the TfpM OTase is TfpMMo (SEQ ID NO: 56). In certain embodiments, the heterologous transcriptional regulatory sequence is a promotor sequence. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding a TfpM-associated pilin-like protein or glycosylation fragment thereof of this disclosure or a fusion protein of this disclosure comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof operably linked to the nucleotide sequence encoding the a TfpM OTase. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding a TfpM-associated pilin-like protein or glycosylation fragment thereof of this disclosure or a fusion protein of this disclosure comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof 5’ of and operably linked to the nucleotide sequence encoding the a TfpM OTase. In certain embodiments, the fusion protein of the construct also comprises a glycosylation sequence of an OTase other than TfpM, such as for PglB, PglL, PglS (e.g., ComP or a glycosylation fragment thereof). In certain embodiments, the coding sequence of the TfpM-associated pilin-like protein or glycosylation fragment thereof or a fusion protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof is within 2, 5, 10, 20, 30, 40, or 50 nucleotides of the sequence encoding the TfpM OTase. In certain embodiments, the coding sequence of the TfpM-associated pilin-like protein or glycosylation fragment thereof or a fusion protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof overlaps the operably linked nucleotide sequence encoding the a TfpM OTase. In certain embodiments, the TfpM-associated pilin-like protein comprises or consists of a full-length TfpM-associated pilin-like protein. In certain embodiments, the TfpM- associated pilin-like protein comprises or consists of a glycosylation fragment of a TfpM- associated pilin-like-protein that is less than a full-length TfpM-associated pilin-like protein. In certain embodiments, the pilin-like-protein glycosylation fragment has a length of from 3 to 138 amino acids in length, has a length of from 10 to 138 amino acids in length, has a length 32080280 - 94 - Atty. Dkt. No.: 64100-234947 of from 20 to 138 amino acids in length, 50 to 138 amino acids in length, has a length of from 100 to 138 amino acids in length, or has a length of from 116 to 138 amino acids in length. In certain embodiments, the pilin-like-protein glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 10 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, 50 to 139 amino acids in length, has a length of from 100 to 139 amino acids in length, or has a length of from 116 to 139 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from 3 to 140 amino acids in length, has a length of from 10 to 140 amino acids in length, has a length of from 20 to 140 amino acids in length, 50 to 140 amino acids in length, has a length of from 100 to 140 amino acids in length, or has a length of from 116 to 140 amino acids in length. In certain embodiments, the glycosylation fragment has a length of has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length. In certain embodiments, the glycosylation fragment has a length of from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, the glycosylation fragment has a length of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, the pilin-like-protein glycosylation fragment comprises or consists of PilMo (SEQ ID NO: 57) or PilMo lacking amino acids corresponding to residues 1–28 (PilMoΔ28, SEQ ID NO: 58) or a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, for example, wherein the C-terminus threonine is substituted with serine. In certain embodiments, the TfpM- associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72- O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), and PilCIP102637 (SEQ ID NO: 100). In certain embodiments, the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID 32080280 - 95 - Atty. Dkt. No.: 64100-234947 NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), PilCIP102637 (SEQ ID NO: 100), and a fragment (e.g., C-terminus fragment) of any thereof and/or a variant wherein the C-terminus threonine is substituted with serine. And, in certain embodiments, the pilin- like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (PilMo_DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C-terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148). Further, in certain embodiments, the pilin-like-protein glycosylation fragment consists of Pil20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine. Further, in certain embodiments, the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil15S (SEQ ID NO: 153), Pil14S (SEQ ID NO: 154), Pil13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil11S (SEQ ID NO: 157), Pil10S (SEQ ID NO: 158), Pil9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil4S (SEQ ID NO: 164), or Pil3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine. In certain embodiments, the fusion protein is a fusion protein of this disclosure. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding an additional OTase as described elsewhere herein operably linked to the TpfM OTase. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding the additional OTase 3’ of and operably linked to the TpfM OTase. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding the additional OTase 5’ of and operably linked to the TpfM OTase. In certain embodiments, the coding sequence of the additional OTase is within 10, 20, 30, 40, 50, 75, or 32080280 - 96 - Atty. Dkt. No.: 64100-234947 100 nucleotides of the sequence encoding the TfpM OTase. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding a PglS OTase 3’ operably linked to the TpfM OTase. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding a PglS OTase 3’ of and operably linked to the TpfM OTase. In certain embodiments, the recombinant construct further comprises a nucleotide sequence encoding a PglS OTase 5’ of and operably linked to the TpfM OTase. In certain embodiments, the coding sequence of the PglS OTase is within 10, 20, 30, 40, 50, 75, or 100 nucleotides of the sequence encoding the TfpM OTase. Further provided for herein is a vector comprising the recombinant nucleic acid construct. And further provided for herein is a host cell comprising the recombinant nucleic acid construct or the vector. In certain embodiments, the host cell is a bacterial cell. In certain embodiments, the host cell is Escherichia coli. In certain embodiments, the host cell is from the genus Klebsiella. And, in certain embodiments, the host cell is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. Provided for herein is a method for producing a TfpM OTase comprising culturing the host cell, wherein said vector is an expression vector, and recovering the TfpM OTase. EXAMPLES [0252] Crosslinking of bioconjugates to NP/VLP monomers affords repeated display of desired glycan and protein epitopes on larger scale than single protein molecules (Liu, Y., et al. (2023) Microb Cell Fact 22, 95). The following illustrative Examples describe the design and demonstration of bioconjugate-mi3 and bioconjugate-AP205 assemblies. These bioconjugates were generated using two different bacterial O-linking OTases: Acinetobacter baylyi PglS and Moraxella osloensis TfpM. PglS and TfpM belong to recently characterized O-linking OTase families that have the broadest saccharide substrate range of known OTases (Harding, C.M., et al. (2019) Nat Commun 10, 891; Knoot, C.J., et al. (2023) Glycobiology, Volume 33, Pages 57-74). Notably, PglS and TfpM are both able to transfer glycans with glucose at the reducing end, allowing these enzymes to be used to generate bioconjugate vaccines against a variety of pathogens whose native polysaccharides have this sugar at the reducing end (Harding, C.M., et al. (2019) Nat Commun 10, 891; Feldman, et al. (2019) PNAS, 116 (37) 18655-18663). For this application, E. coli maltose binding protein (MBP) and P. aeruginosa EPA were engineered to contain PglS- or TfpM-specific sequons (Knoot, C.J., et al. (2021) Glycobiology, Volume 31, Pages 1192–1203; Knoot, C.J., et al. (2023) Glycobiology, Volume 33, Pages 57-74) and subsequently glycosylated with O16 O antigen 32080280 - 97 - Atty. Dkt. No.: 64100-234947 from E. coli. The resulting PglS- or TfpM-derived MBP or EPA bioconjugates were covalently linked to NP monomers using the Spytag/Spycatcher technology. Example 1. SpyTagged MBP-O16 bioconjugates. [0253] The SpyTagged MBP-O16 bioconjugates (“first polypeptide”; e.g., Figure 1: “PROTEIN 1”) were produced in CLM24 glycoengineered E. coli strains. The MBP-SpyTag fusion proteins were separately expressed from a pEXT20 expression plasmid and WbbL was expressed from plasmid pMF19, a pEXT21 derivative. Expression of WbbL restores production of O16 O-antigen in most laboratory strains of E. coli. The mi3- and AP205- SpyCatcher fusion proteins (“second polypeptide”; e.g., Figure 1: “PROTEIN 2”) were separately produced in C41(DE3) E. coli. Following culturing of the E. coli expression strains in TB media, the cell pellets were frozen for downstream lysis and protein purification. [0254] SpyTagged O16-bioconjugates were purified from periplasmic cell extracts by using immobilized metal-affinity (Ni) chromatography (IMAC). The IMAC eluate was concentrated and buffer-exchanged prior to loading on an Akta FPLC instrument for further purification using anion-exchange chromatography. Anion-exchange was used to separate unglycosylated SpyTagged MBP from glycosylated SpyTagged MBP-O16. Fractions containing glycosylated SpyTagged MBP-O16 were pooled, concentrated and quantified using a BCA assay kit. The purified conjugates were stored at -80 °C in Tris-buffered saline (TBS) until used for the isopeptide bond formation reaction. [0255] Mi3 and AP205 nanoparticles (NPs) were purified by whole-cell lysis via sonication. The lysates were centrifuged at 18,000 xG prior to loading on IMAC resin as above. The eluate was concentrated and loaded on an FLPC size-exclusion column to separated fully assembled VLPs and NPs from unassembled, free monomers. Fractions containing the intact, assembled VLPs or NPs were determined based on reference to the masses of known standards run on the same column. These fractions were pooled, concentrated, quantified and stored in TBS at 4 °C until used for the isopeptide bond formation reaction. [0256] For the isopeptide formation reactions, two purified proteins (SpyTagged O16- bioconjugates and VLP/NP) were mixed at ~1:1 or ~2:1 molar ratios of mi3/AP205: SpyTagged O16-bioconjugates in TBS buffer. These reactions were incubated for 1.5 hours (mi3) or 3 hours (AP205) at 22 °C. The reactions were then analyzed using SDS-PAGE Coomassie staining, western blotting, and size-exclusion chromatography using a Sephacryl S- 400 HR column. 32080280 - 98 - Atty. Dkt. No.: 64100-234947 Example 2. Spytagged EPA-O16 bioconjugates. [0257] The Spytagged EPA-O16 bioconjugates were produced and purified in the same manner as MBP-O16 bioconjugates as disclosed elsewhere herein. All versions of the Spytagged EPA were produced from a pEXT20 expression plasmid. [0258] Crosslinking reactions between Spytagged O16 bioconjugates and purified mi3- Spycatcher or AP205-Spycatcher resulted in the production of higher-weight covalently cross- linked proteins as determined using Coomassie protein staining, western blotting, and size- exclusion chromatography. Using E. coli O16 antisera and anti-protein antibodies showed that the higher-weight species was composed of a O16 glycan-linked protein(s) (e.g., Figure 3 and Figure 6). Generally, TfpM-derived Spytagged bioconjugates more completely reacted with Spycatcher-mi3 or AP205 to form isopeptide-bonded assemblies based on the intensity of the cross-linked protein band in Coomassie-stained gels or western blotting and the observation that less of the either the NP/VLP monomer or bioconjugate band remained after the isopeptide bond reaction. [0259] Size-exclusion chromatography showed the bioconjugate-NP/VLP assemblies were larger in mass than those before the isopeptide reaction (Figure 3). [0260] Not all Spytagged-EPA bioconjugates were able to form detectable amounts of crosslinked, isopeptide-bonded species with mi3-Spycatcher or AP205-Spycatcher. Of the three Spytagged EPA variants tested, only EPA-Spytag-v1 resulted in the production of isopeptide-bonded species (Figure 9). Furthermore, only a subset of the protein linker variants of EPA-Spytag-v1 were able to form isopeptide-bonded species with mi3-Spycatcher (Figure 10). [0261] An isopeptide bond-formation time course showed the reaction between EPA- Spytag-v1 and mi3-Spycatcher was essentially >80% complete within 1 hour after starting the reaction based on the relative western blot protein band intensities (Figure 11). Example 3. Immunization with a glycosylated ComP bioconjugate elicits an immune response. [0262] T-cell dependent immune responses to conjugate vaccines are characterized by the secretion of high affinity IgG1 antibody (Avci, F.Y., Li, X., Tsuji, M. & Kasper, D.L. Nat Med 17, 1602-1609 (2011)). The immunogenicity of a CPS14-ComP bioconjugate in a murine vaccination model was evaluated (WO/2020/131236, which in incorporated by reference herein in its entirety). Sera collected from mice vaccinated with a CPS14-ComP bioconjugate had a significant increase in CPS14 specific IgG titers but not IgM titers. Further, secondary 32080280 - 99 - Atty. Dkt. No.: 64100-234947 HRP-tagged anti-IgG subtype antibodies were employed to determine which of the IgG subtypes had elevated titers. IgG1 titers appeared to be higher than the other subtypes. [0263] Next, a second vaccination trial was performed comparing the immunogenicity of a trivalent CPS8-, CPS9V-, and CPS14-ComP bioconjugate to the current standard of care, PREVNAR 13®. Serotypes 9V and 14 are included in PREVNAR 13® and elevated IgG titers could be seen in PREVNAR 13® immunized mice against these two serotypes. The monovalent immunization against serotype 14 also showed significant induction of serotype specific IgG titers, which were similar to the preliminary immunization. Mice receiving the trivalent bioconjugate, all had elevations in serotype specific IgG titers when compared to control as expected, day 49 sera have shown much more elevated IgG tires for serotypes 8 and 14 compared to serotype 9V. Nevertheless, IgG titers against 9V were still significantly higher than the placebo. Example 4. Cloning and Plasmid Assembly [0264] All primers and oligos used in this study are listed in Table 2. Working antibiotic concentrations used for liquid culture and in LB-agar plates were as follows: ampicillin (Amp), 100 µg/mL, kanamycin (Kan), 20 µg/mL, tetracycline (Tet), 10 µg/mL, spectinomycin (Sp), 50 µg/mL. For cloning the tfpM pilin-OTase genes, HiFi gblocks were ordered (Integrated DNA Technologies, IDT) designed with terminal 25 base pair overlaps for Gibson assembly with PCR-linearized plasmid. The plasmid backbone for these fragments was amplified from a pEXT20 plasmid (Dykxhoorn, D. M., et al. (1996) Gene 177, 133-136) encoding the P. aeruginosa EPA gene under control of a tac promoter (pVNM57) (Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203). The EPA gene has a deletion of residue E553 resulting in an inactivated toxin. The linearized plasmid was mixed separately with each of the synthesized tfpM gBlocks and assembled using an NEBuilder HiFi DNA Assembly Kit (New England Biolabs, NEB). After assembly, the plasmids were transformed into E. coli Stellar cells (Takara Bio) by heat shock, out-grown for an hour at 37ºC, and plated on LB-agar supplemented with Amp. Individual colonies were picked and grown in LB media with appropriate antibiotic and plasmids isolated using a GeneJet Plasmid Miniprep Kit (Thermo Fisher). All plasmids were sequence-verified by Sanger sequencing (Genewiz). The plasmid expressing the M. osloensis 1202 EPA-Pil∆28 fusion and TfpMMo was named pVNM227. To generate the PilMo site- directed mutants, the inventors designed overlapping PCR primers that introduced the necessary codon changes in the pilin gene and amplified each fragment from pVNM227 plasmid. The resulting PCR product was DpnI-digested (NEB) for 30 min at 37ºC and gel- 32080280 - 100 - Atty. Dkt. No.: 64100-234947 purified from agarose gel using a Pure-Link Gel Extraction Kit (Thermo Fisher). To insert the truncated pilin gene regions, complementary oligos with terminal 25 bp overlaps homologous to the pVNM227 PCR product were ordered. The oligos were re-suspended in purified water, mixed, and annealed together in thermocycler by heating to 98ºC for 5 min followed by a slow cooling to 4ºC at 0.1ºC/minute. The annealed oligos were diluted 1 to 5 in water and assembled with PCR-linearized pVNM227 using an NEBuilder HiFi DNA Assembly Kit (NEB). The resulting DNA was transformed into Stellar cells and plasmids isolated and verified as described above. The plasmid comprising the construct encoding EPA-Pil20 and TfpM was termed pVNM297. The N-terminally His-tagged EPA-Pil20 variant was constructed by linearizing pVNM297 using PCR and using this fragment in Gibson assembly with complementary annealed oligos containing the 6xHis coding region and terminal homologous regions, resulting in pVNM291. pVNM167 was generated by digesting the previously described EPAiGTcc plasmid (Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203) with SalI. The purified SalI fragment was Gibson assembled with the pglS gene with its native 100 bp 5’ UTR amplified from A. baylyi ADP1 gDNA. pVNM245 was generated from pVNM167 template by separate PCR reactions to amplify products with overhangs for Gibson assembly: (i) the vector backbone with PglS and EPA with one iGT, (ii) the second iGT for integration between E548 and G549 and (iii) the C-terminus of EPA downstream of the iGT. The plasmid pVNM337 was created by amplifying tfpM from pVNM291 with the primers EPA 3’ F1 and pglS-tfpM R1 and cloning the product into PCR-linearized pVNM167, which was amplified with pglS 5’ F1 and EPA 3’ R1. Phylogenetic trees for the TfpM and pilin proteins were generated using the phylogeny.fr server (on the world wide web at phylogeny.fr/) which uses MUSCLE, PhyML, and TreeDyn for sequence alignment, tree calculation, and image generation, respectively. Example 5. Expression of glycans and cloning of K. pneumoniae O2a glycan genes [0265] The S. pneumonia CPS8 glycan was expressed from plasmid pB8 (TetR) (Kay, E. J., et al. (2016) Open Biology 6, 150243), the Salmonella enterica LT2 glycan from plasmid pPR1347 (KanR) (Neal, B. L., et al. (1993) Journal of Bacteriology 175, 7115-7118), the E. coli O16 wbbL gene from plasmid pMF19 (SpR) (Feldman, M. F., et al. (2005) Proceedings of the National Academy of Sciences of the United States of America 102, 3016) and the GBSIII glycan was from a pBBR1MCS2 derivative (Duke, J. A., et al. (2021) ACS Infectious Diseases 7, 3111-3123). Bioconjugation with the K. pneumoniae O2a O-antigen has not previously been reported. To clone the genes that encode the machinery required to synthesize the O2a glycan, 32080280 - 101 - Atty. Dkt. No.: 64100-234947 PCR was used to amplified the wzm, wzt, wbbM, glf, wbbN, and wbbO genes (Clarke, B. R., et al. (2018) Journal of Biological Chemistry 293, 4666-4679) from K. pneumoniae strain NTUH K2044 genomic DNA. K. pneumoniae was cultured to saturation in LB media overnight and genomic DNA was isolated using a Wizard Genomic DNA Purification Kit (Promega). The plasmid backbone for the O2a cluster was amplified from plasmid pBBR1MCS2 (KanR) (Kovach, M. E., et al. (1995) Gene 166, 175-176). Primers for these reactions are listed in Table 2 The PCR products from these reactions were assembled using Gibson assembly with a NEBuilder HiFi DNA Assembly Kit (NEB). Stellar cells were transformed and plasmid isolated and verified as described in the previous section. Example 6. Bioconjugation and western blots [0266] E. coli strains used the bioconjugation experiments were either SDB1 or CLM24 (Feldman, M. F., et al. (2005) Proceedings of the National Academy of Sciences of the United States of America 102, 3016). SDB1 is a W3110 E. coli derivative with mutations in the genes encoding for WecA, the glycosyltransferase that initiates synthesis of the endogenous E. coli O16 antigen and WaaL, the enzyme transferring Und-PP linked glycans to Lipid A-core saccharide to produce LPS. CLM24 is a W3110 derivative with only a deletion of waaL. Elimination of these genes prevents crosstalk of the heterologous bioconjugation system and endogenous E. coli glycosylation pathways. To prepare E. coli strains for bioconjugation, the inventors electroporated plasmids using competent cells prepared as previously described (Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203) followed by out-growth at 37ºC in SOB media. The cells were plated on LB-agar with appropriate antibiotics. The next day, 8 – 10 colonies were picked and inoculated into LB or TB with antibiotics and grown overnight while shaking at 30ºC. The next morning, starter cultures were inoculated into either 30 mL media in a 125 mL Erlenmeyer flask or 1 L media in a 2 L flask to a starting optical density at 600 nm (OD600) of 0.05. Cultures were grown while shaking at 175 RPM until the OD600 reached 0.4 – 0.6 at which point the cultures were induced with 1 mM IPTG. All bioconjugation experiments were performed at 30 ºC unless otherwise noted. After overnight induction, amounting to 20 – 24 hours total growth, the OD600 was measured, and 0.5 OD units of cells pelleted for analysis. [0267] The cell pellets were suspended in 100 µl 1X Laemmli Buffer (Biorad) and boiled for 10 min at 100ºC. The boiled samples were briefly centrifuged at 10,000 rcf and equivalent amounts, normalized to the same OD600 per lane, loaded for SDS-PAGE separation on a 7.5% Mini-Protean TGX gel (Biorad). Proteins were transferred to a nitrocellulose membrane using 32080280 - 102 - Atty. Dkt. No.: 64100-234947 a semi-dry electrode system and blocked with Intercept Blocking Buffer (Li-Cor) for one hour. The membrane was incubated with primary antibodies in 1:1 blocking and TBST for 45 min. For protein detection, commercial rabbit anti-EPA and mouse anti-6xHis antibodies (Millipore- Sigma) were used. Rabbit glycan antibodies for CPS8, GBSIII, and O16 were purchased from SSI Diagnostica. K. pneumoniae rabbit O2a antibodies were a generous gift from Prof. Chris Whitfield (University of Guelph) (Clarke, B. R., et al. (2018) Journal of Biological Chemistry 293, 4666-4679). Salmonella Group B rabbit antibodies were purchased from BD. Primary incubation was followed by three washes with TBST buffer totaling 15 min The membranes were then incubated with secondary antibodies IRDye 680RD goat anti-mouse and/or IRDye 800CW goat anti-rabbit (Li-Cor) in 1:1 blocking buffer and TBST for 30 min. After a final 15- min TBST wash, the membranes were imaged using a Li-Cor Odyssey CLx. Example 7. Lys-C digestion of recombinant M. osloensis PilMo∆28 [0268] In-gel digestion was accomplished according to the protocol of Shevchenko et al (Shevchenko, A., et al. (2006) Nat Protoc 1, 2856-2860) with minor modifications. Gel- separated glycosylated EPA-Pil∆28 was excised and destained with destaining solution (50 mM NH4HCO3, 50% ethanol) twice for 10 min at room temperature with shaking at 750 RPM. The destained band was then dehydrated with 100% ethanol for 10 min and dried by vacuum- centrifugation for 10 min before being rehydrated in 10 mM DTT in 50 mM NH4HCO3. Reduction was carried out for 60 min at 56°C after which the gel band was dehydrated twice with 100% ethanol for 10 min to remove the remaining reduction buffer. The reduced sample was then sequentially alkylated with 55 mM iodoacetamide in 50 mM NH4HCO3 for 45 min at RT in the dark. The alkylated sample was then washed four times for 10 min with 50 mM NH4HCO3 followed by 100% ethanol, followed by 50 mM NH4HCO3 followed by 100% ethanol before being dried by vacuum-centrifugation. The dried alkylated sample was then rehydrated with 20 ng/µl Lys-C endoprotease (Wako Chemicals) in 40 mM NH4HCO3 at 4°C for 1hr. Excess Lys-C was removed, gel pieces were covered in 40mM NH4HCO3 and incubated overnight at 37°C. Peptides were concentrated and desalted using C18 stage tips (Ishihama, Y., et al. (2006) J Proteome Res 5, 988-994; Rappsilber, J., et al. (2007) Nat Protoc 2, 1896-1906) then eluted in buffer B (0.5% acetic acid, 80% acetonitrile (ACN)) before being dried and stored at -20°C prior to LC-MS analysis. Example 8. Analysis of recombinant M. osloensis PilMo∆28 using reversed phase LC-MS 32080280 - 103 - Atty. Dkt. No.: 64100-234947 [0269] The C18-concentrated digest was resuspended in buffer A* (0.1% TFA, 2% ACN) and separated using a two-column chromatography set up comprising a PepMap100 C1820 mm x 75 μm trap and a PepMap C18500 mm x 75 μm analytical column (Thermo Fisher Scientific). The sample was concentrated onto the trap column at 5 μl/minute using 0.1% formic acid (FA) for 5 min and infused into an Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer equipped with a FAIMS Pro interface (Thermo Fisher Scientific) at 300 nl/min via the analytical column using a Dionex Ultimate 3000 UPLC (Thermo Fisher Scientific) by altering the concentration of buffer A (2% DMSO, 0.1% FA) and buffer B (78% ACN, 2% DMSO and 0.1% FA). Identification of potential glycopeptides utilized a 140-minute analytical run while targeted analysis utilized a 60-minute run. Within the identification analytical run the buffer composition was altered from 3% buffer B to 28% buffer B over 120 min, 28% buffer B to 40% buffer B over 9 min, 40% buffer B to 100% buffer B over 3 min, then the composition was held at 100% buffer B for 2 min, and then dropped to 3% buffer B over 2 min and held at 3% buffer B for another 8 min The Lumos™ Mass Spectrometer was operated in a stepped FAIMS data-dependent mode at three different FAIMS CVs, -25, -45 and -65 as previously described (Ahmad Izaham, A. R., et al. (2021) J Proteome Res 20, 599-612), switching between the acquisition of a single Orbitrap MS scan (60k resolution) every 1.5 sec followed by Orbitrap HCD scans (maximum fill time 120 milliseconds, AGC 2 x 105 with a resolution of 30k for Orbitrap MS-MS scans and an NCE of 25, 30,45) at each of the three FAIMS CVs. For targeted characterization of PilMo glycopeptides an analytical run altering the buffer composition from 3% buffer B to 15% buffer B over 30 min, 15% buffer B to 30% buffer B over 10 min, 30% buffer B to 80% buffer B over 5 min, then the composition was held at 100% buffer B for 5 min, and then dropped to 3% buffer B over 1 min and held at 3% buffer B for another 9 min was undertaken. Parallel reaction monitoring using HCD (maximum fill time 250 ms, AGC 2.5 x 105 with a resolution of 30k for the Orbitrap MS-MS scan and an NCE of 15, 30,35) and EThcD (maximum fill time 250 ms, AGC 2.5 x 105 with a resolution of 30k for the Orbitrap MS-MS scan and ETD reaction times controlled using calibration charge dependent ETD parameters (Rose, C. M., et al. (2015) J Am Soc Mass Spectrom 26, 1848-1857) of the +2 charge state of the HexHexA modified glycopeptide 762FLPANCRGT770 (687.2972 m/z) was undertaken with a FAIMS CV of -45. Example 9. Open searching of PilMo∆28 and the annotation of HexHexA-modified C-terminal peptide 32080280 - 104 - Atty. Dkt. No.: 64100-234947 [0270] The identification of PilMo glycosylation events was accomplished using open database searching as previously described (Lewis, J. M., et al. (2021) J Vis Exp,). Briefly, datafiles were processed with MSfragger 3.4 (Polasky, D. A., et al. (2020) Nat Methods 17, 1125-1132; Kong, A. T., et al. (2017) Nat Methods 14, 513-520) in FragPipe (version 17.1) searching against the Moraxella osloensis PilMo sequence (NCBI Accession: WP_156627541.1). Searches were undertaken using “Lys-C” enzyme specificity with carbamidomethylation of cysteine as a fixed modification and oxidation of methionine as a variable modification with a maximum of 2 missed cleavages allowed. To enable identification of potential glycosylation events a mass tolerance, referred to as a delta mass, of 0 to 2000 Da was allowed. Delta masses observed on the C-terminal peptide 762FLPANCRGT770 (SEQ ID NO: 61) were manually inspected to identify potential glycosylation events. Parallel reaction monitoring results corresponding to the HexHexA-modified glycopeptide 762FLPANCRGT770 (SEQ ID NO: 62) was manually extracted using the Freestyle Viewer (1.7 SP1, Thermo Fisher Scientific) the MS/MS data annotated using the Interactive Peptide Spectral Annotator (Brademan, D. R., et al. (2019) Mol Cell Proteomics 18, S193-S201) (on the world wide web at interactivepeptidespectralannotator.com/PeptideAnnotator.html). Annotation of spectra allowed for the modification of the terminal T residue with HexHexA (338.0849 Da) as well as Hex (162.0528 Da). The resulting MS data and search results have been deposited into the PRIDE ProteomeXchange Consortium repository (Perez-Riverol, Y., et al. (2019) Nucleic Acids Res 47, D442-D450; Perez-Riverol, Y., et al. (2015) Proteomics 15, 930-949) and can be accessed with the identifier PXD033468. Example 10. Bioconjugate protein purification [0271] Cells for protein purification were grown in 1 L TB media and bioconjugates isolated using an osmotic shock protocol. After overnight growth and induction, the cells were pelleted by centrifugation, and washed in 0.9% NaCl. The washed cell pellets were suspended in 200 mM Tris-HCl pH 8.5, 100 mM EDTA, 25% sucrose and incubated while rolling for 30 min at 4ºC. Cells were pelleted by centrifugation at 4,700 rcf for 30 min and the resulting pellet suspended in 20 mM Tris-HCl pH 8.5 and incubated while rolling for 45 min at 4ºC. The suspension was centrifuged for 30 min at 18,000 rcf. The supernatant containing the periplasmic fraction was concentrated and either loaded directly on an FPLC anion-exchange column or, for His-tagged EPA-Pil∆28 bioconjugates, purified using Nickel IMAC as previously described (Knoot, C. J., et al. (2021) Glycobiology 31, 1192-1203). The periplasmic extract or IMAC eluate was concentrated and buffer-exchanged into 20 mM Tris-HCl pH 8.0, 32080280 - 105 - Atty. Dkt. No.: 64100-234947 filtered through a 0.2 µm PES filter then loaded on an Äkta pure FPLC instrument (Cytiva) equipped with a SOURCE 15Q 4.6/100 PE anion-exchange column (Cytiva). The bioconjugates were eluted at 2 mL/min using a stepwise gradient with buffer A (20 mM Tris pH 8) and buffer B (20 mM Tris pH 8, 1 M NaCl) from 0% B to 25% in 5% increments at 10 column volumes for each concentration. Bioconjugates for immunization were further purified using a Superdex 200 Increase 10/300 GL column. The concentrated bioconjugates pooled from the anion-exchange column were loaded on a pre-equilibrated Superdex 200 column in PBS buffer and eluted at a flow rate of 0.75 mL/min Fractions containing the purified bioconjugates were pooled, concentrated, and frozen at -80ºC for storage. Protein concentrations for immunization and western blots were determined using a Pierce BCA Protein Assay kit (Thermo Fisher). The ratio of polysaccharide to protein calculate for vaccine dosing was determined using the method described in Duke et al. (Duke, J. A., et al. (2021) ACS Infectious Diseases 7, 3111-3123). Example 11. Murine immunization [0272] All murine immunizations followed ethical regulations for animal testing and research. Experiments were carried out at Washington University School of Medicine in St. Louis according to the institutional guidelines and received approval from the Institutional Animal Care and Use Committee at Washington University in St. Louis. Five-week-old female CD-1 outbred mice (Charles River Laboratories) were subcutaneously injected with 100 μL of a vaccine formulation on days 0, 14, and 28. The vaccination groups were 291 alone (5 μg protein) and GBSIII-291 (5 μg protein, 1 μg polysaccharide). Mice had sera collected on days 0, 14, 28, and 42. All vaccines were formulated with Alhydrogel® 2% aluminum hydroxide gel (InvivoGen) at a 1:9 ratio (50 μL vaccine to 5.5 μL alum in 44.5 μL 1x sterile phosphate buffered saline). Example 12. Enzyme-linked immunosorbent assay (ELISA) [0273] IgG kinetic titers were determined using enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates (TRP Immunomaxi plates) were coated in triplicate overnight with approximately 106 CFU/100 ^L of glycoengineered E. coli expressing the GBSIII capsular polysaccharide in sodium carbonate buffer. The coating E. coli strain was grown the same as referenced above and after overnight induction to induce GBSIII expression was washed and diluted to coat plates. Wells were blocked with 1% BSA in PBS and washed with 0.05% PBS-Tween (PBST), all subsequent washes were the same. Serum from mice was 32080280 - 106 - Atty. Dkt. No.: 64100-234947 diluted to 1:100 and added to wells for 1 hr at room temperature then washed. Total IgG titers were detected by HRP conjugated anti-mouse IgG (GE Lifesciences, 1:5000 dilution) added to wells for 1 hour at room temperature. After washing, plates were developed using 3,3′,5,5′ tetramethyl benzidine (TMB) substrate (Biolegend) and stopped with 2 N H2SO4. The optical densities were determined at 450 nm using a microplate reader (Bio-Tek). Total IgG product was determined using IgG standards to generate a standard curve for data fitting. Standard wells were coated with IgG in sodium carbonate buffer and treated the same as sample wells thereafter. All wells were normalized to blank wells that were treated the same as all samples wells minus receiving the primary mouse sera. Significance was determined using Mann- Whitney nonparametric test with P<0.05. [0274] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents. ***** [0275] Certain embodiments of the present disclosure can be defined in any of the following numbered paragraphs: [0276] 1. A fusion protein comprising: (i) a glycosylation fragment and (ii) a first polypeptide tag, wherein the first polypeptide tag can spontaneously form an isopeptide bond with a second polypeptide tag binding partner; optionally, wherein the glycosylation fragment is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, or 40 amino acids in length; optionally, wherein the glycosylation fragment is not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, 40, 50, 60, 80, or 100 amino acids in length; optionally, wherein the glycosylation fragment is from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, or 30 amino acids in length to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, or 40 amino acids in length; and optionally, wherein the fusion protein comprises a carrier protein. [0277] 2. The fusion protein of Paragraph 1, wherein the fusion protein is a glycoconjugate comprising a saccharide covalently attached to the fusion protein via the glycosylation fragment; optionally, wherein the saccharide is covalently attached to the glycosylation fragment through an N-linkage, O-linkage, or C-linkage; and optionally, wherein said glycoconjugate is immunogenic. 32080280 - 107 - Atty. Dkt. No.: 64100-234947 [0278] 3. The fusion protein of Paragraph 1 or 2, wherein the first polypeptide tag is translationally fused at the N-terminal or C-terminal end of the fusion protein. [0279] 4. The fusion protein of Paragraph 1 or 2, wherein the first polypeptide tag is translationally fused internally within the fusion protein; optionally, wherein the first polypeptide tag is translationally fused internally within the sequence of a carrier protein. [0280] 5. The fusion protein of any one of Paragraphs 1 to 4, wherein the glycosylation fragment is translationally fused at the N-terminal or C-terminal end of the fusion protein. [0281] 6. The fusion protein of any one of Paragraphs 1 to 5, wherein the glycosylation fragment is translationally fused internally within the fusion protein; [0282] optionally, wherein the glycosylation fragment is translationally fused internally within the sequence of a carrier protein. [0283] 7. The fusion protein of any one of Paragraphs 1 to 6, wherein the first polypeptide tag is a SpyTag (SEQ ID NO: 416), SpyTag002 (SEQ ID NO: 417), SpyTag003 (SEQ ID NO: 418), or a DogTag (SEQ ID NO: 419); optionally, wherein the SpyTag, Spytag002, or Spytag003 is translationally fused at the N-terminal or C-terminal end of the fusion protein, optionally, wherein the DogTag is translationally fused internally within the fusion protein. [0284] 8. The fusion protein of any one of Paragraphs 1 to 7, wherein the glycosylation fragment is a ComP glycosylation fragment; optionally, wherein the ComP glycosylation fragment comprises or consists of the amino acid sequence CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 412) or a fragment thereof comprising the amino acids ASA, or a variant thereof comprising the amino acids ASA in positions 11-13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions; optionally, wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of: iGTccΔ0-1 CTGVTQIASGASAATTNVASAQ (SEQ ID NO: 232); iGTccΔ1-0 TGVTQIASGASAATTNVASAQC (SEQ ID NO: 243); iGTccΔ1-1 TGVTQIASGASAATTNVASAQ (SEQ ID NO: 244); iGTccΔ1-2 TGVTQIASGASAATTNVASA (SEQ ID NO: 245); iGTccΔ2-1 GVTQIASGASAATTNVASAQ (SEQ ID NO: 256); iGTccΔ2-2 GVTQIASGASAATTNVASA (SEQ ID NO: 257); 32080280 - 108 - Atty. Dkt. No.: 64100-234947 iGTccΔ2-3 GVTQIASGASAATTNVAS (SEQ ID NO: 258); iGTccΔ3-2 VTQIASGASAATTNVASA (SEQ ID NO: 269); iGTccΔ3-3 VTQIASGASAATTNVAS (SEQ ID NO: 270); iGTccΔ3-4 VTQIASGASAATTNVA (SEQ ID NO: 271); iGTccΔ4-3 TQIASGASAATTNVAS (SEQ ID NO: 282); iGTccΔ4-4 TQIASGASAATTNVA (SEQ ID NO: 283); iGTccΔ4-5 TQIASGASAATTNV (SEQ ID NO: 284); iGTccΔ5-4 QIASGASAATTNVA (SEQ ID NO: 295); iGTccΔ5-5 QIASGASAATTNV (SEQ ID NO: 296); iGTccΔ5-6 QIASGASAATTN (SEQ ID NO: 297); iGTccΔ6-5 IASGASAATTNV (SEQ ID NO: 308); or iGTccΔ6-6 IASGASAATTN (SEQ ID NO: 309), or a variant thereof comprising the amino acids ASA corresponding to positions 11-13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. [0285] 9. The fusion protein of any one of Paragraphs 1 to 7, wherein the glycosylation fragment is a TfpM-associated pilin glycosylation fragment; [0286] optionally, wherein the TfpM-associated pilin glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (SEQ ID NO: 413) or a fragment thereof comprising at least the last three amino acids from the TfpM-associated pilin C-terminus; [0287] or a variant thereof comprising the last three amino acids from the TfpM-associated pilin C-terminus and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. [0288] 10. The fusion protein of any one of Paragraphs 1 to 7, wherein the glycosylation fragment is a PilE glycosylation fragment; optionally, wherein the PilE glycosylation fragment comprises or consists of the amino acid SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) or a fragment thereof comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414; or a variant thereof comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. [0289] 11. The fusion protein of any one of Paragraphs 1 to 7, wherein glycosylation fragment is a PglB glycosylation fragment; optionally, wherein the PglB glycosylation fragment comprises or consists of the consensus motif amino acid sequence X1 X2 N X3 X4, wherein X1 is D or E, X2 is any amino acid except proline, X3 is any amino acid except proline, and X4 is S or T. 32080280 - 109 - Atty. Dkt. No.: 64100-234947 [0290] 12. The fusion protein of any one of Paragraphs 1 to 7, wherein the glycosylation fragment is a PilA glycosylation fragment; optionally, wherein the PilA glycosylation fragment comprises or consists of the PilA pilin disulfide loop region (SEQ ID NO: 415) or a fragment thereof comprising at least the last three amino acids from the PilA C-terminus; or a variant thereof comprising at least the last three amino acids from the PilA terminus and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. [0291] 13. The fusion protein of any one of Paragraphs 1 to 7, wherein the glycosylation fragment is a STT3 glycosylation fragment; optionally, wherein the STT3 glycosylation fragment comprises or consists of the consensus motif amino acid sequence N X1 X2, wherein X1 is any amino acid except proline and X2 is S or T. [0292] 14. The fusion protein of any one of Paragraphs 1 to 7, wherein the glycosylation fragment is a N-linking glycosyltransferase glycosylation fragment; optionally, wherein the N-linking glycosyltransferase glycosylation fragment comprises or consists of the consensus motif amino acid sequence N X1 X2, wherein X1 is any amino acid and X2 is S or T [0293] 15. The fusion protein of any one of Paragraphs 1 to 7, wherein the glycosylation fragment is an O-linking glycosyltransferase glycosylation fragment; optionally, wherein the O-linking glycosyltransferase glycosylation fragment comprises or consists of a fragment of the serine or threonine rich repeats from the serine-rich repeats (SRR) adhesins of streptococci or staphylococci bacteria; optionally, wherein the O-linking glycosyltransferase glycosylation fragment comprises or consists of serine or threonine rich repeats from the adhesin GspB from Streptococcus gordonii. [0294] 16. The fusion protein of any one of Paragraphs 1 to 15, wherein the fusion protein comprises two or more glycosylation fragments; optionally, wherein the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 glycosylation fragments; optionally, wherein the fusion protein comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 glycosylation fragments to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 glycosylation fragments; 32080280 - 110 - Atty. Dkt. No.: 64100-234947 optionally, wherein at least one glycosylation fragment is located at the N-terminal or C-terminal end of the fusion protein and at least one glycosylation fragment is located internally within the fusion protein; optionally, wherein at least two glycosylation fragments are located internally within the fusion protein; and/or optionally, wherein one glycosylation fragment is located at the N-terminal end of the fusion protein and where glycosylation fragment is located at the C-terminal end of the fusion protein. [0295] 17. The fusion protein of Paragraph 16, wherein the two or more glycosylation fragments are the same; wherein at least one of the two or more glycosylation fragments is different; or wherein each of the glycosylation fragments is different; optionally, wherein one glycosylation fragment is a ComP glycosylation fragment and wherein one glycosylation fragment is a TfpM-associated pilin glycosylation fragment; optionally, wherein the fusion protein is a glycoconjugate comprising two or more saccharide covalently attached to the fusion protein via the two or more glycosylation fragments, optionally, wherein the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 covalently attached saccharides; optionally, wherein the fusion protein comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 covalently attached saccharides to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 covalently attached saccharides; and optionally, wherein the two or more saccharides are the same; wherein at least one of the two or more saccharides is different; or wherein each of the saccharides is different. [0296] 18. The fusion protein of any one of Paragraphs 1 to 17, wherein the fusion protein comprises a carrier protein selected from the group consisting of Escherichia coli maltose binding protein, Pseudomonas aeruginosa Exotoxin A (EPA), Pseudomonas aeruginosa PcrV, CRM197, Haemophilus influenzae Protein D, cholera toxin B subunit, or tetanus toxin, and a fragment of any thereof. 32080280 - 111 - Atty. Dkt. No.: 64100-234947 [0297] 19. A composition comprising a polypeptide pair that comprises a first polypeptide and a second polypeptide, wherein the first polypeptide is the fusion protein of any one of Paragraphs 1 to 18, wherein the second polypeptide comprises a second polypeptide tag binding partner to the first polypeptide tag of the first polypeptide, and wherein the first polypeptide is attached to the second polypeptide via an isopeptide bond between the first polypeptide tag and the second polypeptide tag; optionally, where the second polypeptide comprises a monomeric polypeptide that can spontaneously multimerize/self-assemble into a higher-order, multimeric structure; and/or further optionally, wherein said higher-order, multimeric structure is an icosahedron or dodecahedron particle (e.g,. resembling nanocages), virus-like particle, or Adenoviral vector. [0298] 20. The polypeptide pair composition of Paragraph 19, wherein the second polypeptide comprises an Adenoviral capsid structural protein; wherein the second polypeptide comprises a coat protein of the bacteriophage AP205; wherein the second polypeptide comprises a fragment of the 2-keto-3-deoxy- phosphogluconate aldolase (i301); or wherein the second polypeptide comprises a fragment of a mutated 2-keto-3-deoxy- phosphogluconate aldolase (mi3). [0299] 21. The polypeptide pair composition of Paragraph 19 or 20, wherein the second polypeptide tag is a SpyCatcher (SEQ ID NO: 420), SpyCatcher002 (SEQ ID NO: 421), SpyCatcher003 (SEQ ID NO: 422), or a DogCatcher (SEQ ID NO: 423); optionally, wherein the first polypeptide tag is a SpyTag and the second polypeptide tag is a SpyCatcher; wherein the first polypeptide tag is a SpyTag002 and the second polypeptide tag is a SpyCatcher002; wherein the first polypeptide tag is a SpyTag003 and the second polypeptide tag is a SpyCatcher003; or wherein the first polypeptide tag is a DogTag and the second polypeptide tag is a DogCatcher. [0300] 22. The polypeptide pair composition of any one of Paragraphs 19 to 21, wherein the second polypeptide tag is translationally fused at the N-terminal or C- terminal end of the second polypeptide, or 32080280 - 112 - Atty. Dkt. No.: 64100-234947 wherein the second polypeptide tag is translationally fused internally within the second polypeptide. [0301] 23. The polypeptide pair composition of any one of Paragraphs 19 to 22, wherein the first polypeptide is a bioconjugate comprising a saccharide covalently attached to the glycosylation fragment of the first polypeptide; optionally, wherein said composition is immunogenic. [0302] 24. The polypeptide pair composition of any one of Paragraphs 19 to 23, further comprising an adjuvant and/or an excipient. [0303] 25. The polypeptide pair composition of any one of Paragraphs 19 to 24, wherein the composition is a pharmaceutical/therapeutic composition. [0304] 26. The polypeptide pair composition of any one of Paragraphs 19 to 25, wherein the composition is a conjugate vaccine. [0305] 27. A complex comprising two or more of the polypeptide pairs of any one of Paragraphs 19 to 26; optionally, wherein the complex comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, or more complexed polypeptide pairs of any one of Paragraphs 19 to 26; optionally, wherein the complex comprises from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, or 400 complexed polypeptide pairs of any one of Paragraphs 19 to 26 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, or 500 complexed polypeptide pairs of any one of Paragraphs 19 to 26; and/or optionally, wherein the complex is a self-assembled, multimeric higher-order structure. [0306] 28. The complex of Paragraph 27, wherein said self-assembled, multimeric higher- order structure is an icosahedron or dodecahedron particle (e.g,. resembling nanocages), virus- like particle, or Adenoviral vector. [0307] 29. The complex of Paragraph 27 or 28, wherein all of the first polypeptides of the complex comprise the same fusion protein. [0308] 30. The complex of Paragraph 27 or 28, wherein at least two of the first polypeptides of the complex comprise different fusion proteins. 32080280 - 113 - Atty. Dkt. No.: 64100-234947 [0309] 31. The complex any one of Paragraphs 27 to 30, wherein at least one first polypeptide of the complex is a bioconjugate comprising a saccharide covalently attached to the glycosylation fragment of the first polypeptide; optionally, wherein at least about 5%, 10%, 25%, 50%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% of the first polypeptides of the complex are bioconjugates; optionally, wherein from any of about 5%, 10%, 25%, 50%, 75%, 80%, 90%, 95%, 97%, or 98% of the first polypeptides of the complex to about any of about 10%, 25%, 50%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% of the first polypeptides of the complex are bioconjugates; optionally, wherein 100% of the first polypeptides of the complex are bioconjugates; optionally, wherein the complex is immunogenic. [0310] 32. The complex of any one of Paragraphs 27 to 31, wherein two or more of the first polypeptides of the complex are bioconjugates comprising a covalently attached saccharide; optionally, wherein the complex comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 2,000, 2,500, or 5,000 covalently attached saccharides; optionally, wherein the complex comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 2,000, or 2,500 covalently attached saccharides to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 2,000, 2,500, or 5,000 covalently attached saccharides; optionally, wherein all of the saccharides attached to the complex are the same; optionally, wherein at least one of the two or more saccharides attached to the complex is different; optionally, wherein there are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 different saccharides attached to the complex; optionally wherein each of the saccharides attached to the complex is different. [0311] 33. A pharmaceutical/therapeutic composition comprising the complex of any one of Paragraphs 27 to 32 and adjuvant and/or an excipient. 32080280 - 114 - Atty. Dkt. No.: 64100-234947 [0312] 34. The complex of any one of Paragraphs 27 to 32 or the pharmaceutical/therapeutic composition of Paragraph 33, wherein the complex or composition is a conjugate vaccine. [0313] 35. A method of making the polypeptide pair of any one of Paragraphs 19 to 26, the method comprising contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag binding partner. [0314] 36. The method of Paragraph 35, further comprising glycosylating the first polypeptide with a saccharide before contact and isopeptide bond formation with the second polypeptide; optionally, wherein the first polypeptide is glycosylated in vivo before contact and isopeptide bond formation with the second polypeptide; and optionally, wherein the method comprises isolating/purifying the in vivo glycosylated first polypeptide before contact and isopeptide bond formation with the second polypeptide. [0315] 37. The method of Paragraph 35, wherein the first polypeptide is glycosylated after contact and isopeptide bond formation with the second polypeptide. [0316] 38. A method of making the complex of any one of Paragraphs 27 to 32, the method comprising: (i) forming a self-assembled, multimeric higher-order structure of the second polypeptide and then contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag; or (ii) contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag and then forming of a self-assembled, multimeric higher-order structure of the second polypeptide. [0317] 39. The method of Paragraph 38, further comprising glycosylating the first polypeptide with a saccharide, optionally: wherein the first polypeptide is glycosylated before the isopeptide bond is formed between the first polypeptide and the second polypeptide; wherein the first polypeptide is glycosylated after the isopeptide bond is formed between the first polypeptide and the second polypeptide; 32080280 - 115 - Atty. Dkt. No.: 64100-234947 wherein the first polypeptide is glycosylated before it is incorporated into a multimeric, higher-order structure; and/or wherein the first polypeptide is glycosylated after is has been incorporated into a multimeric, higher-order structure. [0318] 40. The method of any one of Paragraphs 36, 37, or 39, wherein the saccharide is transferred to the fusion protein by the action of an N-linking oligosaccharyltransferase (N- OTase), an O-linking oligosaccharyltransferase (O-OTase), an N-linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C-mannosyltransferase (CMT). [0319] 41. The method of any one of Paragraphs 36, 37, 39, or 40: wherein a ComP glycosylation fragment is glycosylated by a PglS OTase; wherein a TfpM-associated pilin glycosylation fragment is glycosylated by a TfpM OTase, optionally, wherein a ComP glycosylation fragment is glycosylated by a PglS OTase and a TfpM-associated pilin glycosylation fragment is glycosylated by a TfpM OTase; wherein a PilE glycosylation fragment is glycosylated by a PglL OTase; wherein a PglB glycosylation fragment is glycosylated by a PglB OTase; wherein a PilA glycosylation fragment is glycosylated by a TfpO or PilO OTase; wherein a STT3 glycosylation fragment is glycosylated by the STT3 catalytic subunit; wherein a PilA_Pa5196-associated pilin glycosylation fragment is glycosylated by a TfpW glycosyltransferase; wherein an N-linking glycosyltransferase glycosylation fragment is glycosylated by an N-linking glycosyltransferase from Actinobacillus pleuropneumoniae; from Haemophilus influenzae; or from Yersinia enterocolitica; and/or wherein an O-linking glycosyltransferase glycosylation fragment is glycosylated by GtfA/GtfB glycosyltransferases. [0320] 42. The method of any one of Paragraphs 36, 37, 39, or 40: wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a PglS OTase; wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpM OTase, optionally, wherein a saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a PglS OTase and another saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpM OTase; wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a PglL OTase; 32080280 - 116 - Atty. Dkt. No.: 64100-234947 wherein the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using a PglB OTase; wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpO or PilO OTase; wherein the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using a STT3 OTase; wherein the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using an AlgB OTase; wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpW glycosyltransferase; wherein the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using an N-linking glycosyltransferase; wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using an O-linking glycosyltransferase; wherein the saccharide is covalently linked to a carbon atom within a glycosylation fragment using a C-mannosyltransferase. [0321] 43. The method of Paragraph 42: wherein the saccharide is covalently linked to an oxygen atom within a ComP glycosylation fragment (e.g., SEQ ID NO: 412 or a variant thereof) using a PglS OTase (e.g., SEQ ID NO: 400); wherein the saccharide is covalently linked to an oxygen atom within a TfpM glycosylation fragment (e.g., SEQ ID NO: 413 or a variant thereof) using a TfpM OTase (e.g., SEQ ID NO: 402); wherein the saccharide is covalently linked to an oxygen atom within a PilE glycosylation fragment (e.g., SEQ ID NO: 414 or a variant thereof) using a PglL OTase (e.g., SEQ ID NO: 404); wherein the saccharide is covalently linked to a nitrogen atom within a PglB glycosylation fragment (e.g., X1 X2 N X3 X4, wherein X1 is D or E, X2 is any amino acid except proline, X3 is any amino acid except proline, and X4 is S or T) using a PglB OTase (e.g., SEQ ID NO: 405); wherein the saccharide is covalently linked to an oxygen atom within a PilA glycosylation fragment (e.g., SEQ ID NO: 415 or a variant thereof) using a TfpO/PilO OTase (e.g., SEQ ID NO: 407); 32080280 - 117 - Atty. Dkt. No.: 64100-234947 wherein the saccharide is covalently linked to a nitrogen atom within a STT3 glycosylation fragment (e.g., N X1 X2, wherein X1 is any amino acid except proline and X2 is S or T) using a STT3 OTase (e.g., SEQ ID NO: 408); wherein the saccharide is covalently linked to a nitrogen atom within an Archaeal AlgB glycosylation fragment (e.g., N X1 X2, wherein X1 is any amino acid except proline and X2 is S or T) using an AlgB OTase (e.g., SEQ ID NO: 409); wherein the saccharide is covalently linked to an oxygen atom within PilA_Pa5196- associated pilin glycosylation fragment (e.g., SEQ ID NO: 426 or a variant thereof) using a TfpW glycosyltransferase (e.g., SEQ ID NO: 424); wherein the saccharide is covalently linked to a nitrogen atom within a N-linking glycosyltransferase sequon (e.g., N X1 X2, wherein X1 is any amino acid and X2 is S or T) using an N-linking glycosyltransferase (e.g., SEQ ID NO: 410); wherein the saccharide is covalently linked to an oxygen atom within an O-linking glycosyltransferase sequon using an O-linking glycosyltransferase (e.g., SEQ ID NO: 411); wherein the saccharide is covalently linked to a carbon atom within a C- mannosyltransferase glycosylation fragment using a C-mannosyltransferase. [0322] 44. The method of any one of Paragraphs 35 to 43, wherein the method is a method of producing a conjugate vaccine. [0323] 45. A system comprising the first polypeptide and the second polypeptide of the composition of any one of Paragraphs 1 to 26; optionally, wherein the first polypeptide is a glycosylated bioconjugate; optionally, wherein the system comprises a multimeric, higher-order structure assembled of the second polypeptide; optionally, wherein the system comprises a saccharide and an N-linking oligosaccharyltransferase (N-OTase), an O-linking oligosaccharyltransferase (O-Otase), an N- linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C- mannosyltransferase (CMT). [0324] 46. An isolated nucleic acid encoding the first polypeptide and/or the second polypeptide of the composition of any one of Paragraphs 1 to 26 and/or the complex of any one of Paragraphs 27 to 32. [0325] 47. A vector comprising the isolated nucleic acid of Paragraph 46. [0326] 48. A host cell comprising the vector of Paragraph 47. 32080280 - 118 - Atty. Dkt. No.: 64100-234947 [0327] 49. A kit comprising two or more components comprising the fusion protein, the first polypeptide, the second polypeptide, a saccharide, an N-linking oligosaccharyltransferase (N-Otase), an O-linking oligosaccharyltransferase (O-Otase), an N-linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C-mannosyltransferase (CMT), the bioconjugate, the multimeric, higher-order structure assembled from the second polypeptide, the isolated nucleic acid, the vector, and the host cell of any of the Paragraphs above. [0328] 50. A method of eliciting an immune response in a subject by administering to said subject an effective amount of any composition, complex, and/or conjugate vaccine of any of the Paragraphs above or a composition, complex, and/or conjugate vaccine of any of the Paragraphs above for use in eliciting an immune response in a subject. [0329] 51. The fusion protein of any one of Paragraphs 1 to 7, wherein the glycosylation fragment is a PilA_Pa5196-associated pilin glycosylation fragment; optionally, wherein the PilA_Pa5196-associated pilin glycosylation fragment comprises or consists of the strands 1 and 2 of the antiparallel beta-sheet domain of PilA_Pa5196 (SEQ ID NO: 426), or a variant thereof having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. [0330] 52. The fusion protein of any one of Paragraphs 1 to 7, wherein the glycosylation fragment comprises means for having a saccharide attached to the glycosylation fragment by a PglS OTase, a TfpM OTase, a PglL OTase, a PglB OTase, a TfpO/PilO OTase, a STT3 OTase, a AlgB OTase, a N-Linking Glycosyltransferase, and/or an O-Linking Glycosyltransferase. [0331] 53. The fusions protein in anyone of the above Paragraphs, comprising an amino acid linker, optionally, wherein the amino acid linker is selected from the group consisting of SGG, SEQ ID NO: 430, SEQ ID NO: 431, SEQ ID NO: 432, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 437, and SEQ ID NO: 438, optionally, wherein the amino acid linker in translationally fused immediately following a leader sequence, a polypeptide tag, a glycosylation fragment, a carrier protein, and or a polyhistidine tag, and/or optionally, wherein the amino acid linker in translationally fused immediately preceding a leader sequence, a polypeptide tag, a glycosylation fragment, a carrier protein, and or a polyhistidine tag. ***** 32080280 - 119 - Atty. Dkt. No.: 64100-234947 [0332] In addition to the above, certain embodiments in the numbered paragraphs above can be further defined in any of the following numbered paragraphs: [0333] 1. A glycoconjugate comprising an oligo- or polysaccharide covalently linked to a fusion protein: wherein the fusion protein comprises a ComP protein (ComP) glycosylation fragment; wherein the ComP glycosylation fragment does not contain a cysteine residue corresponding to the conserved cysteine residue at position 71 of ComP110264 (SEQ ID NO: 201) and/or does not contain a cysteine residue corresponding to the conserved cysteine residue at position 93 of ComP110264 (SEQ ID NO: 201); wherein the ComP glycosylation fragment is located internally within the fusion protein; and wherein the fusion protein is glycosylated with the oligo- or polysaccharide on the ComP glycosylation fragment at serine residue corresponding to the conserved serine residue at position 82 of ComP110264 (SEQ ID NO: 201); optionally, wherein the glycoconjugate is immunogenic; optionally, wherein the ComP glycosylation fragment is solvent (or surface)-exposed; optionally, wherein the ComP glycosylation fragment is integrated into a C10 ^-turn, ^-turn, ^-twist, ^-loop, U turn, reverse turn, chain reversal, or a hairpin loop of the fusion protein. [0334] 2. The glycoconjugate of Paragraph 1, wherein the ComP glycosylation fragment has a length of from 5 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length; optionally, wherein the fragment has at least 1, 2, 3, 4, or 5 amino acid residues N-terminal to the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201 and/or wherein the fragment has at least 1, 2, 3, 4, or 5 amino acid residues C-terminal to the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201. [0335] 3. The glycoconjugate of Paragraph 1 or 2, wherein the amino acid sequence of the ComP glycosylation fragment does not extend in the N-terminus direction beyond the amino acid residue corresponding to position 72 of ComP110264 (SEQ ID NO: 201) and/or does not extend in the C-terminus beyond the amino acid residue corresponding to position 92 of ComP110264 (SEQ ID NO: 201). [0336] 4. The glycoconjugate of any one of Paragraphs 1 to 3, wherein 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: 209 (ComPΔ28110264) SEQ ID NO: 210 (ComPΔ28ADP1), SEQ ID NO: 211 (ComPΔ28GFJ-2), SEQ ID NO: 212 (ComPΔ28P50v1), 32080280 - 120 - Atty. Dkt. No.: 64100-234947 SEQ ID NO: 213 (ComPΔ284466), SEQ ID NO: 214 (ComPΔ28SFC); SEQ ID NO: 215 (ComPΔ28P5312), or SEQ ID NO: 216 (ComPΔ29ANT_H59); optionally, wherein the ComP protein comprises SEQ ID NO: 209 (ComPΔ28110264), SEQ ID NO: 210 (ComPΔ28ADP1), SEQ ID NO: 211 (ComPΔ28GFJ-2), SEQ ID NO: 212 (ComPΔ28P50v1), SEQ ID NO: 213 (ComPΔ284466), SEQ ID NO: 214 (ComPΔ28SFC); SEQ ID NO: 215 (ComPΔ28P5312), or SEQ ID NO: 216 (ComPΔ29ANT_H59). [0337] 5. The glycoconjugate of any one of Paragraphs 1 to 4, wherein the ComP glycosylation fragment comprises or consists of the amino acid consensus sequence of: X1GVX4X5X6X7X8X9ASX12X13TX15NVX18X19X20X21 (SEQ ID NO: 217)wherein: X1 is V, T, A, or I; X4 is Q, T, E, A, or S; X5 is E, Q, T, or L; X6 is I or V; X7 is S, N, A, or G; X8 is S or no amino acid; X9 is G, D, or no amino acid; X12 is N, S, or A; X13 is A, S, or K; X15 is T, S, or K; X18 is A, E, Q, or L; X19 is T, S, or K; X20 is A or S; and X21 is T, Q, A, or V; or a fragment of thereof of at least 5, 6, 7, 8, 9, 10, or 11 amino acids in length comprising the serine (S) residue at position 11 of SEQ ID NO: 217, optionally, wherein the fragment has at least 1, 2, 3, 4, 5, or 6 amino acid residues N-terminal to the serine (S) residue at position 11 of SEQ ID NO: 217 and/or wherein the fragment has at least 1, 2, 3, 4, 5, or 6 amino acid residues C- terminal to the serine (S) residue at position 11 of SEQ ID NO: 217; or a variant of the amino acid consensus sequence of SEQ ID NO: 217 or the fragment thereof, having one, two, or three amino acid substitutions, additions, and/or deletions, wherein the variant maintains the serine (S) residue at position 11 of SEQ ID NO: 217, optionally, wherein the variant has at least 1, 2, 3, 4, 5, or 6 amino acid residues N-terminal to the serine (S) residue at position 11 of SEQ ID NO: 217 and/or wherein the variant has at least 1, 2, 3, 4, 5, or 6 amino acid residues C-terminal to the serine (S) residue at position 11 of SEQ ID NO: 217; optionally, wherein the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein; and optionally, wherein the ComP glycosylation fragment is not glycosylated when located at the N-terminal and/or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal and/or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein. [0338] 6. The glycoconjugate of any one of Paragraphs 1 to 4, wherein the ComP glycosylation fragment comprises or consists of the amino acid consensus sequence of: X1GVX4X5X6X7X8X9ASX12X13TX15NVX18X19X20X21 (SEQ ID NO: 217) wherein: X1 is V, T, A, or I; X4 is Q, T, E, A, or S; X5 is E, Q, T, or L; X6 is I or V; X7 is S, N, A, or G; X8 is S 32080280 - 121 - Atty. Dkt. No.: 64100-234947 or no amino acid; X9 is G, D, or no amino acid; X12 is N, S, or A; X13 is A, S, or K; X15 is T, S, or K; X18 is A, E, Q, or L; X19 is T, S, or K; X20 is A or S; and X21 is T, Q, A, or V; or a fragment of thereof of at least 5, 6, 7, 8, 9, 10, or 11 amino acids in length comprising the serine (S) residue at position 11 of SEQ ID NO: 217, optionally, wherein the fragment has at least 1, 2, 3, 4, 5, or 6 amino acid residues N-terminal to the serine (S) residue at position 11 of SEQ ID NO: 217 and/or wherein the fragment has at least 1, 2, 3, 4, 5, or 6 amino acid residues C- terminal to the serine (S) residue at position 11 of SEQ ID NO: 217; optionally, wherein the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein; and optionally, wherein the ComP glycosylation fragment is not glycosylated when located at the N-terminal and/or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal and/or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein. [0339] 7. The glycoconjugate of any one of Paragraphs 1 to 6, wherein the fusion protein comprises a carrier protein selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae Protein D, and a fragment or fragments thereof; optionally, wherein the Pseudomonas aeruginosa Exotoxin A (EPA) carrier protein comprises the amino acid sequence of SEQ ID NO: 218, or a fragment or fragments thereof; optionally, wherein the CRM197 carrier protein comprises the amino acid sequence of SEQ ID NO: 224, or a fragment or fragments thereof. [0340] 8. The glycoconjugate of Paragraph 7, wherein: (i) the ComP glycosylation fragment is inserted between Ala489 and Arg490 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 219); (ii) the ComP glycosylation fragment is inserted between Glu548 and Gly549 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 220); (iii) the ComP glycosylation fragment is inserted between Ala122 and Gly123 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 221); (iv) the ComP glycosylation fragment is inserted between Thr355 and Gly356 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 222); or (v) the ComP glycosylation fragment is inserted between Lys20 and Asp21 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 223). 32080280 - 122 - Atty. Dkt. No.: 64100-234947 [0341] 9. The glycoconjugate of Paragraph 7, wherein: (i) the ComP glycosylation fragment is inserted between Asn481 and Gly482 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 225); (ii) the ComP glycosylation fragment is inserted between Asp392 and Gly393 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 226); (iii) the ComP glycosylation fragment is inserted between Glu142 and Gly143 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 227); (iv) the ComP glycosylation fragment is inserted between Asp129 and Gly130 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 228); or (v) the ComP glycosylation fragment is inserted between Asn69 and Glu70 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 229). [0342] 10. The glycoconjugate of any one of Paragraphs 1 to 9, 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 fragments; optionally, wherein the fusion protein does not comprise more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five ComP glycosylation fragments. [0343] 11. The glycoconjugate of any one of Paragraphs 1 to 10, wherein the ComP glycosylation fragments are identical. [0344] 12. The glycoconjugate of any one of Paragraphs 1 to 10, wherein the ComP glycosylation fragments differ from each other; optionally, wherein at least three, at least four, or at least five of the ComP glycosylation fragments all differ from each other; optionally, wherein none of the ComP glycosylation fragments are the same. [0345] 13. The glycoconjugate of any one of Paragraphs 1 to 12, wherein the oligo- or polysaccharide is derived from a saccharide produced by bacteria from the genus Streptococcus; optionally, wherein the saccharide is a S. pneumoniae, S. agalactiae, or S. suis capsular polysaccharide; optionally, wherein the saccharide is the serotype 8 capsular polysaccharide from S. pneumoniae.; optionally, wherein the saccharide is the type Ia, Ib, II, III, IV, V, VI, VII, VIII, or X capsular polysaccharide from S. agalactiae. [0346] 14. The glycoconjugate of any one of Paragraphs 1 to 12, wherein the oligo- or polysaccharide is derived from a saccharide produced by the bacteria from the genus Klebsiella; optionally, wherein the saccharide is a K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca capsular polysaccharide; optionally, wherein the saccharide is a K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca O-antigen polysaccharide. [0347] 15. The glycoconjugate of any one of Paragraphs 1 to 14, wherein oligo- or polysaccharide comprises glucose at its reducing end. 32080280 - 123 - Atty. Dkt. No.: 64100-234947 [0348] 16. The glycoconjugate of any one of Paragraphs 1 to 15, wherein the glycoconjugate is produced in vivo; optionally, in a bacterial cell; optionally, in Escherichia coli; optionally, in a bacterium from the genus Klebsiella; optionally, wherein the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0349] 17. The glycoconjugate of any one of Paragraphs 1 to 16, wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of SEQ ID NO: 232- 363, or 364, or a variant thereof having one, two, or three amino acid substitutions, additions, and/or deletions, wherein the variant comprises the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201; optionally, wherein the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein; and optionally, wherein the ComP glycosylation fragment is not glycosylated when located at the N-terminal and/or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal and/or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein. [0350] 18. The glycoconjugate of Paragraph 17, wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of: iGTccΔ0-1 CTGVTQIASGASAATTNVASAQ (SEQ ID NO: 232); iGTccΔ1-0 TGVTQIASGASAATTNVASAQC (SEQ ID NO: 243); iGTccΔ1-1 TGVTQIASGASAATTNVASAQ (SEQ ID NO: 244); iGTccΔ1-2TGVTQIASGASAATTNVASA (SEQ ID NO: 245); iGTccΔ2-1 GVTQIASGASAATTNVASAQ (SEQ ID NO: 256); iGTccΔ2- 2 GVTQIASGASAATTNVASA (SEQ ID NO: 257); iGTccΔ2-3 GVTQIASGASAATTNVAS (SEQ ID NO: 258); iGTccΔ3-2 VTQIASGASAATTNVASA (SEQ ID NO: 269); iGTccΔ3-3 VTQIASGASAATTNVAS (SEQ ID NO: 270); iGTccΔ3-4 VTQIASGASAATTNVA (SEQ ID NO: 271); iGTccΔ4-3 TQIASGASAATTNVAS (SEQ ID NO: 282); iGTccΔ4-4 TQIASGASAATTNVA (SEQ ID NO: 283); iGTccΔ4-5 TQIASGASAATTNV (SEQ ID NO: 284); iGTccΔ5-4 QIASGASAATTNVA (SEQ ID NO: 295); iGTccΔ5-5 QIASGASAATTNV (SEQ ID NO: 296); iGTccΔ5-6 QIASGASAATTN (SEQ ID NO: 297); iGTccΔ6-5 IASGASAATTNV (SEQ ID NO: 308); or iGTccΔ6-6 IASGASAATTN (SEQ ID NO: 309), or the variant thereof. [0351] 19. The glycoconjugate of Paragraph 17, wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of SEQ ID NO: 232-363, or 364, optionally, wherein the ComP glycosylation fragment can be glycosylated when located internally in a 32080280 - 124 - Atty. Dkt. No.: 64100-234947 fusion protein; and optionally, wherein the ComP glycosylation fragment is not glycosylated when located at the N-terminal and/or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal and/or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein. [0352] 20. The glycoconjugate of Paragraph 19, wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of: iGTccΔ0-1 CTGVTQIASGASAATTNVASAQ (SEQ ID NO: 232); iGTccΔ1-0 TGVTQIASGASAATTNVASAQC (SEQ ID NO: 243); iGTccΔ1-1 TGVTQIASGASAATTNVASAQ (SEQ ID NO: 244); iGTccΔ1-2 TGVTQIASGASAATTNVASA (SEQ ID NO: 245); iGTccΔ2-1 GVTQIASGASAATTNVASAQ (SEQ ID NO: 256); iGTccΔ2-2 GVTQIASGASAATTNVASA (SEQ ID NO: 257); iGTccΔ2-3 GVTQIASGASAATTNVAS (SEQ ID NO: 258); iGTccΔ3-2 VTQIASGASAATTNVASA (SEQ ID NO: 269); iGTccΔ3-3 VTQIASGASAATTNVAS (SEQ ID NO: 270); iGTccΔ3-4 VTQIASGASAATTNVA (SEQ ID NO: 271); iGTccΔ4-3 TQIASGASAATTNVAS (SEQ ID NO: 282); iGTccΔ4-4 TQIASGASAATTNVA (SEQ ID NO: 283); iGTccΔ4-5 TQIASGASAATTNV (SEQ ID NO: 284); iGTccΔ5-4 QIASGASAATTNVA (SEQ ID NO: 295); iGTccΔ5-5 QIASGASAATTNV (SEQ ID NO: 296); iGTccΔ5-6 QIASGASAATTN (SEQ ID NO: 297); iGTccΔ6-5 IASGASAATTNV (SEQ ID NO: 308); or iGTccΔ6-6 IASGASAATTN (SEQ ID NO: 309). [0353] 21. The glycoconjugate of any one of Paragraphs 1 to 20, wherein the bioconjugate is a conjugate vaccine; optionally, wherein the conjugate vaccine is a vaccine against Streptococcus pneumoniae serotype 8. [0354] 22. The glycoconjugate of Paragraph 21, wherein when the conjugate vaccine induces an immune response when administered to a subject. [0355] 23. The glycoconjugate of Paragraph 22, wherein the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype- specific antibody response. [0356] 24. The glycoconjugate of Paragraph 23, wherein the antibody response is an IgG or IgM response. [0357] 25. The glycoconjugate of Paragraph 24, wherein the antibody response is an IgG response; optionally an IgG1 response. 32080280 - 125 - Atty. Dkt. No.: 64100-234947 [0358] 26. The glycoconjugate of any one of Paragraphs 21 to 25, wherein the conjugate vaccine generates immunological memory in a subject administered the vaccine. [0359] 27. A ComP glycosylation fragment comprising or consisting of an isolated fragment of a ComP protein, wherein the ComP glycosylation fragment does not contain a cysteine residue corresponding to the conserved cysteine residue at position 71 of ComP110264 (SEQ ID NO: 201) and/or does not contain a cysteine residue corresponding to the conserved cysteine residue at position 93 of ComP110264 (SEQ ID NO: 201); and wherein the ComP glycosylation fragment comprises the serine residue corresponding to the conserved serine residue at position 82 of ComP110264 (SEQ ID NO: 201); optionally, wherein the ComP glycosylation fragment is immunogenic. [0360] 28. The ComP glycosylation fragment of Paragraph 27, wherein the ComP glycosylation fragment has a length of from 5 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length; optionally, wherein the fragment has at least 1, 2, 3, 4, 5, or 6 amino acid residues N-terminal to the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201 and/or wherein the fragment has at least 1, 2, 3, 4, 5, or 6 amino acid residues C-terminal to the serine residue corresponding to the conserved serine residue at position 82 of SEQ ID NO: 201. [0361] 29. The ComP glycosylation fragment of Paragraph 27 or 28, wherein the amino acid sequence of the ComP glycosylation fragment does not extend in the N-terminus direction beyond the amino acid residue corresponding to position 72 of ComP110264 (SEQ ID NO: 201) and/or does not extend in the C-terminus beyond the amino acid residue corresponding to position 92 of ComP110264 (SEQ ID NO: 201). [0362] 30. The ComP glycosylation fragment of any one of Paragraphs 27 to 29, wherein 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: 209 (ComPΔ28110264), SEQ ID NO: 210 (ComPΔ28ADP1), SEQ ID NO: 211 (ComPΔ28GFJ-2), SEQ ID NO: 212 (ComPΔ28P50v1), SEQ ID NO: 213 (ComPΔ284466), SEQ ID NO: 214 (ComPΔ28SFC); SEQ ID NO: 215 (ComPΔ28P5312), or SEQ ID NO: 216 (ComPΔ29ANT_H59); optionally, wherein the ComP protein comprises SEQ ID NO: 209 (ComPΔ28110264), SEQ ID NO: 210 (ComPΔ28ADP1), SEQ ID NO: 211 (ComPΔ28GFJ-2), SEQ ID NO: 212 32080280 - 126 - Atty. Dkt. No.: 64100-234947 (ComPΔ28P50v1), SEQ ID NO: 213 (ComPΔ284466), SEQ ID NO: 214 (ComPΔ28SFC); SEQ ID NO: 215 (ComPΔ28P5312), or SEQ ID NO: 216 (ComPΔ29ANT_H59). [0363] 31. The ComP glycosylation fragment of any one of Paragraphs 27 to 30, wherein the ComP glycosylation fragment comprises or consists of the amino acid consensus sequence of: X1GVX4X5X6X7X8X9ASX12X13TX15NVX18X19X20X21 (SEQ ID NO: 217) wherein: X1 is V, T, A, or I; X4 is Q, T, E, A, or S; X5 is E, Q, T, or L; X6 is I or V; X7 is S, N, A, or G; X8 is S or no amino acid; X9 is G, D, or no amino acid; X12 is N, S, or A; X13 is A, S, or K; X15 is T, S, or K; X18 is A, E, Q, or L; X19 is T, S, or K; X20 is A or S; and X21 is T, Q, A, or V; or a fragment of thereof of at least 5, 6, 7, 8, 9, 10, or 11 amino acids in length comprising the serine (S) residue at position 11 of SEQ ID NO: 217, optionally, wherein the fragment has at least 1, 2, 3, 4, 5, or 6 amino acid residues N-terminal to the serine (S) residue at position 11 of SEQ ID NO: 217 and/or wherein the fragment has at least 1, 2, 3, 4, 5, or 6 amino acid residues C- terminal to the serine (S) residue at position 11 of SEQ ID NO: 217; or a variant of the amino acid consensus sequence of SEQ ID NO: 217 or the fragment thereof, having one, two, three, four, five, six, or seven amino acid substitutions, additions, and/or deletions, wherein the variant maintains the serine (S) residue at position 11 of SEQ ID NO: 217, optionally, wherein the variant has at least 1, 2, 3, 4, 5, or 6 amino acid residues N-terminal to the serine (S) residue at position 11 of SEQ ID NO: 217 and/or wherein the variant has at least 1, 2, 3, 4, 5, or 6 amino acid residues C-terminal to the serine (S) residue at position 11 of SEQ ID NO: 217; optionally, wherein the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein; and optionally, wherein the ComP glycosylation fragment is not glycosylated when located at the N-terminal or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein. [0364] 32. The ComP glycosylation fragment of any one of Paragraphs 27 to 30, wherein the ComP glycosylation fragment comprises or consists of the amino acid consensus sequence of: X1GVX4X5X6X7X8X9ASX12X13TX15NVX18X19X20X21 (SEQ ID NO: 217) wherein: X1 is V, T, A, or I; X4 is Q, T, E, A, or S; X5 is E, Q, T, or L; X6 is I or V; X7 is S, N, A, or G; X8 is S or no amino acid; X9 is G, D, or no amino acid; X12 is N, S, or A; X13 is A, S, or K; X15 is T, S, or K; X18 is A, E, Q, or L; X19 is T, S, or K; X20 is A or S; and X21 is T, Q, A, or V; or a fragment of thereof of at least 5, 6, 7, 8, 9, 10, or 11 amino acids in length comprising the serine (S) residue at position 11 of SEQ ID NO: 217, optionally, wherein the fragment has at least 1, 32080280 - 127 - Atty. Dkt. No.: 64100-234947 2, 3, 4, 5, or 6 amino acid residues N-terminal to the serine (S) residue at position 11 of SEQ ID NO: 217 and/or wherein the fragment has at least 1, 2, 3, 4, 5, or 6 amino acid residues C- terminal to the serine (S) residue at position 11 of SEQ ID NO: 217; optionally, wherein the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein; and optionally, wherein the ComP glycosylation fragment is not glycosylated when located at the N-terminal or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal or C- terminal end of a fusion protein in comparison to when it is located internally in the fusion protein. [0365] 33. The ComP glycosylation fragment of Paragraph 27, wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of SEQ ID NO: 232- 363, or 364, or a variant thereof having one, two, or three amino acid substitutions, additions, and/or deletions, wherein the variant comprises the serine residue corresponding to the conserved serine (S) residue at position 82 of SEQ ID NO: 201; optionally, wherein the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein; and optionally, wherein the ComP glycosylation fragment is not glycosylated when located at the N-terminal or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal or C- terminal end of a fusion protein in comparison to when it is located internally in the fusion protein. [0366] 34. The ComP glycosylation fragment of Paragraph 33, wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of: iGTccΔ0-1 CTGVTQIASGASAATTNVASAQ (SEQ ID NO: 232); iGTccΔ1-0 TGVTQIASGASAATTNVASAQC (SEQ ID NO: 243); iGTccΔ1-1 TGVTQIASGASAATTNVASAQ (SEQ ID NO: 244); iGTccΔ1-2 TGVTQIASGASAATTNVASA (SEQ ID NO: 245); iGTccΔ2-1 GVTQIASGASAATTNVASAQ (SEQ ID NO: 256); iGTccΔ2-2 GVTQIASGASAATTNVASA (SEQ ID NO: 257); iGTccΔ2-3 GVTQIASGASAATTNVAS (SEQ ID NO: 258); iGTccΔ3-2 VTQIASGASAATTNVASA (SEQ ID NO: 269); iGTccΔ3-3 VTQIASGASAATTNVAS (SEQ ID NO: 270); iGTccΔ3-4 VTQIASGASAATTNVA (SEQ ID NO: 271); iGTccΔ4-3 TQIASGASAATTNVAS (SEQ ID NO: 282); iGTccΔ4-4 TQIASGASAATTNVA (SEQ ID NO: 283); iGTccΔ4-5 TQIASGASAATTNV (SEQ ID NO: 284); iGTccΔ5-4 QIASGASAATTNVA (SEQ ID NO: 295); iGTccΔ5-5 QIASGASAATTNV (SEQ ID NO: 296); iGTccΔ5-6 QIASGASAATTN 32080280 - 128 - Atty. Dkt. No.: 64100-234947 (SEQ ID NO: 297); iGTccΔ6-5 IASGASAATTNV (SEQ ID NO: 308); or iGTccΔ6-6 IASGASAATTN (SEQ ID NO: 309), or the variant thereof. [0367] 35. The ComP glycosylation fragment of Paragraph 33, wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of SEQ ID NO: 232- 363, or 364, optionally, wherein the ComP glycosylation fragment can be glycosylated when located internally in a fusion protein; and optionally, wherein the ComP glycosylation fragment is not glycosylated when located at the N-terminal or C-terminal end of a fusion protein or is glycosylated at least 50% less, 60% less, 70% less, 80% less, 90% less, 95% less, or 99% less when located at the N-terminal or C-terminal end of a fusion protein in comparison to when it is located internally in the fusion protein. [0368] 36. The ComP glycosylation fragment of Paragraph 35, wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of: iGTccΔ0-1 CTGVTQIASGASAATTNVASAQ (SEQ ID NO: 232); iGTccΔ1-0 TGVTQIASGASAATTNVASAQC (SEQ ID NO: 243); iGTccΔ1-1 TGVTQIASGASAATTNVASAQ (SEQ ID NO: 244); iGTccΔ1-2 TGVTQIASGASAATTNVASA (SEQ ID NO: 245); iGTccΔ2-1 GVTQIASGASAATTNVASAQ (SEQ ID NO: 256); iGTccΔ2-2 GVTQIASGASAATTNVASA (SEQ ID NO: 257); iGTccΔ2-3 GVTQIASGASAATTNVAS (SEQ ID NO: 258); iGTccΔ3-2 VTQIASGASAATTNVASA (SEQ ID NO: 269); iGTccΔ3-3 VTQIASGASAATTNVAS (SEQ ID NO: 270); iGTccΔ3-4 VTQIASGASAATTNVA (SEQ ID NO: 271); iGTccΔ4-3 TQIASGASAATTNVAS (SEQ ID NO: 282); iGTccΔ4-4 TQIASGASAATTNVA (SEQ ID NO: 283); iGTccΔ4-5 TQIASGASAATTNV (SEQ ID NO: 284); iGTccΔ5-4 QIASGASAATTNVA (SEQ ID NO: 295); iGTccΔ5-5 QIASGASAATTNV (SEQ ID NO: 296); iGTccΔ5-6 QIASGASAATTN (SEQ ID NO: 297); iGTccΔ6-5 IASGASAATTNV (SEQ ID NO: 308); or iGTccΔ6-6 IASGASAATTN (SEQ ID NO: 309). [0369] 37. A fusion protein comprising the ComP glycosylation fragment of any of Paragraphs 27 to 36, wherein the ComP glycosylation fragment is located internally within the fusion protein; optionally, wherein the fusion protein is glycosylated by an oligo- or polysaccharide at a serine residue on the glycosylation fragment corresponding to the serine ComP glycosylation fragment residue at position 82 of SEQ ID NO: 201 (ComP110264). [0370] 38. The fusion protein of Paragraph 37, wherein the oligo- or polysaccharide is derived from a saccharide produced by bacteria from the genus Streptococcus; optionally, 32080280 - 129 - Atty. Dkt. No.: 64100-234947 wherein the saccharide is a S. pneumoniae, S. agalactiae, or S. suis capsular polysaccharide; optionally, wherein the saccharide is the serotype 8 capsular polysaccharide from S. pneumoniae; optionally, wherein the saccharide is the type Ia, Ib, II, III, IV, V, VI, VII, VIII, or X capsular polysaccharide from S. agalactiae. [0371] 39. The fusion protein of Paragraph 37, wherein the oligo- or polysaccharide is derived from a saccharide produced by the bacteria from the genus Klebsiella; optionally, wherein the saccharide is a K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca capsular polysaccharide; optionally, wherein the saccharide is a K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca O-antigen polysaccharide. [0372] 40. The fusion protein of any one of Paragraphs 37 to 39, wherein oligo- or polysaccharide comprises glucose at its reducing end. [0373] 41. The fusion protein of any one of Paragraphs 37 to 40, wherein the glycosylated fusion protein is produced in vivo; optionally, in a bacterial cell; optionally, in Escherichia coli; optionally, in a bacterium from the genus Klebsiella; optionally, wherein the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0374] 42. The fusion protein of any one of Paragraphs 37 to 41, wherein the fusion protein comprises a carrier protein selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, Haemophilus influenzae Protein D, and a fragment or fragments thereof; optionally, wherein the Pseudomonas aeruginosa Exotoxin A (EPA) carrier protein comprises the amino acid sequence of SEQ ID NO: 218, or a fragment or fragments thereof; optionally, wherein the CRM197 carrier protein comprises the amino acid sequence of SEQ ID NO: 224, or a fragment or fragments thereof. [0375] 43. The fusion protein of Paragraph 42, wherein: (i) the ComP glycosylation fragment is inserted between Ala489 and Arg490 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 219); (ii) the ComP glycosylation fragment is inserted between Glu548 and Gly549 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 220); (iii) the ComP glycosylation fragment is inserted between Ala122 and Gly123 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 221); (iv) the ComP glycosylation fragment is inserted between Thr355 and Gly356 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 222); or (v) the ComP 32080280 - 130 - Atty. Dkt. No.: 64100-234947 glycosylation fragment is inserted between Lys20 and Asp21 relative to the PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 223). [0376] 44. The fusion protein of Paragraph 42, wherein: (i) the ComP glycosylation fragment is inserted between Asn481 and Gly482 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 225); (ii) the ComP glycosylation fragment is inserted between Asp392 and Gly393 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 226); (iii) the ComP glycosylation fragment is inserted between Glu142 and Gly143 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 227); (iv) the ComP glycosylation fragment is inserted between Asp129 and Gly130 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 228); or (v) the ComP glycosylation fragment is inserted between Asn69 and Glu70 relative to the PDB entity 4AE0 of CRM197 (SEQ ID NO: 229). [0377] 45. The fusion protein of any one of Paragraph s 37 to 44, 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 fragments; optionally, wherein the fusion protein does not comprise more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five ComP glycosylation fragments. [0378] 46. The fusion protein of any one of Paragraphs 37 to 45, wherein the ComP glycosylation fragments are identical. [0379] 47. The fusion protein of any one of Paragraphs 37 to 45, wherein the ComP glycosylation fragments differ from each other; optionally, wherein at least three, at least four, or at least five of the ComP glycosylation fragments all differ from each other; optionally, wherein none of the ComP glycosylation fragments are the same. [0380] 48. A method of in vivo conjugation of an oligo- or polysaccharide to an acceptor polypeptide, the method comprising covalently linking the oligo- or polysaccharide to the acceptor polypeptide with a PglS oligosaccharyltransferase (OTase), wherein the acceptor polypeptide comprises the ComP glycosylation fragment of any one of Paragraphs 27 to 36. [0381] 49. The method of Paragraph 48, wherein the PglS OTase is PglS110264 (SEQ ID NO: 365), PglSADP1 (SEQ ID NO: 366), PglSGFJ-2 (SEQ ID NO: 367), PglS50v1 (SEQ ID NO: 368), PglS4466 (SEQ ID NO: 369), PglSSFC (SEQ ID NO: 370), PglSP5312 (SEQ ID NO: 371), or PglSANT_H59 (SEQ ID NO: 372). 32080280 - 131 - Atty. Dkt. No.: 64100-234947 [0382] 50. The method of Paragraph 48 or 49, wherein the oligo- or polysaccharide is linked to the ComP glycosylation fragment at a serine (S) residue corresponding to the serine residue at position 82 of SEQ ID NO: 201 (ComP110264). [0383] 51. The method of any one of Paragraphs 48 to 50, wherein the in vivo conjugation occurs in a host cell. [0384] 52. The method of Paragraph 51, wherein the host cell is a bacterial cell; optionally, in Escherichia coli; optionally, in a bacterium from the genus Klebsiella; optionally, wherein the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0385] 53. The method of Paragraph 51 of 52, comprising culturing a host cell that comprises: (a) a genetic cluster encoding for the proteins required to synthesize the oligo- or polysaccharide; (b) a PglS OTase; and (3) the acceptor polypeptide. [0386] 54. The method of any one of Paragraphs 48 to 53, wherein production of the oligo- or polysaccharide is enhanced by the K. pneumoniae transcriptional activator rmpA (K. pneumoniae NTUH K-2044) or a homolog of the K. pneumoniae transcriptional activator rmpA (K. pneumoniae NTUH K-2044). [0387] 55. The method of any one of Paragraphs 48 to 54, wherein the method produces a conjugate vaccine. [0388] 56. A host cell comprising (a) a genetic cluster encoding for the proteins required to synthesize an oligo- or polysaccharide; (b) a PglS OTase; and (3) an acceptor polypeptide comprising the ComP glycosylation fragment of any one of Paragraphs 27 to 36. [0389] 57. The host cell of Paragraph 56, wherein the acceptor polypeptide is a fusion protein. [0390] 58. The host cell of Paragraph 56 or 57, wherein the host cell comprises a nucleic acid encoding the PglS OTase. [0391] 59. The host cell of any one of Paragraphs 56 to 58, wherein the host cell comprises a nucleic acid encoding the acceptor polypeptide. [0392] 60. An isolated nucleic acid encoding the ComP glycosylation fragment of any one of Paragraphs 27 to 36 and/or the fusion protein of any one of Paragraphs 37 to 47. [0393] 61. The isolated nucleic acid of Paragraph 60, wherein the nucleic acid is a vector. [0394] 62. A host cell comprising the isolated nucleic acid of Paragraph 60 or 61. [0395] 63. A composition comprising the conjugate vaccine of any one of Paragraphs 21 to 26 or the fusion protein of any one of Paragraphs 37 to 47, and an adjuvant. 32080280 - 132 - Atty. Dkt. No.: 64100-234947 [0396] 64. A method of inducing a host immune response against a bacterial pathogen, the method comprising administering to a subject in need of the immune response an effective amount of the conjugate vaccine of any one of Paragraphs 21 to 26, the fusion protein of any one of Paragraphs 37 to 47, or the composition of Paragraph 63. [0397] 65. The method of Paragraph 64, wherein the immune response is an antibody response. [0398] 66. The method of Paragraph 64, wherein the immune response is selected from the group consisting of an innate response, an adaptive response, a humoral response, an antibody response, cell mediated response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system cross-talk, and a combination of two or more of said immune responses. [0399] 67. The method of Paragraph 64, wherein the immune response is selected from the group consisting of an innate response, a humoral response, an antibody response, a T cell response, and a combination of two or more of said immune responses. [0400] 68. A method of preventing or treating a bacterial disease and/or infection in a subject comprising administering to a subject in need thereof the conjugate vaccine of any one of Paragraphs 21 to 26, the fusion protein of any one of Paragraphs 37 to 47, or the composition of Paragraph 63. [0401] 69. The method of Paragraph 68, wherein the infection is a localized or systemic infection of skin, soft tissue, blood, or an organ, or is auto-immune in nature. [0402] 70. The method of Paragraph 69, wherein the disease is pneumonia. [0403] 71. The method of Paragraph 69, wherein the infection is a systemic infection and/or an infection of the blood. [0404] 72. The method of any one of Paragraphs 68 to 71, wherein the subject is a human. [0405] 73. The method of any one of Paragraph s 68 to 72, wherein the composition is administered via intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration. [0406] 74. A method of producing a pneumococcal conjugate vaccine against pneumococcal infection, the method comprising:(a) isolating the glycoconjugate of any one of Paragraphs 1 to 26 or a glycosylated fusion protein of any one of Paragraphs 37 to 47; and (b) combining the isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant. 32080280 - 133 - Atty. Dkt. No.: 64100-234947 [0407] 75. The glycoconjugate, glycosylated fusion protein, or conjugate vaccine of any of the above paragraphs for use in inducing a host immune response against a bacterial pathogen and/or preventing or treating a bacterial disease and/or infection in a subject. ***** [0408] In addition to all of the above, certain embodiments in the numbered paragraphs above can be further defined in any of the following numbered paragraphs: [0409] 1. A glycoconjugate comprising an oligo- or polysaccharide covalently linked to an acceptor protein, wherein the acceptor protein comprises or consists of a TfpM-associated pilin-like protein or glycosylation fragment thereof and the oligo- or polysaccharide is covalently linked to the TfpM-associated pilin-like protein or glycosylation fragment thereof, and wherein the TfpM-associated pilin-like protein or glycosylation fragment thereof comprises a C-terminus serine or threonine residue and the oligo- or polysaccharide is covalently linked to the C-terminus serine or threonine; optionally, wherein the TfpM-associated pilin-like protein glycosylation fragment comprises at least the last three amino acids from the pilin-like protein C-terminal end; optionally, wherein the acceptor protein is a fusion protein comprising the TfpM- associated pilin-like protein or glycosylation fragment thereof translationally fused to a heterologous carrier protein and the TfpM-associated pilin-like protein or glycosylation fragment thereof is the C-terminus-most sequence of the acceptor protein such that the acceptor protein comprises a C-terminus serine or threonine residue and the oligo- or polysaccharide is covalently linked to the C-terminus serine or threonine; optionally, wherein the oligo- or polysaccharide comprises a glucose at its reducing end; and/or optionally, wherein the glycoconjugate is immunogenic. [0410] 2. The glycoconjugate of Paragraph 1, wherein the TfpM-associated pilin-like- protein glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, has a length of from 116 to 139 amino acids in length, has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length, 32080280 - 134 - Atty. Dkt. No.: 64100-234947 wherein the TfpM-associated pilin-like protein glycosylation fragment comprises a C- terminus serine or threonine residue. [0411] 3. The glycoconjugate of Paragraph 1 or 2, (a) wherein the TfpM-associated pilin-like-protein or glycosylation fragment thereof is: (i) PilMo (SEQ ID NO: 57), (ii) PilMo lacking amino acids corresponding to residues 1–28 (PilMoΔ28, SEQ ID NO: 58), or (iii) a polypeptide comprising at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, wherein the TfpM-associated pilin-like protein comprises a C-terminus serine or threonine residue, optionally, wherein the C-terminus threonine is substituted with serine; (b) wherein the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), and PilCIP102637 (SEQ ID NO: 100); (c) wherein the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), PilCIP102637 (SEQ ID NO: 100), and a fragment of any thereof that contains a C-terminus serine or threonine residue, or a variant wherein the C-terminus threonine is substituted with serine, optionally, wherein the TfpM- associated pilin-like protein glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end; and/or (d) wherein the pilin-like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (PilMo_DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C- terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148), 32080280 - 135 - Atty. Dkt. No.: 64100-234947 optionally, (e) wherein the pilin-like-protein glycosylation fragment consists of Pil20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C- terminus threonine, further optionally, wherein the glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end, further optionally, wherein the pilin- like-protein glycosylation fragment consists of Pil20[A] (SEQ ID NO: 166), Pil19[A] (SEQ ID NO: 167), Pil18[A] (SEQ ID NO: 168), Pil17[A] (SEQ ID NO: 169), Pil16[A] (SEQ ID NO: 170), Pil15[A] (SEQ ID NO: 171), Pil14[A] (SEQ ID NO: 172), Pil13[A] (SEQ ID NO: 173), Pil12[A] (SEQ ID NO: 174), Pil11[A] (SEQ ID NO: 175), Pil10[A] (SEQ ID NO: 176), Pil9[A] (SEQ ID NO: 177), Pil8[A] (SEQ ID NO: 178), Pil7[A] (SEQ ID NO: 179), Pil6[A] (SEQ ID NO: 180), Pil5[A] (SEQ ID NO: 181), or Pil4[A] (SEQ ID NO: 182), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine, wherein the glycosylation fragment comprises at least the last four amino acids from the pilin C-terminal end; or optionally, (f) wherein the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil15S (SEQ ID NO: 153), Pil14S (SEQ ID NO: 154), Pil13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil11S (SEQ ID NO: 157), Pil10S (SEQ ID NO: 158), Pil9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil4S (SEQ ID NO: 164), or Pil3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine, further optionally, wherein the glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end, further optionally, wherein the pilin-like-protein glycosylation fragment consists of Pil20S[A] (SEQ ID NO: 183), Pil19S[A] (SEQ ID NO: 184), Pil18S[A] (SEQ ID NO: 185), Pil17S[A] (SEQ ID NO: 186), Pil16S[A] (SEQ ID NO: 187), Pil15S[A] (SEQ ID NO: 188), Pil14S[A] (SEQ ID NO: 189), Pil13S[A] (SEQ ID NO: 190), Pil12S[A] (SEQ ID NO: 191), Pil11S[A] (SEQ ID NO: 192), Pil10S[A] (SEQ ID NO: 193), Pil9S[A] (SEQ ID NO: 194), Pil8S[A] (SEQ ID NO: 195), Pil7S[A] (SEQ ID NO: 196), Pil6S[A] (SEQ ID NO: 197), Pil5S[A] (SEQ ID NO: 198), or Pil4S[A] (SEQ ID NO: 199), or a 32080280 - 136 - Atty. Dkt. No.: 64100-234947 variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine, wherein the glycosylation fragment comprises at least the last four amino acids from the pilin C-terminal end. [0412] 4. The glycoconjugate of any one of Paragraphs 1 to 3, wherein the acceptor protein is a fusion protein comprising a heterologous carrier protein and the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and a fragment of any thereof. [0413] 5. The glycoconjugate of any one of Paragraphs 1 to 4, wherein the acceptor protein is a fusion protein and the fusion protein further comprises an additional glycosylation sequence of an OTase other than TfpM oligosaccharyltransferase (OTase) in addition to the TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus, optionally wherein the additional glycosylation sequence is an internal glycosylation fragment of ComP, further optionally, wherein the ComP glycosylation fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least the amino acids ASA in positions 11-13; optionally, wherein the additional glycosylation sequence is also covalently linked to an oligo- or polysaccharide. [0414] 6. The glycoconjugate of Paragraph 5, 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 additional glycosylation sequences; optionally, wherein the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five additional glycosylation sequences; optionally, wherein the additional glycosylation sequences are identical; optionally, wherein the additional glycosylation sequences differ from each other; and/or optionally, wherein at least three, at least four, or at least five of the additional glycosylation sequences all differ from each other; and/or optionally, wherein none of the additional glycosylation sequences are the same. [0415] 7. The glycoconjugate of any one of Paragraphs 1 to 6, wherein the oligo- or polysaccharide covalently linked to the pilin-like protein or glycosylation fragment thereof has a size of at least three repeating units of oligo- or polysaccharide structure and/or has a size of at least ten monosaccharides. 32080280 - 137 - Atty. Dkt. No.: 64100-234947 [0416] 8. The glycoconjugate of any one of Paragraphs 1 to 7, (i) wherein the oligo- or polysaccharide is produced by bacteria of the genus Streptococcus and the polysaccharide is capsular polysaccharide, optionally, wherein is S. pneumoniae or S. agalactiae, and optionally, wherein the S. agalactiae capsular polysaccharide is Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX; (ii) wherein the oligo- or polysaccharide is produced by bacteria of the genus Klebsiella and the polysaccharide is a capsular polysaccharide or O-antigen polysaccharide, optionally wherein the bacteria is K. pneumoniae; or (iii) wherein the oligo- or polysaccharide is produced by bacteria of the genus Salmonella and the polysaccharide is O-antigen polysaccharide; optionally wherein the bacteria is S. enterica and the S. enterica polysaccharide is a Group B O-antigen. [0417] 9. The glycoconjugate of any one of Paragraphs 1 to 8, wherein the glycoconjugate is produced in vivo; optionally, in a bacterial cell; optionally, in Escherichia coli; optionally, in a bacterium from the genus Klebsiella; and/or optionally, wherein the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0418] 10. The glycoconjugate of any one of Paragraphs 1 to 9, wherein the bioconjugate is a conjugate vaccine that induces an immune response when administered to a subject; optionally, wherein the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response; optionally, wherein the antibody response is an IgG or IgM response; optionally, wherein the antibody response is an IgG response; optionally an IgG1 response; and/or optionally, wherein the conjugate vaccine generates immunological memory in a subject administered the vaccine. [0419] 11. A pilin-like-protein glycosylation fragment comprising or consisting of an isolated fragment of a TfpM-associated pilin-like protein, (a) wherein the TfpM-associated pilin-like-protein or glycosylation fragment thereof is: (i) PilMo (SEQ ID NO: 57), (ii) PilMo lacking amino acids corresponding to residues 1–28 32080280 - 138 - Atty. Dkt. No.: 64100-234947 (PilMoΔ28, SEQ ID NO: 58), or (iii) a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, wherein the TfpM-associated pilin-like protein contains a C-terminus serine or threonine residue, optionally, wherein the C-terminus threonine is substituted with serine, (b) wherein the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), and PilCIP102637 (SEQ ID NO: 100); (c) wherein the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), PilCIP102637 (SEQ ID NO: 100), and a fragment of any thereof that contains a C-terminus serine or threonine residue, or a variant wherein the C-terminus threonine is substituted with serine, optionally, wherein the TfpM- associated pilin-like protein glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end; and/or (d) wherein the pilin-like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (PilMo_DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C- terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148), optionally, (e) wherein the pilin-like-protein glycosylation fragment consists of Pil20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), 32080280 - 139 - Atty. Dkt. No.: 64100-234947 Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C- terminus threonine, further optionally, wherein the glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end, further optionally, wherein the pilin- like-protein glycosylation fragment consists of Pil20[A] (SEQ ID NO: 166), Pil19[A] (SEQ ID NO: 167), Pil18[A] (SEQ ID NO: 168), Pil17[A] (SEQ ID NO: 169), Pil16[A] (SEQ ID NO: 170), Pil15[A] (SEQ ID NO: 171), Pil14[A] (SEQ ID NO: 172), Pil13[A] (SEQ ID NO: 173), Pil12[A] (SEQ ID NO: 174), Pil11[A] (SEQ ID NO: 175), Pil10[A] (SEQ ID NO: 176), Pil9[A] (SEQ ID NO: 177), Pil8[A] (SEQ ID NO: 178), Pil7[A] (SEQ ID NO: 179), Pil6[A] (SEQ ID NO: 180), Pil5[A] (SEQ ID NO: 181), or Pil4[A] (SEQ ID NO: 182), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine, wherein the glycosylation fragment comprises at least the last four amino acids from the pilin C-terminal end; or optionally, (f) wherein the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil15S (SEQ ID NO: 153), Pil14S (SEQ ID NO: 154), Pil13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil11S (SEQ ID NO: 157), Pil10S (SEQ ID NO: 158), Pil9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil4S (SEQ ID NO: 164), or Pil3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine, further optionally, wherein the glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end, further optionally, wherein the pilin-like-protein glycosylation fragment consists of Pil20S[A] (SEQ ID NO: 183), Pil19S[A] (SEQ ID NO: 184), Pil18S[A] (SEQ ID NO: 185), Pil17S[A] (SEQ ID NO: 186), Pil16S[A] (SEQ ID NO: 187), Pil15S[A] (SEQ ID NO: 188), Pil14S[A] (SEQ ID NO: 189), Pil13S[A] (SEQ ID NO: 190), Pil12S[A] (SEQ ID NO: 191), Pil11S[A] (SEQ ID NO: 192), Pil10S[A] (SEQ ID NO: 193), Pil9S[A] (SEQ ID NO: 194), Pil8S[A] (SEQ ID NO: 195), Pil7S[A] (SEQ ID NO: 196), Pil6S[A] (SEQ ID NO: 197), Pil5S[A] (SEQ ID NO: 198), or Pil4S[A] (SEQ ID NO: 199), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine, wherein the glycosylation fragment comprises at least the last four amino acids from the pilin C-terminal end. [0420] 12. The pilin-like-protein glycosylation fragment of Paragraph 11, wherein the pilin-like-protein glycosylation fragment has a length of from 3 to 139 amino acids in length, 32080280 - 140 - Atty. Dkt. No.: 64100-234947 has a length of from 20 to 139 amino acids in length, has a length of from 116 to 139 amino acids in length, has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length, wherein the TfpM-associated pilin-like protein glycosylation fragment comprises a C- terminus serine or threonine residue. [0421] 13. A fusion protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof translationally fused to a heterologous carrier protein, wherein the TfpM-associated pilin-like protein or glycosylation fragment comprises a C-terminus serine or threonine residue, wherein the TfpM-associated pilin-like protein or glycosylation fragment is the C- terminus-most sequence of the fusion protein, and wherein the fusion protein comprises a C-terminus serine or threonine residue; optionally, wherein the fusion protein is glycosylated by an olio- or polysaccharide covalently linked to the C-terminus serine or threonine; optionally, wherein the fusion protein is glycosylated by an oligo- or polysaccharide comprising glucose at its reducing end covalently linked to the C-terminus serine or threonine. [0422] 14. The fusion protein of Paragraph 13, wherein the pilin-like-protein glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, has a length of from 116 to 139 amino acids in length, has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length, wherein the TfpM-associated pilin-like protein glycosylation fragment comprises a C- terminus serine or threonine residue. [0423] 15. The fusion protein of Paragraph 13 or 14, (a) wherein the TfpM-associated pilin-like-protein or glycosylation fragment thereof is: (i) PilMo (SEQ ID NO: 57), (ii) PilMo lacking amino acids corresponding to residues 1–28 (PilMoΔ28, SEQ ID NO: 58), or (ii) a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or 32080280 - 141 - Atty. Dkt. No.: 64100-234947 SEQ ID NO: 58, wherein the TfpM-associated pilin-like protein contains a C-terminus serine or threonine residue, optionally, wherein the C-terminus threonine is substituted with serine; (b) wherein the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), and PilCIP102637 (SEQ ID NO: 100); (c) wherein the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), PilCIP102637 (SEQ ID NO: 100), and a fragment of any thereof that contains a C-terminus serine or threonine residue, or a variant wherein the C-terminus threonine is substituted with serine, optionally, wherein the TfpM- associated pilin-like protein glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end; and/or (d) wherein the pilin-like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (PilMo_DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C- terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148), optionally, (e) wherein the pilin-like-protein glycosylation fragment consists of Pil20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C- 32080280 - 142 - Atty. Dkt. No.: 64100-234947 terminus threonine, further optionally, wherein the glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end, further optionally, wherein the pilin- like-protein glycosylation fragment consists of Pil20[A] (SEQ ID NO: 166), Pil19[A] (SEQ ID NO: 167), Pil18[A] (SEQ ID NO: 168), Pil17[A] (SEQ ID NO: 169), Pil16[A] (SEQ ID NO: 170), Pil15[A] (SEQ ID NO: 171), Pil14[A] (SEQ ID NO: 172), Pil13[A] (SEQ ID NO: 173), Pil12[A] (SEQ ID NO: 174), Pil11[A] (SEQ ID NO: 175), Pil10[A] (SEQ ID NO: 176), Pil9[A] (SEQ ID NO: 177), Pil8[A] (SEQ ID NO: 178), Pil7[A] (SEQ ID NO: 179), Pil6[A] (SEQ ID NO: 180), Pil5[A] (SEQ ID NO: 181), or Pil4[A] (SEQ ID NO: 182), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine, wherein the glycosylation fragment comprises at least the last four amino acids from the pilin C-terminal end; or optionally, (f) wherein the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil15S (SEQ ID NO: 153), Pil14S (SEQ ID NO: 154), Pil13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil11S (SEQ ID NO: 157), Pil10S (SEQ ID NO: 158), Pil9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil4S (SEQ ID NO: 164), or Pil3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine, further optionally, wherein the glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end, further optionally, wherein the pilin-like-protein glycosylation fragment consists of Pil20S[A] (SEQ ID NO: 183), Pil19S[A] (SEQ ID NO: 184), Pil18S[A] (SEQ ID NO: 185), Pil17S[A] (SEQ ID NO: 186), Pil16S[A] (SEQ ID NO: 187), Pil15S[A] (SEQ ID NO: 188), Pil14S[A] (SEQ ID NO: 189), Pil13S[A] (SEQ ID NO: 190), Pil12S[A] (SEQ ID NO: 191), Pil11S[A] (SEQ ID NO: 192), Pil10S[A] (SEQ ID NO: 193), Pil9S[A] (SEQ ID NO: 194), Pil8S[A] (SEQ ID NO: 195), Pil7S[A] (SEQ ID NO: 196), Pil6S[A] (SEQ ID NO: 197), Pil5S[A] (SEQ ID NO: 198), or Pil4S[A] (SEQ ID NO: 199), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine, wherein the glycosylation fragment comprises at least the last four amino acids from the pilin C-terminal end. [0424] 16. The fusion protein of any one of Paragraphs 13 to 15, wherein the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and a fragment of any thereof. 32080280 - 143 - Atty. Dkt. No.: 64100-234947 [0425] 17. The fusion protein of any one of Paragraphs 13 to 16, wherein the fusion protein further comprises an additional glycosylation sequence of an OTase other than TfpM oligosaccharyltransferase (OTase) in addition to the TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus, optionally wherein the additional glycosylation sequence is an internal glycosylation fragment of ComP, further optionally, wherein the ComP glycosylation fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least the amino acids ASA in positions 11-13; optionally, wherein the additional glycosylation sequence is also covalently linked to an oligo- or polysaccharide. [0426] 18. The fusion protein of Paragraph 17, 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 additional glycosylation sequence; optionally, wherein the fusion protein does not comprise more than two, more than three, more than five, more than ten, more than fifteen, more than twenty, or more than twenty five additional glycosylation sequence; optionally, wherein the additional glycosylation sequence are identical; optionally, wherein the additional glycosylation sequence differ from each other; and/or optionally, wherein at least three, at least four, or at least five of the additional glycosylation sequences all differ from each other; and/or optionally, wherein none of the additional glycosylation sequence are the same. [0427] 19. The fusion protein of any one of Paragraphs 13 to 18, wherein the oligo- or polysaccharide covalently linked to the pilin-like protein or glycosylation fragment thereof has a size of at least three repeating units of oligo- or polysaccharide structure and/or has a size of at least ten monosaccharides. [0428] 20. The fusion protein of any one of Paragraphs 13 to 19, (i) wherein the oligo- or polysaccharide is produced by bacteria of the genus Streptococcus and the polysaccharide is capsular polysaccharide, optionally, wherein is S. pneumoniae or S. agalactiae, and optionally, wherein the S. agalactiae capsular polysaccharide is Ia, Ib, II, III, IV, V, VI, VII, VIII, or IX; 32080280 - 144 - Atty. Dkt. No.: 64100-234947 (ii) wherein the oligo- or polysaccharide is produced by bacteria of the genus Klebsiella and the polysaccharide is a capsular polysaccharide or O-antigen polysaccharide, optionally wherein the bacteria is K. pneumoniae; or (iii) wherein the oligo- or polysaccharide is produced by bacteria of the genus Salmonella and the polysaccharide is O-antigen polysaccharide; optionally wherein the bacteria is S. enterica and the S. enterica polysaccharide is a Group B O-antigen. [0429] 21. The fusion protein of any one of Paragraphs 13 to 20, wherein the glycosylated fusion protein is produced in vivo; optionally, in a bacterial cell; optionally, in Escherichia coli; optionally, in a bacterium from the genus Klebsiella; and/or optionally, wherein the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0430] 22. The fusion protein of any one of Paragraphs 13 to 21, wherein when the fusion protein is a vaccine that induces an immune response when administered to a subject; optionally, wherein the immune response elicits long term memory (memory B and T cells), is an antibody response, and is optionally a serotype-specific antibody response; optionally, wherein the antibody response is an IgG or IgM response; optionally, wherein the antibody response is an IgG response; optionally an IgG1 response; and/or optionally, wherein the fusion protein generates immunological memory in a subject administered the vaccine. [0431] 23. A method of producing a glycoconjugate, the method comprising covalently linking an oligo- or polysaccharide to an acceptor protein comprising or consisting of a TfpM- associated pilin-like protein or glycosylation fragment thereof using a TfpM oligosaccharyltransferase (OTase); wherein the pilin-like protein or glycosylation fragment comprises a C-terminus serine or threonine residue, the acceptor protein comprises a C-terminus serine or threonine residue, and the oligo- or polysaccharide is covalently linked to the C-terminus serine or threonine residue of the acceptor protein; optionally, wherein the oligo- or polysaccharide comprises a glucose at its reducing end; 32080280 - 145 - Atty. Dkt. No.: 64100-234947 optionally, wherein the acceptor protein is a fusion protein of any one of Paragraphs 13 to 22; optionally, wherein the method is a method of in vivo conjugation of an oligo- or polysaccharide to an acceptor protein; and/or optionally, wherein the glycoconjugate is immunogenic. [0432] 24. The method of Paragraph 23, wherein the TfpM OTase comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to TfpMMo (SEQ ID NO: 56), TfpMDSM16617 (SEQ ID NO: 63), TfpMZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpMYH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpMAI40 (SEQ ID NO: 70), TfpMF78 (SEQ ID NO: 71), TfpMS71 (SEQ ID NO: 72), TfpMANC4282 (SEQ ID NO: 73), TfpMCIP102159 (SEQ ID NO: 74), TfpMjunii-65 (SEQ ID NO: 75), TfpMYZS-X (SEQ ID NO: 76), TfpMCIP102637 (SEQ ID NO: 77), TfpMT-3-2 (SEQ ID NO: 78), TfpMBI730 (SEQ ID NO: 79), TfpMA3K91 (SEQ ID NO: 80), and/or TfpM72-O-c (SEQ ID NO: 81); optionally, wherein the TfpM OTase is TfpMMo (SEQ ID NO: 56), TfpMDSM16617 (SEQ ID NO: 63), TfpMZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpMYH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpMAI40 (SEQ ID NO: 70), TfpMF78 (SEQ ID NO: 71), TfpMS71 (SEQ ID NO: 72), TfpMANC4282 (SEQ ID NO: 73), TfpMCIP102159 (SEQ ID NO: 74), TfpMjunii-65 (SEQ ID NO: 75), TfpMYZS-X (SEQ ID NO: 76), TfpMCIP102637 (SEQ ID NO: 77), TfpMT-3-2 (SEQ ID NO: 78), TfpMBI730 (SEQ ID NO: 79), TfpMA3K91 (SEQ ID NO: 80), or TfpM72-O-c (SEQ ID NO: 81); optionally, wherein the TfpM OTase is TfpMMo (SEQ ID NO: 56). [0433] 25. The method of Paragraph 23 or 24, wherein the pilin-like-protein glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, has a length of from 116 to 139 amino acids in length, has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length, wherein the TfpM-associated pilin-like protein glycosylation fragment comprises a C- terminus serine or threonine residue. [0434] 26. The method of any one of Paragraphs 23 to 25, 32080280 - 146 - Atty. Dkt. No.: 64100-234947 (a) wherein the TfpM-associated pilin-like-protein or glycosylation fragment thereof is: (i) PilMo (SEQ ID NO: 57), (ii) PilMo lacking amino acids corresponding to residues 1–28 (PilMoΔ28, SEQ ID NO: 58), or (ii) a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, wherein the TfpM-associated pilin-like protein contains a C-terminus serine or threonine residue, optionally, wherein the C-terminus threonine is substituted with serine; (b) wherein the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), and PilCIP102637 (SEQ ID NO: 100); (c) wherein the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), PilCIP102637 (SEQ ID NO: 100), and a fragment of any thereof that contains a C-terminus serine or threonine residue, or a variant wherein the C-terminus threonine is substituted with serine, optionally, wherein the TfpM- associated pilin-like protein glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end; and/or (d) wherein the pilin-like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (PilMo_DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C- terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148), optionally, (e) wherein the pilin-like-protein glycosylation fragment consists of Pil20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), Pil16 (SEQ ID NO: 136), Pil15 (SEQ ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ 32080280 - 147 - Atty. Dkt. No.: 64100-234947 ID NO: 110), Pil12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C- terminus threonine, further optionally, wherein the glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end, further optionally, wherein the pilin- like-protein glycosylation fragment consists of Pil20[A] (SEQ ID NO: 166), Pil19[A] (SEQ ID NO: 167), Pil18[A] (SEQ ID NO: 168), Pil17[A] (SEQ ID NO: 169), Pil16[A] (SEQ ID NO: 170), Pil15[A] (SEQ ID NO: 171), Pil14[A] (SEQ ID NO: 172), Pil13[A] (SEQ ID NO: 173), Pil12[A] (SEQ ID NO: 174), Pil11[A] (SEQ ID NO: 175), Pil10[A] (SEQ ID NO: 176), Pil9[A] (SEQ ID NO: 177), Pil8[A] (SEQ ID NO: 178), Pil7[A] (SEQ ID NO: 179), Pil6[A] (SEQ ID NO: 180), Pil5[A] (SEQ ID NO: 181), or Pil4[A] (SEQ ID NO: 182), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine, wherein the glycosylation fragment comprises at least the last four amino acids from the pilin C-terminal end; or optionally, (f) wherein the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil15S (SEQ ID NO: 153), Pil14S (SEQ ID NO: 154), Pil13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil11S (SEQ ID NO: 157), Pil10S (SEQ ID NO: 158), Pil9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil4S (SEQ ID NO: 164), or Pil3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine, further optionally, wherein the glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end, further optionally, wherein the pilin-like-protein glycosylation fragment consists of Pil20S[A] (SEQ ID NO: 183), Pil19S[A] (SEQ ID NO: 184), Pil18S[A] (SEQ ID NO: 185), Pil17S[A] (SEQ ID NO: 186), Pil16S[A] (SEQ ID NO: 187), Pil15S[A] (SEQ ID NO: 188), Pil14S[A] (SEQ ID NO: 189), Pil13S[A] (SEQ ID NO: 190), Pil12S[A] (SEQ ID NO: 191), Pil11S[A] (SEQ ID NO: 192), Pil10S[A] (SEQ ID NO: 193), Pil9S[A] (SEQ ID NO: 194), Pil8S[A] (SEQ ID NO: 195), Pil7S[A] (SEQ ID NO: 196), Pil6S[A] (SEQ ID NO: 197), Pil5S[A] (SEQ ID NO: 198), or Pil4S[A] (SEQ ID NO: 199), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine, wherein the glycosylation fragment comprises at least the last four amino acids from the pilin C-terminal end. 32080280 - 148 - Atty. Dkt. No.: 64100-234947 [0435] 27. The method of any one of Paragraphs 23 to 26, wherein the acceptor protein is a fusion protein comprising a heterologous carrier protein and the carrier protein is selected from the group consisting of Pseudomonas aeruginosa Exotoxin A (EPA), CRM197, cholera toxin B subunit, tetanus toxin C fragment, and a fragment of any thereof. [0436] 28. The method of any one of Paragraphs 23 to 27, wherein the acceptor protein is a fusion protein comprising an additional glycosylation sequence of an OTase other than TfpM oligosaccharyltransferase (OTase) in addition to the TfpM-associated pilin-like-protein glycosylation fragment located at its C-terminus, and the method further comprises covalently linking an oligo- or polysaccharide to the additional glycosylation sequence with an OTase other than TfpM OTase; optionally, wherein the acceptor protein is a fusion protein comprising a ComP glycosylation fragment, and the method further comprises covalently linking an oligo- or polysaccharide to the ComP glycosylation fragment using a PglS OTase, optionally, wherein the ComP glycosylation fragment is an internal glycosylation fragment of ComP, further optionally, wherein the ComP glycosylation fragment comprises or consists of CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 59) or a fragment thereof comprising at least the amino acids ASA in positions 11-13. [0437] 29. The method of any one of Paragraphs23 to 28, wherein the conjugation occurs in a host cell. [0438] 30. The method of Paragraph 29, wherein the host cell is a bacterial cell; optionally, in Escherichia coli; optionally, in a bacterium from the genus Klebsiella; or optionally, wherein the bacterial species is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0439] 31. The method of Paragraph 29 or 30, comprising culturing a host cell that comprises: (a) a genetic cluster encoding for the proteins required to synthesize the oligo- or polysaccharide; (b) a TfpM OTase; and (3) the acceptor protein. [0440] 32. The method of any one of Paragraphs 23 to 31, wherein the method produces a conjugate vaccine. [0441] 33. A host cell comprising (a) a genetic cluster encoding for the proteins required to synthesize an oligo- or polysaccharide; (b) a TfpM OTase; and (3) an acceptor protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof. [0442] 34. The host cell of Paragraph 33, wherein the acceptor protein is a fusion protein. 32080280 - 149 - Atty. Dkt. No.: 64100-234947 [0443] 35. The host cell of Paragraph 33 or 34, wherein the host cell comprises a nucleic acid encoding the TfpM OTase; and/or wherein the host cell comprises a nucleic acid encoding the acceptor protein; optionally, wherein the TfpM OTase and the acceptor protein are encoded by the same nucleic acid. [0444] 36. An isolated nucleic acid encoding the pilin-like-protein glycosylation fragment of Paragraph 11 or 12 and/or the fusion protein of any one of Paragraphs 13 to 22. [0445] 37. The isolated nucleic acid of Paragraph 36, wherein the nucleic acid is a vector. [0446] 38. A host cell comprising the isolated nucleic acid of Paragraph 36 or 37, optionally, wherein the host cell is a bacterial cell; and further: optionally, wherein the host cell is Escherichia coli; optionally, wherein the host cell is from the genus Klebsiella; or optionally, wherein the host cell is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0447] 39. A composition comprising the conjugate vaccine of Paragraph 10 or the fusion protein of Paragraph 22, and an adjuvant and/or carrier. [0448] 40. A method of inducing a host immune response against a bacterial pathogen, the method comprising administering to a subject in need of the immune response an effective amount of the conjugate vaccine of Paragraph 10, the fusion protein of Paragraph 22, or the composition of Paragraph 39. [0449] 41. The method of Paragraph 40, wherein the immune response is an antibody response; wherein the immune response is selected from the group consisting of an innate response, an adaptive response, a humoral response, an antibody response, cell mediated response, a B cell response, a T cell response, cytokine upregulation or downregulation, immune system cross-talk, and a combination of two or more of said immune responses; and/or 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 said immune responses. [0450] 42. A method of preventing or treating a bacterial disease and/or infection in a subject comprising administering to a subject in need thereof the conjugate vaccine of Paragraph 10, the fusion protein of Paragraph 22, or the composition of Paragraph 39; 32080280 - 150 - Atty. Dkt. No.: 64100-234947 optionally, wherein the subject is a human. [0451] 43. The method of Paragraph 42, wherein the infection is a localized or systemic infection of skin, soft tissue, blood, or an organ, or is auto-immune in nature; wherein the disease is pneumonia; and/or wherein the infection is a systemic infection and/or an infection of the blood. [0452] 44. The method of any one of Paragraphs 40 to 43, wherein the conjugate vaccine, the fusion protein, or the composition is administered via intramuscular injection, intradermal injection, intraperitoneal injection, subcutaneous injection, intravenous injection, oral administration, mucosal administration, intranasal administration, or pulmonary administration. [0453] 45. A method of producing a pneumococcal conjugate vaccine against pneumococcal infection, the method comprising: (a) isolating the glycoconjugate of any one of Paragraphs 1 to 10 or a glycosylated fusion protein of any one of Paragraphs 13 to 22; and (b) combining the isolated glycoconjugate or isolated glycosylated fusion protein with an adjuvant and/or carrier. [0454] 46. The glycoconjugate, glycosylated fusion protein, or conjugate vaccine of any of the above Paragraphs for use in inducing a host immune response against a bacterial pathogen and/or preventing or treating a bacterial disease and/or infection in a subject. [0455] 47. A recombinant nucleic acid construct comprising a nucleotide sequence encoding a TfpM oligosaccharyltransferase (OTase) operably linked to at least one heterologous transcriptional regulatory sequence. [0456] 48. The recombinant construct of Paragraph 47, wherein the TfpM OTase comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to TfpMMo (SEQ ID NO: 56), TfpMDSM16617 (SEQ ID NO: 63), TfpMZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpMYH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpMAI40 (SEQ ID NO: 70), TfpMF78 (SEQ ID NO: 71), TfpMS71 (SEQ ID NO: 72), TfpMANC4282 (SEQ ID NO: 73), TfpMCIP102159 (SEQ ID NO: 74), TfpMjunii-65 (SEQ ID NO: 75), TfpMYZS-X (SEQ ID NO: 76), TfpMCIP102637 (SEQ ID NO: 77), TfpMT-3-2 (SEQ ID NO: 78), TfpMBI730 (SEQ ID NO: 79), TfpMA3K91 (SEQ ID NO: 80), and/or TfpM72-O-c (SEQ ID NO: 81); 32080280 - 151 - Atty. Dkt. No.: 64100-234947 optionally, wherein the TfpM OTase is TfpMMo (SEQ ID NO: 56), TfpMDSM16617 (SEQ ID NO: 63), TfpMZZC3 (SEQ ID NO: 64), TfpMTUM15069 (SEQ ID NO: 65), TfpMAI7 (SEQ ID NO: 66), TfpMVE-C3 (SEQ ID NO: 67), TfpMYH01026 (SEQ ID NO: 68), TfpMCIP102143 (SEQ ID NO: 69), TfpMAI40 (SEQ ID NO: 70), TfpMF78 (SEQ ID NO: 71), TfpMS71 (SEQ ID NO: 72), TfpMANC4282 (SEQ ID NO: 73), TfpMCIP102159 (SEQ ID NO: 74), TfpMjunii-65 (SEQ ID NO: 75), TfpMYZS-X (SEQ ID NO: 76), TfpMCIP102637 (SEQ ID NO: 77), TfpMT-3-2 (SEQ ID NO: 78), TfpMBI730 (SEQ ID NO: 79), TfpMA3K91 (SEQ ID NO: 80), and/or TfpM72-O-c (SEQ ID NO: 81); optionally, wherein the TfpM OTase is TfpMMo (SEQ ID NO: 56). [0457] 49. The recombinant construct of Paragraph 47 or 48, wherein the heterologous transcriptional regulatory sequence is a promotor sequence. [0458] 50. The recombinant construct of any one of Paragraphs 47 to 49, further comprising a nucleotide sequence encoding a TfpM-associated pilin-like protein or glycosylation fragment thereof or a fusion protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof operably linked to the nucleotide sequence encoding the a TfpM OTase; optionally, wherein the coding sequence of the TfpM-associated pilin-like protein or glycosylation fragment thereof or a fusion protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof is within 2, 5, 10, 20, 30, 40, or 50 nucleotides of the sequence encoding the TfpM OTase; optionally, wherein the coding sequence of the TfpM-associated pilin-like protein or glycosylation fragment thereof or a fusion protein comprising a TfpM-associated pilin-like protein or glycosylation fragment thereof overlaps the operably linked nucleotide sequence encoding the a TfpM OTase. [0459] 51. The recombinant construct of Paragraph 50, wherein the pilin-like-protein glycosylation fragment has a length of from 3 to 139 amino acids in length, has a length of from 20 to 139 amino acids in length, has a length of from 116 to 139 amino acids in length, has a length of from 3 to 22 amino acids in length, has a length of from 10 to 22 amino acids in length, has a length of from 11 to 22 amino acids in length, has a length of from 5 to 21 amino acids in length, has a length of from 10 to 21 amino acids in length, or has a length of from 11 to 21 amino acids in length, wherein the TfpM-associated pilin-like protein glycosylation fragment comprises a C- terminus serine or threonine residue. 32080280 - 152 - Atty. Dkt. No.: 64100-234947 [0460] 52. The recombinant construct of Paragraph 50 or 51, (a) wherein the TfpM-associated pilin-like-protein or glycosylation fragment thereof is: (i) PilMo (SEQ ID NO: 57), (ii) PilMo lacking amino acids corresponding to residues 1–28 (PilMoΔ28, SEQ ID NO: 58), or (ii) a polypeptide comprising at least comprises at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 57 or SEQ ID NO: 58, wherein the TfpM-associated pilin-like protein contains a C-terminus serine or threonine residue, optionally, wherein the C-terminus threonine is substituted with serine; (b) wherein the TfpM-associated pilin-like-protein is selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT- 3-2 (SEQ ID NO: 99), and PilCIP102637 (SEQ ID NO: 100); (c) wherein the TfpM-associated pilin-like-protein or the pilin-like-protein glycosylation fragment comprises or consists of an amino acid sequence selected from the group consisting of PilDSM16617 (SEQ ID NO: 82), PilZZC3-9 (SEQ ID NO: 83), PilTUM15069 (SEQ ID NO: 84), PilAI7 (SEQ ID NO: 85), PilVE-C3 (SEQ ID NO: 86), PilYH01026 (SEQ ID NO: 87), PilCIP102143 (SEQ ID NO: 88), PilAI40 (SEQ ID NO: 89), PilF78 (SEQ ID NO: 90), PilS71 (SEQ ID NO: 91), PilANC4282 (SEQ ID NO: 92), Pil72-O-c (SEQ ID NO: 93), PilBI730 (SEQ ID NO: 94), PilA3K91 (SEQ ID NO: 95), PilCIP102159 (SEQ ID NO: 96), Piljunii-65 (SEQ ID NO: 97), PilYZS-X (SEQ ID NO: 98), PilT-3-2 (SEQ ID NO: 99), PilCIP102637 (SEQ ID NO: 100), and a fragment of any thereof that contains a C-terminus serine or threonine residue, or a variant wherein the C- terminus threonine is substituted with serine, optionally, wherein the TfpM-associated pilin- like protein glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end; and/or (d) wherein the pilin-like-protein glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (PilMo_DSL, also referred to as Pil20; SEQ ID NO: 60) or truncated derivatives thereof comprising at least the last three amino acids from the pilin C- terminal end or a variant wherein the C-terminus threonine is substituted with serine (SEQ ID NO: 148), optionally, (e) wherein the pilin-like-protein glycosylation fragment consists of Pil20 (SEQ ID NO: 60), Pil19 (SEQ ID NO: 133), Pil18 (SEQ ID NO: 134), Pil17 (SEQ ID NO: 135), 32080280 - 153 - Atty. Dkt. No.: 64100-234947 Pil16 (SEQ ID NO: 136), Pil15 (SEQ ID NO: 109), Pil14 (SEQ ID NO: 137), Pil13 (SEQ ID NO: 110), Pil12 (SEQ ID NO: 138), Pil11 (SEQ ID NO: 139), Pil10 (SEQ ID NO: 112), Pil9 (SEQ ID NO: 140), Pil8 (SEQ ID NO: 141), Pil7 (SEQ ID NO: 113), Pil6 (SEQ ID NO: 114), Pil5 (SEQ ID NO: 115), Pil4 (SEQ ID NO: 116), or Pil3 (SEQ ID NO: 117), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine, further optionally, wherein the glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end, further optionally, wherein the pilin-like- protein glycosylation fragment consists of Pil20[A] (SEQ ID NO: 166), Pil19[A] (SEQ ID NO: 167), Pil18[A] (SEQ ID NO: 168), Pil17[A] (SEQ ID NO: 169), Pil16[A] (SEQ ID NO: 170), Pil15[A] (SEQ ID NO: 171), Pil14[A] (SEQ ID NO: 172), Pil13[A] (SEQ ID NO: 173), Pil12[A] (SEQ ID NO: 174), Pil11[A] (SEQ ID NO: 175), Pil10[A] (SEQ ID NO: 176), Pil9[A] (SEQ ID NO: 177), Pil8[A] (SEQ ID NO: 178), Pil7[A] (SEQ ID NO: 179), Pil6[A] (SEQ ID NO: 180), Pil5[A] (SEQ ID NO: 181), or Pil4[A] (SEQ ID NO: 182), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus threonine, wherein the glycosylation fragment comprises at least the last four amino acids from the pilin C-terminal end; or optionally, (f) wherein the pilin-like-protein glycosylation fragment consists of Pil20S (SEQ ID NO: 148), Pil19S (SEQ ID NO: 149), Pil18S (SEQ ID NO: 150), Pil17S (SEQ ID NO: 151), Pil16S (SEQ ID NO: 152), Pil15S (SEQ ID NO: 153), Pil14S (SEQ ID NO: 154), Pil13S (SEQ ID NO: 155), Pil12S (SEQ ID NO: 156), Pil11S (SEQ ID NO: 157), Pil10S (SEQ ID NO: 158), Pil9S (SEQ ID NO: 159), Pil8S (SEQ ID NO: 160), Pil7S (SEQ ID NO: 161), Pil6S (SEQ ID NO: 162), Pil5S (SEQ ID NO: 163), Pil4S (SEQ ID NO: 164), or Pil3S (SEQ ID NO: 165), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C- terminus serine, further optionally, wherein the glycosylation fragment comprises at least the last three amino acids from the pilin C-terminal end, further optionally, wherein the pilin-like- protein glycosylation fragment consists of Pil20S[A] (SEQ ID NO: 183), Pil19S[A] (SEQ ID NO: 184), Pil18S[A] (SEQ ID NO: 185), Pil17S[A] (SEQ ID NO: 186), Pil16S[A] (SEQ ID NO: 187), Pil15S[A] (SEQ ID NO: 188), Pil14S[A] (SEQ ID NO: 189), Pil13S[A] (SEQ ID NO: 190), Pil12S[A] (SEQ ID NO: 191), Pil11S[A] (SEQ ID NO: 192), Pil10S[A] (SEQ ID NO: 193), Pil9S[A] (SEQ ID NO: 194), Pil8S[A] (SEQ ID NO: 195), Pil7S[A] (SEQ ID NO: 196), Pil6S[A] (SEQ ID NO: 197), Pil5S[A] (SEQ ID NO: 198), or Pil4S[A] (SEQ ID NO: 199), or a variant thereof having one, two, three, four, or five amino acid substitutions and maintaining the C-terminus serine, wherein the 32080280 - 154 - Atty. Dkt. No.: 64100-234947 glycosylation fragment comprises at least the last four amino acids from the pilin C-terminal end. [0461] 53. The recombinant construct of any one of Paragraphs 50 to 52, wherein the fusion protein is a fusion protein of any one of Paragraphs 13 to 22. [0462] 54. The recombinant construct of any one of Paragraphs 50 to 53, further comprising a nucleotide sequence encoding a PglS OTase operably linked to the TpfM OTase; optionally, wherein the coding sequence of the PglS OTase is within 10, 20, 30, 40, 50, 75, or 100 nucleotides of the sequence encoding the TfpM OTase. [0463] 55. A vector comprising the recombinant nucleic acid construct of any one of Paragraphs 47 to 54. [0464] 56. A host cell comprising the recombinant nucleic acid construct of any one of Paragraphs 47 to 54, or the vector of Paragraph 55; optionally, wherein the host cell is a bacterial cell; and further: optionally, wherein the host cell is Escherichia coli; optionally, wherein the host cell is from the genus Klebsiella; or optionally, wherein the host cell is K. pneumoniae, K. varricola, K. michinganenis, or K. oxytoca. [0465] 57. A method for producing a TfpM OTase, the method comprising culturing the host cell of Paragraph 56, wherein said vector of Paragraph 54 is an expression vector, and recovering the TfpM OTase. [0466] 58. A glycoconjugate comprising an oligo- or polysaccharide covalently linked to an acceptor protein, wherein the acceptor protein comprises means for being covalently linked to the oligo- or polysaccharide by a TfpM OTase. [0467] 59. The glycoconjugate of Paragraph 58, wherein the glycoconjugate is the glycoconjugate of any one of Paragraphs 1 to 10. [0468] 60. A fusion protein comprising means for being covalently linked to an oligo- or polysaccharide by a TfpM OTase, wherein said means is translationally fused to a heterologous carrier protein, wherein the means is located at the C-terminus-most sequence of the fusion protein. [0469] 61. The fusion protein of Paragraph 60, wherein the fusion protein is the fusion protein of any one of Paragraphs 13 to 22. 32080280 - 155 - Atty. Dkt. No.: 64100-234947 [0470] 62. The glycoconjugate of Paragraph 58 or 59 or the fusion protein of Paragraph 60 or 61, for use in any of the methods above. ***** [0471] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the appended claims and their equivalents. 32080280 - 156 - Atty. Dkt. No.: 64100-234947 SEQUENCES >TfpM_Mo (SEQ ID NO: 56) MFNLIKHFKNKELFVALYFLLFIVGFSIYSFNFYNELRVLEVFLLIGLGLYGLIHKKNYFSKYEF CFLIFTLLGFLFWSNYSYIFYELILFYLLYKAFFNLNYNEFLSKLLIWLSFSIFLMLPIAITEFILS GVYPNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQEKFKNIYLIFLFLAFLSILLDGGRSATLA YSVFIACVCIFNHAARFKLLFTYVATWLAYFSILFFSSHSASIVGSIVRESTSGRYELWVSAFQC WSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYF VISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFQHAASFHPSTYEASTVSQVFGYIAYI SIALAFIYLFFYTSALSESLPSTPPRFWEYGYQLF >Pil_Mo (SEQ ID NO: 57) MNAQKGFTLIELMIVVAIIGILAAIAIPAYQDYTIRAKISEGLTLSNGLKTAIAESFQSKGPSSM ACTDATTCASIGASPMDATALAGNKNVASITSDAAGVITIVYKPAVVPTGSNNLTLTPVGAD GTTALNLSAAASAGSQVNWRCGGTGTTVAAKFLPANCRGT >Pil_Mo_ ^28 (SEQ ID NO: 58) AYQDYTIRAKISEGLTLSNGLKTAIAESFQSKGPSSMACTDATTCASIGASPMDATALAGNKN VASITSDAAGVITIVYKPAVVPTGSNNLTLTPVGADGTTALNLSAAASAGSQVNWRCGGTGT TVAAKFLPANCRGT >PglS Sequon (SEQ ID NO: 59) CTGVTQIASGASAATTNVASAQC > Pil20 sequon / Pil_Mo_DSL (SEQ ID NO: 60) CGGTGTTVAAKFLPANCRGT >TfpM_DSM16617 (SEQ ID NO: 63) MLNFNKHLKNKELFIALYFLTFILGLSIHISSNFYNESRVLETFLLLSLVFNKNLHLKKIEYIFIIF IIFHLLFLRNSQFIIFEILLYYLLYKAFLIINYNELLAKFIIWISFSIFSILPIEIVKYIHNSIYSNWYP TPWNIRVYNSYFLVLSVFAVWFYLKQEKFKIIYLFFLFLAFLSILLDGGRSATLAYSVFIACVCI FNRAVRFKLLFTYAATWLTYFSILFFSSHSASNVRSIVRESTSGRYELWVSAFQCWSQNPIFGC GFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYFVISAFIAVSID LSFSGIHIYPVTQVALLWLFVFLLKNPEFEHAASFHPSTYEASRVSQVFGYIAYISIALAFIYLFF YTSALSESLASTPPRFWEYGYQLF >TfpM_ZZC3 (SEQ ID NO: 64) MLNFNKYLKNKELFIALYFLSFILGLSIHISSNFYNESRVLETFLLLSLVFNKNLHLKKIEYIFIIF IIFHLFFLRNSQFIIFEILLYYLLYKAFLVINYNELLAKFIIWISFSIFSMLPIEIVKYIHNSIYSNWY PTPWNIRVYNSYFFVLSIFAIWFYLKQEKFKNIYLIFLFLAFLSILLDGGRSATLAYSVFIACVCI FNRAARFKLLFTYAATWLTYFSILFFSSHSASNVRSIVRESTSGRYELWVSAFQCWSQNPIFGC GFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYFVISAFIAVSID LSFSGIHIYPVTQVALLWLFVFLLKNPEFQHAASFHPSTYEASRVSQVFGYIAYISIALAFIYLFF YTSALSESLPSTPPRFWEYGYQLF >TfpM_TUM15069 (SEQ ID NO: 65) MFNLIKHFKNKELFVALYFFLFILGVSINGFNFYNELRVVEVFLLIFLGFYGLINKKNYFSKYE VCFLIFTLLGFLFWSNYSYIFYELILFYLLYKAFLNLNYNEFLSKFVIWLSFLIFLMLPIAITEFIA SGVYQNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQEKFKNIYLVFLFLAFLSILLDGGRSATL AYSVFIACVCIFNHAARFKLLFTYVATWLAYFSILFFSSHSASIVGSIVRESTSGRYELWVSAF QCWSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKEN YFVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFEHAASFHPSTYEASRVSQVFGYI AYISIALAFIYLFFYTSALSESLASTPPRFWEYGYQLF 32080280 - 157 - Atty. Dkt. No.: 64100-234947 >TfpM_AI7 (SEQ ID NO: 66) MFKYIKHFKNKELFIALYFLLFIVGFTIYSFNFYNELRAIEFFLLIFFGFHSLINKNYVSKYEFCF LIFIFLGFLFWSNYSYIFYELILFYLLYKAFLNLNYNEFLSKFVIWLSFLIFLMLPIAITEFILSGV YQNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQEKFKNIYLIFLFLAFLSILLDGGRSATLAYS VFIACVCMFNRAVRFKLLFTYAATWLVYFSILFISSHSASGVRSIVRESTSGRYELWVNAFQC WSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYF VISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFEHAASFHPSTYEASRVSQVFGYIAYI LIALAFIYLFSYTSALSESLPSTPPRFWEYGYQLF >TfpM_VE-C3 (SEQ ID NO: 67) MNFLVHFKNKELFVALYFLLFTVGFSIYSFNFYNELRALEFFLLIGLGLYGLIHKKNYFSKYEF CFLIFTLLGFLFWSNYYYIFFELILFYLLYKAFLNLNYNEFLSKLLIWLSFSIFLMLPIAITEFILS GVYQNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQEKFKNIYLIFLFLAFLSILLDGGRSATLA YSVFIACVCMFNRAARFKLLFTSAATWLTYFSILFFSSHSASNVRSIVRESTSGRYELWVSAFQ CWSQNPIFGCGFYQLDHYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENY FVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFQHAASFHPSTYEASRVSQVFGYIA YISIALAFIYLFFYTSALSESLPSTPPRFWEYGYQLF >TfpM_YH01026 (SEQ ID NO: 68) MFNLIKHFKNKELFIALYFLLFIVGFSIYSFNFYNELRVLEVFIFIGLVLFGLIHKKNYFSKYEFC FLIFTLLGFLFWSNYSYIFYELILFYLLYKAFLNLNYNEFISKLVIWLSFSIFLMLPIAITEFMLSG LYQNWYPMPWNIRVYNSYFLVLSIFAVWFYFKQEKFINIYLVFLFLAFLSILLDGGRSATLAY SVFIACVCIFNRAARFKLLFIYVATWLTYFSILFFSSHSASNVRSIVRESTSGRYELWVSAFQC WSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVICNILKRIKWNLKENYFV ISAFIAVSIDLSFSGIHIYPITQVALLWLFVFLLKNPEFQHAASFHPSTYEASRVSQVFGYIAYILI ALAFIYLFFYTSALSESLPSTPPRFWEYGYQLF >TfpM_CIP102143 (SEQ ID NO: 69) MFNLIKHLKNKELFVALYFLLFIVGFSIYSSNFYNELRALEFFLLIGLGLYVLIHKKNYFSKYEL CFLIFTLLGFLFWSNYSYIFYELILFYLLYKAFLNLNYNEFLSKLLIWLSFSIFLMLPIAITEFIISG VYQNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQEKFKNIYLIFLFLAFLSILLDGGRSATLAY SVFIACVCIFNHAARFKLLFTYAVTWLAYFSILFFSSHSTSIVGSIVRESTSGRYELWVSAFQC WSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYF VISAFIAVSIDLSFSGIHIYPITQVALLWLFVFLLKNPEFQHATSFHPPIYEASKVSQVFEFMVYI LIALAFIYLFFYTSALSESLASTPPRFWEYGYQLF >TfpM_AI40 (SEQ ID NO: 70) MFSVVSHLKNKELFIALYFLLFISGISIYSYNFYNELRVSEVFLLIGLGLCGLTHKKNYFSKYEF YFFIFILLGFLFWSNYNYILYEFILFYLLYQAFLNLNYSEFLSKFVIWLSFSIFLMFPIAVAEFLVS GVYQNWYPMPWNIRIYNSYFLVFSIFSVWFYLKEEKFKNIYLFFLFLAFLSILLDGGRSATLAY SIFIACICMFNRTARLKILFTYMATWLTYFSILFFSSNSASGVRSIARESTSGRYELWVSAFQCW LQNPLFGCGFYQLDLYSGIIPAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYFVIS AFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFQHAASFHSSTYEASRVSQVFGYIVCILIA LAFIYLFFYTSALSESLPSTPPRFWEYGYQLF >TfpM_F78 (SEQ ID NO: 71) MSNLIKYLKNKELFIALYFFIFILGISIQLSFNFYNEARVLEILLLLCLGLYSFINNENLFFKKELV FLFFISFGFFYWFNFQIVFYEILLFYLLYKAFFFLKYNAIVSKLIVFSSFFIFIFLPMSLWEYLTTG KYQNWYPLPWNIRIYNSYFLIFSIFAIWFFLKEKYKSIYLAFIFLAFLSILLDGGRSAALAYTVFI GLLSIFNRLARLKLIFIYSLTWLAYFLIIYFSSQSGSSLRYIARDSTSGRYDLWLNAFQCWLQSP ILGCGFYQLDKYSNLSAHPHNLFIQILTETGLIGLSFLLYIIFIILRNISWKFKENYFVISALIAVFI DLSFSGIHIYPITQVALLWLFVFLLKNPEFQHATYFSPSACEASKKSQFLEFIVYILIALAFIYLFF NTSALSESLPSTPPRFWEYGYQIF 32080280 - 158 - Atty. Dkt. No.: 64100-234947 >TfpM_S71 (SEQ ID NO: 72) MSNLIRHLKNKELFLALYFLTFILGVSLGVSYNFYNEARVLEVLLLLGFGFYSVFSKEIFFSKK EYLFLVVFISYLFFLKNSQFIIFDILLFYLLYKSFFILNYNLVVSKIIVLSSFLIFMTFPLSLLGYW GDGVYRNWYPMPWNIRVYNSYFLILLIFSTWLLMRGNRYTWVYLLFTYLSLLSILLDGGRSA LLAYSTFFIIVIIFNKKVRLKLIFIYIISWLSFLLIVFSAGIASDGISIARVTTSRRSDLWMHALQC WIESPIFGCGFYQLGAYENLSAHPHNLFIQILTETGVMGFSFLALIIFGVLRNISWNIKENYFVI AAFFAIGVDLSFSGIHIYPITQVGLLWLFVFLLKNPEFRHAKYFSDILVQNPKSVWVVNFIIYLII TCAFIYLFVNTSALSESLAVTPPRFWEYGYQLF >TfpM_ANC4282 (SEQ ID NO: 73) MLNLIKNKELFIALYFLIFNLGFSVHISSNFYNEARLLEIFLLLSLGIFSGFVKNIVFHKIEYIFLL FFIFSIFFLKNQPFIFFEILLFYLLFKAFFALNYNSKISKAIILLSFLIFLMFPVSILHYLNSGLYQN WYPMPWNIRIYNSYFLIFSIFAIWFYLKEDKYKNIYLIFIFLAFLSILLDGGRSATLAYTIFIVIVC IFNRLERFKLLLIYCSTWLAYFSIVYFSSQSASTLRSITRESTSGRYELWLNAFQCWLENPILGC GFYQLDKYPSLSAHPHNLFVQILTETGLIGFIFLSFIIFKVVKNISWNFKQNYFVLAALFSVAIE LSFSGIHIYPVTQVALLWLFVFLLKNPEFSHASYFNYLKIKNSKIDKLIQLFIYLFILIIFIYLFINT SVLSENLPSTPPRFWEYGYQLF >TfpM_CIP102519 (SEQ ID NO: 74) MFNLIKHIKNKELFLALYFLLFSVGFSIYSFNFYNELRALEFFLLIGLGLYGLIHKKNYFSKYEV CFLIFTFLGFSFWSNYSYIFYELILFYLLYKAFLNLNYNEFLSKLVIWLSFSIFLMLPIAITEFML NGIYQNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQDKFKNIYLIFLFLAFLSILLDGGRSATL AYSVFIACVCMFNRAARFKLLFTYAVTWLVYFSILFFSSHSDSNVSSIVRESTSGRYELWVSA FQCWSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKE NYFVISAFIAVSIDLSFSGIHIYPITQVALLWLFVFLLKNPEFEHAASFYPSTYEASRVSQVFGYI AYILIALAFIYLFFYTSALSESLPSTPPRFWEYGYQLF >TfpM_junii_65 (SEQ ID NO: 75) MNFLVHFKNKELFVALYFLLFSVGFSIYSFNFYNELRVLEIFLFIGLGLNSLTHKKNYFSKYEF CFLIFTLLGFLFWSNYSYIFFELILFYLLYKAFLNLNYNEFLSKLVIWLSFSIFLMLPIAIKEFIAS GVYQNWYPMPWNIRVYNSYFFILSIFAIWFYSKQEKFKNIYLIFLFLAFLSILLDGGRSATLAY SVFIACVCMFNRAARFKLLFTYAVTWLAYFSILFFSSHSTSIVGSIVRESTSGRYELWVSAFQC WSQNPIFGCGFYQLEQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENYF VISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFQHAASFHPSTYEASRVSQVFGYIVYI LIALAFIYLFSYTSALSESLPSTPPRFWEYGYQLF >TpfM_YZS-X (SEQ ID NO: 76) MFNLIKHFKNKELFIALYFLLFIAGFSIDSFNFYNELRIFEVFLLIGLGFYGLTHKKKYFSKYEF CFFIFILLGFLCWSNYSYILYEFILFYLLYKAFFDLNYNEFLSKLIIWLSFSIFLMFPIAVAEFLVS GVYQNWYPMPWNIRIYNSYFLVFSIFSVWFYLKEEKFKNIYLFFLFLAFLSILLDGGRSANLA YSIFIACICMFNRTARLKILFTYIATWLTYFSILFFSSNSASGVRSIARESTSGRYELWVSAFQC WLQNPLFGCGFYQLDLYSGIIPAHPHNLFIQILTETGLIGFGFLILVIWNILKRIKWNFKENYFVI PALIAVSIDLSFSGIHIYPITQIALLWLFVFLLKNPEFQYAASFNQSTHKGSKKSKNFELIVYILIA LAFIYLFFHTSALSESLPSTPPRFWEYGYRLF >TfpM_CIP102637 (SEQ ID NO: 77) MSNLIKHLKNKELLVALYFFLFILGVSINGFNFYNELRTLEVFLLIGLGFYSLIQKKSYFSKYEF CFLIFTLLGFLFWSNYSYIFYELILFYLLYKAFLNLNYNEFLSKLVIWLSFSIFLMLPIAITEFML SGVYQNWYPMPWNIRVYNSYFLVLSIFAIWFYLKQEKFNNIYLIFLFLAFLSILLDGGRSTALA YSVFIACVCMFNRAVRFKLLFTYAATWLTYFSILFFSSHSTSSVRSIVRESTSGRYELWVSAFQ CWSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETGLIGFGFLILVIWNILKRIKWNLKENY FVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFLLKNPEFQRAASFHPSTYEASRVSQVFGYIA YILIALAFIYLFFYTSALSESLPSTPPRFWEYGYQLF 32080280 - 159 - Atty. Dkt. No.: 64100-234947 >TfpM_T-3-2 (SEQ ID NO: 78) MMKAIKLLAHKESFIGLYFLLFIMGVTVGSGYGIYNESRIAEIALLLGLGAHACFNKYYIVTK VEYLFFVFIIIGSFFWSNSFFIIIDLLLVYLLYKSFFFLEYRPLLTKIIVLASFLIFLLLPVAIWDYIT SGIYTSNWYLLRLNIRIYNSYFLIMSIFAVWLYLTEKNYKKLYLSFIFLAFLSILMDGGRSATL AYTAFVIIICIFRRPVSWQLGFAYSMSWLTYLTINHLASLNAVETLGLGIARATTSQRYDIWM NAVQCWVQNPIWGCGFYQLDSTRNLASHPHNLFLQVLSETGLIGFGFLLAIIFSILKNISWNLN KDYFVIAALLAVVIETLLSGIHIYPITQIALLWLFIFLLKNPIFPHTLYFNSLIASFASNSYLSITV YLILTIFFLYFFINTSALIDPELLTRPRFLENGYNIF >TfpM_BI730 (SEQ ID NO: 79) MSTIVKRLKNKELFIALYFLLFILGFTIGITPKFYNEFRILQVTLLLNFGLHNIIHKHGYISRAELL FFVYIGIASLFWQNYEFIVIDLLLAYLLYKTFFLLKYNELATKVIVFFSLLIFPLLPLSVFDYIST GTYYPIWHPMPWNIRIYDSYLLIVSIFAVWFYITETKYKKIYLLFLLLAFFSVLLNGGRSATLA YTVFIAVIVVFNRIVRWQILATYAIAWLAYISISYLAISNLSMASPIGLQIARTTTSLRYDLWMN AIECWIQSPLVGCGFYQLYRYENLGAHPHNLLLQILTETGLIGFGFLLAIVVTILKHIDWQLKR SYFVIAALLAIGVDTSLSGTHIYPITQMALLWLLVFLLKNPVFQHAAYFNRVPYITSVTDMVV SIVVYFSLTVIFIYLFLNTSVLFDSLMLATPPRFWEYGYQLF >TfpM_A3K91 (SEQ ID NO: 80) MYKKQLLNNQQMTVSIIKALANKESFVALYSLIFIVGVTANSGYGIYNESRIFEVALLLGLGA HACFNKYYIVTKVEYLFFVFIIIGSFFWSNSFFIIIDLLLVYLLYKSFFFLEYRPLLTKIIVLASFLI FLLLPVAIWDYITSGIYTSNWYLLRLNIRIYNSYFLIMSIFAVWLYLTEKNYKKLYLSFIFLAFL SILMDGGRSATLAYTAFVIIICIFRRPVSWQLGFAYSMSWLTYLTINHLASLNAVETLDLGIAR ATTSQRYDIWMNAVQCWVQNPIWGCGFYQLDSTRNLAAHPHNLFLQVLSETGLIGFWFLLA IIFFILKNISWNLNKDYFVIASLLAVVIETLLSGIHIYPITQIALLWLFVFLLKSSNFSHAIYFHSSI PSFVVNRYFSVTMFLVLVILFIYFFINTSALLDNEIMTRPRFLENGYNIF >TfpM_72-O-c (SEQ ID NO: 81) MYNKNPLSKQHKLQTFIKSVRLLFNKELSIGLYFLLFILGMNTGSGHDSYNEFRVFQVTLLLVI GVSTWCYRRLFITKLELLFFAFIAFGSFFWQQPIFVLNDVLLVYLLYKSFYLLNYQPLLSKLIV LSSLLIFLLLPVALWNYIDTGKYSPIWYPLPWNMRVYDSYFLIISVFAVWFYLTEKQYRFLYL LFLFLAFLAVLLDGGRSVTLAYTVFIAIISLFHRRARWRLVLMYAMSWLTYIIVTYTANTSVT SLRIARDTRSDRYDLWINAFQCWSQHPLFGCGFYQLDKYPNIAAHPHNLFIQVLTETGLIGFG FLAFIIFKVAKNINWDLKQNYFVIAALLAISIDMSLSGVHIYPVTQIALLWLFVFLLKNPEFSHA HHFNKVVQQKKAIDNILPLIIYLSLTIWFIYLFTNTSSFLPGTPLTPPRFWVYGYQLW >Pil_DSM16617 (SEQ ID NO: 82) MNTMQKGFTLIELMIVVAIIGILAAIAIPAYQDYTVRARVSEGITTASAMKATVSENILNAGAL VAGTPSTAGSACVGVTEISGGTGNVASATCGASKAGQIIVTMGATTAKSVPVYLTPSYTASA VTWSCSTTAGNEKYVPSECRKVGT >Pil_ZZC3-9 (SEQ ID NO: 83) MNAQKGFTLIELMIVVAIIGILAAIAIPAYQDYTVRARVSEGITTASAMKATVSENILNAGALV AGAPSTAGSACVGVTEISGGTGNVASATCGASKPGQIIVTMGATTAKSVPVYLTPSYTASAV TWSCSTTTGNEKYVPSECRKVGT >Pil_TUM15069 (SEQ ID NO: 84) MNTMQKGFTLIELMIVVAIIGILAAIAIPAYQDYTIRAKISEGLTLSNGLKTAIAESFQSKGPSS MACTDATTCASIGASPMDATALAGNKNVASITSSTAGVITIAYKPAVVPNGSNNLTLTPVGA DGTTALDLSAAASAGSQVNWRCGGTGTTVAAKFLPANCRGT 32080280 - 160 - Atty. Dkt. No.: 64100-234947 >Pil_AI7 (SEQ ID NO: 85) MNAQKGFTLIELMIVVAIIGILAAVAIPAYQDYTTRAKVSEVITAGAACKTSVAEYYQSTGSL PLNTEQAGCSSNATPMVKSLAVASGIITVTASDALAAKFSTSTQNTYVLEPTATTAAAPLTWS CTGSTIEGKYLPAECRGT >Pil_VE-C3 (SEQ ID NO: 86) MKSMQKGFTLIELMIVIAIIGILAAIAIPAYTDYTARAKITEAVGALASAKTSVSEYYTSMGKM PADAAAAGINTAPAGSYVDNVAYAKTSDTVSTVTATIKNVNSTADTKKFKLTGTGSVAGVT WACATVDLDQKYLPANCRST >Pil_YH01026 (SEQ ID NO: 87) MNAQKGFTLIELMIVVAIIGILAAIAIPAYQDYTIRAKISEGLTLSNGLKTAIAESFQSKGPSSME CNNAATCALIGASPMDATALGGNKNVTSITSSEAGVITIAYKPAVVPAGANNLLLTPVGADG TTALNLSAAASAGSQVNWQCGGTNGTTVAAKFLPANCRGT >Pil_CIP102143 (SEQ ID NO: 88) MIVVAIIGILAAVAIPAYQDYTTRAKVSEVITAGAACKTSVAEYYQSTGSLPVNTEQAGCSSN ATPMVKSLAVDKGVITVTASDSLAAKFSTSTKNTYVLRPTATSAAAPLTWSCTGSTIEGKYLP AECRGT >Pil_AI40 (SEQ ID NO: 89) MNAQKGFTLIELMIVVAIIGILAAIAIPAYQDYTTRAKMSEVVNFAAAAKSAVSECAISTGDL DECDSNQKAGLAPAADLTSTYVESVTVGADGLITLAIQGTNVTALDNGSLTMEPTLDPVAGV TWVCKISSNTLNKYVPANCRAT >Pil_F78 (SEQ ID NO: 90) MNTVQKGFTLIELMIVIAIIGILAAIAIPAYTDYTVRARVSEAMTTASAMKATVSENIMNAGG TSIVATNKNCAGIPAFTATKNVATAACTDKTGVILVTTTEAAKSVPLTLTPTYSGGNVSWRCS TTSSFDKYVPSECRST >Pil_S71 (SEQ ID NO: 91) MNAQKGFTLIELMIVVAIIGILAAVAIPAYQDYTTRAKVSEVITAGAACKTSVAEFYQSSGELP GTLEQAGCSSNATPMVASLDVGADGVITVTASTDLAAKFSDSAKNTYVLAPTATTAAAPLT WSCTGSTIEGKYLPAECRGT >Pil_ANC4282 (SEQ ID NO: 92) MNAQKGFTLIELMIVVAIIGILAAIAIPAYQDYTTRAKVTEVMNYAAAAKSAVSECLSSTGVT TSCDTNAEAGLEAATSLTSPYVTSVTVGTGGSITAVVKGTNATSGNVALDGASLVLTPALSN AGVAWTCKISNVALNKFVPQTCRST >Pil_72-O-c (SEQ ID NO: 93) MNAQKGFTLIELMIVIAIIGILAAIAIPAYQNYIAKSQAAEAFTLMGGAKTTINSNLQNNSCTN TDDDTQNTVNGKYGVLTIGGAVAQDSNPTAATGCTMSYLFKGTGVSSQLADLVIEAGLNNN GTLAIDDTATTVDDELLPKSFAS >Pil_BI730 (SEQ ID NO: 94) MNAQKGFTLIELMIVVAIIGILAAIAIPQYQNYIAKSQVSRAMGETSSVKTAVETCLNEGKGA AGACPLGITTSNIQATTVAGSAPKADGTNAAELTETTAIVATFGTGAAKTLQETGKNSVTWT RDATGSWKCNSTVQAKYAPAGCSAT >Pil_A3K91 (SEQ ID NO: 95) MNAQKGFTLIELMIVVAIIGILAAIAIPAYQNYIAKSQASEAFTLIDGAKAEVNTNLEGNSCTN ATAAKNTIAGKYGSLVIAGTAASDASPTASTGCTLTYTFKGTGVSSQLASKVIGATLLNNGTL TKNATTTTVDADILPKSFT 32080280 - 161 - Atty. Dkt. No.: 64100-234947 >Pil_CIP102159 (SEQ ID NO: 96) MNTMQKGFTLIELMIVVAIIGILAAIAIPAYQDYTIRSRVAEALTALSSAKATVSENIANNGGVI AAGACAGYTNQTTATANVASTSCTDTTGVVSATTTTKAGGFVITMTPTVNADKTVVWKCT VPAASFKYAPAECRGT >Pil_junii_65 (SEQ ID NO: 97) MKSMQKGFTLIELMIVVAIIGILAAIAIPAYQDYTTRAKMSEVVNFAAAAKSAVSECAISTGTL SDCNTNAKAGLEAAADLKSTYVESVTVGNNGVITLEIKGTNVTALDAANLTMTPTLDNKAG VSWVCKISSNTLNKYVPANCRST >Pil_YZS-X (SEQ ID NO: 98) MNTVQKGFTLIELMIVVAIIGILAAIAIPAYTDYTARAKVTEAVGALASAKTSVSEYFTSQGV MPTNATQAGINTAPAGQYVSAVAYTKTSDTVSKISATLANINSEANTKTIVLEGTGDTAKGV SWVCKGGTAPGKFLPANCRGT >Pil_T-3-2 (SEQ ID NO: 99) MNAQKGFTLIELMIVIAIIGILAAIAIPAYQNYIAKAQVAEAFTLADGIKTSVGTNLQSGTCFAS GAATAATTDTIEGKYGTATTVADTTTGSNGCGIKYTFKSSGVSNKLTSKVIGMAITENGVLK KSTVTTTDAPADLLPQSFT >Pil_CIP102637 (SEQ ID NO: 100) MECNVLMIVVAIIGILAAIAIPAYTDYTARAKVTEAVGALASAKTSVSEFFTSQGVMPSDADA AGINKTPAGDYVAGVTYTRTDPTHAVVAVELKNINSDANGTTFQLNATGDTAKGVSWTCSS AGTKPTPEKFLPANCRGT >EPA-Pil_Mo_ ^28 (SEQ ID NO: 101) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG VLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLV PIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAI SHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYR VLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCG YPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERF VRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFST RGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIA GDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHP LPLRLDAITGPEEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDY ASQPGKPPREDLKAYQDYTIRAKISEGLTLSNGLKTAIAESFQSKGPSSMACTDATTCASIGAS PMDATALAGNKNVASITSDAAGVITIVYKPAVVPTGSNNLTLTPVGADGTTALNLSAAASAG SQVNWRCGGTGTTVAAKFLPANCRGT >EPA-Pil20 (SEQ ID NO: 102) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG VLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLV PIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAI SHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYR VLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCG YPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERF VRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFST RGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIA GDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHP LPLRLDAITGPEEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDY ASQPGKPPREDLKGGGGCGGTGTTVAAKFLPANCRGT 32080280 - 162 - Atty. Dkt. No.: 64100-234947 >EPA_PglS sequon_Pil20 (SEQ ID NO: 103) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG VLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLV PIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAI SHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYR VLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCG YPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERF VRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFST RGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIA GDPALAYGYAQDQEPDACTGVTQIASGASAATTNVASAQCVRGRIRNGALLRVYVPRSSLP GFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLTILGWPLAERTVVIPSAIPTDP RNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLKGGGGCGGTGTTVAAKFLPANCRGT >EPA_PglS sequon 2X (SEQ ID NO: 104) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG VLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLV PIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAI SHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYR VLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCG YPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERF VRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFST RGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIA GDPALAYGYAQDQEPDACTGVTQIASGASAATTNVASAQCVRGRIRNGALLRVYVPRSSLP GFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEECTGVTQIASGASAATTNVASAQCG GRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK >Dsba_secretion_signal_sequence (SEQ ID NO: 142) MKKIWLALAGLVLAFSASA >Dsba_EPA_exotoxin_A _ΔE553 (SEQ ID NO: 143) (EPA carrier without any sequons. This “bare” version has a non-native N-terminal DsbA secretion signal sequence and has a deletion of residue E553 which inactivates the toxin) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQG VLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLV PIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAI SHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYR VLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCG YPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERF VRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDISFST RGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIA GDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHP LPLRLDAITGPEEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDY ASQPGKPPREDLK >FlgI_secretion_signal_sequence (SEQ ID NO: 144) MIKFLSALILLLVTTAAQA >FlgI_CRM197_G52E (SEQ ID NO: 145) (Inactivated (G52E) form of CRM197 with an N-terminal non-native FlgI secretion signal sequence) MIKFLSALILLLVTTAAQAGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQG NYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKK ELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFE TRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNK MSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWAVNVAQVID 32080280 - 163 - Atty. Dkt. No.: 64100-234947 SETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIG FAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHD IKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGN GVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKS >DsbA_tetanus_toxin_C-fragment_TTc (SEQ ID NO: 146) MKKIWLALAGLVLAFSASAAKNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVIT YPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYG TNEYSIISSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVF ITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFRIFCKALN PKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNG KLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNEHIVGYPKDGNAFNNLDRI LRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILI ASNWYFNHLKDKILGCDWYFVPTDEGWTND >DsbA_cholera_toxin_B_subunit (SEQ ID NO: 147) MKKIWLALAGLVLAFSASANGTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITF KNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN
Figure imgf000166_0001
32080280 - 164 - Atty. Dkt. No.: 64100-234947
Figure imgf000167_0001
>PDB entity 1IKQ of Pseudomonas aeruginosa Exotoxin A (EPA) (SEQ ID NO: 218) AEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDA LKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNI KVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHA GVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKI YRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGW EQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQAR LALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERN YPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSI VFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRS SLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLTILGWPLAERTVV IPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLK >ssDsbA-EPA-iGT-SITE1-6xHis – DsbA signal sequence and hexahistidine tag underlined, the ComP glycosylation fragment insertion site is located between the underlined, bolded amino acid residues (SEQ ID NO: 219) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIAD TNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQA RGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATF FVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVY NYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAH QACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALAS PGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVA AGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEE RGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEP DARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGP EEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPG KPPREDLKHHHHHH >ssDsbA-EPA-iGT-SITE2-6xHis – DsbA signal sequence and hexahistidine tag underlined, ComP glycosylation fragment insertion site is located between the underlined, bolded amino acid residues (SEQ ID NO: 220) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIAD TNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQA RGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATF FVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVY NYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAH QACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALAS PGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVA AGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEE 32080280 - 165 - Atty. Dkt. No.: 64100-234947 RGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEP DARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGP EEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPG KPPREDLKHHHHHH >ssDsbA-EPA-iGT-SITE3-6xHis – DsbA signal sequence and hexahistidine tag underlined, ComP glycosylation fragment insertion site is located between the underlined, bolded amino acid residues (SEQ ID NO: 221) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIAD TNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQA RGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATF FVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVY NYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAH QACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALAS PGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVA AGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEE RGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEP DARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGP EEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPG KPPREDLKHHHHHH > ssDsbA-EPA-iGT-SITE4-6xHis – DsbA signal sequence and hexahistidine tag underlined, ComP glycosylation fragment insertion site located between the underlined, bolded amino acid residues (SEQ ID NO: 222) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIAD TNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQA RGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATF FVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVY NYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAH QACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALAS PGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPV AAGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLE ERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQE PDARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITG PEEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQP GKPPREDLKHHHHHH > ssDsbA-EPA-iGT-SITE5-6xHis – DsbA signal sequence and hexahistidine tag underlined, ComP glycosylation fragment insertion site located between the underlined, bolded amino acid residues (SEQ ID NO: 223) MKKIWLALAGLVLAFSASAAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIAD TNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQA RGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATF FVRAHESNEMQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVY NYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAH QACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALAS PGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVA AGECAGPADSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEE RGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEP DARGRIRNGALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGP 32080280 - 166 - Atty. Dkt. No.: 64100-234947 EEEGGRLTILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPG KPPREDLKHHHHHH > PDB entity 4AE0 of CRM197 (SEQ ID NO: 224) GADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYDDDWKEFYST DNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKELGLSLTE PLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEINFETR GKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLDWDVIRDKTKTKIESLKEHGPI KNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSELKTVTGTNPVFAGANYAAWA VNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSIALSSLMV AQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPGHKTQPFLHDGYAVS WNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTIPGKLDVNKSKTHISVNGRKI RMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHRSSSEKIHSNEISSDSIGVLGYQ KTVDHTKVNSKLSLFFEIKS >ssFlgI-CRM197-iGT-SITE1-6xHis – FlgI signal sequence and hexahistidine tag underlined, ComP glycosylation fragment insertion site located between the underlined, bolded amino acid residues (SEQ ID NO: 225) MIKFLSALILLLVTTAAQAGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKP KSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVL ALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYI NNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLD WDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSEL KTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADG AVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRP AYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTI PGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHR SSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSHHHHHH > ssFlgI-CRM197-iGT-SITE2-6xHis – FlgI signal sequence and hexahistidine tag underlined, ComP glycosylation fragment insertion site located between the underlined, bolded amino acid residues (SEQ ID NO: 226) MIKFLSALILLLVTTAAQAGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKP KSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVL ALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYI NNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLD WDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSEL KTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADG AVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRP AYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTI PGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHR SSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSHHHHHH > ssFlgI-CRM197-iGT-SITE3-6xHis – FlgI signal sequence and hexahistidine tag underlined, ComP glycosylation fragment insertion site located between the underlined, bolded amino acid residues (SEQ ID NO: 227) MIKFLSALILLLVTTAAQAGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKP KSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVL ALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYI NNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLD 32080280 - 167 - Atty. Dkt. No.: 64100-234947 WDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSEL KTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADG AVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRP AYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTI PGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHR SSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSHHHHHH > ssFlgI-CRM197-iGT-SITE4-6xHis – FlgI signal sequence and hexahistidine tag underlined, ComP glycosylation fragment insertion site located between the underlined, bolded amino acid residues (SEQ ID NO: 228) MIKFLSALILLLVTTAAQAGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKP KSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVL ALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYI NNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLD WDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSEL KTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADG AVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRP AYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTI PGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHR SSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSHHHHHH > ssFlgI-CRM197-iGT-SITE5-6xHis – FlgI signal sequence and hexahistidine tag underlined, ComP glycosylation fragment insertion site located between the underlined, bolded amino acid residues (SEQ ID NO: 229) MIKFLSALILLLVTTAAQAGADDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKP KSGTQGNYDDDWKEFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVL ALKVDNAETIKKELGLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYI NNWEQAKALSVELEINFETRGKRGQDAMYEYMAQACAGNRVRRSVGSSLSCINLD WDVIRDKTKTKIESLKEHGPIKNKMSESPNKTVSEEKAKQYLEEFHQTALEHPELSEL KTVTGTNPVFAGANYAAWAVNVAQVIDSETADNLEKTTAALSILPGIGSVMGIADG AVHHNTEEIVAQSIALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRP AYSPGHKTQPFLHDGYAVSWNTVEDSIIRTGFQGESGHDIKITAENTPLPIAGVLLPTI PGKLDVNKSKTHISVNGRKIRMRCRAIDGDVTFCRPKSPVYVGNGVHANLHVAFHR SSSEKIHSNEISSDSIGVLGYQKTVDHTKVNSKLSLFFEIKSHHHHHH >PglS110264 (SEQ ID NO: 365) MNFLISKLKFYVLFIGIVCFCLTFILPNTSYFSSSLFKEIVVVLGFLILLTNQILSLKEIILP KKAPLLFILFLFLFLFLFFQYLFKLIISFQDLFFNLIYISVFFLSIIFGLNSKKYNQIILIHWI LFSLIFSALISFLIGLNQKIRIIESPYLFGVSYNGRATANLGQPNQLSTLTLMAFFSLFYL KKYYKINKLFFYSIIISLIFCNVLTQSRSAWLSVILISIFFITKFPDKKNVLSVFCLNLVF WLSTILIPFFFNYFYPIGNSYTTLDRMKLSSSRFDIWPQLFLATFDKPFLGYGAGQVGL AQIESISNVSTRGEWFTYSHNIFLDFVIWYGWIVGSLVSFFIISLLIKISKSDLNRNETYL FVIILVFFFHCLLEYPYSYFYFLIPIGIISGFLLKLKSDDIFVLKKMYLCIVVFLSWLLFTL FTYQLIELGEKKESYSLQYLFKSSVKPIQSNLFILDGYSEKLDIEYLDYCYLIKNKDKE FFRRVAYRYPSTVSVSKYYSTQSDNLKNAENIVQAYQVISNRVYQPHIKKCNN >PglSADP1 (SEQ ID NO: 366) MNSIFKKIKNYTIVSGVFFLGSAFIIPNTSNLSSTLYKELIAVLGLLILLTVKSFDYKKILI PKNFYWFLFVIFIIFIQLIVGEIYFFQDFFFSISFLVILFLSFLLGFNERLNGDDLIVKKIA WIFIIVVQISFLIAINQKIEIVQNFFLFSSSYNGRSTANLGQPNQFSTLILITLFLLCYLRE 32080280 - 168 - Atty. Dkt. No.: 64100-234947 KNSLNNMVFNILSFCLIFANVMTQSRSAWISVILISLLYLLKFQKKIELRRVIFFNIVFW TLVYCVPLLFNLIFFQKNSYSTFDRLTMGSSRFEIWPQLLKAVFHKPFIGYGWGQTGV AQLETINKSSTKGEWFTYSHNLFLDLMLWNGFFIGLIISILILCFLIELYSSIKNKSDLFL FFCVVAFFVHCLLEYPFAYTYFLIPVGFLCGYISTQNIKNSISYFNLSKRKLTLFLGCC WLGYVAFWVEVLDISKKNEIYARQFLFSNHVKFYNIENYILDGFSKQLDFQYLDYCE LKDKYQLLDFKKVAYRYPNASIVYKYYSISAEMKMDQKSANQIIRAYSVIKNQKIIKP KLKFCSIEY >PglSGFJ-2 (SEQ ID NO: 367) MINILNKFKDCLIIIGLGCLCLAFFLPNTSNFSSSLFKEFFAVLGFLFILTVQFFFLKKIV VPSKLFILFILFIFLFIQYVFNLIINFQDLFFNLIYISIFFLSIIFGLNSKKYNNSVLIHWILFS LIFSALVSFLISLNQKIRIIESPYLFCVSYNGRATANLGQPNQLSTLTLMAFFSLFYLKK YYKINKLFFYTIIISLIFCNVLTQSRSAWLSVILISTFFITKFPDKKNVLSVFCLNLVFWL STILIPFFFNYFYPIGNSYTTLDRMKLSSSRFDIWPQLFLATFDKPFLGYGAGQVGLAQI ESISNASTRGEWFTYSHNIFLDFVIWYGWIVGSLVSFFIISLLIKISKSDLNRNKTYLFIII LVFFFHCLLEYPYSYFYFLIPIGIISGFLLKLKSDGVFVLKKIYLCIVIFLSWLLFALFTY QLIELDEKKESYSLQYLFKSSVKPIQSNLFILDGYSEKLDIEYLDYCYLIKNRDKEFFR RVAYRYPSTVSVSKYYSTQSDNLKNAENIVQAYQVISNRVYQPNIKKCNN >PglS50v1 (SEQ ID NO: 368) MRLYLSFLLLGLSYLSPNSSLLWPNSLQDFFAILSLILLLLTFNLNNFLINKYLFLVFLL LISIPVIQYNLKIIYFKQELFLSCLYITIFFSSIFLGSSIHNSQKVFIKFSIFFLVIGVLCVLIQ IFQWIAVYSSIFINDLNSSRLSANIGQPNQLASLLSISLISCLILYKNKKIKVLIFSTCSVLI IFGIVLTQSRTSWLIFILIILFSYFKKNLKLTKYVTIFSTIFYGLLITYPFFYNSIHKKDISII QRLNSDYSRLDIWQQMLFAIIERPWFGYGWNQTSVAQTEISLYHTTSIWIEYSHNLFL DFLIWNGIPLGIILITIIIFWFIYMYVNIKDLNSFMILIIISSFFIHCLLEFPFAYAYFIFPIGL YIGIINKRYLKYNYFNFNNWNYIFGLIIIFLLFFIVKDYIKITEKHKEYSLKYFSDNSILP NKLDIYLLDSLNVKEDIQYLDICYLIKIYNSEEIRNNFLRYPTNKSAVSLYYISLYNKN VSLETISFMKWKFQNLDLNTLKINKRCNTL >PglS4466 (SEQ ID NO: 369) MSFYCYKYLNLFGIFLMGVAYFSTITLFFSTTFYKEIFAVAGLLFFLTALCFQYKIVST QLLLFNLALLLIPMIQYAFGIIFFLQDALLSTVYLCIFLCSILVGVNFKANHQTNILNIFL AMLVFVGCISVLMAFNQRFMWFNSYLLFSSSYGNRATANLAQPNQLSTLLIMSLFSL FYLYQAQKIKKIIMYGITFILLIGIVMTQSRSAWASCIVLSALYLYYYHQKQDIINVIKL NVVFIGLTLCIPFLLNVLTYSQASTAIDRLQGGSTRFKIWPQLLHAVMEQPWTGYGW GQVDVAQLSTMTPTSTKKELFTYSHNLFLDLLLWNGLVLGTLLSLLIIYILYRCYMNL QYKQDLLLFLGFMAFFVHSCLEYPYAYTYFLIPAGMFLGYVSYQQNIKEVLIQINKKL YVIFLILLCIIFGCFLIEVNHLNEKSDLYARQNLFHEKVDFNDQKFYFLDGYSTSLDFQ TIPYCNLVQYYPLITFKQIAYRYPSALTIAKYWLFSQKQQQMVRDAEQLRQAYVLLT KSGQHTFNNKVCN >PglSSFC (SEQ ID NO: 370) MQLILIILGLSYLNPNSFLPWPNAMQDFCAMVALILLTATQFIKKNIQINKNTFYLFLFI LSIPIIQFLFNILFFKQELFLSILYISIFFLSIIYGINQKEASNRIIKVSFFFVSVGIVCVFIQII QWTNIYYSPFILESNYLRPSANLGQPNNLATLLFICLFSNLYIFKNKKINTSFYISINIFII FGIALTQSRTSWIVFIALLILSHFKKELKLFKTIMINSILFFILVLITPYITLFYHGKGLTII ERINSDYSRLSIWKQIIIAITNKPLTGYGWNQTSVAQTQISLKYPIKVWLEYSHNMFLD ILVWTGIPIGLLIITLINKWLFKTYQNIKNTNQLIIFFIIISFFIHCMFEFPFAYAYFLIPVGI YIGFLNKQDYNIITINIFTILLFLLISTLLTIITIDYMVLSEKRNNYSTKYLFSKKISPLESN 32080280 - 169 - Atty. Dkt. No.: 64100-234947 IKILDALDLHNDILFLNDCYILKNKSIKNIKHIFYRYPTNKNIVIYYRFSLYYKNSSKEV IEYMKLKYPNFDSNQSKYNMCN >PglSP5312 (SEQ ID NO: 371) MPIFYFILGLSYLSPIFMQPWVSAFQDLCAIIAIILLMSIQSYRKNIEIDRRVLYVFGFIV CIPLVQYLFGILFFTQELVLSLIYISVFFLSIISGANFNRSYKNEEKLSFFFVFIGLSCVFIQ LIQWSGLYHSALILDSSSRRPFANIGQPNNLATLLFIGFFSNILLFKNNRLKAKFYFLIS AVLMTGIVLTQSRTSWLVFVSVLLLAFFKSKLELFSIMLKSSVLFFCLVLILPYITLFFH DQGLTVTERISSDSSRLYIWKQMLIAIMDKPWFGYGWNQTSVAQTSVTLKYPLDIWL EYSHNLFLDLIVWTGIPIGLSIIGIIIIWFLQTFKKINTLNQLLYFFIIAAFLIHCMLEYPFA YAYFLVPIGLYVGMLHQQLYETKNLKFKSLVITLVSILIITIIIISRDYFVLSDKRTIYTS ESLFSEQVKPAFSKVLVLDALDVNNDILFLNRCYVLKKNTIENFKSNFYRYPTRMNL VMYYKSTIYYEKNSRDAERYMTAWYPDYKQNLSQYDICS >ComPP5312 (SEQ ID NO: 207) MNAQKGFTLIELMIVIAIIGILAAIALPAYTDYTTRARVSEALTTASAMKATVSENIISK GGTSIDEDSACIGVATVGSDASAATKNVQKSVCDKGVITVTTTPDAKSVPLILTPSYS GDGVEWTCTTTADKKYVPAECR >ComP Δ28P5312 (SEQ ID NO: 215) MNAQKGFTLIELMIVIAIIGILAAIALPAYTDYTTRARVSEALTTASAMKATVSENIISK GGTSIDEDSACIGVATVGSDASAATKNVQKSVCDKGVITVTTTPDAKSVPLILTPSYS GDGVEWTCTTTADKKYVPAECR >PglSANT_H59 (SEQ ID NO: 372) MLIFYIMLGLSYLSPNIFLPWLNALQDLFAIFALIILVSKQSYRKDIEIDERVIYVFGLIA LIPLVQYLFGLLFFTQELVLSLIYISAFFLSIISGINLTKSFKEIEKISFSFIFISLSCVLLQLI QWSNIYHSALLLDSSSRRPFANIGQPNNLATLLFIGFFSNILLFKNNKIKIYLYLLVSAT LMTGIVLTQSRTSWLVFIAVLFITFLKKKLNLFSTMLKSSIAFLFLVLTLPYITLFFHDQ GLTVIERISSDSSRLYIWKQMLIAIIDKPWFGYGWNQTSVAQTSVTLKYPLNIWLEYS HNLFLDIIVWTGIPIGISIITIIIIWFLQTFKKINTPNQLIYFLIITAFFIHCMLEFPFAYAYF LLPVGLYVGILHQQVYETKNSKVKGLVMTIVTVLIVAVIIISRDYFLFNNKRTIYASKN LFSQQIQPISSKILLLNALDINNDILFLDECYVLKNNKFKVLRNSFYRYPTNKNLITYY KSAIYNNQNTQYPEKYMQKEYSNFKSSPAIYNNCSKL >ComPANT_H59 (SEQ ID NO: 208) MNTAQKGFTLIELMIVIAIIGILAAIAIPAYSDYTARARVTEAVTTASSMKATVSENIIS KGGTTIGAGSCAGVSLIGASNKTKNVLSSTCTDTTGVILVTTTADAKSVPLTLTPTYT GDAVTWKCTTTSDFTKYVPAECRPH >ComPΔ29ANT_H59 (SEQ ID NO: 216) MNTAQKGFTLIELMIVIAIIGILAAIAIPAYSDYTARARVTEAVTTASSMKATVSENIIS KGGTTIGAGSCAGVSLIGASNKTKNVLSSTCTDTTGVILVTTTADAKSVPLTLTPTYT GDAVTWKCTTTSDFTKYVPAECRPH iGTcc CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 230); iGTss STGVTQIASGASAATTNVASAQS (SEQ ID NO: 231); iGTccΔ0-1 CTGVTQIASGASAATTNVASAQ (SEQ ID NO: 232; iGTccΔ0-2 CTGVTQIASGASAATTNVASA (SEQ ID NO: 233); 32080280 - 170 - Atty. Dkt. No.: 64100-234947 32080280
Figure imgf000173_0001
Atty. Dkt. No.: 64100-234947 iGTccΔ3-8 VTQIASGASAAT (SEQ ID NO: 275); ID 32080280
Figure imgf000174_0001
Atty. Dkt. No.: 64100-234947 iGTccΔ7-1 ASGASAATTNVASAQ (SEQ ID NO: 316); iGTccΔ7-2 ASGASAATTNVASA (SEQ ID NO: 317); iGTccΔ7-3 ASGASAATTNVAS (SEQ ID NO: 318); iGTccΔ7-4 ASGASAATTNVA (SEQ ID NO: 319); iGTccΔ7-5 ASGASAATTNV (SEQ ID NO: 320); iGTccΔ7-6 ASGASAATTN (SEQ ID NO: 321); iGTccΔ7-7 ASGASAATT (SEQ ID NO: 322); iGTccΔ7-8 ASGASAAT (SEQ ID NO: 323); iGTccΔ7-9 ASGASAA (SEQ ID NO: 324); iGTccΔ7-10 ASGASA (SEQ ID NO: 325); iGTccΔ7-11 ASGAS (SEQ ID NO: 326); iGTccΔ8-0 SGASAATTNVASAQC (SEQ ID NO: 327); iGTccΔ8-1 SGASAATTNVASAQ (SEQ ID NO: 328); iGTccΔ8-2 SGASAATTNVASA (SEQ ID NO: 329); iGTccΔ8-3 SGASAATTNVAS (SEQ ID NO: 330); iGTccΔ8-4 SGASAATTNVA (SEQ ID NO: 331); iGTccΔ8-5 SGASAATTNV (SEQ ID NO: 332); iGTccΔ8-6 SGASAATTN (SEQ ID NO: 333); iGTccΔ8-7 SGASAATT (SEQ ID NO: 334); iGTccΔ8-8 SGASAAT (SEQ ID NO: 335); iGTccΔ8-9 SGASAA (SEQ ID NO: 336); iGTccΔ8-10 SGASA (SEQ ID NO: 337); iGTccΔ9-0 GASAATTNVASAQC (SEQ ID NO: 338); iGTccΔ9-1 GASAATTNVASAQ (SEQ ID NO: 339); iGTccΔ9-2 GASAATTNVASA (SEQ ID NO: 340); iGTccΔ9-3 GASAATTNVAS (SEQ ID NO: 341); iGTccΔ9-4 GASAATTNVA (SEQ ID NO: 342); iGTccΔ9-5 GASAATTNV (SEQ ID NO: 343); iGTccΔ9-6 GASAATTN (SEQ ID NO: 344); iGTccΔ9-7 GASAATT (SEQ ID NO: 345); iGTccΔ9-8 GASAAT (SEQ ID NO: 346); iGTccΔ9-9 GASAA (SEQ ID NO: 347); iGTccΔ10-0 ASAATTNVASAQC (SEQ ID NO: 348); iGTccΔ10-1 ASAATTNVASAQ (SEQ ID NO: 349); iGTccΔ10-2 ASAATTNVASA (SEQ ID NO: 350); iGTccΔ10-3 ASAATTNVAS (SEQ ID NO: 351); iGTccΔ10-4 ASAATTNVA (SEQ ID NO: 352); iGTccΔ10-5 ASAATTNV (SEQ ID NO: 353); iGTccΔ10-6 ASAATTN (SEQ ID NO: 354); iGTccΔ10-7 ASAATT (SEQ ID NO: 355); iGTccΔ10-8 ASAAT (SEQ ID NO: 356); 32080280 - 173 - Atty. Dkt. No.: 64100-234947 iGTccΔ11-0 SAATTNVASAQC (SEQ ID NO: 357); iGTccΔ11-1 SAATTNVASAQ (SEQ ID NO: 358); iGTccΔ11-2 SAATTNVASA (SEQ ID NO: 359); iGTccΔ11-3 SAATTNVAS (SEQ ID NO: 360); iGTccΔ11-4 SAATTNVA (SEQ ID NO: 361); iGTccΔ11-5 SAATTNV (SEQ ID NO: 362); iGTccΔ11-6 SAATTN (SEQ ID NO: 363); iGTccΔ11-7 SAATT (SEQ ID NO: 364). >ComP glycosylation fragment (Representative full-length ComP, non-limiting) (SEQ ID NO: 399) MNAQKGFTLIELMIVIAIIGILAAIAIPAYTDYTVRSRVTEGLTTASAMKATVSENIMN AGGTSMPSSGNCTGVTQIASGASAATTNVASAQCSDSDGVITVTMTDKAKGVSIKLT PSFSSTGSVGWKCTTSSDKKYVPSECRGT >PglS OTase (Representative, non-limiting) (SEQ ID NO: 400) MNSIFKKIKNYTIVSGVFFLGSAFIIPNTSNLSSTLYKELIAVLGLLILLTVKSFDYKKILI PKNFYWFLFVIFIIFIQLIVGEIYFFQDFFFSISFLVILFLSFLLGFNERLNGDDLIVKKIA WIFIIVVQISFLIAINQKIEIVQNFFLFSSSYNGRSTANLGQPNQFSTLILITLFLLCYLRE KNSLNNMVFNILSFCLIFANVMTQSRSAWISVILISLLYLLKFQKKIELRRVIFFNIVFW TLVYCVPLLFNLIFFQKNSYSTFDRLTMGSSRFEIWPQLLKAVFHKPFIGYGWGQTGV AQLETINKSSTKGEWFTYSHNLFLDLMLWNGFFIGLIISILILCFLIELYSSIKNKSDLFL FFCVVAFFVHCLLEYPFAYTYFLIPVGFLCGYISTQNIKNSISYFNLSKRKLTLFLGCC WLGYVAFWVEVLDISKKNEIYARQFLFSNHVKFYNIENYILDGFSKQLDFQYLDYCE LKDKYQLLDFKKVAYRYPNASIVYKYYSISAEMKMDQKSANQIIRAYSVIKNQKIIKP KLKFCSIEY TfpM glycosylation fragment (Representative, non-limiting) (SEQ ID NO: 401) AYQDYTIRAKISEGLTLSNGLKTAIAESFQSKGPSSMACTDATTCASIGASPMDATAL AGNKNVASITSDAAGVITIVYKPAVVPTGSNNLTLTPVGADGTTALNLSAAASAGSQ VNWRCGGTGTTVAAKFLPANCRGT >TfpM OTase (Representative, non-limiting) (SEQ ID NO: 402) MFNLIKHFKNKELFVALYFLLFIVGFSIYSFNFYNELRVLEVFLLIGLGLYGLIHKKNY FSKYEFCFLIFTLLGFLFWSNYSYIFYELILFYLLYKAFFNLNYNEFLSKLLIWLSFSIFL MLPIAITEFILSGVYPNWYPMPWNIRVYNSYFLVLSIFAVWFYLKQEKFKNIYLIFLFL AFLSILLDGGRSATLAYSVFIACVCIFNHAARFKLLFTYVATWLAYFSILFFSSHSASIV GSIVRESTSGRYELWVSAFQCWSQNPIFGCGFYQLDQYSGVISAHPHNLFIQVLTETG LIGFGFLILVIWNILKRIKWNLKENYFVISAFIAVSIDLSFSGIHIYPVTQVALLWLFVFL LKNPEFQHAASFHPSTYEASTVSQVFGYIAYISIALAFIYLFFYTSALSESLPSTPPRFW EYGYQLF >PglL glycosylation fragment (Representative, non-limiting) (SEQ ID NO: 403) MAEGQKSAVTEYYLNHGEWPGNNTSAGVATSSEIKGKYVKSVEVKNGVVTAQMAS SNVNNEIKGKKLSLWAKRQNGSVKWFCGQPVTRDNASADAVKADTAANGKQIDTK HLPSTAPTRKSTPN 32080280 - 174 - Atty. Dkt. No.: 64100-234947 >PglL OTase (Representative, non-limiting) (SEQ ID NO: 404) MPAETTVSGAHPAAKLPIYILPCFLWIGIVPFTFALKLKPSPDFYHDAAAAAGLIVLLF LTAGKKLFDVKIPAISFLLFAMAAFWYLQARLMNLIYPGMNDIVSWIFILLAVSAWA CRSLVAHFGQERIVTLFAWSLLIGSLLQSCIVVIQFAGWEDTPLFQNIIVYSGQGVIGHI GQRNNLGHYLMWGILAAAYLNGQRKIPAALGVICLIMQTAVLGLVNSRTILTYIAAI ALILPFWYFRSDKSNRRTMLGIAAAVFLTALFQFSMNTILETFTGIRYETAVERVANG GFTDLPRQIEWNKALAAFQSAPIFGHGWNSFAQQTFLINAEQHNIYDNLLSNLFTHSH NIVLQLLAEMGISGTLLVAATLLTGIAGLLKRPLTPASLFLICTLAVSMCHSMLEYPL WYVYFLIPFGLMLFLSPAEASDGIAFKKAANLGILTASAAIFAGLLHLDWTYTRLVN AFSPATDDSAKTLNRKINELRYISANSPMLSFYADFSLVNFALPEYPETQTWAEEATL KSLKYRPHSATYRIALYLMRQGKVAEAKQWMRATQSYYPYLMPRYADEIRKLPVW APLLPELLKDCKAFAAAPGHPEAKPCK >PglB glycosylation fragment X1 X2 N X3 X4, wherein X1 is D or E, X2 is any amino acid except proline, X3 is any amino acid except proline, and X4 is S or T >PglB OTase (Representative, non-limiting) (SEQ ID NO: 405) MLKKEYLKNPYLVLFAMIVLAYVFSVFCRFYWVWWASEFNEYFFNNQLMIISNDGY AFAEGARDMIAGFHQPNDLSYYGSSLSTLTYWLYKITPFSFESIILYMSTFLSSLVVIPII LLANEYKRPLMGFVAALLASVANSYYNRTMSGYYDTDMLVIVLPMFILFFMVRMIL KKDFFSLIALPLFIGIYLWWYPSSYTLNVALIGLFLIYTLIFHRKEKIFYIAVILSSLTLSN IAWFYQSAIIVILFALFALEQKRLNFMIIGILGSATLIFLILSGGVDPILYQLKFYIFRSDE SANLTQGFMYFNVNQTIQEVENVDFSEFMRRISGSEIVFLFSLFGFVWLLRKHKSMIM ALPILVLGFLALKGGLRFTIYSVPVMALGFGFLLSEFKAILVKKYSQLTSNVCIVFATI LTLAPVFIHIYNYKAPTVFSQNEASLLNQLKNIANREDYVVTWWDYGYPVRYYSDV KTLVDGGKHLGKDNFFPSFSLSKDEQAAANMARLSVEYTEKSFYAPQNDILKSDILQ AMMKDYNQSNVDLFLASLSKPDFKIDTPKTRDIYLYMPARMSLIFSTVASFSFINLDT GVLDKPFTFSTAYPLDVKNGEIYLSNGVVLSDDFRSFKIGDNVVSVNSIVEINSIKQGE YKITPIDDKAQFYIFYLKDSAIPYAQFILMDKTMFNSAYVQMFFLGNYDKNLFDLVIN SRDAKVFKLKI >PilA glycosylation fragment (Pa 1244) (Representative, non-limiting) (SEQ ID NO: 406) MKAQKGFTLIELMIVVAIIGILAAIAIPQYQDYTARTQVTRAVSEVSALKTAAESAILE GKEIVSSATPKDTQYDIGFTESTLLDGSGKSQIQVTDNQDGTVELVATLGKSSGSAIK GAVITVSRKNDGVWNCKITKTPTAWKPNYAPANCPKS >TfpO/PilO OTase (Representative, non-limiting) (SEQ ID NO: 407) MRIWLAWERMGRASRTILLLLISILLLSPVVYCGVSKNWHDQQRILQLVVLSGSSLLL LFSSRLSFARRMVQVTLLVILGLGSVSAFLSANPSWAFKEWSVFAGLMLFSFNISASP EWVRRIALWGVVVLGGFFCYQFLLSYLAAFVSGLRELNPRVLLSGFSNVRTMGQFQ AMLLPLMAALGLYLRETGRFRLSWLVMLLLAIQWCISFALAGRGLWLGFAVAHLAL CWIGPVGRRFLIVQLSAAFVGLALYFLLMVALPTWLGIDMTLMSGMRSGLSLRDVL WRDAWGMFVAHPLLGVGPMHFSAVPNSVGAHPHQMLLQWFAEWGGAAGLLVVG LMTLGLLRGARYLREQGDPMDAGLWLALVSVLVLAQVDGVFVMPFTQTVLALLVG IAMARWSKPVVPSPAQRWLCRGLAVVVIVVLGRVLLLEVPGLTAAEERYLEIHGGG EAPRFWIQGWIPM 32080280 - 175 - Atty. Dkt. No.: 64100-234947 >STT3 glycosylation fragment N X1 X2, wherein X1 is any amino acid except proline and X2 is S or T >STT3 OTase (Representative, non-limiting) (SEQ ID NO: 408) MGSDRSCVLSVFQTILKLVIFVAIFGAAISSRLFAVIKFESIIHEFDPWFNYRATKYLVN NSFYKFLNWFDDRTWYPLGRVTGGTLYPGLMTTSAFIWHALRNWLGLPIDIRNVCV LFAPLFSGVTAWATYEFTKEIKDASAGLLAAGFIAIVPGYISRSVAGSYDNEAIAITLL MVTFMFWIKAQKTGSIMHATCAALFYFYMVSAWGGYVFITNLIPLHVFLLILMGRYS SKLYSAYTTWYAIGTVASMQIPFVGFLPIRSNDHMAALGVFGLIQIVAFGDFVKGQIS TAKFKVIMMVSLFLILVLGVVGLSALTYMGLIAPWTGRFYSLWDTNYAKIHIPIIASV SEHQPVSWPAFFFDTHFLIWLFPAGVFLLFLDLKDEHVFVIAYSVLCSYFAGVMVRL MLTLTPVICVSAAVALSKIFDIYLDFKTSDRKYAIKPAALLAKLIVSGSFIFYLYLFVFH STWVTRTAYSSPSVVLPSQTPDGKLALIDDFREAYYWLRMNSDEDSKVAAWWDYG YQIGGMADRTTLVDNNTWNNTHIAIVGKAMASPEEKSYEILKEHDVDYVLVIFGGLI GFGGDDINKFLWMIRISEGIWPEEIKERDFYTAEGEYRVDARASETMRNSLLYKMSY KDFPQLFNGGQATDRVRQQMITPLDVPPLDYFDEVFTSENWMVRIYQLKKDDAQGR TLRDVGELTRSSTKTRRSIKRPELGLRV >AlgB glycosylation fragment N X1 X2, wherein X1 is any amino acid except proline and X2 is S or T >AlgB OTase (Representative, non-limiting) (SEQ ID NO: 409) MSDEQTKYSPSIAELARDWYHIPVLSTIILVMLWIRLRSYDAFIREGTVFFSGNDAWY HLRQVEYTVRNWPATMPFDPWTEFPFGRTAGQFGTIYDQLVATAALVVGLGSPSSD LVAKSLLVAPAVFGALTVIPTYLIGKRLGGRLGGLFGAVILMLLPGTFLQRGLVGFAD HNIVEPFFMGFAVLAIMIALTVADREKPVWELVAARDLDALREPLKWSVLAGVATAI YMWSWPPGILLVGIFGLFLVLKMASDYVRGRSPEHTAFVGAISMTVTGLLMFIPIEEP GFGVTDFGFLQPLFSLGVALGAVFLAALARWWESNDVDERYYPAVVGGTMLVGIV LFSLVLPSVFDSIARNFLRTVGFSAGAATRTISEAQPFLAANVLQSNGQTAVGRIMSE YGFTFFTGALAAVWLVAKPLVKGGNSRKIGYAVGSLALIGVLFLIPALPAGIGSALGV EPSLVSLTIVTALIVGAVMQADYESERLFVLVWAAIITSAAFTQVRFNYYLAVVVAV MNAYLLREALGIDFVGLANVERFDDISYGQVAAVVIAVLLILTPVLIIPIQLGNGGVSQ TAMQASQTGPGTVTQWDGSLTWMQNNTPAEGEFGGESNRMEYYGTYEYTDDFDY PDGAYGVMSWWDYGHWITVLGERIPNANPFQGGATEAANYLLAEDEQQAESVLTS MGDDGEGDQTRYVMVDWQMASTDAKFSAPTVFYDESNISRSDFYNPMFRLQEQGE QTTVAAASSLKDQRYYESLMVRLYAYHGSAREASPIVVDWEERTSADGSTTFRVTPS DGQAVRTFDNMSAAEEYVANDPTSQIGGIGTFPEERVSALEHYRLVKSSNSSALRSG SYQRSLISEGNTYGLQPQALVPNNPAWVKTFERVPGATVDGSGAPANTTVTARVQM RDLTTGTNFTYTQQAQTDADGEFTMTLPYSTTGYDEYGPDNGYTNVSVRAAGGYA FTGPTSVTGNSTIVSYQAENVAVDEGLVNGAEDGTVQVTLERNEQELDLPGDSSSED SSSEDGTSDGSQTNESASTSTSASVDASAVSAAA > N-linking glycosyltransferase sequon N X1 X2, wherein X1 is any amino acid and X2 is S or T >N-Linking Glycosyltransferase (Representative, non-limiting) (SEQ ID NO: 410) MENENKPNVANFEAAVAVKDYEKACSELLLILSQLDSNFGGIQEIEFEYPVQLQDLE QEKIVYFCTRMATAITTLFSDPVLEISDLGVQRFLVYQRWLALIFASSPFVNADHILQT YNREPNRKNSLEIHLDSSKSSLIKFCILYLPESNVNLNLDVMWNISPELCASLCFALQS PRFIGTSTAFNKRATILQWFPRHLDQLKNLNNIPSAISHDVYMHCSYDTSVNKHDVK 32080280 - 176 - Atty. Dkt. No.: 64100-234947 RALNHVIRRHIESEYGWKDRYVAHIGYRNNKPVMVVLLEHFHSAHSIYRTHSTSMIA AREHFYLIGLGSPSVDQAGQEVFDEFHLVAGDNMKQKLEFIRSVCESNGAAIFYMPSI GMDMTTIFASNTRLAPIQAIALGHPATTHSDFIEYVIVEDDYVGSEACFSETLLRLPKD ALPYVPSALAPEKVDYLLRENPEVVNIGIASTTMKLNPYFLEALKAIRDRAKVKVHF HFALGQSNGITHPYVERFIKSYLGDSATAHPHSPYHQYLRILHNCDMMVNPFPFGNT NGIIDMVTLGLVGVCKTGAEVHEHIDEGLFKRLGLPEWLIANTVDEYVERAVRLAEN HQERLELRRYIIENNGLNTLFTGDPRPMGQVFLEKLNAFLKEN >O-Linking Glycosyltransferase (Representative, non-limiting) (SEQ ID NO: 411) MTVYNINLGIGWASSGVEYAQAYRAQILRRIQQPAKFIFMDMILADNIQHLTENIGFL DEEIIWLYNYFTDIKIAPTTVTLDQVLAQVAGQPERSEKEGKIVRYFYPQDDQFITCYL RQEDQDSVEHVEYVSRGRLIRKDYFSYVRYASEYFAPHNDAATLYQRRFYHEDGSV AYDMLIEDGQEKLYRFPDRIFYSKAELVRYFLQCLQLQADDVVILDRETGIGQVVFE ESQKAKLGVVVHAEHFSENASSDDYILWNNFYDYQFTNADKVDFFIVATEAQKRILE QQFQHYSDKQPQIATIPVGSLDQLTYPKEPRKPYSMITASRLATEKHIDWLVAATVQ AHAQLPELTLDIYGKGSEEDKLRRRIEEAGAQDYIRLKGHADLSQIYAGYELYLTAST SEGFGLTLMEAVGSGLPLIGFDVRYGNQTFIDDGKNGYLLPVSSNHVEDQIIAAFVEK IIALFSQGRQQEMSQHSYQVAENYLTSRVEAAWTQLLKEVRDDSAL >ComP glycosylation fragment (Representative sequon, non-limiting) (SEQ ID NO: 412) CTGVTQIASGASAATTNVASAQC >PilMo pilin disulfide loop region (Representative, non-limiting) (SEQ ID NO: 413) CGGTGTTVAAKFLPANCRGT >PilE glycosylation fragment (Representative, non-limiting) (SEQ ID NO: 414) SAVTEYYLNHGEWPGNNTSAGVATSSEIK >PilA pilin glycosylation disulfide loop region (Representative, non-limiting) (SEQ ID NO: 415) CKITKTPTAWKPNYAPANCPKS >SpyTag (SEQ ID NO: 416) AHIVMVDAYKPTK >SpyTag002 (SEQ ID NO: 417) VPTIVMVDAYKRYK >SpyTag003 (SEQ ID NO: 418) RGVPHIVMVDAYKRYK >DogTag (SEQ ID NO: 419) PATYEFTDGKHYITNEPIPPK >SpyCatcher (SEQ ID NO: 420) VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWI SD GQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI 32080280 - 177 - Atty. Dkt. No.: 64100-234947 >SpyCatcher002 (SEQ ID NO: 421) VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWI SD GHVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGEATKGDAHT >SpyCatcher003 (SEQ ID NO: 422) VTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWI SD GHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT >DogCatcher (SEQ ID NO: 423) KLGEIEFIKVDKTDKKPLRGAVFSLQKQHPDYPDIYGAIDQNGTYQDVRTGEDGK LTFTNLSDGKYRLIENSEPPGYKPVQNKPIVSFRIVDGEVRDVTSIVPQ >TfpW_Pa5196 (SEQ ID NO: 424) MKSLWVYLQGGGKKNVLLWVGVLCAVVWGLYGRALFFGYVWDDSILFLDKTDLL NSSISWKVLSEPVLPGTSYLRPLVFLSFFVEFQIFGQSPFVSHAVNIAIFNLNVLLVFWL GSTLARHTERKYGLLLAFLAALFYAMHPALIESTVWASGRFDLLTTFFILAGLVVYL ADIKSTVLRAFLLCLCMAGALLSKELGIVFPVLLLCLWLAKECDCSTQRGGALLVQA LFRNGWMLVALIITAFGYLVVRKHSAGGIYHSPVGLSYIADVVLGQRLPLETLKFYL LQSFLPFQSINVSHPLNEVFERSVYVLLAGQMVVLILVLTVIYQALYRRSAAAWMFF AYLVCLLPVLHIIPLTIGENLGHERFLTAPLAFLALALVFVRYDRVLSKVGLSGAPLLR VGLVAGLLWLGMAGFTIYSILPMWTSDASLWAWAYKTHPESKYARYDYLYGALRG GYVELLDKEVSRLQKKYGGLEVPEQILYAHRLISAGDSEGLNYLEGVLQVLPRFHDA ENGRVALNDFKHLSSMQIGGAYAVYAEGLSMLRGDAERALKYNKIAEWYLATGEV IPLMYQRSAILYALGDYGQAARIYQDQEGLHYYMSQAVKKDMKRLVEGFCKEKGF PSDPCGELRERGVISKESEARQG >PilA_Pa5196 (SEQ ID NO: 425) MKAQKGFTLIELMIVVAIIGILAAVAIPAYQDYITRGQVTEAVSLGGGLKSPLAEYGA DKNAWPTLVAPTATPGAGQLNATLVGKYSSVDSTIASGYPNGQITVTMTQGKASGK KLTFSTQDGGSSWACGNASIDGFAGTGTTIDAKYLPNACKP >PilA_Pa5196_Strands_1_2 (SEQ ID NO: 426) GKYSSVDSTIASGYPNGQITVTMTQG >EPA-Spytag-v1 Sequence (MBP secretion signal: underlined, SpyTag: bolded, ComP glycosylation fragment: bolded and underlined, linked: italicized) (SEQ ID NO: 427) MKIKTGARILALSALTTMMFSASALAGGSRGVPHIVMVDAYKRYKSGGAEEAFDL WNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDN ALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELN AGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMA QAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNP AKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGY PVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAA ESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFL GDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRA RSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSSLPGFYRT GLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLTILGWPLAERTVVIPSAIPTDP RNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLKSGGGNCTGVTQIASGASAA TTNVASAQCGSGHHHHHH 32080280 - 178 - Atty. Dkt. No.: 64100-234947 >EPA-Spytag-v2 Sequence (MBP secretion signal: underlined, SpyTag: bolded, ComP glycosylation fragment: bolded and underlined) (SEQ ID NO: 428) MKIKTGARILALSALTTMMFSASALAGGSGNCTGVTQIASGASAATTNVASAQCSG GAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGND ALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSN IKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHA GVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKI YRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGW EQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQAR LALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERN YPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSI VFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRS SLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLTILGWPLAERTVV IPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLKSGGRGVPHIVM VDAYKRYKGSGHHHHHH >EPA-Spytag-v3 Sequence (MBP secretion signal: underlined, SpyTag: bolded, ComP glycosylation fragment: bolded and underlined) (SEQ ID NO: 429) MKIKTGARILALSALTTMMFSASALAGGSHHHHHHSGGGNCTGVTQIASGASAATT NVASAQCSGGAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHY SMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWL VPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNE MQPTLAISHAGVSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCN LDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETF TRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGE AIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPA DSGDALLERNYPTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGY HGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRN GALLRVYVPRSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLTI LGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLKG SGRGVPHIVMVDAYKRYK >EPA-Spytag Linker L1 SGG >EPA-Spytag Linker L2 (SEQ ID NO: 430) GGGGGG >EPA-Spytag Linker L3 (SEQ ID NO: 431) GGGGGGGG >EPA-Spytag Linker L4 (SEQ ID NO: 432) GGGGS >EPA-Spytag Linker L5 (SEQ ID NO: 433) EAAAK >EPA-Spytag Linker L6 (SEQ ID NO: 434) PAPAPPAPAP 32080280 - 179 - Atty. Dkt. No.: 64100-234947 >EPA-Spytag Linker L7 (SEQ ID NO: 435) EAAAKEAAAK >EPA-Spytag Linker L8 (SEQ ID NO: 436) GGGGSPAPAP >EPA-Spytag Linker L9 (SEQ ID NO: 437) GGGGSGGGGS >EPA-Spytag Linker L10 (SEQ ID NO: 438) EAAAKGGGGS >PilE glycosylation fragment (Representative, non-limiting) (SEQ ID NO: 439) WPGNNTSAGV >EPA-Spytag-v1 with Linker L7 Sequence (MBP secretion signal: underlined, SpyTag: bolded, ComP bolded and underlined, linker: italicized) (SEQ ID
Figure imgf000182_0001
EEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDAL KLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIK VFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAG VSVVMAQAQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIY RVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWE QLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARL ALTLAAAESERFVRQGTGNDEAGAASADVVSLTCPVAAGECAGPADSGDALLERNY PTGAEFLGDGGDISFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIV FGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVPRSS LPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLTILGWPLAERTVVI PSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPREDLKSGGGNCTGVTQI ASGASAATTNVASAQCGSGHHHHHH 32080280 - 180 - Atty. Dkt. No.: 64100-234947 REFERENCES Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109, E690-697 (2012). https://doi.org:10.1073/pnas.1115485109 Keeble, A. H. et al. Approaching infinite affinity through engineering of peptide-protein interaction. Proc Natl Acad Sci U S A 116, 26523-26533 (2019). https://doi.org:10.1073/pnas.1909653116 Keeble, A. H. & Howarth, M. Power to the protein: enhancing and combining activities using the Spy toolbox. Chem Sci 11, 7281-7291 (2020). https://doi.org:10.1039/d0sc01878c Brune, K. D. et al. Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization. Sci Rep 6, 19234 (2016). https://doi.org:10.1038/srep19234 Bruun, T. U. J., Andersson, A. C., Draper, S. J. & Howarth, M. Engineering a Rugged Nanoscaffold To Enhance Plug-and-Display Vaccination. ACS Nano 12, 8855-8866 (2018). https://doi.org:10.1021/acsnano.8b02805 Liu, Y., Pan, C., Wang, K. et al. Preparation of a Klebsiella pneumoniae conjugate nanovaccine using glycol-engineered Escherichia coli. Microb Cell Fact 22, 95 (2023). https://doi.org/10.1186/s12934-023-02099-x Szymanski, C. M., Yao, R., Ewing, C. P., Trust, T. J. & Guerry, P. Evidence for a system of general protein glycosylation in Campylobacter jejuni. Mol Microbiol 32, 1022-1030, doi:10.1046/j.1365-2958.1999.01415.x (1999). Wacker, M. et al. N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science 298, 1790-1793, doi:10.1126/science.298.5599.1790 (2002). Nothaft, H. & Szymanski, C. M. Protein glycosylation in bacteria: sweeter than ever. Nat Rev Microbiol 8, 765-778, doi:10.1038/nrmicro2383 (2010). Schaffer, C. & Messner, P. Emerging facets of prokaryotic glycosylation. FEMS Microbiol Rev 41, 49-91, doi:10.1093/femsre/fuw036 (2017). Harding, C. M. & Feldman, M. F. Glycoengineering bioconjugate vaccines, therapeutics, and diagnostics in E. coli. Glycobiology 29, 519-529, doi:10.1093/glycob/cwz031 (2019). Valvano, M. A. Export of O-specific lipopolysaccharide. Front Biosci 8, s452-471, doi:10.2741/1079 (2003). Hug, I. & Feldman, M. F. Analogies and homologies in lipopolysaccharide and glycoprotein biosynthesis in bacteria. Glycobiology 21, 138-151, doi:10.1093/glycob/cwq148 (2011). 32080280 - 181 - Atty. Dkt. No.: 64100-234947 Raetz, C. R. & Whitfield, C. Lipopolysaccharide endotoxins. Annu Rev Biochem 71, 635-700, doi:10.1146/annurev.biochem.71.110601.135414 (2002). Wacker, M. et al. Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems. Proc Natl Acad Sci U S A 103, 7088-7093, doi:10.1073/pnas.0509207103 (2006). Faridmoayer, A. et al. Extreme substrate promiscuity of the Neisseria oligosaccharyl transferase involved in protein O-glycosylation. J Biol Chem 283, 34596-34604, doi:10.1074/jbc.M807113200 (2008). Feldman, M. F. et al. Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli. Proc Natl Acad Sci U S A 102, 3016-3021, doi:10.1073/pnas.0500044102 (2005). Kowarik, M. et al. Definition of the bacterial N-glycosylation site consensus sequence. EMBO J 25, 1957-1966, doi:10.1038/sj.emboj.7601087 (2006). Faridmoayer, A., Fentabil, M. A., Mills, D. C., Klassen, J. S. & Feldman, M. F. Functional characterization of bacterial oligosaccharyltransferases involved in O-linked protein glycosylation. J Bacteriol 189, 8088-8098, doi:10.1128/JB.01318-07 (2007). Vik, A. et al. Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae. Proc Natl Acad Sci U S A 106, 4447-4452, doi:10.1073/pnas.0809504106 (2009). Pan, C. et al. Biosynthesis of Conjugate Vaccines Using an O-Linked Glycosylation System. MBio 7, e00443-00416, doi:10.1128/mBio.00443-16 (2016). Harding, C. M. et al. Acinetobacter strains carry two functional oligosaccharyltransferases, one devoted exclusively to type IV pilin, and the other one dedicated to O-glycosylation of multiple proteins. Mol Microbiol 96, 1023-1041, doi:10.1111/mmi.12986 (2015). Harding, C. M. et al. A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host. Nat Commun 10, 891, doi:10.1038/s41467-019-08869-9 (2019). Wacker, M. et al. Prevention of Staphylococcus aureus infections by glycoprotein vaccines synthesized in Escherichia coli. J Infect Dis 209, 1551-1561, doi:10.1093/infdis/jit800 (2014). Hatz, C. F. et al. Safety and immunogenicity of a candidate bioconjugate vaccine against Shigella dysenteriae type 1 administered to healthy adults: A single blind, partially randomized Phase I study. Vaccine 33, 4594-4601, doi:10.1016/j.vaccine.2015.06.102 (2015). Huttner, A. et al. Safety, immunogenicity, and preliminary clinical efficacy of a vaccine against extraintestinal pathogenic Escherichia coli in women with a history of recurrent urinary tract 32080280 - 182 - Atty. Dkt. No.: 64100-234947 infection: a randomised, single-blind, placebo-controlled phase 1b trial. Lancet Infect Dis 17, 528-537, doi:10.1016/S1473-3099(17)30108-1 (2017). Sun, P. et al. Design and production of conjugate vaccines against S. Paratyphi A using an O- linked glycosylation system in vivo. NPJ Vaccines 3, 4, doi:10.1038/s41541-017-0037-1 (2018). van den Dobbelsteen, G. et al. Immunogenicity and safety of a tetravalent E. coli O-antigen bioconjugate vaccine in animal models. Vaccine 34, 4152-4160, doi:10.1016/j.vaccine.2016.06.067 (2016). Geno, K. A. et al. Pneumococcal Capsules and Their Types: Past, Present, and Future. Clin Microbiol Rev 28, 871-899, doi:10.1128/CMR.00024-15 (2015). Pan, Y. J. et al. Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp. Sci Rep 5, 15573, doi:10.1038/srep15573 (2015). Berti, F. et al. Structure of the type IX group B Streptococcus capsular polysaccharide and its evolutionary relationship with types V and VII. J Biol Chem 289, 23437-23448, doi:10.1074/jbc.M114.567974 (2014). Feldman, M. F. et al. A promising bioconjugate vaccine against hypervirulent Klebsiella pneumoniae. Proc Natl Acad Sci U S A 116, 18655-18663, doi:10.1073/pnas.1907833116 (2019). Ihssen, J. et al. Production of glycoprotein vaccines in Escherichia coli. Microb Cell Fact 9, 61, doi:10.1186/1475-2859-9-61 (2010). Gerber, S. et al. Mechanism of bacterial oligosaccharyltransferase: in vitro quantification of sequon binding and catalysis. J Biol Chem 288, 8849-8861, doi:10.1074/jbc.M112.445940 (2013). Kay, E. J., Yates, L. E., Terra, V. S., Cuccui, J. & Wren, B. W. Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli. Open Biol 6, 150243, doi:10.1098/rsob.150243 (2016). Garcia-Quintanilla, F., Iwashkiw, J. A., Price, N. L., Stratilo, C. & Feldman, M. F. Production of a recombinant vaccine candidate against Burkholderia pseudomallei exploiting the bacterial N-glycosylation machinery. Front Microbiol 5, 381, doi:10.3389/fmicb.2014.00381 (2014). Berti, F. & Adamo, R. Antimicrobial glycoconjugate vaccines: an overview of classic and modern approaches for protein modification. Chem Soc Rev 47, 9015-9025, doi:10.1039/c8cs00495a (2018). 32080280 - 183 - Atty. Dkt. No.: 64100-234947 Goffin, P., Dewerchin, M., De Rop, P., Blais, N. & Dehottay, P. High-yield production of recombinant CRM197, a non-toxic mutant of diphtheria toxin, in the periplasm of Escherichia coli. Biotechnol J 12, doi:10.1002/biot.201700168 (2017). Wedekind, J. E. et al. Refined crystallographic structure of Pseudomonas aeruginosa exotoxin A and its implications for the molecular mechanism of toxicity. J Mol Biol 314, 823-837, doi:10.1006/jmbi.2001.5195 (2001). Harding, C. M., and Feldman, M. F. (2019) Glycoengineering bioconjugate vaccines, therapeutics, and diagnostics in E. coli. Glycobiology 29, 519-529. Nothaft, H., and Szymanski, C. M. (2010) Protein glycosylation in bacteria: sweeter than ever. Nature Reviews Microbiology 8, 765-778. Raetz, C. R. H., and Whitfield, C. (2002) Lipopolysaccharide Endotoxins. Annual Review of Biochemistry 71, 635-700. Faridmoayer, A., Fentabil, M. A., Haurat, M. F., Yi, W., Woodward, R., Wang, P. G., and Feldman, M. F. (2008) Extreme Substrate Promiscuity of the Neisseria Oligosaccharyl Transferase Involved in Protein O-Glycosylation. Journal of Biological Chemistry 283, 34596- 34604. DiGiandomenico, A., Matewish, M. J., Bisaillon, A., Stehle, J. R., Lam, J. S., and Castric, P. (2002) Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity. Molecular Microbiology 46, 519-530. Feldman, M. F., Wacker, M., Hernandez, M., Hitchen, P. G., Marolda, C. L., Kowarik, M., Morris, H. R., Dell, A., Valvano, M. A., and Aebi, M. (2005) Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 102, 3016. Kay, E. J., Yates, L. E., Terra, V. S., Cuccui, J., and Wren, B. W. (2016) Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli. Open Biology 6, 150243. Kay, E., Cuccui, J., and Wren, B. W. (2019) Recent advances in the production of recombinant glycoconjugate vaccines. npj Vaccines 4, 16. (2022) GSK 2022 Infectious Disease Pipeline. https://www.gsk.com/en-gb/research-and- development/our-pipeline/?infectious-diseases. (2022) Johnson & Johnson Infectious Diseases and Vaccines, Global Public Health Pipeline https://www.investor.jnj.com/pharmaceutical-pipeline-information. Schäffer, C., and Messner, P. (2017) Emerging facets of prokaryotic glycosylation. FEMS Microbiology Reviews 41, 49-91. Giltner Carmen, L., Nguyen, Y., and Burrows Lori, L. (2012) Type IV Pilin Proteins: Versatile Molecular Modules. Microbiology and Molecular Biology Reviews 76, 740-772. 32080280 - 184 - Atty. Dkt. No.: 64100-234947 Harvey, H., Bondy-Denomy, J., Marquis, H., Sztanko, K. M., Davidson, A. R., and Burrows, L. L. (2018) Pseudomonas aeruginosa defends against phages through type IV pilus glycosylation. Nature Microbiology 3, 47-52. Yakovlieva, L., Fülleborn, J. A., and Walvoort, M. T. C. (2021) Opportunities and Challenges of Bacterial Glycosylation for the Development of Novel Antibacterial Strategies. Frontiers in Microbiology 12. Marceau, M., Forest, K., Béretti, J.-L., Tainer, J., and Nassif, X. (1998) Consequences of the loss of O-linked glycosylation of meningococcal type IV pilin on piliation and pilus-mediated adhesion. Molecular Microbiology 27, 705-715. Willcocks, S. J., Denman, C., Cia, F., McCarthy, E., Cuccui, J., and Wren, B. W. (2020) Virulence of the emerging pathogen, Burkholderia pseudomallei, depends upon the O-linked oligosaccharyltransferase, PglL. Future Microbiology 15, 241-257. Nguyen, L. C., Taguchi, F., Tran, Q. M., Naito, K., Yamamoto, M., Ohnishi-Kameyama, M., Ono, H., Yoshida, M., Chiku, K., Ishii, T., Inagaki, Y., Toyoda, K., Shiraishi, T., and Ichinose, Y. (2012) Type IV pilin is glycosylated in Pseudomonas syringae pv. tabaci 6605 and is required for surface motility and virulence. Molecular Plant Pathology 13, 764-774. Tan Rommel, M., Kuang, Z., Hao, Y., Lee, F., Lee, T., Lee Ryan, J., Lau Gee, W., and McCormick, B. A. (2015) Type IV Pilus Glycosylation Mediates Resistance of Pseudomonas aeruginosa to Opsonic Activities of the Pulmonary Surfactant Protein A. Infection and Immunity 83, 1339-1346. Faridmoayer, A., Fentabil Messele, A., Mills Dominic, C., Klassen John, S., and Feldman Mario, F. (2007) Functional Characterization of Bacterial Oligosaccharyltransferases Involved in O-Linked Protein Glycosylation. Journal of Bacteriology 189, 8088-8098. Castric, P. (1995) pilO, a gene required for glycosylation of Pseudomonas aeruginosa 1244 pilin. Microbiology 141, 1247-1254. Horzempa, J., Comer, J. E., Davis, S. A., and Castric, P. (2006) Glycosylation Substrate Specificity of Pseudomonas aeruginosa 1244 Pilin. Journal of Biological Chemistry 281, 1128- 1136. Harvey, H., Habash, M., Aidoo, F., and Burrows Lori, L. (2009) Single-Residue Changes in the C-Terminal Disulfide-Bonded Loop of the Pseudomonas aeruginosa Type IV Pilin Influence Pilus Assembly and Twitching Motility. Journal of Bacteriology 191, 6513-6524. Qutyan, M., Henkel, M., Horzempa, J., Quinn, M., and Castric, P. (2010) Glycosylation of Pilin and Nonpilin Protein Constructs by Pseudomonas aeruginosa 1244. Journal of Bacteriology 192, 5972-5981. Harding, C. M., Nasr, M. A., Kinsella, R. L., Scott, N. E., Foster, L. J., Weber, B. S., Fiester, S. E., Actis, L. A., Tracy, E. N., Munson Jr, R. S., and Feldman, M. F. (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. Molecular Microbiology 96, 1023-1041. 32080280 - 185 - Atty. Dkt. No.: 64100-234947 Harding, C. M., Nasr, M. A., Scott, N. E., Goyette-Desjardins, G., Nothaft, H., Mayer, A. E., Chavez, S. M., Huynh, J. P., Kinsella, R. L., Szymanski, C. M., Stallings, C. L., Segura, M., and Feldman, M. F. (2019) A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host. Nature Communications 10, 891. Knoot, C. J., Robinson, L. S., and Harding, C. M. (2021) A minimal sequon sufficient for O- linked glycosylation by the versatile oligosaccharyltransferase PglS. Glycobiology 31, 1192- 1203. Comer Jason, E., Marshall Mark, A., Blanch Vincent, J., Deal Carolyn, D., and Castric, P. (2002) Identification of the Pseudomonas aeruginosa 1244 Pilin Glycosylation Site. Infection and Immunity 70, 2837-2845. Vik, Å., Aas, F. E., Anonsen, J. H., Bilsborough, S., Schneider, A., Egge-Jacobsen, W., and Koomey, M. (2009) Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae. Proceedings of the National Academy of Sciences 106, 4447. Hayes, A. J., Lewis, J. M., Davies, M. R., and Scott, N. E. (2021) Burkholderia PglL enzymes are Serine preferring oligosaccharyltransferases which target conserved proteins across the Burkholderia genus. Communications Biology 4, 1045. Porstendörfer, D., Gohl, O., Mayer, F., and Averhoff, B. (2000) ComP, a Pilin-Like Protein Essential for Natural Competence in Acinetobacter sp. Strain BD413: Regulation, Modification, and Cellular Localization. Journal of Bacteriology 182, 3673-3680. Dykxhoorn, D. M., St. Pierre, R., and Linn, T. (1996) A set of compatible tac promoter expression vectors. Gene 177, 133-136. Ruan, X., Loyola, D. E., Marolda, C. L., Perez-Donoso, J. M., and Valvano, M. A. (2012) The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases*. Glycobiology 22, 288-299. Musumeci, M. A., Faridmoayer, A., Watanabe, Y., and Feldman, M. F. (2014) Evaluating the role of conserved amino acids in bacterial O-oligosaccharyltransferases by in vivo, in vitro and limited proteolysis assays. Glycobiology 24, 39-50. Chick, J. M., Kolippakkam, D., Nusinow, D. P., Zhai, B., Rad, R., Huttlin, E. L., and Gygi, S. P. (2015) A mass-tolerant database search identifies a large proportion of unassigned spectra in shotgun proteomics as modified peptides. Nature Biotechnology 33, 743-749. Polasky, D. A., Yu, F., Teo, G. C., and Nesvizhskii, A. I. (2020) Fast and comprehensive N- and O-glycoproteomics analysis with MSFragger-Glyco. Nature Methods 17, 1125-1132. Lin, C.-w., Haeuptle, M. A., and Aebi, M. (2016) Supercharging Reagent for Enhanced Liquid Chromatographic Separation and Charging of Sialylated and High-Molecular-Weight Glycopeptides for NanoHPLC–ESI-MS/MS Analysis. Analytical Chemistry 88, 8484-8494. Liu, B., Furevi, A., Perepelov, A. V., Guo, X., Cao, H., Wang, Q., Reeves, P. R., Knirel, Y. A., Wang, L., and Widmalm, G. (2020) Structure and genetics of Escherichia coli O antigens. FEMS Microbiology Reviews 44, 655-683. 32080280 - 186 - Atty. Dkt. No.: 64100-234947 Curd, H., Liu, D., and Reeves Peter, R. (1998) Relationships among the O-Antigen Gene Clusters ofSalmonella enterica Groups B, D1, D2, and D3. Journal of Bacteriology 180, 1002- 1007. Whitfield, C., Perry, M. B., MacLean, L. L., and Yu, S. H. (1992) Structural analysis of the O- antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c). Journal of Bacteriology 174, 4913-4919. Pinto, V., and Berti, F. (2014) Exploring the Group B Streptococcus capsular polysaccharides: The structural diversity provides the basis for development of NMR-based identity assays. Journal of Pharmaceutical and Biomedical Analysis 98, 9-15. Geno, K. A., Gilbert Gwendolyn, L., Song Joon, Y., Skovsted Ian, C., Klugman Keith, P., Jones, C., Konradsen Helle, B., and Nahm Moon, H. (2015) Pneumococcal Capsules and Their Types: Past, Present, and Future. Clinical Microbiology Reviews 28, 871-899. Neal, B. L., Brown, P. K., and Reeves, P. R. (1993) Use of Salmonella phage P22 for transduction in Escherichia coli. Journal of Bacteriology 175, 7115-7118. Duke, J. A., Paschall, A. V., Robinson, L. S., Knoot, C. J., Vinogradov, E., Scott, N. E., Feldman, M. F., Avci, F. Y., and Harding, C. M. (2021) Development and Immunogenicity of a Prototype Multivalent Group B Streptococcus Bioconjugate Vaccine. ACS Infectious Diseases 7, 3111-3123. Clarke, B. R., Ovchinnikova, O. G., Kelly, S. D., Williamson, M. L., Butler, J. E., Liu, B., Wang, L., Gou, X., Follador, R., Lowary, T. L., and Whitfield, C. (2018) Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae. Journal of Biological Chemistry 293, 4666-4679. Kovach, M. E., Elzer, P. H., Steven Hill, D., Robertson, G. T., Farris, M. A., Roop, R. M., and Peterson, K. M. (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175-176. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., and Mann, M. (2006) In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat Protoc 1, 2856-2860. Ishihama, Y., Rappsilber, J., and Mann, M. (2006) Modular stop and go extraction tips with stacked disks for parallel and multidimensional Peptide fractionation in proteomics. J Proteome Res 5, 988-994. Rappsilber, J., Mann, M., and Ishihama, Y. (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2, 1896- 1906. Ahmad Izaham, A. R., Ang, C. S., Nie, S., Bird, L. E., Williamson, N. A., and Scott, N. E. (2021) What Are We Missing by Using Hydrophilic Enrichment? Improving Bacterial Glycoproteome Coverage Using Total Proteome and FAIMS Analyses. J Proteome Res 20, 599-612. 32080280 - 187 - Atty. Dkt. No.: 64100-234947 Rose, C. M., Rush, M. J., Riley, N. M., Merrill, A. E., Kwiecien, N. W., Holden, D. D., Mullen, C., Westphall, M. S., and Coon, J. J. (2015) A calibration routine for efficient ETD in large- scale proteomics. J Am Soc Mass Spectrom 26, 1848-1857. Lewis, J. M., Coulon, P. M. L., McDaniels, T. A., and Scott, N. E. (2021) The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides. J Vis Exp, Polasky, D. A., Yu, F., Teo, G. C., and Nesvizhskii, A. I. (2020) Fast and comprehensive N- and O-glycoproteomics analysis with MSFragger-Glyco. Nat Methods 17, 1125-1132. Kong, A. T., Leprevost, F. V., Avtonomov, D. M., Mellacheruvu, D., and Nesvizhskii, A. I. (2017) MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry- based proteomics. Nat Methods 14, 513-520. Brademan, D. R., Riley, N. M., Kwiecien, N. W., and Coon, J. J. (2019) Interactive Peptide Spectral Annotator: A Versatile Web-based Tool for Proteomic Applications. Mol Cell Proteomics 18, S193-S201. Perez-Riverol, Y., Csordas, A., Bai, J., Bernal-Llinares, M., Hewapathirana, S., Kundu, D. J., Inuganti, A., Griss, J., Mayer, G., Eisenacher, M., Perez, E., Uszkoreit, J., Pfeuffer, J., Sachsenberg, T., Yilmaz, S., Tiwary, S., Cox, J., Audain, E., Walzer, M., Jarnuczak, A. F., Ternent, T., Brazma, A., and Vizcaino, J. A. (2019) The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res 47, D442- D450. Perez-Riverol, Y., Alpi, E., Wang, R., Hermjakob, H., and Vizcaino, J. A. (2015) Making proteomics data accessible and reusable: current state of proteomics databases and repositories. Proteomics 15, 930-949. 32080280 - 188 -

Claims

Atty. Dkt. No.: 64100-234947 CLAIMS What is claimed is: 1. A fusion protein comprising: (i) a glycosylation fragment and (ii) a first polypeptide tag, wherein the first polypeptide tag can spontaneously form an isopeptide bond with a second polypeptide tag binding partner; optionally, wherein the glycosylation fragment is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, or 40 amino acids in length; optionally, wherein the glycosylation fragment is not more than 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, 40, 50, 60, 80, or 100 amino acids in length; optionally, wherein the glycosylation fragment is from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, or 30 amino acids in length to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 30, or 40 amino acids in length; and optionally, wherein the fusion protein comprises a carrier protein. 2. The fusion protein of Claim 1, wherein the fusion protein is a glycoconjugate comprising a saccharide covalently attached to the fusion protein via the glycosylation fragment; optionally, wherein the saccharide is covalently attached to the glycosylation fragment through an N-linkage, O-linkage, or C-linkage; and optionally, wherein said glycoconjugate is immunogenic. 3. The fusion protein of Claim 1, wherein the first polypeptide tag is translationally fused at the N-terminal or C-terminal end of the fusion protein. 4. The fusion protein of Claim 1, wherein the first polypeptide tag is translationally fused internally within the fusion protein; optionally, wherein the first polypeptide tag is translationally fused internally within the sequence of a carrier protein. 5. The fusion protein of Claim 1, wherein the glycosylation fragment is translationally fused at the N-terminal or C-terminal end of the fusion protein. 32080280 - 189 - Atty. Dkt. No.: 64100-234947 6. The fusion protein of Claim 1, wherein the glycosylation fragment is translationally fused internally within the fusion protein; optionally, wherein the glycosylation fragment is translationally fused internally within the sequence of a carrier protein. 7. The fusion protein of Claim 1, wherein the first polypeptide tag is a SpyTag (SEQ ID NO: 416), SpyTag002 (SEQ ID NO: 417), SpyTag003 (SEQ ID NO: 418), or a DogTag (SEQ ID NO: 419); optionally, wherein the SpyTag, Spytag002, or Spytag003 is translationally fused at the N-terminal or C-terminal end of the fusion protein, optionally, wherein the DogTag is translationally fused internally within the fusion protein. 8. The fusion protein of Claim 1, wherein the glycosylation fragment is a ComP glycosylation fragment; optionally, wherein the ComP glycosylation fragment comprises or consists of the amino acid sequence CTGVTQIASGASAATTNVASAQC (SEQ ID NO: 412) or a fragment thereof comprising the amino acids ASA, or a variant thereof comprising the amino acids ASA in positions 11-13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions; optionally, wherein the ComP glycosylation fragment comprises or consists of an amino acid sequence of:
Figure imgf000192_0001
32080280 - 190 - Atty. Dkt. No.: 64100-234947 iGTccΔ5-5 QIASGASAATTNV (SEQ ID NO: 296); iGTccΔ5-6 QIASGASAATTN (SEQ ID NO: 297); iGTccΔ6-5 IASGASAATTNV (SEQ ID NO: 308); or iGTccΔ6-6 IASGASAATTN (SEQ ID NO: 309), or a variant thereof comprising the amino acids ASA corresponding to positions 11-13 of SEQ ID NO: 412 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. 9. The fusion protein of Claim 1, wherein the glycosylation fragment is a TfpM- associated pilin glycosylation fragment; Optionally, wherein the TfpM-associated pilin glycosylation fragment comprises or consists of the PilMo pilin disulfide loop region (SEQ ID NO: 413) or a fragment thereof comprising at least the last three amino acids from the TfpM-associated pilin C-terminus; or a variant thereof comprising the last three amino acids from the TfpM-associated pilin C-terminus and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. 10. The fusion protein of Claim 1, wherein the glycosylation fragment is a PilE glycosylation fragment; optionally, wherein the PilE glycosylation fragment comprises or consists of the amino acid SAVTEYYLNHGEWPGNNTSAGVATSSEIK (SEQ ID NO: 414) or a fragment thereof comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414; or a variant thereof comprising at least the amino acids WPGNNTSAGV (SEQ ID NO: 439) in positions 13 to 22 of SEQ ID NO: 414 and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. 11. The fusion protein of Claim 1, wherein the glycosylation fragment is a PglB glycosylation fragment; optionally, wherein the PglB glycosylation fragment comprises or consists of the consensus motif amino acid sequence X1 X2 N X3 X4, wherein X1 is D or E, X2 is any amino acid except proline, X3 is any amino acid except proline, and X4 is S or T. 32080280 - 191 - Atty. Dkt. No.: 64100-234947 12. The fusion protein of Claim 1, wherein the glycosylation fragment is a PilA glycosylation fragment; optionally, wherein the PilA glycosylation fragment comprises or consists of the PilA pilin disulfide loop region (SEQ ID NO: 415) or a fragment thereof comprising at least the last three amino acids from the PilA C-terminus; or a variant thereof comprising at least the last three amino acids from the PilA terminus and having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. 13. The fusion protein of Claim 1, wherein the glycosylation fragment is a STT3 glycosylation fragment; optionally, wherein the STT3 glycosylation fragment comprises or consists of the consensus motif amino acid sequence N X1 X2, wherein X1 is any amino acid except proline and X2 is S or T. 14. The fusion protein of Claim 1, wherein the glycosylation fragment is a N-linking glycosyltransferase glycosylation fragment; optionally, wherein the N-linking glycosyltransferase glycosylation fragment comprises or consists of the consensus motif amino acid sequence N X1 X2, wherein X1 is any amino acid and X2 is S or T 15. The fusion protein of Claim 1, wherein the glycosylation fragment is an O-linking glycosyltransferase glycosylation fragment; optionally, wherein the O-linking glycosyltransferase glycosylation fragment comprises or consists of a fragment of the serine or threonine rich repeats from the serine-rich repeats (SRR) adhesins of streptococci or staphylococci bacteria; optionally, wherein the O-linking glycosyltransferase glycosylation fragment comprises or consists of serine or threonine rich repeats from the adhesin GspB from Streptococcus gordonii. 16. The fusion protein of Claim 1, wherein the fusion protein comprises two or more glycosylation fragments; 32080280 - 192 - Atty. Dkt. No.: 64100-234947 optionally, wherein the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 glycosylation fragments; optionally, wherein the fusion protein comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 glycosylation fragments to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 glycosylation fragments; optionally, wherein at least one glycosylation fragment is located at the N-terminal or C-terminal end of the fusion protein and at least one glycosylation fragment is located internally within the fusion protein; optionally, wherein at least two glycosylation fragments are located internally within the fusion protein; and/or optionally, wherein one glycosylation fragment is located at the N-terminal end of the fusion protein and where glycosylation fragment is located at the C-terminal end of the fusion protein. 17. The fusion protein of Claim 16, wherein the two or more glycosylation fragments are the same; wherein at least one of the two or more glycosylation fragments is different; or wherein each of the glycosylation fragments is different; optionally, wherein one glycosylation fragment is a ComP glycosylation fragment and wherein one glycosylation fragment is a TfpM-associated pilin glycosylation fragment; optionally, wherein the fusion protein is a glycoconjugate comprising two or more saccharide covalently attached to the fusion protein via the two or more glycosylation fragments, optionally, wherein the fusion protein comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 covalently attached saccharides; optionally, wherein the fusion protein comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 covalently attached saccharides to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 covalently attached saccharides; and optionally, wherein the two or more saccharides are the same; wherein at least one of the two or more saccharides is different; or 32080280 - 193 - Atty. Dkt. No.: 64100-234947 wherein each of the saccharides is different. 18. The fusion protein of Claim 1, wherein the fusion protein comprises a carrier protein, optionally, wherein the carrier protein isselected from the group consisting of Escherichia coli maltose binding protein, Pseudomonas aeruginosa Exotoxin A (EPA), Pseudomonas aeruginosa PcrV, CRM197, Haemophilus influenzae Protein D, cholera toxin B subunit, or tetanus toxin, and a fragment of any thereof. 19. A composition comprising a polypeptide pair that comprises a first polypeptide and a second polypeptide, wherein the first polypeptide is the fusion protein of any one of Claims 1 to 18, wherein the second polypeptide comprises a second polypeptide tag binding partner to the first polypeptide tag of the first polypeptide, and wherein the first polypeptide is attached to the second polypeptide via an isopeptide bond between the first polypeptide tag and the second polypeptide tag; optionally, where the second polypeptide comprises a monomeric polypeptide that can spontaneously multimerize/self-assemble into a higher-order, multimeric structure; and/or further optionally, wherein said higher-order, multimeric structure is an icosahedron or dodecahedron particle (e.g,. resembling nanocages), virus-like particle, or Adenoviral vector. 20. The polypeptide pair composition of Claim 19, wherein the second polypeptide comprises an Adenoviral capsid structural protein; wherein the second polypeptide comprises a coat protein of the bacteriophage AP205; wherein the second polypeptide comprises a fragment of the 2-keto-3-deoxy- phosphogluconate aldolase (i301); or wherein the second polypeptide comprises a fragment of a mutated 2-keto-3-deoxy- phosphogluconate aldolase (mi3). 21. The polypeptide pair composition of Claim 19, wherein the second polypeptide tag is a SpyCatcher (SEQ ID NO: 420), SpyCatcher002 (SEQ ID NO: 421), SpyCatcher003 (SEQ ID NO: 422), or a DogCatcher (SEQ ID NO: 423); 32080280 - 194 - Atty. Dkt. No.: 64100-234947 optionally, wherein the first polypeptide tag is a SpyTag and the second polypeptide tag is a SpyCatcher; wherein the first polypeptide tag is a SpyTag002 and the second polypeptide tag is a SpyCatcher002; wherein the first polypeptide tag is a SpyTag003 and the second polypeptide tag is a SpyCatcher003; or wherein the first polypeptide tag is a DogTag and the second polypeptide tag is a DogCatcher. 22. The polypeptide pair composition of Claim 19, wherein the second polypeptide tag is translationally fused at the N-terminal or C- terminal end of the second polypeptide, or wherein the second polypeptide tag is translationally fused internally within the second polypeptide. 23. The polypeptide pair composition of Claim 19, wherein the first polypeptide is a bioconjugate comprising a saccharide covalently attached to the glycosylation fragment of the first polypeptide; optionally, wherein said composition is immunogenic. 24. The polypeptide pair composition of Claim 19, further comprising an adjuvant and/or an excipient. 25. The polypeptide pair composition of Claim 19, wherein the composition is a pharmaceutical/therapeutic composition. 26. The polypeptide pair composition of Claim 19, wherein the composition is a conjugate vaccine. 27. A complex comprising two or more of the polypeptide pairs of Claim 19; 32080280 - 195 - Atty. Dkt. No.: 64100-234947 optionally, wherein the complex comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, or more complexed polypeptide pairs of any one of claims 19 to 26; optionally, wherein the complex comprises from any of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, or 400 complexed polypeptide pairs of any one of claims 19 to 26 to any of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400, or 500 complexed polypeptide pairs of any one of claims 19 to 26; and/or optionally, wherein the complex is a self-assembled, multimeric higher-order structure. 28. The complex of Claim 27, wherein said self-assembled, multimeric higher-order structure is an icosahedron or dodecahedron particle (e.g,. resembling nanocages), virus-like particle, or Adenoviral vector. 29. The complex of Claim 27, wherein all of the first polypeptides of the complex comprise the same fusion protein. 30. The complex of Claim 27, wherein at least two of the first polypeptides of the complex comprise different fusion proteins. 31. The complex of Claim 27, wherein at least one first polypeptide of the complex is a bioconjugate comprising a saccharide covalently attached to the glycosylation fragment of the first polypeptide; optionally, wherein at least about 5%, 10%, 25%, 50%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% of the first polypeptides of the complex are bioconjugates; optionally, wherein from any of about 5%, 10%, 25%, 50%, 75%, 80%, 90%, 95%, 97%, or 98% of the first polypeptides of the complex to about any of about 10%, 25%, 50%, 75%, 80%, 90%, 95%, 97%, 98%, or 99% of the first polypeptides of the complex are bioconjugates; optionally, wherein 100% of the first polypeptides of the complex are bioconjugates; optionally, wherein the complex is immunogenic. 32080280 - 196 - Atty. Dkt. No.: 64100-234947 32. The complex of Claim 27, wherein two or more of the first polypeptides of the complex are bioconjugates comprising a covalently attached saccharide; optionally, wherein the complex comprises at least 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 2,000, 2,500, or 5,000 covalently attached saccharides; optionally, wherein the complex comprises from any of 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 2,000, or 2,500 covalently attached saccharides to any of 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 2,000, 2,500, or 5,000 covalently attached saccharides; optionally, wherein all of the saccharides attached to the complex are the same; optionally, wherein at least one of the two or more saccharides attached to the complex is different; optionally, wherein there are at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33 different saccharides attached to the complex; optionally wherein each of the saccharides attached to the complex is different. 33. A pharmaceutical/therapeutic composition comprising the complex of Claim 27 and adjuvant and/or an excipient. 34. The complex of Claim 27 or the pharmaceutical/therapeutic composition of Claim 33, wherein the complex or composition is a conjugate vaccine. 35. A method of making the polypeptide pair of Claim 19, the method comprising contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag binding partner. 36. The method of Claim 35, further comprising glycosylating the first polypeptide with a saccharide before contact and isopeptide bond formation with the second polypeptide; 32080280 - 197 - Atty. Dkt. No.: 64100-234947 optionally, wherein the first polypeptide is glycosylated in vivo before contact and isopeptide bond formation with the second polypeptide; and optionally, wherein the method comprises isolating/purifying the in vivo glycosylated first polypeptide before contact and isopeptide bond formation with the second polypeptide. 37. The method of Claim 35, wherein the first polypeptide is glycosylated after contact and isopeptide bond formation with the second polypeptide. 38. A method of making the complex of Claim 27, the method comprising: (i) forming a self-assembled, multimeric higher-order structure of the second polypeptide and then contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag; or (ii) contacting the first polypeptide and the second polypeptide under conditions that allow the first polypeptide tag to spontaneously form an isopeptide bond with the second polypeptide tag and then forming of a self-assembled, multimeric higher-order structure of the second polypeptide. 39. The method of Claim 38, further comprising glycosylating the first polypeptide with a saccharide, optionally: wherein the first polypeptide is glycosylated before the isopeptide bond is formed between the first polypeptide and the second polypeptide; wherein the first polypeptide is glycosylated after the isopeptide bond is formed between the first polypeptide and the second polypeptide; wherein the first polypeptide is glycosylated before it is incorporated into a multimeric, higher-order structure; and/or wherein the first polypeptide is glycosylated after is has been incorporated into a multimeric, higher-order structure. 40. The method of claim of Claim 36, wherein the saccharide is transferred to the fusion protein by the action of an N-linking oligosaccharyltransferase (N-OTase), an O-linking oligosaccharyltransferase (O-OTase), an N-linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C-mannosyltransferase (CMT). 32080280 - 198 - Atty. Dkt. No.: 64100-234947 41. The method of Claim 36: wherein a ComP glycosylation fragment is glycosylated by a PglS OTase; wherein a TfpM-associated pilin glycosylation fragment is glycosylated by a TfpM OTase, optionally, wherein a ComP glycosylation fragment is glycosylated by a PglS OTase and a TfpM-associated pilin glycosylation fragment is glycosylated by a TfpM OTase; wherein a PilE glycosylation fragment is glycosylated by a PglL OTase; wherein a PglB glycosylation fragment is glycosylated by a PglB OTase; wherein a PilA glycosylation fragment is glycosylated by a TfpO or PilO OTase; wherein a STT3 glycosylation fragment is glycosylated by the STT3 catalytic subunit; wherein a PilA_Pa5196-associated pilin glycosylation fragment is glycosylated by a TfpW glycosyltransferase; wherein an N-linking glycosyltransferase glycosylation fragment is glycosylated by an N-linking glycosyltransferase from Actinobacillus pleuropneumoniae; from Haemophilus influenzae; or from Yersinia enterocolitica; and/or wherein an O-linking glycosyltransferase glycosylation fragment is glycosylated by GtfA/GtfB glycosyltransferases. 42. The method of Claim 36: wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a PglS OTase; wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpM OTase, optionally, wherein a saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a PglS OTase and another saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpM OTase; wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a PglL OTase; wherein the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using a PglB OTase; wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpO or PilO OTase; wherein the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using a STT3 OTase; 32080280 - 199 - Atty. Dkt. No.: 64100-234947 wherein the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using an AlgB OTase; wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using a TfpW glycosyltransferase; wherein the saccharide is covalently linked to a nitrogen atom within a glycosylation fragment using an N-linking glycosyltransferase; wherein the saccharide is covalently linked to an oxygen atom within a glycosylation fragment using an O-linking glycosyltransferase; wherein the saccharide is covalently linked to a carbon atom within a glycosylation fragment using a C-mannosyltransferase. 43. The method of Claim 42: wherein the saccharide is covalently linked to an oxygen atom within a ComP glycosylation fragment (e.g., SEQ ID NO: 412 or a variant thereof) using a PglS OTase (e.g., SEQ ID NO: 400); wherein the saccharide is covalently linked to an oxygen atom within a TfpM glycosylation fragment (e.g., SEQ ID NO: 413 or a variant thereof) using a TfpM OTase (e.g., SEQ ID NO: 402); wherein the saccharide is covalently linked to an oxygen atom within a PilE glycosylation fragment (e.g., SEQ ID NO: 414 or a variant thereof) using a PglL OTase (e.g., SEQ ID NO: 404); [0472] wherein the saccharide is covalently linked to a nitrogen atom within a PglB glycosylation fragment (e.g., X1 X2 N X3 X4, wherein X1 is D or E, X2 is any amino acid except proline, X3 is any amino acid except proline, and X4 is S or T) using a PglB OTase (e.g., SEQ ID NO: 405); wherein the saccharide is covalently linked to an oxygen atom within a PilA glycosylation fragment (e.g., SEQ ID NO: 415 or a variant thereof) using a TfpO/PilO OTase (e.g., SEQ ID NO: 407); wherein the saccharide is covalently linked to a nitrogen atom within a STT3 glycosylation fragment (e.g., N X1 X2, wherein X1 is any amino acid except proline and X2 is S or T) using a STT3 OTase (e.g., SEQ ID NO: 408); 32080280 - 200 - Atty. Dkt. No.: 64100-234947 wherein the saccharide is covalently linked to a nitrogen atom within an Archaeal AlgB glycosylation fragment (e.g., N X1 X2, wherein X1 is any amino acid except proline and X2 is S or T) using an AlgB OTase (e.g., SEQ ID NO: 409); wherein the saccharide is covalently linked to an oxygen atom within PilA_Pa5196- associated pilin glycosylation fragment (e.g., SEQ ID NO: 426 or a variant thereof) using a TfpW glycosyltransferase (e.g., SEQ ID NO: 424); wherein the saccharide is covalently linked to a nitrogen atom within a N-linking glycosyltransferase sequon (e.g., N X1 X2, wherein X1 is any amino acid and X2 is S or T) using an N-linking glycosyltransferase (e.g., SEQ ID NO: 410); wherein the saccharide is covalently linked to an oxygen atom within an O-linking glycosyltransferase sequon using an O-linking glycosyltransferase (e.g., SEQ ID NO: 411); wherein the saccharide is covalently linked to a carbon atom within a C- mannosyltransferase glycosylation fragment using a C-mannosyltransferase. 44. The method of Claim 35, wherein the method is a method of producing a conjugate vaccine. 45. A system comprising the first polypeptide and the second polypeptide of the composition of Claim 1; optionally, wherein the first polypeptide is a glycosylated bioconjugate; optionally, wherein the system comprises a multimeric, higher-order structure assembled of the second polypeptide; optionally, wherein the system comprises a saccharide and an N-linking oligosaccharyltransferase (N-OTase), an O-linking oligosaccharyltransferase (O-Otase), an N-linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C- mannosyltransferase (CMT). 46. An isolated nucleic acid encoding the first polypeptide and/or the second polypeptide of the composition of Claim 26. 47. A vector comprising the isolated nucleic acid of Claim 46. 48. A host cell comprising the vector of Claim 47. 32080280 - 201 - Atty. Dkt. No.: 64100-234947 49. A kit comprising two or more components comprising the fusion protein, the first polypeptide, the second polypeptide, a saccharide, an N-linking oligosaccharyltransferase (N- Otase), an O-linking oligosaccharyltransferase (O-Otase), an N-linking glycosyltransferase (NGT), an O-linking glycosyltransferase (OGT), and/or a C-mannosyltransferase (CMT), the bioconjugate, the multimeric, higher-order structure assembled from the second polypeptide, the isolated nucleic acid, the vector, and the host cell of any of the Claims above. 50. A method of eliciting an immune response in a subject by administering to said subject an effective amount of any composition, complex, and/or conjugate vaccine of any of the Claims above or a composition, complex, and/or conjugate vaccine of any of the Claims above for use in eliciting an immune response in a subject. 51. The fusion protein of Claim 1, wherein the glycosylation fragment is a PilA_Pa5196- associated pilin glycosylation fragment; optionally, wherein the PilA_Pa5196-associated pilin glycosylation fragment comprises or consists of the strands 1 and 2 of the antiparallel beta-sheet domain of PilA_Pa5196 (SEQ ID NO: 426), or a variant thereof having one, two, three, four, five, or six amino acid substitutions, additions, and/or deletions. 52. The fusion protein of Claim 1, wherein the glycosylation fragment comprises means for having a saccharide attached to the glycosylation fragment by a PglS OTase, a TfpM OTase, a PglL OTase, a PglB OTase, a TfpO/PilO OTase, a STT3 OTase, a AlgB OTase, a N-Linking Glycosyltransferase, and/or an O-Linking Glycosyltransferase. 53. The fusion protein in anyone of the above claims, comprising an amino acid linker, optionally, wherein the amino acid linker is selected from the group consisting of SGG, SEQ ID NO: 430, SEQ ID NO: 431, SEQ ID NO: 432, SEQ ID NO: 433, SEQ ID NO: 434, SEQ ID NO: 435, SEQ ID NO: 436, SEQ ID NO: 437, and SEQ ID NO: 438, optionally, wherein the amino acid linker in translationally fused immediately following a leader sequence, a polypeptide tag, a glycosylation fragment, a carrier protein, and or a polyhistidine tag, and/or 32080280 - 202 - Atty. Dkt. No.: 64100-234947 optionally, wherein the amino acid linker in translationally fused immediately preceding a leader sequence, a polypeptide tag, a glycosylation fragment, a carrier protein, and or a polyhistidine tag. 32080280 - 203 -
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