Synthesis of bioconjugate vaccines in prokaryotic cell lysates

Abstract

Methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins are disclosed, which can be used in vaccines, including antibacterial vaccines. Glycosylated proteins include bacterial polysaccharides conjugated to carriers, which can be used to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. Suitable carriers include, but are not limited to, Haemophilus influenzae protein D (PD), Neisseria meningitidysporin protein (PorA), Corynebacterium diphtheriae toxin (CRM197), Clostridium tetani toxin (TT), and Escherichia coli maltose-binding protein and variants thereof.

Description

[Technical field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with United States Government support under MCB1413563 awarded by the National Science Foundation. The United States Government has certain rights in this invention. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 791,425, filed January 11, 2019, which is incorporated by reference in its entirety.

[0003] The field of the invention relates to the in vitro synthesis of N-glycosylated proteins in prokaryotic cell lysates. In particular, the field of the invention relates to the use of N-glycosylated proteins synthesized in vitro in prokaryotic cell lysates as vaccine conjugates against pathogens such as bacteria. [Background technology]

[0004] background Conjugate vaccines are one of the safest and most effective methods for the prevention of life-threatening bacterial infections. Bioconjugate vaccines are a type of conjugate vaccine produced by protein glycan conjugation technology (PGCT), in which polysaccharide antigens are attached to genetically engineered carrier proteins by N-glycosylation using bacterial oligosaccharyl transferase (OST) in live Escherichia coli cells. Bioconjugate vaccines have the potential to significantly reduce the time and cost required to produce antibacterial vaccines. However, PGCT is limited by: i) the length of the in vivo process development schedule; and ii) the fact that FDA-approved carrier proteins, such as toxins from Clostridium tetani and Corynebacterium diptheriae, have not yet been demonstrated to be compatible with N-linked glycosylation in live E. coli. Here, we applied cell-free glycoprotein synthesis (CFGpS) technology to enable rapid in vitro production of bioconjugate vaccines against pathogenic strains of Escherichia coli and Franscicella tularensis in a reaction lasting 20 h. Due to the modular nature of the CFGpS system, this cell-free strategy could also be easily applied to produce bioconjugates with FDA-approved carrier proteins or booster vaccines against pathogenic bacteria whose surface antigen gene clusters are known. We showed that this system can be lyophilized and maintains bioconjugate synthesis capacity, demonstrating the potential for on-demand vaccine production and development in resource-poor environments. This work represents the first demonstration of bioconjugate vaccine production in E. coli lysate and has promising applications as a portable prototyping or manufacturing platform for antibacterial vaccine candidates. Summary of the Invention

[0005] overview Methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins are disclosed. The glycosylated proteins can be utilized in vaccines, including antibacterial vaccines. The glycosylated proteins include bacterial polysaccharides conjugated to a carrier, which can be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. Suitable carriers include, but are not limited to, Haemophilus influenzae protein D (PD), Neisseria meningitidis porin protein (PorA), Corynebacterium diptheriae toxin (CRM197), Clostridium tetani toxin (TT), and Escherichia coli maltose binding protein, or variants thereof. Glycosylated proteins may be synthesized in a cell-free glycoprotein synthesis (CFGpS) system using prokaryotic cell lysates enriched for components for glycoprotein synthesis, such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs), including OSTs and LLOs associated with the synthesis of bacterial O-antigens. Thus, prokaryotic cell lysates may be prepared from genetically engineered prokaryotes engineered to express heterologous OSTs and / or heterologous glycan synthesis pathways for the production of LLOs. The disclosed lysates may be described as modular and may be combined to prepare glycosylated proteins in the disclosed CFGpS system. [Brief description of the drawings]

[0006] [Figure 1]FIG. 1 is a diagram showing the interrelationships of the N-linked glycosylation pathway of C. jejuni expressed in E. coli from Guarino C., and DeLisa MP, Glycobiology, 2012 May 22(5):596-601, the contents of which are incorporated herein by reference in their entirety.

[0007] [Diagram 2] Strategies for the production of conjugate and bioconjugate vaccines from Ihssen et al., Microb. Cell. Fact. 2010 9:61, pages 1-13, the contents of which are incorporated herein by reference in their entirety. The schematic illustrates the production of an example vaccine against Franciscella tularensis.

[0008] [Diagram 3] Application of CFGpS technology for in vitro production of bioconjugate vaccines. Example of in vivo and in vitro workflow for production of anti-F. tularensis bioconjugates. The ability to produce bioconjugates in vitro allows for rapid prototyping of novel vaccine candidates.

[0009] [Figure 4]Rapid synthesis of glycoproteins carrying various bacterial O-antigens in mixed lysates CFGpS. S30 lysates were prepared from CLM24 cells expressing the C. jejuni OST (CjOST lysate), the Franciscella tularensis O-antigen (FtO-PS) biosynthetic pathway (FtLLO lysate), or the Escherichia coli O78 antigen (EcO78-PS) biosynthetic pathway (EcO78LLO lysate). (A) FtLLO lysate was mixed with CjOST lysate in CFGpS reactions containing either sfGFP21-DQNAT-6xHis or MBP-4xDQNAT-6xHis DNA templates. When both CjOST and FtLLO lysates were present in the CFGpS reaction, FtO-PS was covalently linked to R4-DQNAT and MBP-4xDQNAT, as indicated by the ladder-like pattern observed in the Western blot assay (lanes 2, 4). (B) EcO78LLO lysates were mixed with CjOST lysates in CFGpS reactions containing either sfGFP21-DQNAT-6xHis or MBP-4xDQNAT-6xHis DNA templates. When both CjOST and EcO78LLO lysates were present in the CFGpS reaction, EcO78-PS was covalently linked to sfGFP-21-DQNAT and MBP-4xDQNAT, as indicated by the ladder-like pattern observed in the Western blot assay (lanes 2, 4). The bioconjugate was also cross-reactive with a commercial antiserum against E. coli O78 strain (data not shown). These results demonstrate the modularity of LLO in the mixed lysate CFGpS and the potential of the CFGpS technology for the rapid synthesis of antibacterial vaccines. Abbreviations: CLM24 pSF CjOST; FtLLO lysate: CLM24 pGAB2; α-FtO-Antigen: FB11 mAb specific for F. tularensis O-antigen glycan.

[0010] [Diagram 5]Lyophilized CFGpS reactions for point-of-use vaccine production. (A) Scheme for portable bioconjugate production and distribution in resource-limited environments. (B) Lyophilized CFGpS reactions maintain bioconjugate synthesis activity. CFGpS reactions were prepared and contained either CjOST lysate or FtLLO lysate alone, or a mixture of both CjOST and FtLLO lysates. These reactions were then lyophilized and reconstituted with 15 μL of nuclease-free water. Premix reactions (lanes 1, 2, 5, 6) were run as is after reconstitution, while reactions containing CjOST lysate or FtLLO lysate alone were mixed after reconstitution (lanes 3, 4, 7). DQNAT sequons are synthesized and FtO-PS is attached to the target protein when both CjOST lysate or FtLLO lysate are present in the reaction (lanes 2, 4, 6, 7). Bioconjugate synthesis capacity was preserved after lyophilization, demonstrating the potential of CFGpS for portable bioconjugate manufacturing and distribution in resource-poor regions. Abbreviations: CjOST lysate: CLM24 pSF CjOST; FtLLO lysate: CLM24 pGAB2; α-FtO-Antigen: FB11 mAb specific for F. tularensis O-antigen glycan. Red (e.g., lanes 1, 3, and 5): αHis.

[0011] [Figure 6] Structure of O-antigens produced in selectively enriched S30 lysates. (A) Structure of F. tularensis Schu S4 O-antigen. (B) Structure of enterotoxigenic E. coli O78 antigen.

[0012] [Figure 7]Mixtures of E. coli lysates allow for rapid prototyping of various OST enzymes. (A) Immunoblot analysis of cell-free glycoprotein synthesis reactions performed using a lysate mixing strategy whereby CjLLO lysate from cells expressing the glycan biosynthetic pathway is mixed with CjPglB lysate from cells expressing CjPglB, and the resulting lysate mixture is prepared with plasmid DNA encoding either scFv13-R4DQNAT or sfGFP217-DQNAT. (B) Immunoblot analysis of reactions performed using CjLLO lysate mixed with extracts from cells expressing one of the following OSTs: CjPglB, CcPglB, DdPglB, DgPglB, or DvPglB. Mixed lysates were prepared with plasmid DNA encoding either sfGFP217-DQNAT (D) or sfGFP217-AQNAT (A). Blots were probed with anti-His antibody to detect acceptor protein (upper panel) and hR6 serum against C. jejuni glycan (lower panel). Arrows denote the aglycosylated (g0) and singly glycosylated (g1) forms of the acceptor protein. Molecular weight (MW) markers are indicated on the left. Results are representative of at least triplicate biological replicates. Figure taken from doi.org / 10.1038 / s41467-018-05110-x.

[0013] [Figure 8]Mixtures of E. coli lysates can be used to synthesize conjugate vaccines. Cell-free reactions performed using a lysate mixing strategy whereby LLO lysate from cells expressing the E. coli O78 polysaccharide antigen (EcO78-PS) biosynthetic pathway is mixed with OST lysate from cells expressing either PglB from C. jejuni or C. coli. The resulting lysate mixture is primed with plasmid DNA encoding either sfGFP217-DQNAT. Blots were probed with anti-His antibodies to detect the acceptor protein (left) and anti-EcO78-PS antiserum (right). We observed that either CjPglB or CcPglB successfully conjugated EcO78-PS to sfGFP217-DQNAT. Molecular weight (MW) markers are indicated on the left. An asterisk (*) indicates nonspecific binding of the EcO78-PS antiserum. Results are representative of at least triplicate biological repeats.

[0014] [Figure 9]Scale-up of cell-free glycosylation reactions with various oligosaccharide structures. Immunoblot analysis of in vitro glycosylation reaction products generated using organic solvent-extracted LLO from cells expressing the biosynthetic pathways for the following glycans: a. native C. lari hexasaccharide N-glycan; b. modified C. lari hexasaccharide N-glycan; c. native W. succinogenes hexasaccharide N-glycan; d. E. coli O9 "primer adaptor" Man3GlcNAc structure; e. F. tularensis O-PS Qui4NFm-(GalNAcAN)2-QuiNAc structure; and f. eukaryotic Man3GlcNAc2 N-glycan structure. Blots were probed with anti-His antibodies to detect the acceptor protein and one of the following: hR6 serum, which cross-reacts with native and modified C. lariglycan, or ConA lectin, which binds to internal and non-reducing terminal α-mannosyl groups of Man3GlcNAc and Man3GlcNAc2 glycans. FB11 antibody, which reacts specifically with the Ft-OPS structure. Because the structural determination of the N-glycan of W. succinogenes is currently incomplete and there are no antibodies available, protein products carrying this N-glycan were only probed with anti-His antibodies. Arrows denote the aglycosylated (g0) and singly glycosylated (g1) forms of the scFv13-R4DQNAT protein. Molecular weight (MW) markers are indicated on the left. Results are representative of at least triplicate biological repeats. Figure adapted from doi.org / 10.1038 / s41467-018-05110-x.

[0015] [Figure 10]The cell-free glycoprotein synthesis platform is modular and can be used to synthesize bioconjugates using a variety of bacterial O-polysaccharides. E. coli lysates were prepared from cells expressing CjPglB and either the (A) F. tularensis SchuS4 O-antigen, (B) E. coli O78 antigen, or (C) E. coli O7 antigen biosynthetic pathways and used to synthesize sfGFP217-DQNAT bioconjugates. In (A), anti-His signal is shown in red and anti-F. tularensis O-antigen signal is shown in green. In (B) and (C), the blot images are anti-His. Images are representative of at least triplicate biological repeats. Dashed lines indicate from the same blot with the same exposure.

[0016] [Figure 11]One-pot cell-free protein synthesis using extracts selectively enriched for OST and LLO. (a) Western blot analysis of scFv13-R4DQNAT or sfGFP217-DQNAT produced by cell-free glycosylation lysates selectively enriched for CjPglB and CjLLO. (b) Top - Ribbon representation of human erythropoietin (PDB code 1BUY) with α-helices and flexible loops colored in red and green, respectively. Glycosylation sites modeled by mutating the native sequon at N24 (22-AENIT-26), N38 (36-NENIT-40), or N83 (81-LVNSS-85) relative to DQNAT, with asparagine residues in the respective sequons colored in blue. Glycoengineered hEPO variants in which the native sequons of N24 (22-AENIT-26), N38 (36-NENIT-40), or N83 (81-LVNSS-85) were individually mutated to the optimal bacterial sequon, DQNAT (shown in blue). Bottom - Western blot analysis of hEPO glycovariants produced by cell-free glycosylation reaction lysates. Reactions were set up with plasmids pJL1-hEPO22-DQNAT-26 (N24), pJL1-hEPO36-DQNAT-40 (N38), or pJL1-hEPO81-DQNAT-85 (N83) as indicated. All control reactions (lane 1 of each panel) were performed using CjLLO enriched lysates lacking CjPglB. Blots were probed with anti-hexa-histidine antibody (anti-His) to detect acceptor proteins or with hR6 serum (anti-glycan) to detect N-glycans. Arrows denote aglycosylated (g0) and singly glycosylated (g1) forms of protein targets. Asterisks denote bands corresponding to non-specific serum antibody binding. Molecular weight (MW) markers are indicated on the left. Figure taken from Jaroentomeechai et al., Nat. Comm., July 12, 2018, doi.org / 10.1038 / s41467-018-05110-x, the contents of which are incorporated herein by reference in their entirety.

[0017] [Figure 12] On-demand and reproducible production of bioconjugates against F. tularensis using cell-free glycoprotein synthesis lysates. (a) Glycosylation reaction of sfGFP217-DQNAT with FtO-PS was observed only when CjPglB, FtO-PS, and the preferred sequon were present in the reaction (lane 3). No sfGFP217-DQNAT synthesis was observed when plasmid DNA was omitted. (b) On-demand synthesis of bioconjugate vaccines with immunostimulatory carriers, including carriers used in licensed vaccines. Bioconjugates were purified using Ni-NTA agarose from 1 mL cell-free glycoprotein synthesis reactions lasted for ~1 h at 30 °C. Dashed lines indicate samples are from the same blot with the same exposure. Molecular weight ladders are shown on the left of each image. Figures are from Jaroentomeechai et al., Nat. Comm., July 12, 2018, doi.org / 10.1038 / s41467-018-05110-x, the contents of which are incorporated by reference in their entirety.

[0018] [Figure 13] Crude lysates prepared from various E. coli strains are suitable for in vitro protein glycosylation. Lysates prepared from several chassis strains, including rE. coli 705, BL21(DE3), and CLM24, were used in reactions supplemented with purified CjPglB, organic solvent-extracted CjLLO, and primed with plasmid pJL1-scFv13-R4DQNAT. Efficient glycosylation reactions were observed with reactions continued for 16 h. Blots were probed with anti-hexa-histidine antibody (anti-His) to detect acceptor proteins or with hR6 serum (anti-glycan) to detect N-glycans. Arrows denote the aglycosylated (g0) and singly glycosylated (g1) forms of scFv13-R4DQNAT. Molecular weight (MW) markers are indicated on the left.

[0019] [Figure 14] Detoxified cell-free reactions yield bioconjugates. Lipid A reformulation does not affect glycosylation activity in cell-free glycoprotein synthesis reactions. Lysates were prepared from either wild-type CLM24, CLM24 ΔlpxM, or CLM24 ΔlpxM-expressing FtLpxE cells that also express CjPglB and FtO-PS. Near-identical glycosylation activity was observed for each of the lysates from the engineered strains. Images are representative of at least triplicate biological replicates. Figures are from Stark et al., bioRxiv, Jun. 24, 2019, doi.org / 10.1101 / 681841, the contents of which are incorporated herein by reference in their entirety.

[0020] [Figure 15] Schematic showing one embodiment of the iVAX platform that enables on-demand and portable production of antibacterial vaccines. The in vitro bioconjugate vaccine expression (iVAX) platform provides a rapid means to develop and distribute vaccines against bacterial pathogens. Expression of pathogen-specific polysaccharides (e.g., CPS, O-PS) and bacterial oligosaccharyltransferase enzymes in engineered nonpathogenic E. coli with detoxified lipid A results in a low-endotoxin lysate that contains all of the machinery required for the synthesis of bioconjugate vaccines. Reactions catalyzed by iVAX lysates can be used to produce bioconjugates containing approved carrier proteins and can be lyophilized without loss of activity for refrigeration-free transport and storage. Lyophilized reactions can be activated at the point of care by simple rehydration and used to reproducibly synthesize immunologically active bioconjugates within ∼1 hour.

[0021] [Figure 16]In vitro synthesis of approved conjugate vaccine carrier proteins. (a) All four carrier proteins used in the FDA approved conjugate vaccine were synthesized in soluble form in vitro as measured by 14C-leucine incorporation. These include H. influenzae protein D (PD), N. meningitidysporin protein (PorA), and genetically detoxified mutants of C. diphtheria toxin (CRM197) and C. tetani toxin (TT). Additional immunostimulatory carriers including E. coli maltose binding protein (MBP), and fragment C (TTc) and light chain (TTlight) domains of TT were also synthesized in soluble form. Values ​​represent the mean and error bars represent the standard deviation of biological replicates (n=3). (b) Full-length products were observed for all proteins tested via Western blot. Different exposures are indicated by solid lines. Molecular weight ladder is shown on the left. See also FIG. 21.

[0022] [Figure 17]Highly reproducible protein glycosylation with FtO-PS in iVAX lysates. (a) iVAX lysates were prepared from cells expressing CjPglB and the biosynthetic pathway encoding FtO-PS. (b) Glycosylation of sfGFP217-DQNAT with FtO-PS was observed only when CjPglB, FtO-PS, and the preferred sequon were present in the reaction (lane 3). No sfGFP217-DQNAT synthesis was observed when plasmid DNA was omitted. (c) Biological replicates of iVAX reactions producing sfGFP217-DQNAT using the same (left) or different (right) lots of iVAX lysates demonstrated the reproducibility of the reactions and lysate preparations. The top panel shows the signal from probing with an anti-hexa-histidine antibody (αHis) to detect the carrier protein, the middle panel shows the signal from probing with a commercial anti-FtO-PS antibody (αFtO-PS), and the bottom panel shows the combined αHis and αFtO-PS signals. Images are representative of at least triplicate biological replicates unless replicates are explicitly indicated. Dashed lines indicate that samples are from the same blot with the same exposure. Molecular weight ladders are shown to the left of each image. See also FIG. 22.

[0023] [Figure 18]On-demand production of bioconjugates against F. tularensis using iVAX. (a) iVAX reactions were prepared from lysates containing CjPglB and FtO-PS and primed with plasmids encoding immunostimulatory carriers, including those used in licensed vaccines. (b) On-demand synthesis of anti-F. tularensis bioconjugate vaccines for all carrier proteins tested was observed. Bioconjugates were purified using Ni NTA agarose from 1 mL iVAX reactions lasting ~1 h. The top panel shows the signal from probing with an anti-hexa-histidine antibody (αHis) to detect the carrier proteins, and the middle panel shows the signal from probing with a commercial anti-FtO-PS antibody (αFtO-PS), and the bottom panel shows the combined αHis and αFtO-PS signals. Images are representative of at least triplicate biological replicates. Dashed lines indicate that samples are from the same blot with the same exposure. A molecular weight ladder is shown to the left of each image, see also Figures 23 and 24.

[0024] [Figure 19]Detoxified, lyophilized iVAX reactions yield bioconjugates. (a) iVAX lysates were detoxified by deletion of expression of lpxM and F. tularensis LpxE in the source strain for lysate production. (b) The resulting lysates showed significantly reduced endotoxin activity as measured by two-tailed t-tests; *p=0.019 and **p=0.003. (c) iVAX reactions producing sfGFP217-DQNAT were run immediately or after lyophilization and rehydration. (d) Glycosylation activity was preserved after lyophilization, demonstrating the potential of iVAX reactions for portable biosynthesis of bioconjugate vaccines. The top panel shows the signal from probing with an anti-hexa-histidine antibody (αHis) to detect the carrier protein, the middle panel shows the signal from probing with a commercial anti-FtO-PS antibody (αFtO-PS), and the bottom panel shows the combined αHis and αFtO-PS signals. Images are representative of at least triplicate biological replicates. A molecular weight ladder is shown to the left of each image. See also FIG. 25.

[0025] [Figure 20]iVAX-derived bioconjugates raise FtLPS-specific antibodies in mice. (a) Lyophilized iVAX reactions assembled using detoxified lysates were used to synthesize anti-F. tularensis bioconjugates for immunization studies. (b) Six groups of BALB / c mice were immunized subcutaneously with PBS or 7.5 μg of purified, cell-free synthetic aglycosylated MBP4xDQNAT, FtO-PS-conjugated MBP4xDQNAT, aglycosylated PD4xDQNAT, or FtO-PS-conjugated PD4xDQNAT. FtO-PS-conjugated MBP4xDQNAT prepared with live E. coli cells using PCGT was used as a positive control. Each group consisted of six mice, except for the PBS control group, which consisted of five mice. Mice were immunized with identical doses of antigen on days 21 and 42. FtLPS-specific IgG titers were measured by ELISA at endpoint (day 70) on individual mouse sera (black dots) using fixed F. tularensis LPS as antigen. The mean titers for each group are also shown (red line). The iVAX-derived bioconjugates produced significantly higher levels of FtLPS-specific IgG compared to all other groups (**p<0.01, Tukey-Kramer HSD). (c) IgG1 and IgG2a subtype titers measured by ELISA from endpoint sera revealed that the iVAX-derived bioconjugates stimulated the production of FtO-PS-specific IgG1 compared to all other groups tested (**p<0.01, Tukey-Kramer HSD). These results indicate that the iVAX bioconjugates produced a Th2-biased immune response that is typical of most conjugate vaccines. Values ​​represent the mean and error bars represent the standard error of FtLPS-specific IgGs detected by ELISA. See also Figure 26.

[0026] [Figure 21]In vitro synthesis of approved conjugated vaccine carrier proteins is possible over a range of temperatures and can be easily optimized. See also Figure 16. (a) All carriers expressed with similar solubility, except CRM197, were obtained at 25°C, 30°C, and 37°C, as measured by 14C-leucine incorporation. Values ​​represent the mean and error bars represent the standard deviation of biological replicates (n=3). (b) Soluble expression of PorA was improved with the addition of lipid nanodiscs to the reaction. (c) Expression of full-length TT was enhanced by (i) performing in vitro protein synthesis under oxidizing conditions to improve assembly of disulfide-bonded heavy and light chains in full-length TT, and (ii) running the reaction for only 2 hours to minimize protease degradation. (d) CRM197 and (e) TT arising in the CFPS reaction are detected with α-DT and αTT antibodies, respectively, and compared in size to commercially purified DT and TT protein standards (50 ng of standards were added). Images are representative of at least triplicate biological replicates. Dashed lines indicate that samples are from the same blot with the same exposure. A molecular weight ladder is shown to the left of each image.

[0027] [Figure 22]Glycosylation in iVAX reactions occurs within 1 hour across a range of temperatures. See also Figure 17. The kinetics of FtO-PS glycosylation reactions at 30°C (left), 37°C, 25°C, and room temperature (~21°C) (right) are compared and show that protein synthesis and glycosylation occur within the first hour of the iVAX reaction. These results demonstrate that the iVAX platform synthesizes bioconjugates across a range of permissive temperatures. The top panel shows the signal from probing with an anti-hexa-histidine antibody (αHis) to detect the carrier protein, and the middle panel shows the signal from probing with a commercial anti-FtO-PS antibody (αFtO-PS), and the bottom panel shows the combination of αHis and αFtO-PS signals. Images are representative of at least triplicate biological replicates. A molecular weight ladder is shown to the left of each image.

[0028] [Diagram 23] Production of bioconjugates against F. tularensis using live E. coli PGCT. See also Figure 18. (a) Bioconjugates were produced by PGCT in CLM24 cells expressing CjPglB, the biosynthetic pathway of FtO-PS, and a panel of immunostimulatory carriers including those used in licensed vaccines. (b) We observed low level expression of PorA, membrane proteins, and reduced glycan addition and conjugation of high molecular weight FtO-PS species in all carriers compared to iVAX-derived samples. The top panel shows the signal from probing with an anti-hexa-histidine antibody (αHis) to detect carrier proteins, and the middle panel shows the signal from probing with a commercial anti-FtO-PS antibody (αFtO-PS), and the bottom panel shows the combined αHis and αFtO-PS signals. Images are representative of at least triplicate biological replicates. Molecular weight ladders are shown on the left side of each image.

[0029] [Figure 24]The iVAX platform is modular and can be used to synthesize a variety of clinically relevant outputs of bioconjugates. See also Figure 18. (a) Protein synthesis and glycosylation reactions using FtO-PS were measured in iVAX reactions yielding MBP4xDQNAT and PD4xDQNAT. After ~1 h, the reactions yielded ~40 μg mL-1 of protein as measured by 14C-leucine incorporation, of which ~20 μg mL-1 was glycosylated with FtO-PS as measured by densitometry. Values ​​represent the mean and error bars represent the standard error of biological replicates (n=2). To demonstrate modularity, iVAX lysates were prepared from cells expressing CjPglB and the biosynthetic pathway of either (b) E. coli O78 antigen or (c) E. coli O7 antigen and used to synthesize bioconjugates of PD4xDQNAT (left) or sfGFP217-DQNAT (right). The structure and composition of the repeating monomeric unit of each antigen are shown. Both polysaccharide antigens are compositionally and, in the case of O7 antigen, structurally distinct compared to F. tularensis O-antigen. Blots show signals from probing with an anti-hexa-histidine antibody (αHis) to detect the carrier protein. When commercially available anti-O-PS serum or antibodies were available, they were used to confirm the identity of the conjugated O-antigen (α-EcO78 blot, panel b). Asterisks denote bands resulting from nonspecific serum antibody binding. Images are representative of at least triplicate biological replicates. The dashed lines indicate that the samples were from the same blot with the same exposure. A molecular weight ladder is shown to the left of each image.

[0030] [Diagram 25]Detoxified iVAX lysates reproducibly scaled for bioconjugate and both lysate production and lyophilization reactions. See also FIG. 19. (a) iVAX lysates containing CjPglB and FtO-PS were prepared from wild-type CLM24, CLM24 ΔlpxM, or CLM24 ΔlpxM cells expressing FtLpxE. Near-identical sfGFP217-DQNAT glycosylation was observed for each of the lysates from the engineered strains. (b) To generate material for immunizations, fermentations to produce endotoxin-edited iVAX lysates were scaled from 0.5 L to 10 L. We observed similar levels of sfGFP217-DQNAT glycosylation for lysates from 0.5 L to 10 L cultures and across different batches of lysate resulting from the 10 L fermentation. (c) For immunization, we prepared two lots of FtO-PS-conjugated MBP4xDQNAT and PD4xDQNAT from 5 mL lyophilized iVAX reactions. We observed similar levels of purified protein (~200 μg) and FtO-PS modification (>50%, as measured by densitometry) across both carrier and bulk materials. The top panel shows the signal from probing with an anti-hexa-histidine antibody (αHis) to detect carrier protein, and the middle panel shows the signal from probing with a commercial anti-FtO-PS antibody (αFtO-PS), and the bottom panel shows the combined αHis and αFtO-PS signals. Images are representative of at least triplicate biological replicates. A molecular weight ladder is shown on the left.

[0031] [Figure 26]FtLPS-specific antibody titers in vaccinated mice over time. See also FIG. 20. Six groups of BALB / c mice were immunized subcutaneously with PBS or 7.5 μg of purified, cell-free synthetic aglycosylated MBP4xDQNAT, FtO-PS-conjugated MBP4xDQNAT, aglycosylated PD4xDQNAT, or FtO-PS-conjugated PD4xDQNAT. FtO-PS-conjugated MBP4xDQNAT prepared in live E. coli cells using PCGT was used as a positive control. Each group consisted of six mice, except for the control group, which consisted of five mice. Mice were challenged with the same dose of antigen on days 21 and 42. FtLPS-specific IgG titers were measured by ELISA in sera collected on days −1, 35, 49, 63, and 70 after the initial immunization. The iVAX-derived bioconjugates produced significantly higher levels of FtLPS-specific IgG compared to the PBS control group in serum collected on days 35, 49, and 70 of the study (**p<0.01, Tukey-Kramer HSD). Values ​​represent the mean and error bars represent the standard error of FtLPS-specific IgGs detected by ELISA.

[0032] [Figure 27] Table 1 shows a cost analysis for the iVAX reaction. A 1 mL iVAX reaction produces two 10 μg vaccine doses and can be assembled for $11.75. In Table 1, amino acid costs comprise 2 mM of each of 20 standard amino acids purchased individually from Sigma. Lysate costs are based on a single employee producing 50 mL of lysate from a 10 L fermentation, assuming 30 lysate batches per year and a 5 year equipment life. Ingredient sources are also included in the table if it is possible to purchase directly from a supplier. Homemade ingredients must be prepared following the procedures described in the methods section as they cannot be purchased directly.

[0033] [Figure 28]Table 2 shows the primers used to construct and verify the CLM24 ΔlpxM strain. KO primers were used to amplify the kanamycin resistance cassette from pKD4 with homology to lpxM. Seq primers were used for colony PCR and sequencing confirmation of the knockout strain.

[0034] [Figure 29] Table 3 lists the plasmids used in the examples.

[0035] [Diagram 30] Table 4 lists the antibodies and antisera used in the examples.

[0036] [Diagram 31]iVAX-derived vaccines are efficacious against lethal F. tularensis challenge. (a) Groups of five BALB / c mice were immunized intraperitoneally with PBS or 7.5 μg of purified, cell-free synthetic aglycosylated MBP4xDQNAT, PD4xDQNAT, EPADNNNS-DQNRT, or FtO-PS-conjugated MBP4xDQNAT, PD4xDQNAT, or EPADNNNS-DQNRT. FtO-PS-conjugated MBP4xDQNAT, PD4xDQNAT, and EPADNNNS-DQNRT prepared in live E. coli cells using PCGT were used as positive controls. Mice were challenged with identical doses of antigen on days 21 and 42. On day 66, mice were challenged intranasally with 6000 cfu (50 times the intranasal LD50) of F. tularensis subgenus holarctica live vaccine strain Rocky Mountain Laboratories and observed for survival over an additional 25 days. Kaplan-Meier curves for immunization with (b) MBP4xDQNAT, (c) PD4xDQNAT, and (d) EPADNNNS-DQNRT as carrier proteins are shown. These results indicate that iVAX-derived vaccines protect mice from lethal pathogen challenge as effectively as vaccines synthesized using state-of-the-art PGCT approaches (*p=0.0476, **p=0.0079, Fisher's exact test; ns: not significant). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description The invention is described herein using several definitions, which are set forth below and throughout the application.

[0038] definition

[0039] The disclosed subject matter may be further described using the following definitions and terminology: The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0040] As used in this specification and claims, the singular forms "a," "an," and "the" include the plural forms unless the content clearly dictates otherwise. For example, the terms "gene" or "oligosaccharide" should be construed to mean "one or more genes" and "one or more oligosaccharides," respectively, unless the content clearly dictates otherwise. As used herein, the term "plurality" means "two or more."

[0041] As used herein, the terms "about," "approximately," "substantially," and "significantly" are understood by those of ordinary skill in the art and have some degree of variability depending on the context in which they are used. Where there are uses of a term that are not clear to those of ordinary skill in the art, given the context in which they are used, "about" and "approximately" will mean ±10% or less of the particular term, and "substantially" and "significantly" will mean more than ±10% of the particular term.

[0042] The terms "include" and "including" used in the English specification have the same meaning as "comprise" and "comprising". The terms "comprise" and "comprising" should be interpreted as "open" transitional phrases that allow the addition of additional components to the components recited in the claims. The terms "consist" and "consisting of" should be interpreted as "closed" transitional phrases that do not allow the inclusion of additional components other than the components recited in the claims. "Consisting essentially of" should be interpreted as partially open, allowing the inclusion of only additional components that do not fundamentally change the nature of the subject matter of the invention recited in the claims.

[0043] The phrase "such as" should be interpreted as "including, for example." Moreover, the use of any and all exemplary language, including, but not limited to, "such as," is intended only to facilitate a better understanding of the invention and does not pose limitations on the scope of the invention unless otherwise asserted.

[0044] Moreover, in instances where a convention similar to "at least one of A, B, and C, etc." is used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together). It will be further understood by those of ordinary skill in the art that virtually any disjunction and / or phrase expressing two or more alternative terms, whether in the description or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A or B" or "A and B."

[0045] All language such as "up to," "at least," "greater than," and "less than" refers to a range, inclusive of the recited numbers, which may be subsequently divided into subranges as discussed above. The range includes each individual member. Thus, for example, a group having 1 to 3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, etc.

[0046] The modal verb "may" refers to a preferred use or selection of one or more alternatives, or a choice among several described embodiments or features contained within the same. When no alternatives or choices are disclosed with respect to a particular embodiment or feature contained within the same, the modal verb "may" refers to a positive action regarding how to make or use an aspect of the described embodiment or feature contained within the same, or a definitive decision to use a specific technique with respect to the described embodiment or feature contained within the same. In this latter context, the modal verb "may" has the same meaning and connotation as the modal verb "can."

[0047] As used herein, the terms "bind" and "binding", "interact" and "occupy" refer to covalent, non-covalent and steric interactions. A covalent interaction is a chemical bond between two atoms or radicals formed by the sharing of an electron pair (single bond), two sets of electron pairs (double bond) or three sets of electron pairs (triple bond). Covalent interactions are also known in the art as electron pair interactions or electron pair bonds. Non-covalent interactions include, but are not limited to, van der Waals interactions, hydrogen bonds, weak chemical bonds (due to short-range non-covalent forces), hydrophobic interactions, ionic bonds, etc. A review of binding interactions can be found in Alberts et al., in Molecular Biology of the Cell, 3d edition, Garland Publishing, 1994. Steric interactions are generally understood to include those where the structure of the compound allows the moiety to be available by virtue of its three-dimensional structure, as opposed to any attractive forces between the compound and that moiety.

[0048] Polynucleotides and Synthesis

[0049] The terms "nucleic acid" and "oligonucleotide" as used herein refer to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base. No distinction in length is intended between the terms "nucleic acid", "oligonucleotide" and "polynucleotide". These terms are used interchangeably. These terms refer only to the primary structure of the molecule. Thus, these terms include double-stranded and single-stranded DNA, as well as double-stranded and single-stranded RNA. For use in the present invention, oligonucleotides can also include nucleotide analogs modified in the base, sugar or phosphate backbone, as well as non-purine or non-pyrimidine nucleotide analogs.

[0050] Oligonucleotides can be prepared by any suitable method, including direct chemical synthesis by methods such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68:109-151; the diethyl phosphoramidite method of Beaucage et al., 1981, Tetrahedron Letters 22:1859-1862; and the solid support method of U.S. Patent No. 4,458,066, each of which is incorporated herein by reference. A review of methods for synthesizing oligonucleotide and modified nucleotide conjugates is given in Goodchild, 1990, Bioconjugate Chemistry 1(3): 165-187, which is incorporated herein by reference.

[0051] The term "amplification reaction" refers to any chemical reaction, including an enzymatic reaction, that results in more copies of a template nucleic acid sequence or results in transcription of a template nucleic acid. Amplification reactions include reverse transcription, polymerase chain reaction (PCR), including real-time PCR (see U.S. Pat. Nos. 4,683,195 and 4,683,202; see PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)), and ligase chain reaction (LCR) (see Barany et al., U.S. Pat. No. 5,494,810). Exemplary "amplification reaction conditions" or "amplification conditions" typically include either two-step or three-step cycles. Two-step cycles include a high temperature denaturation step, followed by a hybridization / extension (or ligation) step. Three-step cycles include a denaturation step, followed by a hybridization step, followed by a separate extension or ligation step.

[0052] The terms "target," "target sequence," "target region," and "target nucleic acid," as used herein, are synonymous and refer to a region or sequence of a nucleic acid that is to be amplified, sequenced, or detected.

[0053] The term "hybridization" as used herein refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between completely complementary nucleic acid strands or between "substantially complementary" nucleic acid strands that contain minor regions of mismatch. Conditions under which hybridization of completely complementary nucleic acid strands is highly favorable are referred to as "stringent hybridization conditions" or "sequence-specific hybridization conditions." Stable duplexes of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of mismatch allowed can be controlled by appropriately adjusting the hybridization conditions. Those skilled in the art of nucleic acid technology can empirically determine duplex stability following guidelines provided by the art (see, e.g., Sambrook et al., 1989, Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York; Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26(3 / 4):227-259; and Owczarzy et al., 2008, Biochemistry, 47: 5336-5353, which are incorporated herein by reference), taking into account several variables including, for example, the length and base pair composition of the oligonucleotide, ionic strength, and the frequency of mismatched base pairs.

[0054] The term "primer" as used herein refers to an oligonucleotide that can act as an initiation point for DNA synthesis under appropriate conditions, including conditions that allow synthesis of a primer extension product complementary to a nucleic acid strand to be induced in the presence of four different nucleoside triphosphates and an agent for extension (e.g., DNA polymerase or reverse transcriptase), in a suitable buffer, and at a suitable temperature.

[0055] The primer is preferably a single-stranded DNA. The suitable length of the primer depends on the intended use of the primer, but typically ranges from about 6 to about 225 nucleotides, including intermediate regions such as 15 to 35 nucleotides, 18 to 75 nucleotides, and 25 to 150 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template. The primer does not need to reflect the exact sequence of the template nucleic acid, but must be sufficiently complementary to hybridize with the template. The design of suitable primers for amplification of a desired target sequence is well known in the art and described in the literature cited herein.

[0056] Primers may incorporate additional features that allow for detection or immobilization of the primer, but do not change the basic property of the primer, which is to act as an initiation point for DNA synthesis. For example, a primer may not hybridize to the target nucleic acid, but may facilitate cloning or detection of the amplification product, or may direct the transcription of RNA (e.g., by inclusion of a promoter) or a 5'-UTR or poly(A) such as an Internal Ribosome Entry Site (IRES). n The primer may contain additional nucleic acid sequences at its 5' end that allow for translation of the protein (by inclusion of a 3'-UTR element such as the sequence (where n is in the range of about 20 to about 200). The region of the primer that is sufficiently complementary to the template to hybridize is referred to herein as the hybridizing region.

[0057] As used herein, a primer is "specific" for a target sequence if, when used in an amplification reaction under sufficiently stringent conditions, the primer hybridizes primarily to the target nucleic acid. Typically, a primer is specific for a target sequence if the stability of the primer-target duplex is greater than the stability of the duplex formed between the primer and any other sequence found in the sample. Those skilled in the art will recognize that various factors, such as salt conditions and primer base composition and mismatch position, affect the specificity of a primer, and routine experimental confirmation of primer specificity is required in many cases. Hybridization conditions can be selected that allow the primer to form a stable duplex only with the target sequence. Thus, the use of target-specific primers under appropriately stringent amplification conditions allows selective amplification of target sequences that contain target primer binding sites.

[0058] As used herein, "polymerase" refers to an enzyme that catalyzes the polymerization of nucleotides. "DNA polymerase" catalyzes the polymerization of deoxyribonucleotides. Known DNA polymerases include, for example, Pyrococcus furiosus (Pfu) DNA polymerase, E. coli DNA polymerase, T7 DNA polymerase, and Thermus aquaticus (Taq) DNA polymerase, among others. "RNA polymerase" catalyzes the polymerization of ribonucleotides. The above examples of DNA polymerases are also known as DNA-dependent DNA polymerases. RNA-dependent DNA polymerases are also within the scope of DNA polymerases. Reverse transcriptase (including viral polymerases encoded by retroviruses) is an example of an RNA-dependent DNA polymerase. Known examples of RNA polymerases ("RNAP") include, for example, T3 RNA polymerase, T7 RNA polymerase, SP6 RNA polymerase, and E. coli RNA polymerase, among others. The above examples of RNA polymerases are also known as DNA-dependent RNA polymerases. The polymerase activity of any of the above enzymes can be measured by means well known in the art.

[0059] The term "promoter" refers to a cis-acting DNA sequence that directs RNA polymerase and other trans-acting transcription factors to initiate RNA transcription from a DNA template that contains that cis-acting DNA sequence.

[0060] As used herein, the term "sequence-defined biopolymer" refers to a biopolymer having a specific primary sequence. A sequence-defined biopolymer can correspond to a genetic code-defined biopolymer when a gene encodes a biopolymer having a specific primary sequence.

[0061] As used herein, "expression template" or "nucleic acid template" refers to a nucleic acid that serves as a substrate for transcribing at least one RNA that can be translated into a polypeptide or protein. Expression templates include nucleic acids composed of DNA or RNA. Suitable sources of DNA for use as nucleic acid for expression templates include genomic DNA, cDNA, and RNA that can be converted into cDNA. Genomic DNA, cDNA, and RNA can be from any biological source, such as tissue samples, biopsies, swabs, sputum, blood samples, stool samples, urine samples, exfoliation, among others. Genomic DNA, cDNA, and RNA can be from host cell or viral origin, and can be from any species, including extant and extinct organisms. As used herein, "expression template", "nucleic acid template" and "transcription template" have the same meaning and are used interchangeably.

[0062] In certain exemplary embodiments, vectors are provided, such as expression vectors, that include a nucleic acid encoding one or more rRNA or reporter polypeptides and / or proteins described herein. As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be attached. Such vectors are also referred to herein as "expression vectors." In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably. However, the disclosed methods and compositions are intended to include other forms of expression vectors, such as viral vectors (e.g., repeat-defective retroviruses, adenoviruses, and adeno-satellite viruses), that serve equivalent functions.

[0063] In certain exemplary embodiments, a recombinant expression vector comprises a nucleic acid sequence (e.g., a nucleic acid sequence encoding one or more rRNAs or reporter polypeptides and / or proteins described herein) in a form suitable for expression of the nucleic acid sequence in one or more of the methods described herein, which means that the recombinant expression vector comprises one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed. In the context of a recombinant expression vector, "operably linked" is intended to mean that a nucleotide sequence encoding one or more rRNAs or reporter polypeptides and / or proteins described herein is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription and / or translation system). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

[0064] Oligonucleotides and polynucleotides may optionally include one or more non-standard nucleotides, nucleotide analogs, and / or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, S 2T, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosyl enosine, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5 -Methoxyaminomethyl-2-thiouracil, β-D-mannosyl quenosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, quenosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, 2,6-diaminopurine, etc. Nucleic acid molecules may also be modified at the base moiety (e.g., at one or more atoms normally available that can form a hydrogen bond with a complementary nucleotide and / or at one or more atoms that are not normally capable of forming a hydrogen bond with a complementary nucleotide), the sugar moiety, or the phosphate backbone.

[0065] As used herein, "deletion" refers to the removal of one or more nucleotides relative to a native polynucleotide sequence. The modified strains disclosed herein may include a deletion in one or more genes (e.g., a deletion in gmd and / or a deletion in waaL). Preferably, the deletion results in a non-functional gene product. As used herein, "insertion" refers to the addition of one or more nucleotides relative to a native polynucleotide sequence. The modified strains disclosed herein may include an insertion in one or more genes (e.g., an insertion in gmd and / or an insertion in waaL). Preferably, the deletion results in a non-functional gene product. As used herein, "substitution" refers to the replacement of a nucleotide of a native polynucleotide sequence with a nucleotide that is not native to the polynucleotide sequence. The modified strains disclosed herein may include a substitution in one or more genes (e.g., a substitution in gmd and / or a substitution in waaL). Preferably, the substitution results in a non-functional gene product, for example, if the substitution introduces a premature stop codon (e.g., TAA, TAG, or TGA) in the coding sequence of the gene product. In some embodiments, the engineered strains disclosed herein may contain more than one substitution, for example, if the substitution introduces multiple premature stop codons (e.g., TAATAA, TAGTAG, or TGATGA).

[0066] In some embodiments, the engineered strains disclosed herein may be engineered to contain and express one or more heterologous genes. As understood in the art, a heterologous gene is a gene that does not naturally occur in the engineered strain when the strain occurs in nature. A gene that is heterologous to E. coli is a gene that does not occur in E. coli and may be a gene that occurs naturally in another microorganism (e.g., a gene from C. jejuni) or does not naturally occur in other known microorganisms (i.e., a man-made gene).

[0067] Peptides, Polypeptides, Proteins, and Synthetic Methods

[0068] As used herein, the terms "peptide," "polypeptide," and "protein" refer to molecules comprising polymeric chains of amino acid residues joined by amide linkages. The term "amino acid residue" includes, but is not limited to, amino acid residues included in the group consisting of alanine (Ala or A), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term "amino acid residue" may also include non-standard or unnatural amino acids. The term "amino acid residue" may include α-, β-, γ-, and δ-amino acids.

[0069] In some embodiments, the term "amino acid residue" may include non-standard or unnatural amino acid residues included in the group consisting of homocysteine, 2-aminoadipic acid, N-ethylasparagine, 3-aminoadipic acid, oxylysine, β-alanine, β-amino-propionic acid, allo-oxylysine acid, 2-aminobutyric acid, 3-hydroxyproline, 4-aminobutyric acid, 4-hydroxyproline, piperidinic acid, 6-aminocaproic acid, isodesmosine, 2-aminoheptanoic acid, allo-isoleucine, 2-aminoisobutyric acid, N-methylglycine, sarcosine, 3-aminoisobutyric acid, N-methylisoleucine, 2-aminopimelic acid, 6-N-methyllysine, 2,4-diaminobutyric acid, N-methylvaline, desmosine, norvaline, 2,2'-diaminopimelic acid, norleucine, 2,3-diaminopropionic acid, ornithine, and N-ethylglycine. The term "amino acid residue" may include the L- or D-isomers of any of the aforementioned amino acids.

[0070] Other examples of non-standard or unnatural amino acids include, but are not limited to, p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp β-serine, L-Dopa, fluorinated phenylalanine, isopropyl phenylalanine, and phenylalanine. Propyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, non-natural analogues of the amino acid tyrosine; non-natural analogues of the amino acid glutamine; non-natural analogues of the amino acid phenylalanine; non-natural analogues of the amino acid serine; non-natural analogues of the amino acid threonine; methyl unnatural analogues of onine amino acids; unnatural analogues of leucine amino acids; unnatural analogues of isoleucine amino acids; alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acids, or combinations thereof; amino acids with photoactivatable crosslinkers; spin Labelled amino acids;Fluorescent amino acids;Metal-binding amino acids;Metal-containing amino acids;Radioactive amino acids;Photocaged and / or photoisomerizable amino acids;Biotin or biotin analogue-containing amino acids;Keto-containing amino acids;Polyethylene glycol or polyether-containing amino acids;Heavy atom substituted amino acids;Chemically cleavable or photocleavable amino acids;Amino acids with long side chains;Amino acids containing toxic groups;Sugar-substituted amino acids;Carbon-linked sugar-containing amino acids;Redox-active amino acids;α-hydroxy-containing acids;Aminothioacids;α,α-disubstituted amino acids;β-amino acids;γ-amino acids, cyclic amino acids other than proline or histidine, and aromatic amino acids other than phenylalanine, tyrosine or tryptophan;

[0071] As used herein, a "peptide" is defined as a short polymer of amino acids, typically 20 amino acids or less, and more typically 12 amino acids or less in length (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). In some embodiments, peptides contemplated herein may contain about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids or less. Polypeptides, also called proteins, are typically ≧100 amino acids in length (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks / Cole, 110). Polypeptides contemplated herein include, but are not limited to, 100, 101, 102, 103, 104, 105, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 275, about 300, about 325, about 350, about 375, about 400, about 425, about 450, about 475 , about 500, about 525, about 550, about 575, about 600, about 625, about 650, about 675, about 700, about 725, about 750, about 775, about 800, about 825, about 850, about 875, about 900, about 925, about 950, about 975, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1750, about 2000, about 2250, about 2500 or more amino acid residues.

[0072] The peptides contemplated herein may be further modified to include non-amino acid moieties. Modifications may include, but are not limited to, acylation (e.g., acylation-O (ester), N-acylation (amide), S-acylation (thioester)), acetylation (e.g., addition of an acetyl group at the N-terminus or lysine residue of a protein), formylation lipoylation (e.g., lipoic acid, addition of a C8 functional group), myristoylation (e.g., addition of myristate, C14 saturated acid), palmitoylation (e.g., addition of palmitate, C16 saturated acid), alkylation (e.g., addition of an alkyl group such as methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., addition of an isoprenoid group such as farnesol or geranylgeraniol), C-terminal amidation, glycosylation (e.g., addition of a glycosyl group to asparagine, hydroxylysine, serine, or threonine resulting in a glycoprotein). Distinct from glycosylation are the non-enzymatic additions of sugars, polysialylation (e.g., addition of polysialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., addition of a phosphate group, usually to serine, tyrosine, threonine, or histidine). Modifications may include the addition of glycosylation tags (e.g., 4xDQNAT, optionally at the C-terminus) and / or histidine tags (e.g., 6xHis).

[0073] Reference may be made herein to peptides, polypeptides, proteins, and variants thereof. Reference amino acid sequences may include, but are not limited to, any of the amino acid sequences of SEQ ID NOs: 1-10. Variants contemplated herein may have amino acid sequences that include conservative amino acid substitutions relative to the reference amino acid sequence. For example, variant peptides, polypeptides, or proteins contemplated herein may include conservative amino acid substitutions and / or non-conservative amino acid substitutions relative to the reference peptide, polypeptide, or protein. "Conservative amino acid substitutions" are those substitutions that are predicted to interfere the least with the properties of the reference peptide, polypeptide, or protein, and "non-conservative amino acid substitutions" are those substitutions that are predicted to interfere the most with the properties of the reference peptide, polypeptide, or protein. In other words, conservative amino acid substitutions substantially preserve the structure and function of the reference peptide, polypeptide, or protein. The following table provides a list of exemplary conservative amino acid substitutions.

[0074] Table 1 [Table 1]

[0075] A conservative amino acid substitution generally maintains: (a) the structure of the peptide, polypeptide, or protein backbone in the region of the substitution, e.g., as a beta-sheet or alpha-helical structure, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and / or (c) the bulk of the side chain. A non-conservative amino acid substitution generally disrupts: (a) the structure of the peptide, polypeptide, or protein backbone in the region of the substitution, e.g., as a beta-sheet or alpha-helical structure, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and / or (c) the bulk of the side chain.

[0076] Variants containing deletions relative to the reference amino acid sequence of a peptide, polypeptide, or protein are contemplated herein. A "deletion" refers to a change in an amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides relative to the reference sequence. A deletion removes at least 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 amino acid residues or nucleotides. Deletions may include internal or terminal deletions (e.g., N- or C-terminal truncations of a reference polynucleotide, 5'- or 3'-terminal truncations of a reference polypeptide).

[0077] Variants comprising fragments of a reference amino acid sequence of a peptide, polypeptide, or protein are contemplated herein. A "fragment" is a portion of an amino acid sequence that is identical in sequence to the reference sequence but is shorter in length. A fragment may comprise the full length of the reference sequence minus at least one amino acid residue. For example, a fragment may comprise 5-1000 contiguous amino acid residues of the reference polypeptide, respectively. In some embodiments, a fragment may comprise at least 5, 10, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 250, or 500 contiguous amino acid residues of the reference polypeptide. Fragments may be preferentially selected from particular regions of the molecule. The term "at least a fragment" encompasses the full length polypeptide.

[0078] Variants that include insertions or additions to a reference amino acid sequence of a peptide, polypeptide, or protein are contemplated herein. The words "insertion" and "addition" refer to a change in an amino acid or sequence that results in the addition of one or more amino acid residues. An insertion or addition can refer to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, or 200 amino acid residues.

[0079] Fusion proteins are also contemplated herein. A "fusion protein" refers to a protein formed by the fusion of at least one peptide, polypeptide, protein, or variant thereof disclosed herein to at least one heterologous protein peptide, polypeptide, or protein (or a fragment or variant thereof). The heterologous protein(s) may be fused at the N-terminus, C-terminus, or both termini of the peptide or variant.

[0080] "Homology" refers to sequence similarity, or, interchangeably, sequence identity, between two or more polypeptide sequences. Homology, sequence similarity, and percentage sequence identity can be measured using methods in the art or described herein.

[0081] The phrases "percent identity" and "% identity", when applied to polypeptide sequences, refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm. Methods of polypeptide sequence alignment are well known. Some alignment techniques take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the charge and hydrophobicity at the substituted position, thereby preserving the structure (and therefore function) of the polypeptide. Percent identity of amino acid sequences can be measured as understood in the art (e.g., U.S. Pat. No. 7,396,664, incorporated herein by reference in its entirety). A commonly used and freely available suite of sequence comparison algorithms is provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, SF et al. (1990) J. Mol. Biol. 215:403 410), which is available from several sources including NCBI, Bethesda, Md. at its website. The BLAST software suite includes various sequence analysis programs, including "blastp," which is used to align a known amino acid sequence with other amino acid sequences from various databases.

[0082] Percent identity may be measured over the entire length of a defined polypeptide sequence, e.g., as defined by a particular SEQ ID NO: (e.g., any of SEQ ID NOs:1-10), or over a shorter length, e.g., over the length of a fragment obtained from a larger, defined polypeptide sequence, e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 contiguous residues. It will be understood that such lengths are exemplary only, and that any fragment length supported by the sequences presented herein in the tables, figures, or sequence listing may be used to describe the length over which percentage identity may be measured.

[0083] A "variant" of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide over a particular length of one of the polypeptide sequences using blastp with the "BLAST2 sequences" tool available at the National Center for Biotechnology Information's website (see Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250). Such a set of polypeptides may exhibit, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more sequence identity over a particular defined length of one of the polypeptides. A "variant" may have substantially the same functional activity (e.g., glycosylase activity or other activity) as the reference polypeptide. "Substantially isolated or purified" amino acid sequences are contemplated herein. The term "substantially isolated or purified" refers to amino acid sequences that have been removed from their natural environment and are at least 60% free, preferably at least 75% free, more preferably at least 90% free, and even more preferably at least 95% free from other components with which they are naturally associated. Mutant polypeptides contemplated herein include any of the mutant polypeptides of SEQ ID NOs: 1-10.

[0084] As used herein, "translation template" refers to the RNA product of transcription from an expression template that can be used by ribosomes to synthesize a polypeptide or protein.

[0085] The term "reaction mixture" as used herein refers to a solution containing the reagents needed to carry out a given reaction. A reaction mixture is also called a kit when it contains all the reagents needed to carry out a reaction. The components of a reaction mixture may be stored separately in separate containers, each containing one or more of all the components. The components may be packaged separately for commercialization, and a useful commercial kit may contain one or more of the reaction components for a reaction mixture.

[0086] The steps of the methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Steps can be repeated or repeated any number of times to achieve a desired goal, unless otherwise indicated herein or clearly contradicted by context.

[0087] Preferred embodiments of the invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of the preferred embodiments may become apparent to those skilled in the art upon reading the above description. The inventors anticipate that such variations will be utilized by those skilled in the art, and the inventors anticipate that the invention will be practiced differently than as specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present invention unless otherwise indicated herein or clearly contradicted by context.

[0088] Cell-free Protein Synthesis (CFPS)

[0089] The strains and systems disclosed herein can be applied to cell-free protein synthesis methods known in the art, e.g., U.S. Patent Nos. 4,496,538; 4,727,136; 5,478,730; 5,556,769; 5,623,057; 5,665,563; 5,679,352; 6,168,931; 6,248,334; 6,53 See Nos. 1,131; 6,869,774; 6,994,986; 7,118,883; 7,189,528; 7,338,789; 7,387,884; 7,399,610; 8,703,471; and 8,999,668. See also U.S. Patent Application Publication Nos. 2015-0259757, 2014-0295492, 2014-0255987, 2014-0045267, 2012-0171720, 2008-0138857, 2007-0154983, 2005-0054044, and 2004-0209321. See also U.S. Patent Application Publication Nos. 2005-0170452; 006-0211085; 2006-0234345; 2006-0252672; 2006-0257399; 2006-0286637; 2007-0026485; 2007-0178551. See also PCT International Application Publication Nos. 2003 / 056914; 2004 / 013151; 2004 / 035605; 2006 / 102652; 2006 / 119987; 2007 / 120932.See also Jewett, MC, Hong, SH, Kwon, YC, Martin, RW, and Des Soye, BJ 2014, “Methods for improved in vitro protein synthesis with proteins containing non-standard amino acids,” U.S. Patent Application Serial No. 62 / 044,221; Jewett, MC, Hodgman, CE, and Gan, R. 2013, “Methods for yeast cell-free protein synthesis,” U.S. Patent Application Serial No. 61 / 792,290; Jewett, MC, JA Schoborg, and CE Hodgman. 2014, “Substrate Replenishment and Byproduct Removal Improve Yeast Cell-Free Protein Synthesis,” U.S. Patent Application Serial No. 61 / 953,275; and Jewett, MC, Anderson, MJ, Stark, JC, Hodgman, CE 2015, “Methods for activating natural energy See, U.S. Patent Application Serial No. 62 / 098,578, “A novel method for the synthesis of glycoproteins in a yeast-based cell-free protein synthesis system,” U.S. Patent Application Serial No. 62 / 098,578. See also, Guarino, C., & DeLisa, MP (2012) A prokaryote-based cell-free translation system that efficiently synthesizes glycoproteins. Glycobiology, 22(5), 596-601, each of which is incorporated by reference in its entirety.

[0090] In certain exemplary embodiments, one or more of the methods described herein are carried out in a vessel, e.g., in a vessel. The term "vessel" as used herein refers to any container suitable for holding one or more reactants described herein (e.g., for use in one or more transcription, translation, and / or glycosylation steps). Examples of vessels include, but are not limited to, microtiter plates, test tubes, microfuge tubes, beakers, flasks, multi-well plates, cuvettes, flow systems, microfibers, microscope slides, and the like.

[0091] In certain exemplary embodiments, physiologically compatible (but not necessarily native) ions and buffers are utilized for transcription, translation, and / or glycosylation, e.g., potassium glutamate, ammonium chloride, etc. Physiological cytosolic salts are well known to those of skill in the art.

[0092] The strains and systems disclosed herein may be applied to cell-free protein methods to prepare glycosylated macromolecules (e.g., glycosylated peptides, glycosylated proteins, and glycosylated lipids). Glycosylated proteins that may be prepared using the disclosed strains and systems may include proteins with N-linked glycosylation (i.e., glycans attached to the nitrogen of asparagine and / or arginine side chains) and / or O-linked glycosylation (i.e., glycans attached to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine, and / or hydroxyproline). Glycosylated lipids may include O-linked polysaccharides via oxygen atoms, such as ceramides.

[0093] Glycosylated macromolecules disclosed herein may include unbranched and / or branched sugar chains composed of monomers known in the art, such as, but not limited to, glucose (e.g., β-D-glucose), galactose (e.g., β-D-galactose), mannose (e.g., β-D-mannose), fucose (e.g., α-L-fucose), N-acetyl-glucosamine (GlcNAc), N-acetyl-galactosamine (GalNAc), neuraminic acid, N-acetylneuraminic acid (i.e., sialic acid), and xylose, which may be attached to a glycosylated macromolecule and grown onto a glycan chain or donor molecule (e.g., donor lipid and / or donor nucleotide) by respective glycosyltransferases (e.g., oligosaccharyltransferase, GlcNAc transferase, GalNAc transferase, galactosyltransferase, and sialyltransferase). The glycosylated macromolecules disclosed herein may include glycans known in the art.

[0094] The disclosed cell-free protein synthesis system may utilize components that are unpurified and / or at least partially isolated and / or purified. As used herein, the term "unpurified" may refer to components obtained by disrupting and lysing cells, at most by minimally purifying the unpurified components from the disrupted and lysed cells, for example, by centrifuging the disrupted and lysed cells, and recovering the unpurified components from the supernatant and / or pellet after centrifugation. The term "isolated or purified" refers to components that have been removed from their natural environment and are at least 60% free, preferably at least 75% free, more preferably at least 90% free, and even more preferably at least 95% free from other components with which they are naturally associated.

[0095] Cell-free glycoprotein synthesis in prokaryotic cell lysates enriched for components for glycosylation (CFGpS)

[0096] Compositions and methods for performing cell-free glycoprotein synthesis (CFGpS) are disclosed. In some embodiments, the compositions and methods enrich for components for glycosylation and include or utilize prokaryotic cell lysates prepared from genetically modified strains of prokaryotes.

[0097] In some embodiments, the genetically modified prokaryote is a genetically modified Escherichia coli strain or other prokaryote suitable for preparing a lysate for CFGpS. Optionally, the modified strain of Escherichia coli is derived from rEc.C321. Preferably, the modified strain includes a genomic modification (e.g., deletion of a gene that renders the gene unmodifiable) that preferably results in a lysate capable of high-yield cell-free protein synthesis. Also, preferably, the modified strain includes a genomic modification (e.g., deletion of a gene that renders the gene unmodifiable) that preferably results in a lysate that contains sugar precursors for glycosylation at a relatively high concentration (e.g., compared to a strain that does not have the genomic modification). In some embodiments, a lysate prepared from a modified strain contains sugar precursors at a concentration of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or more than a lysate prepared from an unmodified strain. By way of example, and not by way of limitation, strains of bacterial origin useful in the present methods, kits, and systems include recombinant strains of E. coli, such as recombinant E. coli strains lacking one or more gene products selected from prfA, endA, gor, rne, and lpxM. Suitable recombinant strains for the disclosed methods, kits, and systems include, but are not limited to, rE. coli ΔprfA ΔendA Δgor Δrne(705), E. coli BL21(DE3), E. coli CLM24, E. coli CLM24 ΔlpxM, E. coli CLM24 ΔlpxM CH-IpxE, E. coli CLM24 ΔlpxM CH-IpxE TT-IpxE, E. coli CLM24 ΔlpxM CH-IpxE TT-IpxE KL-IpxE, and E. coli CLM24 ΔlpxM CH-IpxE TT-IpxE KL-IpxE KO-IpxE.

[0098] In some embodiments, the modified strain includes a modification that results in an increased concentration of monosaccharides (e.g., glucose, mannose, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), galactose, sialic acid, neuraminic acid, fucose) utilized in glycosylation. Thus, the modification may inactivate an enzyme that metabolizes a monosaccharide or polysaccharide utilized in glycosylation. In some embodiments, the modification inactivates a dehydratase or carbon-oxygen lyase enzyme (EC 4.2) (e.g., via deletion of at least a portion of the gene encoding that enzyme). In particular, the modification may inactivate GDP-mannose-4,6-dehydratase (EC 4.2.1.47). When the modified strain is E. coli, the modification may include an inactivation modification of the gmd gene (e.g., via deletion of at least a portion of the gmd gene). The sequence of the E. coli gmd gene is provided herein as SEQ ID NO:1, and the amino acid sequence of the E. coli GDP-mannose-4,6-dehydratase is provided as SEQ ID NO:2.

[0099] In some embodiments, the modified strain comprises a modification that inactivates an enzyme utilized in the glycosyltransferase pathway. In some embodiments, the modification inactivates an oligosaccharide ligase enzyme (e.g., via deletion of at least a portion of the gene encoding the enzyme). In particular, the modification may inactivate an O-antigen ligase, which optionally conjugates an O-antigen to the lipid A core oligosaccharide. The modification may comprise an inactivation modification of the waaL gene (e.g., via deletion of at least a portion of the gene for waaL). The sequence of the E. coli waaL gene is provided herein as SEQ ID NO:3, and the amino acid sequence of the E. coli O-antigen ligase is provided as SEQ ID NO:4.

[0100] In some embodiments, the modified strain includes a modification that inactivates a dehydratase or carbon-oxygen lyase enzyme (e.g., via deletion of at least a portion of the gene encoding the enzyme), and the modified strain includes a modification that inactivates an oligosaccharide ligase enzyme (e.g., via deletion of at least a portion of the gene encoding the enzyme). The modified strain may include an inactivation or deletion of both gmd and waaL.

[0101] In some embodiments, the modified strain may be modified to express one or more orthogonal or heterologous genes. In particular, the modified strain may be genetically engineered to express orthogonal or heterologous genes associated with glycoprotein synthesis, such as glycosyltransferases (GTs) involved in the lipid-linked oligosaccharide (LLO) pathway. In some embodiments, the modified strain may be modified to express an orthogonal or heterologous oligosaccharyltransferase (EC 2.4.1.119) (OST). Oligosaccharyltransferases or OSTs are enzymes that transfer oligosaccharides from lipids to proteins.

[0102] In particular, the modified strain may be genetically engineered to express orthogonal or heterologous genes of a glycosylation system (e.g., an N-linked glycosylation system and / or an O-linked glycosylation system). The N-linked glycosylation system of Campylobacter jejuni was transferred to E. coli (Wacker et al., "N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli," Science 2002, Nov 29; 298(5599):1790-3, the contents of which are incorporated herein by reference in their entirety). In particular, the modified strain may be modified to express one or more genes at the pgl locus of C. jejuni or one or more genes at a homologous pgl locus. Genes at the pgl locus include pglG, pglF, pglE, wlaJ, pglD, pglC, pglA, pglB, pglJ, pglI, pglH, pglK, and gne, and are used to synthesize lipid-linked oligosaccharides (LLOs) and transfer the oligosaccharide moiety of LLO to proteins via oligosaccharyltransferase.

[0103] Suitable orthogonal or heterologous oligosaccharyltransferases (OSTs) that can be expressed in the engineered strains include the Campylobacter jejuni oligosaccharyltransferase PglB. The gene for C. jejuni OST is also called pglB, and its sequence is provided as SEQ ID NO:5, and the amino acid sequence of C. jejuni PglB is provided as SEQ ID NO:6. PglB catalyzes the transfer of oligosaccharides to D / EYNXS / T motifs (Y, X≠P) present on proteins. Additional non-limiting examples of OST enzymes useful in the methods, kits, and systems disclosed herein include, but are not limited to, Campylobacter coli PglB, Campylobacter lari PglB, Desulfovibrio desulfuricans PglB, Desulfovibrio gigas PglB, and Desulfovibrio vulgaris PglB.

[0104] A crude cell lysate may be prepared from the modified strains disclosed herein. A crude cell lysate may be prepared from various modified strains disclosed herein, and the crude cell lysates may be combined to prepare a mixed crude cell lysate. In some embodiments, one or more crude cell lysates may be prepared from one or more modified strains that preferably contain a genomic modification (e.g., deletion of a gene that renders the gene unmodifiable) that results in a lysate containing sugar precursors for glycosylation at a relatively high concentration (e.g., in comparison to a strain that does not have the genomic modification). In some embodiments, one or more crude cell lysates may be prepared from one or more modified strains that have been modified to express one or more orthogonal or heterologous genes or gene clusters associated with glycoprotein synthesis. Preferably, the crude cell lysate or mixed crude cell lysate is enriched in glycosylation components, such as lipid-linked oligosaccharides (LLOs), glycosyltransferases (GTs), oligosaccharyltransferases (OSTs), or any combination thereof. More preferably, the crude cell lysate or mixed crude cell lysate is enriched in Man3GlcNAc2LLO, representing the core eukaryotic glycan, and / or Man3GlcNAc4Gal2Neu5Ac2LLO, representing the fully sialylated human glycan. By way of example, and not by way of limitation, glycan structures useful in the disclosed methods, kits, and systems include, but are not limited to, Francisella tularensis SchuS4 O-polysaccharide, Escherichia coli O78 O-polysaccharide, Escherichia coli O-7 O-polysaccharide, Escherichia coli O-9 O-polysaccharide primer, Campylobacter jejuni heptasaccharide N-glycan, Campylobacter lari PglB hexasaccharide N-glycan, modified Campylobacter lari PglB hexasaccharide N-glycan, Wolinella succinogenes hexasaccharide N-glycan, and eukaryotic Man3GlcNac2 N-glycan structures.

[0105] The disclosed crude cell lysates may be used in a cell-free glycoprotein synthesis (CFGpS) system to synthesize a variety of glycoproteins. The glycoproteins synthesized in the CFGpS system may include prokaryotic glycoproteins and eukaryotic proteins, including human proteins. The CFGpS system may be utilized in an in vitro glycoprotein synthesis method by performing the following steps using the crude cell lysate or mixture of crude cell lysates disclosed herein: (a) performing cell-free transcription of the gene of the target glycoprotein; (b) performing cell-free translation; and (c) performing cell-free glycosylation. The method may be performed in a single vessel or in multiple vessels. Preferably, the steps of the synthesis method may be performed using a single reaction vessel. The disclosed method may be used to synthesize a variety of glycoproteins, including prokaryotic and eukaryotic glycoproteins.

[0106] Bioconjugate vaccine manufacturing

[0107] Although protein-glycan coupling technology (PGCT) represents a simplified and cost-effective strategy for bioconjugate vaccine production, it has three main limitations. First, process development timelines, design-build-test (DBT) cycles of glycosylation pathways, and bioconjugate production are all limited by cell growth. Second, it remains to be demonstrated whether FDA-approved carrier proteins, such as toxins from Clostridium tetani and Corynebacterium diphtheriae, can be adapted for N-linked glycosylation in live E. coli. Third, selected non-natural glycans are known to be transferred with low efficiency by the C. jejuni oligosaccharyltransferase (OST), PglB.

[0108] A modular, in vitro platform for the production of bioconjugates has the potential to address all of these limitations. Here, we demonstrate that bioconjugates against pathogenic strains of Francisella tularensis and Escherichia coli can be produced by coordinated in vitro transcription, translation, and N-glycosylation in a cell-free glycoprotein synthesis (CFGpS) reaction lasting just 20 h. This system has the potential to shorten the timeline for process development and distribution of novel antibacterial vaccines by weeks to days. Furthermore, due to the modular nature of the CFGpS platform and the fact that cell-free systems have demonstrated advantages for the production of membrane proteins compared to live cells, this method may be easily adapted to produce bioconjugates using FDA-approved carrier proteins, such as Clostridium tetani and Corynebacterium diphtheriae toxins, which are membrane-localized. This may be easily accomplished by feeding the CFGpS reaction with plasmids encoding these carrier proteins. Furthermore, due to the modular nature of CFGpS, the in vitro approach may be used to prototype other native or engineered homologs of the prototype C. jejuni OST to identify candidate OSTs with improved efficiency for the transfer of the O-antigen LLO of interest. This can be accomplished by concentrating the OSTs of interest in lysates and mixing them with LLO lysates in a mixed lysate CFGpS reaction, as we have previously described (see WO2017 / 117539, the contents of which are incorporated herein by reference in their entirety). Finally, we have demonstrated that cell-free bioconjugate synthesis reactions can be lyophilized and maintain bioconjugate synthesis capability, demonstrating the potential of the CFGpS system for on-demand, portable, and low-cost production or development of novel vaccines. This novel method for in vitro bioconjugate vaccine production has demonstrated advantages for rapid, modular, and portable vaccine prototyping and production compared to existing methods.Our technology enables the rapid production of bioconjugate vaccines directed against user-specified bacterial targets for therapeutic development or basic research.

[0109] The inventors are not aware of any prokaryotic cell-free systems capable of producing glycoprotein or bioconjugate vaccines. Although there are commercially available eukaryotic cell lysate systems for cell-free glycoprotein production (Promega, ThermoFisher), these systems do not involve overexpression of orthogonal glycosylation machinery and therefore do not allow for modular, user-specified glycosylation in the way that our system does. For these reasons, we feel that there is still substantial commercial potential for our invention.

[0110] The disclosed method for in vitro bioconjugate vaccine production has demonstrated advantages over existing methods for rapid, modular, and portable vaccine prototyping and production. This system addresses limitations of existing manufacturing approaches, making it an attractive alternative or complementary strategy for antibacterial vaccine production. In light of the growing health care concerns caused by antibiotic-resistant bacterial infections, this method has the potential to be highly beneficial for the development, production, and distribution of novel vaccines against various pathogenic strains. Advantages of the disclosed method include: on-demand expression of bioconjugate vaccines; prototyping of novel bioconjugate vaccine candidates; prototyping of novel bioconjugate vaccine manufacturing pathways; distribution of bioconjugate vaccines to resource-poor settings.

[0111] In summary, we disclose the first prokaryotic cell-free system capable of coordinated, cell-free transcription, translation, and glycosylation of glycoprotein vaccines. The disclosed system enables the production of bioconjugate vaccines in 20 hours. The modularity of the system allows for the rapid prototyping of vaccine candidates with novel glycosylation pathways and various carrier proteins. Suitable carriers include, but are not limited to, Haemophilus influenzae protein D (PD) (SEQ ID NO: 7), Neisseria meningitidysporin protein (PorA) (SEQ ID NO: 8), Corynebacterium diphtheria toxin (CRM197) (SEQ ID NO: 9), Clostridium tetani toxin (TT) (SEQ ID NO: 10), fragment C of TT (TTc), light chain domain of TT (TTlight), Escherichia coli maltose binding protein, human glucagon peptide fused to MBP, Superfolder Green Fluorescent Protein (GFP), single chain variable antibody fragment (scFv), human erythropoietin mutants, Campylobacter jejuni AcrA, and Pseudomonas aeruginosa exotoxin A (EPA), or mutants thereof. The components and products of the system may be lyophilized, which enables the potential for wide and rapid distribution of vaccine production technology. The disclosed system reduces the time required to produce bioconjugates in prokaryotic cell lysates from weeks to days, which may provide a competitive advantage in commercializing the technology.

[0112] The disclosed bioconjugates may be formulated as vaccines that may optionally include additional agents to induce and / or potentiate an immune response, such as adjuvants. The term "adjuvant" refers to a compound or mixture that enhances the immune response. Adjuvants serve as tissue depot agents that slowly release antigens and also as lymphoid activators that nonspecifically enhance the immune response. Examples of adjuvants that can be utilized in the disclosed compositions include, but are not limited to, copolymer adjuvants (e.g., Pluronic L121® brand poloxamer 401, CRL1005, or low molecular weight copolymer adjuvants such as Polygen® adjuvants), poly(I:C), R-848 (a Th1-like adjuvant), resiquimod, imiquimod, PAM3CYS, aluminum phosphate (e.g., AlPO4), loxoribine, BCG (Bacille Calmette-Guerin), and Corynebacterium parvum. Examples of potential effective human adjuvants include CpG oligodeoxynucleotides (ODN), cholera toxin-derived antigens (e.g., CTA1-DD), lipopolysaccharide adjuvants, complete Freund's adjuvant, incomplete Freund's adjuvant, saponins (e.g., Quil-A), mineral gels such as aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, peptides, surfactants such as hydrocarbon emulsions in oil or water (e.g., MF59 or Montanide ISA720 available from Novartis Vaccines), keyhole limpet hemocyanin, and dinitrophenol. Illustrative Embodiments

[0113] The following embodiments are illustrative and are not intended to limit the scope of the claimed subject matter.

[0114] EMBODIMENT 1 A method of synthesizing an N-glycosylated recombinant protein carrier, which may optionally be utilized as a bioconjugate immunogen or vaccine, comprising performing coordinated transcription, translation, and N-glycosylation of a recombinant protein carrier, thereby providing an N-glycosylated recombinant protein carrier, which may optionally be utilized as a bioconjugate immunogen or vaccine, wherein the N-glycosylated recombinant protein carrier comprises: (i) a consensus sequence, NXS / T (optionally inserted into the protein carrier), where X may be any natural or unnatural amino acid except proline; and (ii) at least one antigenic polysaccharide from at least one bacterium N-linked to the recombinant protein carrier, where the at least one antigenic polysaccharide is optionally at least one bacterial O-antigen, optionally from one or more strains of E. coli or Francisella tularensis; further, the bioconjugate vaccine may optionally comprise an adjuvant.

[0115] EMBODIMENT 2 2. The method of embodiment 1, wherein the carrier protein is an engineered variant of E. coli maltose binding protein (MBP).

[0116] EMBODIMENT 3 2. The method of embodiment 1, wherein the carrier protein is selected from a detoxified mutant of a toxin from Clostridium tetani and a detoxified mutant of a toxin from Corynebacterium diphtheriae.

[0117] EMBODIMENT 4 2. The method of embodiment 1, wherein the carrier protein is selected from Haemophilus influenzae protein D (PD) and Neisseria meningitidysporin protein (PorA), and variants thereof.

[0118] EMBODIMENT 5 The method of any one of embodiments 1-4, wherein said method utilizes an oligosaccharyltransferase (OST) that is a naturally occurring bacterial homolog of C. jejuni PglB.

[0119] EMBODIMENT 6 5. The method of any one of embodiments 1 to 4, wherein said method utilises OST, an engineered variant of C. jejuni PglB.

[0120] EMBODIMENT 7 5. The method of any one of embodiments 1 to 4, wherein said method utilises OST, a naturally occurring archaeal OST.

[0121] EMBODIMENT 8 5. The method of any one of embodiments 1 to 4, wherein the method utilises an OST that is a naturally occurring single subunit eukaryotic OST, such as that found in Trypanosoma bruceii.

[0122] EMBODIMENT 9 A method for crude cell lysate preparation, in which an orthogonal gene or gene cluster is expressed in a source strain for the crude cell lysate, resulting in a lysate enriched in glycosylated components (lipid-linked oligosaccharides (LLO), oligosaccharyl transferase (OST), and both LLO and OST), and optionally resulting in a separate lysate enriched in LLO (e.g., LLO associated with O-antigen) and a separate lysate enriched in OST (e.g., for which LLO is a substrate), and optionally combining the separate lysates to perform cell-free protein synthesis of a carrier protein that is glycosylated with a glycan component of LLO by the enzymatic activity of OST, and further optionally purifying the glycosylated carrier protein, and optionally administering the glycosylated carrier protein as an immunogen.

[0123] EMBODIMENT 10 10. The method of embodiment 9, wherein the source strain overexpresses a gene encoding oligosaccharyl transferase (OST).

[0124] EMBODIMENT 11 The method of embodiment 9 or 10, wherein the source strain overexpresses a synthetic glycosyltransferase pathway that results in the production of an O-antigen from an O-antigen, optionally an F. tularensis Schu S4 lipid-linked oligosaccharide (FtLLO).

[0125] EMBODIMENT 12 11. The method of embodiment 9 or 10, wherein the source strain overexpresses a synthetic glycosyltransferase pathway that results in the production of an O-antigen from an O-antigen, optionally an enterotoxigenic E. coli O78 lipid-linked oligosaccharide (EcO78LLO).

[0126] EMBODIMENT 13 13. The method of any one of embodiments 9 to 12, wherein the source strain overexpresses the glycosyltransferase pathway and OST, resulting in the production of LLO and OST.

[0127] EMBODIMENT 14 11. The method of embodiment 9 or 10, wherein the source strain overexpresses the O-antigen glycosyltransferase pathway from a pathogenic strain, resulting in the production of O-antigen lipid-linked oligosaccharides (LLOs).

[0128] EMBODIMENT 15 A method for cell-free production of a bioconjugate immunogen or vaccine, which involves mixing as one or more steps a crude cell lysate (e.g., any crude cell lysate described in any one of embodiments 9-14).

[0129] EMBODIMENT 16 16. The method of embodiment 15, wherein the bioconjugate immunogen or vaccine comprises an immunogenic carrier that is a protein or peptide.

[0130] EMBODIMENT 17 The method of embodiment 15, wherein the bioconjugate immunogen or vaccine comprises an immunogenic carrier that is a protein or peptide comprising a protein or peptide selected from Haemophilus influenzae protein D (PD), Neisseria meningitidysporin protein (PorA), Corynebacterium diphtheria toxin (CRM197), Clostridium tetani toxin (TT), Escherichia coli maltose binding protein, and variants thereof.

[0131] EMBODIMENT 18 18. The method of any one of the preceding embodiments, wherein the components of the method are capable of maintaining bioconjugate synthesis capability when lyophilized and rehydrated.

[0132] EMBODIMENT 19 The method of any one of embodiments 15 to 18, wherein the goal is on-demand vaccine production.

[0133] EMBODIMENT 20 The method of any one of embodiments 15-19, wherein the goal is vaccine production in resource-limited environments.

[0134] EMBODIMENT 21 A kit for synthesizing an N-glycosylated carrier protein in vitro, the kit comprising one or more of the following components: (i) a first component comprising a cell lysate comprising an orthogonal oligosaccharyl transferase (OST); (ii) a second component comprising a cell lysate comprising an O-antigen (e.g., a lipid-linked oligosaccharide (LLO) comprising an O-antigen); (iii) a third component comprising a transcription template and, optionally, a polymerase for synthesizing mRNA from the transcription template encoding the carrier protein (the carrier protein comprising an inserted and / or naturally occurring consensus sequence, NXS / T, where X can be any natural or unnatural amino acid except proline).

[0135] EMBODIMENT 22 22. The kit of embodiment 21, wherein one or more of the first component, the second component, and the third component maintain biological activity when lyophilized and rehydrated.

[0136] EMBODIMENT 23 The kit of embodiment 21 or 22, wherein the first component, a cell lysate, is prepared from a source strain (e.g., E. coli) that overexpresses a gene encoding an orthogonal OST (e.g., C. jejuni PglB).

[0137] EMBODIMENT 24 The kit according to any one of embodiments 21 to 23, wherein the second component, the cell lysate, is produced from a source strain overexpressing a synthetic glycosyltransferase pathway (e.g., the biosynthetic machinery for producing Francisella tularensis Schu S4 O-antigen (FtLLO lysate) or the biosynthetic machinery for producing enterotoxigenic E. coli O78 lipid-linked oligosaccharides (EcO78LLO lysate)).

[0138] EMBODIMENT 25 A method for cell-free production of a glycoprotein, which may optionally be a bioconjugate suitable for use as a bioconjugate immunogen or vaccine, comprising: (a) mixing a first cell lysate comprising an orthogonal oligosaccharyl transferase (OST) with a second cell lysate comprising an O-antigen (e.g., lipid-linked oligosaccharide (LLO)) to prepare a cell-free protein synthesis reaction; (b) transcribing and translating a carrier protein (the carrier protein comprising an insertion and / or a naturally occurring consensus sequence, NXS / T, where X can be any natural or non-natural amino acid except proline) in the cell-free protein synthesis reaction (e.g., by optionally adding a carrier protein and / or a transcription template for a polymerase to the cell-free protein synthesis reaction); and (c) glycosylating the carrier protein in a cell-free protein synthesis reaction with a bacterial O-antigen.

[0139] EMBODIMENT 25 25. The method of embodiment 24, wherein the second cell lysate comprises the O-antigen as part of a lipid-linked oligosaccharide (LLO).

[0140] EMBODIMENT 26 26. The method of embodiment 24 or 25, further comprising formulating the glycoprotein as a vaccine composition, optionally comprising an adjuvant.

[0141] EMBODIMENT 27 A bioconjugate immunogen or vaccine prepared by any of the above methods and / or kits.

[0142] EMBODIMENT 28 A vaccination method comprising administering the vaccine of embodiment 27 to a subject in need thereof. EXAMPLES

[0143] The following examples are illustrative and are not intended to limit the scope of the claimed subject matter.

[0144] Example 1 - A method for rapid in vitro synthesis of bioconjugate vaccines by recombinant production of N-glycosylated proteins in prokaryotic cell lysates

[0145] summary

[0146] Conjugate vaccines are one of the safest and most effective methods for the prevention of life-threatening bacterial infections [1-10]. Bioconjugate vaccines are a type of conjugate vaccine produced by protein glycan conjugation technology (PGCT), in which polysaccharide antigens are attached to genetically engineered carrier proteins by N-glycosylation using bacterial oligosaccharyl transferase (OST) in live Escherichia coli cells

[11] . Bioconjugate vaccines have the potential to significantly reduce the time and cost required to produce antibacterial vaccines. However, PGCT is limited by: i) the length of the in vivo process development schedule; and ii) the fact that FDA-approved carrier proteins, such as toxins from Clostridium tetani and Corynebacterium diphtheriae, have not yet been demonstrated to be compatible with N-linked glycosylation in live E. coli. Here, we applied cell-free glycoprotein synthesis (CFGpS) technology to enable rapid in vitro production of bioconjugate vaccines against pathogenic strains of Escherichia coli and Francisella tularensis in a reaction lasting 20 h. Due to the modular nature of the CFGpS system, this cell-free strategy could also be easily applied to produce bioconjugates using FDA-approved carrier proteins or booster vaccines against pathogenic bacteria whose surface antigen gene clusters are known

[11] [9][7,10][3,5,6,8]. We showed that this system can be lyophilized and maintains bioconjugate synthesis capacity, demonstrating the potential for on-demand vaccine production and development in resource-poor environments. This work represents the first demonstration of bioconjugate vaccine production in E. coli lysate and has promising applications as a portable prototyping or manufacturing platform for antibacterial vaccine candidates.

[0147] Background and Significance

[0148] Glycosylation, or the addition of glycans (sugars) to proteins, is the most abundant post-translational modification in nature and plays a crucial role in protein folding and activity [1-4]. When it was first discovered in the 1930s

[12] , glycosylation was thought to be limited to eukaryotes. However, glycosylation was also discovered in archaea in the 1970s [13, 14] and in bacteria in the late 1990s and early 2000s [15, 16], establishing glycoproteins as the major post-translational modification in all domains of life. A wide diversity of glycan structures, including both linear and highly branched polysaccharide chains, has been described

[17] , and the information content has been exponentially enhanced compared to other polypeptide modifications

[18] .

[0149] As a result of its role in protein structure and information storage, glycosylation is involved in a variety of biological processes. In eukaryotes, glycoproteins are involved in immune recognition and response, intracellular trafficking, and cell-cell communication [19-22]. Furthermore, alterations in glycosylation have been shown to correlate with pathologies, including cancer [23-25], inflammation [26-29], and Alzheimer's disease

[30] . In prokaryotes, glycosylation is known to play an important role in virulence and host invasion [31-33]. Based on the critical role of glycosylation in numerous biological processes, it has been proposed that a central dogma of biology be constructed that includes glycans as a central element

[34] .

[0150] The most common forms of glycosylation are asparagine-linked (N-linked) and serine (Ser) or threonine (Thr)-linked (O-linked)

[35] . N-linked glycosylation is characterized by the addition of a glycan moiety to the side chain nitrogen of an asparagine (Asn) residue by oligosaccharyltransferases (OSTs), which recognize the consensus sequence Asn-X-Ser / Thr, where X is any amino acid except proline [36, 37]. This process occurs in the endoplasmic reticulum and aids in protein folding, quality control, and trafficking

[38] . O-linked glycosylation occurs in the Golgi apparatus following the addition of N-glycans. Unlike N-linked glycosylation, there is no known consensus sequence for O-linked glycosylation [39, 40]. Despite the importance of glycans in biology, glycoscience is currently considered an underexplored field. A 2012 National Research Council report to the National Academies highlighted the great need for transformational change in glycoscience.

[41] The discovery of bacterial glycosylation pathways has enabled new discoveries about this important post-translational modification,[42, 43] but new synthetic and analytical tools are needed to advance the field.

[0151] Since the recent discovery of bacterial glycosylation, proteins carrying N- and O-linked glycans have been found in a large number of bacteria [44, 45]. The best-tested bacterial glycosylation system is the pgl pathway from Campylobacter jejuni, which has been shown to be functionally expressed in Escherichia coli (Figure 1)

[46] . In C. jejuni, proteins are N-glycosylated with a 1.406 kDa GlcGalNAc5Bac heptasaccharide (Glc: glucose, GalNAc: N-acetylgalactosamine, Bac: bacillosamine). GT assembles the heptasaccharide onto the lipid anchor undecaprenol pyrophosphate (Und-PP), which is then used as a substrate by OST (PglB) for N-linked glycosylation [47-49]. This pathway is considerably simpler than the eukaryotic glycosylation pathway and has been exploited to improve our understanding of the mechanism of N-linked glycosylation [42, 43].

[0152] Glycosylation is often involved in the synthesis of bacterial cell walls, although not all bacteria synthesize glycoproteins. Lipopolysaccharide (LPS) molecules are the main part of the outer membrane of many Gram-negative bacteria and consist of a lipid anchor, an oligosaccharide core, and a variable polysaccharide region known as the O-antigen

[32] . Capsular polysaccharides (CPS) are another type of surface polysaccharide that is similar in structure to LPS, except that in this case, the polysaccharide region is directly linked to the lipid A or phospholipid anchor

[31] . LPS O-antigens (OPS) and CPS are among the main tools used by bacterial pathogens for survival in and hostile host environments [50-53]. As a result, elucidation of the mechanisms of CPS and O-PS biosynthesis is of interest for the development of antibiotic and antibacterial vaccines.

[0153] The emergence of antibiotic-resistant strains of bacteria necessitates the development of novel strategies for the treatment and prevention of life-threatening bacterial infections. In 2013, the US Centers for Disease Control and Prevention published a report citing antibiotic resistance as one of the most significant health threats in the United States. Conjugate vaccines, consisting of CPS or O-PS antigens covalently linked to carrier proteins, are the safest and most effective preventative measures against bacterial infections and are used to reduce the incidence of Streptococcus pneumoniae, Neisseria meningitides, and Haemophilus influenzae infections [1-10]. Because polysaccharide antigens cannot directly activate naive T cells, they need to be conjugated to carrier proteins to trigger sustained immunological memory

[54] . However, existing technologies for producing conjugate vaccines are complex, involve multiple processing and purification steps, and, moreover, the resulting products are not clearly defined (Figure 2, top) [2]. Furthermore, these processes are laborious and can require large-scale fermentation of the pathogenic bacteria, making conjugate vaccines prohibitively expensive for vaccination campaigns in developing countries.

[0154] The production of recombinant O-antigen protein conjugates in live E. coli cells has recently been carried out using the bacterial N-glycosylation machinery (Figure 2, bottom)

[11] . These so-called bioconjugate vaccines have the potential to reduce the cost and time required for antibacterial vaccine production. Bioconjugates have been developed against several bacterial targets, including Francisella tularensis [4], Pseudomonas aeruginosa

[11] , Salmonella enterica [9], Shigella dynsenteriae [7, 10], Shigella flexneri [8], Staphylococcus aureus [5], Brucella abortus [3], and Burkholderia pseudomallei [6]. In vitro methods for bioconjugate production have the potential to shorten the process development timeline for novel antibacterial vaccines by months or weeks

[55] .

[0155] Cell-free protein synthesis (CFPS) is an emerging field that allows for the production of proteins in unpurified cell lysates [55, 56]. CFPS technology was first used 50 years ago by Nirenberg and Matthaei to decipher the genetic code

[57] . In the late 1960s and early 1970s, CFPS was used to help elucidate the regulatory mechanisms of the E. coli lactose

[58] and tryptophan

[59] operons. In the last two decades, CFPS platforms have experienced an upsurge in development to meet the growing demand for recombinant protein expression technologies

[55] .

[0156] CFPS offers several advantages for recombinant protein expression. In particular, the open reaction environment allows for the addition or removal of protein synthesis substrates as well as precise, on-line reaction monitoring. Furthermore, the CFPS reaction environment can be fully targeted and optimized for the production of a protein product of interest. CFPS effectively separates the cellular objective (growth and reproduction) from the engineer's objective (protein overexpression and easy product purification), which has proven advantageous for the production of complex proteins and protein assemblies, including membrane proteins [60-63], bispecific antibodies

[64] , antibody-drug conjugates

[65] , and virus-like particle vaccines [66-68]. Overall, the CFPS technology allows for shortened protein synthesis schedules and enhanced flexibility in the addition or removal of substrates compared to in vivo approaches. In particular, the E. coli CFPS system is unique due to i) its high batch yields, up to 2.3 g / L of green fluorescent protein (GFP) reported

[69] , ii) the inexpensive required substrates [70-72], and iii) the low cost of production of 10 6 It has been widely adopted due to its ability to linearly scale up reaction volumes beyond L

[73] .

[0157] Glycosylation is possible in several eukaryotic CFPS systems, including ICE, CHO extracts, and human leukemia cell line extracts [74-77]. However, these platforms utilize endogenous machinery to perform glycosylation, meaning that i) the possible glycan structures are limited to those naturally synthesized by the host cell and ii) the glycosylation process is carried out in a "black box", which makes it difficult to modify or control. The development of a highly active E. coli CFPS platform has prompted recent efforts to enable glycoprotein production in E. coli lysates by the addition of orthogonal glycosylation components. In one study, Guarino and DeLisa demonstrated that it was possible to produce glycoproteins in E. coli CFPS by adding purified lipid-linked oligosaccharides (LLOs) and C. jejuni OST to the CFPS reaction. Yields of 50-100 μg / mL of AcrA and C. jejuni glycoproteins were achieved

[78] . Despite these recent advances, bacterial cell-free glycosylation systems have been limited by their inability to co-activate efficient protein synthesis and glycosylation. We have recently developed a cell-free glycoprotein synthesis (CFGpS) system that addresses this limitation by enabling modular, coordinated transcription, translation, and N-glycosylation of proteins in E. coli lysates in which glycosylation enzymes are selectively enriched (see WO2017 / 117539, the contents of which are incorporated herein by reference in their entirety). Here, we have applied this technology platform to the production of bioconjugate vaccines to obtain a methodology for rapid, modular in vitro expression of bioconjugates.

[0158] Results and Discussion

[0159] Cell-free glycoprotein synthesis (CFGpS) for bioconjugate vaccine production. Although protein-glycan coupling technology (PGCT) represents a simplified and cost-effective strategy for bioconjugate vaccine production, it has three main limitations. First, process development timelines, design-build-test (DBT) cycles of glycosylation pathways, and bioconjugate production are all limited by cell growth. Second, it remains to be demonstrated whether FDA-approved carrier proteins, such as the toxins from Clostridium tetani and Corynebacterium diphtheriae, can be adapted for N-linked glycosylation in live E. coli. Third, selected non-natural glycans are known to be transferred with low efficiency by the C. jejuni OST, PglB [9].

[0160] A modular, in vitro platform for the production of bioconjugates has the potential to address all of these limitations. Here, we demonstrate that bioconjugates against pathogenic strains of Francisella tularensis and Escherichia coli can be produced by coordinated in vitro transcription, translation, and N-glycosylation in a cell-free glycoprotein synthesis (CFGpS) reaction lasting just 20 h. This system has the potential to shorten the timeline for process development and distribution of novel antibacterial vaccines by weeks to days. Furthermore, due to the modular nature of the CFGpS platform and the fact that cell-free systems have demonstrated advantages for the production of membrane proteins compared to live cells [60-63], this method may be easily adapted to produce bioconjugates using FDA-approved carrier proteins, such as membrane-localized Clostridium tetani and Corynebacterium diphtheriae toxins. This may be easily achieved by feeding the CFGpS reaction with plasmids encoding these carrier proteins. Furthermore, due to the modular nature of CFGpS, the in vitro approach may be used to prototype other native or engineered homologs of the prototypic C. jejuni OST, such as those recently described [9, 79, 80], to identify candidate OSTs with improved efficiency for the transfer of the O-antigen LLO of interest. This can be accomplished by concentrating the OSTs of interest in lysates and mixing them with LLO lysates in a mixed lysate CFGpS reaction, as we have previously described (Jewett lab, unpublished data, US Provisional Patent Application No. 62273124). Finally, we have demonstrated that cell-free bioconjugate synthesis reactions can be lyophilized and maintain bioconjugate synthesis capability, demonstrating the potential of the CFGpS system for on-demand, portable, and low-cost production or development of novel vaccines. This novel method for in vitro bioconjugate vaccine production has demonstrated advantages for rapid, modular, and portable vaccine prototyping and production compared to existing methods.

[0161] A bioconjugate vaccine against F. tularensis is synthesized in vitro via CFGpS. Francisella tularensis is a Gram-negative bacterium that causes tularemia. F. tularensis is considered one of the most infectious bacteria known to humans: a single airborne bacillus leads to infection in 40–50% of individuals, with a mortality rate of up to 30%

[81] . The gene site for O-antigen biosynthesis was recently characterized

[82] and used to create a bioconjugate vaccine in live E. coli cells [4]. We decided to use this pathway to produce an anti-F. tularensis bioconjugate vaccine using CFGpS. We hypothesized that an anti-F. tularensis bioconjugate vaccine could be synthesized in vitro using CFGpS technology (Figure 3).

[0162] We hypothesized that Und-PP-linked polysaccharide antigens are enriched in S30 lysates via overexpression of the orthogonal glycosylation machinery in an E. coli CFGpS chassis strain lacking WaaL O-antigen ligase. To test this hypothesis, we produced lysates from CLM24 cells overexpressing C. jejuni OST, PglB (CjOST lysate) and the biosynthetic machinery to produce Francisella tularensis Schu S4 O-antigen (FtLLO lysate). This 17 kb gene cluster from F. tularensis Schu S4 contains 15 predicted open reading frames (ORFs) and produces oligosaccharides containing the repeating unit GalNAcAN2QuiNAcQui4NFm (GalNAcAN: 2-acetamido-2-deoxy-D-galacturonamide, QuiNAc: 2-acetamido-2,6-dideoxy-D-glucose, Qui4NFm: 4,6-dideoxy-4-formamido-D-glucose) (Figure 6A)

[82] . To encourage the formation of soluble inverted membrane vesicles carrying membrane-bound components such as FtLLO and CjOST in the crude lysate, the lysate was prepared by high-pressure homogenization.

[0163] We hypothesized that an anti-F. tularensis bioconjugate vaccine could be synthesized in vitro using CFGpS technology. To produce a bioconjugate carrying F. tularensis O polysaccharide (FtO-PS), CjPglB lysate was mixed with FtLLO lysate in a CFGpS reaction, which continued for 20 h at 30 °C. These reactions were carried out using either i) a superfolder green fluorescent protein modified to contain a DQNAT sequon in a flexible linker inserted at residue T216 and a C-terminal His tag (sfGFP-21-DQNAT-6xHis) or ii) a modified maltose binding protein construct with four C-terminal repeats of the DQNAT sequon and a C-terminal His tag (MBP- 4xDQNAT -6xHis) were included in the plasmids encoding either MBP- 4xDQNAT A bioconjugate consisting of E. coli O-antigen linked to -6xHis was recently shown to elicit humoral and cellular immune responses in mice

[83] . Manganese chloride (MnCl2) and n-dodecyl-β-D-maltopyranoside (DDM) detergent were added to the reaction mixture within the first hour of the CFGpS reaction at final concentrations of 25 mM MnCl2, 0.1% w / v DDM to optimize CjPglB activity (Jewett lab, unpublished data). After the CFGpS reaction was allowed to continue for a total of 20 hours at 30°C, glycosylation activity was assayed by immunoblotting using anti-His antibodies and a commercially available monoclonal antibody specific for FtO-PS. The ladder-like pattern observed when FtLLO and CjPglB lysates were mixed is suggestive of FtLLO addition and a consequence of variability in O-PS chain length due to the action of Wzy polymerase (Figure 4A) [4, 11]. These results demonstrate, for the first time, the incorporation of coordinated cell-free carrier protein synthesis and O-antigen addition. Furthermore, we showed that cell-free bioconjugate synthesis occurred within just 20 h, making this cell-free platform an attractive option for rapid production and prototyping of bioconjugate vaccine candidates.

[0164] Bioconjugate vaccines against E. coli O78 and other pathogenic strains can be readily synthesized due to the modular nature of the CFGpS platform. To demonstrate the generality and modularity of the CFGpS approach for bioconjugate production, we sought to produce bioconjugates against additional pathogenic bacteria. We decided to synthesize bioconjugates against the enterotoxigenic E. coli strain O78, since its biosynthetic pathway has been previously described

[84] and there are commercial antisera specific for this strain. The E. coli O78 gene cluster was cloned and encoded within the plasmid pMW07-O78. Expression of the resulting E. coli O78 gene cluster resulted in the production of an O-antigen with the repeating unit GlcNAc2Man2 (GlcNAC: N-acetylglucosamine; Man: mannose) (Figure 6B).

[0165] We prepared lysates enriched for E. coli O78 antigen LLO (EcO78LLO lysate) from CLM24 cells expressing pMW07-O78 using the same method described previously. We then mixed EcO78LLO lysate with CjOST lysate in CFGpS reactions containing plasmids encoding either sfGFP21-DQNAT-6xHis or MBP-4xDQNAT-6xHis. CFGpS reactions were performed as described previously. Glycosylation activity was assayed by immunoblotting with an anti-His antibody and a commercial antiserum against E. coli O78 strains. Again, a ladder-like pattern was observed when both EcO78LLO lysate and CjOST lysate were present in the reaction, indicating good conjugation of EcO78-PS (Figure 4B). The bioconjugate was also cross-reactive with anti-EcO78 serum (data not shown). These results demonstrate the modular nature of the in vitro approach and provide evidence that the CFGpS platform can be used to produce bioconjugate vaccines targeting many different pathogenic bacterial strains. Attractive candidates include pathogenic bacteria whose surface antigen gene clusters are known: Pseudomonas aeruginosa

[11] , Salmonella enterica [9], Shigella dysenteriae [7, 10], Shigella flexneri [8], Staphylococcus aureus [5], Brucella abortus [3], and Burkholderia pseudomallei [6]. These results demonstrate the potential of the in vitro system for rapid production or prototyping of vaccine candidates against a variety of pathogenic bacteria. Moreover, since synthesis can be achieved within just 20 h with a variety of O-PS antigens, this cell-free platform represents an attractive option for rapid production and prototyping of novel bioconjugate vaccine candidates.

[0166] Lyophilized CFGpS reactions maintain bioconjugate synthesis capability. A major limitation of conventional conjugate vaccines is their high cost due to their time-consuming manufacturing process. By comparison, bioconjugate vaccines are less expensive to produce, however, production time is affected by the time required to ferment and purify proteins from live cells. We wanted to test whether our CFGpS reactions could be lyophilized for future room temperature storage and distribution to allow for faster production and broader vaccination campaigns and development efforts compared to in vivo approaches. To test whether this was possible, CFGpS reactions were prepared containing either CjOST lysate only or FtLLO lysate only, or a mixture of both CjOST and FtLLO lysates. These reactions were then lyophilized and reconstituted with 15 μL of nuclease-free water. Reactions contained plasmids encoding either a short chain antibody fragment with a C-terminal DQNAT sequon followed by a His tag (scFv13-R4-DQNAT-6xHis) or MBP-4xDQNAT-6xHis. Premix reactions (lanes 1, 2, 5, 6) were run as is after reconstitution, while reactions containing CjOST lysate or FtLLO lysate alone were mixed following reconstitution (lanes 3, 4, 7). The DQNAT sequon is synthesized and FtO-PS is attached to the target protein when both CjOST lysate or FtLLO lysate are present in the reaction (lanes 2, 4, 6, 7). These results confirmed that CFGpS reaction mixtures can be lyophilized without loss of bioconjugate synthesis capacity, highlighting the potential of our technology for portable, on-demand vaccine production and long-term, refrigerated-free storage.

[0167] conclusion

[0168] We describe herein a novel method for coordinated in vitro transcription, translation, and conjugation of vaccine antigens to carrier proteins. This in vitro approach uniquely (i) deinterferes with cell viability from glycosylation activity and allows for reduced cellular metabolic burden by in vitro reconstitution of glycosylation components, (ii) enables design-build-test (DBT) iterative methods for individual glycosylation components, and (iii) allows for construction of glycosylation pathways within a range of well-defined experimental conditions, including chemical and physical procedures not possible in cells. Furthermore, the system has obvious and commercially attractive applications for producing bioconjugates using FDA-approved carrier proteins such as toxins from Clostridium tetani and Corynebacterium diphtheriae. This novel method for in vitro bioconjugate vaccine production has demonstrated advantages for rapid, modular, and portable vaccine prototyping and production compared to existing methods.

[0169] References for Example 1

[0170] 1. Maue, AC, F. Poly, and P. Guerry, A capsule conjugate vaccine approach to prevent diarrheal disease caused by Campylobacter jejuni. Hum Vaccin Immunother, 2014. 10(6): p. 1499-504.

[0171] 2. Anderson, P., Antibody responses to Haemophilus influenzae type b and diphtheria toxin induced by conjugates of oligosaccharides of the type b capsule with the nontoxic protein CRM197. Infect Immun, 1983. 39(1): p. 233-8.

[0172] 3. Iwashkiw, J.A., et al., Exploiting the Campylobacter jejuni protein glycosylation system for glycoengineering vaccines and diagnostic tools directed against brucellosis. Microb Cell Fact, 2012. 11: p. 13.

[0173] 4. Cuccui, J., et al., Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis. Open Biol, 2013. 3(5): p. 130002.

[0174] 5. Wacker, M., et al., Prevention of Staphylococcus aureus infections by glycoprotein vaccines synthesized in Escherichia coli. J Infect Dis, 2014. 209(10): p. 1551-61.

[0175] 6. Garcia-Quintanilla, F., et al., Production of a recombinant vaccine candidate against Burkholderia pseudomallei exploiting the bacterial N-glycosylation machinery. Front Microbiol, 2014. 5: p. 381.

[0176] 7. Ravenscroft, N., et al., Purification and characterization of a Shigella conjugate vaccine, produced by glycoengineering Escherichia coli. Glycobiology, 2015.

[0177] 8. Kampf, M.M., et al., In vivo production of a novel glycoconjugate vaccine against Shigella flexneri 2a in recombinant Escherichia coli: identification of stimulating factors for in vivo glycosylation. Microb Cell Fact, 2015. 14: p. 12.

[0178] 9. Ihssen, J., et al., Increased efficiency of Campylobacter jejuni N-oligosaccharyltransferase PglB by structure-guided engineering. Open Biol, 2015. 5(4).

[0179] 10. Ihssen, J., et al., Production of glycoprotein vaccines in Escherichia coli. Microb Cell Fact, 2010. 9: p. 61.

[0180] 11. Feldman, M.F., et al., Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli. Proc Natl Acad Sci U S A, 2005. 102(8): p. 3016-21.

[0181] 12. Neuberger, A., Carbohydrates in protein: The carbohydrate component of crystalline egg albumin. Biochem J, 1938. 32(9): p. 1435-51.

[0182] 13. Mescher, M.F. and J.L. Strominger, Purification and characterization of a prokaryotic glucoprotein from the cell envelope of Halobacterium salinarium. J Biol Chem, 1976. 251(7): p. 2005-14.

[0183] 14. Sleytr, U.B., Heterologous reattachment of regular arrays of glycoproteins on bacterial surfaces. Nature, 1975. 257(5525): p. 400-2.

[0184] 15. Szymanski, C.M., et al., Evidence for a system of general protein glycosylation in Campylobacter jejuni. Mol Microbiol, 1999. 32(5): p. 1022-30.

[0185] 16. Linton, D., et al., Identification of N-acetylgalactosamine-containing glycoproteins PEB3 and CgpA in Campylobacter jejuni. Mol Microbiol, 2002. 43(2): p. 497-508.

[0186] 17. Spiro, R.G., Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds. Glycobiology, 2002. 12(4): p. 43R-56R.

[0187] 18. Laine, R.A., The Information-Storing Potential of the Sugar Code. Glycosciences: Status and Perspectives, 1997: p. 1-14.

[0188] 19. Raman, R., et al., Glycomics: an integrated systems approach to structure-function relationships of glycans. Nat Methods, 2005. 2(11): p. 817-24.

[0189] 20. Ohtsubo, K. and J.D. Marth, Glycosylation in cellular mechanisms of health and disease. Cell, 2006. 126(5): p. 855-67.

[0190] 21. Imberty, A. and A. Varrot, Microbial recognition of human cell surface glycoconjugates. Curr Opin Struct Biol, 2008. 18(5): p. 567-76.

[0191] 22. Daniels, M.A., K.A. Hogquist, and S.C. Jameson, Sweet 'n' sour: the impact of differential glycosylation on T cell responses. Nat Immunol, 2002. 3(10): p. 903-10.

[0192] 23. Dube, D.H. and A.R. Bertozzi, Glycans in cancer and inflammation--potential for therapeutics and diagnostics. Nat Rev Drug Discov, 2005. 4: p. 477-88.

[0193] 24. Pinho, S.S. and C.A. Reis, Glycosylation in cancer: mechanisms and clinical implications. Nat Rev Cancer, 2015. 15(9): p. 540-55.

[0194] 25. Ma, Z. and K. Vosseller, Cancer metabolism and elevated O-GlcNAc in oncogenic signaling. J Biol Chem, 2014. 289(50): p. 34457-65.

[0195] 26. Theodoratou, E., et al., The role of glycosylation in IBD. Nat Rev Gastroenterol Hepatol, 2014. 11(10): p. 588-600.

[0196] 27. Albrecht, S., et al., Glycosylation as a marker for inflammatory arthritis. Cancer Biomark, 2014. 14(1): p. 17-28.

[0197] 28. Axford, J.S., Glycosylation and rheumatic disease. Biochim Biophys Acta, 1999. 1455(2-3): p. 219-29.

[0198] 29. Baudoin, L. and T. Issad, O-GlcNAcylation and Inflammation: A Vast Territory to Explore. Front Endocrinol (Lausanne), 2014. 5: p. 235.

[0199] 30. Wang, J.Z., I. Grundke-Iqbal, and K. Iqbal, Glycosylation of microtubule-associated protein tau: an abnormal posttranslational modification in Alzheimer's disease. Nat Med, 1996. 2(8): p. 871-5.

[0200] 31. Guerry, P., et al., Campylobacter polysaccharide capsules: virulence and vaccines. Front Cell Infect Microbiol, 2012. 2: p. 7.

[0201] 32. Kalynych, S., R. Morona, and M. Cygler, Progress in understanding the assembly process of bacterial O-antigen. FEMS Microbiol Rev, 2014. 38(5): p. 1048-65.

[0202] 33. Bacon, D.J., et al., A phase-variable capsule is involved in virulence of Campylobacter jejuni 81-176. Mol Microbiol, 2001. 40(3): p. 769-77.

[0203] 34. Wang, L.X. and B.G. Davis, Realizing the Promise of Chemical Glycobiology. Chem Sci, 2013. 4(9): p. 3381-3394.

[0204] 35. Chauhan, J.S., A. Rao, and G.P. Raghava, In silico platform for prediction of N-, O- and C-glycosites in eukaryotic protein sequences. PLoS One, 2013. 8(6): p. e67008.

[0205] 36. Gavel, Y. and G. von Heijne, Sequence differences between glycosylated and non-glycosylated Asn-X-Thr / Ser acceptor sites: implications for protein engineering. Protein Eng, 1990. 3(5): p. 433-42.

[0206] 37. Lehle, L. and W. Tanner, Glycosyl transfer from dolichyl phosphate sugars to endogenous and exogenous glycoprotein acceptors in yeast. Eur J Biochem, 1978. 83(2): p. 563-70.

[0207] 38. Varki, A., Biological roles of oligosaccharides: all of the theories are correct. Glycobiology, 1993. 3(2): p. 97-130.

[0208] 39. Wilson, I.B., Y. Gavel, and G. von Heijne, Amino acid distributions around O-linked glycosylation sites. Biochem J, 1991. 275 ( Pt 2): p. 529-34.

[0209] 40. Thanka Christlet, T.H. and K. Veluraja, Database analysis of O-glycosylation sites in proteins. Biophys J, 2001. 80(2): p. 952-60.

[0210] 41. Walt, D., et al., Transforming Glycoscience: A Roadmap for the Future. 2012: The National Academies Press.

[0211] 42. Lizak, C., et al., X-ray structure of a bacterial oligosaccharyltransferase. Nature, 2011. 474(7351): p. 350-355.

[0212] 43. Perez, C., et al., Structure and mechanism of an active lipid-linked oligosaccharide flippase. Nature, 2015. 524(7566): p. 433-8.

[0213] 44. Abu-Qarn, M., J. Eichler, and N. Sharon, Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea. Curr Opin Struct Biol, 2008. 18(5): p. 544-50.

[0214] 45. Weerapana, E. and B. Imperiali, Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems. Glycobiology, 2006. 16(6): p. 91R-101R.

[0215] 46. Wacker, M., et al., N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science, 2002. 298(5599): p. 1790-3.

[0216] 47. Glover, K.J., et al., Direct biochemical evidence for the utilization of UDP-bacillosamine by PglC, an essential glycosyl-1-phosphate transferase in the Campylobacter jejuni N-linked glycosylation pathway. Biochemistry, 2006. 45(16): p. 5343-50.

[0217] 48. Glover, K.J., E. Weerapana, and B. Imperiali, In vitro assembly of the undecaprenylpyrophosphate-linked heptasaccharide for prokaryotic N-linked glycosylation. Proc Natl Acad Sci U S A, 2005. 102(40): p. 14255-9.

[0218] 49. Olivier, N.B., et al., In vitro biosynthesis of UDP-N,N'-diacetylbacillosamine by enzymes of the Campylobacter jejuni general protein glycosylation system. Biochemistry, 2006. 45(45): p. 13659-69.

[0219] 50. Murray, G.L., S.R. Attridge, and R. Morona, Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz. Mol Microbiol, 2003. 47(5): p. 1395-406.

[0220] 51. Murray, G.L., S.R. Attridge, and R. Morona, Altering the length of the lipopolysaccharide O antigen has an impact on the interaction of Salmonella enterica serovar Typhimurium with macrophages and complement. J Bacteriol, 2006. 188(7): p. 2735-9.

[0221] 52. Duerr, C.U., et al., O-antigen delays lipopolysaccharide recognition and impairs antibacterial host defense in murine intestinal epithelial cells. PLoS Pathog, 2009. 5(9): p. e1000567.

[0222] 53. Saldias, M.S., X. Ortega, and M.A. Valvano, Burkholderia cenocepacia O antigen lipopolysaccharide prevents phagocytosis by macrophages and adhesion to epithelial cells. J Med Microbiol, 2009. 58(Pt 12): p. 1542-8.

[0223] 54. Lesinski, G.B. and M.A. Westerink, Novel vaccine strategies to T-independent antigens. J Microbiol Methods, 2001. 47(2): p. 135-49.

[0224] 55. Carlson, E.D., et al., Cell-free protein synthesis: applications come of age. Biotechnol Adv, 2012. 30(5): p. 1185-94.

[0225] 56. Hodgman, C.E. and M.C. Jewett, Cell-free synthetic biology: thinking outside the cell. Metab Eng, 2012. 14(3): p. 261-9.

[0226] 57. Nirenberg, M.W. and J.H. Matthaei, The dependence of cell-free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc Natl Acad Sci U S A, 1961. 47: p. 1588-602.

[0227] 58. Chambers, D.A. and G. Zubay, The stimulatory effect of cyclic adenosine 3'5'-monophosphate on DNA-directed synthesis of beta-galactosidase in a cell-free system. Proc Natl Acad Sci U S A, 1969. 63(1): p. 118-22.

[0228] 59. Zalkin, H., C. Yanofsky, and C.L. Squires, Regulated in vitro synthesis of Escherichia coli tryptophan operon messenger ribonucleic acid and enzymes. J Biol Chem, 1974. 249(2): p. 465-75.

[0229] 60. Matthies, D., et al., Cell-free expression and assembly of ATP synthase. J Mol Biol, 2011. 413(3): p. 593-603.

[0230] 61. Kaiser, L., et al., Efficient cell-free production of olfactory receptors: detergent optimization, structure, and ligand binding analyses. Proc Natl Acad Sci U S A, 2008. 105(41): p. 15726-31.

[0231] 62. Wang, X., et al., Peptide surfactants for cell-free production of functional G protein-coupled receptors. Proc Natl Acad Sci U S A, 2011. 108(22): p. 9049-54.

[0232] 63. Bernhard, F. and Y. Tozawa, Cell-free expression--making a mark. Curr Opin Struct Biol, 2013. 23(3): p. 374-80.

[0233] 64. Xu, Y., et al. Production of bispecific antibodies in “Knobs-into-Holes” using a cell-free expression system. in mAbs. 2014. Taylor & Francis.

[0234] 65. Zimmerman, E.S., et al., Production of Site-Specific Antibody-Drug Conjugates Using Optimized Non-Natural Amino Acids in a Cell-Free Expression System. Bioconjugate Chemistry, 2014. 25(2): p. 351-361.

[0235] 66. Ng, P.P., et al., A vaccine directed to B cells and produced by cell-free protein synthesis generates potent antilymphoma immunity. Proc Natl Acad Sci U S A, 2012. 109(36): p. 14526-31.

[0236] 67. Lu, Y., J.P. Welsh, and J.R. Swartz, Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines. Proc Natl Acad Sci U S A, 2014. 111(1): p. 125-30.

[0237] 68. Bundy, B.C., M.J. Franciszkowicz, and J.R. Swartz, Escherichia coli-based cell-free synthesis of virus-like particles. Biotechnol Bioeng, 2008. 100(1): p. 28-37.

[0238] 69. Caschera, F. and V. Noireaux, Synthesis of 2.3 mg / ml of protein with an all Escherichia coli cell-free transcription-translation system. Biochimie, 2014. 99: p. 162-8.

[0239] 70. Calhoun, K.A. and J.R. Swartz, An economical method for cell-free protein synthesis using glucose and nucleoside monophosphates. Biotechnol Prog, 2005. 21(4): p. 1146-53.

[0240] 71. Calhoun, K.A. and J.R. Swartz, Energizing cell-free protein synthesis with glucose metabolism. Biotechnol Bioeng, 2005. 90(5): p. 606-13.

[0241] 72. Lian, Q., H. Cao, and F. Wang, The cost-efficiency realization in the Escherichia coli-based cell-free protein synthesis systems. Appl Biochem Biotechnol, 2014. 174(7): p. 2351-67.

[0242] 73. Zawada, J.F., et al., Microscale to manufacturing scale-up of cell-free cytokine production--a new approach for shortening protein production development timelines. Biotechnol Bioeng, 2011. 108(7): p. 1570-8.

[0243] 74. Stech, M., et al., Cell-free systems: functional modules for synthetic and chemical biology. Adv Biochem Eng Biotechnol, 2013. 137: p. 67-102.

[0244] 75. Brodel, A.K., D.A. Wustenhagen, and S. Kubick, Cell-free protein synthesis systems derived from cultured Mammalian cells. Methods Mol Biol, 2015. 1261: p. 129-40.

[0245] 76. Stech, M., et al., A continuous-exchange cell-free protein synthesis system based on extracts from cultured insect cells. PLoS One, 2014. 9(5): p. e96635.

[0246] 77. Kubick, S., et al., In vitro synthesis of posttranslationally modified membrane proteins. Current Topics in Membranes, 2009. 63(2): p. 25-49.

[0247] 78. Guarino, C. and M.P. DeLisa, A prokaryote-based cell-free translation system that efficiently synthesizes glycoproteins. Glycobiology, 2012. 22(5): p. 596-601.

[0248] 79. Ollis, A.A., et al., Substitute sweeteners: diverse bacterial oligosaccharyltransferases with unique N-glycosylation site preferences. Sci Rep, 2015. 5: p. 15237.

[0249] 80. Ollis, A.A., et al., Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity. Nat Chem Biol, 2014. 10(10): p. 816-22.

[0250] 81. Dennis, D.T., et al., Tularemia as a biological weapon: medical and public health management. Jama, 2001. 285(21): p. 2763-73.

[0251] 82. Prior, J.L., et al., Characterization of the O antigen gene cluster and structural analysis of the O antigen of Francisella tularensis subsp. tularensis. J Med Microbiol, 2003. 52(Pt 10): p. 845-51.

[0252] 83. Ma, Z., et al., Glycoconjugate vaccine containing Escherichia coli O157:H7 O-antigen linked with maltose-binding protein elicits humoral and cellular responses. PLoS One, 2014. 9(8): p. e105215.

[0253] 84. Jansson, P.E., et al., Structural studies of the Escherichia coli O78 O-antigen polysaccharide. Carbohydr Res, 1987. 165(1): p. 87-92.

[0254] 85. Nothaft, H., et al., Study of free oligosaccharides derived from the bacterial N-glycosylation pathway. Proc Natl Acad Sci U S A, 2009. 106(35): p. 15019-24.

[0255] 86. Young, N.M., et al., Structure of the N-linked glycan present on multiple glycoproteins in the Gram-negative bacterium, Campylobacter jejuni. Journal of Biological Chemistry, 2002. 277(45): p. 42530-9.

[0256] 87. Fisher, A.C., et al., Production of secretory and extracellular N-linked glycoproteins in Escherichia coli. Appl Environ Microbiol, 2011. 77(3): p. 871-81.

[0257] 88. Oyston, P.C., A. Sjostedt, and R.W. Titball, Tularaemia: bioterrorism defence renews interest in Francisella tularensis. Nat Rev Microbiol, 2004. 2(12): p. 967-78.

[0258] 89. Chen, L., et al., Outer membrane vesicles displaying engineered glycotopes eliciting protective antibodies. Proc Natl Acad Sci USA, 2016. 113(26): p. E3609-18.

[0259] 90. Kumru, OS, et al., Vaccine instability in the cold chain: mechanisms, analysis and formulation strategies. Biologicals, 2014. 42(5): p. 237-59.

[0260] Example 2 - Broad applicability of the cell-free glycoprotein synthesis platform

[0261] The emerging field of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Herein, we describe a novel cell-free glycoprotein synthesis technology that seamlessly combines asparagine-linked protein glycosylation with protein biosynthesis. The technology utilizes several Escherichia coli strains in source cell lysates that selectively enrich for glycosylated components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-selective glycosylation of target proteins. The platform is highly modular, and the use of multiple individual OSTs allows for the appending of structurally diverse glycans, including several bacterial O-polysaccharides, to many glycoprotein therapeutics and glycoconjugate vaccine carriers. The following table summarizes the unique glycosylation components characterized in the disclosed cell-free glycoprotein synthesis system, namely the E. coli origin strain, OST, and LLO, as well as the glycoprotein acceptor.

[0262] [Table 2-1] [Table 2-2]

[0263] Multiple individual oligosaccharyltransferases (OSTs) can be enriched in E. coli lysates to activate cell-free protein glycosylation. For the initial development of our cell-free glycoprotein synthesis technology, we selected the well-tested bacterial protein glycosylation locus (pgl) from the Gram-negative bacterium Campylobacter jejuni as our model glycosylation system [1]. This gene cluster identifies a conserved motif (D / XX) in the acceptor protein. -1 -NX +1 -S / T, in the formula, X +1 and X -1 It encodes an asparagine-linked (N-linked) glycosylation pathway functionally similar to those in eukaryotes and archaea, involving a single subunit OST, CjPglB, which catalyzes the intrablock transfer of a preassembled GlcGalNAc5Bac heptasaccharide (where Bac is bacillosamine) from the lipid carrier undecaprenyl pyrophosphate (Und-PP) onto asparagine residues in the endoplasmic reticulum (Eu, where Bac is any residue except proline) [3].

[0264] Given the cell-free, straightforward nature, it is possible to prototype functionally interchangeable and alternative biochemical reaction components. One straightforward way this can be achieved is by combining separately prepared lysates, each of which is selectively enriched for a given enzyme [4, 5]. As a proof of concept, separately prepared CjLLO and CjPglB lysates were mixed and then incubated with scFv13-R4 DQNATA mixture was prepared using an anti-β-galactosidase (βgal) single-chain variable fragment (scFv) antibody modified at the C-terminus with a single DQNAT motif [6] and DNA encoding the acceptor. The resulting mixture expressed scFv13-R4 as observed by immunoblots probed with anti-His antibody and hR6 serum that detects C. jejuniglycan. DQNAT (Fig. 7a). scFv13-R4 DQNAT In addition, we also expressed various model acceptor proteins, which were created by grafting the 21 amino acid sequence from the C. jejuni glycoprotein AcrA, and which were expressed as superfolder GFP (sfGFP 217-DQNAT The mixed lysate reaction scheme produced sfGFP at 100% conversion. 217-DQNAT The acceptor protein could be glycosylated (Figure 7a).

[0265] Next, we sought to demonstrate that the mixed lysate approach could be used to rapidly probe the activity of four additional bacterial OSTs. Crude lysates were prepared separately from E. coli CLM24 origin strains heterologously overexpressing one of the following bacterial OSTs: Campylobacter coli PglB (CcPglB), Desulfovibrio desulfuricans PglB (DdPglB), Desulfovibrio gigas PglB (DgPglB), or Desulfovibrio vulgaris PglB (DvPglB). Each OST lysate was mixed with a lysate enriched for CjLLO and then incubated with sfGFP. 217-DQNAT or its target protein sfGFP 217-AQNAT Upon completion of the cell-free glycoprotein synthesis reaction, the expression and glycosylation status of both sfGFPs were followed by immunoblot analysis, which revealed information about the sequon preferences of these homologous enzymes. For example, the mixed lysate containing CcPglB expressed sfGFP 217-AQNAT but not sfGFP 217-DQNATCcPglB was observed to efficiently glycosylate the AQNAT sequon (Fig. 7b). This activity profile for CcPglB was identical to that observed for CjPglB, which was not surprising based on its high sequence similarity to CjPglB (~81%) [7]. In contrast, a lysate mixture containing an OST from Desulfovibrio sp., which has low sequence identity to CjPglB (~15-20%), showed a more moderate sequon preference (Fig. 7b). Specifically, a mixed lysate containing DdOST and DvOST preferentially glycosylated the AQNAT sequon, whereas a DgPglB-enriched extract mixture, which modified both (D / A)QNAT motifs, had nearly the same efficiency. Taken together, these results suggest that various oligosaccharyltransferase enzymes (OSTs) are functionally enriched in our cell-free lysates to produce targeted glycoproteins. We postulate that the range of OSTs used in our cell-free system can be expanded to include those originating from organisms such as protozoa, yeast, and mammals.

[0266] Finally, we showed that O-antigen polysaccharides corresponding to clinically relevant vaccine antigens could be transferred to proteins using our lysate mixing approach with multiple OST enzymes. Here, we focused on the conjugation of E. coli O78 polysaccharide antigens (EcO78-PS) to inform the development of antibacterial vaccines, as O78 strains were the major cause of traveler's diarrhea in developing regions. Crude lysates enriched for CjPglB or CcPglB as described previously were mixed with LLO lysates prepared from cells expressing the biosynthetic pathway for the synthesis of EcO78-PS. These lysate mixtures were then conjugated with sfGFP, a lysate containing the sfGFP-PS gene. 217-DQNATWe supplemented the cells with plasmid DNA encoding EcO78-PS. Immunoblot analysis revealed that both CjPglB and CcPglB transferred EcO78-PS to proteins (Figure 8). These data indicate that a variety of oligosaccharyltransferase enzymes (OSTs) can be used to synthesize conjugate vaccine candidates in our cell-free platform. We further developed this platform to enable on-demand and decentralized production of conjugate vaccines, as described below.

[0267] So far, only the C. jejuni glycosylation pathway has been reconstituted in vitro [8, 9], which left an open question as to whether our system could be reconstituted with various LLOs. Therefore, to expand the scope of glycan structures beyond the C. jejuni heptasaccharide, we performed in vitro glycosylation using lipid-linked oligosaccharide donors extracted from E. coli cells harboring alternative glycan biosynthetic pathways. These included LLOs bearing the following glycan structures: (i) the native C. larihexasaccharide N-glycan

[10] ; (ii) a modified GalNAc5GlcNAc based on the C. larihexasaccharide N-glycan

[11] ; (iii) the native Wallinella succinogenes hexasaccharide N-glycan

[12] ; (iv) a modified E. coli O9 primer-adapter O-PS glycan, Man3GlcNAc; (v) F. tularensis O-PS, Qui4NFm-(GalNAcAN)2-QuiNAc structure

[13] , and (vi) a eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 [6]. scFv13-R4 carrying each of these various glycans DQNAT Glycosylation of PglB was observed to occur only in the presence of CjPglB (Figure 9). Taken together, these results demonstrate that structurally diverse glycans, including those that resemble eukaryotic structures and O-antigen polysaccharides, can be exchanged in a modular manner in cell-free glycosylation reactions.

[0268] Building on the successful incorporation of several glycan structures within our cell-free glycoprotein synthesis platform, we focused our attention on a particular group of glycans, bacterial O-polysaccharides. As antimicrobial resistance is an increasingly serious problem worldwide, strategies to accelerate the production of new antibiotics and vaccines are needed. In particular, conjugate vaccines composed of bacterial O-polysaccharide antigens conjugated to immunogenic carrier proteins are more than 90% effective in preventing bacterial infections with very rare instances of resistance. We hypothesized that we could use our cell-free glycoprotein synthesis technology to prepare bioconjugate vaccine candidates in the context of pathogen-specific polysaccharides. As a proof of concept, we synthesized sfGFP- DQNAT We attempted to express and site-specifically glycosylate FtO-PS. Glycosylation activity was assayed by immunoblotting using anti-His and monoclonal antibodies specific for FtO-PS (Fig. 10A). The ladder-like pattern observed when FtLLO and CjPglB lysates were mixed is indicative of FtLLO addition and results from variability in O-PS chain length due to the action of Wzy polymerase.

[0269] To demonstrate the generality and modularity of our cell-free approach for the production of various conjugate vaccines, we attempted to produce vaccines against two pathogenic strains of E. coli, namely, enterotoxigenic E. coli strains O7 and O78. E. coli strain O7 is the main cause of urinary tract infections, and strain O78 is the main cause of diarrheal symptoms in travelers and the main cause of diarrheal disease in low-income countries. One-pot lysates were prepared from cells expressing CjPglB and the biosynthetic pathway for either E. coli O78 or O7 antigens. Cell-free reactions containing these lysates resulted in ladder-like glycosylation of our sfGFP mutants by immunoblotting with anti-His antibodies (Figure 10B,C) and antisera against E. coli O78 and O7 antigens (data not shown). Importantly, these results demonstrate the potential of our integrated technology for coordinated carrier protein synthesis and pathogen-specific O-antigen loading. Moreover, our cell-free platform represents an attractive option for the rapid production and prototyping of novel conjugate vaccine candidates from various structures of bacterial O-polysaccharides.

[0270] We have demonstrated that our fully integrated cell-free glycoprotein synthesis platform enables the one-pot synthesis of various N-glycoproteins. Using this system, the reaction was converted to scFv13-R4 DQNAT or sfGFP 217-DQNAT This was achieved by preparing plasmid DNA encoding either scFv13-R4 or scFv13-R5, leading to the production of the target protein with 100% protein glycosylation (Figure 11a). DQNAT and sfGFP 217-DQNATThe protein was not affected by N-glycan addition (data not shown) and maintained biological activity as measured by the ability of the scFv to bind antigen substrate in an ELISA format and by the fluorescent signal of sfGFP. More importantly, our one-pot cell-free glycosylation system was similarly performed with reactions prepared with plasmid DNA encoding several human erythropoietin (hEPO) glycovariants, including those in which the native sequon at residues N24 (22-AENIT-26), N38 (36-NENIT-40), or N83 (81-LVNSS-85) was individually mutated to the optimal bacterial sequon, DQNAT (Fig. 11b). Western blot analysis clearly revealed detectable glycosylation of each hEPO glycovariant with 100% glycosylation product for the N24 and N38 sites, and ∼30–40% for the N83 site (Fig. 11b). All three glycosylated hEPO variants maintained biological activity indistinguishable from that measured for the corresponding aglycosylated counterparts (data not shown). Collectively, these findings establish that a one-pot lysate can coactivate protein synthesis and N-glycosylation in a manner that efficiently yields glycosylated proteins, including those of human origin.

[0271] Next, we sought to expand the capabilities of our cell-free system to synthesize authentic glycoprotein acceptors beyond model target-like sfGFP or small molecule cytokine glycoproteins such as hEPO. Specifically, we turned our efforts toward the expression of glycoconjugate vaccine carriers. Expressing conjugate vaccines in vivo in E. coli was limited by the use of carrier proteins that have not yet been approved by the FDA for use as protein adjuvants in the context of conjugate vaccines. FDA-approved carrier proteins, such as tetanus or diphtheria toxin proteins, have yet to be demonstrated to be compatible with N-linked glycosylation in the periplasm of live E. coli cells. In practice, these FDA-approved carriers have proven difficult to express in vivo in E. coli, often requiring purification and refolding of insoluble products from inclusion bodies, fusion of expression partners to enhance soluble expression, or expression of protein fragments to favor full-length proteins. In contrast, cell-free protein synthesis approaches have recently shown promise for difficult-to-express proteins

[14] . The carrier proteins we focused on here include nonacylated H. influenzae protein D (PD), N. meningitidisporin protein (PorA), and genetically detoxified mutants of Corynebacterium diphtheriae toxin (CRM197) and Clostridium tetani toxin (TT). We also tested the expression of the fragment C (TTc) and light chain (TTlight) domains of TT, as well as E. coli MBP. To allow glycosylation, all carriers were modified at their C-terminus with four tandem repeats of an optimal bacterial glycosylation motif, DQNAT

[15] . An internal DQNAT glycosylation site (sfGFP) was added to the GFP-GFP vector. 217-DQNAT )

[16] was used as a model protein to facilitate development of the system. All eight carriers were 14 ~50-650 μg mL as determined by C-leucine incorporation -1 It was synthesized in vitro with a soluble yield of (data not shown).

[0272] We next sought to synthesize conjugated versions of these carrier proteins by combining their in vitro expression with one-pot, cell-free glycosylation. For vaccine targets, we focused on the highly virulent Francisella tularensis strain Schu S4. To glycosylate proteins with FtO-PS, we produced a one-pot lysate from glycoengineered E. coli cells expressing the FtO-PS biosynthetic pathway and the CjPglB enzyme. This lysate, which contained lipid-bound FtO-PS and catalytically active CjPglB, was transformed with sfGFP. 217-DQNAT The plasmid DNA encoding the glycosylated sfGFP was used to catalyze the reaction. 217-DQNAT Efficient synthesis of sfGFP was observed by immunoblotting using anti-His antibody or a monoclonal antibody specific for FtO-PS (FIG. 12a). 217-DQNAT Glycosylation of was observed only in reactions containing an intact glycosylation pathway and the preferred DQNAT glycosylation sequence (Figure 12a).

[0273] Next, we investigated whether an FDA-approved carrier could be similarly combined with FtO-PS in our cell-free reactions. 4xDQNAT , P.D. 4xDQNAT , PorA 4xDQNAT , T.T.c. 4xDQNAT , TTlight 4xDQNAT , and CRM197 4xDQNATFollowing addition of plasmid DNA encoding CjPglB, respective glycosylation with FtO-PS was observed for reactions enriched with lipid-linked FtO-PS and CjPglB, but not with control reactions lacking CjPglB (Figure 12b). We observed conjugation of high molecular weight FtO-PS species (~10-20 kDa) to all protein carriers tested, which is important because longer glycan chain lengths have been shown to enhance the efficacy of conjugate vaccines against F. tularensis

[17] . Taken together, these data indicate that the cell-free glycoprotein synthesis platform can efficiently synthesize and N-glycosylate a variety of glycoprotein therapeutics and glycoconjugate carriers, including those approved by the FDA.

[0274] Prokaryotic cell-free protein synthesis has emerged as a promising tool for recombinant protein production [18, 19]. Among them, Escherichia coli-based lysates, called S30 lysates, are one of the most commonly used and well-tested sources of lysates

[20] . So far, however, only a limited number of E. coli strains have been shown to be suitable for efficient cell-free protein synthesis, and even fewer have been investigated in the context of cell-free protein synthesis coupled to post-translational modifications of proteins [8, 21]. To determine whether various common laboratory E. coli strains, including i) rE. coli 705, ii) BL21(DE3), and iii) CLM24, could be used as chassis strains to support our integrated cell-free protein synthesis and glycosylation, we first prepared crude S30 lysates from these strains using a robust procedure based on sonication

[22] . Next, we performed small-scale batch-wise continuous cell-free protein synthesis using a modified PANOx-SP system

[23] and glycosylation [9] using: (i) purified CjPglB catalyst; (ii) oligosaccharide donor CjLLO

[24] ; and (iii) model acceptor protein scFv13-R4. DQNATThese lysates were supplemented with plasmid DNA encoding [6]. After an overnight reaction, highly efficient glycosylation was observed, as seen in the monoglycosylated (g1) form in anti-His immunoblots and in scFv13-R4 with hR6 serum (Figure 13). DQNAT Complete detection of C. jejuni glycan bound to IgG was achieved in all reactions, as evidenced by a mobility shift of the target protein.

[0275] One major challenge in using E. coli-based biotherapeutics production systems is the concern over lipid A or endotoxin contamination. Lipid A toxins are recognized by toll-like receptor 4 (TLR4) and lead to the production of proinflammatory cytokines such as tumor necrosis factor α and interleukin-1β

[25] , which are necessary to fight infection but whose high levels can cause lethal septic shock

[26] . Because cell-free glycoprotein synthesis reactions rely on lipid-associated components, including OST and LLO, standard detoxification approaches involving the removal of lipid A

[27] may reduce the activity or concentration of our glycosylated components. To address this issue, we applied a strategy for detoxification of lipid A molecules by strain engineering [28, 29]. In particular, we showed that deletion of the acyltransferase gene lpxM and overexpression of the F. tularensis phosphatase LpxE in E. coli resulted in the production of a nearly identical pentaacylated, monophosphorylated lipid A that had significantly reduced toxicity but remained active as an adjuvant

[28] . To generate a detoxified lipid A structure in the context of iVAX, we prepared lysates from a ΔlpxM derivative of CLM24 co-expressing the FtLpxE and FtO-PS glycosylation pathways. Lysates obtained from this strain showed significantly reduced levels of toxicity compared to wild-type CLM24 lysates expressing CjPglB and FtO-PS, as measured by human TLR4 activation in HEK-Blue hTLR4 reporter cells

[29] (data not shown). To verify whether the genome coding on the original strain has a negative effect on protein synthesis and glycosylation efficiency, we modified glycosylated sfGFP with FtO-PS. DQNATWe used S30 lysates prepared from these modified strains to produce E. coli. Efficient protein synthesis and N-glycosylation were observed in all reactions. These data indicate that the genome modification, specific structural editing of lipid A relative to the original strain, does not affect the activity of the membrane-bound CjPglB and FtO-PS components of our cell-free glycosylation reactions (Figure 14). Overall, the results here demonstrate that a variety of prokaryotic lysates, including several common laboratory E. coli and genome-modified E. coli strains, can be utilized to functionally reconstitute our cell-free protein synthesis and N-glycosylation reactions.

[0276] References for Example 2

[0277] 1. Wacker, M., et al., N-linked glycosylation in Campylobacter jejuni and its functional transfer into E. coli. Science, 2002. 298(5599): p. 1790-3.

[0278] 2. Weerapana, E. and B. Imperiali, Asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems. Glycobiology, 2006. 16(6): p. 91R-101R.

[0279] 3. Kowarik, M., et al., Definition of the bacterial N-glycosylation site consensus sequence. The EMBO Journal, 2006. 25(9): p. 1957-1966.

[0280] 4. Dudley, Q.M., A.S. Karim, and M.C. Jewett, Cell‐free metabolic engineering: Biomanufacturing beyond the cell. Biotechnology Journal, 2015. 10(1): p. 69-82.

[0281] 5. Karim, A.S. and M.C. Jewett, A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery. Metab Eng, 2016. 36: p. 116-126.

[0282] 6. Valderrama-Rincon, J.D., et al., An engineered eukaryotic protein glycosylation pathway in Escherichia coli. Nat Chem Biol, 2012. 8(5): p. 434-6.

[0283] 7. Ollis, A.A., et al., Substitute sweeteners: diverse bacterial oligosaccharyltransferases with unique N-glycosylation site preferences. Scientific Reports, 2015. 5: p. 15237.

[0284] 8. Jaroentomeechai, T., et al., Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nat Commun, 2018. 9(1): p. 2686.

[0285] 9. Guarino, C. and M.P. DeLisa, A prokaryote-based cell-free translation system that efficiently synthesizes glycoproteins. Glycobiology, 2012. 22(5): p. 596-601.

[0286] 10. Schwarz, F., et al., Relaxed acceptor site specificity of bacterial oligosaccharyltransferase in vivo. Glycobiology, 2011. 21(1): p. 45-54.

[0287] 11. Schwarz, F., et al., A combined method for producing homogeneous glycoproteins with eukaryotic N-glycosylation. Nat Chem Biol, 2010. 6(4): p. 264-6.

[0288] 12. Jervis, A.J., et al., Characterization of the structurally diverse N-linked glycans of Campylobacter species. J Bacteriol, 2012. 194(9): p. 2355-62.

[0289] 13. Fulop, M., et al., Role of antibody to lipopolysaccharide in protection against low- and high-virulence strains of Francisella tularensis. Vaccine, 2001. 19(31): p. 4465-72.

[0290] 14. Perez, J.G., J.C. Stark, and M.C. Jewett, Cell-free synthetic biology: Engineering beyond the cell. Cold Spring Harb. Perspect. Biol., 2016.

[0291] 15. Chen, M.M., K.J. Glover, and B. Imperiali, From peptide to protein: comparative analysis of the substrate specificity of N-linked glycosylation in C. jejuni. Biochemistry, 2007. 46(18): p. 5579-85.

[0292] 16. Jaroentomeechai, T., et al., Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nat. Commun., 2018. 9(1): p. 2686.

[0293] 17. Stefanetti, G., et al., Glycoconjugate vaccine using a genetically modified O antigen induces protective antibodies to Francisella tularensis. Proc. Natl. Acad. Sci. U. S. A., 2019. 116(14): p. 7062-7070.

[0294] 18. Carlson, E.D., et al., Cell-free protein synthesis: Applications come of age. Biotechnology Advances, 2011. 30(5).

[0295] 19. Salehi, A.S., et al., Escherichia coli-based cell-free extract development for protein-based cancer therapeutic production. Int J Dev Biol, 2016. 60(7-8-9): p. 237-243.

[0296] 20. Chong, S., Overview of cell-free protein synthesis: historic landmarks, commercial systems, and expanding applications. Curr Protoc Mol Biol, 2014. 108: p. 16 30 1-11.

[0297] 21. Oza, J.P., et al., Robust production of recombinant phosphoproteins using cell-free protein synthesis. Nature Communications, 2015. 6: p. 8168.

[0298] 22. Kwon, Y.-C. and M.C. Jewett, High-throughput preparation methods of crude extract for robust cell-free protein synthesis. Scientific Reports, 2015. 5: p. 8663.

[0299] 23. Jewett, M.C. and J.R. Swartz, Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnology and bioengineering, 2004. 86(1): p. 19-26.

[0300] 24. Jaroentomeechai, T., et al., A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases. Methods Enzymol, 2017. 597: p. 55-81.

[0301] 25. Rietschel, E.T., et al., Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J., 1994. 8(2): p. 217-25.

[0302] 26. Russell, J.A., Management of sepsis. N. Engl. J. Med., 2006. 355(16): p. 1699-1713.

[0303] 27. Petsch, D. and F.B. Anspach, Endotoxin removal from protein solutions. J. Biotechnol., 2000. 76(2-3): p. 97-119.

[0304] 28. Chen, L., et al., Outer membrane vesicles displaying engineered glycotopes eliciting protective antibodies. Proc. Natl. Acad. Sci. USA, 2016.

[0305] 29. Needham, BD, et al., Modulating the innate immune response by combinatorial engineering of endotoxin. Proc. Natl. Acad. Sci. USA, 2013. 110(4): p. 1464-9.

[0306] Example 3 - On-Demand, Cell-Free Biological Manufacturing of Conjugate Vaccines at the Point of Care

[0307] overview

[0308] Conjugate vaccines are among the most effective methods to prevent bacterial infections and represent a promising strategy to combat drug-resistant pathogens. However, as mentioned previously, existing manufacturing approaches limit access to conjugate vaccines due to centralized manufacturing and cold chain distribution requirements. To address these limitations, a modular technology for in vitro bioconjugate vaccine expression (iVAX) is described below. iVAX contains a portable, lyophilized lysate from attenuated, non-pathogenic Escherichia coli. Upon rehydration, the iVAX reaction synthesizes clinically relevant doses of bioconjugate vaccines against a variety of bacterial pathogens within an hour. This example surprisingly and unexpectedly shows that iVAX synthesizes a vaccine against the highly virulent pathogen Francisella tularensis subgenus tularensis (type A) strain Schu S4 that elicits pathogen-specific antibodies in mice at significantly higher levels compared to vaccines produced using engineered bacteria. The iVAX platform holds the promise of accelerating the development of novel bioconjugate vaccines with enhanced access through distribution without the need for refrigeration and manufacturing at the point of care.

[0309] Introduction

[0310] Antimicrobial resistance is predicted to threaten up to 10 million lives per year by 2050 (Review on Antimicrobial Resistance, 2014), necessitating new strategies for developing and distributing antibiotics and vaccines. Conjugate vaccines, typically composed of pathogen-specific capsules (CPS) or O-antigen polysaccharides (OPS) associated with immunostimulatory protein carriers, are among the safest and most effective methods to prevent life-threatening bacterial infections (Jin et al., 2017; Trotter et al., 2008; Weintraub, 2003). In particular, the implementation of meningococcal and pneumococcal conjugate vaccines has significantly reduced bacterial meningitis and pneumonia worldwide (Novak et al., 2012; Poehling et al., 2006), in addition to reducing antibiotic resistance of target strains (Roush et al., 2008). However, despite its safety and efficacy, global childhood vaccination coverage for conjugate vaccines remains low at ~30%, with lack of access or limited coverage being the major reasons for the remaining disease burden (Wahl et al., 2018). In addition, in 2018, the WHO prequalification of Typhbar-TCV® to prevent typhoid fever will approve the first conjugate vaccine in approximately 10 years. To address the emergence of drug-resistant pathogens, new technologies are needed to accelerate the development and global distribution of conjugate vaccines.

[0311] Contributing to the slow pace of development and distribution of conjugate vaccines is the fact that these molecules are particularly difficult and expensive to produce. The traditional process for producing conjugate vaccines involves chemical conjugation of carrier proteins with polysaccharide antigens purified from large-scale cultures of pathogenic bacteria. Large-scale fermentation of pathogenic bacteria results in high production costs due to associated biosafety hazards and process development challenges. In addition, chemical conjugation can alter the structure of the polysaccharide, resulting in the loss of protective epitopes (Bhushan et al., 1998). To address these challenges, we have demonstrated that polysaccharide protein “bioconjugates” can be produced in Escherichia coli using protein-glycan coupling technology (PGCT) (Feldman et al., 2005). In this approach, engineered E. coli cells covalently conjugate heterologously expressed CPS or O-PS antigens to carrier proteins via asparagine-linked glycosylation reactions catalyzed by the Campylobacter jejuni oligosaccharyltransferase enzyme PglB (CjPglB) (Cuccui et al., 2013; Garcia-Quintanilla et al., 2014; Ihssen et al., 2010; Ma et al., 2014; Marshall et al., 2018; Wacker et al., 2014; Wetter et al., 2013). Despite this progress, both chemical conjugation and PGCT approaches rely on live bacterial cells and require centralized manufacturing facilities from which the vaccine is distributed through a refrigerated supply chain. Refrigeration is important to avoid spoilage of the conjugate vaccine due to precipitation, as well as the significant loss of pathogen-specific polysaccharides due to both heating and freezing (Frasch, 2009; WHO, 2014).Only one conjugate vaccine, MenAfriVac™, is known to remain active outside the cold chain for up to 4 days, which allowed high vaccine coverage and approximately 50% reduction in costs during vaccination in the meningitis belt of sub-Saharan Africa (Lydon et al., 2014). However, this required significant investments in the development and validation of a heat-stable vaccine. Broadly speaking, the need for cold chain refrigeration creates economic and logistical challenges that limit the reach of vaccination campaigns and provide a barrier to disease eradication, especially in developing regions (Ashok et al., 2017; Wahl et al., 2018).

[0312] Cell-free protein synthesis (CFPS) offers the opportunity to accelerate vaccine development as well as enable decentralized, cold-chain independent biological manufacturing by using cell lysates rather than live cells to synthesize proteins in vitro (Carlson et al., 2012). Importantly, the CFPS platform (i) enables protein manufacturing at the point of care as relevant quantities of protein can be synthesized in vitro within just a few hours, (ii) can be lyophilized for distribution at ambient temperature and reconstituted by simply adding water (Adiga et al., 2018; Pardee et al., 2016), and (iii) avoids biosafety concerns associated with the use of live, living cells outside of a controlled laboratory environment. CFPS has recently been used to enable on-demand and portable manufacturing of aglycosylated protein vaccines (Adiga et al., 2018; Pardee et al., 2016). Moreover, we recently described a cell-free glycoprotein synthesis technology that allows for one-pot production of glycosylated proteins, including human glycoproteins and eukaryotic glycans (Jaroentomeechai et al., 2018). Despite these advances, cell-free and even decentralized manufacturing systems have historically been limited by their inability to synthesize glycosylated proteins at relevant titers and carrying various glycan structures, such as polysaccharide antigens, that are necessary for bioconjugate vaccine production (Perez et al., 2016).

[0313] To address these limitations, the iVAX (in vitro bioconjugate vaccine expression) platform is described below, which allows for the rapid development of biosynthesis of conjugate vaccines in cell-free reactions and independence from the cold chain (Figure 15). iVAX was modified to have the following features. First, iVAX was fast, with the ability to produce multiple individual doses of bioconjugates within an hour. Second, iVAX was robust, yielding equivalent amounts of bioconjugates over a range of operating temperatures. Third, iVAX was modular, offering the ability to rapidly exchange carrier proteins used in licensed conjugate vaccines, including those conjugated with polysaccharide antigens. As described in this example, this modularity was exploited to generate an array of vaccine candidates targeted against a variety of bacterial pathogens, including the highly virulent Francisella tularensis subgenus tularensis (Type A) strain Schu S4, enterotoxigenic (ETEC) E. coli O78, and urinary tract infectious (UPEC) E. coli O7. Fourth, iVAX, derived from a lyophilized cell-free reaction that operates with a just-add-water strategy, is storage stable. Fifth, iVAX is safe and utilizes lipid A modifications that effectively circumvent the high levels of endotoxin present in unmodified E. coli production platforms. These results demonstrate that anti-F. tularensis bioconjugates derived from lyophilized, low-endotoxin iVAX reactions elicited pathogen-specific antibody responses in mice and were superior to bioconjugates produced using established PGCT approaches in live cells. Overall, the iVAX platform offers a new way to deliver the protective benefits of an important class of antibacterial vaccines to both developed and developing countries.

[0314] result

[0315] In vitro synthesis of approved vaccine carrier proteins

[0316] To demonstrate proof-of-principle for cell-free bioconjugate vaccine production, we expressed in vitro a set of carrier proteins currently used in FDA-approved conjugate vaccines. The production of these carrier proteins in soluble conformations in vitro represented an important benchmark, since their expression in live E. coli proved attractive, often requiring purification and refolding of insoluble products from inclusion bodies (Haghi et al., 2011; Stefan et al., 2011), for fusion of expression partners such as maltose binding protein (MBP) to enhance soluble expression (Figueiredo et al., 1995; Stefan et al., 2011), or for the expression of truncated protein variants that favor full-length proteins (Figueiredo et al., 1995). In contrast, cell-free protein synthesis approaches have recently shown promise for difficult-to-express proteins (Perez et al., 2016). Carrier proteins expressed in the in vitro system described herein include nonacylated H. influenzae protein D (PD), N. meningitidysporin protein (PorA), and genetically detoxified mutants of Corynebacterium diphtheriae toxin (CRM197) and Clostridium tetani toxin (TT). We also tested expression of the fragment C (TTc) and light chain (TTlight) domains of TT, as well as E. coli MBP. Although MBP is not a licensed carrier, it has demonstrated immunostimulatory properties (Fernandez et al., 2007) and, when linked to O-PS, was found to generate polysaccharide-specific humoral and cellular immune responses in mice (Ma et al., 2014). Similarly, the TT domain, TTlight, and TTc are not used in licensed vaccines, but are immunostimulatory and individually sufficient for protection against C. tetani challenge in mice (Figueiredo et al., 1995). To enable glycosylation, all carriers were modified at their C-terminus with four tandem repeats of the optimal bacterial glycosylation motif, DQNAT ( Chen et al., 2007 ).A C-terminal 6xHis tag was also included to allow purification and detection via Western blot analysis. An internal DQNAT glycosylation site (sfGFP. 217-DQNAT ) (Jaroentomeechai et al., 2018) were used as model proteins to facilitate development of the system.

[0317] All eight carriers 14 ~50-650 μg mL as determined by C-leucine incorporation -1 In particular, MBP was synthesized in vitro with a soluble yield of 1000 mg / kg / day (Figure 16a). 4xDQNAT and P.D. 4xDQNAT The mutants were each 500 μg mL -1 and 200 μg mL -1 The product was nearly 100% soluble with a yield of 100% and was expressed exclusively as a full-length product by Western blot analysis (Figure 16b). Similar soluble yields were observed with CRM197 4xDQNAT Except for (Figure 21a), all carriers were observed at 25°C, 30°C, and 37°C, which are known to be thermosensitive (WHO, 2014). These results indicate that the described method of cell-free carrier biosynthesis is robust over a 13°C range of temperatures and can be used in environments where precise temperature control is not feasible.

[0318] The unrestricted reaction environment of these cell-free reactions has enabled the manipulation of the chemistry and reaction environment to improve the production of more complex carriers. For example, the membrane protein PorA 4xDQNATIn the case of CRM197, lipid nanodiscs were added to enhance soluble expression (Figure 21b). Nanodiscs provide a cell membrane mimic to co-translationally stabilize hydrophobic regions of membrane proteins (Bayburt and Sligar, 2010). For expression of TT containing an intermolecular disulfide bond, expression was performed under oxidizing conditions for 2 h (Knapp et al., 2007), which improved assembly of heavy and light chains into a full-length product and minimized protease degradation of full-length TT (Figure 21c). In vitro synthesized CRM197 4xDQNAT and T.T. 4xDQNAT were comparable in size to commercially available purified diphtheria toxin (DT) and TT protein standards and reactive with α-DT and αTT antibodies, respectively (Figure 21d, e), indicating that both were produced in immunologically relevant conformations. When administered without conjugated polysaccharide, this is known as CRM197, and TT are FDA-approved vaccine antigens for diphtheria and tetanus, respectively. Similarly, these results highlight the ability of CFPS to express approved conjugated vaccine carrier proteins in soluble conformations over a range of temperatures.

[0319] On-Demand Synthesis of Bioconjugate Vaccines

[0320] Polysaccharide-conjugated versions of these carrier proteins were then synthesized by combining their in vitro expression with one-pot, cell-free glycosylation. As a model vaccine target, the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4, a Gram-negative, coccobacillus and causative agent of tularemia, was used. This bacterium has been classified as a class A bioterrorism agent due to its high mortality rate, infection at low doses, and ability to be aerosolized (Oyston et al., 2004). Although no currently licensed vaccine exists against F. tularensis, several studies have independently confirmed the important role of antibodies directed against F. tularensis LPS, specifically, the O-PS repeating unit, in providing protection against the Schu S4 strain (Fulop et al., 2001; Lu et al., 2012). More recently, Pseudomonas aeruginosa exotoxin A (EPA) produced using PGCT (Cuccui et al., 2013; Marshall et al., 2018) has been reported. DNNNS-DQNRT ) A bioconjugate vaccine containing F. tularensis Schu S4 O-PS (FtO-PS) conjugate to a carrier protein was shown to be protective against challenge with the Schu S4 strain in a rat inhalation model of tularemia (Marshall et al., 2018). In light of these earlier findings, we tested the ability of the iVAX platform to produce on-demand anti-F. tularensis bioconjugate vaccine candidates by conjugating FtO-PS structures to various carrier proteins in vitro.

[0321] FtO-PS is composed of the 826 Da repeating tetrasaccharide unit Qui4NFm-(GalNAcAN)2-QuiNAc (Qui4NFm: 4,6-dideoxy-4-formamido-D-glucose; GalNAcAN: 2-acetamido-2-deoxy-D-galacturonamide; QuiNAc: 2-acetamido-2,6-dideoxy-D-glucose) (Prior et al., 2003). To glycosylate proteins with FtO-PS, we prepared an iVAX lysate from glycoengineered E. coli cells expressing the FtO-PS biosynthetic pathway and the oligosaccharyltransferase enzyme CjPglB (Figure 17a). This lysate, containing lipid-bound FtO-PS and active CjPglB, was visualized by immunofluorescence assay using sfGFP. 217-DQNAT The target protein sfGFP, which contains a mutated glycosylation site (AQNAT) that is not modified by CjPglB (Kowarik et al., 2006), was used to catalyze an iVAX reaction prepared with plasmid DNA encoding the target protein sfGFP. Control reactions, in which the addition of FtO-PS was not expected, were performed with lysates from cells lacking either the FtO-PS pathway or the CjPglB enzyme. 217-DQNAT Plasmids encoding sfGFP or 217-AQNAT Reactions prepared with a plasmid encoding sfGFP containing iVAX lysate were also tested. 217-DQNAT In reactions prepared with a plasmid encoding sfGFP, immunoblotting with anti-His antibody or a commercial monoclonal antibody specific for FtO-PS revealed a ladder-like banding pattern (Figure 17b). This ladder is characteristic of FtO-PS addition and is a result of variability in O-PS chain length due to the action of Wzy polymerase (Cuccui et al., 2013; Feldman et al., 2005; Prior et al., 2003). 217-DQNATGlycosylation of was observed only in reactions containing an intact glycosylation pathway and the preferred DQNAT glycosylation sequence (Figure 17b). This glycosylation profile was highly reproducible across biological replicates from the same lot of lysate (Figure 17c, right) and using different lots of lysate (Figure 17c, left). In vitro protein synthesis and glycosylation were observed after 1 hour, with the amount of conjugated polysaccharide reaching a maximum between 0.75 and 1.25 hours (Figure 22). Similar glycosylation reaction kinetics were observed at 37°C, 30°C, 25°C, and room temperature (~21°C), indicating that the iVAX reaction is robust across a range of temperatures (Figure 22).

[0322] We next investigated whether FDA approved carriers could be conjugated similarly to FtO-PS in the iVAX reaction. 4xDQNAT , P.D. 4xDQNAT , PorA 4xDQNAT , T.T.c. 4xDQNAT , TTlight 4xDQNAT , and CRM197 4xDQNAT Following addition of plasmid DNA encoding FtO-PS, respective glycosylation with FtO-PS was observed for iVAX reactions enriched for lipid-bound FtO-PS and CjPglB, but not for control reactions lacking CjPglB (Figure 18). We observed conjugation of high molecular weight FtO-PS species (~10-20 kDa) to all protein carriers tested, which is important since longer glycan chain lengths have been shown to enhance the efficacy of conjugate vaccines against F. tularensis (Stefanetti et al., 2019). Notably, attempts to synthesize the same panel of bioconjugates using the established PGCT approach in live E. coli yielded less promising results. Specifically, we observed lower levels of FtO-PS glycosylation and lower molecular weight FtO-PS species for all PGCT-derived bioconjugates compared to their iVAX-derived counterparts (Figure 23). The same trend was observed for the most common PGCT carrier protein, EPA. DNNNS-DQNRTSimilar results were observed for PGCT- versus iVAX-derived bioconjugates with similar results (Cuccui et al., 2013; Ihssen et al., 2010; Marshall et al., 2018; Wacker et al., 2014; Wetter et al., 2013) (Figure 18; Figure 23). In addition, only limited expression of the PorA membrane protein was achieved in vivo (Figure 23). Taken together, these data indicate that iVAX offers advantages over PGCT for the production of bioconjugate vaccine candidates with high molecular weight O-PS antigens efficiently conjugated to diverse and potential membrane-bound carrier proteins.

[0323] Next, we determined whether the yield of bioconjugates produced using iVAX would allow for the production of relevant vaccine doses. Recent clinical data show that doses of the bioconjugate vaccine candidate, 1-10 μg, are well tolerated and effective in stimulating the production of antibacterial IgG (Hatz et al., 2015; Huttner et al., 2017; Riddle et al., 2016). To assess expression titers and for further experiments, MBP 4xDQNAT and P.D. 4xDQNAT were generated and these carriers were expressed in vitro with high soluble titers and no cleavage of the products (Figure 16). 14 A reaction lasting ∼1 h was observed with ∼20 μg mL C-leucine incorporation and densitometric analysis. -1 Glycosylated MBP 4xDQNAT and P.D. 4xDQNAT (Figure 24a). It should be noted that the vaccine is currently distributed in vials containing 1-20 doses of vaccine to minimize waste (Humphreys, 2011). Thus, these yield results indicate that multiple doses per mL can be synthesized in 1 hour using the iVAX platform.

[0324] To demonstrate the modularity of the iVAX approach for bioconjugate production, we produced bioconjugates carrying O-PS antigens from additional pathogens, including ETEC E. coli strain O78 and UPEC E. coli strain O7. E. coli O78 is a major cause of diarrheal disease in developing countries, especially in children, and is a major cause of traveler's diarrhea (Qadri et al., 2005), whereas O7 strains are a common cause of urinary tract infections (Johnson, 1991). Like FtO-PS, the biosynthetic pathways of EcO78-PS and EcO7-PS have been previously described and confirmed to produce O-PS antigens with the repeating units GlcNAc2Man2 (Jansson et al., 1987) and Qui4NAcMan(Rha)GalGlcNAc (L'vov et al., 1984) (GlcNAc: N-acetylglucosamine; Man: mannose; Qui4NAc: 4-acetamido-4,6-dideoxy-D-glucopyranose; Rha: rhamnose; Gal: galactose), respectively. 4xDQNAT or sfGFP 217-DQNAT Using iVAX lysates from cells expressing CjPglB and either the EcO78-PS or EcO7-PS pathways in reactions prepared with plasmids, carrier glycosylation was observed only when both lipid-bound O-PS and CjPglB were present in the reaction (Figure 24b, c). These results demonstrate the modular production of bioconjugates against multiple bacterial pathogens, enabled by the compatibility of multiple heterologous O-PS pathways with in vitro carrier protein synthesis and glycosylation.

[0325] Endotoxin editing and lyophilization result in a safe and portable iVAX reaction

[0326] A major challenge inherent to the use of any E. coli-based system for biopharmaceutical manufacturing is the presence of lipid A or endotoxin, which is known to contaminate protein products and cause lethal septic shock at high levels (Russell, 2006). As a result, the amount of endotoxin in formulated biopharmaceuticals is regulated by the United States Pharmacopeia (USP), the US Food and Drug Administration (FDA), and the European Medicines Agency (EMEA) (Brito and Singh, 2011). Because the iVAX reaction relies on lipid-associated components such as CjPglB and FtO-PS, standard toxicity removal approaches involving removal of lipid A (Petsch and Anspach, 2000) may reduce activity or concentration of glycosylated components in addition to increased cost and processing complexity.

[0327] To address this issue, we tested previously reported strategies to detoxify lipid A molecules by strain engineering (Chen et al., 2016; Needham et al., 2013). In particular, deletion of the acyltransferase gene lpxM and overexpression of the F. tularensis phosphatase LpxE in E. coli were shown to result in the production of a nearly homogeneous pentaacylated, monophosphorylated lipid A with greatly reduced toxicity, but retaining activity as an adjuvant (Chen et al., 2016). This pentaacylated, monophosphorylated lipid A was structurally identical to the major component of monophosphoryl lipid A (MPL) from Salmonella minnesota R595, an approved adjuvant composed of a mixture of monophosphorylated lipids (Casella and Mitchell, 2008). To produce detoxified lipid A structures in the context of iVAX, we produced lysates from a ΔlpxM derivative of CLM24 co-expressing FtLpxE and the FtO-PS glycosylation pathway (Figure 19a). Lysates obtained from this strain showed significantly reduced levels of toxicity compared to wild-type CLM24 lysates expressing CjPglB and FtO-PS (Figure 19b), as measured by human TLR4 activation in HEK-Blue hTLR4 reporter cells (Needham et al., 2013). Importantly, structural remodeling of lipid A did not affect the activity of the membrane-bound CjPglB and FtO-PS components of the iVAX reaction (Figure 25a). By engineering the chassis strain for lysate production, we were able to produce lysates of <1,000 EU mL, which is within the range of values ​​reported for commercial protein-based vaccine products. -1 iVAX lysates were produced with endotoxin concentrations ranging from 0.288 to 180,000 EU mL -1 )(Brito and Singh, 2011).

[0328] A major limitation of conventional conjugate vaccines is that they must be refrigerated, making it difficult for these vaccines to be distributed to remote or resource-limited settings (WHO, 2014). The ability to lyophilize iVAX reactions for storage and distribution at ambient temperatures could alleviate the logistical challenges associated with the refrigerated supply chain required for conventional vaccines. To investigate this possibility, we lyophilized the detoxified iVAX lysate using two different methods: sfGFP 217-DQNAT The plasmid encoding the target protein was used to produce FtO-PS bioconjugates either by performing the reaction immediately after preparation or by performing the same reaction mixture after lyophilization and rehydration (Figure 19c). 217-DQNAT Conjugation of FtO-PS to MBP was observed with nearly identical modification levels when CjPglB was present (Figure 19d). In addition, detoxified, lyophilized iVAX reactions yielded FtO-PS-conjugated MBP in a manner that was indistinguishable from the non-lyophilized preparation and with good lot-to-lot reproducibility. 4xDQNAT and P.D. 4xDQNAT The volume was adjusted to 5 mL for production of iVAX (Figure 25b, c). The ability to lyophilize the iVAX reaction and produce bioconjugates without specialized equipment highlighted the potential for portable, on-demand vaccine production.

[0329] The in vitro synthesized bioconjugates elicit pathogen-specific antibodies in mice.

[0330] To validate the efficacy of the bioconjugates produced using the iVAX platform, the ability of the iVAX-derived bioconjugates to elicit anti-FtLPS antibodies in mice was tested (Figure 20a). 4xDQNAT or PD 4xDQNATWe found that BALB / c mice fed with PGCT produced high titers of FtLPS-specific IgG antibodies, which were significantly elevated compared to titers measured in the serum of control mice fed PBS or the aglycosylated versions of the respective carrier proteins (Fig. 20b, Fig. 26). 4xDQNAT IgG titers measured in serum from mice fed the antibody were similar to those observed in the control group (Figure 20b, Figure 26), consistent with the much weaker glycosylation of this candidate relative to its iVAX-derived counterpart (Figure 23). 4xDQNAT and P.D. 4xDQNAT Both bioconjugates produced similar levels of IgG production, and neither resulted in observable adverse events in mice, confirming the modularity and safety of the technology for the production of bioconjugate vaccine candidates.

[0331] IgG titers were characterized by analysis of IgG1 and IgG2a subtypes, and iVAX-derived FtO-PS-conjugated MBP 4xDQNAT and P.D. 4xDQNAT Both of these results were comparable to those of all control groups, as well as to the PGCT-derived glycosylated MBP. 4xDQNAT We found that iVAX enhanced the production of IgG1 antibodies by >2 orders of magnitude against iVAX (Figure 20c). This analysis also revealed that both iVAX-derived bioconjugates generated a strongly Th2-biased (IgG1>>IgG2a) response, which is characteristic of most conjugate vaccines (Bogaert et al., 2004). Taken together, these results provide evidence that the iVAX platform delivers vaccine candidates capable of generating strong, pathogen-specific humoral immune responses and recapitulate the Th2 bias that is characteristic of licensed conjugate vaccines.

[0332] Consideration

[0333] In this example, we established iVAX, a cell-free platform for portable, on-demand production of bioconjugate vaccines. We showed that the iVAX reaction can be detoxified to ensure the safety of bioconjugate vaccine products that can be lyophilized for cold-chain independent distribution and reactivated for high-yield bioconjugate production by simply adding water. As a model vaccine candidate, we showed that an anti-F. tularensis bioconjugate derived from a lyophilized, endotoxin-edited iVAX reaction generated pathogen-specific IgG antibodies in mice as part of a Th2-biased immune response, a hallmark of licensed conjugate vaccines.

[0334] The iVAX platform includes several novel features. First, iVAX is modular, as demonstrated herein through the compatibility of (i) carrier proteins, including those used in licensed conjugate vaccines, and (ii) bacterial O-PS antigens from F. tularensis subgenus tularensis (type A) Schu S4, ETEC E. coli O78, and UPEC E. coli O7. This represents the first demonstration of oligosaccharyl transferase-mediated O-PS conjugation to authentic FDA-approved carrier proteins, likely due to historical challenges associated with expression of licensed carriers in live E. coli (Figueiredo et al., 1995; Haghi et al., 2011; Stefan et al., 2011). Further deployment of the O-PS pathway used in iVAX should be able to accommodate the commonly observed clustering of polysaccharide biosynthetic genes within the genomes of pathogenic bacteria (Raetz and Whitfield, 2002). This feature could make iVAX an attractive option for rapid de novo development of bioconjugate vaccine candidates in response to disease pandemics or against emerging drug-resistant bacteria.

[0335] Second, the iVAX reaction is inexpensive, producing ~20 μg bioconjugate mL per hour. -1(Figure 24a) at ~12 mL -1 (Figure 27, Table 1). Assuming a dose size of 10 μg, the iVAX reaction can produce a vaccine dose for ~$6. For comparison, CDC costs per dose of conjugate vaccines range from ~$9.50 for the H. influenzae vaccine ActHIB® to ~$75 and ~$118 for the meningococcal vaccine Menactra® and pneumococcal vaccine Prevnar 13®, respectively (CDC, 2019).

[0336] Third, the iVAX-derived bioconjugates were shown to be more effective in generating FtLPS-specific IgGs than bioconjugates derived from live E. coli cells using PGCT ( FIG. 20 ). Without wishing to be bound by any theory, one possible explanation for this enhanced efficacy is the more extensive glycosylation observed for the in vitro expressed bioconjugates, with greater carbohydrate loading and modification with larger molecular weight FtO-PS species compared to their PGCT-derived counterparts. The reduced presence of high molecular weight O-PS species observed in bioconjugates produced using PGCT may be due to competition between the O-antigen polymerases Wzy and PglB in vivo. In contrast, the in vitro approach of the present disclosure separates O-PS synthesis (which occurs in vivo prior to lysate production) from glycosylation (which occurs in vitro as part of the iVAX reaction). These results are consistent with predictions regarding PGCT-induced anti-F. tularensis bioconjugates showing that increasing the ratio of conjugate FtO-PS to protein in the bioconjugates results in higher protection against F. tularensis Schu S4 in a rat inhalation model of tularemia (Marshall et al., 2018). In addition, a recent study showed that a conjugate vaccine made with high molecular weight (80 kDa) FtO-PS linked to TT showed better protection against intranasal challenge with a live F. tularensis vaccine strain compared to a conjugate made with a low molecular weight (25 kDa) polysaccharide (Stefanetti et al., 2019).

[0337] With the potential portable production of bioconjugate vaccines, iVAX addresses a major gap in both cell-free and decentralized biological manufacturing technologies. The production of glycosylated derivatives has yet to be demonstrated in a cell-based decentralized biological manufacturing platform (Crowell et al., 2018; Perez-Pinera et al., 2016), and existing cell-free platforms using E. coli lysates lack the ability to synthesize glycoproteins (Boles et al., 2017; Murphy et al., 2019; Pardee et al., 2016; Salehi et al., 2016). While glycosylated human erythropoietin has been produced in a freeze-dried Chinese hamster ovary cell lysate (Adiga et al., 2018)-based cell-free biological manufacturing platform, this, as well as the majority of other eukaryotic cell-free and cell-based systems, rely on endogenous protein glycosylation machinery. As a result, these expression platforms offer little control over the produced glycan structures or underlying glycosylation reactions, and significant optimization is often required to achieve acceptable glycosylation profiles (Adiga et al., 2018). In contrast, the iVAX platform is enabled by a lysate derived from E. coli that lacks endogenous protein glycosylation pathways, allowing bottom-up engineering of desired glycosylation activity (Jaroentomeechai et al., 2018). The ability to modify desired glycosylation activity in iVAX will enable the production of an important class of antibacterial vaccines in therapeutic settings.

[0338] iVAX offers a novel approach for rapid development and portable, on-demand biological manufacturing of bioconjugate vaccines. The iVAX platform eases cold chain requirements, which could facilitate delivery of medicines to areas with limited infrastructure and minimize vaccine losses due to spoilage. Additionally, the ability to rapidly manufacture vaccine candidates in vitro provides a unique tool for rapid response to pathogen outbreaks and emergent threats.

[0339] Experimental model and subject details

[0340] bacteria

[0341] E. coli strains were grown in Luria Bertani (LB), LB-Lennox, or 2xYTP medium at 30-37°C in a shaking incubator as specified in the Method Details section below. E. coli NEB 5-alpha (NEB) was used for plasmid cloning and purification. E. coli CLM24 or CLM24 ΔlpxM strains were used to prepare cell-free lysates. E. coli CLM24 was used as a chassis for expression of bioconjugates in vivo using PGCT. CLM24 is a sugar-optimized derivative of W3110 that carries a deletion in the gene encoding WaaL ligase, facilitating the accumulation of preassembled glycans with undecaprenyl diphosphate (Feldman et al., 2005). CLM24 ΔlpxM has an endogenous acyltransferase deletion and serves as a chassis strain for the production of detoxified cell-free lysates.

[0342] Human cell culture

[0343] HEK-Blue hTLR4 cells (Invivogen) were cultured in a humidified incubator at 37°C with 5% CO2 as specified by the manufacturer, with 10% fetal bovine serum, 50 U mL -1 of penicillin, 50 mg mL -1 of streptomycin, and 100 μg mL-1 The cells were cultured in DMEM medium high glucose / L-glutamine supplemented with 100 mg of Normacin™.

[0344] mouse

[0345] Six-week-old female BALB / c mice obtained from Harlan Sprague Dawley were randomly assigned to experimental groups. Mice were housed in groups of 3-5 animals and cages were changed weekly. All animals were observed daily for behavior and physical health, including visible signs of injury and abnormal grooming habits. Mice were also observed 24 and 48 hours after each injection, and no abnormal responses were reported. The study was performed at Cornell University in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All procedures were performed in accordance with Protocol 2012-0132, approved by the Cornell University Institutional Animal Care and Use Committee under relevant university, state, and federal regulations, including the Animal Welfare Act, Public Health Service Policy on Humane Care and Use of Laboratory Animals, New York State Health Law Article 5, Section 504, and Cornell University Policy 1.4.

[0346] Learn how

[0347] Structure of CLM24 ΔlpxM strain

[0348] E. coli CLM24 ΔlpxM was generated using the Datsenko-Wanner gene knockout method (Datsenko and Wanner, 2000). Briefly, CLM24 cells were transformed with the pKD46 plasmid encoding the λ Red system. Transformants were cultured at 50 μg mL -1 25 mL of LB-Lennox medium containing 10 g L -1 of tryptone, 5g L -1 of yeast extract, and 5 g L -1 OD of 0.5-0.7 in 1000 mM NaCl 600 The cells were cultured for 1 h, harvested, washed three times with 25 mL of ice-cold 10% glycerol to make them electrocompetent, and resuspended in a final volume of 100 μL of 10% glycerol. In parallel, the lpxM knockout cassette was created by PCR amplification of the kanamycin resistance cassette from pKD4 using forward and reverse primers with homology to lpxM. Electrocompetent cells were transformed with 400 ng of the lpxM knockout cassette and 30 μg mL of glycerol for selection of resistant colonies. -1 The cells were plated on LB agar with 100 μg / mL kanamycin. The plates were incubated at 37°C to allow cells to recover from the pKD46 plasmid. Colonies that grew on kanamycin were confirmed to have acquired the knockout cassette by colony PCR and DNA sequencing. These confirmed colonies were then transformed with pCP20 to remove the kanamycin resistance gene by Flp-FRT recombination. Transformants were cultured at 50 μg mL -1 Following selection, colonies were grown in submerged culture at 42°C to recover the cells for the pCP20 plasmid. Colonies were confirmed to have both the lpxM and knockout cassette deletions by colony PCR and DNA sequencing, and cultured on LB agar supplemented with 50 μg mL -1It was confirmed that the strain had lost both kanamycin and carbenicillin resistance by replica plating on LB agar plates with carbenicillin or kanamycin of 100 mg / ml. The structure of this strain and all primers used for validation are listed in Figure 28, Table 2.

[0349] All plasmids used in the study are listed in Figure 29, Table 3. Plasmid pJL1-MBP 4xDQNAT , pJL1-PD 4xDQNAT , pJL1-PorA 4xDQNAT , pJL1-TTc 4xDQNAT , pJL1-TTlight 4xDQNAT , pJL1-CRM197 4xDQNAT , and pJL1-TT 4xDQNAT , C-terminally inserted between the NdeI and SalI restriction sites in the pJL1 vector. 4xDQNAT The plasmid pJL1-EPA was generated by PCR amplification of a codon-optimized gene construct purchased from IDT, carrying a -6xHis tag (Fisher et al., 2011), followed by Gibson Assembly. DNNNS-DQNRT was assembled using the same approach but without the addition of the C-terminal 4xDQNAT-6xHis tag. 4xDQNAT , pTrc99s-ssDsbA-PD 4xDQNAT , pTrc99s-ssDsbA-PorA 4xDQNAT , pTrc99s-ssDsbA-TTc 4xDQNAT , pTrc99s-ssDsbA-TTlight 4xDQNAT , and pTrc99s-ssDsbA-EPA DNNNS-DQNRTwere generated by PCR amplification of the respective carrier protein genes and insertion into the pTrc99s vector between the NcoI and HindIII restriction sites by Gibson Assembly. Plasmid pSF-CjPglB-LpxE was assembled by a similar approach but by insertion of the lpxE gene from pE (Needham et al., 2013) between the NdeI and NsiI restriction sites in the pSF vector. The insert was amplified by PCR amplification using Phusion® High-Fidelity DNA Polymerase (NEB) with forward and reverse primers designed using the NEBuilder® Assembly Tool (nebuilder.neb.com) and purchased from IDT. The pJL1 vector (Addgene69496) was digested with the restriction enzymes NdeI and SalI-HF® (NEB). The pSF vector was digested with the restriction enzymes NdeI and NotI (NEB). The PCR products were gel extracted using an EZNA Gel Extraction Kit (Omega Bio-Tek), mixed with Gibson assembly reagents, and incubated for 1 h at 50° C. Plasmid DNA from the Gibson assembly reaction was transformed into E. coli NEB 5-α cells, and the circular construct was diluted at 50 μg ml -1 (Sigma) and selected using kanamycin. Sequence-verified clones were purified using the EZNA Plasmid Midi Kit (Omega Bio-Tek) for use in CFPS and iVAX reactions.

[0350] Preparation of cell-free lysates

[0351] The E. coli CLM24 source strain was cultivated in 2xYTP medium (10 g / L yeast extract, 16 g / L tryptone, 5 g / L NaCl, 7 g / L K2HPO4, 3 g / L KH2PO4, pH 7.2) in shake flasks (1 L scale) or Sartorius Stedim BIOSTAT Cplus bioreactors (10 L scale) at 37 °C. Protein synthesis yields and glycosylation activity were well reproducible across different batches of lysates at both small and large scale. To produce CjPglB enriched lysates, CLM24 cells carrying the plasmid pSF-CjPglB (Ollis et al., 2015) were used as the source strain. To produce FtO-PS enriched lysates, CLM24 carrying the plasmid pGAB2 (Cuccui et al., 2013) was used as the source strain. To produce one-pot lysates containing both CjPglB and FtO-PS, EcO78-PS, or EcO7-PS, CLM24 carrying pSF-CjPglB and one of the following bacterial O-PS biosynthetic pathway plasmids: pGAB2 (FtO-PS), pMW07-O78 (EcO78-PS), and pJHCV32 (EcO7-PS) were used as source strains. CjPglB expression was maintained at an OD of 0.8–1.0 with 0.02% (w / v) L-arabinose. 600 The cells were induced at 30° C. and the cultures were shifted to 30° C. Cells were cultured at a final OD of 3.0. 600The cells were cultured for 1 h at 4°C for 1 min at which time the cells were pelleted by centrifugation at 5,000xg for 15 min at 4°C. The cell pellets were then washed three times with cold S30 buffer (10 mM Tris-acetate pH 8.2, 14 mM magnesium acetate, 60 mM potassium acetate) and pelleted at 5000xg for 10 min at 4°C. After the final wash, the cells were pelleted at 7000xg for 10 min at 4°C, weighed, snap frozen in liquid nitrogen, and stored at -80°C. To generate cell lysates, the cell pellets were homogenously resuspended in 1 mL of S30 buffer per g of wet cell mass. The cells were disrupted by a single pass through an Avestin EmulsiFlex-B15 (1 L scale) or EmulsiFlex-C3 (10 L scale) high pressure homogenizer at 20,000-25,000 psi. The lysate was then centrifuged twice at 30,000×g for 30 min to remove cellular debris. The supernatant was transferred to a clean microcentrifuge tube and incubated at 37° C. for 60 min with shaking at 250 rpm. Following centrifugation (15,000×g) for 15 min at 4° C., the supernatant was collected, aliquoted, snap-frozen in liquid nitrogen, and stored at −80° C. The S30 lysate was active over approximately three freeze-thaw cycles and contained ∼40 g / L of total protein as measured by the Bradford assay.

[0352] Cell-free Protein Synthesis

[0353] CFPS reactions were carried out in 1.5 mL microcentrifuge tubes (15 μL scale), 15 mL conical tubes (1 mL scale), or 50 mL conical tubes (5 mL scale) using a modified PANOx-SP system (Jewett and Swartz, 2004). The CFPS reaction mixture contained the following components: 1.2 mM ATP; 0.85 mM each of GTP, UTP, and CTP; 34.0 μg mL -1 of L-5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid); 170.0 μg mL -1E. coli tRNA mixture; 130 mM potassium glutamate; 10 mM ammonium glutamate; 12 mM magnesium glutamate; 2 mM each of the 20 amino acids; 0.4 mM nicotinamide adenine dinucleotide (NAD); 0.27 mM coenzyme A (CoA); 1.5 mM spermidine; 1 mM putrescine; 4 mM sodium oxalate; 33 mM phosphoenolpyruvate (PEP); 57 mM HEPES; 13.3 μg mL -1 of plasmid; and 27% v / v cell lysate. For reaction volumes ≥1 mL, the plasmid was used at 6.67 μg mL because this low plasmid concentration conserved reagents without affecting protein synthesis yield or kinetics. -1 For PorA expression, 1 μg mL -1 Nanodiscs were supplemented with CRM197, which were either prepared as previously described (Schoborg et al., 2017) or purchased (Cube Biotech). 4xDQNAT For expression of , unless otherwise noted, CFPS was carried out at 25° C. for 20 hours. For all other carrier proteins, CFPS was carried out at 30° C. for 20 hours unless otherwise noted.

[0354] TT containing intermolecular disulfide bonds 4xDQNAT Expression of CFPS was performed under oxidizing conditions, for which lysates were preconditioned with 750 μM iodoacetamide for 30 min at room temperature to covalently bind free sulfhydryl groups (-SH), and the reaction mixture was supplemented with 200 mM glutathione and 10 μM recombinant E. coli DsbC (Knapp et al., 2007) in a 4:1 ratio of oxidized to reduced forms.

[0355] In vitro bioconjugate vaccine expression (iVAX)

[0356] A two-phase scheme was implemented for in vitro expression and glycosylation of carrier proteins in crude lysates. In the first phase, CFPS was performed for 15 min at 25-30 °C as previously described. In the second phase, protein glycosylation was initiated by the addition of MnCl2 and DDM at final concentrations of 25 mM and 0.1% (w / v), respectively, and continued for a total reaction time of 1 h at 30 °C. Protein synthesis yields and glycosylation activity were well reproducible across biological replicates of iVAX reactions at both small and large scales. Reactions were then centrifuged at 20,000 x g for 10 min to remove insoluble or aggregated protein products, and the supernatants were analyzed by SDS-PAGE and Western blotting.

[0357] Aglycosylation and purification of glycosylated carriers from iVAX reactions were performed using Ni-NTA agarose (Qiagen) according to the manufacturer's protocol. Briefly, 0.5 mL of Ni NTA agarose per 1 mL of cell-free reaction mixture was equilibrated with Buffer 1 (300 mM NaCl, 50 mM NaH2PO4) with 10 mM imidazole. The soluble fraction from the iVAX reaction was loaded onto the Ni-NTA agarose and incubated at 4°C for 2-4 hours to allow binding of the 6xHis-tagged protein. Following incubation, the cell-free reaction / agarose mixture was loaded onto a polypropylene column (BioRad) and washed twice with 6 column volumes of Buffer 1 with 20 mM imidazole. Protein was eluted in four fractions, each with 0.3 mL of buffer 1 with 300 mM imidazole per mL of cell-free reaction mixture. All buffers were used and stored at 4°C. Protein was diluted at 1-2 mg mL in sterile 1xPBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) at 4°C. -1 The solution was stored at a final concentration of 100 mM KOH.

[0358] Expression of bioconjugates using protein glycan coupling technology (PGCT) in vivo

[0359] Plasmids encoding bioconjugate carrier protein genes preceded by the DsbA leader sequence for translocation to the periplasm were transformed into CLM24 cells carrying pGAB2 and pSF-CjPglB. CLM24 carrying pGAB2 alone was used as a negative control. Transformed cells were cultured in 5 mL of LB medium (10 g L -1 of yeast extract, 5g L -1 of tryptone, 5g L -1 The cells were cultured overnight at 37°C in 100 mL of LB (137 mM NaCl). The next day, the cells were subcultured in 100 mL of LB and grown at 37°C for 6 hours, after which the culture was supplemented with 0.2% arabinose and 0.5 mM IPTG to induce expression of CjPglB and the bioconjugate carrier protein, respectively. Protein expression was then carried out for 16 hours at 30°C, at which point the cells were harvested. The cell pellet was resuspended in 1 mL of sterile PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4) and lysed using a Q125 Sonicator (Qsonica, Newtown, CT) at 40% amplitude with 10 sec on / 10 sec off cycles for a total of 5 minutes. The soluble fraction was isolated following centrifugation at 15,000 rpm for 30 min at 4° C. Proteins were purified from the soluble fraction using Ni-NTA spin columns (Qiagen) according to the manufacturer's protocol.

[0360] Western blot analysis

[0361] Samples were run on 4-12% Bis Tris SDS-PAGE gels (Invitrogen). Following electrophoretic separation, proteins were transferred from the gel to Immobilon P polyvinylidene difluoride (PVDF) membranes (0.45 μm) according to the manufacturer's protocol. The membranes were washed with PBS (80 g L -1 NaCl, 0.2 g L -1 KCl, 1.44 g L -1 Na2HPO4, 0.24g L -1100 ml of KH2PO4, pH 7.4) followed by incubation in Odyssey® Blocking Buffer (LI-COR) for 1 h. After blocking, the membrane was washed with PBST (80 g L -1 NaCl, 0.2 g L -1 KCl, 1.44 g L -1 Na2HPO4, 0.24g L -1 KH2PO4, 1 mL L -1 The membranes were then washed six times with 100 mL of Tween-20, pH 7.4. For iVAX samples, the membranes were probed with both anti-6xHis tag and anti-O-PS-antibodies or antisera specific for the O-antigen of interest, if commercially available. Membranes were probed for at least 1 hour at room temperature with shaking, and then the membranes were washed with PBST and probed with fluorescently labeled secondary antibodies in the same manner as described above. Membranes were imaged using an Odyssey® Fc Imaging System (LI-COR). CRM197 and TT preparations were compared to commercially available DT and TT standards (Sigma) and orthogonally detected by the same SDS-PAGE procedure followed by Western blot analysis using polyclonal antibodies recognizing diphtheria or tetanus toxin, respectively. All antibodies and dilutions used are listed in Figure 30, Table 4.

[0362] TLR4 activation assay

[0363] HEK-Blue hTLR4 cells (Invivogen) were cultured at 37°C in a humidified incubator containing 5% CO2 with 10% fetal bovine serum, 50 U mL -1 of penicillin, 50 mg mL -1 of streptomycin, and 100 μg mL -1 After reaching ~50-80% confluency, cells were cultured at 1.4 x 10 per mL in HEK-Blue detection medium (Invivogen). 5Cells were plated in 96-well plates at a density of 100 ng / mL. Antigen was added at the following concentrations: 100 ng / mL. -1 of purified protein; 100ng μL in lysate -1 Total protein content of purified E. coli O55:B5 LPS (Sigma-Aldrich) and attenuated E. coli O55:B5 (Sigma-Aldrich) was 1.0 ng mL -1 and served as positive and negative controls, respectively. Plates were incubated at 37°C, 5% CO2 for 10-16 h, after which absorbance was measured at 620 nm. Statistical significance was determined using paired t-tests.

[0364] Immunization of mice

[0365] Six groups of 6-week-old female BALB / c mice (Harlan Sprague Dawley) were subcutaneously injected with 100 μL of PBS (pH 7.4) alone or with purified aglycosylated MBP, FtO-PS-conjugated MBP, aglycosylated PD, or FtO-PS-conjugated PD as previously described (Chen et al., 2010). Groups consisted of 6 mice, except for the PBS control group, which consisted of 5 mice. The amount of antigen in each preparation was standardized to 7.5 μg to ensure that equal amounts of aglycosylated protein or bioconjugate were administered each time. The purified protein groups formulated in PBS were mixed with an equal volume of incomplete Freund's adjuvant (Sigma-Aldrich) prior to injection. Prior to immunization, material from each group (5 μL) was streaked onto LB agar plates and incubated overnight at 37°C to ensure sterility and to measure endotoxin activity by TLR4 activation assay. Mice from each group were challenged with the same dose of antigen 21 and 42 days after the first immunization. Blood was obtained by mandibular bleeding on days -1, 21, 35, 49, and 63, and by cardiac puncture at the end of the study on day 70. Mice were observed for behavioral changes and physical health 24 and 48 hours after each injection, and no abnormal responses were reported. This study and all procedures were performed in accordance with protocol 2012-0132 approved by the Cornell University Institutional Animal Care and Use Committee.

[0366] Enzyme-linked immunosorbent assay

[0367] F. tularensis LPS-specific antibodies produced by immunized mice were measured by indirect ELISA using a modification of a previously described protocol (Chen et al., 2010). Briefly, serum was isolated from blood samples collected after centrifugation at 5000xg for 10 min and stored at -20°C; 96-well plates (Maxisorp; Nunc Nalgene) were filled with 5 μg mL of F. tularensis LPS in PBS. -1Plates were coated with F. tularensis LPS (BEI resources) at a concentration of 1:100 and incubated overnight at 4°C. The next day, plates were washed three times with PBST (PBS, 0.05% Tween-20, 0.3% BSA) and blocked overnight at 4°C with 5% nonfat dry milk (Carnation) in PBS. Samples were serially diluted two-fold in triplicate ranging from 1:100 to 1:12,800,000 in blocking buffer and added to the plates for 2 hours at 37°C. Plates were washed three times with PBST and incubated for 1 hour at 37°C in the presence of one of the following HRP-conjugated antibodies (all from Abcam, used at a dilution of 1:25,000): goat anti-mouse IgG, anti-mouse IgG1, and anti-mouse IgG2a. After three additional washes with PBST, 3,3'-5,5'-tetramethylbenzidine substrate (1-Step Ultra TMB-ELISA; Thermo-Fisher) was added and the plate was incubated for 30 min at room temperature in the dark. The reaction was stopped with 2 M H2SO4 and absorbance was quantified by a microplate spectrophotometer (Tecan) at a wavelength of 450 nm. Serum antibody titers were determined by measuring the lowest dilution that resulted in a signal 3 SDs above serum-free background controls. Statistical significance was determined by RStudio 1.1.463 using one-way ANOVA and Tukey-Kramer post hoc justified significance tests.

[0368] Quantification and statistical analysis

[0369] Quantification of cell-free protein synthesis yields

[0370] To quantify the amount of protein synthesized in the iVAX reaction, two approaches were used. Fluorescence units of sfGFP were converted to concentration using a calibration curve reported previously ( Hong et al., 2014 ). Yields of all other proteins were determined by adding 10 μM L to the CFPS mixture to obtain trichloroacetic acid insoluble radioactivity, which was measured using a liquid scintillation counter as previously described ( Kim and Szotz, 2001 ).-1 of 4 C-Leucine (11.1 GBq mmol -1 The assay was performed by adding 100 mM NaCl (PerkinElmer).

[0371] statistical analysis

[0372] Statistical parameters including definitions, n-values, p-values, standard deviations and standard errors are reported in the figures and corresponding figure legends. Analytical techniques are described in the corresponding method details section.

[0373] Availability of Data and Software

[0374] All plasmid constructs used in this study, including the complete DNA sequences, will be deposited with Addgene (constructs 128389-128404).

[0375] References for Example 3

[0376] Adiga, R., Al-adhami, M., Andar, A., Borhani, S., Brown, S., Burgenson, D., Cooper, MA, Deldari, S., Frey, DD, Ge, X., et al. (2018). Point-of-care production of therapeutic proteins of good-manufacturing-practice quality. Nat Biomed Eng.

[0377] Ashok, A., Brison, M., and LeTallec, Y. (2017). Improving cold chain systems: Challenges and solutions. Vaccine 35, 2217-2223.

[0378] Bayburt, T.H., and Sligar, S.G. (2010). Membrane protein assembly into Nanodiscs. FEBS Lett 584, 1721-1727.

[0379] Bhushan, R., Anthony, B.F., and Frasch, C.E. (1998). Estimation of group B streptococcus type III polysaccharide-specific antibody concentrations in human sera is antigen dependent. Infect Immun 66, 5848-5853.

[0380] Bogaert, D., Hermans, P.W., Adrian, P.V., Rumke, H.C., and de Groot, R. (2004). Pneumococcal vaccines: an update on current strategies. Vaccine 22, 2209-2220.

[0381] Boles, K.S., Kannan, K., Gill, J., Felderman, M., Gouvis, H., Hubby, B., Kamrud, K.I., Venter, J.C., and Gibson, D.G. (2017). Digital-to-biological converter for on-demand production of biologics. Nat Biotechnol 35, 672-675.

[0382] Brito, L.A., and Singh, M. (2011). Acceptable levels of endotoxin in vaccine formulations during preclinical research. J Pharm Sci 100, 34-37.

[0383] Carlson, E.D., Gan, R., Hodgman, C.E., and Jewett, M.C. (2012). Cell-free protein synthesis: Applications come of age. Biotechnol Adv 30, 1185-1194.

[0384] Casella, C.R., and Mitchell, T.C. (2008). Putting endotoxin to work for us: monophosphoryl lipid A as a safe and effective vaccine adjuvant. Cell Mol Life Sci 65, 3231-3240.

[0385] CDC (2019). CDC Vaccine Price List.

[0386] Chen, D.J., Osterrieder, N., Metzger, S.M., Buckles, E., Doody, A.M., DeLisa, M.P., and Putnam, D. (2010). Delivery of foreign antigens by engineered outer membrane vesicle vaccines. Proc Natl Acad Sci U S A 107, 3099-3104.

[0387] Chen, L., Valentine, J.L., Huang, C.-J., Endicott, C.E., Moeller, T.D., Rasmussen, J.A., Fletcher, J.R., Boll, J.M., Rosenthal, J.A., Dobruchowska, J., et al. (2016). Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies. Proc Natl Acad Sci U S A.

[0388] Chen, M.M., Glover, K.J., and Imperiali, B. (2007). From peptide to protein: comparative analysis of the substrate specificity of N-linked glycosylation in C. jejuni. Biochemistry 46, 5579-5585.

[0389] Crowell, L.E., Lu, A.E., Love, K.R., Stockdale, A., Timmick, S.M., Wu, D., Wang, Y.A., Doherty, W., Bonnyman, A., Vecchiarello, N., et al. (2018). On-demand manufacturing of clinical-quality biopharmaceuticals. Nat Biotechnol.

[0390] Cuccui, J., Thomas, R.M., Moule, M.G., D'Elia, R.V., Laws, T.R., Mills, D.C., Williamson, D., Atkins, T.P., Prior, J.L., and Wren, B.W. (2013). Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis. Open Biol 3, 130002.

[0391] Datsenko, K.A., and Wanner, B.L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640-6645.

[0392] 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. Proc Natl Acad Sci U S A 102, 3016-3021.

[0393] Fernandez, S., Palmer, D.R., Simmons, M., Sun, P., Bisbing, J., McClain, S., Mani, S., Burgess, T., Gunther, V., and Sun, W. (2007). Potential role for Toll-like receptor 4 in mediating Escherichia coli maltose-binding protein activation of dendritic cells. Infect Immun 75, 1359-1363.

[0394] Figueiredo, D., Turcotte, C., Frankel, G., Li, Y., Dolly, O., Wilkin, G., Marriott, D., Fairweather, N., and Dougan, G. (1995). Characterization of recombinant tetanus toxin derivatives suitable for vaccine development. Infect Immun 63, 3218-3221.

[0395] Fisher, A.C., Haitjema, C.H., Guarino, C., Celik, E., Endicott, C.E., Reading, C.A., Merritt, J.H., Ptak, A.C., Zhang, S., and DeLisa, M.P. (2011). Production of secretory and extracellular N-linked glycoproteins in Escherichia coli. Appl Environ Microbiol 77, 871-881.

[0396] Frasch, C.E. (2009). Preparation of bacterial polysaccharide-protein conjugates: analytical and manufacturing challenges. Vaccine 27, 6468-6470.

[0397] Fulop, M., Mastroeni, P., Green, M., and Titball, R.W. (2001). Role of antibody to lipopolysaccharide in protection against low- and high-virulence strains of Francisella tularensis. Vaccine 19, 4465-4472.

[0398] Garcia-Quintanilla, F., Iwashkiw, J.A., Price, N.L., Stratilo, C., and Feldman, M.F. (2014). Production of a recombinant vaccine candidate against Burkholderia pseudomallei exploiting the bacterial N-glycosylation machinery. Front Microbiol 5, 381.

[0399] Haghi, F., Peerayeh, S.N., Siadat, S.D., and Montajabiniat, M. (2011). Cloning, expression and purification of outer membrane protein PorA of Neisseria meningitidis serogroup B. J Infect Dev Ctries 5, 856-862.

[0400] Hatz, C.F., Bally, B., Rohrer, S., Steffen, R., Kramme, S., Siegrist, C.A., Wacker, M., Alaimo, C., and Fonck, V.G. (2015). 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.

[0401] Hong, S.H., Ntai, I., Haimovich, A.D., Kelleher, N.L., Isaacs, F.J., and Jewett, M.C. (2014). Cell-free protein synthesis from a release factor 1 deficient Escherichia coli activates efficient and multiple site-specific nonstandard amino acid incorporation. ACS Synth Biol 3, 398-409.

[0402] Humphreys, G. (2011). Vaccination: rattling the supply chain (Bulletin of the World Health Organization: World Health Organization).

[0403] Huttner, A., Hatz, C., van den Dobbelsteen, G., Abbanat, D., Hornacek, A., Frolich, R., Dreyer, A.M., Martin, P., Davies, T., Fae, K., et al. (2017). Safety, immunogenicity, and preliminary clinical efficacy of a vaccine against extraintestinal pathogenic Escherichia coli in women with a history of recurrent urinary tract infection: a randomised, single-blind, placebo-controlled phase 1b trial. Lancet Infect Dis.

[0404] Ihssen, J., Kowarik, M., Dilettoso, S., Tanner, C., Wacker, M., and Thony-Meyer, L. (2010). Production of glycoprotein vaccines in Escherichia coli. Microb Cell Fact 9, 61.

[0405] Jansson, P.E., Lindberg, B., Widmalm, G., and Leontein, K. (1987). Structural studies of the Escherichia coli O78 O-antigen polysaccharide. Carbohydr Res 165, 87-92.

[0406] Jaroentomeechai, T., Stark, J.C., Natarajan, A., Glasscock, C.J., Yates, L.E., Hsu, K.J., Mrksich, M., Jewett, M.C., and DeLisa, M.P. (2018). Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nat Commun 9, 2686.

[0407] Jewett, M.C., and Swartz, J.R. (2004). Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnol Bioeng 86, 19-26.

[0408] Jin, C., Gibani, M.M., Moore, M., Juel, H.B., Jones, E., Meiring, J., Harris, V., Gardner, J., Nebykova, A., Kerridge, S.A., et al. (2017). Efficacy and immunogenicity of a Vi-tetanus toxoid conjugate vaccine in the prevention of typhoid fever using a controlled human infection model of Salmonella Typhi: a randomised controlled, phase 2b trial. Lancet 390, 2472-2480.

[0409] Johnson, J.R. (1991). Virulence factors in Escherichia coli urinary tract infection. Clin Microbiol Rev 4, 80-128.

[0410] Kim, D.M., and Swartz, J.R. (2001). Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis. Biotechnol Bioeng 74, 309-316.

[0411] Knapp, K.G., Goerke, A.R., and Swartz, J.R. (2007). Cell-free synthesis of proteins that require disulfide bonds using glucose as an energy source. Biotechnol Bioeng 97, 901-908.

[0412] Kowarik, M., Young, N.M., Numao, S., Schulz, B.L., Hug, I., Callewaert, N., Mills, D.C., Watson, D.C., Hernandez, M., Kelly, J.F., et al. (2006). Definition of the bacterial N-glycosylation site consensus sequence. EMBO J 25, 1957-1966.

[0413] L’vov, V.L., Shashkov, A.S., Dmitriev, B.A., Kochetkov, N.K., Jann, B., and Jann, K. (1984). Structural studies of the O-specific side chain of the lipopolysaccharide from Escherichia coli O:7. Carbohydr Res 126, 249-259.

[0414] Lu, Z., Madico, G., Roche, M.I., Wang, Q., Hui, J.H., Perkins, H.M., Zaia, J., Costello, C.E., and Sharon, J. (2012). Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia. Immunology 136, 352-360.

[0415] Lydon, P., Zipursky, S., Tevi-Benissan, C., Djingarey, M.H., Gbedonou, P., Youssouf, B.O., and Zaffran, M. (2014). Economic benefits of keeping vaccines at ambient temperature during mass vaccination: the case of meningitis A vaccine in Chad. Bull World Health Organ 92, 86-92.

[0416] Ma, Z., Zhang, H., Shang, W., Zhu, F., Han, W., Zhao, X., Han, D., Wang, P.G., and Chen, M. (2014). Glycoconjugate vaccine containing Escherichia coli O157:H7 O-antigen linked with maltose-binding protein elicits humoral and cellular responses. PloS One 9, e105215.

[0417] Marshall, L.E., Nelson, M., Davies, C.H., Whelan, A.O., Jenner, D.C., Moule, M.G., Denman, C., Cuccui, J., Atkins, T.P., Wren, B.W., et al. (2018). An O-antigen glycoconjugate vaccine produced using protein glycan coupling technology is protective in an inhalational rat model of tularemia. J Immunol Res 2018, 8087916.

[0418] Murphy, T.W., Sheng, J., Naler, L.B., Feng, X., and Lu, C. (2019). On-chip manufacturing of synthetic proteins for point-of-care therapeutics. Microsyst Nanoeng 5, 13.

[0419] Needham, B.D., Carroll, S.M., Giles, D.K., Georgiou, G., Whiteley, M., and Trent, M.S. (2013). Modulating the innate immune response by combinatorial engineering of endotoxin. Proc Natl Acad Sci U S A 110, 1464-1469.

[0420] Novak, R.T., Kambou, J.L., Diomande, F.V., Tarbangdo, T.F., Ouedraogo-Traore, R., Sangare, L., Lingani, C., Martin, S.W., Hatcher, C., Mayer, L.W., et al. (2012). Serogroup A meningococcal conjugate vaccination in Burkina Faso: analysis of national surveillance data. Lancet Infect Dis 12, 757-764.

[0421] Ollis, A.A., Chai, Y., Natarajan, A., Perregaux, E., Jaroentomeechai, T., Guarino, C., Smith, J., Zhang, S., and DeLisa, M.P. (2015). Substitute sweeteners: Diverse bacterial oligosaccharyltransferases with unique N-glycosylation site preferences. Sci Rep 5, 15237.

[0422] Oyston, P.C., Sjostedt, A., and Titball, R.W. (2004). Tularaemia: bioterrorism defence renews interest in Francisella tularensis. Nat Rev Microbiol 2, 967-978.

[0423] Pardee, K., Slomovic, S., Nguyen, Peter Q., Lee, Jeong W., Donghia, N., Burrill, D., Ferrante, T., McSorley, Fern R., Furuta, Y., Vernet, A., et al. (2016). Portable, on-demand biomolecular manufacturing. Cell 167, 248-259.e212.

[0424] Perez, J.G., Stark, J.C., and Jewett, M.C. (2016). Cell-free synthetic biology: Engineering beyond the cell. Cold Spring Harb Perspect Biol.

[0425] Perez-Pinera, P., Han, N., Cleto, S., Cao, J., Purcell, O., Shah, K.A., Lee, K., Ram, R., and Lu, T.K. (2016). Synthetic biology and microbioreactor platforms for programmable production of biologics at the point-of-care. Nat Commun 7, 12211.

[0426] Petsch, D., and Anspach, F.B. (2000). Endotoxin removal from protein solutions. J Biotechnol 76, 97-119.

[0427] Poehling, K.A., Talbot, T.R., Griffin, M.R., Craig, A.S., Whitney, C.G., Zell, E., Lexau, C.A., Thomas, A.R., Harrison, L.H., Reingold, A.L., et al. (2006). Invasive pneumococcal disease among infants before and after introduction of pneumococcal conjugate vaccine. JAMA 295, 1668-1674.

[0428] Prior, J.L., Prior, R.G., Hitchen, P.G., Diaper, H., Griffin, K.F., Morris, H.R., Dell, A., and Titball, R.W. (2003). Characterization of the O antigen gene cluster and structural analysis of the O antigen of Francisella tularensis subsp. tularensis. J Med Microbiol 52, 845-851.

[0429] Qadri, F., Svennerholm, A.M., Faruque, A.S., and Sack, R.B. (2005). Enterotoxigenic Escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. Clin Microbiol Rev 18, 465-483.

[0430] Raetz, C.R., and Whitfield, C. (2002). Lipopolysaccharide endotoxins. Annu Rev Biochem 71, 635-700.

[0431] Riddle, M.S., Kaminski, R.W., Di Paolo, C., Porter, C.K., Gutierrez, R.L., Clarkson, K.A., Weerts, H.E., Duplessis, C., Castellano, A., Alaimo, C., et al. (2016). Safety and immunogenicity of a candidate bioconjugate vaccine against Shigella flexneri 2a administered to healthy adults: a single blind, randomized phase I study. Clin Vaccine Immunol.

[0432] Roush, S.W., McIntyre, L., and Baldy, L.M. (2008). Manual for the surveillance of vaccine-preventable diseases. Atlanta: Centers for Disease Control and Prevention, 4.

[0433] Russell, J.A. (2006). Management of sepsis. N Engl J Med 355, 1699-1713.

[0434] Salehi, A.S., Smith, M.T., Bennett, A.M., Williams, J.B., Pitt, W.G., and Bundy, B.C. (2016). Cell-free protein synthesis of a cytotoxic cancer therapeutic: Onconase production and a just-add-water cell-free system. Biotechnol J 11, 274-281.

[0435] Schoborg, J.A., Hershewe, J.M., Stark, J.C., Kightlinger, W., Kath, J.E., Jaroentomeechai, T., Natarajan, A., DeLisa, M.P., and Jewett, M.C. (2017). A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases. Biotechnol Bioeng.

[0436] Stefan, A., Conti, M., Rubboli, D., Ravagli, L., Presta, E., and Hochkoeppler, A. (2011). Overexpression and purification of the recombinant diphtheria toxin variant CRM197 in Escherichia coli. J Biotechnol 156, 245-252.

[0437] Stefanetti, G., Okan, N., Fink, A., Gardner, E., and Kasper, D.L. (2019). Glycoconjugate vaccine using a genetically modified O antigen induces protective antibodies to Francisella tularensis. Proc Natl Acad Sci U S A 116, 7062-7070.

[0438] The Review on Antimicrobial Resistance, C.b.J.O.N. (2014). Antimicrobial Resistance: Tackling a crisis for the health and wealth of nations.

[0439] Trotter, C.L., McVernon, J., Ramsay, M.E., Whitney, C.G., Mulholland, E.K., Goldblatt, D., Hombach, J., and Kieny, M.P. (2008). Optimising the use of conjugate vaccines to prevent disease caused by Haemophilus influenzae type b, Neisseria meningitidis and Streptococcus pneumoniae. Vaccine 26, 4434-4445.

[0440] Wacker, M., Wang, L., Kowarik, M., Dowd, M., Lipowsky, G., Faridmoayer, A., Shields, K., Park, S., Alaimo, C., Kelley, K.A., et al. (2014). Prevention of Staphylococcus aureus infections by glycoprotein vaccines synthesized in Escherichia coli. J Infect Dis 209, 1551-1561.

[0441] Wahl, B., O'Brien, K.L., Greenbaum, A., Majumder, A., Liu, L., Chu, Y., Luksic, I., Nair, H., McAllister, D.A., Campbell, H., et al. (2018). Burden of Streptococcus pneumoniae and Haemophilus influenzae type b disease in children in the era of conjugate vaccines: global, regional, and national estimates for 2000-15. Lancet Glob Health 6, e744-e757.

[0442] Weintraub, A. (2003). Immunology of bacterial polysaccharide antigens. Carbohydr Res 338, 2539-2547.

[0443] Wetter, M., Kowarik, M., Steffen, M., Carranza, P., Corradin, G., and Wacker, M. (2013). Engineering, conjugation, and immunogenicity assessment of Escherichia coli O121 O antigen for its potential use as a typhoid vaccine component. Glycoconj J 30, 511-522.WHO (2014). Temperature Sensitivity of Vaccines.

[0444] Celik, E., Ollis, A.A., Lasanajak, Y., Fisher, A.C., Gur, G., Smith, D.F., and DeLisa, M.P. (2015). Glycoarrays with engineered phages displaying structurally diverse oligosaccharides enable high-throughput detection of glycan-protein interactions. Biotechnol J 10, 199-209.

[0445] Cuccui, J., Thomas, R.M., Moule, M.G., D'Elia, R.V., Laws, T.R., Mills, D.C., Williamson, D., Atkins, T.P., Prior, J.L., and Wren, B.W. (2013). Exploitation of bacterial N-linked glycosylation to develop a novel recombinant glycoconjugate vaccine against Francisella tularensis. Open Biol 3, 130002.

[0446] Datsenko, K.A., and Wanner, B.L. (2000). One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640-6645.

[0447] Jaroentomeechai, T., Stark, J.C., Natarajan, A., Glasscock, C.J., Yates, L.E., Hsu, K.J., Mrksich, M., Jewett, M.C., and DeLisa, M.P. (2018). Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery. Nat Commun 9, 2686.

[0448] Ollis, A.A., Zhang, S., Fisher, A.C., and DeLisa, M.P. (2014). Engineered oligosaccharyltransferases with greatly relaxed acceptor-site specificity. Nat Chem Biol 10, 816-822.

[0449] Valvano, M.A., and Crosa, J.H. (1989). Molecular cloning and expression in Escherichia coli K-12 of chromosomal genes determining the O7 lipopolysaccharide antigen of a human invasive strain of E. coli O7:K1. Infect Immun 57, 937-943.

[0450] Example 3 - iVAX-derived vaccines protect against lethal F. tularensis challenge

[0451] Groups of five BALB / c mice were cultured with either PBS or 7.5 μg of purified, cell-free synthetic aglycosylated MBP. 4xDQNAT , P.D. 4xDQNAT , E.P.A. DNNNS-DQNRT , or FtO-PS-conjugated MBP 4xDQNAT , P.D. 4xDQNAT , or EPA DNNNS-DQNRT The mice were immunized intraperitoneally with FtO-PS-conjugated MBP prepared in live E. coli cells using PCGT. 4xDQNAT , P.D. 4xDQNAT , and E.P.A. DNNNS-DQNRT was used as a positive control. Mice were immunized with the same dose of antigen on days 21 and 42. On day 66, mice were challenged with 6000 cfu (intranasal LD 50 Mice were challenged intranasally with a 50-fold increase in the concentration of F. tularensis subgenus holarctica live vaccine strain, Rocky Mountain Laboratories, and monitored for survival over an additional 25 days (Figure 31a). 4xDQNAT , (Fig. 31c)PD 4xDQNAT and (Figure 31d) EPA DNNNS-DQNRT Figure 1 shows Kaplan-Meier curves for immunization with iVAX. These results indicate that iVAX-derived vaccines protect mice against lethal pathogen challenge as effectively as vaccines synthesized using state-of-the-art PGCT approaches (*p=0.0476, **p=0.0079, Fisher's exact test; ns: not significant).

[0452] In the above description, as will be readily apparent to those skilled in the art, various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The present invention illustratively described herein can be practiced without an element or elements, or a limitation or limitations not specifically disclosed herein. The terms and expressions employed are used as terms of description, not of limitation, and in the use of such terms and expressions, it is not intended to exclude any equivalents of the features or parts thereof described and illustrated, and it is recognized that various modifications can be included within the scope of the present invention. Thus, it should be understood that although the present invention has been described with specific embodiments and optional features, modifications and / or variations of the technical ideas disclosed herein can be conceived by those skilled in the art, and such modifications and variations are considered to be included within the scope of the present invention.

[0453] A number of patent and non-patent literature references have been cited herein. The cited references are incorporated herein by reference in their entirety. In the event of a discrepancy in the definition of a term in this specification as compared to the definition of a term in a cited reference, such term shall be interpreted in accordance with the definition in this specification. Some aspects of the invention are described below. 1. A method for synthesizing an N-glycosylated carrier protein of a polysaccharide by coordinated in vitro transcription, translation, and N-glycosylation, comprising: (i) transcribing and translating a carrier protein in a cell-free protein synthesis reaction, wherein the carrier protein contains an inserted consensus sequence, NXS / T, where X can be any natural or unnatural amino acid except proline; (ii) glycosylating a carrier protein in a cell-free protein synthesis reaction with at least one polysaccharide, wherein the at least one polysaccharide is at least one bacterial O-antigen; The method includes: 2. The method of claim 1, wherein the carrier protein is selected from Haemophilus influenzae protein D (PD), Neisseria meningitidysporin protein (PorA), and mutants thereof. 3. The method of claim 1, wherein the bacterial O-antigen is from E. coli. 4. The method of claim 1, wherein the bacterial O-antigen is from Francisella tularensis. 5. The method of claim 1, further comprising formulating the N-glycosylated carrier protein as a vaccine composition comprising said N-glycosylated carrier protein. 6. The method of claim 1, wherein the vaccine composition further comprises an adjuvant. 7. The method according to claim 1, wherein the glycosylation step utilizes an oligosaccharyltransferase (OST) that is a naturally occurring or modified bacterial oligosaccharyltransferase (OST). 8. The method according to item 1, wherein the glycosylation step utilizes an oligosaccharyltransferase (OST) that is a naturally occurring or modified archaeal oligosaccharyltransferase (OST). 9. The method according to item 1, wherein the glycosylation step utilizes oligosaccharyltransferase (OST), a naturally occurring bacterial homologue of C. jejuni PglB. 10. The method according to item 1, wherein the glycosylation step utilizes OST, an engineered mutant of C. jejuni PglB. 11. The method of claim 1, wherein the glycosylation step utilizes OST, a naturally occurring single-subunit eukaryotic OST. 12. The method of claim 5, further comprising administering the vaccine composition to a subject in need thereof. 13. A kit for synthesizing an N-glycosylated carrier protein in vitro, comprising: (i) a first component comprising a cell lysate containing an orthogonal oligosaccharyltransferase (OST); (ii) a second component comprising a cell lysate containing the O-antigen; (iii) a third component comprising a transcription template and a polymerase for synthesizing mRNA from the transcription template encoding a carrier protein, wherein the carrier protein comprises an inserted consensus sequence, NXS / T, where X can be any natural or unnatural amino acid except proline; Kit including: 14. A kit for synthesizing an N-glycosylated carrier protein in vitro, comprising: (i) a first component comprising a cell lysate containing an orthogonal oligosaccharyltransferase (OST) and an O-antigen; (ii) a second component comprising a transcription template and a polymerase for synthesizing mRNA from the transcription template encoding a carrier protein, wherein the carrier protein comprises an inserted consensus sequence, NXS / T, where X can be any natural or unnatural amino acid except proline; Kit including: 15. The kit according to item 13 or 14, wherein one or more of the first component, the second component, and, if present, the third component, are lyophilized. 16. The kit according to item 13, wherein the first component, the cell lysate, is prepared from a source strain that overexpresses a gene encoding an orthogonal OST. 17. The kit according to item 13, wherein the second component, a cell lysate, is produced from a source strain that overexpresses a synthetic glycosyltransferase pathway. 18. The kit according to item 14, wherein the first component, the cell lysate, is produced from a source strain that overexpresses one or both of (a) a gene encoding an orthogonal OST and (b) a synthetic glycosyltransferase pathway. 19. The kit according to item 13 or 14, wherein the second component cell lysate or the first component cell lysate comprises an O-antigen from F. tularensis Schu S4 lipid-linked oligosaccharides (FtLLO) or an O-antigen from enterotoxigenic E. coli O78 lipid-linked oligosaccharides (EcO78LLO). 20. A method for cell-free production of a glycoprotein, comprising: (a) preparing a cell-free protein synthesis reaction by mixing a first cell lysate containing an orthogonal oligosaccharyltransferase (OST) with a second cell lysate containing an O-antigen; (b) transcribing and translating in a cell-free protein synthesis reaction a carrier protein selected from Haemophilus influenzae protein D (PD), Neisseria meningitidysporin protein (PorA), and variants thereof, wherein the carrier protein contains an inserted consensus sequence, NXS / T, where X can be any natural or unnatural amino acid except proline; and (c) glycosylating the carrier protein in a cell-free protein synthesis reaction using bacterial O-antigen; The method includes: 21. A method for cell-free production of a glycoprotein, comprising: (a) preparing a cell-free protein synthesis reaction by combining a first cell lysate containing an orthogonal oligosaccharyltransferase (OST) with an O-antigen; (b) transcribing and translating in a cell-free protein synthesis reaction a carrier protein selected from Haemophilus influenzae protein D (PD), Neisseria meningitidysporin protein (PorA), and variants thereof, wherein the carrier protein contains an inserted consensus sequence, NXS / T, where X can be any natural or unnatural amino acid except proline; and (c) glycosylating the carrier protein in a cell-free protein synthesis reaction using bacterial O-antigen; The method includes: 22. The method according to item 20 or 21, wherein the second cell lysate or the first cell lysate comprises the O-antigen as part of a lipid-linked oligosaccharide (LLO). 23. The method according to item 20 or 21, further comprising formulating the glycoprotein as a vaccine composition. 24. The method of claim 22, further comprising administering the vaccine composition to a subject in need thereof.

Claims

1. 1. A method for synthesizing an N-glycosylated carrier protein of a polysaccharide by coordinated in vitro transcription, translation, and N-glycosylation, comprising: (i) transcribing and translating a carrier protein in a cell-free protein synthesis reaction, wherein the carrier protein contains an inserted consensus sequence, NXS / T, where X can be any natural or unnatural amino acid except proline; (ii) glycosylating a carrier protein in a cell-free protein synthesis reaction with at least one polysaccharide, wherein the at least one polysaccharide is at least one bacterial O-antigen, and the glycosylation step utilizes an oligosaccharyltransferase (OST); Including, wherein the cell-free protein synthesis reaction is with a lysate derived from one or more genetically modified Escherichia coli (E. coli) strains, wherein the one or more genetically modified E. coli strains (a) lack an lpxM gene product, and (b) contain an orthogonal or heterologous LpxE gene expressed in the genetically modified E. coli strains and present in the lysate. method.

2. 2. The method of claim 1, wherein the carrier protein is selected from Haemophilus influenzae protein D (PD), Neisseria meningitidysporin protein (PorA), and mutants thereof.

3. The method of claim 1, wherein the bacterial O-antigen is from E. coli.

4. The method of claim 1, wherein the bacterial O-antigen is from Francisella tularensis.

5. The method of claim 1, further comprising formulating the N-glycosylated carrier protein as a vaccine composition comprising the N-glycosylated carrier protein.

6. The method of claim 5, wherein the vaccine composition further comprises an adjuvant.

7. The method of claim 1, wherein the OST is a naturally occurring or modified bacterial OST.

8. The method of claim 1, wherein the OST is a naturally occurring or modified archaeal OST.

9. 2. The method of claim 1, wherein the OST is a naturally occurring bacterial homolog of C. jejuni PglB.

10. 2. The method of claim 1, wherein the OST is an engineered mutant of C. jejuni PglB.

11. The method of claim 1, wherein the OST is a naturally occurring single-subunit eukaryotic OST.

12. The method of claim 5, wherein the vaccine composition is for administration to a subject in need thereof.

13. 1. A kit for in vitro synthesis of an N-glycosylated carrier protein, comprising: (i) a first component comprising a cell lysate containing an orthogonal oligosaccharyltransferase (OST); (ii) a second component comprising a cell lysate containing the O-antigen; (iii) a third component comprising a transcription template encoding a carrier protein and a polymerase for synthesizing mRNA from the transcription template encoding the carrier protein, wherein the carrier protein comprises an inserted consensus sequence, NXS / T, where X can be any natural or unnatural amino acid except proline; Including, wherein the cell lysate of (i) and / or (ii) is derived from one or more genetically modified Escherichia coli (E. coli) strains, wherein the genetically modified E. coli strains (a) lack an lpxM gene product, and (b) comprise an orthogonal or heterologous LpxE gene that is expressed in the genetically modified E. coli strain and present in the lysate. kit.

14. 1. A kit for in vitro synthesis of an N-glycosylated carrier protein, comprising: (i) a first component comprising a cell lysate containing an orthogonal oligosaccharyltransferase (OST) and an O-antigen; (ii) a second component comprising a transcription template encoding a carrier protein and a polymerase for synthesizing mRNA from the transcription template encoding the carrier protein, wherein the carrier protein comprises an inserted consensus sequence, NXS / T, where X can be any natural or unnatural amino acid except proline; Including, wherein the cell lysate of (i) and / or (ii) is derived from one or more genetically modified Escherichia coli (E. coli) strains, wherein the genetically modified E. coli strains (a) lack an lpxM gene product, and (b) comprise an orthogonal or heterologous LpxE gene that is expressed in the genetically modified E. coli strain and present in the lysate. kit.

15. 15. The kit of claim 13 or 14, wherein one or more of the first component, the second component, and, if present, the third component, are lyophilized.

16. The kit of claim 13, wherein the first component, a cell lysate, is prepared from a source strain that overexpresses a gene encoding an orthogonal OST.

17. 14. The kit of claim 13, wherein the second component, a cell lysate, is produced from a source strain that overexpresses a synthetic glycosyltransferase pathway.

18. 15. The kit of claim 14, wherein the first component, a cell lysate, is produced from a source strain that overexpresses one or both of: (a) a gene encoding an orthogonal OST; and (b) a synthetic glycosyltransferase pathway.

19. The kit according to claim 13 or 14, wherein the second component cell lysate or the first component cell lysate comprises an O-antigen from F. tularensis Schu S4 lipid-linked oligosaccharides (FtLLO) or an O-antigen from enterotoxigenic E. coli O78 lipid-linked oligosaccharides (EcO78LLO).

20. 1. A method for cell-free production of a glycoprotein, comprising: (a) preparing a cell-free protein synthesis reaction by combining a first cell lysate containing an orthogonal oligosaccharyltransferase (OST) and a second cell lysate containing an O-antigen; (b) transcribing and translating in a cell-free protein synthesis reaction a carrier protein selected from Haemophilus influenzae protein D (PD), Neisseria meningitidysporin protein (PorA), and variants thereof, wherein the carrier protein contains an inserted consensus sequence, NXS / T, where X can be any natural or unnatural amino acid except proline; and (c) glycosylating the carrier protein in a cell-free protein synthesis reaction using bacterial O-antigen; Including, wherein the first cell lysate and / or the second cell lysate are derived from one or more genetically modified Escherichia coli (E. coli) strains, wherein the genetically modified E. coli strains (a) lack an lpxM gene product, and (b) comprise an orthogonal or heterologous LpxE gene that is expressed in the genetically modified E. coli strain and present in the lysate. method.

21. 1. A method for cell-free production of a glycoprotein, comprising: (a) mixing a first cell lysate containing an orthogonal oligosaccharyltransferase (OST) with an O-antigen to prepare a cell-free protein synthesis reaction; (b) transcribing and translating in a cell-free protein synthesis reaction a carrier protein selected from Haemophilus influenzae protein D (PD), Neisseria meningitidysporin protein (PorA), and variants thereof, wherein the carrier protein contains an inserted consensus sequence, NXS / T, where X can be any natural or unnatural amino acid except proline; and (c) glycosylating the carrier protein in a cell-free protein synthesis reaction using bacterial O-antigen; Including, wherein the first cell lysate is derived from one or more genetically modified Escherichia coli (E. coli) strains, wherein the genetically modified E. coli strains (a) lack an lpxM gene product, and (b) contain an orthogonal or heterologous LpxE gene that is expressed in the genetically modified E. coli strain and present in the lysate. method.

22. 22. The method of claim 20 or 21, wherein the O-antigen is part of a lipid-linked oligosaccharide (LLO).

23. 22. The method of claim 20 or 21, further comprising formulating the glycoprotein as a vaccine composition.

24. 24. The method of claim 23, wherein the vaccine composition is for administration to a subject in need thereof.

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