Production of Recombinant Conjugate Vaccine Candidates ...

Production of Recombinant Conjugate Vaccine Candidates against Burkholderia pseudomallei

Mario Feldman, Fatima Garcia Quintanilla, Jeremy Iwashkiw, and Nancy Price University of Alberta (Dept. of Biological Sciences, Feldman Lab) Prepared By: University of Alberta (Dept. of Biological Sciences, Feldman Lab) 116 St and 85 Ave, Edmonton, AB T6G 2R3

PWGSC Contract Number: W7702-08R212 CSA: Chad Stralito, Biologist, DRDC- Suffield Research Centre, 403-544-4072

The scientific or technical validity of this Contract Report is entirely the responsibility of the Contractor and the

contents do not necessarily have the approval or endorsement of Defence R&D Canada.

Defence Research and Development Canada Contract Report

DRDC-RDDC-2014-C44

March 2014

IMPORTANT INFORMATIVE STATEMENTS

The information contained herein is proprietary to Her Majesty and is provided to the recipient on the understanding that it will be used for information and evaluation purposes only. Any commercial use including use for manufacture is prohibited.

© Her Majesty the Queen in Right of Canada, as represented by the Minister of National Defence, 2014

© Sa Majesté la Reine (en droit du Canada), telle que représentée par le ministre de la Défense nationale, 2014

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Production of Recombinant Conjugate Vaccine Candidates against Burkholderia pseudomallei

Contract Project

Chad Stratilo (Scientific Authority)

Defence Research and Development Canada ‐ Suffield Research Centre

Mario Feldman, Fatima Garcia Quintanilla, Jeremy Iwashkiw, and Nancy Price

University of Alberta (Dept. of Biological Sciences, Feldman Lab)

DRDC ‐ Suffield Research Centre & University of Alberta Burkholderia vaccine project

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Preface / Covering Page

The following contract report concerns the contract W7702‐08R212, Production of Recombinant

Conjugate Vaccine Candidates against Burkholderia pseudomallei. This work was funded by ARP 14fa05.

The purpose of this work was to produce recombinant Burkholderia glycan in E. coli; this would allow a

possible protective antigen of Burkholderia pseudomallei to be produced in a much lower bio‐

containment level than extracting the glycan from B. pseudomallei. The other part of this work involved

the production of recombinant outer membrane vesicles (OMVs) in E. coli that would carry the glycan of

B. pseudomallei as well as other protective proteins of B. pseudomallei recombinantly produced in E.

coli.

The contractor was able to produce the recombinant glycan in E. coli and attach it to a carrier protein for

ease of purification. They were also able to produce recombinant OMVs carrying the glycan and other

bacterial proteins.

Both of these vaccine candidates have been tested in DRDC animal models. This work will be captured in

external publications to follow.

The impact of this work is it is the first demonstration of recombinantly produced Burkholderia glycans

in E. coli that were purified and used as a vaccine candidate to protect against a B. pseudomallei

challenge. This demonstrates that the production of glycovaccines against B. pseudomallei is possible in

a recombinant system. This would allow for the production of meaningful amounts of vaccine

candidates in a much safer manner and in a system already approved by regulators.


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Production of Recombinant Conjugate Vaccine Candidates against Burkholderia pseudomallei Defence Research and Development Canada ‐ Suffield Research Centre Chad Stratilo University of Alberta (Dept of Biological Sciences, Feldman Lab) Mario Feldman, Fatima Garcia Quintanilla, Jeremy Iwashkiw, and Nancy Price Project Overview

The purpose of this project was to develop a novel Burkholderia vaccine based on glycoconjugate technologies. The project involved two different vaccine directed approaches. The first project was the development of a protein glycoconjugate consisting of a Burkholderia OPS II glycan and a Campylobacter jejuni protein carrier AcrA. The second project involved exploring the use of outer membrane vesicles isolated from Escherichia coli which consisted of the Burkholderia OPS II glycan.

Project 1: Burkholderia glycoconjugate vaccine

At present there is no known effective B. pseudomallei vaccine. Although both live attenuated and heat killed bacterial vaccines have been shown to be highly effective as vaccine candidates for various bacterial pathogens, such vaccines tested against B. pseudomallei have been shown to provide little to no protection against disease and mortality in mice (Peacock et al., 2012). Another possible vaccine type is the traditional conjugate vaccine, where bacterial surface polysaccharides are chemically conjugated to a carrier protein. Such vaccines have been demonstrated to be highly effective, best demonstrated by the Haemophilus influenzae type b conjugate vaccine, which has nearly eliminated infections in most parts of the world (Pollard et al, 2009). However, manufacturing these conjugate vaccines requires complex synthetic chemistry for attaching the glycan to protein carriers. Also, bacterial polysaccharides are too complex to be synthesized efficiently, making this process economically prohibitive. Additionally, since B. pseudomallei requires class III biosafety facilities, manufacturing conjugates containing glycans from its native host is challenging and possibly hazardous. Finally, chemical attachment of the sugar to the carrier protein can result in large and heterogeneous conjugates, modifying the native structure, and thus decreasing the protective nature of the vaccine.

A novel method of synthesizing conjugate vaccines is exploitation of the protein glycosylation machinery of the bacteria. The cornerstone of bacterial glycosylation is the oligosaccharyltransferase (OTase) enzyme, which covalently attaches glycan structures to either asparagine (N-linked) or serine/threonine (O-linked) residues. OTases have high substrate promiscuity, and thus can transfer a wide range of lipid linked glycan structures to acceptor proteins, in a process called en bloc glycosylation. The best-characterized system is the N-glycosylation system of Campylobacter jejuni. Briefly, a unique initiation glycosyltransferase attaches a nucleotide-activated monosaccharide-1P to undecaprenyl phosphate (Und-P) in the cytoplasmic face of the inner membrane. Subsequently, a series of additional glycosyltransferases attach additional monosaccharides to the first residue, and when completed, the lipid-linked oligosaccharide (LLO) is translocated to the periplasmic face of the inner membrane by a flippase. Finally, PglB (N-OTase) covalently attaches the glycan to asparagine residues in with the sequon D/E-X-N_Y-S/T (X,Y ≠P). Earlier studies have demonstrated that PglB has relaxed glycan specificity, allowing the transfer of a vast array of glycans, including O antigens, to acceptor proteins. Thus, bacterial glycosylation systems can be exploited to synthesize novel glycoconjugates for vaccination and diagnostic purposes. Glycoconjugates produced by this method are significantly less


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expensive, less challenging to produce, and produce less hazardous waste than conventional chemical methods.

In this work, we demonstrate that the operon responsible for the biosynthesis of the B. pseudomallei OPS can be functionally reconstituted in E. coli by developing a double knockout of the waaL ligase and the wecA initiating glycosyltransferase, Both utilized for the production of a N-linked glycoconjugate when co-expressed with an OTase and an engineered acceptor protein derived from C. jejuni.

Cloning and expression of the B. pseudomallei K96243 O-antigen Polysaccharide II (OPS II) Locus in E. coli

Previous work cloned a region consisting of 21 potential open reading frames, and further investigation identified a cluster of 15 genes required for the biosynthesis of B. pseudomallei K96243 OPS II (Fig 1A) (DeShazer, 2004). A previous study demonstrated by NMR analysis identified the structure of the OPS II to be a polymer of a disaccharide repeating structure composed of -3-)-β-D-glucopyranose-(1-3)-α-L-6-deoxy-talopyranose-(1-, with variable O-methyl and O-acetyl modifications (Fig 1B) (Perry, 1995). In order to recombinantly express the B. pseudomallei OPS II in E. coli, the essential 15 genes were subcloned (genes rmlB to wbiI) from the plasmid pCC1FOS-BPF16β_E10 by restriction digest into the arabinose inducible expression vector pBAD24, generating pEQ3 (Figure 1A). Expression of the B. pseudomallei OPS II in E. coli was visualized by Western blot with a polymer of immunoreactive bands being observed, confirming the production of the carbohydrate structure (Fig 1B).

Reconstitution of the Campylobacter jejuni N-Glycosylation System in E. coli to synthesize a glycoconjugate with the B. pseudomallei OPS II

Next, we attempted to generate a N-linked glycoprotein with the OPS II by exploiting the C. jejuni N-glycosylation system as previously described (Wacker, Iwashkiw, etc). In earlier work, N-glycosylated AcrA was synthesized in E. coli by co-expression of C. jejuni PglB and AcrA with an appropriate carbohydrate structure. We therefore expressed PglB (pMAF10), AcrA (pIH18), and the B. pseudomallei OPS II antigen (pEQ3) in both an expression (EPI300) and waaL- strain (CLM24) and tested for glycosylation by Western blot. We were unable to detect any evidence of glycosylation.

Construction of an E. coli wecA- waaL- strain (SDB1)

One issue with exploiting O-antigens using protein glycosylation may be that the precursor can also be used by the WaaL ligase in E. coli, thus siphoning off the substrate, and decreasing the glycosylation efficiency (Fig 2). Additionally, in E. coli strains expressing their own O-antigens and in addition to a WaaL ligase, our glycan of interest would be outcompeted, and the small amount that is produced could be transferred to lipid A (Fig. 2A). In theory, when the initiating enzyme (wecA) for the native O-antigen is deleted, only our glycan of interest is produced on the undecaprenyl-diphosphate (Und-PP) carrier, increasing its production, but the glycan could be preferentially utilized by the WaaL ligase, thus decreasing the production of glycoprotein (Fig 2B). However, when both wecA and waaL are deleted, we hypothesize an increased efficiency of protein glycosylation (Fig 2C).

In order to optimize the glycosylation of the protein acceptor AcrA with OPS II in E. coli, we constructed an E. coli wecA- waaL- mutant, SDB1. Using the KEIO strain collection (Baba et al., 2006), the wecA mutation was transduced into CLM24, an E. coli waaL mutant (Feldman 2005), creating SDB1. To confirm the double mutation, we tested SDB1 transformed with pJHCV32 by looking at LPS extractions (Fig 3). The plasmid pJHCV32 constitutively expresses the E. coli O7 O-antigen, but is lacking an initiating glycosyltransferase (WecA). Expression in trans of either WecA or WaaL individually in this background did not produce LPS (lane 1-8). However, when both genes were expressed simultaneously


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in SDB1, we observe the characteristic polymerization of O-antigen previously observed for the E. coli O7 LPS (Lane 9-10). We observe complementation regardless of induction of the genes, which is due to the leaky nature of the expression vectors.

Analysis of Expression of B. pseudomallei OPS II

To analyze the production of B. pseudomallei OPS II in E. coli, pEQ3 was expressed in different E. coli strains and LPS extractions were performed and visualized by Western blot against the OPS II glycan (Fig 4). Without induction of the OPS II by arabinose, no signal was detected in any of the three strains. For SDB1, regardless of induction of the OPS II, no signal was observed, as expected (lane 1, 2). In EPI300, when OPS II was induced, a weak signal was observed corresponding to the glycan attached to lipid A. However in CLM37 (wecA-), a significant increase in the amount of glycan being produced and transferred to the Lipid A is observed, supporting our hypothesis that there may be competition for the undecaprenyl pyrophosphate intermediate (lane 4). This confirms that pEQ3 does express the OPS II antigen, and that our strain SDB1 does not have ligase activity, suggesting it could be a more efficient strain for producing glycoconjugate proteins.

In vivo synthesis and purification of an N-linked glycoconjugate with the Burkholderia OPS II glycan

In order to create an N-linked glycoconjugate, we transformed the E. coli strain SDB1 with the pEQ3 (OPS II), pMAF10 (PglB), and pIH18 (AcrA). Cultures were inoculated in LB media and grown at 37°C with shaking. After 2h of incubation, the cultures were induced with 0.1 mM isopropyl 1-thio-β-D-galactopyranoside and 0.2% (w/v) arabinose. To increase the glycosylation yield in SDB1 we also added MnCl2 (4 mM). Five hours after induction at 37°C, an additional 0.2% (w/v) arabinose was added to ensure PglB expression. Cells were harvested by centrifugation after an overnight induction period and the periplasmic extract containing the glycoproteins was extracted using a lysozyme treatment. For purification, the periplasmic fraction was equilibrated with 1/9 volumes of 10 X loading buffer (0.1 M Imidazole, 3 M NaCl, 0.2 M Tris-HCl pH 8.0) and subjected to a Ni2+ affinity chromatography as described by Iwashkiw et al (2012). To determine if AcrA was glycosylated, we analyzed the purified protein by Western blot with antibodies specific to either AcrA (Fig 5A) or OPS II (Fig 5B) and when visualized together, we observed an overlap of the signal, highly suggesting glycosylation (Fig 5C).

Mass Spectrometry Analysis of the Purified Glycosylated AcrA confirms OPS II attached

Next, the purified glycosylated AcrA was typically digested in solution to identify the glycan structure. Manual analysis of the LC-ESI-Q-TOF data revealed a peak of 1152.063+ that corresponded to the previously identified second glycosylation site (AVFDNNNSTLLPGAFATITSEGFIQK; m/z 2754.1) of AcrA with the addition of an m/z 700 glycan (Fig 6). Manual peak annotation identified the glycan to be a tetramer of Peptide-188-162-188-162. The mass of 188 Da is consistent with an O-acetylated deoxytalose residue, and our mass spectrometry analysis is consistent with the previously published data demonstrating the OPS II of B. pseudomallei to be a polymer of a dimer of deoxytalose and glucose (Perry, 1995).

The purified AcrA containing the B. pseudomallei OPS II antigen was sent to Defence Research and Development Canada – Suffield Research Centre for murine immunizations and challenges.


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Project 2: Outer Membrane vesicles (OMVs) with Burkholderia OPS II vaccine

Gram negative bacteria are able to produce outer membrane vesicles (OMV). These vesicles, for a long time thought to be cell debris, constituted of a novel secretion pathway. OMVs mainly composed of LPS, outer membrane and periplasmic proteins, and phospholipids, are formed by blebbing of the outer membrane. Due to their immunogenic properties, self-adjuvanticity, ability to be taken up by mammalian cells, and capacity for enhancement by recombinant engineering, OMVs are attractive candidates for vaccine delivery platforms. OMV vaccines are best exemplified by the MeNZB vaccine, which has been effectively and safely used against bacterial meningitis in New Zealand, leading to a drastic reduction in the incidence of this type of infection in the country. OMVs have also been safely used in humans in other countries. The Feldman lab has been developing a promising new platform for antibacterial vaccines consisting of glycoengineered OMVs (geOMVs) (Fig 7). The proof of principle is the Streptococcus pneumoniae vaccine in which engineered E. coli cells produce OMV expressing S. pneumoniae capsular polysaccharide serotype 14. In E. coli, the capsule was incorporated as an O chain in the LPS. geOMVs produced from these cells were used to vaccinate mice and showed that these geOMVs can elicit a specific immunogenic IgG response that is comparable to current commercially available Streptococcus vaccines (i.e., Prevnar 13). As an additional collaboration with Defence Research and Development Canada – Suffield Research Centre, we also applied the geOMV technology platform to incorporate other glycans such as the Burkholderia pseudomallei OPS II antigen. Here we describe the development of a Burkholderia geOMV vaccine.

geOMVs expressing Burkholderia OPS II antigen

Using the constructed pEQ3 plasmid which expresses the Burkholderia OPS II, pEQ3 was introduced into the E. coli strains CLM37 and EPI300, each harbouring pWEL2. The plasmid pWEL2 overexpressed PagL, a lipid A deacylase, which has been shown to detoxify the endotoxin of OMVs (Asensio et al 2011). The strains were cultured overnight in LB media with 0.1% IPTG and 0.4% arabinose to stimulate plasmid expression. The supernatant was collected and filtered, and OMVs were collected by ultracentrifugation as previously described (Morein et al., 1984). Samples of collected geOMVS were subsequently digested with proteinase K, subjected to SDS-PAGE and silver staining. Western blots analysis were performed for visualization of OPS II glycans (Fig 8). geOMVs isolated from both EPI300 and CLM37 E. coli backgrounds could be visualized.

Packaging antigenic Burkholderia proteins into geOMVs

As successfully marketed OMVs vaccines have incorporated the packaging of recombinant proteins into the vesicles (i.e., 4CMenB BexSero), we explored the possibility to package Burkholderia recombinant proteins into E. coli geOMVs. We chose the Burkholderia PotF as the recombinant antigenic protein of choice and cloned the wildtype protein into pACT3, generating pNLP81 (Fig 9A).

The expression of wildtype PotF (pNLP81) in concert with Burkholderia OPS II (pEQ3) and PagL (pWEL2) was visualized by Western blot analysis (Figure 10A). We noted that the PotF expression in CLM37 strain was stronger than in EPI300 strain, and therefore we propagated and isolated vesicles from the CLM37 strain background (Fig 10B). We observed that PotF was present in the geOMVs, however in very low quantities. A proteinase K digestion of the geOMVs confirmed the low PotF amounts.

Collected geOMVs from the CLM37 strain carrying pWEL2 and pEQ3 were sent to Defence Research and Development Canada- Suffield Research Centre for murine immunizations and challenges.

As Burkholderia PotF does not contain any glycosylation site motifs, additional directives were undertaken to generate Burkholderia PotF with glycotags in which engineered PotF may be glycosylated.


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Two engineered PotF were generated. The first engineered PotF consisted of a single glycotag introduced by way of site directed mutagenesis which was cloned into expression vector pACT3, pNLP82. A second engineered PotF was synthetically produced (GenScript, Piscataway, NJ, USA) containing three glycotags with the protein (Fig 9B). The triple glycotage potF was also subcloned in pACT3 expression vector, pNLP83. Analysis of PotF expression showed that pNLP82 appeared to have low PotF expression compared to wildtype PotF (pNLP81) and the triple glycotag potF (pNLP83) had problems with protein stability (Fig 9A). Further directives to troubleshoot such issues were not explored due to time constraints of the contract. In addition, no attempts were made to determine if the engineered PotF with glycotags could be glycosylated by PglB. All three potF plasmids (pNLP81, pNLP82, and pNLP83) were sent to Defence Research and Development Canada – Suffield Research Centre in contractual agreement.

Efficiency of Burkholderia glycoconjugate vaccine vs. geOMV vaccine

Both B. pseudomallei vaccines were sent to Defence Research and Development Canada – Suffield Research Centre for vaccinology studies. Methods and data are not presented here, however overall preliminary data shared by DRDC’s Suffield Research Centre suggests that the both Burkholderia vaccines demonstrated IgG immune response by ELISA towards whole cell extracts of B. pseudomallei. In mice challenge experiments, the glycoconjugate vaccine elicited a stronger protective effect compared to the geOMV vaccine. As geOMVs are currently an untested novel vaccine platform compared to glycoconjugate vaccines, it is suggested that future efforts should be focused on improving the B. pseudomallei glycoconjugate vaccine to increase its effectiveness.


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List of strains and plasmids sent to DRDC’s Suffield Research Centre

Strain/Plasmid Information Antibiotic Resistancea

E. coli SDB1 pEQ3, pMAF10, pIH18 strain expressing glycosylated AcrA Amp, Tmp, Spc

E. coli SDB1 pBAD24, pMAF10, pIH18 strain expressing unglycosylated AcrA Amp, Tmp, Spc

pNLP81 wildtype potF cloned into pACT3 Cam pNLP82 single glycotag potF cloned into pACT3 Cam pNLP83 triple glycotag potF cloned into pACT3 Cam

a Amp = ampicillin, Tmp = trimethoprim; Spc = spectinomycin; Cam = chloramphenicol

List of plasmids for in text reference (not sent to DRDC’s Suffield Research Centre)

Plasmid Information Antibiotic Resistanceb

pBAD24 Cloning and expression vector, arabinose inducible Amp pEXT21 Cloning and expression vector, IPTG inducible Spc pMLBAD Cloning and expression vector, arabinose inducible Tmp

pEQ3 B. pseudomallei type II OPS Amp pIH18 Soluble periplasmic C. jejuni acrA

6xHis cloned into pEXT21 Spc

pMAF10 C. jejuni pglB cloned into pMLBAD Tmp pFLP2 Source of Flp recombinase Amp pACT3 Cloning and expression vector, IPTG inducible Cam pWEL2 Salmonella pagL6xHIS cloned into pEXT21 Spc

b Amp = ampicillin, Tmp = trimethoprim; Spc = spectinomycin; Cam = chloramphenicol


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Figure 1. Description of Burkholderia pseudomallei O-polysaccharide II (OPS II) cluster. (A) Representation of the required 15 genes for the synthesis of OPS II and predicted functions. (B) Expected glycan intermediate structure attached to Undecaprenyl pyrophosphate. (C) Western blot anti-Glycan reveals expression of OPS II (subcloned in pEQ3) in E. coli produces a polymer in whole cell extracts

C

VC pEQ3


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Figure 2. Hypothesis for the lack of glycosylation due to glycan competition and utilization, resulting in the development of the E. coli strain SDB1. (A) When expressing pEQ3 in a WT E. coli background, OPS II is produced at a lower quantity than the native O-antigen, and both structures are preferentially transferred by WaaL to Lipid A, instead of to the acceptor protein by PglB (OTase). (B) If wecA is deleted, the native O-antigen cannot be produced, allowing for a greater production of OPS II. However, OPS II would be still selectively transferred by WaaL over PglB. (C) if both wecA and waaL are deleted, the OPS II structure would be highly produced, and only available to PglB for synthesis of glycoproteins


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Figure 3. Confirmation of a wecA- waaL- double mutant (SDB1) in E. coli W3110. To verify the double mutant, LPS extractions were analyzed by silverstain for the ability to produce exogenously expressed E. coli O7 LPS (pJHCV32). No LPS was observed when either WecA or WaaL was expressed in trans (lanes 1-8). However, when both were coexpressed, a laddering pattern was observed, confirming complementation of the double mutant (lanes 9-10).


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Figure 4. Expression of OPS II and transfer to Lipid A by Western Blot. pEQ3 (OPS II) was expressed in different E. coli backgrounds and LPS extractions were performed. Detection of the OPS II LPS was observed by Western blot. No transfer of OPS II to Lipid A was observed in SDB1, whereas high levels were observed in CLM37 (wecA-), and less amount was observed in the WT background EPI300 when expression was induced by 0.2% Arabinose.

SDB1 CLM37 EPI300

Arabinose 0.2% ‐ + ‐ + ‐ +


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Figure 5. Analysis of purified AcrA by Western Blot. SDB1 transformed with pMAF10 (pglB), pIH18 (AcrA), and pEQ3 (OPS II) were grown (+/-) induction of OPS II, harvested, and AcrA was purified by Ni2+-NTA affinity chromatography. A single band is only observed by anti-His Western blot at ~35 kDa without induction of pEQ3, whereas a high molecular weight ladder is co-detected by both anti-His and anti-Glycan antibodies in cultures induced with 0.2% arabinose, confirming glycosylation of AcrA.


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Figure 6. Mass spectrometry Identification of the B. pseudomallei OPS II glycan attached to AcrA. MS analysis of typically digested glycosylated AcrA revealed a peak of m/z 1152.063+. MS/MS of this peak showed a previously characterized glycosylation site of AcrA (AVFDNNNSTLLPGAFATITSEGFIQK; 2754.1 Da) with a modification of 700.2 Da. Analysis of the modification revealed a tetrameric glycan, with a structure of 188-162-188-162. This structure is consistent with the previously determined structure of B. pseudomallei OPS II being a polymer of dimers of O-acetylated deoxytalose (188 Da) and glucose (162 Da).


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Figure 7. The geOMV platform. Genetic locus encoding glycan structure of interest is cloned into expression vector and introduced into an engineered E. coli strain which generates vesicles that display the glycan. The collected geOMVs can be directly utilized for immunizations.


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Figure 8. geOMVs displaying Burkholderia OPS II glycans. The geOMVs were isolated from E. coli EPI300 and CLM37 strains harbouring pEQ3 (OPS II) and pWEL2 (expresses PagL). Silver staining and Western blots (using anti-OPSII) show the presence of the OPS II glycan. geOMVS displaying pneumococcal capsule (CPS14) was utilized as a visualization control.


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Figure 9. The construction of PotF expression plasmids. (A) Western blot analysis of whole cell lysates using anti-HIS of PotF constructs expression in E. coli and (B) the amino acid alignment of the PotF constructs. All constructs were cloned into the expression vector pACT3; pNLP81, wild-type potF ; pNLP82, a potF mutant consisting of a single glycotag (Q226FNSS230 to D226INSS230, highlighted in green); pNLP83, a potF mutant synthetically engineered with 3 glycotags (highlighted in yellow, green, and red).


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Figure 10. Packaging of Burkholderia PotF in geOMVs. (A) Western blot of whole cell lysates (WCL) of E. coli EPI300 and CLM37 strains expressing PotF (pNLP81), PagL (pWEL2), and Burkholderia OPS II glycan (pEQ3). (B) Western blot of isolated WCL geOMVs from CLM37 strain carrying pNLP81, pWEL2, and pEQ3. PotF and PagL visualized by anti-HIS (red) and Burkholderia glycan by anti-OPS II (green) antibodies. PK: proteinase K digestion.

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