Deciphering minimal antigenic epitopes associated with Burkholderia pseudomallei and Burkholderia mallei lipopolysaccharide O-antigens

Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm), the etiologic agents of melioidosis and glanders, respectively, cause severe disease in both humans and animals. Studies have highlighted the importance of Bp and Bm lipopolysaccharides (LPS) as vaccine candidates. Here we describe the synthesis of seven oligosaccharides as the minimal structures featuring all of the reported acetylation/methylation patterns associated with Bp and Bm LPS O-antigens (OAgs). Our approach is based on the conversion of an l-rhamnose into a 6-deoxy-l-talose residue at a late stage of the synthetic sequence. Using biochemical and biophysical methods, we demonstrate the binding of several Bp and Bm LPS-specific monoclonal antibodies with terminal OAg residues. Mice immunized with terminal disaccharide–CRM197 constructs produced high-titer antibody responses that crossreacted with Bm-like OAgs. Collectively, these studies serve as foundation for the development of novel therapeutics, diagnostics, and vaccine candidates to combat diseases caused by Bp and Bm.

│ Analysis of LPS antigens purified from wild type and mutant strains of B. pseudomallei. LPS antigens (2 μg/lane) were separated on 12% Tris-Glycine gels and visualized by (A) silver staining. For Western immunoblotting, LPS antigens were electrophoretically transferred to nitrocellulose membranes and probed with (B) mAb Pp-PS-W or (C) mAb 3D11. Wild type LPS was purified from B. pseudomallei RR2808 while OacA mutant LPS was purified from B. pseudomallei Bp RR4744. Data not shown: Similar to mAb 3D11, mAbs 4C7 and 9C1-2 only reacted with RR4744 LPS. Likewise, B. mallei LPS only reacted with mAbs 3D11, 4C7 and 9C1-2. Based on these results, Bp RR4744 OPS and B. mallei OPS antigens appeared to share a common epitope (see Figure 2). were separated on 4-12% Bis-Tris Bolt gels and electrophoretically transferred to nitrocellulose. SOC-6 was detected by chemiluminescence using a 1/2000 dilution of mAb 3D11 and a 1/5000 dilution of an anti-mouse IgG-HRP conjugate. Results similar to mAb 3D11 were observed using mAbs 4C7 and 9C1-2 (data not shown). In contrast, SOC-7 was detected by chemiluminescence using a 1/400 dilution of mAb Pp-PS-W and a 1/5000 dilution of an anti-mouse IgM-HRP conjugate (data not shown). were immunized with OC-2808. ELISAs were used to quantitate immune serum IgG titers. Colored dots represent the mean endpoint titers for individual mice against the various target antigens.

General methods
All starting materials and reagents were purchased from commercial sources, and used as received without further purification. Air and water sensitive reactions were performed in heat gun-dried glassware under Ar atmosphere. Moisture sensitive reagents were introduced via a dry syringe. Anhydrous solvents were supplied over molecular sieves, and used as received.

General procedures
Synthesis of trichloroacetimidate donors. 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)iridium(I) hexafluorophosphate (0.02-0.1 equiv) was dissolved in anhydrous THF (5 mL⋅mmol -1 ) and the red solution was degassed under Ar. Hydrogen was bubbled through the solution for 5 min, and then the yellow solution was once again degassed under Ar. A solution of allyl taloside (1.0 equiv) in anhydrous THF (5.0 mL⋅mmol -1 ) was added. The mixture was stirred for 2 h at rt under Ar. Then, a solution of iodine (2.0-2.5 equiv) in THF/H2O (6.0 mL⋅mmol -1 , 4:1 v/v) was added to the mixture, which was stirred for another 1 h at rt. The excess of iodine was quenched by adding a freshly prepared 10% Na2S2O3(aq) solution and stirred until the color turned bright yellow (∼5 min). The aqueous phase was extracted with EtOAc (3 ×). The combined organic layers were washed with a saturated NaHCO3(aq) solution and brine. The solvents of the dried solution (MgSO4) were concentrated under reduced pressure. The residue was purified by silica gel flash chromatography to give the corresponding hemiacetal as an α/β mixture. To a cooled (0 °C) solution of the hemiacetal (1.0 equiv) in DCM/acetone (14 mL⋅mmol -1 , 8:3 v/v) were added DBU (0.3 equiv) or Cs2CO3 (0.2 equiv) followed by CCl3CN (5.0-6.0 equiv). The mixture was stirred for 1 h at rt, then the suspension was filtered over Celite and rinsed with DCM. The solvents were concentrated under reduced pressure. The residue was purified by silica gel flash chromatography to give the trichloroacemidate donor, the α-anomer being the major compound.
Synthesis of protected disaccharides. Acceptor 13 (1.0 equiv) and donor 8-12 (2.0 equiv) were dried for 2 h under high vacuum and then dissolved in anhydrous Et2O (20 mL⋅mmol -1 ). The solution was cooled to -10 °C and TMSOTf (0.01-0.2 equiv) was added keeping rigorous anhydrous conditions. The mixture was stirred at -10 °C for 10 min under Ar, and then quenched with a few drops of Et3N. The suspension was filtered over Celite, rinsed with DCM and the filtrate was concentrated under reduced pressure. The residue was purified by combi-flash chromatography to give the target disaccharide as a pure α-anomer.
Deprotection of PMB group. To a solution of disaccharide 15-17 (1.0 equiv) in DCM/H2O (22 mL⋅mmol -1 , 10:1 v/v) was added DDQ (2.0 equiv) and the deep-green mixture was stirred for 2 h at rt. The reaction was quenched by adding a saturated NaHCO3(aq) solution, stirred until the color turned bright yellow (∼10 min), and diluted with EtOAc. The organic phase was washed with a saturated NaHCO3(aq) solution and brine. The solvents of the dried solution (MgSO4) were concentrated under reduced pressure. The residue was purified by silica gel flash chromatography to give the corresponding alcohol.
Hydrogenolysis using the H-Cube system. The oligosaccharide (1.0 equiv) was dissolved in DCE (10 mL⋅mmol -1 ), then MeOH (250 mL⋅mmol -1 ) followed by concentrated HCl (2.0 equiv) were added. The solution was passed without delay through a 20% Pd(OH)2/C cartridge (CatCart30) using a H-Cube continuous flow system in control mode (10 bars). The temperature was set at 40 °C, and the flow rate was fixed at 1.0 mL⋅mmol -1 . After one run, the cartridge was rinsed with MeOH and the solutions were concentrated under reduced pressure keeping the bath temperature below 40 °C. The residue was subjected to C18 reversed-phase flash chromatography (H2O/MeOH 10:0 to 6:4) followed by freeze-drying to give the target oligosaccharide in the form of a hydrochloride salt.
Hydrogenolysis under heterogeneous conditions. The oligosaccharide (1.0 equiv) was dissolved in anhydrous DCE (10 mL⋅mmol -1 ), then anhydrous MeOH (250 mL⋅mmol -1 ) followed by concentrated HCl (1.0 equiv) were added. The solution was degassed with Ar and Pd black (1 mg⋅mg -1 of compound) was added. The suspension was stirred under an atmosphere of H2 at 40 °C for 16 h. The mixture was filtered over Celite to remove the catalyst, and the cake was rinsed with MeOH. The solutions were concentrated under reduced pressure keeping the bath temperature below 40 °C. The soluble part of the residue was dissolved in D2O, filtered over Celite using a pipette, rinsed with D2O and the solutions were concentrated under reduced pressure to give the target oligosaccharide in the form of a hydrochloride salt.

Biotinylation of oligosaccharides.
A solution of the free oligosaccharide (1.0 equiv) and 6biotinylamidohexanoic acid N-hydroxysuccinimidoyl ester (2.0 equiv) in DMF (22.5 mL⋅mmol -1 ), Et3N (2.5 mL⋅mmol -1 ), and H2O (25.0 mL⋅mmol -1 ) was stirred for 1 h at rt. The solvents were concentrated under reduced pressure. The resulting residue was dissolved in EtOH and the soluble fraction was purified by silica gel flash chromatography (DCM/MeOH) to give the biotinylated oligosaccharide.

SDS-PAGE and Western immunoblotting.
Glycoconjugate samples were solubilized in 1X SDS-PAGE sample buffer and heated to 100 °C for 5 min prior to electrophoresis on 4-12% Bis-Tris Bolt gels (Life Technologies). Proteins were visualized via staining with Coomassie Blue R-250. For Western immunoblot analyses, the glycoconjugate samples and CRM197 were separated on the same 4-12% gels and electrophoretically transferred to nitrocellulose membranes. The membranes were blocked with 3% skim milk in high salt Tris-buffered saline (HS-TBS; 20 mM Tris, 500 mM NaCl, pH 7.5) for 60 min at room temperature and then incubated overnight at 4 °C with 1/400 -1/2000 dilutions of a B. pseudomallei (Pp-PS-W) or B. mallei OPS-specific mAbs (4C7, 3D11 and 9C1-2). To facilitate detection, the membranes were incubated for 1 h at room temperature with 1/5000 dilutions of an anti-mouse IgG horse radish peroxidase conjugate (SouthernBiotech). The blots were then visualized using Pierce ECL Western Blotting Substrate (Pierce).
Immunofluorescence staining and microscopy. B. mallei ATCC 23344 was cultured at 37 °C with aeration (200 rpm) in LB Lennox broth (Fisher Scientific) supplemented with 4% glycerol. Mid log phase bacteria were pelleted by centrifugation, fixed with 2.5% paraformaldehyde for 15 min then washed extensively with PBS and then blocked with PBS containing 10% normal goat serum (PBS-G; Invitrogen) for 20 min. Bacteria were stained with CRM197, OC-4744 or SOC-6 mouse antiserum (from mice represented by green dots in Fig 8a and 8b) diluted 1/500 in PBS-G for 30 min, washed three times with PBS and then incubated with Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) diluted 1/1000 in PBS-G for 30 min. Stained bacteria were then washed three times with PBS, rinsed two times with water and mounted onto glass slides with ProLong Gold (Invitrogen) medium. Fluorescence and bright field microscopy was performed using a Nikon Eclipse 90i imaging system using a CFI Plan APO VC 100X/1.4 oil objective (Nikon Instruments Inc.). Images were acquired using NIS-Elements Advanced Research software (Nikon Instruments Inc.). All manipulations of B. mallei were conducted in CDC-approved and -registered biosafety level 3 facility at the University of South Alabama in accordance with standard select agent operating practices in compliance with the rules and regulations of the U.S. Federal Select Agent Program.
Immunogenicity evaluation. Groups of 6-8 week old female C57BL/6 mice (Charles River) were immunized subcutaneously on days 0, 21 and 35 with 10 μg of the OAg-CRM197 glycoconjugate OC-2808 formulated in saline plus Alhydrogel 2% (500 μg/mouse; Brenntag) and PolyI:C (PIC; 30 μg/mouse; InvivoGen). Terminal bleeds were conducted 14 days after the third immunization for the assessment of antibody responses. All procedures involving mice were performed according to protocols approved by the University of South Alabama Institutional Animal Care and Use Committee and were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Route B (Pfitzner-Moffatt procedure):
To a solution of DMSO (4.4 mL, 61.4 mmol, 5.0 equiv) in anhydrous DCM (123 mL) at -10 °C under Ar were added sequentially with stirring PDCP (5.5 mL, 36.8 mmol, 3.0 equiv) and Et3N (8.6 mL, 61.4 mmol, 5.0 equiv). Then a solution of alcohol S1 (3.0 g, 12.3 mmol, 1.0 equiv) in DCM (61 mL) was added dropwise during 1 h. The reaction mixture was stirred at -10 °C for 10 min, then allowed to slowly warm up to rt. After 30 min, water (100 mL) was added. The organic phase was separated and the aqueous phase was extracted with DCM (3 × 40 mL). The combined organic phases were washed with brine. The solvents of the dried solution (MgSO4) were concentrated under reduced pressure. To a cooled (-10 °C) solution of the ketone in MeOH (123 mL) was slowly added NaBH4 (558 mg, 22.1 mmol, 1.8 equiv). The mixture was stirred from -10 to 0 °C under Ar for 1 h. Then, the reaction mixture was diluted with DCM (200 mL) and the organic layer was washed with water (120 mL). The aqueous layer was back extracted with DCM (3 × 50 mL). The combined organic phases were washed with brine, dried (MgSO4) and then concentrated under reduced pressure. The residue was purified by silica gel flash chromatography (PE/EtOAc 9:1 to 8:2) to give alcohol S2 (