Constructing conjugate vaccine against Salmonella Typhimurium using lipid-A free lipopolysaccharide

Background Salmonella enterica serotype Typhimurium is a nontyphoidal and common foodborne pathogen that causes serious threat to humans. There is no licensed vaccine to prevent the nontyphoid bacterial infection caused by S. Typhimurium. Methods To develop conjugate vaccines, the bacterial lipid-A free lipopolysaccharide (LFPS) is prepared as the immunogen and used to synthesize the LFPS–linker–protein conjugates 6a–9b. The designed bifunctional linkers 1–5 comprising either an o-phenylenediamine or amine moiety are specifically attached to the exposed 3-deoxy-D-manno-octulosonic acid (Kdo), an α-ketoacid saccharide of LFPS, via condensation reaction or decarboxylative amidation. In addition to bovine serum albumin and ovalbumin, the S. Typhimurium flagellin (FliC) is also used as a self-adjuvanting protein carrier. Results The synthesized conjugate vaccines are characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and fast performance liquid chromatography (FPLC), and their contents of polysaccharides and protein are determined by phenol–sulfuric acid assay and bicinchoninic acid assay, respectively. Enzyme-linked immunosorbent assay (ELISA) shows that immunization of mouse with the LFPS–linker–protein vaccines at a dosage of 2.5 μg is sufficient to elicit serum immunoglobulin G (IgG) specific to S. Typhimurium lipopolysaccharide (LPS). The straight-chain amide linkers in conjugates 7a–9b do not interfere with the desired immune response. Vaccines 7a and 7b derived from either unfractionated LFPS or the high-mass portion show equal efficacy in induction of IgG antibodies. The challenge experiments are performed by oral gavage of S. Typhimurium pathogen, and vaccine 7c having FliC as the self-adjuvanting protein carrier exhibits a high vaccine efficacy of 74% with 80% mice survival rate at day 28 post the pathogen challenge. Conclusions This study demonstrates that lipid-A free lipopolysaccharide prepared from Gram-negative bacteria is an appropriate immunogen, in which the exposed Kdo is connected to bifunctional linkers to form conjugate vaccines. The decarboxylative amidation of Kdo is a novel and useful method to construct a relatively robust and low immunogenic straight-chain amide linkage. The vaccine efficacy is enhanced by using bacterial flagellin as the self-adjuvanting carrier protein. Graphical abstract


Synthesis of PS−protein conjugates 8a and 8b (Scheme S3)
Traceless Staudinger ligation is a biorthogonal conjugation method that enables the reaction of phosphinothioester and azide to form an amide bond. [41] This method was applied to prepare PS− The size-exclusion chromatography on a Superdex-200 pg column enabled fractionation of LFPS to give an overview analysis of the polysaccharides and protein contaminants. The S.
Typhimurium LFPS (23 mg) was separated into three fractions in a period of 2 h by using 10 mM NaCl aqueous solution as eluent at a flow rate of 1 mL/min. Fraction-1 PS with high molecular weights (MW) was least retained in the Superdex column. The polysaccharide component of each fraction was monitored by refractive index (RI) detector, and the protein contaminant was detected by UV absorption at 280 nm. PSA assay was used to quantify the content of polysaccharides in each fraction. The total weight of the collected polysaccharides from three fractions was 12 mg (52% yield), comparable to the previously reported result. [28] Using pullulan as standard, the molecular weights of LFPS for fraction-1, 2 and 3 were estimated to be 85.8±31.8 kDa, 37.2±20.9 kDa and 10.4±5.8 kDa, respectively. The high-mass portion of LFPS induced higher IgG antibodies. Column: Superdex-200. Eluent: 10 mM NaCl aqueous solution at a flow rate of 1 mL/min. *** p < 0.001. S10 Figure S4. Non-selective and site-selective modifications of flagellin (FliC). [45,46] Lysine residues in all the D0, D1, D2 and D3 domains in a FliC monomer can react with imidazole-1sulfonyl azide. In high concentration of Na2SO4, FliC monomers undergo self-assembling to form flagellin filament. The chemical modifications mainly occur in the exposed D2 and D3 domains, while the D0 and D1 domains are compactly sheltered by each other inside the flagellar filament. The site-selective modified flagellin filament can be depolymerized in phosphate-buffered saline (pH 7) with brief heating at 65 ℃. S11 Figure S5. SDS-PAGE diagram of PS-FliC conjugates (7c, 8b and 9b) using 9% polyacrylamide gel and Coomassie blue staining. Most S. Typhimurium PS-FliC conjugates cannot enter the stacking gel due to their large molecular masses. PBS was negative control. *** p < 0.001. S15

Experimental Section
Materials.
All the reagents and solvents were reagent grade and were used without further purification unless otherwise specified. CH2Cl2 was distilled from CaH2. THF was distilled from sodium.

Instrumentation.
Melting points were recorded on a Yanaco micro apparatus and are uncorrected. Infrared (IR) spectra were recorded on a Thermo Nicolet iS5 FT-IR spectrometer (ThermoFisher). The

Isolation of S. Typhimurium lipopolysaccharide (LPS).
This experiment was conducted in Chang Gung Memorial Hospital. The bacterium S.
Typhimurium (strain SL1344) was cultured in lysogeny broth at 37 ℃ for 16 h. The culture was added into fresh lysogeny broth with a dilution factor 1:100, and cultured at 37 ℃ for 5 h to reach an OD600 of 0.6−0.8. The LPS was isolated by a modified procedure according to Darveau−Hancock method. After the sample had cooled to 0 ℃, it was centrifuged at 12,000 g for 15 min at 0 to 4 ℃. The sample had to be kept as close to 0 ℃ as possible after the precipitate was formed and during the centrifugation. The pellet obtained was suspended in 35 mL of 2% sodium dodecylsulfate (SDS)-0.1 M tetrasodium ethylenediaminetetraacetic acid (EDTA), dissolved in 10 mM Tris-HCl (pH 8), and sonicated as described above (at this stage the solution was usually clear and the pH is about 9.5); however, the pH of the solution could be lowered to pH 7 by dropwise addition of 4 M HCl, to avoid slight saponification of lipids. The solution was then incubated at 85 ℃ for 30 min to ensure the denaturation of SDS-resistant proteins. After cooling, the pH was raised (if previously lowered) to 9.5 by addition of 4 M NaOH. Pronase (35 μg/mL) was added, and the sample was incubated for 16 h at 37 ℃ with constant shaking. After incubation, LPS was precipitated with 2 volumes of 0.375 M MgCl2 in 95% ethanol at 0 ℃ as described above. The supernatant was followed by centrifugation at 12,000 g for 15 min at 0 to 4 ℃. The S17 pellet after centrifugation was resuspended in 25 mL of 10 mM Tris-HCl (pH 8), sonicated as described above, and centrifuged in a clinical centrifuge at 1,000 rpm for 5 min to remove insoluble Mg 2+ -EDTA crystals. The supernatant was then centrifuged at 100,000 g for 20 h at 15 ℃ in the presence of 25 mM MgCl2. The pellet containing the LPS was resuspended in distilled water, and the DNA and protein contamination was checked by the UV absorbance at 260 nm and Coomassie brilliant blue assay.

Preparation of S. Typhimurium lipid-A free polysaccharide (LFPS).
S. Typhimurium LPS (200 mg) was dissolved in 1% AcOH (10 mL) and stirred at 100 ℃ for 2 h. The mixture was dialyzed with 3.5K membrane against H2O four times at 1, 2, 4, and 16 h. After ultracentrifugation at 150,000 g for 5 h, the pellet of lipid A was removed, and the supernatant was purified by ultracentrifugation at 150,000 g for 17 h. The supernatant was lyophilized to get the desired LFPS. Alternatively, the dialyzed sample was subjected to ultracentrifugation once and purification by gel chromatography to give LFPS.

Non-selective azido modification of flagellin monomer and bovine serum albumin.
As a representative procedure, azido-modified FliC (FliC/N3, 30b) was prepared by adding an aqueous solution of imidazole-1-sulfonyl azide hydrochloride (60 mM, 5 μL) to a phosphate buffered saline (PBS solution, 60 μL, 50−167 mM) containing monomeric S. Typhimurium The content of carbohydrate was measured by phenol-sulfuric acid assay. A sample (25 μL) was pipetted into a well of 96-well plate, and freshly prepared phenol solution (30 μL 4% aqueous solution) was added. The mixture was gently shaken for 10 min, and concentrated H2SO4 (180 μL) was added rapidly. The mixture was shaken for another 5 min, and allowed to stand at room temperature for 25 min. A distinct change from colorless to yellow solution was observed. The UV absorbance at λ = 490 nm was measured with a plate reader, and the content of carbohydrate was quantified by interpolation to a standard line of mannose.

Bicinchoninic acid assay (BCA).
Bicinchoninic acid (BCA) assay was conducted following the procedure provided by with NaOH. After incubation for 15 min, the absorbance at 412 nm was measured, and the content of thiol was quantified by interpolation of a standard curve of 2-nitro-5-thiobenzoate dianion (TNB 2− ).

Size-Exclusive Chromatography (SEC).
An Agilent 1100 high-pressure liquid chromatograph (HPLC) in our lab was modified in a way similar to fast performance liquid chromatograph (FPLC) for fractionation of LFPS. The stainless-steel pipelines in conventional HPLC system was changed into that of Teflon or polyether ether ketone (PEEK) material, which can sustain moderate pressure without deformation and also avoid adsorption of proteins. Size exclusion chromatography was conducted on an HiLoad 16/600 Superdex 200 prep grade column (GE Healthcare, Pittsburgh, USA). This instrumental system was equipped with RI and UV detectors.

S19
In one experiment, S. Typhimurium LFPS sample (23 mg) was chromatographed on the Superdex-200 column by elution with 10 mM NaCl aqueous solution at a flow rate of 1 mL/min.

Fast performance liquid chromatography (FPLC).
An Agilent 1100 HPLC system was equipped with RI and UV detectors and a size-

LPS-induced human macrophage cell line assay.
THP-1 cells were treated with 100, 500 or 1,000 ng/mL of sample for 6 h. The secretion of TNF-α and IL-6 was determined by ELISA (R&D system) according to the manual. Briefly, ELISA plates were coated with capture antibody at room temperature for 14 h. The coated plates were washed 3 times with PBS containing 0.05% Tween 20 (PBST-20) and then blocked with 1% BSA in PBS at room temperature. After blocking for 1 h, plates were washed as above.
Appropriate dilution of the cell supernatant was added to the well and incubated for 2 h at room temperature. After washing as above, detection antibody was added to the well for further 2 h incubation. Streptavidin-HRP was added to the well and incubated at room temperature for 20 min. Following the washing steps as described above, a TMB substrate solution was added to the well for 20 minutes and the reaction was stopped with 2 M H2SO4. The optical density at 450 nm of each well was measured.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
The LFPS-protein conjugates sample were subjected to SDS-PAGE to determine the molecular weights. In another operation, SDS-PAGE was performed with standard procedures in a 1.5 mm, 9% polyacrylamide gel. Briefly, a protein sample was mixed with loading buffer (sample/loading buffer = 4:1 (v/v); GeneMark, cat. GM-47b). The sample was vortexed briefly and heated at 95−100 ℃ for 5−10 min. The sample (20 μL) was loaded into the well. The electrophoresis was run at 60 V through the stacking part of the gel and up to 150 V after the proteins have migrated into the resolving gel until the dye front reached the bottom of gel. The gel was rinsed with distilled water (dH2O) to remove the running buffer, and then placed in fixing solution (isopropanol/dH2O/acetic acid = 2.5:6.5:1 (v/v)) for 1−2 h with gentle shaking. After the fixing solution was poured off, the gel was rinsed with dH2O, and Coomassie blue G-250 was applied for 1−2 h with gentle shaking. After the staining solution was removed, the gel was rinsed with dH2O, and placed in a destaining solution (10% acetic acid) with gentle shaking until the clearly visible bands appeared (ca. 1−3 h).

Evaluation of hTLR5 activity.
The ability of FliC-based slef-adjuvanting vaccines to stimulate TLR5 was evaluated by a SEAP reporter cellular assay using an HEK-Blue hTLR5 cell line (InvivoGen). According to the protocol, a suspension of HEK-Blue hTLR5 cells was prepared at a concentration of 1.4×10 4 cells/mL in the HEK-Blue detection medium. Then, the cell suspension was added to a 96-well plate (180 μL/well, ≈ 25,000 cells per well). The native S. Typhimurium flagellin was used as a positive control, and 1× PBS buffer was used as a negative control. In the presence of different concentrations of the indicated FliC samples (50, 10 and 1 ng/mL), the cells were incubated at 37 ℃ for 24 h; then, TLR5 activation was evaluated by the absorbance at λ = 620 nm due to SEAP-catalyzed hydrolysis of the substrate. The data are presented as mean ± standard deviation (n = 5). The comparison of paired samples was performed by using Student's t-test.
The immunological assays were performed with general sandwich ELISA to measure the titer of linker-specific, hapten-specific, and carrier-specific antibodies. Sera were taken from immunized BALB/c mice by the retro-orbital bleeding method. ELISA plates (Corning, cat. 9018) were coated with distinct testing antigen at 4 ℃ for 14 h (5 μg LFPS, 2 μg linker-BSA conjugate, or 2 μg native FliC per well). The coated plates were blocked with 2% BSA at room temperature for 2 h. Plates were washed 3 times in PBS containing 0.05% Tween 20 (PBST-20). Then an appropriate diluted solution of mice serum was added to the well. Plates were incubated at room temperature for 2 h. After washing as described above, HRP-conjugated goat anti-mouse antibody (Millipore) was added to each well, and the plates were incubated at room temperature for 1 h. Following the washing steps as described above, a 3,3',5,5'-S21 tetramethylbenzidine (TMB) substrate solution was added to the well for 20 minutes, and the reaction was stopped with 2 M H2SO4. The optical density at 450 nm of each well was measured.

Mice immunization experiment.
The animal study was carried out in the laboratory animal center of Chang Gung University and compliance with the policy of animal care and use. Each group in the immunization experiments has 5 mice (BALB/c mice aged 6-8 weeks). BALB/c mice were immunized with the LFPS-protein conjugate (e.g. PS-A-B-BSA, 7a) at a dose of 2.5 or 5 μg for four times on weeks 0, 2, 4 and 6. Sera were collected from immunized mice by eye-bleeding method (bleeding from the retroorbital venous plexus of mice) before immunization and after immunization on week 8. For the first immunization on week 0, the LFPS-protein conjugate was mixed with equal volume of Freund's complete adjuvant (Sigma). For the rest of immunization, the LFPS-protein conjugate (or LFPS) was mixed with equal volume of Freund's incomplete adjuvant (Sigma).
Alternatively, BALB/c mice aged 6-8 weeks were randomly assigned to one control group (n = 5) and three experimental groups of 10 mice. Mice were immunized with the LFPS-protein conjugate (e.g. PS-A-B-FliC, 7c) at a dose of 2.5 μg by subcutaneous administration for three times on weeks 0, 2, and 4. No additive adjuvant was used for both initial vaccination and booster immunization. Sera were collected from immunized mice by retro-orbital bleeding method (bleeding from the retro-orbital venous plexus of mice) before immunization and after immunization on week 6.

Serum antibody titer test.
Anti-LFPS antibodies in BALB/c mice were determined by ELISA. Mouse sera were taken from immunized BALB/c mice by eye-bleeding method. ELISA plates were coated with 3 μg antigen (e.g. purified S. Typhimurium LFPS) at 4 ℃ for 16 h. The coated plates were blocked with 2% BSA at room temperature for 2 h. Plates were washed 3 times in PBS containing 0.05% Tween 20 (PBST-20). Then appropriate diluted solution of mouse serum was added to the well. Plates were incubated for 2 h at room temperature. After washing as described above, horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Millipore) was added to each well and the plates were incubated at room temperature for 2 h. Following the washing steps as described above, a 3,3′,5,5′-tetramethylbenzidine (TMB) solution was added to the well for 20 min, and the reaction was stopped with 2 M H2SO4. The optical density at 450 nm of each well was measured.

Bacterial challenge test.
The immunized mice received oral or intravenous challenge with virulent pathogen (e.g.

S.
Typhimurium SL1344) equivalent to 10 6 CFU (oral challenge) and 500 CFU (intravenous S22 challenge) on day 14 after the last immunization. The mortality was recorded daily for 14 days.

Mice survival analysis.
The Kaplan-Meier method and the log-rank test were used to compare the survival of mice in the immunized and control groups. The survival times were also modeled using Cox proportional hazards regression to estimate the hazard ratios and protective factors of vaccination with distinct vaccines compared to the controls. [50] Data analysis was performed using IBM SPSS Statics (version 24).

N-(3-Aminopropyl) 3,4-diaminobenzamide (13, as the hydrochloric salt).
Compound 12 (180 mg, 0.58 mmol) was dissolved in EtOAc (2 mL), followed by dropwise addition of 3 M HCl (5 mL). The mixture was stirred at room temperature for 2 h, and then concentrated under reduced pressure. The crude product was purified by C18 reverse-phase silica gel column chromatography with elution of water, and then lyophilized to afford the desired produce 13 (130 mg, 91% yield

S26
A suspension of S. Typhimurium LFPS (92.6 mg, 4.0 μmol, based on the average molecular weight of 23 kDa) in DMSO (2.5 mL) was sonicated at room temperature for 10 min to dissolve all solid particles. A solution of N-(6-aminohexyl)-2-nitrobenzenesulfonamide (19, linker A/Ns as the TFA salt, 14.4 mg, 35 μmol) in DMSO (0.5 mL) was added to a vial containing iodine (19.9 mg, 78.3 μmol) and Cs2CO3 (57.8 mg, 177 μmol). The mixture was added to the aboveprepared DMSO solution of LFPS, and stirred at room temperature for 23 h under an atmosphere of argon. The reaction was quenched by addition of Na2S2O3 (24.8 mg,156.9 μmol) with stirring at room temperature for 10 min to give a crude product of PS-A/Ns (20).

S27
A suspension of S. Typhimurium PS-A/NH2 (21, 113.6 mg) in DMF (5 mL) was sonicated at room temperature for 5 min to completely dissolve the solid particles. The PS-A/NH2 solution was added dropwise (5 mL/15 min) to bis(4-nitrophenyl) adipate (34 mg, 87.5 μmol) in DMF (1 mL). The mixture was stirred at room temperature for 23 h under an atmosphere of argon, and then concentrated under reduced pressure to give a yellow oil. After successive washes with CH2Cl2 (3 mL), yellow solids were obtained. The solids were collected by centrifugation (5,000 g, 2 min), and then washed with CH2Cl2 (3 mL, 2×). After removal of CH2Cl2 under reduced pressure, the PS-A-B/Np product 23 was obtained as pale-yellow powder (96.2 mg).

Preparation of PS-A-B-BSA (7a) and PS-A-B-OVA (7b) conjugates.
To a solution of BSA (1.4 mg) in phosphate buffer solution (PB, 0.5 mL, 400 mM, pH 7) was added S. Typhimurium PS-A-B/Np (23, 10.7 mg) in PB/DMF (2:1) solution (2 mL, 400 mM pH 8-9). The mixture was diluted by addition of PB solution (400 mM, pH 7) to a final volume of 7 mL. The mixture was gently shaken at room temperature for 65 h under an atmosphere of argon to furnish the PS-A-B-BSA conjugate (7a). The crude product was concentrated by spinning filtration against 1× PBS (pH 7) through an Amicon Ultra-15 10 kDa centrifugal filter (5,000 g, 30 min, 3×), sterilized through a 0.22 μm PES filter (Acrodisc Supor, Pall), and purified by FPLC (1× PBS) to give pure PS-A-B-BSA product (7a). The conjugate was assessed for the contents of carbohydrate and protein using PSA method and BCA assay, respectively.
NaH (60% dispersion in oil, 91.7 mg, 2.29 mmol) was placed in an oven-dried roundbottomed flask containing a stirring bar, and then sealed and connected to an argon balloon.
Anhydrous DMF (1.2 mL) was added dropwise while the flask was cooled in an ice-bath. No bubbling was observed during addition of DMF. A solution of borane diphenylphosphine complex (458 mg, 2.29 mmol) in anhydrous DMF (0.6 mL) was added dropwise to the suspension of NaH in DMF with stirring until bubbling ceased. A solution of S-bromomethyl ethanethioate (374 mg, 2.21 mmol) in anhydrous DMF (0.6 mL) was then added to the mixture, which was cooled in an ice bath. The mixture was stirred for 10 min, allowed to warm to room temperature, and stirred for 24 h. EtOAc (5 mL) was added to the mixture, and the insoluble solids were filtered off using a cotton filter. The filtrate was collected, concentrated under reduced pressure, and subjected to silica gel column chromatography using a gradient of EtOAc/hexane (1:10, 100 mL; 1:4, 400 mL) to give diphenylphosphinomethyl thioacetate borane complex (247 mg, 39%). The product was stored at 4 ℃ until use. C15H18BOPS; Diphenylphosphinomethyl thioacetate borane complex (110.6 mg, 0.38 mmol) was dissolved in anhydrous MeOH (1.6 mL), followed by addition of a suspension of sodium methoxide in anhydrous MeOH (30% (w/w), 100 μL). The mixture was stirred at room temperature for 10 min under an atmosphere of argon. An aqueous HCl solution (1 M) was