Novel Solutions for Vaccines and Diagnostics To Combat Brucellosis

Brucellosis is diagnosed by detection of antibodies in the blood of animals and humans that are specific for two carbohydrate antigens, termed A and M, which are present concurrently in a single cell wall O-polysaccharide. Animal brucellosis vaccines contain these antigenic determinants, and consequently infected and vaccinated animals cannot be differentiated as both groups produce A and M specific antibodies. We hypothesized that chemical synthesis of a pure A vaccine would offer unique identification of infected animals by a synthetic M diagnostic antigen that would not react with antibodies generated by this vaccine. Two forms of the A antigen, a hexasaccharide and a heptasaccharide conjugated to tetanus toxoid via reducing and nonreducing terminal sugars, were synthesized and used as lead vaccine candidates. Mouse antibody profiles to these immunogens showed that to avoid reaction with diagnostic M antigen it was essential to maximize the induction of anti-A antibodies that bind internal oligosaccharide sequences and minimize production of antibodies directed toward the terminal nonreducing monosaccharide. This objective was achieved by conjugation of Brucella O-polysaccharide to tetanus toxoid via its periodate oxidized terminal nonreducing monosaccharide, thereby destroying terminal epitopes and focusing the antibody response on internal A epitopes. This establishes the method to resolve the decades-long challenge of how to create effective brucellosis vaccines without compromising diagnosis of infected animals.


Figure S1
Oligosaccharides available from other studies were activated and conjugated to BSA according to methods reported elsewhere to provide glycoconjugates S28-S37. 1,2 The number of hapten groups per BSA was 10-15.

Figure S2
Pentasaccharide methyl glycosides were synthesized as previously described 3,4 and used as inhibitors of sera raised to vaccine 2.
To the solution of benzoyl protected compound (0.9 g, 2.63 mmol) dissolved in anhydrous DMF (10 mL) was added NaH (0.12 g, 2.89 mmol) at 0 o C. The mixture was stirred at 0 o C for 45 min, and then BnBr (0.37 mL, 3.16 mmol) were added. After stirring for another 12 h when TLC showed that the reaction was completed, it was quenched with H 2 O at 0 o C, and the mixture was diluted with EtOAc. The aqueous layer was extracted with EtOAc (5 × 25 mL), and the organic S8 phases were combined and dried over Na 2 SO 4 . The desired product S13 (1.093 g, 96.1%) was obtained upon flash column chromatography (ethyl acetatehexane gradient elution) of the condensed product. Analytical data for S13: Rf = 0.6 (ethyl acetate/hexane, 1/3. 5 benzyl N-benzyl(5-bromopentanyl)carbamate (12).
Sodium methoxide (~0.8 mL, 0.5 M solution) was added to a solution of S13 (1.0 g, 2.32 mmol) in CH 3 OH (15 mL) until pH ~9 and the resulting mixture was stirred under argon for 6 h at 21 o C. After that, the reaction mixture was neutralized with Amberlite IR 120 (H+) ion exchange resin, the resin was filtered off and rinsed successively with CH 3 OH. The combined filtrate was concentrated in vacuo and this crude material was directly used for bromination.
To the solution of deprotected compound (0.96 g, 2.92 mmol) dissolved in anhydrous CH 2 Cl 2 (15 mL) were added CBr 4 (1.85 g, 5.55 mmol) and PPh 3 (1.54 g, 5.86 mmol) at 0 o C. The reaction was allowed to warmup to room temperature and stirring for another 3 h. When TLC showed the reaction was completed, it was quenched with H 2 O at 0 o C, mixture was then diluted with CH 2 Cl 2 (~50 mL) and washed with water (2 x 10 mL), sat. aq. NaHCO 3 (15 mL), and brine (15 mL

S17
To the mixture was added NIS (1.83 g, 8.11 mmol). After cooling to -10 ºC, TMSOTf (0.16 mL 0.893 mmol) was added and the reaction was allowed to warmup to room temperature. When TLC showed the reaction was completed, saturated aqueous NaHCO 3 (15 mL) and CH 2 Cl 2 were then added, and the resulting mixture was passed through celite to remove molecular sieves. The combined filtrates were washed with aqueous Na 2 S 2 O 3 (20%) [30 mL] and water (20 mL). After extraction of the aqueous layer with CH 2 Cl 2 (3x15), the combined organic phase was dried over Na 2 SO 4 , concentrated in vacuum, and purified by silica gel column chromatography (Ethyl acetate /Hexane gradient elution) to give trisaccharide 16 (3.09 g, 88.9%) as a sticky liquid.

IX. PREPARATION OF OPS-TETANUS TOXOID GLYCOCONJUGATE
The sLPS from B. abortus S99 and B. suis biovar 2 (strain Thomsen) was purified by hot-phenol extraction 10 (11) and the OPS was liberated from this product my mild acid hydrolysis. 11 The precipitated Lipid A was removed as the pellet following centrifugation at 17,000 g for 30 mins. The supernatant was buffer exchanged into water by size exclusion chromatography using sephadex G-25 which also removed low molecular weight impurities. The purified OPS, at 2 mg/ml, was oxidised by incubation in 10 mM sodium metaperiodate in 50 mM sodium acetate buffer at pH 5.5 at 4ºc for 1h in the dark. The OPS was then desalted using sephadex G-25 (PD-10 column, GE Healthcare) to buffer exchange into water, removing residual sodium metaperiodate. Oxidised OPS, 5 mg/ml, was incubated in PBS with 0.5 M ammonium chloride and 0.1 M sodium cyanoborohydride at 37ºc for 24h after which the OPS was desalted into water using sephadex-G25 and freeze dried. The oxidised and aminated OPS was reconstituted to 5 mg/ml with a 10% concentration of DMSO in PBS containing 5 mg/ml DSG (disuccinimidal glutarate) and incubated for 45 mins at room temperature on a rotary shaker then desalted back into PBS using a Zeba 40 kDa column according to the manufacturer's instructions (Pierce) to remove unconjugated DSG. The OPS-DSG conjugate was added to tetanus toxoid (TT) at final concentrations of 2.5 and 0.5 mg/ml respectively. This solution was incubated for 2h at room temperature on a rotary shaker after which a final concentration of 2 mg/ml glycine was added and this was further incubated for 15 mins. The OPS-TT conjugate was separated from the non-conjugated OPS and glycine by SEC-HPLC using an Agilent Infinity Bioinert HPLC system fitted with a 30 cm Tosoh TSK-gel G3000 PWxl, bore size 7 mm (cat: 05762) size exclusion chromatography column plus guard column comprised of the same matrix. The mobile phase was PBS pH 7.4 (50 mM sodium phosphate, 150 mM sodium chloride) and flow rate was 0.8 ml/min. The fraction eluting at 6.5 to 8.8 mins was collected as this contained the OPS conjugated tetanus toxoid only. The unconjugated OPS eluted from 8.8 to 11.0 mins whereas 75% of the OPS-TT conjugate eluted between 6.5 and 8.8 mins. The effect of the HPLC separation is visible in the SDS-PAGE silver stain image ( Figure  S3) as the TT light chain fragment is lost from the final preparation used for immunisation. The B. abortus and B. suis OPS-TT conjugates were evaluated by SDS-PAGE silver staining and Western blotting to verify their anti-OPS antibody reactivity ( Figure S3). For SDS-PAGE TT and glycoconjugates were diluted to 0.2mg/ml in sample buffer (Invitrogen, Life Technologies) then heated for 5 minutes at 80 °C in a water bath. The antigens were loaded into NuPAGE® Novex Tris-Acetate Gels (Invitrogen, Life technologies) 10 µl per well. HiMark™ Unstained Protein Standard (Invitrogen, Life technologies) was also loaded into the gels for silver staining and HiMark™ Prestained Protein Standard (Invitrogen, Life technologies) for the Western blot gels, 7.5 µl per well. The gels were run at 110 volts in an electrophoresis tank with MOPS running buffer (Invitrogen, Life Technologies) for 90 minutes. After gel electrophoresis, the gels were stained using a silver staining kit (Biorad). Initially the gels were fixed with in-house 40% methanol, 10% acetic acid fixative for 30 minutes. Then oxidising concentrate (Biorad) was diluted 1 in 10 in deionised water and added to the gels, the gels were incubated for 5 minutes on a rocker. The gels were then washed with deionised water for 15 minutes with frequent changes of water. Silver reagent (Biorad) was diluted 1 in 10 in deionised water and incubated with the gels for 20 minutes on a rocker. The gels were rinsed for 30 seconds then incubated with developing concentrate (Biorad) for 15 minutes until bands were visualised. The gels were then incubated with 5% acetic acid stopper solution (in-house preparation) for 30 minutes and then scanned. For Western blot the gels were run then they were placed on a nitrocellulose membrane (Invitrogen) and the antigens were transferred to the membrane using the iBlot™ dry transfer system (Invitrogen). The nitrocellulose membranes were blocked overnight at 4-8 °C with blocking buffer (Candor). The membranes were then incubated with mouse anti-Brucella OPS monoclonal antibody clones BrG11 (Fzmb, Germany), specific to A epitopes, or BM40 12 (APHA, Weybridge), specific to M epitopes, at 20 µg/ml in Low-cross buffer (Candor) for 90 minutes at room temperature. Then the membranes were washed three times, for fifteen minutes with washing buffer (Candor), on a rocker at room temperature. The membranes were then incubated with anti-mouse Ig:alkaline phosphatase at 1/1000 in Low-cross buffer (Candor) for 90 minutes then washed three times, for 15 minutes. The membranes were incubated with BCIP/NBT tablets (Sigma) until bands were visualised. Then the membranes were allowed to dry and scanned. The same method was used for Western blot with bovine polyclonal sera which was applied at a 1/100 dilution and developed with Protein G:alkaline phosphatase. The silver stain images (lanes 2-6) show the increase in size of the main TT protein when conjugated to B. abortus S99 OPS although the minimum size remains the same. There is a small increase in size visible due to conjugation with B. suis OPS. Size exclusion fractionation by HPLC leads to the elimination of the light chain TT fragment in both the OPS-TT preparations. The Western blots confirm that the TT has been conjugated with OPS. Polyclonal anti-Brucella sera, derived from a B. abortus infected cow binds to both glycoconjugates, although more to the B. abortus OPS-TT, with a very low background response to the unconjugated TT. A S35 monoclonal antibody specific to 'A' OPS epitopes (a series of 5 or more α1,2 linked D-Rha4NFo units) binds to both glycoconjugates, again more so to the B. abortus OPS-TT. A monoclonal antibody specific to 'M' epitopes (a short series of D-Rha4NFo units incorporating a single α1,3 link) bound only to B. abortus OPS-TT (with a weak background reaction to TT only). This is consistent with the known structure of the two OPS antigens in which the B. abortus S99 antigen contains low proportion (2%) of α1,3 links, the remainder α1,2 linked whereas the B. suis biovar 2 OPS is exclusively α1,2 linked. The lack of α1,3 links is considered to be unique to B. suis biovar 2 and means that the D-RhaN4Fo polymer is identical to that of the unrelated bacteria Y. enterocolitica O:9. The conjugation of tetanus toxoid with OPS was also evaluated by MALDI-ToF using an Applied biosystems/MDS SCIEX 4800 MALDI TOF/TOF analyser. Sample was added in 0.5 µl to the plate and allowed to dry then covered with 0.5 µl of 10 mg/ml sinapic acid. Data was collected in linear mode. The peak mass of unconjugated TT was 152,375 m/z (figure S4). The peak mass for the B. abortus S99 OPS-tetanus conjugate was 156,320 m/z ( Figure S5) and for B. suis biovar 2 OPS-TT conjugate it was 154,114 m/z ( Figure S6), although peak broadening towards higher masses were evident, especially with the B. abortus OPS-TT conjugate. Based on this, the OPS content for each conjugate was therefore 2.5% for B. abortus OPS-TT and 1.1% for B. suis OPS-TT, equivalent to an average of approximately 13 and 20 D-Rha4NFo units per TT respectively.
However, given that the average length of an OPS polymer is 96-100 units 13 it is probable that the conjugation of TT has been poor and that the distribution of the number of OPS molecules that have been conjugated per TT is in the range of 0-4. It is likely that conjugation has favoured shorter OPS molecules as these would more rapidly diffuse within a reaction mixture. Enrichment of longer OPS molecules prior to conjugation would increase the glycan content of the conjugate, as might adoption of a different conjugation technique. For example, the use of a squarate linker such as 3,4-dibutoxy-3-cyclobutene-1,2-dione would reduce the likelihood of any intramolecular linking of the two aldehydes on the oxidised terminal D-Rha4NFo due to the reduced activity of the linker once the first active site has conjugated. However, this may necessitate the use of DSG as the linker for the diagnostic antigens.

X. IMMUNIZATION OF MICE
Vaccine formulation: Alum was suspended in PBS at 50 mg/mL concentration and thimerosal (0.01% w/v) was added and stored at 4°C. Conjugate solutions were prepared at 1 mg/mL of PBS. Alum ( 14 L) was mixed with the tetanus toxoid conjugates (144 mL) in 5:1 weight ratio, diluted with 2.85 mL of PBS and the mixture was allowed to rock overnight before administering to animals. 14 Immunization with glycoconjugates 2 and 9 : Female CD1 mice (Charles River, Canada) age 6-8 weeks in groups of 10 were immunised three times at 21 day intervals. Each mouse received 250 µl distributed 150 µl intraperitoneally and 100 µl subcutaneously. Pre bleeds were collected prior to immunisation and mice were euthanized at day 10 after the final injection and final bleeds were collected. Blood was incubated at 37° C for one hour then spun at 1500 g for 10 min. Clear serum was collected and stored at -20°C until use.

Immunisation with OPS-Tetanus toxoid conjugate
Two groups of 8 female CD1 mice of 7 weeks of age were immunized on days 1, 21 and 35 with 5 g each of conjugate administered subcutaneously in a 100 µl volume of PBS without S39 adjuvant. Prebleeds were collected prior to immunization and post vaccination bleeds were taken on days 19, 33 and 49.

XI.
ELISA DATA INDIRECT ELISA Immunoassays: Antibody titres against glycoconjugate coated plates and plates coated with purified LPS were determined according to a published protocol 15 with minor modification. Briefly, polystyrene microtiter plates were incubated with the coating glycoconjugate antigen (1 μg/mL, 100 μL/well) at 4°C overnight, then washed (5×) with PBST (0.05% Tween-20 in phosphate buffer saline, PBS). LPS 1g/mL in sodium carbonate buffer.

Figure S7
Antibody titres of sera raised to vaccines 2 and 9 titred against the immunizing hapten conjugated to BSA and the three sLPS of B. abortus, B. melitensis and Yersenia enterocolitica O:9.

S40
Mouse sera was diluted 1:100 murine sera in 0.1% BSA in PBST added to the coated well (100 μL/well) at serial √10 dilutions in the same buffer. After incubation at room temperature for 2 h, the plates were washed (5×) with PBST. Then the plate was incubated with 100 μL/well of HRPO labelled goat anti-mouse IgG antibody (KPL) (1:5000 dilution of a 1.0 mg/mL stock) for 30 min at room temperature, then washed (5×) with PBST. Peroxidase substrate, 3,3',5,5'tetramethylbenzidine (TMB) with H 2 O 2 , was added. After 15 min the reaction was quenched by addition of phosphoric acid (1M, 100 μL/well). Plates were read at 450 nm and the data were processed using Origin software. End point dilution (x 0 ) was recorded as the serum dilution giving an absorbance 0.2 above background and serum titer was calculated as the reciprocal of x 0 . All the data were processed using Origin 9 and Graphpad Prism software. For the synthetic antigens the sera were also diluted at 1/31.62. Monoclonal antibody BM40, specific to M epitopes, was diluted to 5 µg/ml in casein buffer (Sigma) and added to the plates, 100 µl per well, as the positive control. A positive serum control, mouse sera from a mouse immunised with 1,2-Hexasaccharide, and a negative serum control from a normal (non-immunised) mouse were also included, 100 µl per well, as controls.
The plates were incubated for 30 minutes at room temperature, on a rotator at 120 rpm, then washed four times with PBS-Tween, 200 µl per well and tapped dry on blotting paper. Antimouse immunoglobulins HRP conjugate (Dako) was diluted 1 in 1000 in casein buffer and 100 µl/well was added to the plates. The plates were incubated for 60 minutes for the synthetic antigens and tetanus toxoid and 30 minutes for sLPS and whole cell antigens at room temperature, on a rotator at 120 rpm, then washed four times with PBS-Tween, 200 µl per well and tapped dry on blotting paper. Substrate buffer (pH4.0) (Fluka) with 2,2′-Azino-bis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (Sigma) and 3% hydrogen peroxide (Sigma) was added to the plates, 100 µl per well, and incubated at room temperature for 20 minutes. The reaction was slowed with 0.1M sodium azide, 100 µl per well, and the plates were read at 405 nm absorbance. Data was calculated as the blanked mean of duplicate wells as a percentage of the BM40 positive control wells tested with Disaccharide as this was added to every test plate.
The optical densities (ODs) for each sample and dilution were blanked by subtracting the OD for control wells to which no sera had been added but were otherwise processed as described above. The quantitative data for the samples were then normalised by expressing the ODs as a percentage of the positive control. The end titres were calculated (using GraphPad Prism 6) as the dilution at which the signal (expressed as a percentage of the positive control) was equal to the positive/negative threshold. This threshold was calculated as the mean of the pre-bleed samples plus 1.96 times the standard deviation of the pre-bleed samples.