O:2-CRM(197) conjugates against Salmonella Paratyphi A

Abstract

Enteric fevers remain a common and serious disease, affecting mainly children and adolescents in developing countries. Salmonella enterica serovar Typhi was believed to cause most enteric fever episodes, but several recent reports have shown an increasing incidence of S. Paratyphi A, encouraging the development of a bivalent vaccine to protect against both serovars, especially considering that at present there is no vaccine against S. Paratyphi A. The O-specific polysaccharide (O:2) of S. Paratyphi A is a protective antigen and clinical data have previously demonstrated the potential of using O:2 conjugate vaccines. Here we describe a new conjugation chemistry to link O:2 and the carrier protein CRM(197), using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the O:2 chain unmodified. The new conjugates were tested in mice and compared with other O:2-antigen conjugates, synthesized adopting previously described methods that use CRM(197) as carrier protein. The newly developed conjugation chemistry yielded immunogenic conjugates with strong serum bactericidal activity against S. Paratyphi A.

Figure 1. Structure of S. Paratyphi A O-antigen chain linked to the core region ; .

Figure 2. O:2-ADH-SIDEA-CRM₁₉₇ conjugation scheme.

Same reaction was also performed with the shorter linker CDH instead of ADH.

Figure 3. SDS-PAGE analysis of conjugation mixtures in comparison to unconjugated CRM₁₉₇.

Lane 1: marker, lane 2: CRM₁₉₇, lane 3: O:2-(CDAP)ADH-CRM₁₉₇ conjugation mixture, lane 4: O:2-(EDAC)ADH-CRM₁₉₇ conjugation mixture, lane 5: O:2-ADH-SIDEA-CRM₁₉₇ conjugation mixture, lane 6: O:2-CDH-SIDEA-CRM₁₉₇ conjugation mixture, lane 7: O:2-SIDEA-CRM₁₉₇ conjugation mixture. Ten µg of protein were loaded per conjugation mixture, 5 µg for CRM₁₉₇.

Figure 4. Gel filtration profiles on Sephacryl S-300 HR column (1.6×90

cm) of O:2-ADH-SIDEA-CRM₁₉₇ conjugation mixture (dash dot line; 880 µg of total protein injected; kd of 0.20), free O:2 (dotted line; 6.7 mg of sugar injected; kd of 0.33) and free CRM₁₉₇ (dashed line; 1 mg of protein injected; kd of 0.61) at 214 nm. Elution with 50 mM NaH₂PO₄, 0.15 M NaCl, pH 7.2 at a flow rate of 0.5 mL/min. Arrows indicate the fractions pooled to generate purified conjugate.

Figure 5. HPLC-SEC analysis of A) O:2; B) O:2-ADH; C) O:2-ADH-SIDEA (RI detection).

Samples run on TosoHaas TSK gel 3000 PW_XL column; eluent: 0.1 M NaH₂PO₄, 0.1 M NaCl, 5% CH₃CN, pH 7.2; flow rate: 0.5 mL/min. Column void volume: 10.85 min.; total volume: 23.54 min. O:2 molecular weight distribution remains unchanged during the O:2 derivatization steps with ADH and SIDEA (all the samples have Kd of 0.17).

Figure 6. ¹H NMR spectra of A) O:2 (68% OAc); B) O:2-ADH (70% OAc); C) O:2-ADH-SIDEA (68% OAc); D) O:2-ADH-SIDEA-CRM₁₉₇ (67% OAc).

O-acetylation level quantified by comparing acetate (released after treatment with NaOD, at 1.91 ppm) and Rha-H6 peaks at 1.40 ppm, and expressed as molar % of O-acetyl with respect to OAg chain repeating units (being Rha present only in the OAg chain, at one sugar per repeating unit). O-acetylation level is maintained at the same level after conjugation.

Figure 7. HPLC-SEC profiles (fluorescence emission detection) of O:2-ADH-SIDEA-CRM₁₉₇ (dotted line; 28.7 µg/mL of protein; Kd of 0.39) in comparison to free CRM₁₉₇ (dashed line; 50 mg/mL; Kd of 0.69) and free O:2 (solid line; 1 mg/mL of sugar; Kd of 0.55).

80 µL of each samples run on TosoHaas TSK gel 6000+5000 PW columns; eluent: 0.1 M NaH₂PO₄, 0.1 M NaCl, 5% CH₃CN, pH 7.2; flow rate: 0.5 mL/min. Column void volume: 23.65 min.; total volume: 49.07 min. Free O:2 is not detected by fluorescence emission.

Figure 8. SDS-PAGE analysis.

Lane 1: marker, lane 2: CRM₁₉₇ (5 µg), lane 3: conjugation mixture of O:2(TNBS)-SIDEA + CRM₁₉₇ (10 µg of protein), lane 4 conjugation mixture of O:2(TNBS)-ADH-SIDEA + CRM₁₉₇(10 µg of protein).

Figure 9. Anti-O:2 IgG (panel A) and Anti-CRM₁₉₇ (panel B) serum IgG ELISA units detected in sera of CD-1 mice immunized with O:2-conjugates and unconjugated O:2.

Mice were immunized three times at two week-intervals with the indicated doses of material. Mice were bled and sera collected on the indicated days. Individual animals are represented by the scatter plots; bars represent the group geometric mean.

Figure 10. SBA assay performed with pooled mouse sera from different immunization groups and CVD1901.

Data are presented as percentage of CFU recovered in test sera with active BRC (and in control serum with inactive BRC) compared with CFU present in negative control, per anti-O:2 ELISA antibody unit of each serum pool.