Effect of conjugation methodology on the immunogenicity and protective efficacy of meningococcal group C polysaccharide–P64k protein conjugates

Abstract

Neisseria meningitidis serogroup C polysaccharide (CCPS) was conjugated to the carrier protein P64k using two different conjugation procedures, condensation mediated by carbodiimide with adipic acid dihydrazide as spacer and the reductive amination method. BALB/c mice were immunized with the resultant polysaccharide-protein conjugates and the immune response was evaluated. All conjugates assayed generated at least 10-fold higher antibody titers than the free polysaccharide. The reductive amination method rendered the best conjugate (CCPS-P64kR) that was able to elicit antibody titers statistically higher than the titer elicited by the plain CCPS (P<0.001). The sera of the group immunized with CCPS-P64kR showed a three-fold higher bactericidal response than the sera of the group immunized with the plain CCPS and they were able to protect against challenge with meningococci in the infant rat protection model. In addition, three different conjugates were obtained from polysaccharides with molecular relative sizes of 2000-4000 Da, 4000-10,000 Da or 10,000-50,000 Da, but no differences were detected in the immune response obtained against the three conjugates. Our experiments demonstrate that it is possible to generate a protective, T-cell-dependent response against CCPS using the P64k protein as carrier.

Development of a glycoconjugate vaccine to prevent meningitis in Africa caused by meningococcal serogroup X

“…The use of shorter saccharides has allowed the production of more defined and less crosslinked conjugates, with advantages in relation to reproducibility and vaccine characterization. Parameters, including conjugation chemistry (random against selective carbohydrate modification), linker used for coupling and saccharide chain length, can have an impact on the immunogenicity of the corresponding conjugate vaccines (53)(54)(55)(56). The effect of varying such parameters is antigen dependent and was investigated in the current study to optimize the design of a vaccine against MenX.…”

Immunization and chemical conjugation of Bm95 obtained from Pichia pastoris enhances the immune response against vaccinal protein and Neisseria meningitidis capsular polysaccharide

“…Therefore, a rational approach for using Bm95 as a novel carrier molecule for preventing infections caused by polysaccharide encapsulated bacteria was conceived. 42 The results obtained in this study showed high immunogenicity of the Bm95-based conjugate (MenCpSBm-ra) compared with the meningococcal C polysaccharide. Additionally, an enhanced immune response typical of the T-cell-dependent immune response against Men CpS was observed.…”

“…The mice were selected at random to form five groups with a total of five animals per group, and were maintained under controlled lighting conditions at 22°C, with access to water and laboratory chow ad libitum, as previously described. 42 The Bm95 conjugates and polysaccharides were mixed with incomplete Freund's adjuvant (Sigma-Aldrich, St Louis, MO, USA). The mice in group A were immunized with conjugated MenCpSBm-ra.…”

“…42 Microtiter plates (Nunc Immunoplate I, Gibco Ltd., Paisley, UK) were activated using poly-L-lysine at a concentration of 2 µg/mL in carbonate/ bicarbonate (0-0.5 M) coating buffer (pH 9.5). Three washes were done with Milli Q water (18.2 mΩ ⋅ cm; EMD Millipore, Bedford, MA, USA).…”

Synergistic Effect of Coadministration of <em>Haemophilus influenzae</em> and <em>Neisseria meningitidis</em> Vaccines on Immune Responses to Meningitis in Mouse Model

“…For assaying meningococcal ACWY antibody (IgG) levels, plates were activated by adding 100 µl of 2 µg/ml poly L-lysine (Sigma-Aldrich, USA) in carbonate buffer at 37°C for 2 hours following previously detailed method [24]. After washing, plates were coated with 100µg/ml polysaccharide (NIBSC, London, UK) in PBS and incubated at 2-8°C overnight.…”