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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.

P64k is a meningococcal protein from Neisseria meningitidis that has been obtained by recombinant DNA technology. Recombinant P64k has been extensively characterized by physicochemical and immunological methods. Lately this protein has been found to act as a versatile immunological carrier for weak antigens in mice. In the present work, a Phase I clinical trial was carried out in healthy volunteers who received three inoculations of either placebo or recombinant P64k (20 or 50 microg). No severe adverse events occurred during the trial. Only mild adverse events in ten volunteers were observed. At 1 month after the third dose, 15 out of 18 volunteers (83.3%) who received the recombinant antigen had a P64k-specific antibody titre > or =1:100, as detected by ELISA. A fourth dose, given 9 months after the third one, elicited a potent booster immune response in P64k vaccinees. Accordingly, these P64k formulations were considered safe and immunogenic in healthy human volunteers.

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