Memory immune response generated in Cercopithecus aethiops against meningococcal polysaccharide serogroup C conjugate vaccine.

The chemical conjugation of bacterial polysaccharide to carrier proteins has proved to be an efficient tool to improve the immunological response against these bacterial antigens. In this study, we characterized the antibody response generated in a non-human primate model against the meningococcal capsular polysaccharide serogroup C (CCPS) conjugated to the P64k protein. Similar to licensed vaccines the CCPS conjugate is able to generate a good memory immune response with antibody titers threefold higher than the free CCPS. Three different ELISA protocols were used to measure the antibody response and the importance of the coating antigen was demonstrated. The ELISA using the derivatized CCPS showed the best results and had a high correlation with the bactericidal activity. The antibodies elicited showed a high protective capacity when assayed in the infant rat protection model.

Recombinant Opc protein from Neisseria meningitidis reconstituted into liposomes elicits opsonic antibodies following immunization.

The reconstitution of recombinant bacterial outer membrane proteins (OMPs) into their native conformations after purification has been the major problem in their use as effective vaccines. Liposomes have been shown to be an attractive approach, providing a native-like environment for these antigens. The meningococcal recombinant Opc (rOpc) protein, produced as inclusion bodies in Escherichia coli, was incorporated into phospholipid vesicles consisting of dipalmitoyl phosphatidylcholine and cholesterol. The incorporation of rOpc into the lipid bilayer was demonstrated, and the reconstitution of some native epitopes was tested using a set of monoclonal antibodies. Subcutaneous immunization of Balb/c mice with rOpc-containing vesicles resulted in the generation of a high level of specific antibodies. The elicited antibodies reacted with the native meningococcal protein and showed opsonic activity.

Mapping of monoclonal antibodies specific to P64k: a common antigen of several isolates of Neisseria meningitidis.

P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.

Expression in Escherichia coli of the lpdA gene, protein sequence analysis and immunological characterization of the P64k protein from Neisseria meningitidis.

By making use of recombinant DNA technology it is possible to characterize meningococcal outer membrane proteins (OMPs) capable of stimulating a host immune response. The lpdA gene, which codes for an OMP (P64k) from Neisseria meningitidis, was cloned in Escherichia coli. The recombinant protein was recognized by sera from patients convalescing from meningococcal disease. The monoclonal antibodies obtained against the recombinant protein recognized the natural protein on a Western blot, and monoclonal antibody 114 was assayed in ELISA with a panel of 85 N. meningitidis strains. The protein was recognized in 81 strains (95.3%); the strains that were not recognized were neither epidemic nor isolated from systemic disease. The complete amino acid sequence of P64k was obtained by automatic sequencing and MS.

Recombinant Opc meningococcal protein, folded in vitro, elicits bactericidal antibodies after immunization.

The meningococcal Opc protein has been expressed as inclusion bodies in Escherichia coli. After cell disruption and successive washing of the insoluble fraction, insoluble proteins were solubilized in presence of the chaotropic agent guanidium hydrochloride. The extract was applied to a Reverse Phase High Performance Liquid Chromatography (RP-HPLC)-C4 column, for further purification. The obtained recombinant Opc protein was refolded in vitro, by the addition of several compounds to the resuspended solution. Over time, the progress of renaturation was tested by immunoblot with the human monoclonal antibody LuNm03 against the meningococcal Opc protein. LuNm03 recognizes a conformational epitope on the native meningococcal Opc protein. Having established the optimal conditions of renaturation. Balb/c mice were immunized to study the humoral immune response. The human at immune response elicited in mice was measured by ELISA and immunoblot, while the functional activity of these antibodies was assayed in a bactericidal test. According to our results, it was possible to obtain a recombinant Opc protein folded in vitro, with a conformation suitable enough to generate functional antibodies in mice, capable of killing meningococci in the presence of human complement.