Safety and preliminary immunogenicity of the recombinant outer membrane protein P64k of Neisseria meningitidis in human volunteers.

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.

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.

Anti-PorA antibodies elicited by immunization with peptides conjugated to P64k.

To increase the humoral immune response against two cyclic synthetic peptides, derived from variable regions within the outer membrane meningococcal protein PorA (subtypes 19 and 15), we conjugated the peptides to P64k, a novel carrier protein from the same bacterium expressed in Escherichia coli. In addition, one of these peptides was restricted to a linear conformation before it was chemically coupled to the carrier. The conjugates were administered to mice in a three-dose immunization schedule, resulting in a potent anti-peptide immune response, which suggested that chemical conjugation to this carrier provided T-cell help. Antisera directed to the three conjugates reacted with Neisseria meningitidis outer membrane PorA upon immunoblot analysis. Moreover, in two out of three conjugates, the anti-peptide sera reacted with native meningococcal outer membrane vesicles in ELISA.

B-cell epitope mapping of the Neisseria meningitidis P64k protein using overlapping peptides.

A common meningococcal antigen designated P64k has been identified, cloned and expressed in Escherichia coli. The recombinant antigen is highly immunogenic in several animal species and its immunogenicity in healthy human volunteers is under investigation. Recently, P64k has been used as an immunological carrier for weak immunogens. To characterize the B-cell epitopes on P64k, recognized by immune sera obtained from mice, rabbits and monkeys, multiple overlapping peptides were synthesized and screened for antibody binding. Peptides covering the complete sequence of the P64k protein, 59 in all, of 20 amino acids each (overlapped by 10 residues), were synthesized. A number of continuous epitopes were detected with all sera, when immune and pre-immune bleeds were compared. For mouse and monkey sera, a few major antigenic peptides were identified, while the recognition of the rabbit serum was much more heterogeneous. Despite variation in the exact location of continuous epitopes defined by different anti-P64k sera, we found an immunogenic core region within the molecule, composed of amino acids Asp(524)-Gly(533). Consistently, in this protein segment there was an amino acid stretch located in a beta-hairpin loop, which is exposed to the solvent in the previously determined three-dimensional structure of the protein. This region is protruding and accessible to a sphere with a radius of 9 A.

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.

Influence of several adjuvants on the immune response against a recombinant meningococcal high molecular weight antigen.

Studying outer membrane proteins as vaccine candidates, our group has previously isolated, cloned, and expressed in Escherichia coli the gene encoding for a high molecular weight protein (P64k), common to many meningococcal strains. To continue the characterisation of this meningococcal antigen, we have evaluated its immunogenicity in mice alone or combined with several commercially-available adjuvants. We used as an adjuvant aluminium hydroxide (Alhydrogel and Rehydragel), aluminium phosphate, Algammulin, crude saponin, the saponin Quil A, dimethyl-dioctadecyl ammonium bromide (DDA), Freund's adjuvant, and Montanide 888. The antibody titres against the recombinant protein and whole meningococci elicited with these adjuvants were compared. We found that Quil A produced the highest titres against the recombinant P64k. Algammulin and the quaternary ammonium compound DDA induced the highest levels of antibodies against meningococci. We analysed the recognition of a set of linear peptides by antisera prepared against the protein combined with some of the adjuvants. The responses depended on the adjuvant used and the results have been confirmed by epitope mapping using overlapping peptides synthesised on pins.

B cell responses to a peptide epitope. I. The cellular basis for restricted recognition.

Primary humoral responses in BALB/c mice to a variety of peptide constructs containing a common 15-amino acid residue antigenic determinant (PS1) in conjunction with one or more Th cell epitopes were examined. In all cases, the mature IgG response was found to focus primarily on a tetrapeptide sequence, Asp-Pro-Ala-Phe. The dominance of this segment was independent of the position of the PS1 determinant in the peptide sequence and was also observed in constructs with a random secondary structure. In contrast to the mature IgG response, the early primary IgM response was constituted by multiple specificities that collectively spanned a major proportion of the PS1 sequence. However, subsequent progression of this response entailed a strict selection for only those Abs directed against the Asp-Pro-Ala-Phe segment and apparently occurred at or around the time of the IgM to IgG class switch. Studies of murine responses to peptide analogs containing single amino acid substitutions within the Asp-Pro-Ala-Phe sequence revealed that emergence of this segment as the dominant epitope was a consequence of active suppression of B cells directed against alternate determinants. Positive selection of this subset of Abs correlated with overall higher avidity for epitope binding and was the outcome of a competitive process enforced by the limiting amounts of Th cell help available in the early stages of the primary response.