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.
González
S
S
Division of Vaccines, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba. sonia.gonzalez@cigb.edu.cu
Viña
L
L
Nazábal
C
C
Chinea
G
G
Caballero
E
E
Musacchio
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D002522:Chlorocebus aethiops / Q000276:immunology; D018604:Epitope Mapping / Q000379:methods*; D005260:Female; D008562:Membrane Glycoproteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009345:Neisseria meningitidis / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology; D010750:Phosphoproteins / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation; D011817:Rabbits
D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; C026742:plastin