Analysis of B-cell epitopes in the variable C-terminal region of the Mycobacterium leprae 70-kilodalton heat shock protein.
The C-terminal region of the Mycobacterium leprae 70-kDa heat-shock protein is the major target for the humoral immune response to this protein and contains M. leprae-specific sequences. To examine B-cell responses to this region more closely, we constructed and expressed a recombinant fragment of the M. leprae P70 gene that encodes the C-terminal 142 residues (C-142) and synthesized a series of 10 overlapping peptides to encompass this region. The affinities of three monoclonal antibodies (MAbs) reactive with this region of P70 were measured, and the binding site of the highest-affinity MAb was determined to lie between residues 498 and 515. This reactivity was confirmed by a fluid-phase inhibition enzyme-linked immunosorbent assay. By contrast, sera from leprosy patients which were strongly reactive with the C-142 fragment failed to bind directly to the conjugated or unconjugated peptides. To determine whether the M. leprae-specific C-terminal 70 residues could stimulate B-cell responses, the reactivity of hyperimmune anti-M. leprae P70 antisera with the peptides was examined. Rabbit polyclonal anti-M. leprae P70 antisera recognized epitopes between residues 498 and 515 and in the M. leprae-specific region between residues 567 and 591. The latter, in turn, when coupled to ovalbumin, was able to generate a strong anti-P70 response specific for mycobacterial, but not human, HSP70. Three strains of mice immunized with either C-142 or P70 recognized epitopes in the region between residues 487 and 532, but the response varied with the strain and immunogen. These data demonstrate that two regions in the C-terminal portion of M. leprae P70 contain linear B-cell epitopes recognized by MAbs or hyperimmune serum. Sera from leprosy patients, however, react predominantly with conformational determinants in the immunodominant C-terminal part of the protein.
Peake
P W
PW
Centenary Institute of Cancer Medicine and Cell Biology, University of Sydney, Australia.
Britton
W J
WJ
Davenport
M P
MP
Roche
P W
PW
McKenzie
K R
KR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000917:Antibody Formation; D000918:Antibody Specificity; D001402:B-Lymphocytes / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics*; D005798:Genes, Bacterial; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000276:immunology*; D006801:Humans; D007918:Leprosy / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009166:Mycobacterium leprae / Q000276:immunology*; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins