Characterization of B- and T-cell responses and HLA-DR4 binding motifs of the latex allergen Hev b 6.01 (prohevein) and its post-transcriptionally formed proteins Hev b 6.02 and Hev b 6.03.
BACKGROUND
Multiple immunoglobulin E (IgE)-binding proteins in natural rubber latex extracts have been identified. In the case of Hev b 6 a differentiation was made between the precursor protein prohevein (Hev b 6.01) and its two post-transcriptionally formed proteins, the N-terminal hevein (Hev b 6.02) and the C-terminal domain (Hev b 6.03). All three components act as independent allergens. The aim of this study was a detailed analysis of the T-cell responses and the IgE-binding capacity of Hev b 6.01, Hev b 6.02 and Hev b 6.03 by using these allergens as recombinant maltose-binding fusion (MBP) proteins and the usage of synthetic modified hevein peptides.


METHODS
Latex-allergic health care workers (HCWs) suffering from rhinitis, conjunctivitis, contact urticaria and/or asthma with increased specific IgE-antibodies to latex were tested for their IgE-binding capacity and T-cell reactivity (by proliferation response) to the recombinant MBP-rHev b 6.01, MBP-rHev b 6.02, MBP-rHev b 6.03, to native Hev b 6.02, to modified hevein peptides and wheat germ agglutinin (WGA). For testing of the human leucocyte antigen (HLA) class II restriction of MBP-rHev b 6.01 induced peripheral blood mononuclear cell (PBMC) responses, monoclonal antibodies against HLA-DR, HLA-DP or HLA-DQ were added.


RESULTS
Seventeen of 18 (94%) serum samples from latex-allergic HCWs had increased levels of specific IgE to MBP-rHev b 6.01, 16 (89%) to MBP-rHev b 6.02 and 13 (72%) to MBP-rHev b 6.03. A significant difference existed between the specific IgE-values of MBP-rHev b 6.02 and MBP-rHev b 6.03 (P < 0.01). Proliferation responses of PBMC of the same 18 latex-allergic patients were positive for MBP-rHev b 6.01 and MBP-rHev b 6.03 in 83 and 67% of the tested PBMC suspension, whereas the proliferation responses induced with MBP-rHev b 6.02 or native Hev b 6.02 were very low (5.6 and 22.2%). Sera from nine additional latex-allergic patients showed specific IgE binding to the native Hev b 6.02, but none of these sera showed specific IgE binding to the modified Hev b 6.02-peptides [whereby all eight cysteine residues were substituted by serine (C --> S) or by alanine (C --> A)]. Proliferation responses induced by the modified Hev b 6.02 peptides were not significantly different from that induced by Hev b 6.02. Potential HLA-DR4Dw4(DRB1*0401)-restricted T-cell epitopes of Hev b 6.01 predicted by two computer algorithms were only found in the Hev b 6.03-part of Hev b 6.01.


CONCLUSIONS
In the Hev b 6.01 precursor the regions responsible for IgE binding and those for inducing the T-cell proliferation responses are settled in different parts of the protein. The Hev b 6.02 domain is responsible for IgE binding and carries discontinuous B-cell epitopes whereas Hev b 6.03 is a better inducer of a proliferation response and contains HLA-DR4-binding motifs.
Raulf-Heimsoth
M
M
Research Institute for Occupational Medicine of the Berufsgenossenschaften (BGFA), Institute of the Ruhr-University Bochum, Bochum, Germany.
Rozynek
P
P
Brüning
T
T
Rihs
H-P
HP
D000328:Adult; D000465:Algorithms; D000485:Allergens / Q000276:immunology*; D020816:Amino Acid Motifs / Q000276:immunology; D000595:Amino Acid Sequence; D052179:Antigens, Plant; D023181:Antimicrobial Cationic Peptides / Q000276:immunology*; D001402:B-Lymphocytes / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005260:Female; D015804:HLA-DR4 Antigen / Q000276:immunology*; D006282:Health Personnel; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood / Q000276:immunology; D007840:Latex / Q000276:immunology*; D020315:Latex Hypersensitivity / Q000097:blood / Q000276:immunology; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D037121:Plant Lectins / Q000276:immunology*; D010940:Plant Proteins / Q000276:immunology*; D011237:Predictive Value of Tests; D011499:Protein Processing, Post-Translational; D013601:T-Lymphocytes / Q000276:immunology*
D000485:Allergens; D052179:Antigens, Plant; D023181:Antimicrobial Cationic Peptides; D018984:Epitopes, T-Lymphocyte; D015804:HLA-DR4 Antigen; C097982:Hev b 6.01 protein, Hevea brasiliensis; D007840:Latex; D010446:Peptide Fragments; D037121:Plant Lectins; D010940:Plant Proteins; C045875:hevein; D007073:Immunoglobulin E