The humoral response to human factor VIII in hemophilia A mice.
BACKGROUND
Inhibitory antibodies (Abs) to factor VIII (FVIII inhibitors) constitute the most significant complication in the management of hemophilia A. The analysis of FVIII inhibitors is confounded by polyclonality and the size of FVIII.


OBJECTIVE
The goal of this study was to dissect the polyclonal response to human FVIII in hemophilia A mice undergoing a dosage schedule that mimics human use.


METHODS
Splenic B-cell hybridomas were obtained following serial i.v. injections of submicrogram doses of FVIII. Results of a novel, anti-FVIII domain-specific enzyme-linked immunosorbent assay were compared to Ab isotype and anti-FVIII inhibitory activity.


RESULTS
The robust immune response resulted in the production of approximately 300 hybridomas per spleen. We characterized Abs from 506 hybridomas, representing the most comprehensive analysis of a protein antigen to date. Similar to the human response to FVIII, anti-A2 and anti-C2 Abs constituted the majority of inhibitors. A novel epitope was identified in the A2 domain by competition ELISA. Anti-A2 and anti-C2 Abs were significantly associated with IgG(1) and IgG(2a) isotypes, respectively. Because the IgG(2a) isotype is associated with enhanced Fc receptor-mediated effector mechanisms, this result suggests that anti-C2 Abs and inflammation may be linked. Additionally, we identified a novel class of Abs with dual specificity for the A1 and A3 domains. Forty per cent of the Abs had no detectable inhibitory activity, indicating that they are prominent and potentially pathologically significant.


CONCLUSIONS
The expanded delineation of the humoral response to FVIII may lead to improved management of hemophilia A through mutagenesis of FVIII B-cell epitopes.
Healey
J F
JF
Aflac Cancer Center and Blood Disorders Service, Department of Pediatrics, Children's Healthcare of Atlanta and Emory University, Atlanta, GA 30322, USA.
Parker
E T
ET
Barrow
R T
RT
Langley
T J
TJ
Church
W R
WR
Lollar
P
P
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000097:blood / Q000276:immunology*; D000917:Antibody Formation; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D002460:Cell Line; D003029:Coagulants / Q000008:administration & dosage / Q000276:immunology*; D006224:Cricetinae; D004195:Disease Models, Animal; D004334:Drug Administration Schedule; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018604:Epitope Mapping / Q000379:methods; D005169:Factor VIII / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006467:Hemophilia A / Q000097:blood / Q000188:drug therapy / Q000276:immunology*; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D007132:Immunoglobulin Isotypes / Q000096:biosynthesis / Q000097:blood / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D011993:Recombinant Fusion Proteins / Q000276:immunology; D015203:Reproducibility of Results; D013154:Spleen / Q000166:cytology / Q000378:metabolism; D013552:Swine; D014162:Transfection
D000911:Antibodies, Monoclonal; D003029:Coagulants; D007132:Immunoglobulin Isotypes; D011993:Recombinant Fusion Proteins; D005169:Factor VIII