Neutralizing and Non-Neutralizing Anti-FVIII Antibodies in Black and White Hemophilia A Subjects: A Natural History Profile

The most common complication in hemophilia A (HA) treatment, affecting 25-30% of severe HA patients, is the development of alloimmune inhibitors that foreclose the ability of infused factor VIII (FVIII) to participate in coagulation. Inhibitors confer significant pathology on affected individuals and present major complexities in their management. Inhibitors are more common in African American patients, and it has been hypothesized that this is a consequence of haplotype (H)-treatment product mismatch. F8 gene haplotypes H1-H5 are defined by combinations of nonsynonymous SNPs encoding FVIII sequence variants D1241E, M2238V and R484H. F8 haplotypes H2-H5 are more prevalent in individuals with black African ancestry, while >90% of the white population has the H1 haplotype. This study used a validated Luminex-based assay to determine total anti-FVIII antibody titers in plasma from 395 HA (189 black, 206 white) and 23 non-HA control subjects, measuring their binding to recombinant full-length H1 and H2 and B-domain-deleted (BDD) H1/H2, H3 and H4 FVIII proteins. Inhibitor titers were determined using a chromogenic Bethesda assay with the Nijmegen modification. Linear B-cell epitopes recognized by antibodies in human plasma samples were characterized using commercial peptide microarrays with imprinted 15-mer peptides spanning the FVIII A1, A2, C1 and C2 domains, with binding interactions detected using fluorescent-labeled anti-human IgG antibodies. Neither total nor inhibitory antibody titers correlated with F8 haplotype. FVIII peptides with the D1241E and M3348V polymorphisms showed low antibody reactivity, indicating they do not comprise linear B-cell epitopes. Similarly, antibodies from subjects with H3 and H5 haplotypes, who were necessarily infused with FVIII products having a different haplotype than that of their endogenous, (dysfunctional) F8 sequence, did not show haplotype-correlated differential binding to the three BDD-FVIII or full-length FVIII proteins, indicating the polymorphic M2238V or D1241E sites do not correspond to immunodominant, conformational B-cell epitopes. Interestingly, the BDD-FVIII proteins were significantly more reactive with antibodies in plasma than were two commercial full-length recombinant FVIII products. Overall, results of this study indicated that low-titer FVIII-reactive antibodies are readily detected in most HA subjects and in a majority of healthy non-HA controls. The observed stronger immunoreactivity of BDD-FVIII suggests that B-domain removal exposes novel B-cell epitopes, perhaps through conformational rearrangements of FVIII domains.