Discontinuous Epitopes On FVIII A2 Domain Mapped by Computer-Designed Synthetic Peptides

The development of alloantibodies (AlloAbs) directed against the factor VIII (FVIII) is actually the main iatrogenic complication in hemophilia A (HA). Immune tolerance induction (ITI) is the only validated treatment to eradicate inhibitors. ITI is based on the infusion of high doses of therapeutic FVIII. It has been recently suggested that epitope specificity during ITI could be related to the outcome of this therapy. Anti-FVIII immune response is polyclonal, complex, dynamic over time and preferentially directed against C2 and A2 domains of FVIII. Tools for fine epitope mapping are useful to identify new epitopes and follow the over time evolution of epitope specificity. The aim of our work was to identify new epitopes on FVIII A2 domain using computer-designed peptides mimicking discontinuous epitopes. We used a multiplex assay based on Luminex™ technology to select the most reactive peptide (1).

All the peptides were predicted by a bioinformatical tool named PEPOP using a new protocol of prediction. Starting from the tridimensional structure of the FVIII, PEPOP were able to work out the sequence of 40 peptides mimicking discontinuous epitopes distributed on the A2 domain surface. Seven negative control peptides were predicted in unaccessible solvant area. A previously published peptide mimicking a linear epitope (residues 484–508) were also synthetized and tested (2). The capacity of peptides to block the binding of the anti-A2 inhibitors on beads coated with the A2 domain obtained by thrombin cleavage of FVIII was assessed. This multiplexed inhibition assay were based on Luminex™ technology. We used a pool of 10 plasma with anti-A2 domain inhibitors as a source of antibodies.

The inhibition assays realized with pool of anti-A2 antibodies made it possible to identify 2 peptides mimicking discontinuous epitopes on the A2 domain. The inhibition rate of the two new identified peptides reaches to 50% and 26% at the maximal concentration. We also confirm that the previously published peptide is able to block the binding of anti-A2 inhibitors in our population. The localization of the discontinuous epitopes mimicked by peptides within the tridimensional structure of the A2 domain showed than these epitopes are only represented on a restricted area.

We identified two peptides mimicking new discontinuous epitopes on the A2 domain and predicted by a new algorithm of PEPOP. According to the literature, immune response against A2 domain seems to be more restricted. Several residues involved in the new identified discontinuous epitopes are crucial for thrombin, protein S, FIXa and low-density lipoprotein receptor-related protein (LRP) interactions. Tools for fine epitope mapping are essential to identify epitopes on FVIII domains, with a particular concern for discontinuous epitopes. Further studies are needed to validate the concept that fine epitope mapping at the epitope level could be useful to predict ITI outcome.

No relevant conflicts of interest to declare.