Thesis Mariarosaria Miranda

Abstract

One of the main challenge in the treatment of hemophilia A is the formation of inhibitory antibodies against factor VIII (FVIII), limiting the efficacy of replacement therapies. Addressing this requires a comprehensive understanding of how the immune system recognizes and responds to FVIII, with the goal of translating this knowledge into novel strategies to prevent or mitigate immune complications. Investigation of FVIII peptide presentation on HLA-DP molecules, particularly HLA-DP4, identified promiscuous peptides that provide a foundation for epitope-based interventions aimed at promoting immune tolerance. Concurrently, red blood cells (RBCs) have been explored as a delivery system for FVIII peptides fused to the TAT peptide, demonstrating efficient antigen delivery to immune cells and supporting RBCs as an effective platform for hemophilia therapy. Enhancement of antigen targeting through FVIII fusion proteins linked to Annexin A5 (AnxA5) engages the apoptotic clearance pathways; these fusion constructs efficiently enter the antigen presentation pathways, suggesting a route to induce tolerogenic immune responses by mimicking clearance mechanisms. Furthermore, examination of FVIII-containing immune complexes revealed that targeting the inhibitory Fcγ receptor IIb (FcγRIIb) may reduce dendritic cell activation, highlighting an additional mechanism to suppress anti-FVIII responses. Characterization of the spectrum of anti-FVIII antibodies in a large hemophilia A cohort provided deeper insight into the role of non-neutralizing antibodies (NNA), suggesting that certain antibody subclasses may serve as markers of inhibitor risk or immune regulation. Together, these studies present a coherent picture of FVIII immunogenicity and highlight multiple avenues for developing targeted strategies to enhance immune tolerance.