SUBLINGUAL IMMUNOTHERAPY INDUCES TOLERANCE TO FACTOR VIII IN HEMOPHILIA A MICE

Abstract

Hemophilia A (HA) is a rare X-linked recessive genetic bleeding disorder characterized by the absence or decreased amount of functional coagulation Factor VIII (FVIII) in the blood. The clinical manifestations of HA include uncontrolled bleeding events, spontaneous muscle bleeding, and joint bleeding leading to the painful swelling of joints. There is currently no cure for HA, the treatment for HA patients is centered around preventing and managing bleeding episodes. Replacement therapy has been the most common world-wide prophylactic therapy involving intravenous (IV) infusions of FVIII to restore healthy FVIII concentrations in the blood. The development of inhibitors, which are anti-FVIII polyclonal IgG alloantibodies, is a serious side effect of replacement therapy. These inhibitory antibodies target and neutralize FVIII, making it no longer effective at participating in the coagulation cascade. Sublingual immunotherapy (SLIT) is a promising approach to treating allergies and has been shown to significantly reduce the immune responses to allergens. Previous studies exploring SLIT have demonstrated a CD25+FoxP3+ regulatory T-cell (Treg) dependent tolerance in response to sublingually administered antigens. This study investigated whether the use of SLIT with FVIII could induce tolerance to IV-administered FVIII in a Treg dependent mechanism within HA mice. HA mice received three weeks of SLIT with either 1 IU, 5 IU, or 10 IU FVIII, and control mice received SLIT with PBS. Mice were then challenged with FVIII injections of either 25IU/Kg or 50IU/Kg FVIII to stimulate the immune system to produce antibodies. The plasma from blood samples collected throughout the experiment was subjected to an ELISA to measure FVIII-specific IgG levels. To study the mechanisms involved in SLIT, an anti-CD25 antibody was used to deplete CD25+FoxP3+ Tregs to explore their involvement in the tolerance effect. SLIT with 5IU and 10IU FVIII significantly reduced the average anti-FVIII IgG titers in HA mice compared to the control mice, whereas SLIT with 1IU FVIII resulted in similar average anti-FVIII IgG titers as the control mice. The 10IU group produced a slightly lower average anti-FVIII IgG titer than the 5IU group, hinting towards a dose-dependent response. The depletion of CD25+FoxP3+ Tregs with Anti-CD25 injections along with SLIT with FVIII resulted in the previously observed tolerance to FVIII being eliminated.