Investigating persistent factor VIII-specific IgM in the humoral immune response to factor VIII

Abstract Hemophilia A is an X-linked bleeding disorder resulting in events due to insufficient levels of factor VIII (FVIII). The most significant complication the management hemophilia formation polyclonal neutralizing IgG (i.e., inhibitors) which impede FVIII procoagulant activity. In a (FVIII KO) murine model injected with multiple doses FVIII, FVIII-specific IgM were found persist despite established response. attempt characterize role immunity, 18 IgMs purified from immortal hybridomas and binding was confirmed via ELISA. demonstrated weak static ELISA mean optical densities (OD) at 405 nm 0.33 ± 0.43 compared anti-FVIII OD 1.85 0.02. novel fluid-phase ELISA, developed our lab, utilizing N-hydroxysuccinimide (NHS)-activated magnetic beads indirectly conjugated increased IgM-FVIII 1.15 0.60. also porcine cross-reactivity. To evaluate effect on antibody titers vivo, KO mice received 5 weekly injections either or 4 separate immune complexes (IC) IgM-FVIII. Mice IC showed slightly higher median than alone. These findings suggest potential for persistent propagating inhibitor development. Further studies determining complement activation, as well epitope specificity clonality computational methods RNA-sequencing, may provide additional mechanistic insight into humoral response FVIII. Hemostasis Thrombosis Research Society- Student Award (EY), NIH K99HL150595 (GB), Georgia Clinical Scientist Development Grant (GB).