Characterization of Antibodies Induced by Human Factor VIII in a Murine Knockout Model of Hemophilia A

Abstract

SummaryTo investigate the usefulness of factor VIII (FVIII) knockout mice as an animal model of hemophilia A, we characterized the antibody response in FVIII knockout mice to recombinant human FVIII, administered intravenously or subcutaneously with or without adjuvant, and compared results to those in normal mice. Anti-factor VIII antibodies were detected after both intravenous and subcutaneous administration, with the highest titers after subcutaneous administration plus adjuvant. Depending on the administration strategy, knockout mice formed antibodies more rapidly and developed higher titers of inhibitory antibodies (Bethesda) than normal mice, suggesting differences in epitope specificity. Blotting thrombin cleavage products separated by gel electrophoresis showed that both strains developed antibodies against the nonfunctional B domain as well as against functional domains of factor VIII. The antibodies were mainly of the IgG1 subclass and resembled type I antibodies in hemophilia A.

Lower Inhibitor Development in Hemophilia A Mice following Administration of Recombinant Factor VIII-O-Phospho-L-serine Complex

“…The subcutaneous route of administration was investigated mainly to amplify the immune response. Further, Reipert et al (30) have reported that the IgG subtype levels were identical after subcutaneous and intravenous administration in hemophilia A mice, suggesting an identical mechanism of immune response for subcutaneous and intravenous routes. Blood samples were obtained in acid citrate dextrose buffer at various time points up to 6 weeks by cardiac puncture.…”

PEGylation of a Factor VIII–Phosphatidylinositol Complex: Pharmacokinetics and Immunogenicity in Hemophilia A Mice

“…Hemophilia A mice mount an IgG-based immune response similar to that of patients with hemophilia A (43). Immunizations were administered subcutaneously, and it has been shown that inhibitory titers to FVIII generated via this route are comparable to those following i.v.…”

Suppression of FVIII Inhibitor Formation in Hemophilic Mice by Delivery of Transgene Modified Apoptotic Fibroblasts

“…Anti-FVIII and anti-OVA IgG titers were determined by enzyme-linked immunosorbent assay as previously described. 47,48 Briefly, the solid phase of MaxiSorp flat-bottom 96-well plates (Nalge Nunc International, Rochester, NY) were coated with rhFVIII (0.2 µg in 200 µl/well) or OVA (0.4 µg in 200 µl/well) and then incubated with serial dilutions of mouse serum. The bound anti-FVIII or anti-OVA antibodies were detected by probing with an AP-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL) and developed using phosphatase substrate (Sigma-Aldrich).…”