Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells.
Hemophilia A is the X-linked bleeding disorder caused by deficiency of coagulation factor VIII (FVIII). To address serious complications of inhibitory antibody formation in current replacement therapy, we created tobacco transplastomic lines expressing FVIII antigens, heavy chain (HC) and C2, fused with the transmucosal carrier, cholera toxin B subunit. Cholera toxin B-HC and cholera toxin B-C2 fusion proteins expressed up to 80 or 370 µg/g in fresh leaves, assembled into pentameric forms, and bound to GM1 receptors. Protection of FVIII antigen through bioencapsulation in plant cells and oral delivery to the gut immune system was confirmed by immunostaining. Feeding of HC/C2 mixture substantially suppressed T helper cell responses and inhibitor formation against FVIII in mice of 2 different strain backgrounds with hemophilia A. Prolonged oral delivery was required to control inhibitor formation long-term. Substantial reduction of inhibitor titers in preimmune mice demonstrated that the protocol could also reverse inhibitor formation. Gene expression and flow cytometry analyses showed upregulation of immune suppressive cytokines (transforming growth factor β and interleukin 10). Adoptive transfer experiments confirmed an active suppression mechanism and revealed induction of CD4(+)CD25(+) and CD4(+)CD25(-) T cells that potently suppressed anti-FVIII formation. In sum, these data support plant cell-based oral tolerance for suppression of inhibitor formation against FVIII.
Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL; and.
Alexandra
A
Sherman
Department of Biochemistry and Department of Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA.
Jin
J
Su
Department of Biochemistry and Department of Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA.
Shina
S
Lin
Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL; and.
Xiaomei
X
Wang
Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL; and.
Roland W
RW
Herzog
Department of Biochemistry and Department of Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA.
Henry
H
Daniell
D000284:Administration, Oral; D000818:Animals; D052179:Antigens, Plant / Q000276:immunology; D004195:Disease Models, Animal; D004337:Drug Carriers / Q000008:administration & dosage; D004339:Drug Compounding / Q000379:methods; D016503:Drug Delivery Systems / Q000379:methods*; D005169:Factor VIII / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D006467:Hemophilia A / Q000235:genetics* / Q000628:therapy*; D007108:Immune Tolerance; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D059828:Plant Cells / Q000378:metabolism; D014026:Nicotiana
D052179:Antigens, Plant; D004337:Drug Carriers; D005169:Factor VIII