Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells

Abstract

• Factor VIII antigens can be expressed in chloroplasts and bioencapsulated in plant cells.• Oral delivery of plant cells expressing FVIII domains suppresses and reverses inhibitor formation in mice with hemophilia A by induction of CD4 1 regulatory T 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 mg/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 b and interleukin 10). Adoptive transfer experiments confirmed an active suppression mechanism and revealed induction of CD4 1 CD25 1 and CD4 1 CD25 2 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. (Blood. 2014;124(10):1659-1668

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…Likewise, oral delivery of myelin basic protein reduced Ab plaques in advanced mouse and human Alzheimer's brains (Kohli et al, 2014). Delivery of coagulation factors to hemophilic mice induced oral tolerance and suppressed inhibitor formation and anaphylaxis Sherman et al, 2014;Wang et al, 2015a). The aforementioned examples illustrate the significance of this novel, cost-effective protein drug-delivery concept.…”

“…The native sequence of FVIII HC was amplified using the pAAV-TTR-hF8 mini plasmid (Sherman et al, 2014) as the PCR template. The codon-optimized HC sequence obtained using Codon Optimizer version 2.1 was synthesized by GenScript.…”

“…However, expression of larger human proteins is a major challenge. For example, cholera nontoxic B subunit (CNTB)-fused native human blood-clotting factor VIII heavy chain (FVIII HC;86.4 kD) or ANGIOTENSIN CONVERTING ENZYME2 (92.5 kD) were expressed at very low levels (Shenoy et al, 2014;Sherman et al, 2014). Likewise, the expression of viral vaccine antigens is quite unpredictable, with high, moderate, or extremely low expression levels (Birch-Machin et al, 2004;Lenzi et al, 2008;Waheed et al, 2011aWaheed et al, , 2011bInka Borchers et al, 2012;Hassan et al, 2014).…”

“…Therefore, transgenes inserted into chloroplast genomes are expressed at high levels, up to 70% of total leaf protein (De Cosa et al, 2001;Ruhlman et al, 2010). A wide range of proteins, from very small antimicrobial peptides (Lee et al, 2011) or hormones (Boyhan and Daniell, 2011;Kwon et al, 2013) to very large proteins encoded by bacterial, viral, fungal, animal, and human genes, have been expressed successfully in plant chloroplasts (DeGray et al, 2001;Daniell et al, 2009;Verma et al, 2010;Shenoy et al, 2014;Sherman et al, 2014;Shil et al, 2014). Most importantly, expressed proteins are highly stable when lyophilized plant cells are stored at ambient temperature (Kwon et al, 2013;Lakshmi et al, 2013;Kohli et al, 2014;Jin and Daniell, 2015).…”

Immunology: Oral solutions

“…The researchers behind the bio engineered lettuce have shown that inhibitor formation and severe allergic reactions can be prevented in mice by feeding the animals with a product based on these plants 1,2 . If the strategy also works in Anita and her kennel mates -and ultimately in humans -it could form the basis of the first product to protect against the immune responses associated with haemophilia treatment.…”