Oral Tolerance: Another Reason to Eat Your Veggies!
Authors: Romain Hardet, Federico Mingozzi
Journal: Molecular Therapy (2017)

Development of inhibitory antibodies directed against infused clotting factors (termed inhibitors) is a major complication in the current treatment of hemophilia.1Di Michele D.M. Immune tolerance induction in haemophilia: evidence and the way forward.J. Thromb. Haemost. 2011; 9: 216-225Crossref PubMed Scopus (48) Google Scholar Inhibitor formation occurs in about 30% of hemophilia A and 2%–5% of hemophilia B patients, making protein replacement therapy ineffective and requiring expensive and inconvenient bypass treatments to manage bleeds. Development of inhibitors in hemophilia B is also associated with several side effects, such as nephropathic syndrome and anaphylactic reactions, which complicate immune tolerance induction (ITI).1Di Michele D.M. Immune tolerance induction in haemophilia: evidence and the way forward.J. Thromb. Haemost. 2011; 9: 216-225Crossref PubMed Scopus (48) Google Scholar In this issue of Molecular Therapy, Herzog and colleagues2Herzog R.W. Nichols T.C. Su J. Zhang B. Sherman A. Merricks E.P. Raymer R. Perrin G.Q. Hӓger M. Wiinberg B. Daniell H. Oral tolerance induction in hemophilia B dogs fed with transplastomic lettuce.Mol Ther. 2017; 25 (this issue): 512-522Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar tackle the issue of inhibitors and demonstrate for the first time that oral tolerance to coagulation factor IX (F.IX) can be achieved in a large animal model of hemophilia B.3Weiner H.L. da Cunha A.P. Quintana F. Wu H. Oral tolerance.Immunol. Rev. 2011; 241: 241-259Crossref PubMed Scopus (464) Google Scholar To do so, they used lyophilized transplastomic lettuce containing bioencapsulated cholera toxin subunit B fused to F.IX (CTB-F.IX), which can be easily produced in large quantities and is stable over time. Oral tolerance refers to a state of peripheral immune unresponsiveness induced by the gut-associated lymphoid tissue (GALT) toward antigens administered via the oral route (Figure 1). Briefly, after transport across the epithelium of the small intestine, ingested antigens are taken up by antigen-presenting cells (APCs). The unique immune environment of the GALT fosters a tolerogenic presentation by resident APCs to antigen-specific T lymphocytes, which results in induction of tolerance. Oral tolerance appears to be mediated by various immune mechanisms (anergy, clonal deletion, or generation of regulatory T cells), depending on the levels of antigen that become bioavailable through the gut tract.3Weiner H.L. da Cunha A.P. Quintana F. Wu H. Oral tolerance.Immunol. Rev. 2011; 241: 241-259Crossref PubMed Scopus (464) Google Scholar For example, in the context of hemophilia, tolerance in mice is mediated by subsets of regulatory T cells that are CD4+CD25+ and CD4+CD25−LAP+.4Wang X. Su J. Sherman A. Rogers G.L. Liao G. Hoffman B.E. Leong K.W. Terhorst C. Daniell H. Herzog R.W. Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells.Blood. 2015; 125: 2418-2427Crossref PubMed Scopus (52) Google Scholar Previous proof-of-concept studies showed that oral tolerance to both clotting factor VIII and F.IX could be achieved in mice fed an antigen produced in tobacco leaves.5Verma D. Moghimi B. LoDuca P.A. Singh H.D. Hoffman B.E. Herzog R.W. Daniell H. Oral delivery of bioencapsulated coagulation factor IX prevents inhibitor formation and fatal anaphylaxis in hemophilia B mice.Proc. Natl. Acad. Sci. USA. 2010; 107: 7101-7106Crossref PubMed Scopus (118) Google Scholar, 6Sherman A. Su J. Lin S. Wang X. Herzog R.W. Daniell H. Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells.Blood. 2014; 124: 1659-1668Crossref PubMed Scopus (84) Google Scholar Similarly, using a model of gene transfer targeted to the skeletal muscle, we have recently shown that oral tolerance can be used to induce anergy and clonal deletion of transgene-specific CD8+ T cells.7Hardet R. Chevalier B. Dupaty L. Naïmi Y. Riou G. Drouot L. Jean L. Salvetti A. Boyer O. Adriouch S. Oral-tolerization prevents immune responses and improves transgene persistence following AAV-mediated gene transfer.Mol. Ther. 2016; 24: 87-95Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar Together, these studies reflect both the large spectrum of immune mechanisms underlying oral tolerance and the potential broad applicability of the concept. Because of its specificity and its long-lasting effect, oral tolerance is potentially safer and more effective than conventional immunosuppression and represents an elegant approach for treating pathologies such as allergy and autoimmunity. However, while this strategy showed therapeutic efficacy in several murine models and in humans for desensitizing against food allergy,8Burks A.W. Jones S.M. Wood R.A. Fleischer D.M. Sicherer S.H. Lindblad R.W. Stablein D. Henning A.K. Vickery B.P. Liu A.H. et al.Consortium of Food Allergy Research (CoFAR)Oral immunotherapy for treatment of egg allergy in children.N. Engl. J. Med. 2012; 367: 233-243Crossref PubMed Scopus (534) Google Scholar the results obtained in clinical trials aimed at treating autoimmunity were modest.9Meyer T. Ullrich R. Zeitz M. Oral tolerance induction in humans.Exp. Mol. Pathol. 2012; 93: 449-454Crossref PubMed Scopus (9) Google Scholar A number of different factors could contribute to the difficulty of translating the approach to the clinic. First, few basic studies in humans can be found in the literature,9Meyer T. Ullrich R. Zeitz M. Oral tolerance induction in humans.Exp. Mol. Pathol. 2012; 93: 449-454Crossref PubMed Scopus (9) Google Scholar and our knowledge of the parameters influencing the efficacy of oral tolerance (optimal dose per antigen, regimen duration, role of HLA, etc.) are thus limited. Second, autoimmune diseases are extremely complex and are associated with generalized immune system dysregulation, with poor understanding of the triggering antigen(s). Importantly, results in humans raise a key question as to the clinical context in which oral tolerance might be successfully applied. Based on the results obtained in autoimmunity trials, oral tolerance seems more likely to lead to clinical success in a prophylactic, rather than curative, fashion. Indeed, preventive oral tolerance could be a major advance in the clinical management of hemophilia or Pompe disease, in which anti-drug antibodies (ADAs) can have devastating effects. For autoimmunity, or in the context of established immunity to a foreign antigen, one could envision combining oral tolerance induction with other strategies such as tolerogenic immunomodulation10Moghimi B. Sack B.K. Nayak S. Markusic D.M. Mah C.S. Herzog R.W. Induction of tolerance to factor VIII by transient co-administration with rapamycin.J. Thromb. Haemost. 2011; 9: 1524-1533Crossref PubMed Scopus (76) Google Scholar or liver-mediated gene transfer.11Mingozzi F. Liu Y.L. Dobrzynski E. Kaufhold A. Liu J.H. Wang Y. Arruda V.R. High K.A. Herzog R.W. Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer.J. Clin. Invest. 2003; 111: 1347-1356Crossref PubMed Scopus (375) Google Scholar, 12Follenzi A. Battaglia M. Lombardo A. Annoni A. Roncarolo M.G. Naldini L. Targeting lentiviral vector expression to hepatocytes limits transgene-specific immune response and establishes long-term expression of human antihemophilic factor IX in mice.Blood. 2004; 103: 3700-3709Crossref PubMed Scopus (176) Google Scholar Another important question addressed in the study published in this issue of Molecular Therapy is the cost and scalability of the proposed treatment, which are important considerations for successful translation to humans. In the context of ADAs, conventional methods will not easily produce sufficient quantities of protein to enable oral tolerance induction protocols. Herzog and colleagues2Herzog R.W. Nichols T.C. Su J. Zhang B. Sherman A. Merricks E.P. Raymer R. Perrin G.Q. Hӓger M. Wiinberg B. Daniell H. Oral tolerance induction in hemophilia B dogs fed with transplastomic lettuce.Mol Ther. 2017; 25 (this issue): 512-522Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar demonstrate that large quantities of antigen can be produced in plants with good manufacturing practice (GMP)-compliant methods. Indeed, expression of F.IX and other proteins, such as acid alpha glucosidase (GAA),13Su J. Sherman A. Doerfler P.A. Byrne B.J. Herzog R.W. Daniell H. Oral delivery of Acid Alpha Glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of Pompe mice.Plant Biotechnol. J. 2015; 13: 1023-1032Crossref PubMed Scopus (46) Google Scholar in chloroplasts allows for easy and reliable scale up, low processing cost, and convenient long-term storage in the lyophilized form.14Su J. Zhu L. Sherman A. Wang X. Lin S. Kamesh A. Norikane J.H. Streatfield S.J. Herzog R.W. Daniell H. Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B.Biomaterials. 2015; 70: 84-93Crossref PubMed Scopus (101) Google Scholar Furthermore, the destructive action of gastric acids and gut enzymes is avoided thanks to the plant cell wall that envelops and protects the protein from degradation. Clearly, while this is a very important advance in the field of protein production and delivery and in our ability to address the critically important problem of ADAs, much remains to be done for the safe and effective translation of the approach to humans. Clinical protocols will have to be designed to provide the optimal chances for success, for example, to tolerize naive subjects prior to protein replacement therapy. This may be challenging for some indications, such as infantile Pompe disease, in which enzyme replacement therapy in the first months of life is key to a positive clinical outcome. Treatment of cross-reactive immune material (CRIM)-negative Pompe patients is in fact almost invariably associated with the production of ADAs,15Kishnani P.S. Goldenberg P.C. DeArmey S.L. Heller J. Benjamin D. Young S. Bali D. Smith S.A. Li J.S. Mandel H. et al.Cross-reactive immunologic material status affects treatment outcomes in Pompe disease infants.Mol. Genet. Metab. 2010; 99: 26-33Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar requiring the use of prophylactic immunosuppression. Thus, in CRIM-negative patients at high-risk for ADA formation, oral tolerance trials could be envisioned initially in combination with conventional pharmacological immunomodulation. Additionally, whereas Herzog and colleagues2Herzog R.W. Nichols T.C. Su J. Zhang B. Sherman A. Merricks E.P. Raymer R. Perrin G.Q. Hӓger M. Wiinberg B. Daniell H. Oral tolerance induction in hemophilia B dogs fed with transplastomic lettuce.Mol Ther. 2017; 25 (this issue): 512-522Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar address the issue of potential toxicities associated with the cholera toxin subunit B uptake domain, the safety of the approach will have to be carefully assessed in humans. While several important questions remain, the demonstration of feasibility and scalability of induction oral tolerance in hemophilia dogs using plant-derived proteins should be considered as a major breakthrough in the management of ADAs. Hopefully, the potential benefit of these advances on protein- and gene-based therapies will soon be evaluated in the clinic. Oral Tolerance Induction in Hemophilia B Dogs Fed with Transplastomic LettuceHerzog et al.Molecular TherapyDecember 22, 2016In BriefAnti-drug antibodies substantially complicate hemophilia treatment. In this article, authors report an antigen-specific immune tolerance induction protocol based on oral delivery of factor IX expressed in lettuce chloroplasts. Effectiveness and safety of the protocol are demonstrated by successful suppression of inhibitory antibody formation in hemophilia B dogs upon long-term delivery. Full-Text PDF Open Access