Coagulation factor replacement therapy for the X-linked bleeding disorder Haemophilia, characterized by a deficiency of coagulation protein factor VIII (FVIII), is severely complicated by antibody (inhibitors) formation. The development of FVIII inhibitors drastically alters the quality of life of the patients and is associated with a tremendous increase in morbidity as well as treatment costs. The ultimate goal of inhibitor control is antibody elimination. Immune tolerance induction (ITI) is the only clinically established approach for developing antigen-specific tolerance to FVIII. This work aims to establish a novel cost-effective strategy to produce FVIII molecules in fusion with cholera toxin B (CTB) subunit at the N terminus using the Bacillus subtilis expression system for oral tolerance, as the current clinical immune tolerance protocols are expensive. Regions of B-Domain Deleted (BDD)-FVIII that have potential epitopes were identified by employing Bepipred linear epitope prediction; 2 or more epitopes in each domain were combined and cDNA encoding these regions were fused with CTB and cloned in the Bacillus subtilis expression vector pHT43 and expression analysis was carried out. The expressed CTB-fused FVIII epitope domains showed strong binding affinity towards the CTB-receptor GM1 ganglioside. To conclude, Bacillus subtilis expressing FVIII molecules might be a promising candidate for exploring for the induction of oral immune tolerance.

Centre de Recherche des Cordeliers Institut National de la Santé et de la Recherche Médicale CNRS Sorbonne Université Université de Paris Paris F 75006 France

Centre for Bio Separation Technology Tamil Nadu Vellore 632014 India

See more in PubMed

Amuguni H, Tzipori S (2012) Bacillus subtilis A temperature resistant and needle free delivery system of immunogens © 2012 Landes Bioscience. Do not distribute © 2012 Landes Bioscience. Do Not Distribute Hum Vaccin Immunother 8:979–986 DOI PMC

Baldauf KJ, Royal JM, Hamorsky KT, Matoba N (2015) Cholera toxin B: One subunit with many pharmaceutical applications. Toxins (basel) 7:974–996. https://doi.org/10.3390/toxins7030974 PubMed DOI

Batsuli G, Meeks SL, Herzog RW, Lacroix-Desmazes S (2016) Innovating immune tolerance induction for haemophilia. Haemophilia 22:31–35. https://doi.org/10.1111/hae.12989 PubMed DOI

Berntorp E (2023) Immune tolerance induction in development. Blood 141:1901–1902. https://doi.org/10.1182/blood.2022019465 PubMed DOI

Bin SJ, Holmgren J, Czerkinsky C (1994) Cholera toxin B subunit: An efficient transmucosal carrier-delivery system for induction of peripheral immunological tolerance. Proc Natl Acad Sci USA 91:10795–10799. https://doi.org/10.1073/pnas.91.23.10795 DOI

Chavin SI (1984) Factor VIII: Structure and function in blood clotting. Am J Hematol 16:297–306. https://doi.org/10.1002/ajh.2830160312 PubMed DOI

Chunfeng G, Xiaozhou L, Gang W et al (2013) Expression of cholera toxin B-lumbrokinase fusion protein in Pichia pastoris - The use of transmucosal carriers in the delivery of therapeutic proteins to protect rats against thrombosis. Appl Biochem Biotechnol 169:636–650. https://doi.org/10.1007/s12010-012-0004-2 PubMed DOI

Dimichele DM (2012) Immune tolerance in haemophilia: The long journey to the fork in the road. Br J Haematol 159:123–134. https://doi.org/10.1111/bjh.12028 PubMed DOI

Franchini M, Mannucci PM (2012) Past, present and future of hemophilia: a narrative review. Orphanet J Rare Dis 7:1–8. https://doi.org/10.1186/1750-1172-7-24 DOI

Hardet R, Mingozzi F (2017) Oral tolerance: another reason to eat your veggies! Mol Ther 25:311–313. https://doi.org/10.1016/j.ymthe.2017.01.001 PubMed DOI PMC

Herzog RW, Nichols TC, Su J et al (2017) Oral tolerance induction in hemophilia B dogs fed with transplastomic lettuce. Mol Ther 25:512–522. https://doi.org/10.1016/j.ymthe.2016.11.009 PubMed DOI PMC

Hu B, Li C, Lu H et al (2011) Immune responses to the oral administration of recombinant Bacillus subtilis expressing multi-epitopes of foot-and-mouth disease virus and a cholera toxin B subunit. J Virol Methods 171:272–279. https://doi.org/10.1016/j.jviromet.2010.11.023 PubMed DOI

Jiang H, Bian Q, Zeng W et al (2019) Oral delivery of Bacillus subtilis spores expressing grass carp reovirus VP4 protein produces protection against grass carp reovirus infection. Fish Shellfish Immunol 84:768–780. https://doi.org/10.1016/j.fsi.2018.10.008 PubMed DOI

Kim YC, Zhang AH, Su Y et al (2015) Engineered antigen-specific human regulatory T cells: Immunosuppression of FVIII-specific T- and B-cell responses. Blood 125:1107–1115. https://doi.org/10.1182/blood-2014-04-566786 PubMed DOI PMC

Kosloski MP, Miclea RD, Balu-Iyer SV (2009) Role of glycosylation in conformational stability, activity, macromolecular interaction and immunogenicity of recombinant human factor VIII. AAPS J 11:424–431. https://doi.org/10.1208/s12248-009-9119-y PubMed DOI PMC

Krishnamoorthy S, Liu T, Drager D et al (2016) Recombinant factor VIII Fc (rFVIIIFc) fusion protein reduces immunogenicity and induces tolerance in hemophilia A mice. Cell Immunol 301:30–39. https://doi.org/10.1016/J.CELLIMM.2015.12.008 PubMed DOI

Kwon KC, Sherman A, Chang WJ et al (2018) Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice. Plant Biotechnol J 16:1148–1160. https://doi.org/10.1111/pbi.12859 PubMed DOI

Lenting PJ, van Mourik JA, Mertens K (1998) The life cycle of coagulation factor VIII in view of its structure and function. Blood 92:3983–3996. https://doi.org/10.1182/blood.v92.11.3983 PubMed DOI

Li W, Feng J, Li J et al (2019) Surface display of antigen protein VP8* of porcine rotavirus on Bacillus subtilis spores using COtB as a fusion partner. Molecules. https://doi.org/10.3390/molecules24203793 PubMed DOI PMC

Mancuso ME, Cannavò A (2015) Immune tolerance induction in hemophilia. Clin Investig (lond) 5:321–335. https://doi.org/10.4155/cli.14.122 DOI

Mimoun A, Bou-Jaoudeh M, Delignat S et al (2023) Transplacental delivery of therapeutic proteins by engineered immunoglobulin G: a step toward perinatal replacement therapy. J Thromb Haemost. https://doi.org/10.1016/j.jtha.2023.05.021 PubMed DOI

Moghimi B, Sack BK, Nayak S et al (2011) Induction of tolerance to factor VIII by transient co-administration with rapamycin. J Thromb Haemost 9:1524–1533. https://doi.org/10.1111/j.1538-7836.2011.04351.x PubMed DOI PMC

Mou C, Zhu L, Yang J et al (2016) Immune responses induced by recombinant Bacillus Subtilis expressing the hemagglutinin protein of H5N1 in chickens. Sci Rep 6:1–11. https://doi.org/10.1038/srep38403 DOI

Neef J, van Dijl JM, Buist G (2021) Recombinant protein secretion by Bacillus subtilis and lactococcus lactis: Pathways, applications, and innovation potential. Essays Biochem 65:187–195. https://doi.org/10.1042/EBC20200171 PubMed DOI PMC

Pinheiro-Rosa N, Torres L, de Oliveira M, A, et al (2021) Oral tolerance as antigen-specific immunotherapy. Immunother Adv 1:1–21. https://doi.org/10.1093/immadv/ltab017 DOI

Ruhlman T, Ahangari R, Devine A et al (2007) Expression of cholera toxin B-proinsulin fusion protein in lettuce and tobacco chloroplasts - Oral administration protects against development of insulitis in non-obese diabetic mice. Plant Biotechnol J 5:495–510. https://doi.org/10.1111/j.1467-7652.2007.00259.x PubMed DOI PMC

Sack BK, Herzog RW, Terhorst C, Markusic DM (2014) Development of gene transfer for induction of antigen-specific tolerance. Mol Ther - Methods Clin Dev 1:14013. https://doi.org/10.1038/mtm.2014.13 PubMed DOI PMC

Sánchez J, Holmgren J (2008) Cholera toxin structure, gene regulation and pathophysiological and immunological aspects. Cell Mol Life Sci 65:1347–1360. https://doi.org/10.1007/s00018-008-7496-5 PubMed DOI PMC

Sarkar D, Biswas M, Liao G et al (2014) Ex vivo expanded autologous polyclonal regulatory T cells suppress inhibitor formation in hemophilia. Mol Ther - Methods Clin Dev 1:14030. https://doi.org/10.1038/mtm.2014.30 PubMed DOI PMC

Schep SJ, Schutgens REG, Fischer K, Boes ML (2018) Review of immune tolerance induction in hemophilia A. Blood Rev 32:326–338. https://doi.org/10.1016/j.blre.2018.02.003 PubMed DOI

Scott DW, Pratt KP, Miao CH (2013) Progress toward inducing immunologic tolerance to factor VIII. Blood 121:4449–4456. https://doi.org/10.1182/blood-2013-01-478669 PubMed DOI PMC

Shafaati M, Ghorbani M, Mahmoodi M et al (2022) Expression and characterization of hemagglutinin–neuraminidase protein from Newcastle disease virus in Bacillus subtilis WB800. J Genet Eng Biotechnol. https://doi.org/10.1186/s43141-022-00357-w PubMed DOI PMC

Shen BW, Spiegel PC, Chang CH et al (2008) The tertiary structure and domain organization of coagulation factor VIII. Blood 111:1240–1247. https://doi.org/10.1182/blood-2007-08-109918 PubMed DOI PMC

Sherman A, Su J, Lin S et al (2014) Suppression of inhibitor formation against FVIII in a murine model of hemophilia a by oral delivery of antigens bioencapsulated in plant cells. Blood 124:1659–1668. https://doi.org/10.1182/blood-2013-10-528737 PubMed DOI PMC

Sherman A, Biswas M, Herzog RW (2017) Innovative approaches for immune tolerance to factor VIII in the treatment of hemophilia A. Front Immunol. https://doi.org/10.3389/fimmu.2017.01604 PubMed DOI PMC

Souza CCD, Guimarães JM, Pereira SDS, Mariúba LAM (2021) The multifunctionality of expression systems in Bacillus subtilis: Emerging devices for the production of recombinant proteins. Exp Biol Med 246:2443–2453. https://doi.org/10.1177/15353702211030189 DOI

Sricharunrat T, Pumirat P, Leaungwutiwong P (2018) Oral tolerance: recent advances on mechanisms and potential applications. Asian Pacific J allergy Immunol 36:207–216. https://doi.org/10.12932/AP0848 DOI

Steinitz KN, Van Helden PM, Binder B et al (2012) CD4+ T-cell epitopes associated with antibody responses after intravenously and subcutaneously applied human FVIII in humanized hemophilic E17 HLA-DRB1*1501 mice. Blood 119:4073–4082. https://doi.org/10.1182/blood-2011-08-374645 PubMed DOI

Stratmann T (2015) Cholera toxin subunit b as adjuvant—an accelerator in protective immunity and a break in autoimmunity. Vaccines 3:579–596. https://doi.org/10.3390/vaccines3030579 PubMed DOI PMC

Sun J, Czerkinsky C, Holmgren J (2010) Mucosally induced immunological tolerance, regulatory T cells and the adjuvant effect by cholera toxin B subunit 71(1):1–11. https://doi.org/10.1111/j.1365-3083.2009.02321.x DOI

Tordesillas L, Berin MC (2018) Mechanisms of oral tolerance. Clin Rev Allergy Immunol 55:107–117. https://doi.org/10.1007/s12016-018-8680-5 PubMed DOI PMC

Volkers P, Hanschmann KM, Calvez T et al (2019) Recombinant factor VIII products and inhibitor development in previously untreated patients with severe haemophilia A: Combined analysis of three studies. Haemophilia 25:398–407. https://doi.org/10.1111/hae.13747 PubMed DOI

Wang X, Sherman A, Liao G et al (2013) Mechanism of oral tolerance induction to therapeutic proteins. Adv Drug Deliv Rev 65:759–773. https://doi.org/10.1016/j.addr.2012.10.013 PubMed DOI

Wang X, Su J, Sherman A et al (2015a) Plant-based oral tolerance to hemophilia therapy employs a complex immune regulatory response including LAP+CD4+ T cells. Blood 125:2418–2427. https://doi.org/10.1182/blood-2014-08-597070 PubMed DOI PMC

Wang X, Su J, Sherman A et al (2015b) Plant-based oral tolerance for hemophilia results from a complex immune regulatory mechanism including LAP+ CD4+ T cells. Blood 125:2418–2428. https://doi.org/10.1182/blood-2014-08-597070.The PubMed DOI PMC

Wickramasuriya SS, Park I, Lee Y et al (2021) Oral delivery of Bacillus subtilis expressing chicken NK-2 peptide protects against Eimeria acervulina infection in broiler chickens. Front Vet Sci 8:1–10. https://doi.org/10.3389/fvets.2021.684818 DOI

Xiao Y, Kwon KC, Hoffman BE et al (2016) Low cost delivery of proteins bioencapsulated in plant cells to human non-immune or immune modulatory cells. Biomaterials 80:68–79. https://doi.org/10.1016/j.biomaterials.2015.11.051 PubMed DOI

Zhang S, Mou C, Cao Y et al (2020) Immune response in piglets orally immunized with recombinant Bacillus subtilis expressing the capsid protein of porcine circovirus type 2. Cell Commun Signal 18:1–14. https://doi.org/10.1186/s12964-020-0514-4 DOI