Modification of the cholera toxin B subunit coding sequence to enhance expression in plants

This study examined the immunogenic properties of the fusion protein fimbria 2 ofBordetella pertussis(Fim2)—cholera toxin B subunit (CTB) in the intranasal murine model of infection. To this endB. pertussisFim2 coding sequence was cloned downstream of the cholera toxin B subunit coding sequence. The expression and assembly of the fusion protein into pentameric structures (CTB-Fim2) were evaluated by SDS-PAGE and monosialotetrahexosylgaglioside (GM1-ganglioside) enzyme-linked immunosorbent assay (ELISA). To evaluate the protective capacity of CTB-Fim2, an intraperitoneal or intranasal mouse immunization schedule was performed with 50 μg of CTB-Fim2. Recombinant (rFim2) or purified (BpFim2) Fim2, CTB, and phosphate-buffered saline (PBS) were used as controls. The results showed that mice immunized with BpFim2 or CTB-Fim2 intraperitoneally or intranasally presented a significant reduction in bacterial lung counts compared to control groups (P<0.01orP<0.001,resp.). Moreover, intranasal immunization with CTB-Fim2 induced significant levels of Fim2-specific IgG in serum and bronchoalveolar lavage (BAL) and Fim2-specific IgA in BAL. Analysis of IgG isotypes and cytokines mRNA levels showed that CTB-Fim2 results in a mixed Th1/Th2 (T-helper) response. The data presented here provide support for CTB-Fim2 as a promising recombinant antigen againstBordetella pertussisinfection.

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Oral administration to NOD mice of diabetic autoantigens: Pure and linked to cholera toxin B subunit

Immunology Letters ◽

10.1016/s0165-2478(97)88813-x ◽

1997 ◽

Vol 56 (1-3) ◽

pp. 484

Author(s):

I Bache

Keyword(s):

Oral Administration ◽

Cholera Toxin ◽

Nod Mice ◽

Cholera Toxin B Subunit ◽

Toxin B ◽

Cholera Toxin B ◽

B Subunit

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Spray-Dried Formulation of Epicertin, a Recombinant Cholera Toxin B Subunit Variant That Induces Mucosal Healing

Pharmaceutics ◽

10.3390/pharmaceutics13040576 ◽

2021 ◽

Vol 13 (4) ◽

pp. 576

Author(s):

Micaela A. Reeves ◽

Joshua M. Royal ◽

David A. Morris ◽

Jessica M. Jurkiewicz ◽

Nobuyuki Matoba ◽

...

Keyword(s):

Gastric Acid ◽

Cholera Toxin ◽

Oral Formulation ◽

Size Exclusion ◽

Cholera Toxin B Subunit ◽

Toxin B ◽

Cholera Toxin B ◽

B Subunit ◽

Enteric Coated ◽

Spray Dried

Epicertin (EPT) is a recombinant variant of the cholera toxin B subunit, modified with a C-terminal KDEL endoplasmic reticulum retention motif. EPT has therapeutic potential for ulcerative colitis treatment. Previously, orally administered EPT demonstrated colon epithelial repair activity in dextran sodium sulfate (DSS)-induced acute and chronic colitis in mice. However, the oral dosing requires cumbersome pretreatment with sodium bicarbonate to conserve the acid-labile drug substance while transit through the stomach, hampering its facile application in chronic disease treatment. Here, we developed a solid oral formulation of EPT that circumvents degradation in gastric acid. EPT was spray-dried and packed into enteric-coated capsules to allow for pH-dependent release in the colon. A GM1-capture KDEL-detection ELISA and size-exclusion HPLC indicated that EPT powder maintains activity and structural stability for up to 9 months. Capsule disintegration tests showed that EPT remained encapsulated at pH 1 but was released over 180 min at pH 6.8, the approximate pH of the proximal colon. An acute DSS colitis study confirmed the therapeutic efficacy of encapsulated EPT in C57BL/6 mice upon oral administration without gastric acid neutralization pretreatment compared to vehicle-treated mice (p < 0.05). These results provide a foundation for an enteric-coated oral formulation of spray-dried EPT.

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