Transgenic plants expressing a Clostridium difficile spore antigen as an approach to develop low‐cost oral vaccines

Abstract

Gastrointestinal infections caused by Clostridium difficile lead to significant impact in terms of morbidity and mortality, causing from mild symptoms, such as a lowgrade fever, watery stools, and minor abdominal cramping as well as more severe symptoms such as bloody diarrhea, pseudomembrane colitis, and toxic megacolon.Vaccination is a viable approach to fight against C. difficile and several efforts in this direction are ongoing. Plants are promising vaccine biofactories offering low cost, enhanced safety, and allow for the formulation of oral vaccines. Herein, the CdeM protein, which is a spore antigen associated with immunoprotection against C. difficile, was selected to begin the development of plant-based vaccine candidates. The vaccine antigen is based in a fusion protein (LTB-CdeM), carrying the CdeM antigen, fused to the carboxi-terminus of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) as a mucosal immunogenic carrier. LTB-CdeM was produced in plants using a synthetic optimized gene according codon usage and mRNA stability criteria. The obtained transformed tobacco lines produced the LTB-CdeM antigen in the range of 52-90 μg/g dry weight leaf tissues. The antigenicity of the plant-made LTB-CdeM antigen was evidenced by GM1-ELISA and immunogenicity assessment performed in test mice revealed that the LTB-CdeM antigen is orally immunogenic inducing humoral responses against CdeM epitopes. This report constitutes the first step in the development of plant-based vaccines against C. difficile infection. K E Y W O R D S C. difficile infection, chimeric antigen, heat labile E. coli enterotoxin B subunit, molecular farming, oral immunization 1 | INTRODUCTION Clostridium difficile is a spore-forming, Gram-positive bacteria that is the leading cause of antibiotic-associated infectious diarrhea in the United States and is one the most common hospital bacterial acquired infection. 1 C. difficile infection (CDI) is a gastrointestinal disease believed to be causally related to perturbations in the intestinal microbiota and is the largest single cause of morbidity and mortality among hospitalacquired infections. 2,3 CDI is generally acquired when patients with predisposing factors, such as advanced age and antibiotic use, are exposed to C. difficile spores emanating from other hospitalized infected patients. 4 CDI cause mild symptoms, such as a low-grade fever, watery Sergio Rosales-Mendoza and Tomás Cervantes-Rincón contributed equally to this study.

Understanding host immune responses in Clostridioides difficile infection: Implications for pathogenesis and immunotherapy

“…To this end, the CdeM protein, serving as a spore antigen, has been chosen as a carrier for mucosal immunization in vaccine development. Administration of this antigen orally has been shown to induce humoral responses against CDI in mice [ 68 ]. In the United Kingdom, Bradshaw et al found that specific traits of lipoprotein CD0873 have potential as a vaccine against C. difficile .…”

Non-Toxin-Based Clostridioides difficile Vaccination Approaches

“…The conjugate of exosporium protein CdeM fused to the carboxy-terminus of the B subunit of the Escherichia coli heat-labile enterotoxin (LTB) was used to design a plant-based vaccine against CD. Transformed tobacco plants efficiently produced the LTB-CdeM antigen that was orally immunogenic [ 17 ]. CdeM was already shown to induce a high level of protection in CDI animal models [ 18 ].…”

Antibodies induced by oral immunization of mice with a recombinant protein produced in tobacco plants harboring Bordetella pertussis epitopes

“…In the other hand, recently the use of plants as bioreactors has been explored as an alternative and less expensive platform capable of incorporating transgenes and expressing heterologous proteins that are immunogenic in mice (Soria-Guerra et al 2007Brodzik et al 2009). Plants provide many advantages over recombinant mammalian systems, including the absence of potential contamination with viruses and prions, a much higher scalable capacity, and very low costs of large-scale production (Kwon and Daniell, 2016;Ward et al 2020;Rosales-Mendoza et al 2021).…”

“…Plants provide many advantages over recombinant mammalian systems, including the absence of potential contamination with viruses and prions, a much higher scalable capacity, and very low costs of large-scale production (Kwon and Daniell, 2016 ; Ward et al 2020 ; Rosales-Mendoza et al 2021 ).…”