Low Cost Tuberculosis Vaccine Antigens in Capsules: Expression in Chloroplasts, Bio-Encapsulation, Stability and Functional Evaluation In Vitro

Abstract

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading fatal infectious diseases. The development of TB vaccines has been recognized as a major public health priority by the World Health Organization. In this study, three candidate antigens, ESAT-6 (6kDa early secretory antigenic target) and Mtb72F (a fusion polyprotein from two TB antigens, Mtb32 and Mtb39) fused with cholera toxin B-subunit (CTB) and LipY (a cell wall protein) were expressed in tobacco and/or lettuce chloroplasts to facilitate bioencapsulation/oral delivery. Site-specific transgene integration into the chloroplast genome was confirmed by Southern blot analysis. In transplastomic leaves, CTB fusion proteins existed in soluble monomeric or multimeric forms of expected sizes and their expression levels varied depending upon the developmental stage and time of leaf harvest, with the highest-level of accumulation in mature leaves harvested at 6PM. The CTB-ESAT6 and CTB-Mtb72F expression levels reached up to 7.5% and 1.2% of total soluble protein respectively in mature tobacco leaves. Transplastomic CTB-ESAT6 lettuce plants accumulated up to 0.75% of total leaf protein. Western blot analysis of lyophilized lettuce leaves stored at room temperature for up to six months showed that the CTB-ESAT6 fusion protein was stable and preserved proper folding, disulfide bonds and assembly into pentamers for prolonged periods. Also, antigen concentration per gram of leaf tissue was increased 22 fold after lyophilization. Hemolysis assay with purified CTB-ESAT6 protein showed partial hemolysis of red blood cells and confirmed functionality of the ESAT-6 antigen. GM1-binding assay demonstrated that the CTB-ESAT6 fusion protein formed pentamers to bind with the GM1-ganglioside receptor. The expression of functional Mycobacterium tuberculosis antigens in transplastomic plants should facilitate development of a cost-effective and orally deliverable TB booster vaccine with potential for long-term storage at room temperature. To our knowledge, this is the first report of expression of TB vaccine antigens in chloroplasts.

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

“…Strategies discussed here will allow us to develop optimal materials in lettuce chloroplasts to facilitate clinical translation, which we know from multiple prior studies to be equivalent in delivery of bioencapsulated antigen to the GALT. 19,32,35 Further studies of the duration of oral FVIII administration are also needed. Thus far, the data suggest that more continuous oral delivery is required for FVIII than what we had found for FIX.…”

“…Regeneration of FVIII transplastomic tobacco plants was performed as described earlier. [33][34][35] Characterization of FVIII expression in leaf tissues of transplastomic plants…”

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…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). Therefore, oral delivery of proinsulin or exendin-4 reduced blood sugar levels similar to injected proteins (Boyhan and Daniell, 2011;Kwon et al, 2013).…”

An overview of tuberculosis plant-derived vaccines

“…Hemolysis assay performed with purified CTB-ESAT-6 detected partial hemolysis of red blood cells, confirming the functional activity of the ESAT-6 antigen. Although this is a relevant report constituting the first attempt on exploring the expression of TB antigens in chloroplasts, immunogenicity assessment in test animals is a critical pending objective to determine the potential of these vaccine prototypes [76]. CTB is well recognized as a mucosal adjuvant that facilitates translocation of antigens into the subephitelium and thus a proper mucosal immunogenicity is expected for this candidate, being of relevance in the induction of mucosal immune responses in easily administered vaccines.…”