Chloroplast-derived vaccine antigens confer dual immunity against cholera and malaria by oral or injectable delivery

Department of Molecular Biology and Microbiology, College of Medicine, University of Central Florida, Orlando, FL 32816-2364, USA.
Plant Biotechnology Journal (Impact Factor: 5.75). 02/2010; 8(2):223-42. DOI: 10.1111/j.1467-7652.2009.00479.x
Source: PubMed


Cholera and malaria are major diseases causing high mortality. The only licensed cholera vaccine is expensive; immunity is lost in children within 3 years and adults are not fully protected. No vaccine is yet available for malaria. Therefore, in this study, the cholera toxin-B subunit (CTB) of Vibrio cholerae fused to malarial vaccine antigens apical membrane antigen-1 (AMA1) and merozoite surface protein-1 (MSP1) was expressed in lettuce and tobacco chloroplasts. Southern blot analysis confirmed homoplasmy and stable integration of transgenes. CTB-AMA1 and CTB-MSP1 fusion proteins accumulated up to 13.17% and 10.11% (total soluble protein, TSP) in tobacco and up to 7.3% and 6.1% (TSP) in lettuce, respectively. Nine groups of mice (n = 10/group) were immunized subcutaneously (SQV) or orally (ORV) with purified antigens or transplastomic tobacco leaves. Significant levels of antigen-specific antibody titres of immunized mice completely inhibited proliferation of the malarial parasite and cross-reacted with the native parasite proteins in immunoblots and immunofluorescence studies. Protection against cholera toxin challenge in both ORV (100%) and SQV (89%) mice correlated with CTB-specific titres of intestinal, serum IgA and IgG1 in ORV and only IgG1 in SQV mice, but no other immunoglobulin. Increasing numbers of interleukin-10(+) T cell but not Foxp3(+) regulatory T cells, suppression of interferon-gamma and absence of interleukin-17 were observed in protected mice, suggesting that immunity is conferred via the Tr1/Th2 immune response. Dual immunity against two major infectious diseases provided by chloroplast-derived vaccine antigens for long-term (>300 days, 50% of mouse life span) offers a realistic platform for low cost vaccines and insight into mucosal and systemic immunity.

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    • "for bioactivity such as pentamer assembly, disulphide bond formation, cyclization, protein lipidation and N-terminal methionine excision, but glycosylation does not occur in chloroplasts (Boyhan and Daniell, 2011; Davoodi-Semiromi et al., 2010; Kohli et al., 2014; Kwon et al., 2013a; Scotti et al., 2012; Sherman et al., 2014; Shil et al., 2014; Verma et al., 2010). The expression of a thioredoxin in chloroplasts enhanced protein solubility, proper folding and disulphide bond formation (Sanz-Barrio et al., 2011). "
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    • "In the past two decades, the concept of chloroplast genetic engineering has been advanced to achieve hyperexpression of foreign proteins, with recent advances in conferring novel agronomic traits (Clarke and Daniell, 2011; Jin et al., 2012; Lee et al., 2011), biomass/biofuel enhancement (Agrawal et al., 2011; Jin et al., 2011; Verma et al., 2010a, 2013) or for various biomedical applications for oral delivery of vaccines (Arlen et al., 2008; Davoodi-Semiromi et al., 2010; Lakshmi et al., 2013), autoantigens (Ruhlman et al., 2007; Sherman et al., 2014; Verma et al., 2010b) or biopharmaceuticals (Boyhan and Daniell, 2010; Kohli et al., 2014; Kwon et al., 2013a,c; Ruhlman et al., 2010; Shenoy et al., 2014; Shil et al., 2014). While application of RNAi technology through plant nuclear transformation has several limitations, delivery of small RNA prepared in more efficient systems as human therapeutics is also severely limited by their methods of delivery (Rothschild, 2014). "
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    • "Some of valuable improvements, e.g. enhancing yield (Ichikawa et al., 2010), carotenoid profile (Harada et al., 2013), vitamin E quality (Yabuta et al., 2013), and also the accumulation of pharmaceutical proteins (Ruhlman et al., 2007; Davoodi-Semiromi et al., 2010; Ruhlman et al., 2010; Boyhan and Daniell, 2011; Lim et al., 2011; Sharifi Tabar et al., 2013) were achieved in lettuce by chloroplast transformation using particle bombardment. The success of these genetic manipulation methods depend on that plants can be regenerated from transformed cells in a reliable and efficient manner. "
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