Dengue virus tetra-epitope peptide expressed in lettuce chloroplasts for potential use in dengue diagnosis

Abstract

Dengue virus causes about 100 million cases of dengue disease per year in the world. Laboratory diagnosis is done mainly by serological techniques, which in many cases use crude virus extracts that may cause cross-reactions to other flaviviruses. These undesirable cross-reactions can be reduced or eliminated by using recombinant proteins based on restricted epitopes. Aiming to decrease flaviviral cross-reactions and non-specific interactions in dengue serological assays, a plant expression system was chosen for recombinant antigen production as a reliable and inexpensive dengue diagnostic tool. In the present report, the lettuce plastid transformation system was applied to achieve efficient and stable tetra-epitope peptide antigen production, and its reactivity was evaluated. For this purpose, one putative epitope at positions 34 to 57 of E protein within the junction site of domains I and II of dengue virus (DENV) 1 to 4 serotypes linked by glycine linkers was expressed in lettuce chloroplasts. The potential immunoreactivity for the four DENV serotypes was evaluated using sera from patients of positive and negative dengue cases. Results indicated an overall sensitivity of 71.7% and specificity of 100%. No cross-reactions with the sera of yellow fever-positive or healthy individuals vaccinated against yellow fever were observed. This novel approach may provide an alternative system for the large-scale production of dengue recombinant antigens useful for serodiagnosis.

Plant‐made oral vaccines against human infectious diseases—Are we there yet?

“…Therefore, edible leafy vegetables are ideal for biopharmaceutical applications. The lettuce chloroplast system has been successfully used to express a number of vaccines and biopharmaceuticals (Boyhan and Daniell, 2011;Davoodi-Semiromi et al, 2010;Kanagaraj et al, 2011;Maldaner et al, 2013). Chloroplasts permit high gene expression levels and facilitate several post-translational modifications that are required 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).…”

Production of tetravalent dengue virus envelope protein domain III based antigens in lettuce chloroplasts and immunologic analysis for future oral vaccine development

“…In the present study, we have successfully expressed the tetravalent antigen in chloroplasts of the edible crop lettuce, assayed its stability upon passage through the gastrointestinal tract and determined its antigenicity in experimental animals. Further efforts in producing other dengue antigens in plants have also been reported (Kanagaraj et al ., ; Maldaner et al ., ). Our work adds to a growing number of studies in plant molecular farming that have demonstrated that production of vaccines and antibodies in plants is a very attractive alternative to traditional production systems.…”

“…Since the first report of transplastomic lettuce plants, the evaluation of different integration sites and transformation strategies have led to the successful expression of several recombinant proteins in lettuce plastids (Boyhan and Daniell, 2011;Davoodi-Semiromi et al, 2010;Ichikawa et al, 2010;Kanagaraj et al, 2011;Kanamoto et al, 2006;Maldaner et al, 2013;Ruhlman et al, 2007). Most importantly, lettuce is the only system so far that has been shown to be feasible for commercial scale production of clinical grade biopharmaceuticals (Su et al, 2015).…”

Recent developments in therapeutic protein expression technologies in plants

“…Several plant transient expression rely on viral vector systems. In the 1990s scientists were using Dengue virus tetraepitope peptide (cE-DI/IIp) Tobacco psbA promoter/ pRL1001 Lettuce plastid transformation Maldaner et al (2013) Agrobacterium to do transient expression in plants which does not rely on viral vectors (Kapila et al 1997). The study of plant viruses revealed their latent ability to carry foreign genes.…”