Production of dengue virus envelope protein domain III-based antigens in tobacco chloroplasts using inducible and constitutive expression systems

Abstract

Dengue fever is a disease in many parts of the tropics and subtropics and about half the world's population is at risk of infection according to the World Health Organization. Dengue is caused by any of the four related dengue virus serotypes DEN-1, -2, -3 and -4, which are transmitted to people by Aedes aegypti mosquitoes. Currently there is only one vaccine (Dengvaxia Ò ) available (limited to a few countries) on the market since 2015 after half a century's intensive efforts. Affordable and accessible vaccines against dengue are hence still urgently needed. The dengue envelop protein domain III (EDIII), which is capable of eliciting serotype-specific neutralizing antibodies, has become the focus for subunit vaccine development. To contribute to the development of an accessible and affordable dengue vaccine, in the current study we have used plant-based vaccine production systems to generate a dengue subunit vaccine candidate in tobacco. Chloroplast genome engineering was applied to express serotypespecific recombinant EDIII proteins in tobacco chloroplasts using both constitutive and ethanol-inducible expression systems. Expression of a tetravalent antigen fusion construct combining EDIII polypeptides from all four serotypes was also attempted. Transplastomic EDIIIexpressing tobacco lines were obtained and homoplasmy was verified by Southern blot analysis. Northern blot analyses showed expression of EDIII antigen-encoding genes. EDIII protein accumulation levels varied for the different recombinant EDIII proteins and the different expression systems, and reached between 0.8 and 1.6 % of total cellular protein. Our study demonstrates the suitability of the chloroplast compartment as a production site for an EDIII-based vaccine candidate against dengue fever and presents a Gateway Ò plastid transformation vector for inducible transgene expression.

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

“…M: spectra multicolour broad range protein ladder (Thermo Scientific, molecular weight of the marker bands indicated in kDa). The band migrating at 70 kDa results from non‐specific binding of the antibody to a plant protein of unknown identity (Gottschamel et al ., ).…”

“…In order to produce a dengue antigen that covers all four dengue virus serotypes, transplastomic plants expressing the tetravalent antigen EDIII‐1‐4 (Gottschamel et al ., ) in the chloroplast were generated. To compare expression levels and help ascertain the stability of the linker regions in the tetravalent antigen, a transplastomic line expressing only the EDIII from DENV‐1 was also generated.…”

“…The tetravalent antigen EDIII‐1‐4 was only detected as a single band corresponding to the full‐length antigen, indicating that the linker regions are stable in the chloroplast and no significant proteolytic cleavage occurs. The EDIII‐1‐4 accumulation was quantified in TP extracts by using purified E. coli ‐produced tetravalent antigen (Gottschamel et al ., ) as a standard. These assays (Figure S1) established that the EDIII‐1‐4 fusion protein was present at 3.45% ± 0.05 of the TP content in the transplastomic lettuce plants.…”

“…Sera of a rabbit immunized with lettuce‐produced EDIII ‐1‐4 antigen at 63 days post vaccination ( EDIII ‐1‐4) were tested for binding to E. coli ‐produced EDIII ‐1‐4 antigen and lettuce‐produced EDIII ‐1‐4 antigen. Rab neg ctrl is serum from non‐vaccinated rabbit; Rab Pos Ctrl represents a rabbit polyclonal antibody against a synthetic EDIII peptide (Gottschamel et al ., ) used as a positive control. All sera were used in a 1 : 50 dilution.…”

Rapid production of therapeutic proteins using plant system

“…Using this approach a whole range of therapeutic proteins has been produced in the plant tissue. Depending upon the use of a specific promoter, expression can be achieved uniformly throughout the plant or it can be limited up to the expression in plant seed, organs and sub-cellular organelles [28][29][30][31][32][33] . Apart from this, the focus of future research is in the production of oral vaccines in the edible plants/ fruits, such as tomatoes and bananas.…”