Transient Expression of Dengue Virus NS1 Antigen in Nicotiana benthamiana for Use as a Diagnostic Antigen

Abstract

Dengue is a viral disease that represents a significant threat to global public health since billions of people are now at risk of infection by this mosquito-borne virus. The implementation of extensive screening tests is indispensable to control this disease, and the Dengue virus non-structural protein 1 (NS1) is a promising antigen for the serological diagnosis of dengue fever. Plant-based systems can be a safe and costeffective alternative for the production of dengue virus antigens. In this work, two strategies to produce the dengue NS1 protein in Nicotiana benthamiana leaves were evaluated: Targeting NS1 to five different subcellular compartments to assess the best subcellular organelle for the expression and accumulation of NS1, and the addition of elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags to NS1. The transiently expressed proteins in N. benthamiana were quantified by Western blot analysis. The NS1 fused to ELP and targeted to the ER (NS1 ELP-ER) showed the highest yield (445 mg/kg), approximately a forty-fold increase in accumulation levels compared to the non-fused protein (NS1-ER), representing the first example of transient expression of DENV NS1 in plant. We also demonstrated that NS1 ELP-ER was successfully recognized by a monoclonal anti-dengue virus NS1 glycoprotein antibody, and by sera from dengue virus-infected patients. Interestingly, it was found that transient production of NS1-ER and NS1 ELP-ER using vacuum infiltration of whole plants, which is easier to scale up, rather than syringe infiltration of leaves, greatly improved the accumulation of NS1 proteins. The generated plant made NS1, even without extensive purification, showed potential to be used for the development of the NS1 diagnostic tests in resource-limited areas where dengue is endemic.

Efficient Plant Production of Recombinant NS1 Protein for Diagnosis of Dengue

“…These same vectors were used to the expression of monoclonal antibodies and reached a yield between 1.2 and 1.4 g/kg. Recently, Marques et al (2020) demonstrated the transient expression of the dengue virus NS1 protein in N. benthamiana for diagnostic purposes. Using an elastin-like polypeptide (ELP) as a fusion tag, the authors obtained 445 mg/kg yield, more than double that obtained in this work, but less than other works that used the transient expression approach.…”

Contributions of the international plant science community to the fight against infectious diseases in humans—part 2: Affordable drugs in edible plants for endemic and re‐emerging diseases

“…The NS1 protein from DENV-2 was expressed in tobacco as a diagnostic reagent for ELISA tests because it has a greater sensitivity than DENV-1 with only a slightly lower specificity (Amaro et al, 2015;Marques et al, 2019;). The first dengue VLP vaccines were produced based on DENV-3 polyprotein in lettuce chloroplasts (Kanagaraj et al, 2011) and cross-reacted with the anti-dengue primary polyclonal antibody.…”

“…As is the case for other endemic diseases that affect primarily developing countries, plants offer a promising platform for vaccine and antibody production because of the low costs, ease of distribution and the particular efficacy of VLP candidates. Recombinant proteins based on consensus envelope protein domain III (cEDIII) and non‐structural protein 1 (NS1) have been produced by transient expression in N. benthamiana (Kim et al ., 2018 ; Marques et al ., 2019 ; Pang et al ., 2019 ) and in transgenic tobacco (Amaro et al ., 2015 ; Gottschamel et al ., 2016 ; Kim et al ., 2010 , 2015 , 2017b ;), lettuce (van Eerde et al ., 2019 ; Kanagaraj et al ., 2011 ; Maldaner et al ., 2013 ), rice (Kim et al ., 2013 , 2014 , 2016 ) and potato (Kim et al ., 2017a ). Transient expression produces soluble antigens with maximum yields of 600 mg/kg fresh leaf biomass (Martinez et al ., 2010 ) although an HBcAg‐cEDIII fusion protein was mostly insoluble and the soluble fraction was not detected by Western blot (Pang et al ., 2019 ).…”

Development and Optimization of an Enzyme Immunoassay to Detect Serum Antibodies against the Hepatitis E Virus in Pigs, Using Plant-Derived ORF2 Recombinant Protein

“…Plant-derived antigens have demonstrable utility in the serological detection of a number of infectious diseases, such as the Dengue virus [ 44 , 45 ], West Nile Virus [ 46 ], Rift Valley fever virus [ 47 ], and Hepatitis E virus [ 15 ]. These studies demonstrate that plants are among the most rapid, prolific, and flexible producers of diagnostic immunogenic reagents.…”