A Plant-Produced Antigen Elicits Potent Immune Responses against West Nile Virus in Mice

Abstract

We described the rapid production of the domain III (DIII) of the envelope (E) protein in plants as a vaccine candidate for West Nile Virus (WNV). Using various combinations of vector modules of a deconstructed viral vector expression system, DIII was produced in three subcellular compartments in leaves of Nicotiana benthamiana by transient expression. DIII expressed at much higher levels when targeted to the endoplasmic reticulum (ER) than that targeted to the chloroplast or the cytosol, with accumulation level up to 73 μg DIII per gram of leaf fresh weight within 4 days after infiltration. Plant ER-derived DIII was soluble and readily purified to > 95% homogeneity without the time-consuming process of denaturing and refolding. Further analysis revealed that plant-produced DIII was processed properly and demonstrated specific binding to an anti-DIII monoclonal antibody that recognizes a conformational epitope. Furthermore, subcutaneous immunization of mice with 5 and 25 μg of purified DIII elicited a potent systemic response. This study provided the proof of principle for rapidly producing immunogenic vaccine candidates against WNV in plants with low cost and scalability.

Immunization of Zika virus envelope protein domain III induces specific and neutralizing immune responses against Zika virus

“…Furthermore, they have shown neutralizing potency against African, Asian and American strains of ZIKV and some of them protected mice against a lethal ZIKV challenge [8]. These results and the ability of EDIII of flaviviruses to independently fold into a functional domain [12] suggest that recombinant zEDIII is an appealing vaccine candidate. In this study, zEDIII was facilely produced in large quantity in E. coli .…”

“…zEDIII was expressed in E. coli, refolded using an oxidative protocol, and purified by immobilized metal anion chromatography (IMAC) with a Ni His.Bind column as described previously [12]. Details of these methods are provided in Supplementary material.…”

Plant‐produced Zika virus envelope protein elicits neutralizing immune responses that correlate with protective immunity against Zika virus in mice

“…PzE purified from plant extracts was immobilized on microtitre plates and incubated with ZV1 and ZV54, mAbs that have been shown to specifically bind conformational epitopes on zEDII and zEDIII, respectively (Dai et al ., ; Zhao et al ., ), followed by an HRP‐conjugated goat anti‐mouse‐IgG antibody (Southern Biotech). E16, a WNV EDIII‐specific mAb that recognizes the equivalent epitope of ZV54 (He et al ., ) was used as a negative control.…”

Plant‐produced candidate countermeasures against emerging and reemerging infections and bioterror agents

“…Domain III (DIII) of WNV E glycoprotein, containing the cell receptor‐binding motif and the majority of epitopes inducing strong virus‐neutralizing antibodies and protection (Oliphant et al ., ), is a target of WNV subunit vaccine development. Recently, DIII has been transiently produced in N. benthamiana plants using Magnifection (Gleba et al ., ), with maximum accumulation detected at 4 days postinfiltration (He et al ., ). Of subcellular localizations tested for DIII expression (the cytosol, ER and chloroplasts), target accumulation was highest in the ER (~73 mg/kg of fresh leaf tissue).…”

“…The plant‐derived DIII was soluble, with correct conformation and epitope display, as demonstrated by specific binding to a monoclonal antibody hE16 recognizing a native WNV DIII conformational epitope. Subcutaneous immunization of mice with four doses of alum‐adjuvanted purified DIII elicited potent antigen‐specific serum IgG titres that bound to the native form of the viral DIII antigen and were exclusively of the IgG1 isotype, indicating a Th2‐type immune response (He et al ., ). The importance of IgG1 in the WNV protective immunity has previously been demonstrated by fractionation of WNV‐neutralizing human immunoglobulins.…”