Engineering, production and characterization of Spike and Nucleocapsid structural proteins of SARS–CoV-2 inNicotiana benthamianaas vaccine candidates against COVID-19

Abstract

The COVID-19 pandemic, which is caused by SARS-CoV-2 has rapidly spread to more than 216 countries and has put global public health at high risk. The world urgently needs a cost-effective and safe SARS-CoV-2 coronavirus vaccine, antiviral and therapeutic drugs to control the COVID-19 pandemic. In this study, we engineered the Nucleocapsid (N) and Spike protein (S) variants (Receptor binding domain, RBD and S1 domain) of SARS-CoV-2 genes and produced in Nicotiana benthamiana plant. The purification yields were at least 20 mg of pure protein/kg of plant biomass for each target protein. The S protein variants of SARS-CoV-2 showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. The purified plant produced N and S variants were recognized by N and S protein specific monoclonal and polyclonal antibodies demonstrating specific reactivity of mAb to plant produced N and S protein variants. In addition, IgG responses of plant produced N and S antigens elicited significantly high titers of antibody in mice. This is the first report demonstrating production of functional active S1 domain and Nucleocapsid protein of SARC-CoV-2 in plants. In addition, in this study, for the first time, we report the co-expression of RBD with N protein to produce a cocktail antigen of SARS-CoV-2, which elicited high-titer antibodies compared to RBD or N proteins. Thus, obtained data support that plant produced N and S antigens, developed in this study, are promising vaccine candidates against COVID-19.

Plant-Produced Glycosylated and In Vivo Deglycosylated Receptor Binding Domain Proteins of SARS-CoV-2 Induce Potent Neutralizing Responses in Mice

“…Flexible approaches are required for successful production of functionally active recombinant proteins in plants with high yields. In this study, we achieved the production of functionally active glycosylated and in vivo deglycosylated variants of RBD (consisting of amino acids R319–S591) of SARS-CoV-2 structural protein at a high level and in a highly soluble form in the plant N. benthamiana using a transient expression platform [ 69 ]. Production of the glycosylated form of RBD (aa F318–C617), alone [ 48 ] or fused with immunoglobulin Fc domain [ 31 ], using a transient plant expression system in N. benthamiana , has been recently reported.…”

N-Glycosylation of the SARS-CoV-2 Receptor Binding Domain Is Important for Functional Expression in Plants

“…We expressed an RBD variant that was previously produced in mammalian or insect cells ( Stadlbauer et al, 2020 ; Klausberger et al, 2021 ) and observed low expression levels and high amounts of homodimers. Recombinant SARS-CoV-2 RBD variants have recently been produced in N. benthamiana using different transient expression systems ( Diego-Martin et al, 2020 ; Mamedov et al, 2020 ; Rattanapisit et al, 2020 ; Makatsa et al, 2021 ). Diego-Martin et al (2020) used a MagnICON-based expression vector to produce a His-tagged RBD variant carrying the same amino acid region (R319-F541) as present in our RBD.…”

“…The overall yield of this longer RBD variant was in the same range (8 μg/g fresh weight) as observed for the other plant produced RBDs. Mamedov et al (2020) reported the expression of an RBD variant consisting of amino acids R319-S591. Like in the RBD-215 variant, there is an even number of cysteine residues in the amino acid sequence and the yield after purification (10–20 μg/g fresh weight, using the pEAQ- HT expression vector) was comparable to our yield.…”

Producing Vaccines against Enveloped Viruses in Plants: Making the Impossible, Difficult

“…Similarly, Kentucky BioProcessing has also announced that it is developing a plant-produced subunit vaccine candidate, which is currently being tested in a phase 1–2 clinical trial (NCT04473690). No scientific data on either of these candidate vaccines has yet been published; however, there have been pre-published reports by a separate group of the transient expression of S1 (the surface-exposed region of the S glycoprotein) and nucleocapsid (N) proteins in plants for potential use as subunit vaccine candidates [ 69 ].…”