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

Abstract

The COVID-19 pandemic, caused by SARS-CoV-2, has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs cost-effective and safe SARS-CoV-2 vaccines, antiviral, and therapeutic drugs to control it. In this study, we engineered the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and produced it in the plant Nicotiana benthamiana in a glycosylated and deglycosylated form. Expression levels of both glycosylated (gRBD) and deglycosylated (dRBD) RBD were greater than 45 mg/kg fresh weight. The purification yields were 22 mg of pure protein/kg of plant biomass for gRBD and 20 mg for dRBD, which would be sufficient for commercialization of these vaccine candidates. The purified plant-produced RBD protein was recognized by an S protein-specific monoclonal antibody, demonstrating specific reactivity of the antibody to the plant-produced RBD proteins. The SARS-CoV-2 RBD showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. In mice, the plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity. To our knowledge, this is the first report demonstrating that mice immunized with plant-produced deglycosylated RBD form elicited high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2 infection. Thus, obtained data support that plant-produced glycosylated and in vivo deglycosylated RBD antigens, developed in this study, are promising vaccine candidates for the prevention of COVID-19.

Investigating Constraints Along the Plant Secretory Pathway to Improve Production of a SARS-CoV-2 Spike Vaccine Candidate

“…This has also been combined with the co-expression of human furin to support proteolytic processing in planta , where this would not otherwise occur ( Margolin et al, 2020c ). The impetus behind these approaches is to produce recombinant viral glycoproteins in as close to their native conformation as possible and while many promising plant-derived SARS-CoV-2 vaccine candidates have been reported these have mostly focused on the receptor binding domain ( Rattanapisit et al, 2020 ; Maharjan et al, 2021 ; Mamedov et al, 2021 ; Shin et al, 2021 ) or chimaeric spike-derived antigens ( Ward et al, 2021 ) rather than a native spike that recapitulates the virion-associated glycoprotein structure.…”

Production and Characterization of Nucleocapsid and RBD Cocktail Antigens of SARS-CoV-2 in Nicotiana benthamiana Plant as a Vaccine Candidate against COVID-19

“…Since the S protein of SARS-CoV, MERS-CoV, and SARS-CoV-2 contains neutralizing epitopes, it has been shown to be a leading candidate for vaccine developments. In this regard, a number of studies demonstrated that the S protein induced potent humoral and cellular immune responses in tested animal models [1][2][3][4][5]. Most COVID-19 vaccines, which are currently approved or under development (mRNA, DNA, viral vector-based, subunits, and protein-based vaccine) were designed and developed on the basis of the S protein [4][5][6][7][8][9][10][11].…”

“…In this regard, a number of studies demonstrated that the S protein induced potent humoral and cellular immune responses in tested animal models [1][2][3][4][5]. Most COVID-19 vaccines, which are currently approved or under development (mRNA, DNA, viral vector-based, subunits, and protein-based vaccine) were designed and developed on the basis of the S protein [4][5][6][7][8][9][10][11]. The genome size of SARS-CoV-2 is ~30 kb, one-third of which encodes the S (spike), N (nucleocapsid), E (envelop), and M (matrix) structural proteins.…”

“…Notably, among a number of expression systems, transient expression in plants is a promising platform for the production of recombinant proteins. This system allows the cost-effective production of a variety of complex recombinant proteins, including vaccine candidates, therapeutic proteins, enzymes, and antibodies, in a short period of time, with high yield and fully functional activity [4,[27][28][29][30][31][32]. In this study, we report the expression and production of N protein and its co-expression with RBD in N. benthamiana plant to produce cocktail antigens of SARS-CoV-2 as a vaccine candidate, which may have broader-spectrum protection.…”

Development of an Inactivated Vaccine against SARS CoV-2

“…ERUCoV-VAC-specific neutralizing antibody was identified using a microneutralization test (MNT) as described previously [29]. The titre of the neutralising antibody was determined as the highest dilution of serum at which the infectivity was neutralised in 50% of the cells in the wells.…”