Scalable agroinfiltration-based production of SARS-CoV-2 antigens for use in diagnostic assays and subunit vaccines.

Agroinfiltration is a method used in biopharming to support plant-based biosynthesis of therapeutic proteins such as antibodies and viral antigens involved in vaccines. Major advantages of generating proteins in plants is the low cost, massive scalability and the rapid yield of the technology. Herein, we report the agroinfiltration-based production of glycosylated SARS-CoV-2 Spike receptor-binding domain (RBD) protein. We show that it exhibits high-affinity binding to the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) and displays folding similar to antigen produced in mammalian expression systems. Moreover, our plant-expressed RBD was readily detected by IgM, IgA, and IgG antibodies from the serum of SARS-CoV-2 infected and vaccinated individuals. We further demonstrate that binding of plant-expressed RBD to ACE2 is efficiently neutralized by these antibodies. Collectively, these findings demonstrate that recombinant RBD produced via agroinfiltration exhibits suitable biochemical and antigenic features for use in serological and neutralization assays, and in subunit vaccine platforms

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Biochemical evaluation of plant-expressed RBD by circular dichroism (CD).

A) Spectral profiles of mammalian standard (black) and plant (blue) expressed RBD in PBS buffer pH 7.4 at 37°C (200–250 nm). Data expressed in molar circular dichroism (Δε) calculated from averaging 5 independent spectra of each sample. B) Secondary structure content in percentage calculated for mammalian and plant-derived RBD in PBS buffer pH 7.4 at 37°C (200–250 nm). Content was calculated using BeStSel analysis server by direct analysis of the raw data (mDeg) from the CD system. Molecular weights for both proteins were considered as 35 kDa.</p

Expression and purification of SARS-CoV-2 RBD in <i>Nicotiana benthamiana</i>.

(A) Agro-infiltrated Nicotiana benthamiana growing in greenhouse. (B) Schematic representation of genetic construct used to express SARS-CoV-2 RBD in planta. The SARS-CoV-2 sequence was expressed as a recombinant protein with a dual 8xHis and Twin-Strep II tag, interspersed with Gly-Ser linkers (gold boxes). An ER-retention KDEL sequence was positioned at the C-terminus, and a Thrombin cleavage site (LVPRGS) was included for tag removal. (C) Left panel: Anti-His IB of samples obtained from N. benthamiana 2 to 5 days post-infiltration (dpi) with RBD construct in B. Loading control at 5 dpi reproducibly demonstrates reduced abundance of protein due to initiation of tissue necrosis at this time. Right panel: NT control (lane 1) compared to RBD expressed in N. benthamiana with calreticulin (lane 2). Anti-his IB. (D) Co-infiltration of human calreticulin (CRT) increases expression levels of RBD in N. benthamiana. Samples collected 4dpi. Anti-his IB. (E) Anti-S1 IB of purified RBD expressed in N. benthamiana (lane 1) and control RBD expressed in mammalian 293F cells (lane 2). Arrow indicates expected migration of a protein corresponding to 31.3 kDa. (F) CBB-stained SDS-PAGE of purified RBD expressed in N. benthamiana (lane 1) and control RBD expressed in mammalian 293F cells (lane 2). (G) CBB-stained SDS-PAGE of plant-derived RBD treated with (+) and without (-) the amidase Peptide-N-Glycosidase F (PNGaseF), an enzyme that cleaves N-linked glycan chains. SP, signal peptide. IB, immunoblot. CBB, Coomassie Brilliant Blue. NT, non-transformed.</p

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Evaluation of plant-expressed RBD protein functionality, folding and binding kinetics.

A comprehensive assessment of protein function of the RBD produced in N. benthamiana as it pertains to protein folding and binding to ACE2 receptor, recognition and neutralization by antibodies in sera from SARS-CoV-2 exposed individuals. (A) Indirect ELISA demonstrating binding kinetics of soluble human ACE2 to immobilized mammalian RBD (red circle) and plant RBD (blue triangle). (B) Binding and recognition of immobilized mammalian and plant produced RBD by IgG, IgM and IgA polyclonal antibodies in sera of pooled naïve (unvaccinated, uninfected individuals; n>100), pooled convalescent (pooled Conv.) (n>100), convalescent and vaccinated with one dose (Conv. + 1 dose), convalescent and vaccinated with two doses (Conv. + 2 dose), vaccinated with one dose (1 dose), vaccinated with 2 doses (2 doses) of Pfizer (BNT162b2) and naïve (PCR negative confirmed). Pooled sera are samples pooled from a surveillance study of 100 individuals with (pooled convalescent) or without (pooled naïve) prior SARS-CoV-2 infection. While the rest of the samples were collected from single individuals from each category described above. (C) Binding and recognition of immobilized mammalian and plant produced RBD by conformation dependant monoclonal IgG, IgM, and IgA CR3022 antibodies and (D) their half-maximal inhibitory dilution (ID50) values. (E) Relative inhibition percentage of anti-SARS-CoV-2 neutralizing antibodies in blocking immobilized mammalian and plant produced RBD from binding to soluble ACE2 by snELISA. This assay is representative of technical triplicates or quadruplicates and presented as mean ± standard deviation. (F) Reciprocal ID50 values from (E) for mammalian- and plant-expressed RBD.</p

Schematic representations of the different types of ELISA used to characterize plant-expressed RBD.

(A) Indirect ELISA (Kd) set up for the evaluation of binding kinetics of soluble human ACE2 to immobilized RBD. (B) Indirect ELISA (serology) set up for the evaluation of binding and recognition of commercial monoclonal and serum IgG, IgM and IgA antibodies to immobilized RBD. (C) Surrogate neutralization ELISA (snELISA) set up to evaluate relative inhibition of anti-SARS-CoV-2 neutralizing antibodies in blocking immobilized RBD from binding soluble ACE2.</p