Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing global outbreak of coronavirus disease (COVID-19) which is a significant threat to global public health. The rapid spread of COVID-19 necessitates the development of cost-effective technology platforms for the production of vaccines, drugs, and protein reagents for appropriate disease diagnosis and treatment. In this study, we explored the possibility of producing the receptor binding domain (RBD) of SARS-CoV-2 and an anti-SARS-CoV monoclonal antibody (mAb) CR3022 in Nicotiana benthamiana. Both RBD and mAb CR3022 were transiently produced with the highest expression level of 8 μg/g and 130 μg/g leaf fresh weight respectively at 3 days post-infiltration. The plant-produced RBD exhibited specific binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, the plant-produced mAb CR3022 binds to SARS-CoV-2, but fails to neutralize the virus in vitro. This is the first report showing the production of anti-SARS-CoV-2 RBD and mAb CR3022 in plants. Overall these findings provide a proof-of-concept for using plants as an expression system for the production of SARS-CoV-2 antigens and antibodies or similar other diagnostic reagents against SARS-CoV-2 rapidly, especially during epidemic or pandemic situation.

tation and the timeline for recombinant protein production in plant system is given in Fig. 1a,b respectively. The codon-optimized RBD of SARS-CoV-2 was cloned into the geminiviral plant expression vector pBY2e (Fig. 2) and agroinfiltrated into N. benthamiana. The infiltrated leaves were harvested 3 days post-infiltration and the recombinant RBD protein was purified by Ni affinity chromatography. The expression and purification of the recombinant RBD protein were evaluated by SDS-PAGE and western blot analysis. The RBD protein was observed at the expected molecular weight of approximately 38 kDa in SDS-PAGE (Fig. 3a, Lane 2) and western blot (Fig. 3b, Lane 2 and 3). Further a faint band was detected in the western blot at about 120 kDa, which could be protein trimer (Fig. 3b). The expression level of the RBD of SARS-CoV-2 was estimated to be 8 μg per gram leaf fresh weight.
Binding of plant-produced RBD to ACE2, the receptor of SARS-CoV-2. We further tested the binding of the plant-produced RBD to ACE2, which is a SARS-CoV-2 receptor protein. Non-infiltrated wild type (WT) plant protein was used as a negative control to show that there was no cross-reactivity between ACE2 and plant proteins. The result showed that the plant-produced RBD can bind to ACE2, similar to commercial RBD protein (Fig. 4). This result indicates the authenticity and proper folding of the plant-produced RBD protein.
Expression and purification of mAb CR3022 in N. benthamiana. Codon-optimized heavy chain (HC) and light chain (LC) expression cassettes of mAb CR3022 were cloned into pBY2e (Fig. 2). The antibody was transiently expressed in N. benthamiana and purified by protein A affinity chromatography. Non-reducing SDS-PAGE confirmed the tetrameric form of the fully assembled IgG at approximately 150 kDa (Fig. 5a). The assembled antibody was also confirmed by western blot using anti-human gamma (Fig. 5b) and anti-human kappa antibodies conjugated with HRP (Fig. 5c). The expression level of mAb CR3022 was estimated to be 130 μg per gram leaf fresh weight. Purified mAb CR3022 was used for further studies.
Binding of plant-produced mAb CR3022 to RBD of SARS-CoV-2. The binding of the plant-produced mAb CR3022 to RBD protein was examined by ELISA. Human IgG, a plant-produced anti-PD1 antibody, and negative serum were used as negative controls. Convalescent serum collected from a COVID-19 patient was www.nature.com/scientificreports/ used as a positive control. The plant-produced mAb CR3022 and positive serum showed specific binding to the plant-produced RBD protein (Fig. 6a,b) and commercial CHO-produced RBD protein (Fig. 6c,d) but negative controls did not bind to the RBD protein. These results confirmed that the plant-produced mAb CR3022 can specifically bind to RBD protein of SARS-CoV-2.
Binding and neutralization activity of plant-produced mAb CR3022 against SARS-CoV-2 in vitro. An immunofluorescence assay was performed to determine whether mAb CR3022 recognize SARS-CoV-2. SARS-CoV-2 was inoculated onto Vero cells and infected cells were incubated with a known positive serum, CR3022, and a negative control antibody 28 before detection with an anti-human IgG conjugated with FITC. The results showed that the plant produced mAb CR3022 could bind to SARS-CoV-2 in infected cells, similar to the positive control serum (Fig. 7). To test the neutralizing activity of mAb CR3022, Vero cells infected  www.nature.com/scientificreports/ with SARS-CoV-2 developed cytopathic effects at 3 days post infection. Positive serum had a high viral neutralization titer against SARS-CoV-2 (Table 1). In contrast, mAb CR3022 and negative serum had no neutralizing activity against SARS-CoV-2. Overall, the plant-produced CR3022 can bind to SARS-CoV-2 but did not neutralize the virus in vitro.

Discussion
The frequent outbreaks of emerging or re-emerging infectious diseases threaten global health security, as they can have devastating health and economic impact especially in the developing world. The fear and panic over the spread of epidemic diseases can disrupt economy, travel, social activities and tourism, as well as decrease trade which in turn can affect whole societies, economies and political systems. Thus, infectious diseases create a massive burden for the global economy 29 . The recent emergence and rapid spread of the novel coronavirus SARS-CoV-2 that causes COVID-19 has attracted the attention of the whole world. Millions of infected cases have been reported, and the death toll is rising daily 30 . The receptor binding domain (RBD) located within the spike region of SARS-CoV-2 mediates virus entry into host cells by interacting with the host receptor angiotensin converting enzyme 2 (ACE2) 31 . The new virus SARS-CoV-2 is genetically related to SARS-CoV which also utilizes the ACE2 receptor on human cells for its cell attachment and entry 32 . The RBD region located in the spike glycoprotein is essential for membrane fusion and is regarded as a major target of the host antibody response. As a result, antibodies targeting the RBD region have been extensively explored as potential coronavirus therapeutic candidates and also for the development of SARS-CoV-2 diagnostics 3, 33 .
In order to meet the demand for variety of reagents required for the diagnosis and detection of COVID-19, the current study evaluated the potential of plant expression system for the production of the RBD of SARS-CoV-2 and mAb CR3022 specific to the RBD of SARS-CoV-1 and SARS-CoV-2 in order to use as a diagnostic reagent for SARS-COV-2 detection. We used a geminiviral replicon vector derived from the bean yellow dwarf virus 34,35 for the production of both RBD and mAb CR3022. Our results showed that both the RBD of SARS-CoV-2 and mAb    www.nature.com/scientificreports/ The plant-produced RBD protein showed specific binding to plant-produced mAb CR3022 and the SARS-CoV-2 receptor, ACE2, which confirmed its structural integrity. Interestingly, the plant-produced mAb CR3022 could bind to SARS-CoV-2 but failed to neutralize the virus in vitro. It could be due to sequence conservation in the epitopic region of the RBD between SARS-CoV-2 and SARS-CoV, which enables cross-reactive binding of SARS-CoV neutralizing mAb CR3022 with SARS-CoV-2. However, the epitope on the SARS-CoV-2 RBD recognized by CR3022 does not overlap with the receptor ACE2 binding site. This implies that CR3022 does not compete with ACE2 for binding to the RBD 45 . Although, mAb CR3022 alone cannot neutralize SARS-CoV-2 in vitro, the synergistic potential of mAb CR3022 along with other SARS-CoV-2 specific mAbs needs to be investigated for SARS-CoV-2 neutralization.
This study provides a spotlight for the rapid production of recombinant proteins in a plant expression system, which is highly needed during an infectious disease outbreak. Moreover, a specific neutralizing antibody, if identified against SARS-CoV-2 in near future, can be produced in plant system inexpensively in a short time which could be rapidly translated into clinical trials. Earlier studies showed that plant-made antibodies developed for West Nile virus, HIV, rabies lyssavirus, dengue virus and chikungunya virus have shown potent neutralization activity in vitro which demonstrates that plants are a suitable platform for mAb production [46][47][48][49][50] .
Moreover, earlier reports have shown that antibodies against alphavirus, cytomegalovirus and influenza virus that did not show in vitro neutralization activity were able to confer protection in in vivo studies which highlights the importance of in vivo evaluation of non-neutralizing antibodies 45,[51][52][53][54] . Additionally, this mAb could also be utilized in the development of diagnostic assays for SARS-CoV-2, and the results of this study could contribute towards the low-cost development of mAbs specific diagnostic tools for SARS-CoV-2. Altogether, our results convincingly demonstrate the practicability of using a plant expression system for the rapid production of recombinant antigens and antibodies either with diagnostic or therapeutic potential. In particular, this methodology could be suitable for use in developing economies. Furthermore, this study proved the robustness of a plant transient expression system for the production of anti-SARS-CoV mAb CR3022 with high yield which can likely improve the affordability and accessibility of mAb-based diagnosis of COVID-19 in the developing world.
In summary, we have demonstrated the rapid production of SARS-CoV-2 RBD and mAb CR3022 in N. benthamiana. The RBD and mAb CR3022 were purified from plant extracts. Intriguingly, the plant-produced RBD exhibited specific binding to the SARS-CoV-2 receptor ACE2. The plant-produced mAb CR3022 demonstrated binding to SARS-CoV-2 but was not able to neutralize SARS-CoV-2 in vitro. However, the plant-produced mAb CR3022 can be used as a diagnostic reagent for the effective diagnosis of COVID-19. The immunogenicity, Figure 7. Specific binding of plant-produced mAb CR3022 to SARS-CoV-2 in infected Vero E6 cells using immunofluorescence. The plant-produced mAb CR3022, positive serum, and a plant-produced anti-PD1 antibody (as negative control) were incubated with SARS-CoV-2-infected and non-infected Vero E6 cells and signal detected with an FITC-conjugated anti-human IgG antibody (green color). Hoechst33342 was used for counterstaining (blue color). The data are the representative images of triplicate assays. www.nature.com/scientificreports/ neutralizing potential, and protective efficacy of the plant-produced RBD can be studied as a potential vaccine candidate in the future. Our study provides a proof-of-principle of utilizing a plant transient expression system for the rapid production of other similar recombinant proteins that could be either used as detection/diagnostic reagents or as biotherapeutics to tackle COVID-19 or other infectious diseases.

Construction of expression vectors for RBD and mAb CR3022. The Institutional Review Board of
Chulalongkorn University approved the present study.
The coding nucleotide sequence of the RBD region located in spike protein of SARS-CoV-2 (SARS-CoV-2 RBD) (GenBank accession number: YP_009724390.1; F318-C617) was codon-optimized for N. benthamiana and commercially synthesized (Genewiz, Suzhou, China). The RBD was fused with an 8XHis tag at the C-terminus and cloned into a geminiviral vector (pBY2e) by using XbaI and SacI restriction enzymes to create pBY2e-SARS-CoV-2 RBD.
The coding gene fragments of the variable heavy chain (V H ) and variable light chain (V L ) regions of mAb CR3022 (GenBank accession numbers: DQ168569.1 and DQ168570.1) were codon-optimized for expression in N. benthamiana and commercially synthesized (Genewiz, Suzhou, China). The V H and V L chains were fused with human IgG1 C H and C L regions respectively. The resulting full length coding sequences of CR3022 HC and LC were cloned into a geminiviral vector (pBY2e) as described previously 10 by a three fragment ligation: the backbone from pBY2e was obtained from XbaI-SacI digestion; V H and C H were obtained by XbaI-NheI and NheI-SacI digestion, respectively while V L and C L were obtained by XbaI-AflII and AflII-SacI digestion, respectively to create the expression cassettes pBY2e-CR3022 HC and pBY2e-CR3022 LC. Extraction and purification of recombinant RBD and mAb CR3022 from plant leaves. For the RBD protein purification from the infiltrated plants, leaves were harvested at 3 days post-infiltration and extracted with extraction buffer (5 mM imidazole, 20 mM Tris-HCl pH 8.8, 50 mM NaCl) as described previously with some modifications 55 . The crude leaf extract was obtained by homogenization and clarified by centrifugation at 15,000 g for 30 min at 4 °C. The crude extract was purified by Ni-NTA affinity resin (Expedeon, Cambridge, UK). Then the purity of the recombinant protein was analyzed by SDS-PAGE and the bands were visualized by Instant Blue staining (Expedeon, Cambridge, UK) and detected by western blot probed with a rabbit anti-His antibody conjugated with HRP (ab1187; Abcam, UK). The concentration of the purified RBD protein was determined by the Bradford assay.

Transient expression of SARS-CoV-2-RBD and mAb CR3022 in
For the purification of recombinant mAb CR3022, the agroinfiltrated leaves were harvested after 3 days and proteins extracted in extraction buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 ) at pH 7.4 using a previously developed method with some modifications 28 . The crude leaf extract was obtained by homogenization and clarified by centrifugation at 15,000 g for 30 min at 4 °C. The recombinant protein in the clarified plant extract was purified by using protein A resin (Expedeon, Cambridge, UK). The recombinant purified antibody was analyzed by SDS-PAGE and the bands were visualized by Instant Blue staining. For western blot analysis of mAb CR3022, the separated proteins were transferred onto nitrocellulose membranes and detected either with anti-human kappa light chain (2060-05; Southern Biotech, USA) or anti-gamma heavy chain (AP004; The Binding site, UK) antibodies conjugated with horseradish peroxidase (HRP).
Binding of plant-produced RBD to the ACE2 receptor. The binding affinity of the plant-produced RBD to ACE2 was analyzed by ELISA. Briefly a 96-well plate (Greiner Bio-One GmbH, Germany) was coated with 2 μg/ml of commercially available ACE2 (ab273687, Abcam, UK) and incubated overnight at 4ºC. After incubation, the coating buffer was discarded and the plates were blocked with 5% skim milk in 1XPBS for 2 h at 37 °C. The plate was then washed three times with 1X PBST and incubated with the plant-produced RBD or commercial recombinant CHO-derived SARS-CoV-2 spike RBD protein (R & D Systems, USA) for 2 h at 37 °C and non-infiltrated (WT) plant protein was used as a control. Then the plates were washed three times with 1X PBST followed after which an anti 6xHis antibody (ab1187, Abcam, UK) diluted (1:1000) in 1X PBS was added to the plate which was then incubated for 1 h at 37 °C. Finally the plate was washed with 1X PBST, and the signal developed with TMB substrate (Promega, USA) and the absorbance read at 450 nm.
Binding of plant-produced mAb CR3022 to the RBD of SARS-CoV-2. ELISA was performed as described previously to examine the binding of mAb CR3022 with RBD 26 with some modifications. Briefly, 50 μl (2 μg/ml) of the plant-purified SARS-CoV-2 RBD or commercial recombinant CHO-derived SARS-CoV-2 spike RBD-His protein (10534-CV, R & D Systems, USA) was coated on 96-well microplates (Greiner Bio-One GmbH, Binding and neutralization of plant-derived mAb CR3022 against SARS-CoV-2. Neutralizing titers were determined by a microneutralization assay. A positive convalescent serum of a COVID-19 patient was approved for use as a clinical specimen by the Faculty of Medicine Ramathibodi Hospital, Mahidol University. Informed consent was waived by the Institutional Review Board that approved the present study. The mAbs or positive serum were serially diluted twofold and incubated with 100 TCID50 of the SARS-CoV-2 virus for 1 h at 37 °C. The virus and antibodies were then added to a 96-well plate with 1 × 10 4 Vero E6 cells/well in DMEM supplemented with 2% FBS, 100 U/ml of penicillin and 0.1 mg/ml of streptomycin in quadruplicate. Wells were observed for cytopathic effect (CPE) at 3 days post infection, and the 50% neutralization titer was determined as the mAb concentration at which at least 50% of wells revealed no CPE. Anti-SARS-CoV mAb binding was detected by immunofluorescence. Vero E6 cell monolayers in 96 wells were inoculated with 10 TCID 50 SARS-CoV-2 and incubated for 3 days. Uninfected and infected cells were washed three times with PBS, then incubated with ice-cold 1:1 methanol/acetone fixative for 20 min at 4 °C then washed 3 times with PBST. Blocking reagent (2% bovine serum albumin, BSA) was added to the wells, and plates were incubated for 1 h at room temperature. After washing, the mAbs or the positive serum at a dilution factor 1:40 were added and the samples were incubated at 37 °C for 1 h. The antibodies were detected using a 1:1000 dilution of an anti-human IgG antibody conjugated with FITC (Santa Cruz Biotechnology, Inc.). After incubation at 37 °C for 1 h, the plate was washed three times and the DNA staining dye, Hoechst33342 was added. The plate was then subjected to automated image acquisition and analysis using Operetta (PerkinElmer). All sera were heat inactivated at 56 °C for 30 min before use.

Data availability
The datasets used and analyzed in this study are available from the corresponding author upon request.