key: cord-0959128-wxoiby49
authors: Mamedov, Tarlan; Yuksel, Damla; Ilgın, Merve; Gürbüzaslan, İrem; Gulec, Burcu; Mammadova, Gulshan; Say, Deniz; Hasanova, Gulnara
title: Engineering, production and characterization of Spike and Nucleocapsid structural proteins of SARS–CoV-2 in Nicotiana benthamiana as vaccine candidates against COVID-19
date: 2020-12-30
journal: bioRxiv
DOI: 10.1101/2020.12.29.424779
sha: e808373fc7f7eac248c90763316d5164c2fe3dd0
doc_id: 959128
cord_uid: wxoiby49

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.

The novel coronavirus, currently designated as SARS-CoV-2, is a novel and highly pathogenic coronavirus, and spread more than to 222 countries and territories in a short time, and as of December 25, 2020, more than 79,931,215 cases were recorded, and more than 1,765,265 confirmed deaths. Although a number of various types vaccines (mRNA, DNA, and viral vectorbased, protein based subunits, inactivated, attenuated etc.) for COVID-19 is underway and there are currently no drugs, no specific treatment and no vaccines available to treat infections and protect people against fatal SARS-CoV-2 coronavirus. The world urgently needs a safe and effective SARS-CoV-2 coronavirus vaccine, antiviral and therapeutic drugs, cost effective diagnostic reagents and kits to control the COVID-19 pandemic and relieve the human suffering associated with the pandemic that kills thousands of people every day. One of the major observed features of this virus is that SARS-CoV-2 is transmitted from infected people without symptoms, therefore, it increases the challenges of controlling a deadly pandemic without the use of a vaccine. One-third of the virus genome (~30 kb) of SARS-CoV encode mainly structural proteins such as spike (S) glycoprotein, nucleocapsid protein (N), small envelop protein (E) and matrix protein (M). When reported on January 2020, in Spike protein, SARS-CoV-2 had about 79.6 % sequence identity with that of SARS-CoV 1,2 . Now the sequence identity is less than 76%, as the spike protein of the new coronavirus SARS-CoV-2 has acquired more than 725 mutations. S protein of coronavirus consist of S1 and S2 domains and plays a key role in virus binding, fusion and entry to host cells. S protein has been demonstrated as a leading target for vaccine and neutralizing antibody development. In fact, most of vaccines, such as mRNA, DNA, viral vector-based, subunits, protein based vaccines against have been developed based on the gene encoding S protein [3] [4] [5] [6] [7] . S1 domain contains a receptorbindingdomain (RBD), which binds specifically to angiotensin-converting enzyme (AEC2), areceptorfor both SARS-CoV and SARS-CoV-2.The S protein of SARS-CoV-2 coronavirus is cycteine-rich protein and nine cysteine residues are found in the RBD, eight of which involve in forming four pairs of disulfide bridges 8 . In addition, the S protein has 22 potential Nglycosylation sites, of which two of them are in the RBD 9 . Thus, correct formation of disulfide bridges and status of glycosylation would be essential for functional activity of recombinant S protein-based vaccines, produced using different expression system. The structure of SARS-CoV-2 spike RBD was recently determined by crystal structure analysis at 2.45 Å resolution 1 . It was demonstrated that the overall ACE2-binding mode of the SARS-CoV-2 RBD is nearly identical to that of the SARS-CoV RBD, which also utilizes ACE2 as the cell receptor. It should be noted that since the RBD is the critical region for receptor binding, therefore RBD based vaccines could be great promise for developing vaccines and also highly potent cross-reactive therapeutic agents. It has been recently demonstrated that RBD-Fc-based COVID-19 vaccine candidate elicited high titer of RBD-specific antibodies with neutralizing activity against SARS-CoV-2 infections 3 . As mentioned above, N protein has been shown to be more conserved compared with S protein and share 90% amino acid homology with SARC-CoV and stable with fewer observed mutations [10] [11] [12] [13] . Since the N-protein was found to be more conservative and showed strong immunogenicity, the N-protein could be a strong potential candidate for a COVID-19 vaccine 14 .

Numerous studies in recent years have demonstrated plant expression systems as promising expression platforms for the cost-effective, rapid and safe production of various recombinant proteins. Plant expression systems have several advantages over other expression systems currently in use. This system has been successfully used for rapid and cost-effective production of a variety of recombinant proteins, vaccine candidates, therapeutic proteins, enzymes, antibodies etc. [15] [16] [17] [18] [19] [20] [21] [22] . Recently, a number of flexible approaches have been developed, which enabled the successfully production of pharmaceutically important complex proteins including human proteins and enzymes in plants 15, [19] [20] [21] . Thus, plant expression system could be ideal platform for cost effective, safe and rapid production of structural proteins of SARS-CoV-2 as a vaccine candidates against COVID-19. In previous efforts, a number of studies have been conducted on the transgenic (stable) expression of the spike protein of SARS-CoV in plants. Nterminal fragment of SARS-CoV S protein (S1) was produced in tomato and low-nicotine tobacco plants and demonstrated immunogenicity in mice after parenteral or oral administration 23 . Several groups have reported development of S protein recombinant plantbased vaccines against different coronoviruses for oral delivery that elicit protective immunity against virus challenge [24] [25] [26] . Recombinant SARS-CoV spike protein was also expressed in plant cytosol and chloroplasts 27 . However, transgenic plant approach has some concerns, which are mainly associated with the long development time. Additionally low target accumulation levels and the possibility of gene flow from transgenic plants to wild types are of concern 28 . Plant benthamina plant was very low, 8 μg/g, which is unsatisfactory to be economical for commercialization. In this study, we report the high production of functionally active structural proteins of SARC-CoV-2 such as RBD (glycosylated and deglycosylated variants), and glycosylated variant of S1 domain, and N proteins in the N. benthamiana plant using a transient expression platform. Notably, neither the S1 domain of SARS-CoV nor the SARS-CoV-2 was produced using a transient plant expression system elsewhere.

The purification yields were at least 20 mg pure protein/kg plant biomass for each target protein.

On SDS-PAGE and western blotting, plant produced N protein molecule appears as a doublet protein with a MM of 48 and 24 kDa (Figure 1 ). Both molecular forms of plant produced N proteins were recognized by anti-FLAG antibody ( Figure 1B ) and by N protein specific mAb ( Figure 1C ), demonstrating specific reactivity of mAb to the two forms and also confirm that epitope for mAb still present in ~24 kDa fragment. N protein of SARS-CoV-2 has 5 potential Nglycosylation sites. When N protein was co-expressed with Endo H, there was no protein band shift in the western blotting suggesting that plant produced N protein is not N-glycosylated. To understand the role of glycosylation, we produced both glycosylated and non-glycosylated variants of RBD and S1 protein in N. benthamiana plant. proteins were purified using anti-Flag column. SDS-PAGE analysis of the cocktail protein purified from N. benthamiana plant is presented in Figure 3 .Deglycosylated variants were produced using the in vivo Endo H deglycosylation strategy, which we recently developed 19 .

Deglycosylated and glycosylated variants of plant produced S1 proteins were purified using HisPur™ Ni-NTA resin. On SDS-PAGE and western blotting, plant produced glycosylated S1

(gS1) protein appears as a single protein with a MM of ~100 kDa ( Figure 4A ). No clear visible band of Ni-NTA resin purified, Endo H deglycosylated S1 protein (dS1) was observed in SDS-PAGE gels, suggesting degradation of deglycosylated S1 protein. However, on Western blotting, deglycosylated S1 protein with a band of ~ 80 kDa was observed. Plant produced, purified glycosylated and deglycoslated S1 variants were recognized by anti-His mAb tag ( Figure 4B ) or anti-S protein specific pAb ( Figure 4C ).

Binding of plant-produced gRBD, dRBD and gS1 proteins to ACE2 were performed as described in Methods. As can be seen from Figure 5 , the plant-produced RBD variants and S1 protein demonstrated specific binding to AEC2, the SARS-CoV-2 receptor. Notable, Endo deglycosylated RBD exhibited stronger binding to AEC2 compared with glycosylated counterpart.

The stability analysis of plant produced glycosylated and Endo H in vivo deglycosylated forms of RBD and gS1 of SARC-CoV were examined after incubation at 37 °C for 48, 96, 72 and 96 hours. The procedure of stability analysis was similar to those described previously 21 .These analyses showed that dRBD degradation was very low at incubation at 37 ° C for 24 hours (less than 10 %), however, at the same condition, gRBD degraded significantly (more than 60%).

Plant produced gS1 protein showed better stability compared to gRBD or dRBD and degraded about 50% after 48 hours. Since the yield of Endo H dS1 was very low, we were not able to compare the stability of gS1 with dS1 counterpart.

Mice received two doses from each variant (N, gRBD, dRBD, [N+ RBD (cocktail)] or gS1 proteins) adsorbed to 0.3% Alhydrogel at three-week intervals (0, 21 days). Serum samples were collected on days -1 (pre-bleed) and 42 (post 2nd vaccination) and assessed for anti-N, anti-gRBD, anti-dRBD anti-(N+gRBD) and anti-gS1 antibody responses by an IgG ELISA. IgG responses showed that plant produced N and S protein variants were able to induce significantly higher titers of antibody with alum adjuvant at the 5 μg dose ( Figure 6 ).

SARS-CoV-2 is a novel and highly pathogenic coronavirus, which has caused an outbreak in vaccine development since N protein is more conserved and stable, in fact few mutations was observed over time 2, 10, [12] [13] [14] [40] [41] [42] . It has also been recently shown that the crystal structure of the SARS-CoV-2 nucleocapsid protein is very similar to coronavirus N proteins, which were previously described 43 with high solubility and high yield. Flexible approaches are required for successful production of functional active recombinant proteins in plants with high yield. Thus, plant expression system could be ideal platform for rapid, cost effective, safe and high level production of structural proteins of SARC-COV-2. In this study, we demonstrate the production of functionally active structural proteins SARC-CoV-2, such as RBD variants, S1 domain and nucleocapsid proteins, at a high level and in a highly soluble form in the N. benthamiana plant using a transient expression platform. We purified these protein using anti-Flag and Ni-NTA resin chromatography. The purification yields were at least 20 mg/ kg plant leaf. In this study, we constructed two plasmids, expressing FLAG tagged and His tagged variants of RBD. We found that the expression level and purification yield of RBD-His tagged was low (less than 10 mg/kg leaf biomass), however, the expression level and purification yield of plant produced RBD-FLAG tagged protein was significantly higher, more than 20 mg/kg leaf biomass. The purified plant produced N and S proteins were recognized by N and S protein specific monoclonal antibodies demonstrating specific reactivity of mAb to plant produced N and S protein variants. The plant-produced RBD and S1 protein showed specific binding to AEC2, SARS-CoV-2 receptor. Notable, plant produced Endo H deglycosylated RBD exhibited stronger binding to AEC2 compared with glycosylated counterpart, suggesting native-like folding of dRBD, and negative effect of glycosylation on masking of important epitopes of the RBD.

IgG responses showed that plant produced N, S1 antigens were able to induce significantly higher titers of antibodies with Alhydrogel adjuvant. In this study, we are also reporting for the first time the co-expression of gRBD with protein N to produce a cocktail of SARS-CoV-2 antigens, which elicited high-titer antibodies compared to RBD or N proteins. Thus, N, S1 and benthamianaplants and de-novo synthesized at Biomatik Corp. To transiently express N, S protein (RBD, S1 domain and N+RBD) variants in N. benthamiana plants, the Nicotiana tabacum PR-1a signal peptide (MGFVLFSQLPSFLLVSTLLLFLVISHSCRA) was added to the N-terminus of N, RBD and S1 proteins. In addition, the KDEL sequence (the ER retention signal) and the FLAG epitope (the affinity purification tag for N and RBD proteins) or His6 tag (for S1 domain) were added to the C-terminus. The resulting sequences were inserted into the pEAQ 46 binary expression vectors to obtain pEAQ-N, pEAQ-RBD and pEAQ-S1. These plasmids were then transferred into Agrobacterium AGL1 strain. To express N, RBD, S1 and N+RBD variants in N. benthamiana plant, AGL1 harboring pEAQ-N, pEAQ-RBD and pEAQ-S1 plasmids were infiltrated into N. benthamiana plant leaves. Plants were harvested at 4 dpi (day afterpost infiltration).

SDS-PAGE analysis of plant produced N, RBD and S1 variants were performed on 10% acrylamide gels stained with Coomassie (Gel Code Blue, Pierce Rockford, IL). Western blot analysis was performed after electrophoresis and transfer of the proteins to Polyvinylidene 

To produce deglycosylated variants of N, RBD and S1 domain, AGL1 strain harboring pEAQ-N, pEAQ-RBD and pEAQ-S1 were co-infiltrated with pGeen-Endo H construct 19 .

Purification of plant produced N and RBD (glycosylated and deglycosylated) variants were performed by anti-FLAG affinity chromatography using anti-DYKDDDDK affinity gel (cat. no. 651503, BioLegend) as described previously 21 

Plant produced S1 domain variants (glycoslated and deglycosylated variants) were purified using HisPur™ Ni-NTA Resin (ThermoFisher scientific, Cat. No. 8822) as described previously 19 .

Briefly, 25 grams of frozen plant leaves, infiltrated with the pEAQ-S1-His6-KDEL construct, were ground using mortar and pestle in a phosphate extraction buffer (20 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole, pH 7.4). The plant cell extract was filtered through Miracloth and centrifuged at 20,000 g for 25 minutes and then filtered through a 0.45 μm syringe filter (Millipore). The clarified total protein extract was then purified using HisPur Ni-NTA resin (Cat. No. 8822, Thermo Fisher Scientific) by following a previously used protocol 19 .

Briefly a 96-well plate (Greiner Bio-One GmbH, Germany) was coated with 100 ng of commercially available ACE2 (MBS2563876, MyBiosource, USA) using 100 mM carbonate buffer and incubated overnight at 4ºC. After incubation, the plates were blocked with blocking buffer for 2 h at room temperature. After blocking, various concentrations (50-1000 ng) of plant 

The stability analysis of plant produced gRBD, dRBD and gS1 of SARS-CoV-2 recombinant proteins were examined after incubation at 37 °C for 48, 96, 72 and 96 hours using similar procedure as described previously 21 . Protein samples were diluted to 0.5 mg/ mL with PBS and were transferred into polypropylene Eppendorf low-binding tubes. After incubation at at 37 °C for 48, 96, 72 and 96 hours, samples were analyzed by SDS-PAGE and western blotting. The degradation of gRBD, dRBD or gS1 protein bands were calculated and quantified based on SDS-PAGE and WB analysis using highly sensitive Gene Tools software (Syngene Bioimaging, UK) and ImageJ software as described previously 21 .

Mice received two doses of N, RBD, N+RBD and S1 domain proteins adsorbed to 0.3% Alhydrogel at three-week intervals (0, 21 days). Groups of seven-week-old mice (6 animals/group) were immunized IM with 5 μg of N, RBD, N+RBD and S1 domainvariants at 0 and 21 days. IgG titers were determined by ELISA in sera collected on 21st or 42nd days post vaccination from mice immunized with N, S1 or N+ S1 (cocktail) variants.Serum samples were GraphPad Prism software was used for all statistical analyses. Unpaired two-tailed t-test was used to compare antibody response of N, gRBD, gS1 and N+gRBD induced serums and PBS induced serums as control. Significant was accepted as P < 0.05 and P values were showed as *P < 0.05; **P < 0.01; ***P < 0.001. Each point on the graph were derived from three replica for each dilution. Spike RBD Polyclonal Antibody (B). The image was taken using the highly sensitive GeneGnome XRQ Chemiluminescence imaging system. 

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The authors are grateful to Dr. George P. Lomonossoff (John Innes Centre, Biological Chemistry Department) and Plant Bioscience Limited for kindly providing pEAQ binary expression vector.This study was funded by the Akdeniz University to Tarlan Mamedov.The authors are grateful to Dr. George P. Lomonossoff (John Innes Centre, Biological Chemistry Department) and Plant Bioscience Limited for kindly providing pEAQ binary expression vector. We thank Dr. Munevver Aksoy at Akdeniz University for editorial assistance.