Enhanced binding and inhibition of SARS‐CoV‐2 by a plant‐derived ACE2 protein containing a fused mu tailpiece

Infectious diseases such as Coronavirus disease 2019 (COVID‐19) and Middle East respiratory syndrome (MERS) present an increasingly persistent crisis in many parts of the world. COVID‐19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). The angiotensin‐converting enzyme 2 (ACE2) is a crucial cellular receptor for SARS‐CoV‐2 infection. Inhibition of the interaction between SARS‐CoV‐2 and ACE2 has been proposed as a target for the prevention and treatment of COVID‐19. We produced four recombinant plant‐derived ACE2 isoforms with or without the mu tailpiece (μ‐tp) of immunoglobulin M (IgM) and the KDEL endoplasmic reticulum retention motif in a plant expression system. The plant‐derived ACE2 isoforms bound whole SARS‐CoV‐2 virus and the isolated receptor binding domains of SARS‐CoV‐2 Alpha, Beta, Gamma, Delta, and Omicron variants. Fusion of μ‐tp and KDEL to the ACE2 protein (ACE2 μK) had enhanced binding activity with SARS‐CoV‐2 in comparison with unmodified ACE2 protein derived from CHO cells. Furthermore, the plant‐derived ACE2 μK protein exhibited no cytotoxic effects on Vero E6 cells and effectively inhibited SARS‐CoV‐2 infection. The efficient and rapid scalability of plant‐derived ACE2 μK protein offers potential for the development of preventive and therapeutic agents in the early response to future viral outbreaks.


INTRODUCTION
Coronavirus disease 2019 (COVID- 19) is a deadly infectious disease that was first discovered in December 2019 in Wuhan, China. [1]The World Health Organization declared COVID-19 a pandemic on March 11, 2020. [2]As of September 21, 2023, the global COVID-19 outbreak caused over 770 million confirmed cases and over 6.9 million deaths. [3]e COVID-19 global health crisis is currently the focus of research by an international scientific collaboration.COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a positive-sense single-stranded RNA virus that belongs to the family Coronaviridae and the genus Betacoronavirus. [4,5]RS-CoV-2 is transmitted through human-to-human contact and can spread from respiratory fluid particles when infected individuals sneeze, cough, speak, or breathe. [6,7]SARS-CoV-2 has four structural proteins: spike glycoprotein (S), envelope protein (E), membrane glycoprotein (M), and nucleocapsid phosphoprotein (N). [8,9]The S protein, especially the receptor binding domain (RBD) of the S1 subunit, plays a crucial role in enabling the virus to enter host cells by interacting with the human cell-surface receptor angiotensin-converting enzyme 2 (ACE2). [8][12] The primary function of ACE2 is to reduce blood pressure and inflammation as part of the renin-angiotensin system by converting angiotensin I into angiotensin (1-9) and angiotensin II into angiotensin (1-7). [10,13,14]secondary function of ACE2 is to provide a protective pathway for organs, including the lungs and heart.[15] In addition to its physiological roles, ACE2 is the receptor for the S protein of severe acute respiratory syndrome-associated coronavirus (SARS-CoV), SARS-CoV-2, and human coronavirus NL63 (HCoV-NL63).[16] For this reason, the interaction between ACE2 and the S protein of SARS-CoV-2 has been a target for therapeutic intervention.Although many studies have been conducted to explore the use of ACE2 protein to inhibit SARS-CoV-2 infection, there have been few attempts to increase the binding activity of ACE2 by imposing structural changes.
Like other RNA viruses, SARS-CoV-2 has produced multiple variants.So far, five variants of concern (VOCs) have been identified: Alpha (B.1.1.7;documented in the United Kingdom), Beta (B.1.351;documented in South Africa), Gamma (P.1; documented in Brazil), Delta (B.1.617.2;documented in India), and Omicron (B.1.1.529;documented in multiple countries). [17]Most changes that occur in viruses have little to no impact on the biological prop-erties of the virus, but some can have important effects on viral transmission, disease severity, applicability of diagnostic tools or therapeutic medicines, performance of vaccines, and other public health factors. [18]Compared with the original SARS-CoV-2, most of the emergent VOCs have accumulated mutations in the S protein, resulting in enhanced virulence, transmissibility, and/or resistance to neutralization by antibodies.As of June 24, 2023, Omicron is the dominant variant of SARS-CoV-2, and Omicron subvariants are currently emerging. [18,19]ere has been renewed attention in recent years to the potential use of plant-derived biopharmaceuticals for the treatment of human disease.During the 2014-2016 Ebola outbreak, the plant-derived biopharmaceutical ZMapp™ was used to treat human patients. [20] randomized phase 3 trials, a plant-derived quadrivalent virus-like particle of the influenza vaccine developed by Medicago Inc. (Quebec, Canada) showed immunogenicity, safety, and efficacy in humans.[21,22] Plant expression systems have many advantages, including inexpensive growth conditions (sunlight, water, and nutrients), scalability, no potential human or animal pathogenic contaminants, rapid production, and easy harvesting.[23][24][25][26][27] Furthermore, plant cells produce post-translational modifications similar to those in mammalian cells, including N-glycosylation, which is required for biological activity, although there are differences in composition.[23,25,27,28] Importantly, transient plant expression systems also offer speed and flexibility.[29] The mu tailpiece of IgM (μ-tp) is an 18 amino acid C-terminal extension of the IgM heavy chain that contains a critical penultimate cysteine that facilitates the disulfide bonds required for oligomerization.[30][31][32][33] To rapidly produce more effective and inexpensive preventive and therapeutic agents against SARS-CoV-2, we fused μ-tp to ACE2 protein iso- proteins tested.To the best of our knowledge, this is the first recombinant protein to have a fused μ-tp without the Fc region of the antibody.
Our results provide the important insight that the fusion of μ-tp to ACE2 enhances the activity of the receptor protein to bind and inhibit viruses.

Construction of vectors for plant transient expression of ACE2
To construct the ACE2 isoforms, an ACE2 sequence (GenBank accession no.NM_00137415.1)was used in which the original N-terminal signal peptide, and the C-terminal transmembrane domain were removed.The ACE2 N isoform was created by an N-terminal addition of the 29 amino acid plant ER signal peptide (MATQRRANPSSLH-LITVFSLLAAVVSAEV), as described by Lim et al. [25] To form oligomeric proteins, the ACE2 μ isoform (with μ-tp) was created by fusing the μ-tp of IgM (PTLYNVSLVMSDTAGTCY) at the C-terminal end of the ACE2 sequence in ACE2 N. Similarly, the ACE2 K isoform (with KDEL) was created by attaching a KDEL motif at the C-terminal end of the ACE2 sequence in ACE2 N. Finally, the ACE2 μK isoform (with μ-tp & KDEL) was created by attaching a KDEL motif at the C-terminal end of the μ-tp sequence in ACE2 μ.To enable purification of the ACE2 isoforms after their expression in plants, an HA tag and a 6XHis tag were attached to the N-terminus and C-terminus, respectively.All four ACE2 constructs were subcloned under control of an enhanced 35s promoter (P35Sx2Enh) and the tobacco mosaic virus 5′ untranslated region (TMV) into the geminiviral vector pBYR-2fp (Figure 1). [34]

Transient expression of ACE2 proteins in N. benthamiana leaves by agroinfiltration
The plant transient expression vector was transferred into Agrobacterium tumefaciens EHA105 by the freeze-thaw method. [35]combinant A. tumefaciens was then inoculated into Luria-Bertani medium containing 50 µg ml -1 each of kanamycin and rifampicin and incubated 48 h at 28 • C with shaking.Wild-type N. benthamiana plants were germinated from seed and grown under controlled conditions in soil pots with a 16-h light/8-h dark cycle at 25 • C and 40% relative humidity in growth cabinets.For transient expression of the ACE2 proteins, recombinant A. tumefaciens cells were pelleted and resuspended in 1X infiltration buffer (10 mM 2-[N-morpholino] ethanesulfonic acid, pH 5.6; 10 mM MgCl 2 ; 100 mM acetosyringone) to an OD 600 of 0.4. [36]The suspension was then infiltrated into the adaxial side of 6-7-week-old N. benthamiana leaves by syringe or vacuum infiltration.The infiltrated leaves were harvested at 4 days post infiltration (dpi).

Protein purification and SDS-PAGE analysis
For purification of the plant-derived ACE2 proteins, infiltrated N. benthamiana leaves were harvested at 4 dpi and purified using a modified protocol of Gupta and Kim. [37]Three hundred grams of harvested leaves were homogenized in 900 mL pre-chilled 1X extraction buffer (0.

ELISA analysis with inactivated SARS-CoV-2
The wells of 96-well MaxiSorp Nunc-Immuno plates (Sigma-Aldrich, Burlington, MA) were coated with 5×10 3 or 5×10 5 plaque-forming units (PFU) ml -1 per well of inactivated beta strain of SARS-CoV-2 diluted in 0.05 M carbonate-bicarbonate buffer, pH 9.6 (Sigma-Aldrich, Burlington, MA) and incubated overnight at 4 • C. The plates were then washed four times with 200 µL 1X PBS-T and blocked with 5% skim milk for 2 h at RT.After the wash step, 1 µg plant-derived ACE2 μK or ACE2 K protein in 5% skim milk was added as a receptor, and the plates were incubated for 2 h at 37 • C.After washing with 1X PBS-T, the plates were treated with anti-ACE2 antibody (AC18Z, dilution 1:500; Santa Cruz Biotechnology, Dallas, TX) for 2 h at 37

Size exclusion high-performance liquid chromatography (SEC-HPLC) analysis
SEC-HPLC was performed to determine the oligomeric state of plantderived ACE2 μK using the Agilent 1290 Infinity LC system (Agilent, Santa Clara, CA).The analysis was performed on a Bio SEC-5 size exclusion column (7.8 mm × 300 mm, Agilent, Santa Clara, CA) in 1X PBS, pH 7.4.The injection volume was 5 µL with a flow rate of 0.5 ml min -1 .
Detection was performed at 280 nm.AdvanceBio SEC 300 Å Protein Standard was used as a molecular weight standard.

Immunoblot analysis with plant extract and purified proteins
For confirmation of plant-derived protein expression, fresh leaf samples (100 mg) from each agroinfiltrated plant were homogenized in 1X PBS.For normalization of purified protein samples, 1 µg purified samples were prepared.Equal amounts of homogenized leaf sample or purified protein were mixed with 5X loading buffer (1% bromophenol blue, 10% SDS, 50% glycerol, 5% 2-mercaptoethanol, 1 M Tris-HCl) and heated at 100 • C for 5 min.Twenty microliters of each sample were then separated on a 10% SDS-PAGE gel and transferred to a nitrocellulose transfer membrane (Pall Corporation, Port Washington, NY).The membrane was blocked with blocking buffer [5% Blotting-Grade Blocker (Bio-Rad, Hercules, CA) in 1X TBS plus 0.05% v/v Tween 20] and then incubated with anti-ACE2 antibody (AC18Z, dilution 1:500; Santa Cruz Biotechnology, Dallas, TX) in blocking buffer for 2 h at RT.Then, the membrane was incubated with goat anti-mouse IgG H&L, HRP conjugate (dilution 1:10000; Abcam, Cambridge, UK) in blocking buffer for 2 h at RT to detect the primary antibody.The recombinant protein bands were detected using a Clarity Western ECL Substrate (Bio-Rad, Hercules, CA).CHO cell-derived ACE2-Fc and buffer-infiltrated plant leaf extract were used as positive and negative controls, respectively.

Immunoblot analysis with inactivated SARS-CoV-2
To confirm the binding activity of plant-derived ACE2 μK and ACE2 K proteins with inactivated beta strain of SARS-CoV-2, 1.25×10 5 to 5×10 5 PFU ml -1 inactivated virus per well was diluted in 1X PBS.The samples were mixed with 5X loading buffer and heated at 100 • C for 5 min.Then, twenty microliters virus sample was separated on a 7.5% SDS-PAGE gel and transferred to a nitrocellulose transfer membrane (Pall Corporation, Port Washington, NY).The membrane was blocked with blocking buffer [5% Blotting-Grade Blocker (Bio-Rad, Hercules, CA) in 1X TBS plus 0.05% v/v Tween 20 (1X TBS-T)] and then incubated with purified ACE2 μK or ACE2 K protein in blocking buffer overnight at 4 • C.After washing with 1X TBS-T, the membrane was incubated with anti-ACE2 antibody (AC18Z, dilution 1:500; Santa Cruz Biotechnology, Dallas, TX) for 2 h at RT.Then, the membrane was incubated with goat anti-mouse IgG H&L, HRP conjugate (dilution 1:10000; Abcam, Cambridge, UK) in blocking buffer for 2 h at RT to detect the ACE2 antibody.The protein bands were detected using a Clarity Western ECL Substrate (Bio-Rad, Hercules, CA).The negative control was 1X PBS.

Cytotoxicity assay
Monolayered Vero E6 cells were grown on 96-well plates at 5×10 4 cells/well and incubated at 37

Simultaneous-treatment assay
In

Transient expression of ACE2 isoforms in N. benthamiana
Four different ACE2 protein constructs were designed as shown in Figure 1A and cloned into plant transient expression vectors.The ACE2 μ and ACE2 μK isoforms had the μ-tp of IgM fused to the Cterminal end of ACE2 N to enable the proteins to adopt an oligomeric form.In addition, in the ACE2 μK and ACE2 K isoforms, the glycan structures were modified by adding a KDEL peptide at the C-terminal end.To produce plant-derived ACE2 proteins, A. tumefaciens cells containing pBYR-2fp geminiviral vectors with the ACE2 constructs were introduced into N. benthamiana plants by agroinfiltration (Figure 1B). [34,38]The expression of each ACE2 isoform in leaf tissues of the agroinfiltrated plants at 4 dpi was analyzed by immunoblot analysis with an anti-ACE2 antibody (Figure 1C).ACE2 μ and ACE2 μK each showed an approximately 76 kDa protein band, whereas ACE2 N and ACE K each showed a 74 kDa protein band.All four ACE2 isoforms were well expressed in N. benthamiana plants, although ACE2 μK and ACE K showed slightly higher expression than ACE2 μ and ACE2 N.

Purification of recombinant ACE2 proteins from N. benthamiana leaves
To produce large quantities of the plant-derived ACE2 proteins, vacuum infiltration was performed with recombinant A. tumefaciens cells containing each construct.The ACE2 proteins were then purified from freshly harvested leaf extracts (4 dpi) using one-step Ni-NTA affinity chromatography.Each purified protein was confirmed by SDS-PAGE with Coomassie blue staining (Figure 2).The expected bands of ACE2 protein were detected at 74-76 kDa in the eluted fractions.Each ACE2 isoform was purified at the highest concentration in fraction 2 (F2).The size, identity, and normalization of the plant-derived ACE2 proteins were confirmed by SDS-PAGE and immunoblot analysis with specific anti-ACE2 antibodies under reducing conditions (Figure 3A).
The immunoblot results indicated that all the isoforms were well purified, normalized, and properly folded, matching the results of SDS-PAGE with Coomassie blue staining.

Binding of plant-derived ACE2 to the RBD of SARS-CoV-2
The RBD of SARS-CoV-2 is the key structure that binds the hACE2 receptor to enable the virus to enter into host cells. [39,40]To determine if the plant-derived ACE2 proteins were properly folded and capable of interaction with the RBD of SARS-CoV-2, the binding activity of the four ACE2 isoforms with the wild-type SARS-CoV-2 RBD was confirmed by ELISA analysis.The purified ACE2 proteins, CHO cell-derived human ACE2 (CHO-hACE2; as a positive control), and the hdpp4 receptor of MERS-CoV (hdpp4; as a negative control) were immobilized on nickel-coated 96-well plates.Four different dilutions of HRP-conjugated wild-type SARS-CoV-2 RBD were applied to the ELISA plate shown in Figure 3B.The plant-derived ACE2 μ and ACE2 μK isoforms displayed better binding than CHO-hACE2 (Figure 3C).
ACE2 μK had the highest rate of binding among the four plant-derived ACE2 isoforms.Because ACE2 μK had better binding activity than CHO-hACE2, and the binding activity of ACE2 μK containing μ-tp was higher than that of ACE2 K without μ-tp, ACE2 μK and ACE2 K were used for further experiments.

SEC-HPLC analysis of plant-derived ACE2 µK and ACE2 K
SDS-PAGE and SEC-HPLC were performed to identify the size distributions of ACE2 μK and ACE2 K (Figure 4).In SDS-PAGE analysis under non-reducing and reducing conditions, ACE2 μK and ACE2 K produced major protein bands according to the predicted molecular weights of their monomers at 74-76 kDa (Figure 4A).SEC-HPLC was performed with AdvanceBio SEC 300 Å Protein Standard as a size marker (Figure 4B).ACE2 μK eluted as a major peak (80%) approximately corresponding to a monomer molecular weight of 77.7 kDa (8.322 min), with a small peak (20%) corresponding to a trimer molecular weight of 249.7 kDa (7.347 min).ACE2 K showed a major peak corresponding to a monomer molecular weight of 69 kDa (8.421 min).
The molecular weight of each protein was estimated based on the size marker.For the scope of this paper, all experiments were conducted using this mixed form of ACE2 μK.

Binding of ACE2 µK and ACE2 K to SARS-CoV-2 RBD variants
ELISA analysis was performed to investigate differences in the binding activities of purified ACE2 μK and ACE2 K for different SARS-CoV-2 RBD variants (Figure 5A).The plant-derived ACE2 μK and ACE2 K proteins were immobilized on nickel-coated 96-well plates.Four different concentrations of each SARS-CoV-2 RBD variant (Wild-type, Alpha, Beta, Gamma, Delta, and Omicron) were then applied onto the ELISA plate.ACE2 μK showed higher binding activity than ACE2 K or CHO-hACE2 for each of the RBD variants (Figure 5B-G).The relative rates of binding activity between the plant-derived ACE2 proteins and the SARS-CoV-2 RBD variants were as follows: Omicron < Wildtype < Delta < Beta < Gamma < Alpha.Although there was a difference in the degree of binding affinity between ACE2 μK and ACE2 K, both plant-derived ACE2 proteins were confirmed to bind with all of the investigated SARS-CoV-2 RBD variants.

Binding of ACE2 µK and ACE2 K to inactivated SARS-CoV-2 virus
Immunoblot and ELISA analyses were performed to confirm the binding activity of plant-derived ACE2 μK and ACE2 K with whole inactivated SARS-CoV-2 (Figure 6A).Different concentrations of whole inactivated beta strain of SARS-CoV-2 were immobilized on a membrane or 96-well plates, and then the ACE2 isoforms were applied to the membrane or ELISA plate (Figure 6B, 6C).
In these assays, ACE2 μK showed higher binding activity than ACE2 K.

Inhibition of SARS-CoV-2 infection by plant-derived ACE2 µK protein
To determine if plant-derived ACE2 μK or ACE2 K are toxic to cells, cell viability was measured by MTT assay using Vero E6 cells (Figure 7A, 7B).Vero E6 cells were treated for 72 h with purified ACE2 μK or ACE2 K at concentrations ranging from 0 µg ml -1 to 1000 µg ml -1 .MTT assays showed that the viability of the Vero E6 cells was not affected by either plant-derived ACE2 isoform.Because ACE2 is the key receptor for SARS-CoV-2 infection, we tested whether plantderived ACE2 μK and ACE2 K had inhibitory effects on SARS-CoV-2 infection in Vero E6 cells.SARS-CoV-2 was incubated at 4 • C for 1 h with purified ACE2 μK or ACE2 K before inoculation onto Vero E6 cells followed by further incubation at 37 • C for 1 h.The cells were then washed and supplemented with fresh medium to remove any unbound virus.After incubation for another 72 h, the inhibition rate of the plantderived ACE2 isoforms was determined (Figure 7C).ACE2 μK inhibited SARS-CoV-2 infection in a concentration-dependent manner, whereas

DISCUSSION
Currently approved monoclonal antibodies and vaccines for COVID-19 act by blocking the interaction between the viral S protein and the hACE2 receptor. [41,42][48][49][50][51] Therefore, inspired by the inherent oligomeric structures of IgM antibodies, which form either pentamers or hexamers, we attempted to engineer oligomeric forms of ACE2 proteins by fusing them with the μ-tp of IgM.[32][33] We hypothesized and investigated that this fusion would enhance the avidity of the engineered ACE2 proteins for the S protein of SARS-CoV-2, thereby creating more effective viral traps.
Since human growth hormone was first produced in transgenic sunflower callus tissue and tobacco in 1986, plant expression systems have steadily attracted attention as an effective way to produce recombinant proteins.][54][55] Furthermore, the transient plant expression system provides the potential to rapidly produce large amounts of proteins, for instance, in response to early in the pandemic. [27]To further explore the use of these systems as fast, scalable, and cost-effective platforms to enhance the effectiveness of preventive and therapeutic agents against SARS-CoV-2, we produced four different isoforms of hACE2 using transient expression in [58][59] Therefore, the enhanced expression of ACE2 μK and ACE2 K might be due to the effect of the C-terminal KDEL motif.
However, the KDEL motif appeared to have a negligible effect on the binding activity of the protein.This observation can be confirmed in material. [56,57,59]Additionally, because ACE2 μK exhibited higher bind-ing activity than CHO-hACE2 and the inclusion of the μ-tp further enhanced this activity compared to ACE2 K without μ-tp, we opted to employ ACE2 μK and ACE2 K with fused KDEL motifs in subsequent experiments.To investigate the size distribution of ACE2 μK mediated by μ-tp fusion, we conducted SDS-PAGE under both non-reducing and reducing conditions, as well as SEC-HPLC.According to our analyses, ACE2 μK predominantly exists as a mixture, comprising approximately 20% trimers and 80% monomers.[62] All subsequent experiments in this study were carried out using this mixed form of ACE2 μK without any further fractionation.ACE2 μK and ACE2 K were confirmed to bind with all five investigated SARS-CoV-2 RBD variants, although there were differences in the degree of binding.ACE2 μK exhibited the most potent binding to the variant RBDs.
The plant-derived ACE2 μK protein also showed potent binding to SARS-CoV-2 virus and inhibition of SARS-CoV-2 infection in vitro.
These results suggest that the fusion of μ-tp with the ACE2 protein enhanced the binding activity of the protein, resulting in improved inhibition of SARS-CoV-2 infection.Recent studies have been reported enhancing the avidity of ACE2 proteins using various strategies such as oligomeric motifs or Fc region of antibodies. [60]One of the studies utilized the C-terminal domain of T4 fibritin (foldon) and the trimeric domain of a three-helix bundle (3HB) to create trimeric ACE2, which exhibited a 14-to 125-fold increase in infection neutralization efficiency compared to the monomer ACE2. [63]Another approach involving the use of a p53 tetramerization domain resulted in ACE2 tetramers with a 23-fold improvement in neutralization efficiency due to enhanced binding avidity. [64]In particular, a recent study involving ACE2 fused with IgM Fc showed a mixture of monomeric and hexameric ACE2-IgM Fc forms.Upon separation, the hexameric ACE2-IgM-Fc displayed between a 27-and 96-fold enhancement in antiviral activity compared to its monomeric ACE2-IgM-Fc. [60]Our plant-derived ACE2 μK also partially formed a trimeric structure, enhancing binding avidity to SARS-CoV-2 spike proteins and improving antiviral efficiency compared to monomeric ACE2 K.There are numerous reports indicating that the administration of excessive amounts of soluble ACE2 can neutralize SARS-CoV-2 infection by competitively binding to the virus and preventing it from binding to ACE2 on cell membranes. [13,42,65]The ACE2 μK isoform has the added advantages of no risk of unwanted Fcreceptor activation and antibody-dependent enhancement, as well as enhanced binding affinity and inhibition of SARS-CoV-2.
The plant-derived ACE2 μK protein is a receptor trap that directly competes with the process of viral entry into cells.[68] There is also the possibility to produce synergistic effects by mixing ACE2 μK with neutralizing antibodies that bind to viral RBDs outside the ACE2 binding site. [67,69] previous studies, ACE2 treatment protected against acute lung injury (ALI) caused by acid aspiration and sepsis in ACE2-knockout mice. [70,71]Administration of recombinant hACE2 also improved lung injury, alleviated disease, and increased survival in mice infected with the Avian influenza (H5N1) virus. [71]In addition, a recent study demonstrated that the SARS-CoV-2 RBD is very similar or identical in structure to highly immunogenic peptides present in the neuraminidase of the novel 2009 influenza (H1N1) virus. [72]These findings suggest that treatment with plant-derived ACE2 μK might provide a strategy to alleviate and treat ALI and acute respiratory distress syndrome caused by other respiratory diseases and newly emerging pathogens such as H1N1 and H5N1 influenza viruses. [71]ere are many ongoing efforts to prevent and treat COVID-19.
In this study, we showed that enhanced ACE2 proteins designed to prevent and treat SARS-CoV-2 infection can be produced in a rapid, scalable, and affordable manner by using transient expression in N. benthamiana.Although the structure of ACE2 μK was mostly monomeric and partly trimeric, the inclusion of the 18 amino acid μ-tp peptide increased the binding activity and, consequently, the inhibition of μK in animal experiments and to investigate whether the binding activity and inhibitory effects of other proteins can be increased by fusion with μ-tp.Additionally, two different proteins can be transiently co-expressed within a single plant system by agroinfiltration. [73]J chain, a protein that mediates IgM oligomeric assembly, facilitates the formation of IgM pentamers by establishing intermolecular disulfide bonds with the penultimate cysteine residue of the tailpiece from IgM monomers.Therefore, we expect that in further studies, co-expression of ACE2 μK and the J chain could potentially increase the proportion of trimers or oligomers of ACE2 μK, thereby enhancing antiviral efficacy through improved avidity.

AUTHOR CONTRIBUTIONS
forms and produced the recombinant proteins in Nicotiana benthamiana plants.The ACE2 isoforms were well expressed and easily purified from the transient plant system.We confirmed the binding activity and SARS-CoV-2 inhibition of plant-derived ACE2 isoforms using SARS-CoV-2 virus and RBD proteins from SARS-CoV-2 VOCs.The plant-derived ACE2 proteins showed binding activity with SARS-CoV-2 RBDs and whole virus particles and also inhibited SARS-CoV-2 infection of Vero E6 cells.An ACE2 protein fused with μ-tp and the KDEL endoplasmic reticulum (ER) retention motif showed the strongest binding activity and inhibition of SARS-CoV-2 infection among all the ACE2 confirm the binding activity of the four plant-derived ACE2 proteins to SARS-CoV-2 RBD The wells of 96-well Pierce™ Nickel Coated Plates (Thermo fisher scientific, Waltham, MA) were coated with 1 µg per well of plantderived ACE2 protein (ACE2 μ, ACE2 μK, ACE2 N, or ACE2 K).Chinese hamster ovary (CHO) cell-derived human ACE2 protein (CHO-hACE2) was used as a positive control, and the hdpp4 receptor of Middle East respiratory syndrome coronavirus (MERS-CoV) was used as a negative control.The proteins were diluted in 1X PBS and incubated for 1 h at room temperature (RT).The plates were then washed four times with 200 µL 1X PBS + 0.05% v/v Tween 20 (1X PBS-T).After the wash step, 0.1-100 ng horseradish peroxidase (HRP)-conjugated wild-type SARS-CoV-2 RBD in 1X PBS was added, and the plates were incubated for 1 h at RT.The HRP-conjugated RBD, CHO-hACE2, and hdpp4 were a kind gift of DG Jeong (Bionanotechnology Research Center at KRIBB, Korea).The plates were washed again with 1X PBS-T, and TMB One component HRP Microwell Substrate and 450 nm Liquid Stop Solution for TMB Microwell Substrates (BioFX, Owing Mills, MD) were added for detection.Absorbance at 450 nm was measured in the wells using a microplate reader (Tecan, Zurich, Switzerland).At least three technical and biological replicates were performed.2.4.2ELISA analysis to evaluate binding activity of ACE2 K and ACE2 μK to SARS-CoV-2 RBD variants The wells of 96-well Pierce™ Nickel Coated Plates (Thermo fisher scientific, Waltham, MA) were coated with 1 µg per well of plant-F I G U R E 1 Plant expression cassettes, expected proteins, and expression of recombinant ACE2 proteins.(A) Schematics of the gene expression cassettes and expected protein structures of ACE2 μ, ACE2 μK, ACE2 N, and ACE2 K in the plant expression vector pBYR-2fp.P35Sx2Enh, cauliflower mosaic virus enhanced 35s promoter; TMV, 5′-untranslated leader sequence of tobacco mosaic virus; SP, ER signal peptide; μ-tp, mu tailpiece of IgM; KDEL, ER retention motif; Ext3' , terminator of extensin gene.(B) Syringe agroinfiltration of N. benthamiana leaves with A. tumefaciens EHA105.(C) Immunoblot analysis of ACE2 μ, ACE2 μK, ACE2 N, and ACE2 K expressed in N. benthamiana leaf extracts.The recombinant ACE2 proteins were detected by ACE2 antibody.Lanes: +, positive control (CHO cell-derived ACE2-Fc); -, negative control (buffer-infiltrated plant leaf); μ, ACE2 μ; μK, ACE2 μK; N, ACE2 N; K, ACE2 K. derived ACE2 μK or ACE2 K protein.CHO-hACE2 was used as a positive control.The proteins were diluted in 1X PBS and incubated overnight at 4 • C. The plates were then washed four times with 200 µL 1X PBS-T and blocked with 5% Blotting-Grade Blocker (Bio-Rad, Hercules, CA) in 1X PBS-T (5% skim milk) for 1 h at RT.After the wash step, 0.1-100 ng SARS-CoV-2 RBD-Fc protein [SARS-CoV-2 (wild type), SARS-CoV-2 B.1.1.7N501Y (Alpha), SARS-CoV-2 B.1.351(Beta), SARS-CoV-2 P.1 (Gamma), SARS-CoV-2 B.1.617.2 (Delta), or SARS-CoV-2 B.1.1.529(Omicron); (R&D systems, Minneapolis, MN] in 5% skim milk was added, and the plates were incubated for 2 h at RT.After washing with 1X PBS-T, the plates were treated with mouse anti-human IgG1, HRP conjugate (dilution 1:1000; Invitrogen, Waltham, MA) and incubated for 2 h at RT.After another wash with 1X PBS-T, TMB One component HRP Microwell Substrate and 450 nm Liquid Stop Solution for TMB Microwell Substrates (BioFX, Owing Mills, MD) were added for detection.Absorbance in the wells was measured at 450 nm using a microplate reader (Tecan, Zurich, Switzerland).At least three technical and biological replicates were performed.

Vero
E6 cells (an African green monkey cell line) were employed to study cell viability and for simultaneous-treatment assays.Vero E6 cells were kindly provided by the American Type Culture Collection (ATCC CRL-1586; Manassas, VA).A SARS-CoV-2 strain (Beta-CoV/Korea/KCDC03/2020: NCCP 43326) was obtained from the National Culture Collection for Pathogens in Korea.Vero E6 cells were cultured using Dulbecco's modified Eagle's minimum (DMEM) medium (Gibco/BRL; Burlington, ON, Canada) supplemented with 10% fetal bovine serum and 1% antibiotics-antimycotics at 37 • C with 5% CO 2 for 72 h.The SARS-CoV-2 strain was propagated onto confluent Vero E6 cells in the presence of 1 µg ml -1 L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin purchased from Sigma-Aldrich (St. Louis, MO).Infection assays were performed at the Korea Centers for Disease Control and Prevention-approved Biosafety Level 3 facility of the Korea Research Institute of Bioscience and Biotechnology in accordance with institutional biosafety requirements.
• C for 48 h.The cells were then supplemented with media containing serially diluted ACE2 μK or ACE2 K and incubated for 72 h.MTT (3-[4,5-dimethylthiozol-2-yl]−3,5diphenyl tetrazolium bromide) solution was added, and the plates were allowed to stand for 4 h.The formazan crystals were dissolved, and the absorbance was measured on a microplate reader (Bio-Rad model 680) at 540 nm with a subtraction of the background measurement at 655 nm.The percent cell viability was calculated using the following formula: [(OD sample -OD blank ) ÷ (OD control -OD blank )] × 100%.At least three technical and biological replicates were performed to statistically analyze data using Student's t-test.
the simultaneous-treatment assay, SARS-CoV-2 at 0.05 MOI was mixed with various concentrations of ACE2 μK or ACE2 K and incubated at 4 • C for 1 h.The mixtures were then inoculated onto confluent Vero E6 cells and incubated at 37 • C for 1 h.The virus-protein mixtures were then removed and replaced with DMEM medium containing 1 µg ml -1 TPCK-treated trypsin (Sigma-Aldrich, Burlington, MA), and the cells were incubated at 37 • C for 72 h.MTT solution was added, and the cells were incubated at 37 • C for 4 h.The formazan crystals were dissolved, and the absorbance was measured.The inhibition ratio was calculated according to the following equation: Inhibition rate (%) = [(OD sample -OD virus ) / (OD control -OD virus )] × 100%.At least three technical and biological replicates were performed.

F I G U R E 2
Purification of plant-derived recombinant ACE2 proteins from agroinfiltrated plants.Results of purification of recombinant (A) ACE2 μ, (B) ACE2 μK, (C) ACE2 N, and (D) ACE2 K shown by Coomassie-stained SDS-PAGE under reducing conditions.Lanes: M, protein marker; BSA, quantitative control (1 µg); F1∼F6, purified samples obtained from transient plants expressing ACE2 proteins; CT, column through; W, wash waste; S, supernatant after treatment with protamine sulfate; P.S, pellet after treatment with protamine sulfate; TSP, total soluble protein.Twenty microliters of sample were loaded on each well.

F I G U R E 3
ELISA for the binding activity of recombinant ACE2 proteins with SARS-CoV-2 RBD protein.(A) Normalization of purified ACE2 proteins confirmed by SDS-PAGE and Immunoblot analysis.Lanes: +, positive control (CHO cell-derived ACE2); -, negative control (buffer-infiltrated plant leaf); μ, ACE2 μ; μK, ACE2 μK; N, ACE2 N; K, ACE2 K. (B) Schematic of ELISA.(C) Data represent ELISA results for the binding activity of ACE2 isoforms with wild-type SARS-CoV-2 RBD-HRP protein.ELISA wells were coated with ACE2 isoforms as a receptor and treated with HRP-conjugated RBD protein as a detector.CHO-hACE2; positive control (CHO cell-derived ACE2), negative control hdpp4 (MERS-CoV receptor).All data was obtained from at least three technical and biological replicates.Data represent the mean ± standard deviation (SD) from three independent experiments.ACE2 K did not produce significant inhibition at any concentration (Figure 7D, 7E).

F I G U R E 6
Immunoblot and ELISA for the binding activity of plant-derived ACE2 proteins with inactivated SARS-CoV-2.(A) Schematic of immunoblot and ELISA for the binding activity of ACE2 with an inactivated beta strain of SARS-CoV-2.(B) Immunoblot results of recombinant ACE2 proteins with inactivated SARS-CoV-2.(C) ELISA to measure the binding affinity of purified ACE2 proteins with inactivated SARS-CoV-2.ELISA wells were coated with inactivated SARS-CoV-2 as a target and plant-derived recombinant ACE2 K or ACE2 μK as a receptor.ACE2 proteins were detected using an anti-ACE2 antibody and HRP-conjugated anti-mouse IgG H&L.All data was obtained from at least three technical and biological replicates and statistically analyzed.Data represent the mean ± SD from three independent experiments.Statistical differences between ACE2 K and ACE2 μK were represented asterisks (Student's t-test; **P < 0.01; ***P < 0.001).

F I G U R E 7
Cell viability and SARS-CoV-2 inhibition assays of plant-derived ACE2 K and ACE2 μK.(A, B) Histograms represent the percentage of viable cells after treatment with 0, 7.8, 15.6, 31.3,62.5, 125, 250, 500, or 1000 µg ml -1 (A) ACE2 K or (B) ACE2 μK in comparison with untreated controls (100% viability).(C) Schematic of the simultaneous-treatment assay.Beta strain of SARS-CoV-2 was incubated at 4 • C for 1 h with (D) ACE2 K or (E) ACE2 μK at concentrations of 0, 7.8, 15.6, 31.3,62.5, 125, 250, 500, or 1000 µg ml -1 before exposure to Vero E6 cells (0.05 MOI).Histograms are expressed as the percentage inhibition of virus infection during incubation with Vero E6 cells for 72 h.All data was obtained from at least three technical and biological replicates.Data show the mean ± SD from three independent experiments.viral infection.Taken together, our results suggest that the efficient and affordable plant-derived ACE2 μK protein can be stockpiled and rapidly and easily scaled up as a preventive and therapeutic agent for future outbreaks of SARS-CoV-2 and other viral pandemics.Our results provide important insight into the fusion of μ-tp can enhance the binding activities of recombinant proteins.Further studies are needed to confirm the effectiveness and safety of plant-derived ACE2 5 M Tris-HCl, pH 8.3; 20% NP-40; 20 Mm MgCl 2 ) and centrifuged at 10000 g for 15 min at 4 • C. The supernatant was filtered by a Miracloth (Biosciences, La Jolla, CA), and 1% protamine sulfate solution was added to make a final concentration of 0.1%.The solution was incubated on ice for 30 min and then centrifuged at 10000 g for 15 min at 4 • C. The supernatant was filtered through a Miracloth and a 0.45 µm membrane filter (VWR International, Radnor, PA)