Plant-Produced S1 Subunit Protein of SARS-CoV-2 Elicits Immunogenic Responses in Mice

SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic. The virus spreads rapidly with a high transmission rate among humans, and hence virus management has been challenging owing to finding specific therapies or vaccinations. Hence, an effective, low-cost vaccine is urgently required. In this study, the immunogenicity of the plant-produced S1 subunit protein of SARS-CoV-2 was examined in order to assess it as a potential candidate for SARS-CoV-2. The SARS-CoV-2 S1-Fc fusion protein was transiently produced in Nicotiana benthamiana. Within four days of infiltration, the SARS-CoV-2 S1-Fc protein was expressed in high quantities, and using protein A affinity column chromatography, plant-produced S1-Fc protein was purified from the crude extracts. The characterization of plant-produced S1-Fc protein was analyzed by SDS-PAGE and Western blotting. Immunogenicity of the purified S1-Fc protein formulated with alum induced both RBD specific antibodies and T cell immune responses in mice. These preliminary results indicated that the plant-produced S1 protein is immunogenic in mice.


Introduction
After the first outbreak in Wuhan, China, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) quickly spread throughout the world. [1]. SARS-CoV-2, a Betacoronavirus [2,3], has a 79.5% similar genome sequence to its related severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) [4,5]. SARS-CoV-2 is a single-strand RNA-enveloped virus belonging to the Coronaviridae family [4,6,7]. Currently, few vaccines to protect against SARS-CoV-2 are available, and many are in development, such as mRNA vaccines, DNA vaccines, subunit vaccines, viral vector vaccines, and inactivated virus vaccines [8][9][10]. SARS-CoV-2 has four primary structural proteins: the spike (S) surface glycoprotein, the membrane (M) protein, the envelope (E) protein, and the nucleocapsid (N) protein. [11]. The most important target for developing a vaccine is the spike protein of SARS-CoV-2, as it is involved in the virus attachment and fusion into the host cell [11,12]. The spike protein has a trimeric form, which contains two parts of the S1 and S2 subunits [11]. The S1 subunit includes two domains: the C-terminal (CTD) and the N-terminal (NTD), the latter of which features a receptor binding domain (RBD) that interacts with the host cell receptor by attaching with angiotensin-converting enzyme 2 (ACE2) [11,12]. The majority of the epitopes that the neutralizing antibodies target are found on the spike protein, especially on the S1 subunit [13] and RBD domain [14][15][16], which can be considered as the major targets for vaccine development.
Due to the rapid spread of SARS-CoV-2 and its variants, vaccines that are both safe and broadly effective against variants are urgently needed. Therefore, it is necessary to select an appropriate expression system for subunit antigen production [17,18]. The available expression system for recombinant protein synthesis are mammalian cells, insect cells, or yeast, but every system also has certain limitations [19]. Hence, this study utilizes plants to produce the S1 subunit of SARS-CoV-2 due to its advantages, such as costeffectiveness, speed, and scalability, which were reviewed in much of the literature [19][20][21]. For these reasons, this study used Nicotiana benthamiana to produce the recombinant SARS-CoV-2 S1-Fc protein by combining the SARS-CoV-2 S1 with the Fc domain of human immunoglobulin G1 (IgG1). Briefly, SARS-CoV-2 S1-Fc was cloned and expressed in plants using a geminiviral expression vector. The SARS-CoV-2 S1-Fc protein produced by plants was characterized, purified, quantified, and its immunogenicity was tested in mice.

Construction of Plant Expression Vector for SARS-CoV-2 S1-Fc
The SARS-CoV-2 S1 (Genbank accession number: YP_009724390.1) was modified to include a peptide linker at the C-terminus that allows it to anneal with the Fc region of human immunoglobulin G1 (IgG1) (GenBank accession number: 4CDH_A). The SARS-CoV-2 S1 nucleotide sequence was amplified from the total DNA S1-His plasmid by using the polymerase chain reaction (PCR) with specific primers pairs (XbaI-SP F 5 TCT AGA ACA ATG GGC TGG 3 and BamHI-GS R 5 CGG GAT CCA CCA CCA CCA GAG ATA TCT CTA GCC CTT CTA GGA G 3 (Bionics, Gwangju, South Korea). To generate the SARS-CoV-2 S1-Fc geminiviral expression vector pBYR2eK2Md (pBYR2e), the human Fc region was digested with BamHI/SacI and ligated into the geminiviral vector containing the SARS-CoV-2 S1 for plant expression (Figure 1).

Quantification of Plant-Produced SARS-CoV-2 S1-Fc Protein
The yield of purified plant-produced SARS-CoV-2 S1-Fc protein was determined by ELISA. Briefly, 96-well microplate (Greiner Bio-One, Frickenhausen, Germany) was coated with 2 µg/mL monoclonal antibody H4 diluted in 1× PBS buffer and incubated overnight at 4 • C. The 96-well microplate was washed three times with PBST buffer (0.05% Tween 20 in 1× PBS). Then the 96-well microplate was blocked with 5% skim milk (BD Difco, NJ, USA) in 1× PBS for 2 h at 37 • C. After the blocking and washing step, the SARS-CoV-2 spike protein RBD (GenScript, Piscataway, NJ, USA) and samples were added into the microplate and incubated for 2 h at 37 • C. Then the plate was washed and incubated with SARS-CoV-2 Spike antibody (HRP) (Sino Biological, Beijing, China) diluted 1:1000 in 1× PBS for 1 h at 37 • C. Finally, the plate was washed before being developed using a TMB One Solution (Promega, Madison, WI, USA) and incubated for at least 2 min. The reaction was terminated with 50 µL of 1M sulfuric acid (H 2 SO 4 ), and the absorbance at 450 nm was measured using a 96-well plate reader (BMG Labtech, Ortenberg, Germany).

Mice Immunization with the SARS-CoV-2 S1-Fc Protein
The protocol of mice immunization was endorsed by the Institutional Animal Care and Use Committee, Faculty of Medicine, Chulalongkorn University (Protocol No. 013/2564). On days 0 and 21, four-week-old female ICR mice (n = 5 per group) were intramuscularly (IM) injected with 10 µg of plant-produced SARS-CoV-2 S1-Fc protein formulated with 0.1 mg aluminum hydroxide gel adjuvant (Croda, Frederikssund, Denmark). To evaluate the SARS-CoV-2-specific antibody response, mice sera were obtained before the first inoculation (baseline) and 14 days after each administration. The mice were euthanized 14 days following the second immunization (day 35), and splenocytes were collected for investigation of SARS-CoV-2 RBD-specific T-cell responses.

Evaluation of Immunological Responses in Mice
SARS-CoV-2 RBD specific total antibody responses were determined using Sf9 insect cells SARS-CoV-2 spike protein RBD (GenScript, NJ, USA) as coating antigen and goat anti-mouse IgG HRP conjugate antibody (Jackson ImmunoResearch, Westgrove, PA, USA) as the detection antibody. The endpoint titers were determined by following the method, as described previously [16]. The cells secreting mouse IFN-γ by SARS-CoV-2-specific cells were evaluated using an ELISpot test for mouse IFN-γ (Mabtech, Stockholm, Sweden) by following the protocol as described previously [16], and the results are given in terms of spot-forming cells (SFCs)/10 6 splenocytes.

Statistical Analysis
The statistical analyses were carried out using GraphPad Prism 9.0 (GraphPad Software, Inc., CA, USA). To calculate the results of total IgG and IgG subclasses, two-way analysis of variance (ANOVA), Tukey test, and multiple comparison tests were used. Mann-Whitney test was used to calculate the IFN-γ ELISpot assay results. All p values < 0.05 were defined as significant.

SARS-CoV-2 S1-Fc Expression in N. benthamiana
The SARS-CoV-2 S1-Fc fusion protein was cloned into the geminiviral plant expression vector named pBYR2e. Agrobacterium harboring pBYR2e-SARS-CoV-2 S1-Fc was agroinfiltrated into N. benthamiana plants (Figure 1). The plants infiltrated with Agrobacterium containing pBYR2e-SARS-CoV-2 S1-Fc construct exhibited necrosis in comparison to control plants (Figure 2a). For the time-course experiment, at 2, 4, 6, 8, and 10 dpi, the infiltrated leaves were collected. The yield of SARS-CoV-2 S1-Fc protein was quantified by ELISA. The optimal expression of SARS-CoV-2 S1-Fc protein was obtained four days after infiltration, and the protein accumulated up to 30 µg/g fresh leaf weight (Figure 2b). cells SARS-CoV-2 spike protein RBD (GenScript, NJ, USA) as coating antigen and goat anti-mouse IgG HRP conjugate antibody (Jackson ImmunoResearch, Westgrove, PA, USA) as the detection antibody. The endpoint titers were determined by following the method, as described previously [16]. The cells secreting mouse IFN-γ by SARS-CoV-2specific cells were evaluated using an ELISpot test for mouse IFN-γ (Mabtech, Stockholm, Sweden) by following the protocol as described previously [16], and the results are given in terms of spot-forming cells (SFCs)/10 6 splenocytes.

Statistical Analysis
The statistical analyses were carried out using GraphPad Prism 9.0 (GraphPad Software, Inc., CA, USA). To calculate the results of total IgG and IgG subclasses, twoway analysis of variance (ANOVA), Tukey test, and multiple comparison tests were used. Mann-Whitney test was used to calculate the IFN-γ ELISpot assay results. All p values < 0.05 were defined as significant.

Purification and Characterization of SARS-CoV-2 S1-Fc from N. Benthamiana Leaves
The plant-produced SARS-CoV-2 S1-Fc was purified from a crude extract of N. benthamiana leaves using single-step protein A affinity chromatography. The SARS-CoV-2 S1-Fc purified from plants was concentrated and filtered using a 0.22 µm syringe filter (Pall Corporation, NY, USA). The purified SARS-CoV-2 S1-Fc protein was characterized

Purification and Characterization of SARS-CoV-2 S1-Fc from N. Benthamiana Leaves
The plant-produced SARS-CoV-2 S1-Fc was purified from a crude extract of N. benthamiana leaves using single-step protein A affinity chromatography. The SARS-CoV-2 S1-Fc purified from plants was concentrated and filtered using a 0.22 µm syringe filter (Pall Corporation, NY, USA). The purified SARS-CoV-2 S1-Fc protein was characterized by SDS-PAGE and Western blot analysis. The expected band at the size of 100-150 kDa and 250 kDa was observed under reducing (Figure 2c; Lane 1) and non-reducing conditions in the InstantBlue-stained SDS gel (Figure 2c; Lane 2). Western blot analysis with anti-human gamma chain-HRP conjugated antibody confirmed the molecular weight of SARS-CoV-2 S1-Fc at 100-150 kDa and 250 kDa under reducing and non-reducing conditions, respectively (Figure 2d; Lane 1 and Lane 2; Figure S1). The yield of plant-produced SARS-CoV-2 S1-Fc protein was measured using ELISA and determined to be 3.9 mg/mL.

Immunogenicity in Mice
On days 0 and 21, mice were immunized with 10 µg of plant-produced SARS-CoV-2 S1-Fc with alum adjuvant, and sera were collected on days 0, 14, and 35, as shown in Figure 3a. The evaluation of SARS-CoV-2 RBD-specific antibodies was analyzed by ELISA using commercial Sf9-produced SARS-CoV-2 RBD-His as a capture antigen. To measure IgG response, mice were immunized with 10 µg of plant-produced SARS-CoV-2 S1-Fc with alum or alum alone as a control. The sera were collected on days 0, 14, and 35. The SARS-CoV-2 RBD-specific total IgG of S1-Fc immunized mice was detected after 14 days of second immunization (geometric mean end-point titer (GMT) = 919) which was significantly higher than the control group with p < 0.01, but not at 14 days after first immunization ( Figure 3b). Moreover, the mice sera were also subjected to IgG subtypes evaluation. The RBD-specific-IgG1 (Figure 3c) and -IgG2a (Figure 3d) titers, induced by the SARS-CoV-2 S1-Fc protein, were not significantly different compared with the control group. However, but RBD-specific-IgG1 titers (GMT = 1600), was found to be higher than SARS-CoV-2 RBD-specific IgG2a titers (GMT = 459.47) with a IgG1/IgG2a ratio of 3.38 folds (Figure 3e).

IFN-γ ELISpot Assay
The immunized mice splenocytes were isolated and evaluated for RBD-specific IFNγ secretion using IFN-γ ELISpot assay on day 35. The findings showed that compared to the control group, the plant-produced SARS-CoV-2 S1-Fc considerably increased the IFNγ secretion with p < 0.01 (Figure 4, Table S1).

IFN-γ ELISpot Assay
The immunized mice splenocytes were isolated and evaluated for RBD-specific IFN-γ secretion using IFN-γ ELISpot assay on day 35. The findings showed that compared to the control group, the plant-produced SARS-CoV-2 S1-Fc considerably increased the IFN-γ secretion with p < 0.01 ( Figure 4, Table S1).

Discussion
Since the emergence of SARS-CoV-2, researchers have been working towards the development of vaccines in order to decrease mortality and morbidity [12,24]. Earlier studies indicated that the SARS-CoV-2 spike protein can induce a potent immune response and neutralize antibodies, and it is considered suitable for the development of a recombinant vaccine [12,25,26]. The S protein is found on the surface of the virus particles involved in the virus entry into the host cell [12]. However, this study is interested in the S1 subunit because the S1 subunit has considerable neutralization epitopes than the S2 subunit [13]. Moreover, the receptor binding domain (RBD) from the S1 subunit induced significant immune response and neutralizing antibodies which were evident from several studies [14,15]. The full length of the S1 subunit also can stimulate the immune

Discussion
Since the emergence of SARS-CoV-2, researchers have been working towards the development of vaccines in order to decrease mortality and morbidity [12,24]. Earlier studies indicated that the SARS-CoV-2 spike protein can induce a potent immune response and neutralize antibodies, and it is considered suitable for the development of a recombinant vaccine [12,25,26]. The S protein is found on the surface of the virus particles involved in the virus entry into the host cell [12]. However, this study is interested in the S1 subunit because the S1 subunit has considerable neutralization epitopes than the S2 subunit [13]. Moreover, the receptor binding domain (RBD) from the S1 subunit induced significant immune response and neutralizing antibodies which were evident from several studies [14,15]. The full length of the S1 subunit also can stimulate the immune response and the neutralization antibodies to block the viral infection [27][28][29]. As a result, the S1 subunit in the S protein could be a target for developing an effective SARS-CoV-2 vaccine.
The number of COVID-19 cases is rapidly increasing; hence, a platform that can produce antigens in a short time to make the recombinant vaccine is required. Plants are a suitable platform to use during an emergency situation because, in comparison to other expression systems, it has many advantages, such as cost, safety, and speed of production, and it has been largely used for producing biopharmaceuticals over the past decade [20,[30][31][32][33]. Many research groups have evaluated and thoroughly studied the feasibility of plant-derived biopharmaceuticals and vaccines [34,35]. In particular, plants are used as an expression system for antibodies, vaccines, diagnostic reagents, human serum albumin, cytokines, and growth factors production [36]. The capacity of plants to produce recombinant biopharmaceuticals against SARS-CoV-2 has also been documented [37][38][39][40][41][42][43]. Notably, the plant-derived biopharmaceutical taliglucerase alfa (ELELYSO TM ), used to treat Gaucher disease in adults, was approved by Food and Drug Administration (FDA) [44].
Our study demonstrated the expression of SARS-CoV-2 S1-Fc protein in N. benthamiana plants and tested the efficacy in mice. The results showed that the SARS-CoV-2 S1 subunit fused with Fc domain can be expressed in N. benthamiana plants with high yield obtained within four days after infiltration. The infiltrated leaves showed necrosis compared to the control. Furthermore, the characterization of plant-produced SARS-S1-Fc protein indicated that the expected size of approximately 100-150 kDa and 250 kDa in reducing and nonreducing conditions, respectively, was observed in Western blot. In addition, the results depicted that some of the proteins are degraded during processing, and hence, further optimization is required to enhance protein accumulation and prevent protein degradation during downstream processing.
The plant-produced SARS-CoV-2 S1-Fc protein was prepared using alum as an adjuvant, and the findings indicated the S1 protein induces an antigen-specific antibody response. Previous report showed that the SARS-CoV-2 S1 subunit protein can stimulate the SARS-CoV-2 S1-specific IgG titer higher than the SARS-CoV-2 RBD protein when using the SARS-CoV-2 S1 protein and SARS-CoV-2 RBD protein as a coating antigen in ELISA [28]. Further, alum adjuvant can enhance the Th-2 (IgG1) immune response better than the Th-1 (IgG2a) immune response [45], which confirmed in the present study that the SARS-CoV-2 RBD-specific IgG1 antibody responses were increased more than the SARS-CoV-2 RBD-specific IgG2a antibody responses [27,45].
Nevertheless, RBD-specific IFN-γ secretion analyzed by ELISpot assay indicated that the plant-produced SARS-CoV-2 S1-Fc with alum was immunogenic in mice, as shown in Figure 4, and it has a significant IFN-γ secretion higher than the control group. However, additional experiments, such as neutralizing antibody analysis and viral challenge experiments, are warranted to support these results.
In summary, our research revealed that the S1 subunit of SARS-CoV-2 fused with Fc region can be produced in plants as an expression system with a high yield and can be obtained within four dpi. Further, the plant-produced vaccine-induced antigen-specific antibodies and T-cell responses in mice. These findings provide a basis to further advance the development of a plant-produced subunit SARS-CoV-2 vaccine.