Development of novel monoclonal antibodies against nsp12 of SARS-CoV-2

P3X63Ag8U.1 (P3U1) and SV40-transformed human embryonic kidney cell line HEK-293T (293T) cells were obtained from the American Type Culture Collection (Manassas, VA). P3U1 cells were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) that was supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.). 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, streptomycin (100 μg/ml), and penicillin (100 U/ml).

Anti-β-actin (AC-15) mouse mAb (Merck, Darmstadt, Germany) and anti-FLAG mouse mAb (M2) (Merck) were used for western blotting analysis. mAbs against nsp12 of SARS-CoV-2 (RdMab-2, -13, and -20) were developed in this study (see below).

Three female BALB/c mice (6-weeks-old) were purchased from CLEA Japan (Tokyo, Japan). The animals were housed under specific pathogen-free conditions. The Animal Care and Use Committee of Tohoku University approved all animal experiments (Permit number: 2019NiA-001). We designed three peptides (#1–3) of nsp12 as immunogens to immunize mice around the NiRAN domain (Fig. 1A, B). To develop mAbs against nsp12 of SARS-CoV-2, three synthesized peptides, such as ₃₄AFDIYNDKVAGFAKFLKTNC₅₃, ₇₄RHTFSNYQHEETIYNLLKDC₈₃, and ₂₄₈TRALTAESHVDTDLTKPYIC₂₆₆, which were keyhole limpet hemocyanin (KLH)-conjugated (Eurofins Genomics, Tokyo, Japan) were immunized intraperitoneally (i.p.) with Imject Alum (Thermo Fisher Scientific Inc.) into BALB/c mice (100 μg of each peptide/one mouse). The procedure included three additional immunization procedures (100 μg of each peptide), followed by a final booster intraperitoneal injection (100 μg of each peptide) 2 days before its spleen cells were harvested. The harvested spleen cells were subsequently fused with P3U1 cells, using polyethylene glycol 1500 (PEG1500; Roche Diagnostics, Indianapolis, IN). Then, hybridomas were grown in an RPMI medium supplemented with hypoxanthine, aminopterin, and thymidine for selection (Thermo Fisher Scientific Inc.). Cultured supernatants were finally screened using enzyme-linked immunosorbent assay (ELISA) for the detection of nsp12 peptides [15]. Of 24 clones (RdMab-1 to -24), three clones (RdMab-2, 13, 20) were cultured using Hybridoma-SFM medium (Thermo Fisher Scientific Inc.), and were purified with Ab-Capcher (ProteNova, Kagawa, Japan).

The plasmid vectors, pCMV-FLAG-CoV-nsp12 and -CoV-2-nsp12, containing a FLAG-tagged nsp12 expression cassette driven by a CMV promoter, was designed and constructed by VectorBuilder Inc. (Chicago, IL). The SARS-CoV and -CoV-2 nsp12 genes (GenBank: NC_004718 and MN908947, respectively) cloned into the plasmid vectors were codon optimized for expression in human cells.

To overexpress SARS-CoV and -CoV-2 nsp12 transiently, 293T cells were transfected with plasmid vectors carrying a FLAG-tagged nsp12 expression cassette, pCMV-FLAG-CoV-nsp12 and -CoV-2-nsp12 using Lipofectamine 2000 (Thermo Fisher Scientific). After 72 h of incubation, cells were lysed in RIPA buffer (Nacalai Tesque) for western blotting analysis.

Western blotting assay was performed as previously described [16]. Briefly, whole-cell extracts were prepared and 30 μg of total protein per lane was loaded onto 4–20% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gels. After electrophoresis under reducing conditions, bands of protein were transferred to nitrocellulose membranes (Amersham; Cytiva, Marlborough, MA). After blocking with 5% skim milk prepared in TBS-T (Tween-20, 0.1%), the membrane was incubated with 5 μg/mL of RdMab-2, -13, and -20, 1:5000 dilution of anti-FLAG (clone M2; Merck), or 1:10,000 dilution of anti-β-actin (clone AC-15; Merck), followed by incubation in the presence of horseradish peroxidase (HRP)-labeled anti-mouse IgG antibody (1:5000; Cell Signaling Technology, Danvers, MA).

Immunoprecipitation assays were performed as previously described [15]. Briefly, 293T cells were transfected with a FLAG-tagged nsp12-expressing plasmid (pCMV-FLAG-CoV-2-nsp12). After 72 h of incubation, 1 × 10⁷ cells were lysed in 1 ml of Lysis buffer A (0.5% NP-40, 20 mM Tris–HCl (pH 7.4), and 150 mM NaCl). After sonication, lysates were cleared of insoluble material by centrifugation at 21,000×g at 4 °C for 15 min. FLAG-nsp12 proteins were co-immunoprecipitated using Pierce Protein A Plus Agarose (Thermo Fisher Scientific) and 20 µg of anti-nsp12 mAb (RdMab-2) from the lysate, and then precipitated immune complexes were eluted in 2 × SDS loading buffer (2% β-mercaptoethanol, 20% glycerol, 4% SDS, and 100 mM Tris–HCl (pH 6.8)). The eluted proteins were detected by western blotting analysis. To detect the precipitated FLAG-nsp12 proteins, anti-FLAG mouse mAb (1:5000; M2) and Mouse TrueBlot ULTRA Anti-Mouse Ig HRP (1:4000; Rockland, Gilbertsville, PA) were used for western blotting analysis.

Immunofluorescence cell staining was performed as previously described [16]. Briefly, cells were fixed with 4% formaldehyde in PBS, permeabilized with 0.2% TritonX-100 in PBS, and blocked with 2% bovine serum albumin in PBS. The cells were incubated with 10 μg/mL the primary antibody RdMab-2, followed by incubation in the presence of Alexa488-labeled goat anti-mouse IgG (1:1000; Thermo Fisher Scientific). The cells were mounted in ProLong Glass Antifade Mountant with NucBlue Stain (Thermo Fisher Scientific) and imaged under a fluorescent microscope (IX81, Olympus, Tokyo, Japan).

Structural analyses using electron cryo-microscopy have shown that SARS-CoV-2 nsp12 protein has a closed, right-handed structure, which consists of palm, finger, and thumb domains (Fig. 1A, B) [8, 9]. Comparative analysis of structures of nsp12 encoded by SARS-CoV and SARS-CoV-2 suggested that SARS-CoV-2 nsp12 has a characteristic β-hairpin motif in the NiRAN domain, which is structurally different from that of SARS-CoV [9]. We hypothesized that antibodies recognizing the NiRAN domain in SARS-CoV-2 nsp12 could discriminate between nsp12 of SARS-CoV and SARS-CoV-2. Therefore, we designed and utilized three peptides (#1–3) of nsp12 around the NiRAN domain, but not recombinant nsp12 protein, as immunogens to immunize mice (Fig. 1A, B) and subsequently established hybridomas producing anti-nsp12 mAbs from the mouse spleen. We first screened the culture supernatants by ELISA for binding to nsp12 peptides, which selected 24 mAb clones (RdMab-1-24) (Table 1). To examine whether these putative clones binds to the nsp12 protein, we transiently expressed FLAG-tagged nsp12 of SARS-CoV-2 in 293T cells and then performed western blotting analysis using the culture supernatants of hybridomas producing these clones. We confirmed that anti-FLAG mAb (M2) recognized the FLAG-tagged nsp12 of SARS-CoV-2 in the position of the predicted size (~ 100 kDa) (Additional File 1: Fig. S1). The screening of 24 mAb clones by western blotting analysis demonstrated that 6 clones (RdMab-1, -2, -13, -15, -16, and -20) could detect SARS-CoV-2 nsp12 (Additional File 1: Fig. S1). To further evaluate whether they discriminate between nsp12 of SARS-CoV and SARS-CoV-2, we performed western blotting analysis using 293T cells transiently expressing the FLAG-tagged nsp12 of SARS-CoV or SARS-CoV-2, suggesting that the culture supernatants of three clones (RdMab-2, -13, and -20) could specifically detect SARS-CoV-2 nsp12 (Additional File 1: Fig. S2). Next, we purified these clones (RdMab-2, -13, and -20) from the hybridoma supernatants, which recognized SARS-CoV-2 nsp12 but not that of SARS-CoV (Fig. 1C). Together, we established three clones (RdMab-2, -13, and -20) which could specifically detect SARS-CoV-2 nsp12.

We next performed immunoprecipitation assay using lysates of 293T cells transiently expressing the FLAG-tagged nsp12 of SARS-CoV-2. Immunoprecipitation with RdMab-2 successfully isolated the FLAG-tagged nsp12 of SARS-CoV-2 from the lysates (Fig. 2), whereas RdMab-13 and -20 failed to isolate it by immunoprecipitation (data not shown). We confirmed using an isotype matched irrelevant control that the immune-complex precipitated by the RdMab-2 is specific signal (Fig. 2). Finally, we applied RdMab-2 to immunostaining analysis. No signals were detected in non-transfected 293T cells (Fig. 3, upper). When we transiently overexpressed the nsp12 in 293T cells, we found discrete signal of nsp12, which was spreading throughout the cytoplasm (Fig. 3, lower). This results are consistent with previous reports that the RdRP reaction of CoVs, including SARS-CoV, is conducted in the cytoplasm [17‐19]. Together, these findings indicated the utility of RdMab-2 for immunoprecipitation and immunofluorescence cell staining assays.