In vitro selection of anti-SARS-CoV-2 S1 VHHs

We used VHH-cDNA display for in vitro selection of VHHs against the SARS-CoV-2 S1 protein (Fig 1A). cDNA display yields functional VHHs, whose coding RNA is linked via a puromycin linker [13–15]. This procedure selects the coding sequences for high-affinity VHHs from a diverse (10¹³⁻¹⁴) DNA library [16]. To select the VHH candidates targeting SARS-CoV-2 S1 proteins, DNA libraries at Round 2 (R2) and Round 3 (R3) of in vitro selection were sequenced, and anti-SARS-CoV-2 VHH candidates were translated.

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Fig 1. Isolation and characterization of K-874A.

(A) Schematic showing in vitro selection of VHHs against SARS-CoV-2 S1 protein using VHH-cDNA display. In vitro transcription and translation of VHH-cDNA library form VHH linked to its mRNA with a puromycin linker. cDNA of linked mRNA was reverse-transcribed and VHH-cDNA complex was produced. High-affinity VHH-cDNA complex to immobilized S1 protein was isolated, and its cDNA was amplified. Three rounds of selection were performed, cDNA libraries from rounds 2 and 3 were sequenced, and anti-SARS-CoV-2 VHH candidates were translated. (B) Frequency distribution of amino acid sequences corresponding to VHH antibody candidates targeting SARS-CoV-2 S1 subunits in the selected VHH libraries. Sample ID “1” with the highest frequency (39.5%) is clone K-874A. (C) Binding affinity of K-874A to SARS-CoV-2 S1 subunits. Biolayer interferometry sensorgram measures the apparent binding affinity of K-874A-6xHis to immobilized SARS-CoV-2 S1 fused with sheep Fc. Binding curves for different concentrations of K-874A are shown in different colors. The black curves are 1:1 fits of the data. (D) Direct antigen ELISA measuring the binding affinity of FLAG-tagged K-874A to immobilized S1-6xHis subunits of beta-coronaviruses (HCoV-HKU1, HCoV-OC43, SARS-CoV-1, SARS-CoV-2) (left). Each immobilized S1-6xHis subunit was detected by anti-His antibody (right). Error bars are mean ± SD (N = 3). Data are from a representative experiment of three independent experiments.

https://doi.org/10.1371/journal.ppat.1009542.g001

From the frequency distribution of VHH clones in the selected library, clone K-874A appeared most frequently (39.5%) after three rounds of in vitro selection, indicating that K-874A has a high affinity for S1 proteins (Fig 1B). Biolayer interferometry assay showed the binding affinity of K-874A to SARS-CoV-2 S1 protein is 1.4 nM (K_a (1/Ms) = 6.72E+04, K_d (1/s) = 9.42E-05) (Fig 1C). When a direct antigen enzyme-linked immunosorbent assay (ELISA) was performed using FLAG-tagged K-874A and immobilized recombinant His-tagged S1 protein, we found that K-874A bound with high affinity to the S1 protein of SARS-CoV-2 but not to other coronaviruses, including human CoV (HCoV-HKU1, HCoV-OC43) and SARS-CoV-1, even when all S1-6xHis subunit were immobilized (Fig 1D). These results indicate that K-874A binds strongly and specifically to the S1 protein of SARS-CoV-2.

K-874A VHH prevents SARS-CoV-2 fusion with host cell

With its strong binding affinity and specificity to SARS-CoV-2, we investigated K-874A as a potential therapeutic drug for COVID-19. We infected African green monkey kidney (VeroE6) cells expressing transmembrane serine protease 2 (VeroE6/TMPRSS2) with SARS-CoV-2 (KUH003) and determined how well K-874A inhibited SARS-CoV-2 infection in the cells by quantitative real-time polymerase chain reaction (qRT-PCR). The half-maximal inhibitory concentration (IC₅₀) calculated from RNA copies was 5.74±2.6 μg/mL (Fig 2A).

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Fig 2. K-874A VHH neutralizes SARS-CoV-2 by preventing viral fusion with host cell.

(A) Neutralization of SARS-CoV-2 by K-874A VHH. SARS-CoV-2 was pretreated with different concentrations of K-874A and inoculated into VeroE6/TRMPSS2 cells. Fold-reductions in viral RNA copies in culture supernatants were calculated by qRT-PCR. K-874A has an IC₅₀ of 5.74±2.6 μg/ml. Error bars indicate mean ± SD from three independent experiments. (B) The numbers of viral RNA copies in VeroE6/TMRPSS2 cells infected with SARS-CoV-2 (untreated) and SARS-CoV-2 pretreated with K-874A VHH (K-874A-treated) were similar. Number of RNA copies attached or incorporated in the cells were determined by qRT-PCR. Error bars indicate mean ± SD from three independent experiments. (C) Number of viral RNA copies in VeroE6/TMRPSS2 cells infected with untreated SARS-CoV-2 and K-874A-treated SARS-CoV-2 at 0, 3 and 6 hr. RNA levels were estimated by qRT-PCR (N = 3, **P<0.005, Welch’s t test). (D) Immunofluorescence images show viral protein, NSP8, and RNA replication are suppressed in Vero/TMPRSS2 cells infected with K-874A-treated SARS-CoV-2. SARS-CoV-2-infected cells were fixed at 24 hr after infection and treated with anti-NSP8 (red) or anti-dsRNA (green). After fluorescence-conjugated secondary antibody and Hoechest (blue) treatment, images were captured by BZX800. Scale bar = 50 μm. (E) Cell fusion induced by S protein transduction in HiBiT- and LgBiT-expressing VeroE6/TMPRSS2 cells. Treatment with K-874A VHH suppressed cell fusion in a dose-dependent manner. Error bars indicate mean ± SD from three independent experiments. (F) Optical images of VeroE6/TMPRSS2 cells expressing S protein transduced by a lentivirus vector show cell fusion (S+) and no cell fusion when treated with K-874A VHH (S+K-874A+). Inset: magnified view of dotted square shown in left panels. (G) ELISA results show K-874A (left) and ACE2 (right) bound to immobilized recombinant S protein in a dose-dependent manner. Immobilized S protein was detected by serially diluted K-874A (1 to 0.002 μg/well) and horseradish peroxidase (HRP)-conjugated anti-VHH antibody (left), or by serially diluted ACE2 protein (20 to 0.0625 μg/ml) and anti-ACE2 antibody and HRP-conjugated secondary antibody (right). Binding of ACE2 to S protein was unaffected by K-874A (1 μg/well) treatment.

https://doi.org/10.1371/journal.ppat.1009542.g002

To determine whether K-874A VHH neutralizes SARS-CoV-2 by preventing the virus from attaching to the host cell, we compared the early phases of infection in cells infected with K-874A-treated virus and untreated virus. VeroE6/TMPRSS2 cells were incubated with K-874A-treated SARS-CoV-2 and untreated virus for 1 hr, and the number of RNA copies that were attached or incorporated into the cells were determined by qRT-PCR. K-874A treatment did not change the levels of virus attachment or early incorporation in the cells (Fig 2B). At 6 hr post-infection, the number of viral RNA copies in cells infected with untreated virus were increased more than 400 times, but the numbers were remarkably inhibited in cells infected with K-874A treated virus (Fig 2C). Additionally, expression of the viral protein, NSP8, and double-stranded RNA (dsRNA), which is generated during viral RNA replication, were suppressed in cells infected with K-874A-treated viruses (Fig 2D). These results indicated that K-874A did not reduce viral attachment.

We further investigated whether K-874A acts by inhibiting the virus from fusing with the host cell. VeroE6/TMPRSS2 cells express the ACE2 receptor and the TMPRSS2 protease on its surface. To study viral fusion, we transduced the VeroE6/TMPRSS2 cells with a lentivirus coding for the viral S protein into the cells. When the S protein from one VeroE6/TMPRSS2 cell binds to the ACE2 receptor on an adjacent VeroE6/TMPRSS2 cell, the TMPRSS2 protease cleaves and activates the S protein for fusion. To quantify this cell fusion, we used HiBiT technology, which involves the binding of HiBiT (a small 11–amino acid peptide) with a larger subunit, called LgBiT, to form a complex with luciferase activity. VeroE6/TMPRSS2 cells expressing HiBiT or LgBiT were produced and co-cultivated. The HiBiT-LgBiT complex and the resulting luciferase activity form only when the S protein from one cell binds to the ACE2 receptor on an adjacent cell and their membranes are fused. We found that treating VeroE6/TMPRSS2 cells with K-874A suppressed cell fusion in a dose-dependent manner (Fig 2E). Cells without S protein (S ˗) did not fuse, whereas those expressing S protein but not treated with K-874A (S+) did fuse. Optical micrographs confirmed these results (Fig 2F). Direct binding of K-874A or recombinant ACE2 to immobilized recombinant S protein in ELISA further showed that both K-874A (left panel in Fig 2G) and ACE2 (blue line on right panel in Fig 2G) bound to S protein in a dose-dependent manner. However, K-874A did not block ACE2-S protein interaction (red line on right panel in Fig 2G). These findings suggest that K-874A does not prevent the virus from attaching to the ACE2 receptor on the cell surface. Rather, K-874A prevents the virus from entering the cell by blocking the viral membrane from fusing with the host cell.

To estimate the K-874A binding region on the S protein, we reconstructed the cryo-electron microscopy (cryo-EM) structures of the recombinant S protein trimer (S1 Fig) and its complex with K-874A complex (S2 Fig), and fitted each molecular model to the maps (S3G–S3I Fig). As a result, we acquired two types of the complex structures, named type 1 and type 2 (S2 Fig). The ratio of type 1 and type 2 particles was approximately 9:1. The type 1 structure showed that K-874A was located in the vacant space between the NTD and RBD of the S protein (Figs 3A–3D, and S3B, S3E and S3H). In the prefusion state, the RBD moves upwards to bind ACE2 [17,18]. However, the RBD in the type 1 structure did not rise up after binding K-874A and moved laterally to the outside of the S protein trimer (S5A Fig). The NTD moved down after binding of K-874A. Amino acid residues in CDR1, CDR2, and N-terminal of K-874A formed polar bonds with the NTD of the S protein, whereas amino acid residues of CDR2 and CDR3 bonded hydrophobically with the RBD (Fig 3B–3D). In the type 2 structure, K-874A was bound only to the RBD and not to the NTD (S3C, S3F, S3I and S4 Figs). The S protein trimer was in the prefusion state with RBD moving upwards (S5B Fig). All amino acid residues that interact with ACE2 were placed in the upward state of RBD, even when the RBD was bound to K-874A, demonstrating that ACE2 binds to RBD even in the bound state of K-874A (Fig 3E and 3F). These findings demonstrate that K-874A neutralizes SARS-CoV-2 via a different route that did not involve ACE2 binding.

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Fig 3. K-874A-binding region on the S protein in the major cryo-EM map (type 1).

(A) A ribbon model of S protein trimer and K-874A VHHs. The coloring follows the standard designation of K-874A VHH (magenta), NTD (blue) and RBD (green). (B) An enlarged view of RBD, NTD and K-874A in (A). (C) A view of the structure in (B) rotated 90°. Residues that interact with ACE2 are shown in yellow. N501 and E484 (cyan) are amino acid residues frequently mutated. Possible chemical interactions between RBD and K-874A are shown with black dotted lines. (D) Possible chemical interactions between K-874A and NTD shown with black dotted lines. The viewing area was indicated by the dotted square in (B). (E) The binding of K-874A and ACE2 on RBD. (F) A close-up view of the interactions between the peptidase domain (PD) of ACE2, K-874A, RBD, and NTD. K-874A does not interfere the binding of ACE2.

https://doi.org/10.1371/journal.ppat.1009542.g003

K-874A VHH neutralizes B.1.1.7 variant but no other variants

We evaluated the binding affinity and the neutralization effect of K-874A against variants prevalent to date. Calu3 cells were infected with each variant and incubated for an hour. After removing the inoculum and washing, Calu3 cells were incubated with fresh new medium, including K-874A for 2 days. The numbers of RNA copies of Wuhan, KUH003 (D614G), and B.1.1.7 (Alpha) in the medium at day 2 were slightly reduced (S8A Fig), suggesting that newly produced progeny viruses were captured by K-874A and could not infect the cells again. Numbers of RNA copies of B.1.351 (Beta), P.1 (Gamma), and B.1.617.2 (Delta) did not change with or without K-874A. To further investigate the infectivity of each progeny in the culture supernatant, the number of RNA copies was adjusted to 10,000, and the TCID₅₀ was calculated after infecting Vero/TMPRSS2 cells. Infectivity of Wuhan, KUH003 (D614G), and B.1.1.7 (Alpha) were remarkably depressed by K-874A treatment, and the infectivities of B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta) were not (S8B Fig). Furthermore, the binding affinity of K-874A to S protein from each variant was evaluated by ELISA. K-874A bound to Wuhan- or B.1.1.7 (Alpha)-derived-S, but not to B.1.351 (Beta), P.1 (Gamma), B.1.429 (Epsilon), B.1.525 (Eta) and B.1.617 (Kappa) (S8C Fig). Neutralized variants did not have the mutation in K-874A binding motif at amino acids 450–490 of the S protein; however, un-neutralized variants had some mutations. These finding suggested that it would be neutralized if K-874A is strongly bound to S protein and targeting to applicable motif of S proteins may lead to a highly effective VHH to neutralize these un-neutralized variants.

K-874A VHH prevents viral replication in lung organoids

Because SARS-CoV-2 targets lung tissues and induces severe respiratory disease, we evaluated K-874A VHH in a human lung–derived alveolar organoid that is susceptible to SARS-CoV-2 and releases high levels of progeny virus into the culture supernatant [19,20]. To determine if K-874A blocks production of virus progeny in infected alveolar organoids, we incubated the organoid cells with SARS-CoV-2 for 1 hr before treating the infected cells with K-874A-containing medium for 3 days. Untreated cells released >1x10⁷ RNA copies/μL into the culture supernatant at day 3, and VHH treatment reduced the progeny production to 1x10⁴ RNA copies/μL (Fig 4A), indicating that K-874A reduced the production of virus progeny. To test the infectivity of virus progeny produced from K-874A-treated and untreated infected cells, we adjusted each viral progeny to 1x10⁴ RNA copies and incubated with fresh VeroE6/TMPRSS2 cells. Untreated virus progeny showed 565 TCID₅₀/ml, and K-874A-treated virus progeny showed fewer than 10 TCID₅₀/ml (Fig 4B). These data suggested that the K-874A-treated progeny have a lower infectivity. This is likely due to K-874A VHH binding to the S protein of the virus progeny, preventing them from infecting other cells.

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Fig 4. K-874A VHH treatment inhibits SARS-CoV-2 replication in an alveolar organoid model and improves viral symptoms in Syrian hamsters.

(A) Graph shows infected alveolar organoid treated with K-874A-containing medium for 3 days released fewer RNA copies (1x10⁴ RNA copies/μL) into the culture supernatant than organoids that were not treated with K-874A (>1x10⁵ RNA copies/μL). (N = 5, * P<0.05, ** P<0.005, Welch’s t-test). Data are from a representative experiment of two independent experiments. (B) Virus progeny in each supernatant at Day 3 were adjusted to 1x10⁴ RNA copies and the infectivity was compared between K-874A treated and untreated. The infectivity of K-874A untreated was 565±195 TCID₅₀/ml, and K-874A-treated was lower than 10 TCID₅₀/ml. Virus infectivity was determined in VeroE6/TMPRSS2 cells. Error bars indicate mean ± SEM. Black dotted line indicates detection limit. (N = 5, * P<0.05, Welch’s t-test) (C) Time schedule for inoculation and K-874A administration to Syrian hamsters. All animals were infected with 2x10³ TCID₅₀ of SARS-CoV-2. Untreated animals were treated with BSA/PBS (blue circle) on Day 1 and Day 2 post-infection. K-874A-treated groups were treated with either a single dose immediately before infection (K-874A/virus; red square) or 1 day after infection (+ K-874A (1); green triangle), or two doses at Day 1 and Day 2 after infection (+ K-874A (2); purple rhombus). Each dose was 3 mg K-874A per animal. (N = 3 for infected with untreated or treated) (D) Unlike BSA/PBS-treated animals, all K-874A-treated animals showed no weight loss. Weight at Day 0 is 100%. (E) Amounts of viral RNA in the lung homogenates at Day 4, as determined by qRT-PCR, show all K-874A-treated animals had fewer viral RNA copies in their lungs than untreated animals. (N = 3 for infected and N = 2 for uninfected, *P<0.05, Dunnett’s multiple comparison test) (F) qRT-PCR measurements of lung homogenates collected on Day 4 show untreated animals expressed elevated levels of inflammatory cytokines IL-6 and IL-10 but not TNF-α and IL-1β. Cytokine levels in K-874A-treated animals were similar to uninfected animals. (N = 3 for infected and N = 2 for uninfected, **P<0.01, Dunnett’s multiple comparison test).

https://doi.org/10.1371/journal.ppat.1009542.g004

Infected Syrian hamsters recover after K-874AVHH treatment

We further tested K-874A VHH as a potential therapeutic drug for COVID-19 in the Syrian hamster model. When infected with SARS-CoV-2, Syrian hamsters lose weight but spontaneously heal by 14 days post-infection [21]. To determine if K-874A improves the symptoms of COVID-19 infection, we infected 6-week-old male Syrian hamsters with SARS-CoV-2 (2 x 10³ TCID₅₀) and administrated different regimens of K-874A intranasally. The animals were treated with a single dose of K-874A just before the infection or at 1 day after the infection, or multiple doses at 1 and 2 days after the infection (Fig 4C). Each dose of K-874A was 30 mg/kg, which was determined to be the maximum dose that can be administrated. All infected animals treated with K-874A did not lose weight, but infected control animals treated with bovine serum albumin in phosphate-buffered saline (BSA/PBS) lost weight 3 days after the infection (Fig 4D). Further, all K-874A-treated hamsters, regardless of treatment regimen, had fewer viral RNA copies in their lung tissues than BSA/PBS-treated hamsters 4 days after the infection (Fig 4E). Because cytokines are upregulated after an infection [22,23], we compared the mRNA levels of several cytokines in the infected lung tissues of the untreated and K-874A-treated animals. Untreated animals showed elevated levels of IL-6 and IL-10 but not IL-1β and TNF-α, in their lungs 4 days after the infection. All K-874A-treated animals, regardless of the treatment regimen, displayed cytokine levels that were similar to uninfected animals (Fig 4F). When different doses (1.2, 6, or 30 mg/kg) of K-874A were administered in a two-dose regimen (on Days 1 and 2 post-infection) (S9A Fig), animals receiving the lowest K-874A dose displayed the highest weight loss and viral RNA copies in their lung tissues (S9B and S9C Fig). Their IL-6 and IL-10, but not IL-1β and TNF-α, levels were also higher than those receiving higher doses of K-874A (S9D Fig). These data demonstrate that K-874A improved the symptoms of COVID-19 infection and in a dose-dependent manner.

Ethics statement

Animal experiments were approved by the President of Kitasato University through the Institutional Animal Care and Use Committee of Kitasato University (20–031), and performed in accordance with the Guidelines for Animal Experiments of Kitasato University. Human normal lung organoid experiments for SARS-CoV-2 infection were approved by the ethics committees of Keio University School of Medicine and Kitasato Institute Hospital.

Preparation of cDNA display library

A cDNA display library (200 pmol-scale) used for the 1^st round of selection (R1) was synthesized from an initial VHH-encoding DNA library (predicted diversity, 1x10¹³), provided by Epsilon Molecular Engineering Co. Ltd. (Japan). A cDNA display library (6-pmol scale) used at the 2^nd and 3^rd rounds of selection (R2 and R3, respectively) was synthesized from the selected DNA library of each previous round. Translation of the DNA library was performed using T7 RiboMAX Express Large-Scale RNA Production System (Promega, USA), according to the manufacturer’s instructions. The DNase-treated mRNA mixture was purified using Agencourt RNAClean XP beads (Beckman Coulter Genomics). The purified mRNA was hybridized to cnvK (riboG) linker (Epsilon Molecular Engineering) at the 3′-terminal region in 50 mM Tris-HCl (pH 7.5) with 200 mM NaCl under the following annealing conditions: heating at 90°C for 2 min, followed by lowering the temperature to 70°C at a rate of 0.1°C/s, incubating for 1 min, then cooling to 25°C at a rate of 0.1°C/s, incubating for 30 sec, and then stored at 4°C until use. The mixture was irradiated with UV light at 365 nm using a handheld UV lamp (6W, UVGL-58, 254/365 nm, 100V; Analytik Jena) for 5 min to obtain mRNA-linker complex.

The mRNA-linker complex was translated using a Rabbit Reticulocyte Lysate System, Nuclease-Treated (Promega) at 37°C for 15 min. To synthesized mRNA-linker-VHH complex, KCl and MgCl₂ were added to final concentrations of 900 and 75 mM, respectively, and the mixture was incubated at 37°C for 20 min.

EDTA (final concentration: 70 mM) and an equal volume of 20 mM Tris-HCl buffer (pH 8) containing 2 mM EDTA, 2 M NaCl and 0.2% Tween 20 were added, and the mRNA-linker-VHH complex was immobilized in 60 μL of Dynabeads MyOne streptavidin C1 magnetic beads (Thermo Fisher Scientific) at 25°C for 30 min. The beads were washed two times with 60 μL of wash buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA, 1 M NaCl and 0.1% Tween 20). The immobilized library was then reverse-transcribed by ReverTra Ace (TOYOBO) at 42°C for 30 min. The beads were washed two times with 200 μL of wash buffer (20 mM sodium phosphate buffer, 0.5 M NaCl, 5 mM imidazole, and 0.05% Tween 20 (pH 7.4)) and then, 30 μL of RNase T1 (Thermo Fisher Scientific) prepared with the wash buffer was added to the beads. The mixture was incubated for 15 min 37°C to release the cDNA-linker-VHH complex from the beads. The supernatant containing cDNA-linker-VHH complex was purified using His Mag Sepharose Ni beads (GE Healthcare). The supernatant was added to 20 μL of the beads and incubated at 25°C for 30 min. The beads were washed with 200 μL of wash buffer (20 mM sodium phosphate buffer, 0.5 M NaCl and 20 mM imidazole (pH 7.4)), and then incubated in 10 μL of elution buffer (20 mM sodium phosphate buffer, 0.5 M NaCl and 250 mM imidazole, 0.05% Tween 20 (pH 7.4)) at 25°C for 15 min. The elution collected was stored at 16°C until use.

In vitro selection of VHHs using cDNA display

The C-terminal polyhistidine tagged-SARS-CoV-2 S1 subunits (Val16-Arg685) (YP_009724390.1) (#40591-V08H, Sino Biological) were used as target molecules for screening. Microtiter wells of Nunc-Immuno Plate II (Thermo Fisher Scientific) were coated overnight at 4°C with 100 μL of 100 (R1), 10 (R2), or 1 μg/mL (R3) recombinant SARS-CoV-2 S1 subunits-His tag that was prepared with PBS. The S1 subunits-coated wells were blocked with 200 μL of 3% BSA in PBST for 2 hr at room temperature, and then washed at three times with 200 μL of HBST. 100 μL of VHH-cDNA complex library was prepared as follows: 50 μL of HEPES buffer containing 1% BSA was added to 50 μL of VHH-cDNA conjugate library for R1, 50 μL of HBST containing 1% BSA, 25 μL of HBT was added to 50 μL of cDNA display library for R2 and R3, respectively. The VHH-cDNA complex library was added to the target-fixed well, incubated for 1 hr at room temperature. The residual VHH-cDNA conjugates were removed by washing at 10 times with 200 μL of HBST. The target-binding VHH-cDNA complex was eluted by adding 100 μL of 100 mM Tris (pH 11), gently pipetting, and incubating for 10 min at 37°C. The elution was immediately transferred to PCR cocktails prepared with KAPA HiFi HotStart ReadyMix (2x) (Kapa Biosystems, USA), forward primer (5′-GATCCCGCGAAATTAATACGACTCACTATAGGGGAAGTATTTTTACAACAATTACCAACA-3′), and reverse primer (5′-TTTCCACGCCGCCCCCCGTCCT-3′). The PCR cycle conditions consisted of 95°C for 2 min, followed by 22 cycles (R1) or 24 cycles (R2 and R3) of 98°C for 20 sec, 68°C for 15 sec, and 72°C for 20 sec, and then 72°C for 5 min. The PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter Genomics), according to the manufacturer’s instruction. The purified DNA libraries were used for translation, as described above, to prepare cDNA display libraries used at the next selection round.

Sequencing of the DNA library

DNA libraries obtained at R2 and R3 were used as templates in amplicon PCR for Illumina sequencing. The DNA library was quantified using PicoGreen dsDNA reagent kit (Thermo Fisher Scientific) and prepared at 100 ng/mL with nuclease-free water. 1^st PCR was performed with 12.5 μL of KAPA HiFi HotStart ReadyMix (2x) (Kapa Biosystems), 0.5 μL of 10 μM forward primer (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNATGGCTGAGGTGCAGCTCGTG-3′), 0.5 μL of 10 μM reverse primer (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNTGATGATGATGGCTACCACCTCCCG-3′), 9 μL of nuclease-free water, and 2.5 μL of template DNA. The PCR cycle conditions consisted of 95°C for 3 min, followed by 16 cycles of 98°C for 20 sec, 62°C for 15 sec, and 72°C for 20 sec, and then 72°C for 5 min. PCR clean-up was performed with Agencourt AMPure XP beads (Beckman Coulter Genomics). Subsequently, index PCR was performed with 12.5 μL of KAPA HiFi HotStart ReadyMix (2x) (Kapa Biosystems), each 1 μL of 10 μM forward and reverse primers from Nextera XT Index Kit v2 (Illumina), 8 μL of nuclease-free water, and 2.5 μL of template DNA. The PCR cycle conditions consisted of 95°C for 3 min, followed by 8 cycles of 98°C for 20 sec, 55°C for 15 sec, and 72°C for 30 sec, and then 72°C for 5 min. After magnetic bead-based purification of PCR products, the library concentration was measured with PicoGreen dsDNA reagent kit (ThermoFisher Scientific) and prepared to 4 nM with nuclease-free water. The library was diluted to a final concentration of 7 pM and 5% of PhiX DNA (Illumina) was added. Sequencing was performed with the Miseq Reagent Nano kit V2 500 cycle using a MiSeq 2000 (Illumina), according to the manufacturer’s instructions.

Data analysis

Dell Mobile Precision 7520 (Xeon E3-1535M v6 (Quad core, 3.1 GHz, 4.2 GHz turbo), 1TB SSD, memory: 64 GB (4x16 GB), 2,400 MHz DDR4 ECC SDRAM) was used to analyze sequencing data. The raw Illumina paired-end reads that passed through the Q30 filter were merged using PEAR software [27]. with default parameter on Linux. Nucleotide sequences encoding VHHs were extracted using combination of basic Linux command lines specifying typical nucleotide sequences conserved on VHHs frame regions. The VHHs-encoding sequences were translated based on standard genetic code using MEGA X software [28].

Purification of VHHs

Bacillus subtilis 168, which is deficient for nine proteases (i.e., epr, wprA, mpr, nprB, bpr, nprE, vpr, aprE and aprX) [29] and contains a sigma factor for a sporulation (sigF)-deficient mutant was used (JP4336082B2). B. subtilis was precultured in L medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl) and cultured in 2xL-Mal medium (2% tryptone, 1% yeast extract, 1% NaCl, 7.5% maltose hydrate, and 7.5 μg/mL MnSO₄) with 15 μg/mL tetracycline at 30°C 72 hr.

Supernatants were subjected to SDS-PAGE and western blotting. SDS-PAGE used SuperSep Ace 15–20% Tricine Gel (Wako), and then proteins in the gel were transferred to PVDF membrane using Trans-Blot Turbo Mini PVDF Transfer Packs (Bio-Rad) and Trans-Blot Turbo System (Bio-Rad). PVDF membranes were treated with 6xHis Tag Monoclonal Antibody (3D5) HRP (Thermo Fisher Scientific) for 6xHis-tagged proteins, and ANTI-FLAG M2-peroxidase (HRP)-conjugated (Sigma Aldrich) for FLAG (DYKDDDDK)-tagged proteins in iBind Western System (Thermo Fisher Scientific). Each tagged protein was visualized by 1-Step Ultra TMB-Blotting Solution (Thermo Fisher Scientific).

His-tagged K-874A was purified from the supernatants of culture medium using Ni-NTA agarose beads (Wako) and resuspended into PBS with 30 mM Imidazol. The K-874A-FLAG fraction was collected from the supernatants using an Amicon Ultra Centrifugal Filter Unit (Merck) and resuspended into PBS with 30 mM imidazol. The purified VHHs were stored at 4°C until use.

ELISA for evaluation of the binding specificity of K-874A

Microtiter wells of Nunc-Immuno Plate II (Thermo Fisher Scientific) were coated overnight at 4°C with 100 μL of His-tagged recombinant human-CoV-HKU1 and OC43, SARS-CoV-2 (Sino Biological) and SARS-CoV S1 subunits (The Native Antigen Company) diluted in PBS. The S1 subunits-coated wells were blocked with 200 μL of 5% skim milk in PBS for 1 hr at room temperature, and then washed at three times with 200 μL of PBST. 100 μL of 20 μg/mL K-874A-FLAG prepared with PBST were added to the wells, incubated for 1 hr, and then washed at three times with 200 μL of PBST. The binding of K-874A-FLAG to S1 subunits-His tag was detected by incubating for 1 hr at room temperature with mouse monoclonal ANTI-FLAG M2-Peroxidase (Merck) diluted with PBST. The wells were washed three times with PBST, and then 100 μL of substrate buffer prepared with an o-phenylenediamine dihydrochloride substrate tablet (Thermo Fisher Scientific), 10x Stable Peroxide Substrate Buffer (Thermo Fisher Scientific), and water. After incubating for 30 min at room temperature, the absorbance at 450 nm was immediately measured with Microplate Reader Infinite M1000 PRO (TECAN).

Biolayer interferometry

Binding affinities of K-874A and S1 protein were determined using biolayer interferometry (BLI) of Octet RED 384 (Pall Life Sciences, USA). The kinetic buffer was PBST for BLI. The temperature was fixed at 25°C. 50 μl of each measurement solution was added to a 384-well black plate (Fortebio), and the measurements were performed as below. 1) The loading baseline was measured in kinetic buffer for 30 sec. 2) K-874A-6xHis was immobilized on a HIS1K biosensor (Fortebio) and loaded until a 0.3-nm signal was achieved. 3) The measurement baseline was measured in kinetic buffer for 30 sec. 3) The loaded sensors were dipped into twofold serial dilutions from 245.8 nM of the SARS-CoV-2 (2019-nCoV) Spike S1-Fc Recombinant Protein (Sino Biological) to measure a 180-sec specific binding at the association step. 4) The dissociation was obtained by dipping the biosensors once more time into the kinetic buffer for 240 sec. The data were analyzed using Fortebio Octet analysis software (Date Analysis HT Version 11.1.2.48) (Fortebio) and kinetic parameters were determined using a 1:1 monovalent binding model.

Cells and viruses

VeroE6/TMPRSS2 were purchased from JCRB Cell Bank and preserved in our laboratory. Calu3 was gently gifted from Dr. Matsuyama in NIID. SARS-CoV-2, 2019-nCoV JPN/TY/WK-521, B1.1.7 (Alpha), B.1.351(Beta), P.1 (Gamma) and B.1.617.2 (Delta) were provided from National Institute of Infectious Diseases (NIID, Japan). KUH003 (Accession number LC630936) [20] is isolated from nasal swab of COVID-19 patients in Tokyo. KUH003 has D614G.

Neutralization assay

2.5 x10³ TCID₅₀ of SARS-CoV-2 (Multiplicity of Infection: MOI = 0.05) was incubated with serial diluted VHHs for 2 hours at 37°C and subsequently for 24 hr at 4°C. VHH-treated virus was incubated with 5.0x10⁴ of VeroE6/TMPRSS2 cells for 1 hr at 37°C, and the wells were washed to remove unbound viruses. At 24 hr after infection, the culture supernatant was used to determine the number of RNA copies of virus by qRT-PCR (SARS-CoV-2 Detection Kit, TOYOBO), according to manufacturer’s protocol. IC₅₀ was determined 1 day after infection.

Neutralization assay using Calu3 cells

2.5 x10³ TCID₅₀ of each SARS-CoV-2 variant (MOI = 0.05) was incubated with 5.0 x10⁴ of Calu3 cells for 1 hr at 37°C, and the wells were washed to remove unbound viruses. After washing, the cells were incubated with or without 150 μg/ml of K-874A for 2 days. At 2 days after infection, the culture supernatant was used to determine the number of RNA copies of virus by qRT-PCR (SARS-CoV-2 Detection Kit, TOYOBO), according to manufacturer’s protocol. Based on the RNA copies, each variant in the culture supernatant was adjusted 10,000 RNA copies and was determined their TCID₅₀ by infecting to VeroE6/TMPRSS2 cells.

ELISA for evaluation of the binding specificity of K-874A to variants

20 μg/ml of K-874A was immobilized on Pierce Nickle Coated Plates, Clear, 96-well (Thermo Fisher Scientific). PBST was used for washing after each step. K-874A-coated wells were blocked with 5% skim milk in PBST for 1 hr. Recombinant S proteins produced by HEK293 cells were purchased from BioServ. Each S protein was diluted 5, 50, or 500 ng/ml and 100 μl of diluted S protein was incubated for 1 hr. The S protein captured by K-874A was detected with anti-rhodopsin [1D4] (Abcam) mouse monoclonal antibody and anti-mouse IgG goat polyclonal antibody-HRP (Abcam). The o-phenylenediamine dihydrochloride substrate tablet (Thermo Fisher Scientific) dissolved with Stable Peroxide Substrate Buffer (Thermo Fisher Scientific) was used as a substrate. The absorbance at 450 nm was measured with GloMax Explorer System (Promega).

SARS-CoV-2 binding on and early replication in Vero/TMPRSS2 cells

2.5 x10⁴ TCID₅₀ (MOI = 0.5) of SARS-CoV-2 was incubated with 150 μg/ml of VHHs for 2 hr at 37°C and then 24 hr at 4°C and inoculated to 5.0 x10⁴ VeroE6/TMPRSS2 cells for an hr. Similarly, to compare early RNA replication in the infected cells, infected cells were collected at 3 or 6 hr after infection. Total RNA was extracted from the collected cells with Nucleospin RNA (TAKARA). Numbers of RNA copies of the virus were determined by qRT-PCR (SARS-CoV-2 Detection Kit, TOYOBO), according to manufacturer’s protocol.

Cell fusion assay

To quantify the cell fusion induced by S protein, HiBiT technology (Promega) was applied. HiBiT was linked to the C-terminal of ZsGreen (TAKARA) and transduced by lentivirus vector LVSIN-IRES-puro, and LgBiT was transduced by lentivirus vector LVSIN-IRES-Hyg [30]. Equal numbers of ZsGreen-HiBiT-expressing cells and LgBiT-expressing cells were plated on a 96-well plate (1603101, Thermo Fisher Scientific). S protein was transduced by lentivirus vector. At 14 hr after transduction, the culture supernatant was replaced to Opti-MEM (ThermoFisher Scientific), and 25 μL of diluted Nano-Glo live solution (Promega) was added into the each well. After mixing, the relative luminescence was measured by Ensight (Perkin Elmer).

ELISA assay for direct interaction of recombinant ACE2 and S protein

Diluted recombinant S protein (2 μg/mL) (#RP012383LQ, ABclonal) with PBS was incubated to immobilize on the maxisorp plate (464718, Thermo). After blocking with 1% BSA/PBS, 1 μg of K-874A diluted in 1%BSA/PBS was incubated to bind to S protein. Then, serial diluted recombinant ACE2 (from 1 to 0.06 μg/ml) (ab151852, abcam), subsequent rabbit anti-ACE2 antibody (HPA000288, Atlas Antibodies) and HRP-conjugated anti-rabbit IgG (7074S, Cell signaling) were incubated. To detect K-874A binding to immobilized S protein, serial diluted K-874A (1 to 0.002 μg/ml) in 1% BSA/PBS was incubated, and K-874A which bound to S protein was detected by HRP-conjugated anti-VHH antibody (#128-035-232, Jackson Immuno Research). Between each incubation, wells were washed with PBST. To color, o-phenylenediamine dihydrochloride (p6662, Merck) was used as a substrate of HRP and reaction was stopped with 2 M H₂SO₄. Signaling was quantified by Ensight (Perkin Elmer).

Immunofluorescence staining of SARS-CoV-2-infected cells

VeroE6/TMPRSS2 cells were infected SARS CoV-2 and fixed at 24 hr after infection. After treatment with 0.05% Triton-X and 3% BSA/PBS for permeabilization and blocking, cells were incubated with anti-NSP8 (5A10, GeneTex) or anti-dsRNA (rJ2, Merck) for 1 hr and subsequently with Alexa 568-conjugated goat anti-mouse IgG (A110301, Thermo Fisher Scientific) and Hoechest for an hour at room temperature. Images were taken by BZX800 (Keyence).

Infection to alveolar organoid and VHH treatment

Human alveolar-derived organoids were established [20]. Briefly, 3D cultured organoid compound in Matrigel was treated with cell recovery solution to remove the cells from the Matrigel. The cells were infected with SARS-CoV-2 (MOI = 5) for 1 hr at 37°C, washed to remove unbound virus, and compounded into Matrigel. After the Matrigel solidified, VHH (15 μg/mL) was added or not. Culture supernatants were collected at each time point and preserved at -80°C.

VHH treatment of SARS CoV-2-infected Syrian hamsters

2x10³ TCID₅₀ of SARS-CoV-2 was nasally inoculated to 7 weeks age of Syrian hamsters. Then 3 mg of K-874A was nasally administrated just before, 1 day or 1 and 2 days after virus inoculation. Each hamster was weighed every day up to 4 days after inoculation. Lung tissues were homogenized at 4 days after inoculation, and RNA was extracted with Nucleospin RNA (TAKARA) for qRT-PCR.

qRT-PCR for inflammatory cytokines

Primers and probes for hamsters were designed as reported [31]. The primers and probes are listed in Table 1. qRT-PCR experiments were performed on a Light Cycler 96 (Roche) with Luna Universal Probe One-Step RT-qPCR Kit (NEB), according to manufacturer’s instructions. Briefly, 50 ng of RNA was used for each reaction as template. The final concentrations of each test primer and probe set for target gene were 0.4 and 0.2 μM, respectively. The final concentrations of the internal control primer and probe set were 0.2 and 0.1 μM, respectively. Cycling conditions were as follows: 10 min at 50°C (initial reverse transcription), 20 sec at 95°C (inactivation and initial denaturation), and 40 cycles of 3 sec at 95°C, followed by 60 sec at 60°C per cycle.

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Table 1. Detailed sequence information of the primers and probes for qRT-PCR.

https://doi.org/10.1371/journal.ppat.1009542.t001

Cryo-electron microscopy

S protein trimers of SARS-CoV-2 (# SPN-C52H9, Acrobiosystems, 600 μg/mL) and K-874A (11 mg/mL) were mixed at 10:1 and kept at room temperature for 1 day. Aliquot (2.5 μL) of the sample was placed onto a holey-carbon copper grid (Quantifoil Micro Tools, R 1.2/1.3), previously glow-discharged in a plasma ion bombarder (PIB-10, Vacuum Device Inc.). The grid was then blotted (blotting time: 3.5 sec, blotting force: 7) and plunge-frozen at a condition of 95% humidity and 4°C by using a Vitrobot Mark IV (Thermo Fisher Scientific). The cryo-EM data were acquired with a Titan Krios at 300 kV (Thermo Fisher Scientific) and a Gatan K3 camera (Gatan) at a nominal magnification of 64,000, corresponding to 1.11Å per pixel on specimen. Each micrograph was recorded as a movie of 58 frames at the total dose of approximately 40 electrons per Ų. A GIF-quantum energy filter (Gatan) was used with a slit width of 20 eV to remove inelastically scattered electrons. Individual movies were subjected to per-frame drift correction by MotionCor2 [32]. The contrast transfer function parameters of each micrograph were estimated using CTFFIND4 [33] and the following 2D and 3D classification, 3D refinement, and local resolution calculation were performed with RELION3.1 software [34]. A total of 2,252,622 particles were automatically selected from 6,552 micrographs. The selected particles were divided into three groups and sorted by 2D classification to accelerate computation speed. The sorted particles were further divided into two classes by 3D classification. One is the structure in which K-874A binds to both RBD and NTD (type 1), and the other is the structure in which K-874A bind to only RBD (type 2). The ratio of type 1 and type 2 particles was approximately 9:1. In the case of type 1 particles, 3D classification was further performed against all particles classified as type 1, and 3D refinement was performed with only selected good classes. The final 3D reconstruction was computed with 115,297 particles. The global resolution of the final map was estimated to be 3.9Å (gold standard FSC criterion) by imposing C3 symmetry, but the external regions including K-874A, RBD, and NTD were too much noisy. Therefore, the external regions including K-874A, RBD, and NTD were individually extracted from symmetry-expanded particles, and focused refinement was performed. Using the “relion_particle_symmetry_expand” command [34], the regions including K-874A, RBD, and NTD in each particle image were independently expanded, and the generated 3 × 115,297 (345,891) sub-particle images were used for the 3D reconstruction. Next, to make a focused mask, the density including K-874A, RBD, and NTD was extracted from the final type 1 map using UCSF Chimera [35]. The extracted density was used to generate the mask using “Mask creation” command in RELION 3.1. The images containing K-874A, RBD, and NTD were extracted from the symmetry-expanded images with the prepared mask using “Particle subtraction” command in RELION3.1. These masked sub-particles were subjected to 3D classification without shift and rotation, and the sub-particle images (143,647 particles) of the selected good classes were used for 3D refinement by imposing C1 symmetry. The resolution of the subtracted map was estimated to be 5.0 Å based on a gold standard FSC. On the other hand, all type 2 particles from three groups were merged and subjected to 3D classification again. Then, selected good classes were subjected to 3D refinement. The final 3D map was reconstructed from 51,305 particles. The global resolution of the final map was estimated to be 4.4Å (gold standard FSC criterion) by imposing C3 symmetry, but the external region including K-874A, RBD, and NTD was too much noisy as the same as the type 1 particle. Therefore, the region, including K-874A, RBD, and NTD, was subjected to focused refinement in the same way as the type 1 particle. The resolution of the subtracted map was estimated to be 5.0Å, based on a gold standard FSC. The entire procedure and the processing data are summarized in S2 Fig and S1 Table.

The cryo-EM images of S protein trimer without K-874A were collected with a JEM2200FS electron microscope (JEOL) using a side-entry Gatan 626 cryo-specimen holder (Gatan). For image formation, a field-emission gun operated at 200 kV and an in-column (Omega-type) energy filter operating in zero-energy-loss mode with a slit width of 20 eV were used. Images of the frozen hydrated particles were recorded on a direct-detector CMOS camera (DE20, Direct Electron, LP) at a microscope magnification of 40,000×, corresponding to 1.422Å per pixel on the specimen. Using a low-dose method, the total electron dose on the specimen was about 20 electrons per Ų for a 3-sec exposure. The single particle analysis was performed with RELION3.1. The 3D map reconstructed from 14,235 particles was estimated to be 6.9Å, based on a gold standard FSC. The entire procedure and the processing data are summarized in S1 Fig and S1 Table.

Model building

The SWISS-MODEL [36] server was used to generate homology models of K-874A and S protein monomer of SARS-CoV-2 (SPN-C52H9) using the atomic models of VHH-72 (PDB ID: 6WAQ) and 2019-nCoV S protein (PDB ID: 6VSB) as templates, respectively. The amino acid sequence comparisons between SPN-C52H9 and 6VSB and between K-874A and VHH-72 are shown in S6 and S7 Figs. Multiple-sequence alignments of these proteins were performed using the PROMALS3D program [37]. The homology models were manually modified in the individual maps using COOT [38] and refined using PHENIX [39]. Type 1 and Type 2 structures consisting of the K-874A S protein trimer showed almost similar interactions with the same amino acid residues between K-874A and RBD, although the models were built independently. The amino acid residues in Type 1 and Type 2 are indicated in Figs 3C and S4C. The model statistics are summarized in S1 Table.