A Recombinant Fragment of Human Surfactant Protein D Binds Spike Protein and Inhibits Infectivity and Replication of SARS-CoV-2 in Clinical Samples

Abstract

Coronavirus disease (COVID-19) is an acute infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human SP-D (surfactant protein D) is known to interact with the spike protein of SARS-CoV, but its immune surveillance against SARS-CoV-2 is not known. The current study aimed to examine the potential of a recombinant fragment of human SP-D (rfhSP-D) as an inhibitor of replication and infection of SARS-CoV-2. The interaction of rfhSP-D with the spike protein of SARS-CoV-2 and human ACE-2 (angiotensin-converting enzyme 2) receptor was predicted via docking analysis. The inhibition of interaction between the spike protein and ACE-2 by rfhSP-D was confirmed using direct and indirect ELISA. The effect of rfhSP-D on replication and infectivity of SARS-CoV-2 from clinical samples was assessed by measuring the expression of RdRp gene of the virus using quantitative PCR. In silico interaction studies indicated that three amino acid residues in the receptor-binding domain of spike protein of SARS-CoV-2 were commonly involved in interacting with rfhSP-D and ACE-2. Studies using clinical samples of SARS-CoV-2-positive cases (asymptomatic, n = 7; symptomatic, n = 8) and negative control samples (n = 15) demonstrated that treatment with 1.67 μM rfhSP-D inhibited viral replication by ∼5.5-fold and was more efficient than remdesivir (100 μM) in Vero cells. An approximately two-fold reduction in viral infectivity was also observed after treatment with 1.67 μM rfhSP-D. These results conclusively demonstrate that the rfhSP-D mediated calcium independent interaction between the receptor-binding domain of the S1 subunit of the SARS-CoV-2 spike protein and human ACE-2, its host cell receptor, and significantly reduced SARS-CoV-2 infection and replication in vitro.

Keywords: COVID-19; SARS-CoV-2; entry inhibitor; spike protein; surfactant protein D.

Figure 1.

Tripartite interaction between spike protein (S protein) (green), recombinant fragment of human SP-D (surfactant protein D) (rfhSP-D) (red), and ACE-2 (angiotensin-converting enzyme 2) (blue) (A and B). ACE-2 residues Ser19, Lys31, Glu35 and His34 interact with both S protein and rfhSP-D. The interactions between S protein and ACE-2 are deduced from the crystal structure (Protein Data Bank identification 6VW1), and those between rfhSP-D and ACE-2 protein are based on docked complexes. B is a zoomed view of A. Individual intermolecular interactions between (C) S protein (green) and ACE-2 (blue), (D) S protein (green) and rfhSP-D (red), and (E) rfhSP-D (red) and ACE-2 (blue). The S protein residues Tyr449, Gln493, and Gln498 participate in intermolecular interactions with both ACE-2 and rfhSP-D.

Figure 2.

Heatmap representation of effect of single-residue mutations of rfhSP-D (y axis) on binding energy of docked rfhSP-D complexed with (A) ACE-2 and (B) S protein using mCSM-PPI2 web server. Each of the rfhSP-D residues involved in interaction with virus-binding hotspot of ACE-2 and the receptor-binding motif of the S protein were mutated to the standard amino acids, and its effect on binding energy of the complex was assessed. Most of the mutations led to decrease in the binding affinity (red cells). Few of the conserved substitutions led to increase in the binding affinity (blue cells), highlighting the likely functional importance of the mutated residues.

Figure 3.

rfhSP-D binds to the immobilized S protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2); immobilized rfhSP-D binds to human ACE-2 (hACE-2) in a dose-dependent, but calcium-independent, manner. ELISA showing binding of rfhSP-D to the immobilized S protein. Microtiter wells were coated with 0.3 μg/ml (0.54 nM) of S protein. rfhSP-D (20, 10, and 5 μg/ml or 0.334, 0.167, and 0.083 μM in PBS with 5 mM CaCl₂) were added to the wells. Full-length (FL) SP-D (20 μg/ml or 0.038 μM) was also used in a similar manner. BSA (20 μg/ml) was used as a non-specific protein control (mean of the normalized triplicates ± SEM = 0.07 ± 0.006 with polyclonal antibody and 0.025 ± 0.006 with monoclonal antibody). (A) S protein–SP-D binding was detected with either polyclonal or monoclonal antibodies against SP-D. (B) To assess the effect of calcium in the SP-D–S protein interaction, rfhSP-D (20, 10, and 5 μg/ml or 0.334, 0.167, and 0.083 μM) and FL SP-D (20 μg/ml or 0.038 μM), either with or without 10 mM EDTA, was used in a similar manner and probed with polyclonal antibodies against SP-D. (C) The binding of immobilized rfhSP-D to hACE-2 was assessed by coating microtiter wells with 0.1 μg/ml of FL SP-D (1.9 nM) or rfhSP-D (16.7 nM). BSA (0.1 μg/ml) was used as a non-specific protein control (mean of the normalized triplicates ± SEM = 0.006 ± 0.005). Decreasing concentration of hACE-2 (0.12, 0.06, and 0.00 μg/ml or 0.52, 0.26, and 0.0 nM) was added to the wells. The SP-D–hACE-2 binding was detected using streptavidin-HRP. The background was subtracted from all data points. The data were expressed as the mean of triplicates ± SD. Significance was determined using the two-way ANOVA (n = 3) test. **P < 0.05 and ***P < 0.0001. ns = no significance.

Figure 4.

rfhSP-D inhibits the interaction between S protein of SARS-CoV-2 and biotinylated hACE-2 in a calcium-independent manner (A and B). Microtiter wells were coated with 0.3 μg/ml (0.54 nM) of S protein. (A) After blocking, rfhSP-D (5, 1, and 0 μg/ml or 83.5, 16.7, and 0.0 nM) was added and incubated for 1 hour, followed by probing with biotinylated hACE-2. (B) To assess the effect of calcium in the SP-D–mediated inhibition of S protein–hACE-2 interaction, 5 μg/ml or 83.5 nM of rfhSP-D and FL SP-D (5 μg/ml or 9.5 nM) with or without 10 mM EDTA was added. BSA (5 μg/ml) was used as nonspecific protein control (mean of the normalized triplicates ± SEM = 0.297 ± 0.005). S protein–hACE-2 binding was detected with streptavidin-HRP. Background was subtracted from all data points. The data were normalized with 100% S protein–hACE-2 binding being defined as the mean of the absorbance recorded from the control sample (0 μg/ml rfhSP-D). The data were presented as the mean of the normalized triplicates ± SEM. Significance was determined using the one-way ANOVA (n = 3). ***P < 0.0001.

Figure 5.

rfhSP-D inhibits the interaction between the receptor-binding domain (RBD) of the S protein of SARS-CoV-2 and biotinylated hACE-2 in a calcium-independent manner. Microtiter wells were coated with 0.1 μg/ml (2.5 nM) of S protein RBD. After blocking, decreasing concentrations of rfhSP-D (1, 0.5, 0.25, 0.125, and 0 μg/ml or 16.7, 8.35, 4.18, and 2.09 nM in PBS with 5 mM CaCl₂) (A) were incubated for 1 hour, followed by probing with biotinylated hACE-2. To assess the effect of calcium in the rfhSP-D–mediated inhibition of S protein RBD–hACE-2 interaction (B), 5 μg/ml (83.5 nM) of rfhSP-D or FL SP-D (5 μg/ml or 9.5 nM) with or without 10 mM EDTA was used. BSA (5 μg/ml) was used as a nonspecific protein control (mean of the normalized triplicates ± SEM = 0.894 ± 0.006 [this data is not plotted in the figure]). S protein RBD–hACE-2 binding was detected using streptavidin-HRP. Background was subtracted from all data points. The data obtained were normalized with 100% S protein RBD–hACE-2 binding being defined as the mean of the absorbance recorded from the control sample (0 μg/ml rfhSP-D). The data were presented as the mean of the normalized triplicates ± SEM. Significance was determined using the one-way ANOVA (n = 3). ***P < 0.0001.

Figure 6.

Determination of 50% tissue culture infective dose (TCID₅₀) value of the clinical samples in Vero cells using an MTT assay. Vero cells (5 × 10⁴/well) were seeded in complete MEM in 96-well culture plates and grown overnight at 37°C with 5% CO₂. Swab samples of 15 confirmed cases of coronavirus disease (COVID-19), including symptomatic contacts of laboratory-confirmed cases (category [Cat] 2) (n = 2), hospitalized patients with severe acute respiratory infections (Cat 4) (n = 3), asymptomatic direct and high-risk contacts of laboratory-confirmed cases (Cat 5a) (n = 7), and hospitalized patients with symptomatic influenza-like illness (Cat 6) (n = 3) who had tested positive by RT-PCR test for SARS-CoV-2, and 15 controls (at different dilutions/well) were added to the cells and incubated for 1 hour. The supernatants were removed, and the wells were washed twice with sterile PBS. Fresh complete MEM was added to the wells, and the cells were incubated for 96 hours. Viability of the cells was evaluated using an MTT assay. MTT (0.5 mg/ml)-containing medium was added to the wells for 4 hours. The supernatants were removed, and cells were lysed using DMSO. Absorbance was measured at 590 nm. The data obtained were normalized with 100% cell viability being defined as the mean of the absorbance recorded from the control sample (0 TCID₅₀/well), and TCID₅₀ units were evaluated in each sample. The same assay was used to validate the cytopathic effects of 100 TCID₅₀ and 50 TCID₅₀ units of the samples. Data for cytopathic effects of 100 TCID₅₀ units for all 15 cases and 15 controls have been provided in the Table E1. The representative data for cases (n = 2) and controls (n = 2) are presented as the mean of the normalized triplicates ± SEM. Significance was determined using the two-way ANOVA (n = 3) test. **P < 0.01 and ***P < 0.0001.

Figure 7.

rfhSP-D pretreatment of SARS-CoV-2 significantly inhibited its replication. Vero cells (5 × 10⁴/well) were seeded in complete MEM in 96-well culture plates and grown overnight at 37°C under 5% CO₂. Cells were washed with sterile PBS twice. SARS-CoV-2 clinical samples (100 TCID₅₀/well; MOI 0.01), including symptomatic contacts of laboratory-confirmed cases (Cat 2) (n = 2), hospitalized patients with severe acute respiratory infections (Cat 4) (n = 3), asymptomatic direct and high-risk contacts of laboratory-confirmed cases (Cat 5a) (n = 7), and hospitalized patients with symptomatic influenza-like illness (Cat 6) (n = 3) who had tested positive by RT-PCR test for SARS-CoV-2, were preincubated with rfhSP-D (0 μg/ml, 50 μg/ml, and 100 μg/ml or 0, 0.835, and 1.67 μM) in MEM containing 5 mM CaCl₂ for 1 hour at room temperature. The pretreated or untreated virus in the sample was added to the cells and incubated for 1 hour at 37°C under 5% CO₂. The wells were washed with PBS twice, and infection medium (MEM + 0.3% BSA) was added to the cells and incubated for 24 hours at 37°C. The supernatants were collected, and RNA was extracted by Perkin Elmer automated extractor and subjected to qRT-PCR for SARS-CoV-2. For control samples, the volume of the sample taken was equivalent to the volume of the case sample (100 TCID₅₀) in which no RdRp expression was detected. The relative expression of RdRp was calculated using rfhSP-D–untreated cells (0 μM rfhSP-D), infected with respective samples as the calibrator. Data are provided in Table E1, and data of representative cases (n = 2) are presented as the mean of triplicates (n = 3). Error bars represent ± SEM. Significance (compared with 100 μM remdesivir) was determined using the two-way ANOVA test. *P < 0.05 and ***P < 0.0001.

Figure 8.

rfhSP-D pretreatment of SARS-CoV-2 significantly inhibited its infectivity. Vero cells (5 × 10⁵/well) were seeded in complete MEM in 12-well culture plates and grown overnight at 37°C under 5% CO₂. Cells were washed with sterile PBS twice. SARS-CoV-2 clinical samples (500 TCID₅₀/well, multiplicity of infection, 0.05), including symptomatic contacts of laboratory-confirmed cases (Cat 2) (n = 2), hospitalized patients with severe acute respiratory infections (Cat 4) (n = 3), asymptomatic direct and high-risk contacts of laboratory-confirmed cases (Cat 5a) (n = 7), and hospitalized patients with symptomatic influenza-like illness (Cat 6) (n = 3) who had tested positive by RT-PCR test for SARS-CoV-2, were preincubated with rfhSP-D (0 μg/ml, 50 μg/ml, and 100 μg/ml or 0, 0.835, and 1.67 μM) in MEM containing 5 mM CaCl₂ for 1 hour at room temperature and 1 hour at 4°C. This pretreated or untreated virus-containing sample was added to the cells and incubated for 1 hour at 37°C under 5% CO₂. The wells were washed with PBS twice, infection medium (MEM + 0.3% BSA) was added to the cells and incubated for 2 hours at 37°C under 5% CO₂. The cells were scraped, and the media containing scraped cells were collected. RNA was extracted and subjected to RT-PCR for SARS-CoV-2. For control samples, the volume of the sample taken was equivalent to the volume of the case sample (500 TCID₅₀); no RdRp expression was detected. The relative expression of RdRp was calculated by using rfhSP-D untreated cells (0 μM rfhSP-D) infected with respective samples as the calibrator. Data are provided in Table E1, and data for representative cases (n = 2) are presented as the mean of triplicates (n = 3). Error bars represent ± SEM. Significance (compared with control sample [cells + virus]) was determined using the two-way ANOVA test. ***P < 0.0001.

Figure 9.

rfhSP-D binds spike/RBD and hACE-2 and inhibits SARS-CoV-2 infection and replication. Interaction of rfhSP-D with RBD/spike of SARS-CoV-2 and hACE-2 inhibit the interaction of RBD/spike and hACE-2, which is essential for viral entry into the host cells. This dual interaction of rfhSP-D plausibly causes the significant inhibition of infection and replication of SARS-CoV-2 in clinical samples. The figure was created using BioRender.com.