Full-Length Recombinant hSP-D Binds and Inhibits SARS-CoV-2

Abstract

SARS-CoV-2 infection of host cells is driven by binding of the SARS-CoV-2 spike-(S)-protein to lung type II pneumocytes, followed by virus replication. Surfactant protein SP-D, member of the front-line immune defense of the lungs, binds glycosylated structures on invading pathogens such as viruses to induce their clearance from the lungs. The objective of this study is to measure the pulmonary SP-D levels in COVID-19 patients and demonstrate the activity of SP-D against SARS-CoV-2, opening the possibility of using SP-D as potential therapy for COVID-19 patients. Pulmonary SP-D concentrations were measured in bronchoalveolar lavage samples from patients with corona virus disease 2019 (COVID-19) by anti-SP-D ELISA. Binding assays were performed by ELISAs. Protein bridge and aggregation assays were performed by gel electrophoresis followed by silver staining and band densitometry. Viral replication was evaluated in vitro using epithelial Caco-2 cells. Results indicate that COVID-19 patients (n = 12) show decreased pulmonary levels of SP-D (median = 68.9 ng/mL) when compared to levels reported for healthy controls in literature. Binding assays demonstrate that SP-D binds the SARS-CoV-2 glycosylated spike-(S)-protein of different emerging clinical variants. Binding induces the formation of protein bridges, the critical step of viral aggregation to facilitate its clearance. SP-D inhibits SARS-CoV-2 replication in Caco-2 cells (EC₉₀ = 3.7 μg/mL). Therefore, SP-D recognizes and binds to the spike-(S)-protein of SARS-CoV-2 in vitro, initiates the aggregation, and inhibits viral replication in cells. Combined with the low levels of SP-D observed in COVID-19 patients, these results suggest that SP-D is important in the immune response to SARS-CoV-2 and that rhSP-D supplementation has the potential to be a novel class of anti-viral that will target SARS-CoV-2 infection.

Keywords: ACE2; COVID-19; SP-D; anti-inflammatory; spike-protein.

Figure 1

rhSP-D binds to the spike-(S)-protein of SARS-CoV-2. ELISA binding assays were performed, coating the plate wells with S₁-protein. (A) The binding of rhSP-D to the Wuhan variant S₁-protein was determined by anti-SP-D ELISA under different conditions: 5 mM calcium, 200 mM maltose, and 50 mM EDTA; additionally, wells were coated with bovine serum albumin (BSA) instead of S-protein to determine nonspecific binding of rhSP-D and show that the binding to the S-protein was specific. The binding of rhSP-D to S₁-protein in the presence of calcium was significantly different to the binding of rhSP-D in the presence of maltose (p = 0.004) or EDTA (p = 0.02) (Kruskal–Wallis with Dunn’s post hoc test). Applying a one-binding site model the apparent Kd was 1.65 and the apparent B_max was 1.35. Binding of rhSP-D to the S₁-protein variant from Wuhan, compared to a S₁-protein enclosing different mutations, was tested: N501Y (B), D614G (C), E484K + D614G (D) or K417N + E484K + N501Y + D614G (E); binding was significantly different in the last mutant compared to the Wuhan variant (E) p = 0.0005 (Wilcoxon test). (F,G) rhSP-D binding to relevant clinical variants of S₁-protein (Wuhan, Brazil (G), South Africa (G) and U.K. (F)) was determined and rhSP-D bound to all the variants tested; binding was significantly different in the Brazil variant compared to the Wuhan variant (G) p = 0.002 (Friedman with Dunn’s test). n = 4, error bars represent standard deviation.

Figure 2

rhSP-D forms protein bridges with the spike (S)-protein of SARS-CoV-2. (A,B) The fractions obtained from supernatant (S_A and S_B) and eluted fraction from pellet (P) were analyzed by SDS-PAGE and developed by silver-staining to detect S₁-protein (migrates as 100–140 kDa) and rhSP-D (43 kDa). (A) Pre-mix approach: rhSP-D and S₁-protein (Wuhan variant) were incubated before adding maltose-coated beads. The process is described in the schematic diagram. “S_A” supernatant contained rhSP-D bound to S₁-protein, which did not interact with the maltose-beads. The eluted fraction (“P”) comprised the rhSP-D that was bound to both S₁-protein and maltose-beads, forming a protein bridge. Two concentrations of rhSP-D were tested 4 μg (lanes 1–4) and 2 μg (lanes 5–10) with 2 μg S₁-protein (lanes 3, 4, 7, 8, 9, 10) or only buffer as control (lanes 1, 2, 5, 6). The experiment was performed in the presence of 5 mM calcium (lanes 1–8), except elution of the pellet (P), which was carried out with 20 mM EDTA; non-binding control was performed in the presence of 20 mM EDTA (lanes 9–10). (B) The 1st-rhSP-D approach: rhSP-D was first incubated with maltose beads. Then, the excess of rhSP-D was removed (supernatant “S_A”) and S₁-protein (Wuhan variant) was added. “S_B” supernatant contained rhSP-D only bound to S₁-protein, and the eluted fraction (P) contained rhSP-D that remained bound to the maltose beads and also to S₁-protein, forming the protein bridges. Samples contained 2 μg of rhSP-D and 2 μg S₁-protein (lanes 7–12) or buffer (lanes 1–6). Binding of rhSP-D to maltose beads was always performed in the presence of calcium (S_A, lanes: 1, 4, 7, 10). Binding to S₁-protein was performed at 5 mM calcium (S_B, lanes: 2, 8) or 20 mM EDTA as non-binding control (S_B, lanes: 5, 11); elution (P) was carried out with 20 mM EDTA (E, lanes: 3, 6, 9, 12). The bar graph shows the densitometry of the eluted (P) bands in the pre-mix VS 1st-rhSP-D approaches at 4 μg of rhSP-D in the presence of S₁-protein or buffer; ANOVA with Sidak post-test was performed, error bars represent standard deviation, densitometry (n = 2).

Figure 3

ACE2 does not interfere in the binding of rhSP-D to the spike-(S)-protein of SARS-CoV-2. (A,B) An ELISA assay was performed to determine if rhSP-D could decrease the binding of ACE2 to S₁-protein (Wuhan variant). Binding of ACE2 to S₁-protein immobilized on the wells was determined in the presence of different concentrations of rhSP-D 1 μg/mL (blue, circles), 0.5 μg/mL (red, squares) or 0.1μg/mL (green, upward triangles) and 0 μg/mL (pink, downward triangles) (positive control for the binding of ACE2 to S₁-protein). rhSP-D promoted a slight decrease in the interaction between S₁-protein and ACE2. The reduction induced by rhSP-D at 0.5 μg/mL was significant compared to the positive control (no rhSP-D: 0 μg/mL). (C,D) Binding of S₁-protein to rhSP-D (immobilized on the wells) in the presence of different concentrations of ACE2: 3 μg/mL (purple, circles), 0.37 μg/mL (blue, squares) or 0.045 μg/mL (green, upward triangles) and 0 μg/mL (orange, downward triangles) (positive control for binding of S₁-protein to rhSP-D) was determined by ELISA. There was only a discrete reduction in the binding of S₁-protein to rhSP-D at the highest concentration of ACE2 tested (3 μg/mL). A–D, ANOVA with Tukey post-test, p-values in bar graphs when significant. n = 2, error bars represent standard deviation for duplicates.

Figure 4

rhSP-D inhibits SARS-CoV-2 cell replication. Viral titers in cells after infection with SARS-CoV-2 and treatment with rhSP-D at different concentrations. The viral titer in the cell supernatant is reported as CCID50 (50% cell culture infectious dose). Individual data points represent the average of three replicates. The concentration of rhSP-D to inhibit viral replication by 90% (EC₉₀) was 3.7 μg/mL.