Human Surfactant Protein D Binds Spike Protein and Acts as an Entry Inhibitor of SARS-CoV-2 Pseudotyped Viral Particles

Abstract

Human SP-D is a potent innate immune molecule whose presence at pulmonary mucosal surfaces allows its role in immune surveillance against pathogens. Higher levels of serum SP-D have been reported in the patients with severe acute respiratory syndrome coronavirus (SARS-CoV). Studies have suggested the ability of human SP-D to recognise spike glycoprotein of SARS-CoV; its interaction with HCoV-229E strain leads to viral inhibition in human bronchial epithelial (16HBE) cells. Previous studies have reported that a recombinant fragment of human SP-D (rfhSP-D) composed of 8 Gly-X-Y repeats, neck and CRD region, can act against a range of viral pathogens including influenza A Virus and Respiratory Syncytial Virus in vitro, in vivo and ex vivo. In this context, this study was aimed at examining the likely protective role of rfhSP-D against SARS-CoV-2 infection. rfhSP-D showed a dose-responsive binding to S1 spike protein of SARS-CoV-2 and its receptor binding domain. Importantly, rfhSP-D inhibited interaction of S1 protein with the HEK293T cells overexpressing human angiotensin converting enzyme 2 (hACE2). The protective role of rfhSP-D against SARS-CoV-2 infection as an entry inhibitor was further validated by the use of pseudotyped lentiviral particles expressing SARS-CoV-2 S1 protein; ~0.5 RLU fold reduction in viral entry was seen following treatment with rfhSP-D (10 µg/ml). These results highlight the therapeutic potential of rfhSP-D in SARS-CoV-2 infection and merit pre-clinical studies in animal models.

Keywords: SARS-COV-2; angiotensin converting enzyme 2; human pulmonary collectins; innate immunity; spike protein; surfactant protein D.

Figure 1

rfhSP-D and recombinant human full-length SP-D (hFL-SP-D) binding with the spike (S1) (A) and its RBD (B) of the SARS-CoV-2 was determined via direct ELISA. Microtiter wells were coated with SARS-CoV-2 spike S1 protein (5 µg/ml) (HEK 293 cells) or RBD (5 µg/ml) (HEK 293 cells) in carbonate-bicarbonate buffer, pH 9.6 overnight at 4°C. The following day, the wells were blocked with Tris Buffered Saline (TBS) buffer containing 1% BSA and 5mM CaCl₂, pH 7.2-7.4. After washing the wells with TBS, the wells were incubated with a series of two-fold dilutions of rfhSP-D or hFL-SP-D protein in blocking buffer at 4°C overnight. The binding between S1 protein and rfhSP-D was detected using biotinylated mouse anti-Human SP-D detection antibody (1:180), followed by probing with Streptavidin horseradish peroxidase (HRP)-conjugate 1:40. The data were expressed as mean of three independent experiments done in triplicates ± SEM. Significance was determined using the unpaired t test statistical analysis. The error bars show SEM. Control, maltose and EDTA groups compared to 2.5 µg or 5 µg S1 (RBD) in CaCl₂.

Figure 2

Competitive ELISA to show the impact of Maltose and EDTA on rfhSP-D binding to S1 (A) and its RBD (B). Polystyrene microtiter plates were coated with 2µg/ml rfhSP-D, and incubated with SARS-CoV-2 spike S1 protein (2.5 and 5 µg/ml) (sheep-IgG tag) or RBD (His-tag) (2.5 and 5 µg/ml). The binding was detected using anti-sheep IgG HRP antibodies (1:2000) or anti-His antibodies (1:2000). Absorbance at 450nm were recorded by VersaMax™ ELISA Microplate Reader. Significance was determined using the unpaired t test statistical analysis. The error bars show SEM. All group compared to RBD in CaCl₂ (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 3

Expression of ACE2 receptor on HEK293T cells by immunofluorescence microscopy (A), flow cytometry (B) and western blotting (C). (A) HEK293T (0.5x10⁵ cells) and HEK293T-ACE2 cells (0.5x10⁵ cells) were seeded on coverslips, followed by incubation at 37°C under standard culture conditions. After wasing the cells with PBS twice, the ACE2 expression was detected in both cell lines using the ACE2 antibody [SN0754](1:250), followed byincubation for 1 h at room temperature. Following PBS washes, Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (1:500) was added. Following PBS washes, the coverslips were mounted in medium with DAPI on a microscopy slide and viewed under a fluorescence microscope (Olympus). (B) Flow cytometric analysis of ACE2 expression was determined by the shift in the fluorescence intensity using ACE2 antibody [N1N2], N-term (GeneTex) (1:250). The ACE2 expression was detected by CytoFLEX. (C) The ACE2 expression was examined by western blotting using ACE2 antibody [SN0754] (GeneTex) (1:1000).

Figure 4

rfhSP-D treatment inhibits the interaction between SARS-CoV-2 S1 and ACE2 receptor on HEK293T cells. Protein complex was made by tagging SARS-CoV-2 S1 protein (5 ug/ml) with anti-His antibody (10ug/ml), followed by incubation with rfhSP-D (0.625, 1.25, 2.5, 5 or 10 µg/ml) for 2h at room temperature. This complex (S1+ anti-His+rfhSP-D) was added on to HEK293T-ACE2 cells (1x10⁵ cells) at 37°C for 2 h. The cells were collected and washed with FACS buffer twice and incubated with anti-mouse IgG PE conjugate (Genetex, GTX25881) (1:100) for 30 min and washed three times. The cells stained with S1 were detected by CytoFLEX. Significance was determined using the unpaired t test statistical analysis. All groups compared to S1. The error bars show SEM. M=mock (*p < 0.05; **p < 0.01; ****p < 0.0001) (n = 3).

Figure 5

rfhSP-D acts as an entry inhibitor of SARS-CoV-2 infection. (A) The SARS-CoV-2 pseudotyped lentiviral particle and pseudotyped lentiviral particle containing medium were determined the S1 expression by western blotting. (B) Luciferase reporter activity of rfhSP-D treated HEK293T cells (overexpressing ACE2 receptor) transduced with of SARS-CoV-2 S1 pseudotyped lentiviral particles. Significance was determined using the unpaired t test statistical analysis. All groups compared to VSV-S1. The error bars show SEM. M=medium (**p<0.01; ***p<0.001; ****p<0.0001) (n = 3).