Human complement Factor H and Properdin act as soluble pattern recognition receptors and differentially modulate SARS-CoV-2 Infection (preprint)

Severe cases of SARS-CoV-2 infection are characterised by an imbalanced immune response, excessive inflammation, and the development of acute respiratory distress syndrome, which can lead to multiorgan failure and death. Several studies have demonstrated dysregulated complement activity as an indicator of immunopathogenesis in the SARS-CoV-2 infection. Notably, the complement alternative pathway has been implicated in driving the excessive inflammation during severe SARS-CoV-2 infection. Reduced levels of factor H (FH), a down-regulator of the alternative pathway, and increased levels of properdin (Factor P/FP), the only known up-regulator of the alternative pathway, have been observed in individuals with severe COVID-19 infection. The present study investigated the complement activation-independent, and a more direct role of FH and FP against SARS-CoV-2 infection. Using direct ELISA, the interactions of FH and FP with the SARS-CoV-2 spike (S) and receptor binding domain (RBD) were assessed. Using S protein expressing lentiviral pseudotypes, the cell binding and luciferase-based virus entry assays were employed to assess the potential modulatory effects of FH, FP, and recombinant thrombospondin repeats 4 and 5 (TSR4+5) on SARS-CoV-2 cell entry. We also evaluated the immunomodulatory functions of FH and FP in the cytokine response triggered by SARS-CoV-2 pseudotypes via RT-qPCR. SARS-CoV-2 S and RBD proteins were found to bind both FH and FP. Treatment of A549 cells expressing human ACE2 and TMPRSS2 with FP or TSR4+5 resulted in increased cell entry and binding of SARS-CoV-2 pseudotypes. In silico studies revealed that FP increases affinity between SARS-CoV-2 and host ACE2. The impact of FP on viral cell entry and binding was reversed by anti-FP antibody treatment in A549-hACE2+TMPRSS2 cells. However, FH treatment reduced the cell entry and binding of SARS-CoV-2 lentiviral pseudotypes. Furthermore, the A549-hACE2+TMPRSS2 cells challenged with SARS-CoV-2 spike, envelope, nucleoprotein, and membrane protein expressing alphaviral pseudotypes pre-treated with FP or TSR4+5, exhibited upregulation of the transcripts of pro-inflammatory cytokines, such as IL-1{beta}, IL-8, IL-6, TNF-, IFN- and RANTES (as well as NF-kB). Conversely, FH pre-treatment downregulated the expression of these pro-inflammatory cytokines. Treatment of A549-hACE2+TMPRSS2 cells with FP increased S protein-mediated NF-kB activation, while FH treatment reduced it. These findings suggest that FH may act as an inhibitor of SARS-CoV-2 cell entry and binding, thereby attenuating the infection-associated inflammatory response in a complement activation-independent manner. FP may contribute to viral cell entry, binding, and exacerbating the immune response. That may result in potentially influencing the severity of the infection.

Human Surfactant Protein D Facilitates SARS-CoV-2 Pseudotype Binding and Entry in DC-SIGN Expressing Cells, and Downregulates Spike protein Induced Inflammation (preprint)

Pattern recognition receptors are crucial for innate anti-viral immunity, including C-type lectin receptors. Two such examples are Lung surfactant protein D (SP-D) and Dendritic cell-specific intercellular adhesion molecules-3 grabbing non-integrin (DC-SIGN) which are soluble and membrane-bound C-type lectin receptors, respectively. SP-D has a crucial immune function in detecting and clearing pulmonary pathogens; DC-SIGN is involved in facilitating dendritic cell interaction as an antigen-presenting cell with naive T cells to mount an anti-viral immune response. Both SP-D and DC-SIGN have been shown to interact with various viruses, including HIV-1, Influenza A virus and SARS-CoV-2. SARS-CoV-2 is an enveloped RNA virus that causes COVID-19. A recombinant fragment of human SP-D (rfhSP-D) comprising of -helical neck region, carbohydrate recognition domain, and eight N-terminal Gly-X-Y repeats has been shown to bind SARS-CoV-2 Spike protein and inhibit SARS-CoV-2 replication by preventing viral entry in Vero cells and HEK293T cells expressing ACE2. DC-SIGN has also been shown to act as a cell surface receptor for SARS-CoV-2 independent of ACE2. Since rfhSP-D is known to interact with SARS-CoV-2 Spike protein and DC-SIGN, this study was aimed at investigating the potential of rfhSP-D in modulating SARS-CoV-2 infection. Coincubation of rfhSP-D with Spike protein improved the Spike Protein: DC-SIGN interaction. Molecular dynamic studies revealed that rfhSP-D stabilised the interaction between DC-SIGN and Spike protein. Cell binding analysis with DC-SIGN expressing HEK 293T and THP- 1 cells and rfhSP-D treated SARS-CoV-2 Spike pseudotypes confirmed the increased binding. Furthermore, infection assays using the pseudotypes revealed their increased uptake by DC-SIGN expressing cells. The immunomodulatory effect of rfhSP-D on the DC-SIGN: Spike protein interaction on DC-SIGN expressing epithelial and macrophage-like cell lines was also assessed by measuring the mRNA expression of cytokines and chemokines. The RT-qPCR analysis showed that rfhSP-D treatment downregulated the mRNA expression levels of pro-inflammatory cytokines and chemokines such as TNF-, IFN-, IL-1{beta}, IL- 6, IL-8, and RANTES (as well as NF-{kappa}B) in DC-SIGN expressing cells challenged by Spike protein. Furthermore, rfhSP-D treatment was found to downregulate the mRNA levels of MHC class II in DC expressing THP-1 when compared to the untreated controls. We conclude that rfhSP-D helps stabilise the interaction of SARS- CoV-2 Spike protein and DC-SIGN and increases viral uptake by macrophages via DC-SIGN, suggesting an additional role for rfhSP-D in SARS-CoV-2 infection.

Human Surfactant Protein D Binds S1 and Receptor Binding Domain of Spike protein and acts as an entry inhibitor of SARS-CoV-2 Pseudotyped viral particles in vitro (preprint)

Human SP-D is a potent innate immune molecule whose presence at pulmonary mucosal surfaces allows immune surveillance role against pulmonary pathogens. Higher levels of serum SP-D have been reported in patients with severe acute respiratory syndrome coronavirus-1 (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 models. 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 Angiotensin Converting Enzyme 2. 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 rfhSP-D treatment (10 g/ml). The results highlight the therapeutic potential of rfhSP-D in SARS-CoV-2 infection and merits pre-clinical studies in murine models.

A recombinant fragment of Human surfactant protein D binds Spike protein and inhibits infectivity and replication of SARS-CoV-2 in clinical samples (preprint)

Rationale COVID-19 is an acute infectious disease caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Human surfactant protein D (SP-D) is known to interact with spike protein of SARS-CoV, but its immune-surveillance against SARS-CoV-2 is not known. Objective This 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. Methods rfhSP-D interaction with spike protein of SARS-CoV-2 and hACE-2 receptor was predicted via docking analysis. The inhibition of interaction between 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 studied by measuring the expression of RdRp gene of the virus using qPCR. Measurements and Main Results In-silico interaction studies indicated that three amino acid residues in the RBD of spike 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 and symptomatic, n=8 and negative controls n=15) demonstrated that treatment with 5μM rfhSP-D inhibited viral replication by ~5.5 fold and was more efficient than Remdesivir (100 μM). Approximately, a 2-fold reduction in viral infectivity was also observed after treatment with 5μM rfhSP-D. Conclusions These results conclusively demonstrate that the calcium independent rfhSP-D mediated inhibition of binding 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 a significant reduction in SARS-CoV-2 infection and replication in-vitro .