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

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.

The role of angiopoietin-2 and surfactant protein-D levels in SARS-CoV-2-related lung injury: A prospective, observational, cohort study.

INTRODUCTION: Coronavirus disease-2019 (COVID-19) is a respiratory disease whose clinical manifestation ranges from asymptomatic to severe respiratory failure. The purpose of this study was to investigate the place of serum surfactant-D (SP-D) and angiopoetin-2 (Ang-2) levels in predicting severity of disease in patients diagnosed with COVID-19. METHODS: Sixty-four patients diagnosed with COVID-19 between September 2020 and February 2021, 50 patients diagnosed with community-acquired pneumonia and a 50-member healthy control group were included in the study. Plasma samples and clinical data were collected within 72 h after admission, during hospital stay. Serum SP-D and Ang-2 concentrations were measured using the enzyme-linked immunosorbent assay. RESULTS: SP-D and Ang-2 levels were significantly higher in the mild-moderate pneumonia and severe/critical patient groups compared to the asymptomatic and noncomplicated COVID-19 patients (p < 0.001 for all groups). Serum SP-D and Ang-2 levels of severe-critical COVID-19 patients were significantly higher than CAP patients (p < 0.001). Powerful correlation was present between clinical severity of COVID-19 and SP-D and Ang-2 levels (r = 0.885 p < 0.001 and r = 0.913 p < 0.001, respectively). Cut-off values of 37.7 ng/ml (AUC = 0.763, p < 0.001, 95% confidence interval [CI] = 0.667-0.860) for SP-D and 4208.3 pg/ml (AUC = 0.659, p = 0.004, 95% CI = 0.554-0.763) for Ang-2 were identified as predictors of COVID-19 disease at receiver operating characteristic curve analysis. CONCLUSION: SP-D and Ang-2 are predictive factors in differentiating COVID-19 patients and determining severity of disease. These data may be important for the initiation of treatment in the early stage of the disease in patients with COVID-19.

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

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.

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

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.

S1P lyase inhibition protects against sepsis by promoting disease tolerance via the S1P/S1PR3 axis.

BACKGROUND: One-third of all deaths in hospitals are caused by sepsis. Despite its demonstrated prevalence and high case fatality rate, antibiotics remain the only target-oriented treatment option currently available. Starting from results showing that low-dose anthracyclines protect against sepsis in mice, we sought to find new causative treatment options to improve sepsis outcomes. METHODS: Sepsis was induced in mice, and different treatment options were evaluated regarding cytokine and biomarker expression, lung epithelial cell permeability, autophagy induction, and survival benefit. Results were validated in cell culture experiments and correlated with patient samples. FINDINGS: Effective low-dose epirubicin treatment resulted in substantial downregulation of the sphingosine 1-phosphate (S1P) degrading enzyme S1P lyase (SPL). Consequent accumulation and secretion of S1P in lung parenchyma cells stimulated the S1P-receptor type 3 (S1PR3) and mitogen-activated protein kinases p38 and ERK, reducing tissue damage via increased disease tolerance. The protective effects of SPL inhibition were absent in S1PR3 deficient mice. Sepsis patients showed increased expression of SPL, stable expression of S1PR3, and increased levels of mucin-1 and surfactant protein D as indicators of lung damage. INTERPRETATION: Our work highlights a tissue-protective effect of SPL inhibition in sepsis due to activation of the S1P/S1PR3 axis and implies that SPL inhibitors and S1PR3 agonists might be potential therapeutics to protect against sepsis by increasing disease tolerance against infections. FUNDING: This study was supported by the Center for Sepsis Control and Care (CSCC), the German Research Foundation (DFG), RTG 1715 (to M. H. G. and I. R.) and the National Institutes of Health, Grant R01GM043880 (to S. S.).