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.

Surfactant Protein-D: The Potential ARDS Biomarker And Its Role In COVID-19

“…This causes the number of SP-Ds that leak into the systemic circulation is not much because the number of SP-Ds in the lungs from the beginning has decreased. This is supported by a study conducted by Arroyo, who found that COVID-19 patients exhibited decreased pulmonary SP-D serum levels (median = 68.9 ng/mL) compared to SP-D serum levels reported in the literature for healthy subjects, which ranged from 900-1300 ng/mL (Arroyo et al, 2021). This suggests that initial administration of natural surfactants should also improve lung function in adult patients with severe ARDS.…”

Commentary: Pattern Recognition Proteins: First Line of Defense Against Coronaviruses

“…Arroyo et al studied 12 patients > 18 years old who had respiratory failure requiring intubation and mechanical ventilation secondary to COVID-19 ( 5 ). SARS-CoV-2 infection was confirmed with PCR testing.…”

“…Arroyo et al also demonstrated that a full-length recombinant human SP-D bound the Spike protein (S-protein) of the SARS-CoV-2 virus in a calcium-dependent manner involving the carbohydrate recognition domain ( 5 ). This process also showed that protein cross bridges formed when binding the S-protein in the presence of another molecule (maltose-coated beads) suggesting the ability of SP-D to aggregate the virus and potentially facilitate viral clearance.…”

“…Labarrere and Kassab recently wrote an informative paper that includes a very interesting overview of the role Surfactant Protein D (SP-D) plays as part of the innate immune system in the defense against several viral pathogens including SARS-CoV-2 ( 1 ). It has been described that the Carbohydrate Recognition Domain (CRD) of SP-D recognizes and binds to carbohydrates present in certain viruses such as the hemagglutinin of Influenza A Virus ( 2 , 3 ) or the Spike (S)-Protein of SARS-CoV and SARS-Cov-2 ( 4 , 5 ). Binding promotes agglutination and clearance of the virus ( 6 ).…”

“…We would like to point out that in addition to high levels of serum levels being associated with COVID-19 severity, low levels of SP-D in the lung as measured in bronchoalveolar lavage fluid (BALF) have been reported in severe COVID-19 ( 5 ) and have been associated with an increased risk of the acute respiratory distress syndrome (ARDS) ( 12 ). These observations resulted in the filing of a New Investigational Drug (IND) application for a recombinant human surfactant Protein D (rhSP-D) formulation administered as a therapeutic agent for patients with severe COVID-19 with respiratory failure, as well as the initiation of a Phase 1B study this therapy, that is currently enrolling patients (NCT 04659122 ).…”