Structural insights into the binding of SARS-CoV-2, SARS-CoV, and hCoV-NL63 spike receptor-binding domain to horse ACE2.

Severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and human coronavirus (hCoV)-NL63 utilize ACE2 as the functional receptor for cell entry, which leads to zoonotic infection. Horses (Equus caballus) attracted our attention because the spike protein receptor-binding domains (RBDs) of SARS-CoV-2 and SARS-CoV-2-related coronaviruses bind equine ACE2 (eACE2) with high affinity. Here we show that eACE2 binds the RBDs of these three coronaviruses and also SARS-CoV-2 variants but with lower affinities compared with human ACE2 (hACE2). Structural analysis and mutation assays indicated that eACE2-H41 accounts for the lower binding affinity of eACE2 to the RBDs of SARS-CoV-2 variants (Alpha, Beta, and Gamma), SARS-CoV, and hCoV-NL63. Pseudovirus infection assays showed that the SARS-CoV-2 Delta strain (B.1.617.2) displayed a significantly increased infection efficiency in eACE2-expressing HeLa cells. Our results reveal the molecular basis of eACE2 binding to the RBDs of SARS-CoV, SARS-CoV-2, and hCoV-NL63, which provides insights into the potential animal transmission of these ACE2-dependent coronaviruses.

An anti-inflammatory and anti-fibrotic proprietary Chinese medicine nasal spray designated as Allergic Rhinitis Nose Drops (ARND) with potential to prevent SARS-CoV-2 coronavirus infection by targeting RBD (Delta)- angiotensin converting enzyme 2 (ACE2) binding.

BACKGROUND: Since the outbreak of COVID-19 has resulted in over 313,000,000 confirmed cases of infection and over 5,500,000 deaths, substantial research work has been conducted to discover agents/ vaccines against COVID-19. Undesired adverse effects were observed in clinical practice and common vaccines do not protect the nasal tissue. An increasing volume of direct evidence based on clinical studies of traditional Chinese medicines (TCM) in the treatment of COVID-19 has been reported. However, the safe anti-inflammatory and anti-fibrotic proprietary Chinese medicines nasal spray, designated as Allergic Rhinitis Nose Drops (ARND), and its potential of re-purposing for suppressing viral infection via SARS-CoV-2 RBD (Delta)- angiotensin converting enzyme 2 (ACE2) binding have not been elucidated. PURPOSE: To characterize ARND as a potential SARS-CoV-2 entry inhibitor for its possible preventive application in anti-virus hygienic agent. METHODS: Network pharmacology analysis of ARND was adopted to asacertain gene targets which were commonly affected by COVID-19. The inhibitory effect of ARND on viral infection was determined by an in vitro pseudovirus assay. Furthermore, ARND was confirmed to have a strong binding affinity with ACE2 and SARS-CoV-2 spike-RBD (Delta) by ELISA. Finally, inflammatory and fibrotic cell models were used in conjunction in this study. RESULTS: The results suggested ARND not only inhibited pseudovirus infection and undermined the binding affinity between ACE2 and the Spike protein (Delta), but also attenuated the inflammatory response upon infection and may lead to a better prognosis with a lower risk of pulmonary fibrosis. The data in this study also provide a basis for further development of ARND as an antiviral hygienic product and further investigations on ARND in the live virus, in vivo and COVID-19 patients. ARND holds promise for use in the current COVID-19 outbreak as well as in future pandemics. CONCLUSION: ARND could be considered as a safe anti-SARS-CoV-2 agent with potential to prevent SARS-CoV-2 coronavirus infection.

Dynamic Assay for Profiling Anti-SARS-CoV-2 Antibodies and Their ACE2/Spike RBD Neutralization Capacity.

Serological assays have been widely employed during the coronavirus disease 2019 (COVID-19) pandemic to measure antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to track seroconversion in populations. However, currently available assays do not allow determination of neutralization capacity within the assay protocol. Furthermore, commercial serology assays have a high buy-in cost that is inaccessible for many research groups. We have replicated the serological enzyme-linked immunosorbent assay for the detection of SARS-CoV-2 antibody isotypes, developed at the Icahn School of Medicine at Mount Sinai, New York. Additionally, we have modified the protocol to include a neutralization assay with only a minor modification to this protocol. We used this assay to screen local COVID-19 patient sera (n = 91) and pre-COVID-19 control sera (n = 103), and obtained approximate parity with approved commercial anti-nucleoprotein-based assays with these sera. Furthermore, data from our neutralization assay closely aligns with that generated using a spike-based pseudovirus infection model when a subset of patient sera was analyzed.