A Novel Conserved Linear Neutralizing Epitope on the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein.

The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made it challenging to develop broad-spectrum prophylactic vaccines and therapeutic antibodies. Here, we have identified a broad-spectrum neutralizing antibody and its highly conserved epitope in the receptor-binding domain (RBD) of the spike protein (S) S1 subunit of SARS-CoV-2. First, nine monoclonal antibodies (MAbs) against the RBD or S1 were generated; of these, one RBD-specific MAb, 22.9-1, was selected for its broad RBD-binding abilities and neutralizing activities against SARS-CoV-2 variants. An epitope of 22.9-1 was fine-mapped with overlapping and truncated peptide fusion proteins. The core sequence of the epitope, 405D(N)EVR(S)QIAPGQ414, was identified on the internal surface of the up-state RBD. The epitope was conserved in nearly all variants of concern of SARS-CoV-2. MAb 22.9-1 and its novel epitope could be beneficial for research on broad-spectrum prophylactic vaccines and therapeutic antibody drugs. IMPORTANCE The continuous emergence of new variants of SARS-CoV-2 has caused great challenge in vaccine design and therapeutic antibody development. In this study, we selected a broad-spectrum neutralizing mouse monoclonal antibody which recognized a conserved linear B-cell epitope located on the internal surface of RBD. This MAb could neutralize all variants until now. The epitope was conserved in all variants. This work provides new insights in developing broad-spectrum prophylactic vaccines and therapeutic antibodies.

Alpha-Soluble NSF Attachment Protein Prevents the Cleavage of the SARS-CoV-2 Spike Protein by Functioning as an Interferon-Upregulated Furin Inhibitor.

Loss of the furin cleavage motif in the SARS-CoV-2 spike protein reduces the virulence and transmission of SARS-CoV-2, suggesting that furin is an attractive antiviral drug target. However, lack of understanding of the regulation of furin activity has largely limited the development of furin-based therapeutic strategies. Here, we find that alpha-soluble NSF attachment protein (α-SNAP), an indispensable component of vesicle trafficking machinery, inhibits the cleavage of SARS-CoV-2 spike protein and other furin-dependent virus glycoproteins. SARS-CoV-2 infection increases the expression of α-SNAP, and overexpression of α-SNAP reduces SARS-CoV-2 infection in cells. We further reveal that α-SNAP is an interferon-upregulated furin inhibitor that inhibits furin function by interacting with its P domain. Our study demonstrates that α-SNAP, in addition to its role in vesicle trafficking, plays an important role in the host defense against furin-dependent virus infection and therefore could be a target for the development of therapeutic options for COVID-19. IMPORTANCE Some key mutations of SARS-CoV-2 spike protein, such as D614G and P681R mutations, increase the transmission or pathogenicity by enhancing the cleavage efficacy of spike protein by furin. Loss of the furin cleavage motif of SARS-CoV-2 spike protein reduces the virulence and transmission, suggesting that furin is an attractive antiviral drug target. However, lack of understanding of the regulation of furin activity has largely limited the development of furin-based therapeutic strategies. Here, we found that in addition to its canonical role in vesicle trafficking, alpha-soluble NSF attachment protein (α-SNAP) plays an important role in the host defense against furin-dependent virus infection. we identified that α-SNAP is a novel interferon-upregulated furin inhibitor and inhibits the cleavage of SARS-CoV-2 spike protein and other furin-dependent virus glycoproteins by interacting with P domain of furin. Our study demonstrates that α-SNAP could be a target for the development of therapeutic options for COVID-19.

Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2.

Identification of bona fide functional receptors and elucidation of the mechanism of receptor-mediated virus entry are important to reveal targets for developing therapeutics against rabies virus (RABV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our previous studies suggest that metabotropic glutamate receptor subtype 2 (mGluR2) functions as an entry receptor for RABV in vitro, and is an important internalization factor for SARS-CoV-2 in vitro and in vivo. Here, we demonstrate that mGluR2 facilitates RABV internalization in vitro and infection in vivo. We found that transferrin receptor 1 (TfR1) interacts with mGluR2 and internalizes with mGluR2 and RABV in the same clathrin-coated pit. Knockdown of TfR1 blocks agonist-triggered internalization of mGluR2. Importantly, TfR1 also interacts with the SARS-CoV-2 spike protein and is important for SARS-CoV-2 internalization. Our findings identify a novel axis (mGluR2-TfR1 axis) used by RABV and SARS-CoV-2 for entry, and reveal TfR1 as a potential target for therapeutics against RABV and SARS-CoV-2. IMPORTANCE We previously found that metabotropic glutamate receptor subtype 2 (mGluR2) is an entry receptor for RABV in vitro, and an important internalization factor for SARS-CoV-2 in vitro and in vivo. However, whether mGluR2 is required for RABV infection in vivo was unknown. In addition, how mGluR2 mediates the internalization of RABV and SARS-CoV-2 needed to be resolved. Here, we found that mGluR2 gene knockout mice survived a lethal challenge with RABV. To our knowledge, mGluR2 is the first host factor to be definitively shown to play an important role in RABV street virus infection in vivo. We further found that transferrin receptor protein 1 (TfR1) directly interacts and cooperates with mGluR2 to regulate the endocytosis of RABV and SARS-CoV-2. Our study identifies a novel axis (mGluR2-TfR1 axis) used by RABV and SARS-CoV-2 for entry and opens a new door for the development of therapeutics against RABV and SARS-CoV-2.

An influenza virus vector candidate vaccine stably expressing SARS-CoV-2 receptor-binding domain produces high and long-lasting neutralizing antibodies in mice.

Viral infectious pathogens, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus, can cause extremely high infection rates and mortality in humans. Therefore, it is urgent to develop an effective vaccine against coronavirus and influenza virus infection. Herein, we used the influenza virus as a vector to express the SARS-CoV-2 spike receptor-binding domain (RBD) and hemagglutinin-esterase-fusion (HEF) protein of the influenza C virus. We then evaluated the feasibility and effectiveness of this design strategy through experiments in vitro and in vivo. The results showed that the chimeric viruses could stably express the HEF protein and the SARS-CoV-2 spike RBD at a high level. BALB/c mice, infected with the chimeric virus, exhibited mild clinical symptoms, yet produced high specific antibody levels against RBD and HEF, including neutralizing antibodies. Importantly, high neutralizing antibodies could be retained in the sera of mice for at least 20 weeks. Altogether, our data provided a new strategy for developing safe and effective COVID-19 and influenza virus vaccines.

Diltiazem inhibits SARS-CoV-2 cell attachment and internalization and decreases the viral infection in mouse lung.

The continuous emergence of severe acute respiratory coronavirus 2 (SARS-CoV-2) variants and the increasing number of breakthrough infection cases among vaccinated people support the urgent need for research and development of antiviral drugs. Viral entry is an intriguing target for antiviral drug development. We found that diltiazem, a blocker of the L-type calcium channel Cav1.2 pore-forming subunit (Cav1.2 α1c) and an FDA-approved drug, inhibits the binding and internalization of SARS-CoV-2, and decreases SARS-CoV-2 infection in cells and mouse lung. Cav1.2 α1c interacts with SARS-CoV-2 spike protein and ACE2, and affects the attachment and internalization of SARS-CoV-2. Our finding suggests that diltiazem has potential as a drug against SARS-CoV-2 infection and that Cav1.2 α1c is a promising target for antiviral drug development for COVID-19.

Immunogenicity of a recombinant VSV-Vectored SARS-CoV vaccine induced robust immunity in rhesus monkeys after single-dose immunization.

Severe acute respiratory syndrome (SARS) is a highly contagious zoonotic disease caused by SARS coronavirus (SARS-CoV). Since its outbreak in Guangdong Province of China in 2002, SARS has caused 8096 infections and 774 deaths by December 31st, 2003. Although there have been no more SARS cases reported in human populations since 2004, the recent emergence of a novel coronavirus disease (COVID-19) indicates the potential of the recurrence of SARS and other coronavirus disease among humans. Thus, developing a rapid response SARS vaccine to provide protection for human populations is still needed. Spike (S) protein of SARS-CoV can induce neutralizing antibodies, which is a pivotal immunogenic antigen for vaccine development. Here we constructed a recombinant chimeric vesicular stomatitis virus (VSV) VSVΔG-SARS, in which the glycoprotein (G) gene is replaced with the SARS-CoV S gene. VSVΔG-SARS maintains the bullet-like shape of the native VSV, with the heterogeneous S protein incorporated into its surface instead of G protein. The results of safety trials revealed that VSVΔG-SARS is safe and effective in mice at a dose of 1 â€‹× â€‹106 TCID50. More importantly, only a single-dose immunization of 2 â€‹× â€‹107 TCID50 can provide high-level neutralizing antibodies and robust T cell responses to non-human primate animal models. Thus, our data indicate that VSVΔG-SARS can be used as a rapid response vaccine candidate. Our study on the recombinant VSV-vectored SARS-CoV vaccines can accumulate experience and provide a foundation for the new coronavirus disease in the future.

SARS-CoV-2 uses metabotropic glutamate receptor subtype 2 as an internalization factor to infect cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses angiotensin-converting enzyme 2 (ACE2) as a binding receptor to enter cells via clathrin-mediated endocytosis (CME). However, receptors involved in other steps of SARS-CoV-2 infection remain largely unknown. Here, we found that metabotropic glutamate receptor subtype 2 (mGluR2) is an internalization factor for SARS-CoV-2. Our results show that mGluR2 directly interacts with the SARS-CoV-2 spike protein and that knockdown of mGluR2 decreases internalization of SARS-CoV-2 but not cell binding. Further, mGluR2 is uncovered to cooperate with ACE2 to facilitate SARS-CoV-2 internalization through CME and mGluR2 knockout in mice abolished SARS-CoV-2 infection in the nasal turbinates and significantly reduced viral infection in the lungs. Notably, mGluR2 is also important for SARS-CoV spike protein- and Middle East respiratory syndrome coronavirus spike protein-mediated internalization. Thus, our study identifies a novel internalization factor used by SARS-CoV-2 and opens a new door for antiviral development against coronavirus infection.

Construction and immunogenicity evaluation of a recombinantnewcastle disease virus expressing SARS-CoV spike protein

Severe Acute Respiratory Syndrome (SARS) is a zoonotic disease that is acute, feverish and accompanied by respiratory system and even multiple organ infections. Although no SARS infection cases have been reported since 2004, the outbreak of new coronavirus pneumonia (COVID-19) in Wuhan, Hubei Province, my country in December 2019 indicates that SARS coronavirus (SARS-CoV) or SARS-like coronavirus (SARSL-CoV) is highly likely to become widespread in the population again. In this study, a full-length cDNA clone pBRN-FL-SARS-CoV-S expressing the SARS-CoV spike protein (S protein) was first constructed, and the Newcastle Disease Virus (NDV) LaSota vaccine strain reverse genetic operating system was used to rescue The recombinant virus rLa-SARS-CoV-S expressing SARS-CoV S protein was identified. After rLa-SARS-CoV-S was infected with BHK-21 cells at a dose of MOI 0.01 for 36 hours, the SARS-CoV S protein was detected by western blot and laser confocal test. The results showed that the S protein was correct in the infected cells. Expressed and accurately located on the cell membrane. After inoculating 10-day-old SPF chicken embryos with rLa-SARS-CoV-S at a dose of 1x104 EID50, allantoic fluid was collected at different time points and the EID50 was determined. The growth kinetic curve of the virus showed that rLa-SARSCoV-S could The chicken embryo grows at high titer, consistent with the parental virus. Dilute the rLa-SARS-CoV-S and parent virus by 10-fold ratio and inoculate 10-day-old SPF chicken embryos and record the death time of each chicken embryo. Calculate the average chicken embryo death time according to the highest dilution of the virus. Lethal time (MDT), the results showed that the MDT of rLa-SARS-CoV-S was 112.8 h and the MDT of NDV LaSota was 96 h, indicating that the recombinant virus still maintains the low pathogenicity characteristics of the NDV LaSota vaccine strain. RLa-SARS-CoV-S and NDV LaSota were injected intramuscularly with 6-week-old BALB/c mice at a dose of 5x106 EID50 and boosted on the 21st day. At the same time, a PBS control group was set up. The results of the mouse safety test showed that all the mice vaccinated with rLa-SARS-CoV-S survived without any clinical symptoms, and the weight gain was consistent with that of the NDV LaSota group and the control group;the mice were treated on 21 d and 42 d after immunization Blood was collected to prepare serum, and the level of IgG antibodies against SARS-CoV S protein in mice was detected by ELISA. The results showed that the recombinant protein can induce mice to produce higher levels of specific IgG antibodies after initial immunization and booster immunization. The level can be maintained for a longer period of time. The results of this study indicate that rLa-SARS-CoV-S has potential value as a SARS-CoV vaccine candidate, and at the same time provides ideas for the development of SARS-CoV-2 vaccines.