Molecular switches regulating the potency and immune evasiveness of SARS-CoV-2 spike protein (preprint)

What enabled SARS-CoV-2, but not other coronaviruses, to cause a global pandemic? Here we investigated key structural determinants of the pandemic. Using SARS-CoV-1 and bat RaTG13-CoV as comparisons, we identified two molecular switches that regulate the conformations of SARS-CoV-2 spike protein: (i) a furin motif loop turns SARS-CoV-2 spike from a closed conformation to a mixture of open and closed conformations, and (ii) a K417V mutation turns SARS-CoV-2 spike from mixed conformations to an open conformation. We showed that the open conformation favors viral potency by exposing the RBD for receptor binding and viral entry, while the closed conformation supports viral immune evasion by hiding the RBD from neutralizing antibodies. Hence SARS-CoV-2 spike has evolved to reach a balance between potency and immune evasiveness, which contributes to the pandemic spread of SARS-CoV-2.The dynamics between viral potency and invasiveness is likely to further evolve, providing insights into future evolution of SARS-CoV-2.

The Development of a Novel Nanobody Therapeutic for SARS-CoV-2 (preprint)

Combating the COVID-19 pandemic requires potent and low-cost therapeutics. We identified a novel series of single-domain antibodies (i.e., nanobody), Nanosota-1, from a camelid nanobody phage display library. Structural data showed that Nanosota-1 bound to the oft-hidden receptor-binding domain (RBD) of SARS-CoV-2 spike protein, blocking out viral receptor ACE2. The lead drug possessing an Fc tag (Nanosota-1C-Fc) bound to SARS-CoV-2 RBD with a Kd of 15.7picomolar (~3000 times more tightly than ACE2 did) and inhibited SARS-CoV-2 infection with an ND50 of 0.16microgram/milliliter (~6000 times more potently than ACE2 did). Administered at a single dose, Nanosota-1C-Fc demonstrated preventive and therapeutic efficacy in hamsters subjected to SARS-CoV-2 infection. Unlike conventional antibody drugs, Nanosota-1C-Fc was produced at high yields in bacteria and had exceptional thermostability. Pharmacokinetic analysis of Nanosota-1C-Fc documented a greater than 10-day in vivo half-life efficacy and high tissue bioavailability. Nanosota-1C-Fc is a potentially effective and realistic solution to the COVID-19 pandemic.

Biomechanical Characterization of SARS-CoV-2 Spike RBD and Human ACE2 Protein-Protein Interaction (preprint)

The current COVID-19 pandemic has led to a devastating impact across the world. SARS-CoV-2 (the virus causing COVID-19) is known to use receptor-binding domain (RBD) at viral surface spike (S) protein to interact with the angiotensin-converting enzyme 2 (ACE2) receptor expressed on many human cell types. The RBD-ACE2 interaction is a crucial step to mediate the host cell entry of SARS-CoV-2. Recent studies indicate that the ACE2 interaction with the SARS-CoV-2 S protein has higher affinity than its binding with the structurally identical S protein of SARS-CoV-1, the virus causing the 2002-2004 SARS outbreak. However, the biophysical mechanism behind such binding affinity difference is unclear. This study utilizes a combined single-molecule force spectroscopy and steered molecular dynamics (SMD) simulation approach to quantify the specific interactions between CoV-2 or CoV-1 RBD and ACE2. Depending on the loading rates, the unbinding forces between CoV-2 RBD and ACE2 range from 70 to 110 pN, and are 30-50% higher than those of CoV-1 RBD and ACE2 under similar loading rates. SMD results indicate that CoV-2 RBD interacts with the N-linked glycan on Asn90 of ACE2. This interaction is mostly absent in the CoV-1 RBD-ACE2 complex. During the SMD simulations, the extra RBD-N-glycan interaction contributes to a greater force and prolonged interaction lifetime. The observation is confirmed by our experimental force spectroscopy study. After the removal of N-linked glycans on ACE2, its mechanical binding strength with CoV-2 RBD decreases to a similar level of the CoV-1 RBD-ACE2 interaction. Together, the study uncovers the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1, and could aid in the development of new strategies to block SARS-CoV-2 entry. STATEMENT OF SIGNIFICANCEThis study utilizes a combined single-molecule force spectroscopy and steered molecular dynamics simulation approach to quantify the specific interactions between SARS-CoV-2 or SARS-CoV-1 receptor-binding domain and human ACE2. The study reveals the mechanism behind the difference in ACE2 binding between SARS-CoV-2 and SARS-CoV-1, and could aid in the development of new strategies to block SARS-CoV-2 entry.

Yeast-Expressed SARS-CoV Recombinant Receptor-Binding Domain (RBD219-N1) Formulated with Alum Induces Protective Immunity and Reduces Immune Enhancement (preprint)

We developed a severe acute respiratory syndrome (SARS) subunit recombinant protein vaccine candidate based on a high-yielding, yeast-engineered, receptor-binding domain (RBD219-N1) of the SARS beta-coronavirus (SARS-CoV) spike (S) protein. When formulated with Alhydrogel®, RBD219-N1 induced high-level neutralizing antibodies against both pseudotyped virus and a clinical (mouse-adapted) isolate of SARS-CoV. Here, we report that mice immunized with RBD219-N1/Alhydrogel® were fully protected from lethal SARS-CoV challenge (0% mortality), compared to ∼ 30% mortality in mice when immunized with the SARS S protein formulated with Alhydrogel®, and 100% mortality in negative controls. An RBD219-N1 formulation Alhydrogel® was also superior to the S protein, unadjuvanted RBD, and AddaVax (MF59-like adjuvant)-formulated RBD in inducing specific antibodies and preventing cellular infiltrates in the lungs upon SARS-CoV challenge. Specifically, a formulation with a 1:25 ratio of RBD219-N1 to Alhydrogel® provided high neutralizing antibody titers, 100% protection with non-detectable viral loads with minimal or no eosinophilic pulmonary infiltrates. As a result, this vaccine formulation is under consideration for further development against SARS-CoV and potentially other emerging and re-emerging beta-CoVs such as SARS-CoV-2.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Inhibition of SARS-CoV-2 infection (previously 2019-nCoV) by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion (preprint)

The recent outbreak of coronavirus disease (COVID-19) caused by SARS-CoV-2 infection in Wuhan, China has posed a serious threat to global public health. To develop specific anti-coronavirus therapeutics and prophylactics, the molecular mechanism that underlies viral infection must first be confirmed. Therefore, we herein used a SARS-CoV-2 spike (S) protein-mediated cell-cell fusion assay and found that SARS-CoV-2 showed plasma membrane fusion capacity superior to that of SARS-CoV. We solved the X-ray crystal structure of six-helical bundle (6-HB) core of the HR1 and HR2 domains in SARS-CoV-2 S protein S2 subunit, revealing that several mutated amino acid residues in the HR1 domain may be associated with enhanced interactions with HR2 domain. We previously developed a pan-coronavirus fusion inhibitor, EK1, which targeted HR1 domain and could inhibit infection by divergent human coronaviruses tested, including SARS-CoV and MERS-CoV. We then generated a series of lipopeptides and found that the EK1C4 was the most potent fusion inhibitor against SARS-CoV-2 S protein-mediated membrane fusion and pseudovirus infection with IC50s of 1.3 and 15.8 nM, about 241- and 149-fold more potent than that of EK1 peptide, respectively. EK1C4 was also highly effective against membrane fusion and infection of other human coronavirus pseudoviruses tested, including SARS-CoV and MERS-CoV, as well as SARSr-CoVs, potently inhibiting replication of 4 live human coronaviruses, including SARS-CoV-2. Intranasal application of EK1C4 before or after challenge with HCoV-OC43 protected mice from infection, suggesting that EK1C4 could be used for prevention and treatment of infection by currently circulating SARS-CoV-2 and emerging SARSr-CoVs.