Antibodies utilizing VL6-57 light chains target a convergent cryptic epitope on SARS-CoV-2 spike protein driving the genesis of Omicron variants (preprint)

Continued evolution of SARS-CoV-2 generates variants to challenge antibody immunity established by infection and vaccination. A connection between population immunity and genesis of virus variants has long been suggested but its molecular basis remains poorly understood. Here, we identify a class of SARS-CoV-2 neutralising public antibodies defined by their shared usage of VL6-57 light chains. Although heavy chains of diverse genotypes are utilized, convergent HCDR3 rearrangements have been observed among these public antibodies to cooperate with germline VL6-57 LCDRs to target a convergent epitope defined by RBD residues S371-S373-S375. Antibody repertoire analysis identifies that this class of VL6-57 antibodies is present in SARS-CoV-2-naive individuals and is clonally expanded in most COVID-19 patients. We confirm that Omicron specific substitutions at S371, S373 and S375 mediate escape of antibodies of the VL6-57 class. These findings support that this class of public antibodies constitutes immune pressure promoting the introduction of S371L/F-S373P-S375F in Omicron variants. The results provide further molecular evidences to support that antigenic evolution of SARS-CoV-2 is driven by antibody mediated population immunity.

Sequential Glycosylations at the Multibasic Cleavage Site of SARS-CoV-2 Spike Protein Regulate Viral Activation, Assembly, and Infection (preprint)

The multibasic furin cleavage site at the S1/S2 boundary of the spike protein (S protein) is a hallmark of SARS-CoV-2 and is essential for its increased infectivity. O-glycosylation near the furin site catalyzed by host cell glycosyltransferases can theoretically hinder spike protein processing and impede viral infection, but so far such hypothesis has not been tested with authentic viruses. The mechanism for furin activation is not clearly understood either. Here in this study, we discovered that GalNAc-T3 and T7 together initiate clustered O-glycosylations in the multibasic S1/S2 boundary region, which inhibits furin processing of the spike protein and surprisingly suppresses the incorporation of S protein into virus-like-particles (VLPs). Mechanistic analysis revealed that the assembly of spike protein into VLPs relies on protein-protein interaction between the furin-cleaved S protein and a double aspartic motif on the membrane protein of SARS-CoV-2, suggesting a novel mechanism for furin activation of S protein. Interestingly, a point mutation at P681, found in the SARS-CoV-2 variants alpha and delta, resists the glycosylation by GalNAc-T3 and T7 and its inhibitory effect against furin processing. However, an additional mutation at N679 in the most recent omicron variant reverts this resistance, making it both prone to glycosylation in vitro and sensitive to the expression of GalNAc-T3 and T7 in human lung cells. Together, our results suggest a glycosylation-based defense mechanism of host cells against SARS-CoV-2 and reveal the host-pathogen interplay at this critical “battle field” as the virus first escapes and currently surrenders itself to the host cell glycosylation.

Broadly effective ACE2 decoy proteins protect mice from lethal SARS-CoV-2 infection (preprint)

As SARS-CoV-2 variants have been causing increasingly serious drug resistance problem, development of broadly effective and hard-to-escape anti-SARS-CoV-2 agents is in urgent need. Here we describe further development and characterization of two SARS-CoV-2 receptor decoy proteins, ACE2-Ig-95 and ACE2-Ig-105/106. We found that both proteins had potent and robust in vitro neutralization activities against diverse SARS-CoV-2 variants including Omicron, with an average IC50 of up to 37 pM. In a stringent lethal SARS-CoV-2 infection mouse model, both proteins lowered lung viral load by up to ~1000 fold, prevented the emergence of clinical signs in >75% animals, and increased animal survival rate from 0% (untreated) to >87.5% (treated). These results demonstrate that both proteins are good drug candidates for protecting animals from severe COVID-19. In a head-to-head comparison of these two proteins with five previously-described ACE2-Ig constructs, we found that two of these constructs, each carrying five surface mutations in the ACE2 region, had partial loss of neutralization potency against three SARS-CoV-2 variants. These data suggest that extensively mutating ACE2 residues near the RBD-binding interface should be avoided or performed with extra caution. Further, we found that both ACE2-Ig-95 and ACE2-Ig-105/106 could be produced to gram/liter level, demonstrating the developability of them as biologic drug candidates. Stress-condition stability test of them further suggests that more studies are required in the future to improve the stability of these proteins. These studies provide useful insight into critical factors for engineering and preclinical development of ACE2 decoys as broadly effective therapeutics against diverse ACE2-utilizing coronaviruses.

Disulfide stabilization reveals conserved dynamic features between SARS-CoV-1 and SARS-CoV-2 spikes. (preprint)

SARS-CoV-2 spike protein (S) is structurally dynamic and has been observed by cryo-EM to adopt a variety of prefusion conformations that can be categorized as locked, closed and open. The locked conformations feature tightly packed trimers with structural elements incompatible with RBD in "up" position. For SARS-CoV-2 S, it has been shown that the locked conformations are transient under neutral pH. Probably due to their transience, locked conformations remain largely uncharacterized for SARS-CoV-1 S. Intriguingly, locked conformations were the only conformations captured for S proteins of bat and pangolin origin SARS-related coronaviruses. In this study, we introduced x1, x2, and x3 disulfides into SARS-CoV-1 S. Some of these disulfides have been shown to preserve rare locked conformations when introduced to SARS-CoV-2 S. Introduction of these disulfides allowed us to image a variety of locked and other rare conformations for SARS-CoV-1 S by cryo-EM. We identified bound cofactors and structural features that are associated with SARS-CoV-1 S locked conformations. We compare newly determined structures to other available spike structures of Sarbecoviruses to identify conserved features and discuss their possible functions.

Efficient Targeted Delivery of Bifunctional Circular Aptamer ASO Chimera to Suppress the SARS-CoV-2 Proliferation and Inflammation (preprint)

Inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and excessive inflammation is the current task in the prevention and treatment of COVID-19. Here, we designed a dual-function circular aptamerASO chimera (circSApt-NASO) to suppress SARS-CoV-2 replication and inflammation. The chemically unmodified circSApt-NASO exhibited high serum stability by artificial cyclization, significantly enhancing the utility of oligonucleotides. It presents great efficiency in knocking down, demonstrating the superiority of the circular ASO as a novel tool for sequence-specific silencing of gene expression. Furthermore, we propose and demonstrate that the SApt binding to spike protein enables the chimera to be efficiently delivered into the susceptible host cells expressing ACE2 along with the infection of SARS-CoV-2. At high concentrations of SARS-CoV-2, the efficiency of targeted delivery of circSApt-NASO can even be compared to transfection. Among them, the anti-spike aptamer (SApt) that blocks the Spike-TLR4 interaction potently inhibits spike-induced inflammation. The NASO targeting to silence N genes not only display robust anti-N-induced inflammatory activity, but also achieve efficient inhibition of SARS-CoV-2 replication. Therefore, benefiting from the high stability of the cyclization, anti-spike aptamer-dependent and viral infection-mediate targeted delivery, the circSApt-NASO displays robust potential against authentic SARS-CoV-2 and Omicron (B.1.1.529), providing a promising specific anti-inflammatory and anti-proliferative reagent for therapeutic COVID-19 based on the oligonucleotide therapeutics strategy.

Priming conditions shape breadth of neutralizing antibody responses to sarbecoviruses (preprint)

Vaccines that are broadly cross-protective against current and future SARS-CoV-2 variants of concern (VOC) or across the sarbecoviruses subgenus remain a priority for public health. Virus neutralization is the best available correlate of protection. We used sera from cohorts of individuals vaccinated with two or three doses of RNA (BNT162b2) or inactivated SARS-CoV-2 (Coronavac or Sinopharm) vaccines with or without a history of previous SARS-CoV-2 or SARS-CoV-1 (in 2003) infection, to define the magnitude and breath of cross-neutralization in a multiplex surrogate neutralization assay based on virus spike receptor binding domain of multiple SARS-CoV-2 variants of concern (VOC), SARS-CoV-2 related bat and pangolin viruses, SARS-CoV-1 and related bat sarbecoviruses. SARS-CoV-2 or SARS-CoV-1 infection followed by BNT162b2 vaccine, Omicron BA.2 breakthrough infection following BNT162b2 vaccine or a third dose of BNT162b2 following two doses of BNT162b2 or CoronaVac elicited the highest and broadest neutralization across VOCs. Considering breadth and magnitude of neutralization across all sarbecoviruses, those infected with SARS-CoV-1 immunized with BNT162b2 outperformed all other combinations of infection and/or vaccination. These data may inform vaccine design strategies for generating broadly neutralizing antibodies to SARS-CoV-2 variants or across the sarbecovirus subgenus.

Safety and superior immunogenicity of heterologous boosting with an RBD-based SARS-CoV-2 mRNA vaccine in Chinese adults (preprint)

Homologous and heterologous booster with COVID-19 mRNA vaccines represent the most effective strategy to prevent the ongoing Omicron pandemic. The additional protection from these prototype SARS-CoV-2 S-targeting vaccine was attributed to the increased RBD-specific memory B cells with expanded potency and breadth. Herein, we show the safety and immunogenicity of heterologous boosting with the RBD-targeting mRNA vaccine AWcorna (also term ARCoV) in Chinese adults who have received two doses inactivated vaccine. The superiority over inactivated vaccine in neutralization antibodies, as well as the safety profile, support the use of AWcorna as heterologous booster in China.

A population antibody response against SARS-CoV-2 with differential neutralization activities towards the Delta and Omicron variants (preprint)

Population antibody response is believed to be important in selection of new variant viruses. We identified that SARS-CoV-2 infections elicit a population immune response mediated by a lineage of VH1-69 germline antibodies. The representative antibody R1-32 targets a novel semi-cryptic epitope defining a new class of RBD targeting antibodies. Binding to this non-ACE2 competing epitope leading to spike destruction impairing virus entry. Based on epitope location, neutralization mechanism and analysis of antibody binding to spike variants we propose that recurrent substitutions at 452 and 490 are associated with immune evasion of this population antibody response. These substitutions, including L452R found in the Delta variant, disrupt interaction mediated by the VH1-69 specific hydrophobic HCDR2 to impair antibody-antigen association allowing variants to escape. Lacking 452/490 substitutions, the Omicron variant is sensitive to this class of antibodies. Our results provide new insights into SARS-CoV-2 variant genesis and immune evasion.

Combating the SARS-CoV-2 Omicron variant with non-Omicron neutralizing antibodies (preprint)

The highly mutated and transmissible Omicron variant has provoked serious concerns over its decreased sensitivity to the current coronavirus disease 2019 (COVID-19) vaccines and evasion from most anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies (NAbs). In this study, we explored the possibility of combatting the Omicron variant by constructing bispecific antibodies based on non-Omicron NAbs. We engineered ten IgG-like bispecific antibodies with non-Omicron NAbs named GW01, 16L9, 4L12, and REGN10987 by fusing the single-chain variable fragments (scFvs) of two antibodies through a linker and then connecting them to the Fc region of IgG1. Surprisingly, eight out of ten bispecific antibodies showed high binding affinity to the Omicron receptor-binding domain (RBD) and exhibited extreme breadth and potency against pseudotyped SARS-CoV-2 variants of concern (VOCs) including Omicron, as well as authentic Omicron(+R346K) variants. Six bispecific antibodies containing the cross-NAb GW01 neutralized Omicron variant and retained their abilities to neutralize other sarbecoviruses. Bispecific antibodies inhibited Omicron infection by binding to the ACE2 binding site. A cryo-electron microscopy (cryo-EM) structure study of the representative bispecific antibody FD01 in complex with the Omicron spike (S) revealed 5 distinct trimers and one unique bi-trimer conformation. The structure and mapping analyses of 34 Omicron S variant single mutants elucidated that two scFvs of the bispecific antibody synergistically induced the RBD-down conformation into 3-RBD-up conformation, enlarged the interface area, accommodated the S371L mutation, improved the affinity between a single IgG and the Omicron RBD, and hindered ACE2 binding by forming bi-trimer conformation. Our study offers an important foundation for anti-Omicron NAb design. Engineering bispecific antibodies based on non-Omicron NAbs may provide an efficient solution to combat the Omicron variant.

Single Extracellular Vesicles (EV) Proteomic Profiling Altered and Identifies Co-Localization of SARS-CoV-2 Nucleocapsid Protein with CD81/Integrin-Rich EV Subpopulation in Sputum Samples of COVID-19 Patients (preprint)

Understanding the pathogenesis of SARS-CoV-2 is crucial to respond to the current coronavirus disease 2019 (COVID-19) pandemic. Sputum samples from 20 COVID-19 patients and healthy controls were collected, respectively. During the isolation of infectious SARS-CoV-2 virus, EV-like vesicles were associated with virions under a transmission electron microscope. Next, the expression of IL6 and TGF-β increased in EVs derived from the sputum of patients, and these were highly correlated with the expression of the SARS-CoV-2 N protein. Further, proximity barcoding assay (PBA) was used to investigate the immune-related proteins in the EVs, and the relationship between EVs and SARS-CoV-2 N protein in COVID-19 patients’ samples. Particularly, to investigate the differential contribution of the specific EV subsets, the protein expression of a single EV was detected and analyzed for the first time. Among the 40 EV subpopulations, 18 were found to have significant differences. The EV subpopulation regulated by CD81 were most likely to correlate with the changes in the pulmonary microenvironment after SARS-CoV-2 infection. This study provides evidence on the association between EVs and the SARS-CoV-2 virus, give a deep insight into the possible pathogenesis of SARS-CoV-2 infection and the possibility of nanoparticles drug intervention in viral infection.

The adenosine analogue prodrug ATV006 is orally bioavailable and has potent preclinical efficacy against SARS-CoV-2 and its variants (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the COVID-19 pandemic, is rapidly evolving. Due to the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOC), including the currently most prevalent Delta variant, orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously we showed that adenosine analogue 69-0 (also known as GS-441524), possesses potent anti-SARS-CoV-2 activity. Herein, we report that esterification of the 5-hydroxyl moieties of 69-0 markedly improved the antiviral potency. The 5-hydroxyl -isobutyryl prodrug, ATV006, showed excellent oral bioavailability in rats and cynomolgus monkeys and potent antiviral efficacy against different VOCs of SARS-CoV-2 in cell culture and three mouse models. Oral administration of ATV006 significantly reduced viral loads, alleviated lung damage and rescued mice from death in the K18-hACE2 mouse model challenged with the Delta variant. Moreover, ATV006 showed broad antiviral efficacy against different mammal-infecting coronaviruses. These indicate that ATV006 represents a promising oral drug candidate against SARS-CoV-2 VOCs and other coronaviruses.

Plasma cell-free DNA promise disease monitoring and tissue injury assessment of COVID-19 (preprint)

COVID-19 is a huge threat to global health. Due to the lack of definitive etiological therapeutics currently, effective disease monitoring is of high clinical value for better healthcare and management of the large number of COVID-19 patients. In this study, we recruited 37 COVID-19 patients, collected 176 blood samples upon diagnosis and during treatment, and analyzed cell-free DNA (cfDNA) in these samples. We report gross abnormalities in cfDNA of COVID-19 patients, including elevated GC content, altered molecule size and end motif patterns. More importantly, such cfDNA characteristics reflect patient-specific physiological conditions during treatment. Further analysis on tissue origin tracing of cfDNA reveals frequent tissue injuries in COVID-19 patients, which is supported by clinical diagnoses. Hence, we demonstrate the translational merit of cfDNA as valuable analyte for effective disease monitoring, as well as tissue injury assessment in COVID-19 patients.

SARS-CoV-2-Triggered Mast Cell Rapid Degranulation Induces Alveolar Epithelial Inflammation and Lung Injury (preprint)

SARS-CoV-2 infection-induced hyper-inflammation links to the acute lung injury and COVID-19 severity. Identifying the primary mediators that initiate the uncontrolled hypercytokinemia is essential for treatments. Mast cells (MCs) are strategically located at the mucosa and beneficially or detrimentally regulate immune inflammations. Here we showed that SARS-CoV-2-triggeed MC degranulation initiated alveolar epithelial inflammation and lung injury. SARS-CoV-2 challenge induced MC degranulation in ACE-2 humanized mice and rhesus macaques, and a rapid MC degranulation could be recapitulated with Spike-RBD binding to ACE2 in cells; MC degranulation alterred various signaling pathways in alveolar epithelial cells, particularly, led to the production of pro-inflammatory factors and consequential disruption of tight junctions. Importantly, the administration of clinical MC stabilizers for blocking degranulation dampened SARS-CoV-2-induced production of pro-inflammatory factors and prevented lung injury. These findings uncover a novel mechanism for SARS-CoV-2 initiating lung inflammation, and suggest an off-label use of MC stabilizer as immunomodulators for COVID-19 treatments.

Interferon-armed RBD dimer enhances the immunogenicity of RBD for sterilizing immunity against SARS-CoV-2 (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global crisis, urgently necessitating the development of safe, efficacious, convenient-to-store, and low-cost vaccine options. A major challenge is that the receptor-binding domain (RBD)-only vaccine fails to trigger long-lasting protective immunity if used solely for vaccination. To enhance antigen processing and cross-presentation in draining lymph nodes (DLNs), we developed an interferon (IFN)-armed RBD dimerized by immunoglobulin fragment (I-R-F). I-R-F efficiently directs immunity against RBD to DLN. A low dose of I-R-F induces not only high titer long-lasting neutralizing antibodies but also comprehensive T cell responses than RBD, and even provides comprehensive protection in one dose without adjuvant. This study shows that the I-R-F vaccine provides rapid and complete protection throughout upper and lower respiratory tracts against high dose SARS-CoV-2 challenge in rhesus macaques. Due to its potency and safety, this engineered vaccine may become one of the next-generation vaccine candidates in the global race to defeat COVID-19.

SUMOylation restrains ACE2 degradation through TOLLIP-mediated selective autophagy to facilitate the host susceptibility to SARS-CoV-2 infection (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Alongside investigations into the virology of SARS-CoV-2, understanding the host–virus dependencies are vital for the identification and rational design of effective antiviral therapy. Here, we report the dominant SARS-CoV-2 entry receptor, ACE2, conjugates with small ubiquitin-like modifier 3 (SUMO3) through a proteome-wide protein interaction analysis. We further demonstrate that E3 SUMO ligase PIAS4 prompts the SUMOylation and stabilization of ACE2, whereas deSUMOylation enzyme SENP3 reverses this process. Conjugation of SUMO3 with ACE2 at lysine (K) 187 hampers the K48-linked ubiquitination of ACE2, thus suppressing its subsequent cargo receptor TOLLIP-dependent autophagic degradation. Pharmacological intervention of ACE2 SUMOylation blocks the entry of SARS-CoV-2 and viral infection-triggered immune responses. Collectively, our findings suggest selective autophagic degradation of ACE2 orchestrated by SUMOylation and ubiquitination can be targeted to future antiviral therapy of SARS-CoV-2.

Single Extracellular Vesicles (EV) Proteomic Profiling Altered and Identifies Co-Localization of SARS-CoV-2 Nucleocapsid Protein with CD81/Integrin-Rich EV Subpopulation in Sputum Samples of COVID-19 Patients (preprint)

In response to the present coronavirus disease 2019 (COVID-19) pandemic, it is important to understand the infection pathogenesis of SARS-CoV-2. Sputum samples from 20 COVID-19 patients and healthy controls were collected, respectively. During the isolation of infectious SARS-CoV-2 virus, exosome-like vesicles were found associated with virions under transmission electron microscope. Next, the expression of IL6 and TGF-β increased in exosomes derived from the sputum of patients, and these were highly correlated with the expression of the SARS-CoV-2 N protein. Further, proximity barcoding assay (PBA) was used to investigate the immune related proteins in the exosomes, as well as the relationship between exosomes and SARS-CoV-2 N protein in COVID-19 patients’ samples. Particularly, to investigate the differential contribution of the specific exosome subsets, the protein expression of a single exosome was detected and analyzed for the first time. Among the 40 exosome subpopulations, 18 were found to have significant differences. The exosome subpopulation regulated by CD81 were most likely to correlate with the changes in the pulmonary microenvironment after SARS-CoV-2 infection. This study provides evidence on the association between exosomes and SARS-CoV-2 virus and promotes our understanding on possible pathogenesis of SARS-CoV-2 infection.Funding Statement: This work is supported by the emergency grants for prevention and control of SARS-CoV-2 of Ministry of Guangdong province (2020B111133001), the China Postdoctoral Science Project (2020T130025ZX and 2019M652860), the National Key Research and Development Program of China (2016YFC1304101), the Independent project of the State Key Laboratory of Respiratory Diseases (SKLRD-QN-201913), and the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01S155).Declaration of Interests: The authors have declared that no conflict of interest exists.Ethics Approval Statement: The present study obtained the approval of the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (Guangzhou, China).

Large-scale single-cell analysis reveals critical immune characteristics of COVID-19 patients (preprint)

Dysfunctional immune response in the COVID-19 patients is a recurrent theme impacting symptoms and mortality, yet the detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 205 COVID-19 patients and controls to create a comprehensive immune landscape. Lymphopenia and active T and B cell responses were found to coexist and associated with age, sex and their interactions with COVID-19. Diverse epithelial and immune cell types were observed to be virus-positive and showed dramatic transcriptomic changes. Elevation of ANXA1 and S100A9 in virus-positive squamous epithelial cells may enable the initiation of neutrophil and macrophage responses via the ANXA1-FPR1 and S100A8/9-TLR4 axes. Systemic up-regulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis and designing effective therapeutic strategies for COVID-19.

Modeling the Opening SARS-CoV-2 Spike: an Investigation of its Dynamic Electro-Geometric Properties (preprint)

The recent COVID-19 pandemic has brought about a surge of crowd-sourced initiatives aimed at simulating the proteins of the SARS-CoV-2 virus. A bottleneck currently exists in translating these simulations into tangible predictions that can be leveraged for pharmacological studies. Here we report on extensive electrostatic calculations done on an exascale simulation of the opening of the SARS-CoV-2 spike protein, performed by the Folding@home initiative. We compute the electric potential as the solution of the non-linear Poisson-Boltzmann equation using a parallel sharp numerical solver. The inherent multiple length scales present in the geometry and solution are reproduced using highly adaptive Octree grids. We analyze our results focusing on the electro-geometric properties of the receptor-binding domain and its vicinity. This work paves the way for a new class of hybrid computational and data-enabled approaches, where molecular dynamics simulations are combined with continuum modeling to produce high-fidelity computational measurements serving as a basis for protein bio-mechanism investigations.

SARS-CoV-2 viroporin triggers the NLRP3 inflammatory pathway (preprint)

Cytokine storm resulting from a heightened inflammatory response is a prominent feature of severe COVID-19 disease. This inflammatory response results from assembly/activation of a cell-intrinsic defense platform known as the inflammasome. We report that the SARS-CoV-2 viroporin encoded by ORF3a activates the NLRP3 inflammasome, the most promiscuous of known inflammasomes. ORF3a triggers IL-1 beta expression via NFkB, thus priming the inflammasome while also activating it via ASC-dependent and -independent modes. ORF3a-mediated inflammasome activation requires efflux of potassium ions and oligomerization between NEK7 and NLRP3. With the selective NLRP3 inhibitor MCC950 able to block ORF3a-mediated inflammasome activation and key ORF3a residues needed for virus release and inflammasome activation conserved in SARS-CoV-2 isolates across continents, ORF3a and NLRP3 present prime targets for intervention.