An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants (preprint)

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential. Many of its mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of most therapeutic monoclonal antibodies. Here we describe a receptor-blocking human monoclonal antibody, 87G7, that retains ultrapotent neutralization against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) Variants-of-Concern (VOCs). Structural analysis reveals that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protects mice and/or hamsters against challenge with all current SARS-CoV-2 VOCs. Our findings may aid the development of sustainable antibody-based strategies against COVID-19 that are more resilient to SARS-CoV-2 antigenic diversity.

The glycosylated extracellular domain of MUC1 protects against SARS-CoV-2 infection at the respiratory surface (preprint)

Mucins play an essential role in protecting the respiratory tract against microbial infections. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance while transmembrane mucins MUC1, MUC4, and MUC16 restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on SARS-CoV-2 spike-mediated epithelial entry. Human lung epithelial Calu-3 cells have endogenous expression of the SARS-CoV-2 entry receptor ACE2 and express high levels of glycosylated MUC1 on the surface but not MUC4 and MUC16. Removal of the MUC1 extracellular domain (ED) using the O-glycan-specific mucinase StcE greatly enhanced spike binding and viral infection. By contrast, removal of mucin glycans sialic acid and fucose did not impact viral invasion. This study implicates the glycosylated ED of MUC1 as an important component of the host defense that restricts the severity of SARS-CoV-2 infection.

Isolation of cross-reactive monoclonal antibodies against divergent human coronaviruses that delineate a conserved and vulnerable site on the spike protein (preprint)

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry. As such, it is an attractive target for the development of protective antibodies and vaccines. Here we describe two human monoclonal antibodies, 1.6C7 and 28D9, that display a remarkable cross-reactivity against distinct species from three Betacoronavirus subgenera, capable of binding the spike proteins of SARS-CoV and SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both antibodies, derived from immunized transgenic mice carrying a human immunoglobulin repertoire, blocked MERS-CoV infection in cells, whereas 28D9 also showed weak cross-neutralizing potential against HCoV-OC43, SARS-CoV and SARS-CoV-2 in a neutralization-sensitive virus pseudotyping system, but not against authentic virus. Both cross-reactive monoclonal antibodies were found to target the stem helix in the spike protein S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle, that is antibody-accessible. We demonstrate that administration of these antibodies in mice protects from a lethal MERS-CoV challenge in both prophylactic and/or therapeutic models. Collectively, these antibodies delineate a conserved, immunogenic and vulnerabe site on the spike protein which spurs the development of broad-range diagnostic, preventive and therapeutic measures against coronaviruses.

A human monoclonal antibody targeting a conserved pocket in the SARS-CoV-2 receptor-binding domain core (preprint)

SARS-CoV-2 has caused a global outbreak of severe respiratory disease (COVID-19), leading to an unprecedented public health crisis. To date, there has been over thirty-three million diagnosed infections, and over one million deaths. No vaccine or targeted therapeutics are currently available. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralising SARS-CoV-2 and the related 2002/2003 SARS-CoV in vitro, and preventing SARS-CoV-2 induced pneumonia in a hamster model. Here we present the structural basis of its neutralization mechanism. We describe cryo-EM structures of trimeric SARS-CoV and SARS-CoV-2 spike ectodomains in complex with the 47D11 Fab. These data reveal that 47D11 binds specifically to the closed conformation of the receptor binding domain, distal to the ACE2 binding site. The CDRL3 stabilises the N343 glycan in an upright conformation, exposing a conserved and mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. Interestingly, 47D11 preferentially selects for the partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies that target the exposed receptor-binding motif. Taken together, these results expose a cryptic site of vulnerability on the SARS-CoV-2 RBD and provide a structural roadmap for the development of 47D11 as a prophylactic or post-exposure therapy for COVID-19.

Highly potent anti-SARS-CoV-2 multi-DARPin therapeutic candidates (preprint)

Globally accessible preventive and therapeutic molecules against SARS-CoV-2 are urgently needed. DARPin molecules are an emerging class of novel therapeutics based on naturally occurring repeat proteins ([~]15 kDa in size) and can be rapidly produced in bacteria in large quantities. Here, we report the identification of 380 DARPin molecules specifically targeting the SARS-CoV-2 spike protein selected from a naive library of 1012 DARPin molecules. Using extensive biophysical and biochemical characterization, (pseudo)virus neutralization assays and cryo-EM analysis, 11 mono-DARPin molecules targeting either the receptor binding domain (RBD), the S1 N-terminal-domain (NTD) or the S2 domain of the SARS-CoV-2 spike protein were chosen. Based on these 11 mono-DARPin molecules, 31 anti-SARS-CoV-2 multi-DARPin molecules were constructed which can broadly be grouped into 2 types; multi-paratopic RBD-neutralizing DARPin molecules and multi-mode DARPin molecules targeting simultaneously RBD, NTD and the S2 domain. Each of these multi-DARPin molecules acts by binding with 3 DARPin modules to the SARS-CoV-2 spike protein, leading to potent inhibition of SARS-CoV-2 infection down to 1 ng/ml (12 pM) and potentially providing protection against viral escape mutations. Additionally, 2 DARPin modules binding serum albumin, conferring an expected half-life of about 3 weeks in humans, were included in the multi-DARPin molecules. The protective efficacy of one multi-DARPin molecule was studied in a Golden Syrian hamster SARS-CoV-2 infection model, resulting in a significant reduction in viral load and pathogenesis. In conclusion, the multi-DARPin molecules reported here display very high antiviral potency, high-production yield, and a long systemic half-life, and thereby have the potential for single-dose use for prevention and treatment of COVID-19.

Identification of a polymorphism in the N gene of SARS-CoV-2 that adversely impacts detection by a widely-used RT-PCR assay (preprint)

We identify a mutation in the N gene of SARS-CoV-2 that adversely affects annealing of a commonly used RT-PCR primer; epidemiologic evidence suggests the virus retains pathogenicity and competence for spread. This reinforces the importance of using multiple targets, preferably in at least 2 genes, for robust SARS-CoV-2 detection. Article Summary LineA SARS-CoV-2 variant that occurs worldwide and has spread in California significantly affects diagnostic sensitivity of an N gene assay, highlighting the need to employ multiple viral targets for detection.

The SARS-CoV-2 Spike mutation D614G increases entry fitness across a range of ACE2 levels, directly outcompetes the wild type, and is preferentially incorporated into trimers (preprint)

Early in the current pandemic, the D614G mutation arose in the Spike protein of SARS-CoV-2 and quickly became the dominant variant globally. Mounting evidence suggests D614G enhances viral entry. Here we use a direct competition assay with single-cycle viruses to show that D614G outcompetes the wildtype. We developed a cell line with inducible ACE2 expression to confirm that D614G more efficiently enters cells with ACE2 levels spanning the different primary cells targeted by SARS-CoV-2. Using a new assay for crosslinking and directly extracting Spike trimers from the pseudovirus surface, we found an increase in trimerization efficiency and viral incorporation of D614G protomers. Our findings suggest that D614G increases infection of cells expressing a wide range of ACE2, and informs the mechanism underlying enhanced entry. The tools developed here can be broadly applied to study other Spike variants and SARS-CoV-2 entry, to inform functional studies of viral evolution and vaccine development.

Furin Cleavage Site Is Key to SARS-CoV-2 Pathogenesis (preprint)

SARS-CoV-2 has resulted in a global pandemic and shutdown economies around the world. Sequence analysis indicates that the novel coronavirus (CoV) has an insertion of a furin cleavage site (PRRAR) in its spike protein. Absent in other group 2B CoVs, the insertion may be a key factor in the replication and virulence of SARS-CoV-2. To explore this question, we generated a SARS-CoV-2 mutant lacking the furin cleavage site ({Delta}PRRA) in the spike protein. This mutant virus replicated with faster kinetics and improved fitness in Vero E6 cells. The mutant virus also had reduced spike protein processing as compared to wild-type SARS-CoV-2. In contrast, the {Delta}PRRA had reduced replication in Calu3 cells, a human respiratory cell line, and had attenuated disease in a hamster pathogenesis model. Despite the reduced disease, the {Delta}PRRA mutant offered robust protection from SARS-CoV-2 rechallenge. Importantly, plaque reduction neutralization tests (PRNT50) with COVID-19 patient sera and monoclonal antibodies against the receptor-binding domain found a shift, with the mutant virus resulting in consistently reduced PRNT50 titers. Together, these results demonstrate a critical role for the furin cleavage site insertion in SARS-CoV-2 replication and pathogenesis. In addition, these findings illustrate the importance of this insertion in evaluating neutralization and other downstream SARS-CoV-2 assays. ImportanceAs COVID-19 has impacted the world, understanding how SARS-CoV-2 replicates and causes virulence offers potential pathways to disrupt its disease. By removing the furin cleavage site, we demonstrate the importance of this insertion to SARS-CoV-2 replication and pathogenesis. In addition, the findings with Vero cells indicate the likelihood of cell culture adaptations in virus stocks that can influence reagent generation and interpretation of a wide range of data including neutralization and drug efficacy. Overall, our work highlights the importance of this key motif in SARS-CoV-2 infection and pathogenesis. Article SummaryA deletion of the furin cleavage site in SARS-CoV-2 amplifies replication in Vero cells, but attenuates replication in respiratory cells and pathogenesis in vivo. Loss of the furin site also reduces susceptibility to neutralization in vitro.

Epitopes targeted by T cells in convalescent COVID-19 patients (preprint)

There is growing evidence pointing to the protective role of T cells against COVID-19. Vaccines eliciting targeted T cell responses have the potential to provide robust, long-lasting immunity. However, their design requires knowledge of the SARS-CoV-2-specific epitopes that can elicit a T cell response and confer protection across a wide population. Here, we provide a unified description of emerging data of SARS-CoV-2 T cell epitopes compiled from results of 8 independent studies of convalescent COVID-19 patients. We describe features of these epitopes relevant for vaccine design, while indicating knowledge gaps that can, in part, be augmented using prior immunological data from SARS-CoV. The landscape of SARS-CoV-2 T cell epitopes that we describe can help guide SARS-CoV-2 vaccine development as well as future immunological studies. A web-based platform has also been developed to complement these efforts.

Inhibiting coronavirus replication in cultured cells by chemical ER stress (preprint)

Coronaviruses (CoVs) are important human pathogens for which no specific treatment is available. Here, we provide evidence that pharmacological reprogramming of ER stress pathways can be exploited to suppress CoV replication. We found that the ER stress inducer thapsigargin efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types, (partially) restores the virus-induced translational shut-down, and counteracts the CoV-mediated downregulation of IRE1 and the ER chaperone BiP. Proteome-wide data sets revealed specific pathways, protein networks and components that likely mediate the thapsigargin-induced antiviral state, including HERPUD1, an essential factor of ER quality control, and ER-associated protein degradation complexes. The data show that thapsigargin hits a central mechanism required for CoV replication, suggesting that thapsigargin (or derivatives thereof) may be developed into broad-spectrum anti-CoV drugs. One Sentence Summary / Running titleSuppression of coronavirus replication through thapsigargin-regulated ER stress, ERQC / ERAD and metabolic pathways

Global and Local Mutations in Bangladeshi SARS-CoV-2 Genomes (preprint)

Corona Virus Disease-2019 (COVID-19) warrants comprehensive investigations of publicly available Severe Acute Respiratory Syndrome-CoronaVirus-2 (SARS-CoV-2) genomes to gain new insight about their epidemiology, mutations and pathogenesis. Nearly 0.4 million mutations were identified so far in [~]60,000 SARS-CoV-2 genomic sequences. In this study, we compared 207 of SARS-CoV-2 genomes reported from different parts of Bangladesh and their comparison with 467 globally reported sequences to understand the origin of viruses, possible patterns of mutations, availability of unique mutations, and their apparent impact on pathogenicity of the virus in victims of Bangladeshi population. Phylogenetic analyses indicates that in Bangladesh, SARS-CoV-2 viruses might arrived through infected travelers from European countries, and the GR clade was found as predominant in this region. We found 2602 mutations including 1602 missense mutations, 612 synonymous mutations, 36 insertions and deletions with 352 other mutations types. In line with the global trend, D614G mutation in spike glycoprotein was predominantly high (95.6%) in Bangladeshi isolates. Interestingly, we found the average number of mutations in ORF1ab, S, ORF3a, M and N of genomes, having nucleotide shift at G614 (n=459), were significantly higher (p[≤]0.001) than those having mutation at D614 (n=215). Previously reported frequent mutations such as P4715L, D614G, R203K, G204R and I300F were also prevalent in Bangladeshi isolates. Additionally, 87 unique amino acid changes were revealed and were categorized as originating from different cities of Bangladesh. The analyses would increase our understanding of variations in virus genomes circulating in Bangladesh and elsewhere and help develop novel therapeutic targets against SARS-CoV-2.

Multi-species ELISA for the detection of antibodies against SARS-CoV-2 in animals (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with millions of infected humans and hundreds of thousands of fatalities. As the novel disease - referred to as COVID-19 - unfolded, occasional anthropozoonotic infections of animals by owners or caretakers were reported in dogs, felid species and farmed mink. Further species were shown to be susceptible under experimental conditions. The extent of natural infections of animals, however, is still largely unknown. Serological methods will be useful tools for tracing SARS-CoV-2 infections in animals once test systems are validated for use in different species. Here, we developed an indirect multi-species ELISA based on the receptor-binding domain (RBD) of SARS-CoV-2. The newly established ELISA was validated using 59 sera of infected or vaccinated animals including ferrets, raccoon dogs, hamsters, rabbits, chickens, cattle and a cat, and a total of 220 antibody-negative sera of the same animal species. Overall, a diagnostic specificity of 100.0% and sensitivity of 98.31% was achieved, and the functionality with every species included in this study could be demonstrated. Hence, a versatile and reliable ELISA protocol was established that enables high-throughput antibody detection in a broad range of animal species, which may be used for outbreak investigations, to assess the seroprevalence in susceptible species or to screen for reservoir or intermediate hosts.

AI aided design of epitope-based vaccine for the induction of cellular immune responses against SARS-CoV-2 (preprint)

The heavy burden imposed by the COVID-19 pandemic on our society triggered the race towards the development of therapies or preventive strategies. Among these, antibodies and vaccines are particularly attractive because of their high specificity, low probability of drug-drug interaction, and potentially long-standing protective effects. While the threat at hand justifies the pace of research, the implementation of therapeutic strategies cannot be exempted from safety considerations. There are several potential adverse events reported after the vaccination or antibody therapy, but two are of utmost importance: antibody-dependent enhancement (ADE) and cytokine storm syndrome (CSS). On the other hand, the depletion or exhaustion of T-cells has been reported to be associated with worse prognosis in COVID-19 patients. This observation suggests a potential role of vaccines eliciting cellular immunity, which might simultaneously limit the risk of ADE and CSS. Such risk was proposed to be associated with FcR-induced activation of proinflammatory macrophages (M1) by Fu et al. 2020 and Iwasaki et al. 2020. All aspects of the newly developed vaccine (including the route of administration, delivery system, and adjuvant selection) may affect its effectiveness and safety. In this work we use a novel in silico approach (based on AI and bioinformatics methods) developed to support the design of epitope-based vaccines. We evaluated the capabilities of our method for predicting the immunogenicity of epitopes. Next, the results of our approach were compared with other vaccine-design strategies reported in the literature. The risk of immuno-toxicity was also assessed. The analysis of epitope conservation among other Coronaviridae was carried out in order to facilitate the selection of peptides shared across different SARS-CoV-2 strains and which might be conserved in emerging zootic coronavirus strains. Finally, the potential applicability of the selected epitopes for the development of a vaccine eliciting cellular immunity for COVID-19 was discussed, highlighting the benefits and challenges of such an approach.

SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model (preprint)

Effective clinical intervention strategies for COVID-19 are urgently needed. Although several clinical trials have evaluated the use of convalescent plasma containing virus-neutralizing antibodies, the effectiveness has not been proven. We show that hamsters treated with a high dose of human convalescent plasma or a monoclonal antibody were protected against weight loss showing reduced pneumonia and pulmonary virus replication compared to control animals. However, a ten-fold lower dose of convalescent plasma showed no protective effect. Thus, variable and relatively low levels of virus neutralizing antibodies in convalescent plasma may limit their use for effective antiviral therapy, favouring concentrated, purified (monoclonal) antibodies.

The Pattern of Liver Dysfunction in Patients with COVID-19: A Retrospective Study (preprint)

Background. Information about liver dysfunction in patients with COVID-19 is scarce. We aimed to explored the pattern and risk factors of liver dysfunction in patients with COVID-19.Methods. In this retrospective study, we included all consecutive confirmed patients with COVID-19 in Fuyang Second People’s Hospital between January 20 and February 25, 2020 and collected clinical characteristics until discharge. The pattern and risk factors of liver dysfunction, viral shedding and outcome were analyzed.Results. Totally, 146 patients were analyzed. The median age was 44.9 years and 54.1% were men, 43.8% patients presented liver dysfunction (22.6% on admission, 21.2% during hospitalization). The percentage of elevated ALT (15.1% on admission and 24.7% during hospitalization) were significantly higher than ALP (2.1% on admission and 3.4% during hospitalization) (P < 0.001). Four clinical types were identified, type 1 (persistent normal liver function, 56.2%), type 2 (normal liver function on admission developed to liver dysfunction during hospitalization, 21.2%), type 3 (liver dysfunction on admission restored to normal on discharge, 13.0%) and type 4 (persistent liver dysfunction, 9.6%). The median duration of viral shedding was 12.0 (type 1), 15.0 (type 2), 14.0 (type 3) and 18.0 (type 4) days (P < 0.001). Prolonged viral shedding and severity were potential risk factors associated with liver dysfunction. Conclusions. The incidence of liver dysfunction in patients with COVID-19 is common but not severe, which mainly due to SARS-CoV-2-mediated immune injury on hepatocyte rather than cholangiocyte, DILI and underlying chronic liver disease should not be neglect. 

SARS-CoV-2 specific antibody responses in COVID-19 patients (preprint)

A new coronavirus, SARS-CoV-2, has recently emerged to cause a human pandemic. Whereas molecular diagnostic tests were rapidly developed, serologic assays are still lacking, yet urgently needed. Validated serologic assays are important for contact tracing, identifying the viral reservoir and epidemiological studies. Here, we developed serological assays for the detection of SARS-CoV-2 neutralizing, spike- and nucleocapsid-specific antibodies. Using serum samples from patients with PCR-confirmed infections of SARS-CoV-2, other coronaviruses, or other respiratory pathogenic infections, we validated and tested various antigens in different in-house and commercial ELISAs. We demonstrate that most PCR-confirmed SARS-CoV-2 infected individuals seroconverted, as revealed by sensitive and specific in-house ELISAs. We found that commercial S1 IgG or IgA ELISAs were of lower specificity while sensitivity varied between the two, with IgA showing higher sensitivity. Overall, the validated assays described here can be instrumental for the detection of SARS-CoV-2-specific antibodies for diagnostic, seroepidemiological and vaccine evaluation studies.

A human monoclonal 1 antibody blocking SARS-CoV-2 infection (preprint)

The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV). This cross-neutralizing antibody targets a communal epitope on these viruses and offers potential for prevention and treatment of COVID-19.