Filamentous Fungus-Produced Human Monoclonal Antibody Provides SARS-CoV-2 Protection in Hamster and Non-Human Primate Models (preprint)

Monoclonal antibodies are an increasingly important tool for prophylaxis and treatment of acute virus infections like those with SARS-CoV-2. However, their use is largely limited by the length of development, yield and high production costs, as well as the need for continuous adaptation to newly emerging virus variants. Here we have used the filamentous fungus expression system Thermothelomyces heterothallica(C1), which has a natural high biosynthesis capacity for secretory enzymes and other proteins further enhanced by genetic engineering of the wild-type fungus, to produce a human monoclonal IgG1 antibody (HuMab 87G7) that neutralises SARS-CoV-2 variants of concern (VOCs) Alpha, Beta, Gamma, Delta, and Omicron. Like its mammalian cell produced equivalent, C1 produced HuMab 87G7 broadly neutralised SARS-CoV-2 VOCs in vitro and it also provided protection against Omicron and Delta VOCs in both hamsters and non-human primates, respectively. The only notable difference between the two versions was their N-linked glycosylation patterns detected by glyoproteomic analysis. Taken together, these findings demonstrate potential of the C1 expression system as a promising technology platform for the development of HuMabs in preventive and therapeutic medicine.

Sialoglycan binding triggers spike opening in a human coronavirus (preprint)

Coronavirus (CoV) spikes mediate receptor binding and membrane fusion, making them prime targets for neutralising antibodies. In the cases of SARS-CoV, SARS-CoV-2, and MERS-CoV, spikes transition freely between open and closed conformations to balance host cell attachment and immune evasion. The open conformation exposes domain S1B, allowing it to bind to proteinaceous cell surface receptors. It also facilitates protein refolding during spike-mediated membrane fusion. However, with a single exception, the pre-fusion spikes of all other CoVs studied so far have been observed exclusively in the closed state. This raises the possibility of regulation, where spikes more commonly transition to open states in response to specific cues, rather than spontaneously. In our study, using cryo-EM and molecular dynamics simulations, we show that the spike protein of the common cold human coronavirus HKU1 undergoes local and long-range conformational changes upon binding a sialoglycan-based primary receptor to domain S1A. This binding triggers the transition of S1B domains to the open state via allosteric inter-domain cross-talk. Our findings paint a more elaborate picture of CoV attachment, with possibilities of dual receptor usage and priming of entry as a means of immune escape.

A broad-spectrum macrocyclic peptide inhibitor of the SARS-CoV-2 spike protein (preprint)

The ongoing COVID-19 pandemic has had great societal and health consequences. Despite the availability of vaccines, infection rates remain high due to immune evasive Omicron sublineages. Broad-spectrum antivirals are needed to safeguard against emerging variants and future pandemics. We used mRNA display under a reprogrammed genetic code to find a spike-targeting macrocyclic peptide that inhibits SARS-CoV-2 Wuhan strain infection and also pseudoviruses containing spike proteins of SARS-CoV-2 variants or related sarbecoviruses. Structural and bioinformatic analyses reveal a conserved binding pocket between the receptor binding domain and other domains, distal to the ACE2 receptor-interaction site. Collectively, our data reveal a hitherto unexplored site of vulnerability in sarbecoviruses that can be targeted by peptides and potentially other drug-like molecules.

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.

A Highly Potent Antibody Effective Against SARS-CoV-2 Variants of Concern (preprint)

Control of the ongoing SARS-CoV-2 pandemic is endangered by the emergence of viral variants with increased transmission efficiency, resistance to marketed therapeutic antibodies and reduced sensitivity to vaccine-induced immunity. Here, we screened B cells from COVID-19 donors and identified P5C3, a highly potent and broadly neutralizing monoclonal antibody with picomolar neutralizing activity against all SARS-CoV-2 variants of concern (VOC) identified to date. Structural characterization of P5C3 Fab in complex with the Spike demonstrates a neutralizing activity defined by a large buried surface area, highly overlapping with the receptor-binding domain (RBD) surface necessary for ACE2 interaction. We further demonstrate that P5C3 showed complete prophylactic protection in the SARS-CoV-2 infected hamster challenge model. These results indicate that P5C3 opens exciting perspectives either as a prophylactic agent in immunocompromised individuals with poor response to vaccination or as combination therapy in SARS-CoV-2-infected individuals.Funding: This CARE project has received funding from the Innovative MedicinesInitiative 2 Joint Undertaking (JU) under grant agreement No 101005077. The JU receives support from the European Union’s Horizon 2020 research and innovation program and EFPIA and BILL & MELINDA GATES FOUNDATION, GLOBAL HEALTH DRUG DISCOVERYINSTITUTE, UNIVERSITY OF DUNDEE. Furthermore, funding was also provided through the Lausanne University Hospital, through the Swiss Vaccine Research Institute to G.P., and through the EPFL COVID fund to D.T.Conflict of Interest: None to declare. Ethical Approval: Study design and use of subject samples were approved by the Institutional Review Board of the Lausanne University Hospital and the ‘Commission d’éthique du Canton de Vaud’ (CER-VD).

An alphavirus replicon-based vaccine expressing a stabilized Spike antigen induces sterile immunity and prevents transmission of SARS-CoV-2 between cats (preprint)

Early in the global SARS-CoV-2 pandemic concerns were raised regarding infection of other animal hosts and whether these could play a significant role in the viral epidemiology. Infection of animals could be detrimental by causing clinical disease but also of concern if they become a viral reservoir allowing further mutations, plus having the potential to infect other animals or humans. The first reported animals to be infected both under experimental conditions and from anecdotal field evidence were cats described in China early in 2020. Given the concerns this finding raised and the close contacts between humans and cats, we aimed to determine whether a vaccine candidate could be developed that was suitable for use in multiple susceptible animal species and whether this vaccine could reduce infection of cats in addition to preventing spread to other cats. Here we report that a Replicon Particle (RP) vaccine based on Venezuelan equine encephalitis virus (VEEV), known to be safe and efficacious for use in a variety of animals, expressing a stabilised Spike antigen, could induce neutralising antibody titers in guinea pigs and cats. After two intramuscular vaccinations, virus neutralising antibodies were detected in the respiratory tract of the guinea pigs and a cell mediated immune response was induced. The design of the SARS-CoV-2 antigen was shown to be critical in developing a strong neutralising antibody response. Vaccination of cats was able to induce a serum neutralising antibody response which lasted for the course of the experiment. Interestingly, in contrast to control animals, infectious virus could not be detected in oropharyngeal or nasal swabs of vaccinated cats after challenge. Moreover, the challenged control cats spread the virus to in-contact cats whereas the vaccinated cats did not transmit virus. The results show that the RP vaccine induces sterile immunity preventing SARS-CoV-2 infection and transmission. This data suggests that this RP vaccine could be a multi-species vaccine useful for preventing spread to and between other animals should that approach be required.

Elevated mucosal antibody responses against SARS-CoV-2 are correlated with lower viral load and faster decrease in systemic COVID-19 symptoms (preprint)

Mucosal antibodies play a key role in protection against SARS-CoV-2 exposure, but their role during primary infection is not well understood. We assessed mucosal antibody responses during primary infection with SARS-CoV-2 and examined their relationship with viral load and clinical symptoms. Elevated mucosal IgM was associated with lower viral load. RBD and viral spike protein-specific mucosal antibodies were correlated with decreases in systemic symptoms, while older age was associated with an increase in respiratory symptoms. Up to 42% of household contacts developed SARS-CoV-2-specific mucosal antibodies, including children, indicating high transmission rates within households in which children might play an important role.

Dynamics of antibodies to SARS-CoV-2 in convalescent plasma donors (preprint)

The novel SARS-CoV-2 virus emerged in late 2019 and has caused a global health and economic crisis. The characterization of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes as well for treatment options such as transfusion with convalescent plasma or immunoglobin products derived from convalescent plasma. In this study, we measured antibody responses in 844 longitudinal samples from 151 RT-PCR positive SARS-CoV-2 convalescent adults during the first 34 weeks after onset of symptoms. All donors were seropositive at the first sampling moment and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels slowly declined with median half-lifes of 62 and 59 days during 2-5 months after symptom onset, respectively. The rate of decline of antibody levels diminished during extended follow-up. In addition, the magnitude of the IgG response correlated with neutralization capacity measured in a classic plaque reduction assay as well in our in-house developed competition assay. The result of this study gives valuable insight into the longitudinal response of antibodies to SARS-CoV-2.

Complex Systems Analysis Informs on the Spread of COVID-19 (preprint)

The non-linear progression of new infection numbers in a pandemic poses challenges to the evaluation of its management. The tools of complex systems research may aid in attaining information that would be difficult to extract with other means. To study the COVID-19 pandemic, we utilize the reported new cases per day for the globe, nine countries and six US states through October 2020. Fourier and univariate wavelet analyses inform on periodicity and extent of change. Evaluating time-lagged data sets of various lag lengths, we find that the autocorrelation function, average mutual information and box counting dimension represent good quantitative readouts for the progression of new infections. Bivariate wavelet analysis and return plots give indications of containment versus exacerbation. Homogeneity or heterogeneity in the population response, uptick versus suppression, and worsening or improving trends are discernible, in part by plotting various time lags in three dimensions. The analysis of epidemic or pandemic progression with the techniques available for observed (noisy) complex data can aid decision making in the public health response.

RAPPID: a platform of ratiometric bioluminescent sensors for homogeneous immunoassays (preprint)

Heterogeneous immunoassays such as ELISA have become indispensable in modern bioanalysis, yet translation into easy-to-use point-of-care assays is hindered by their dependence on external calibration and multiple washing and incubation steps. Here, we introduce RAPPID (Ratiometric Plug-and-Play Immunodiagnostics), a "mix-and-measure" homogeneous immunoassay platform that combines highly specific antibody-based detection with a ratiometric bioluminescent readout that can be detected using a basic digital camera. The concept entails analyte-induced complementation of split NanoLuc luciferase fragments, photoconjugated to an antibody sandwich pair via protein G adapters. We also introduce the use of a calibrator luciferase that provides a robust ratiometric signal, allowing direct in-sample calibration and quantitative measurements in complex media such as blood plasma. We developed RAPPID sensors that allow low-picomolar detection of several protein biomarkers, anti-drug antibodies, therapeutic antibodies, and both SARS-CoV-2 spike protein and anti-SARS-CoV-2 antibodies. RAPPID combines ratiometric bioluminescent detection with antibody-based target recognition into an easy-to-implement standardized workflow, and therefore represents an attractive, fast, and low-cost alternative to traditional immunoassays, both in an academic setting and in clinical laboratories for point-of-care applications.

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.

Inhibition of SARS-CoV-2 in Vero cell cultures by peptide-conjugated morpholino-oligomers. (preprint)

Background: SARS-CoV-2 is the causative agent of COVID-19 and a pathogen of immense global public health importance. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense agents composed of a phosphordiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking. Objectives and Methods: Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5'-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method and viral growth was measured with quantitative RT-PCR and TCID50 infectivity assays. Results: PPMO designed to base-pair with sequence in the 5'-terminal region or the leader transcription regulatory sequence-region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titers by up to 4-6 log10 in cell cultures at 48-72 hours post-infection, in a non-toxic and dose-responsive manner. Conclusion: The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further pre-clinical development.

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