Quantitation of SARS-CoV-2 neutralizing antibodies with a virus-free, authentic test

Neutralizing antibodies (NAbs), and their concentration in sera of convalescents and vaccinees are a solid correlate of protection from COVID-19. The antibody concentrations in clinical samples that neutralize SARS-CoV-2 are difficult and very cumbersome to assess with conventional virus neutralization tests (cVNTs), which require work with the infectious virus and biosafety level 3 containment precautions. Alternative virus neutralization tests currently in use are mostly surrogate tests based on direct or competitive ELISA formats or use viral vectors with the spike protein as the single structural component of SARS-CoV-2. To overcome these obstacles, we developed a virus-free, safe and very fast (4.5 h) in vitro diagnostic test based on engineered yet authentic SARS-CoV-2 virus-like-particles (VLPs). They share all features of the original SARS-CoV-2 but lack the viral RNA genome and thus are non-infectious. NAbs induced by infection or vaccination, but also potentially neutralizing monoclonal antibodies can be reliably quantified and assessed with ease and within hours with our test, because they interfere and block the ACE2-mediated uptake of VLPs by recipient cells. Results from the VLP neutralization test (VLPNT) show excellent correlation to a cVNT with fully infectious SARS-CoV-2 and allow to estimate the reduced neutralization capacity of COVID-19 vaccinee sera with variants of concern of SARS-CoV-2. Author summaryThe current pandemic caused by SARS-CoV-2 is a major challenge not only for COVID-19 patients, medical staff, healthcare systems and the general public, but also virologists and clinical laboratories. A particular challenge are safety issues which require biological safety level 3 to work with and study the pathogen. An alternative are virus-like particles (VLPs) of SARS-CoV-2, which are authentic in terms of viral structure and function but are harmless bioproducts in nature. We engineered VLPs which are close-to-perfect mimics of SARS-CoV-2 by all structural, biochemical, physical and functional criteria tested. SARS-CoV-2 VLPs were used in virus neutralization tests (VNTs). Because high concentrations of neutralizing antibodies correlate with protection from COVID-19 practical VNTs are urgently needed. We developed an authentic, virus-free, thus safe yet very fast in vitro diagnostic test with SARS-CoV-2 VLPs. Virus neutralizing antibodies induced by natural infection or vaccination but also certain monoclonal antibodies inhibit VLP fusion with recipient cells carrying ACE2. Quantitative results from a conventional neutralization test with fully infectious SARS-CoV-2 and results from the VLP-based neutralization test correlate perfectly. The setup of the test is very flexible and allows to analyze sera for their neutralizing capacity against different variants of concern and in a standardized assay format.

No substantial pre-existing B cell immunity against SARS-CoV-2 in healthy adults

Pre-existing immunity against SARS-CoV-2 may have critical implications for our understanding of COVID-19 susceptibility and severity. Various studies recently provided evidence of pre-existing T cell immunity against SARS-CoV-2 in unexposed individuals. In contrast, the presence and clinical relevance of a pre-existing B cell immunity remains to be fully elucidated. Here, we provide a detailed analysis of the B cell response to SARS-CoV-2 in unexposed individuals. To this end, we extensively investigated the memory B cell response to SARS-CoV-2 in 150 adults sampled pre-pandemically. Comprehensive screening of donor plasma and purified IgG samples for binding and neutralization in various functional assays revealed no substantial activity against SARS-CoV-2 but broad reactivity to endemic betacoronaviruses. Moreover, we analyzed antibody sequences of 8,174 putatively SARS-CoV-2-reactive B cells on a single cell level and generated and tested 158 monoclonal antibodies. None of the isolated antibodies displayed relevant binding or neutralizing activity against SARS-CoV-2. Taken together, our results show no evidence of relevant pre-existing antibody and B cell immunity against SARS-CoV-2 in unexposed adults.

Intranasal administration of a monoclonal neutralizing antibody protects mice against SARS-CoV-2 infection

Despite recent availability of vaccines against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), there is an urgent need for specific anti-SARS-CoV-2 drugs. Monoclonal neutralizing antibodies are an important drug class in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection and their potential to be used as both, prophylactic and therapeutic drugs. Clinically used neutralizing antibodies against respiratory viruses are currently injected intravenously, which can lead to suboptimal pulmonary bioavailability and thus to a lower effectiveness. Here we describe DZIF-10c, a fully human monoclonal neutralizing antibody that binds the receptor-binding domain of SARS-CoV-2 spike protein. DZIF-10c displays an exceptionally high neutralizing potency against SARS-CoV-2 and retains activity against the variants of concern B.1.1.7 and B.1.351. Importantly, not only systemic but also intranasal application of DZIF-10c abolished presence of infectious particles in the lungs of SARS-CoV-2 infected mice and mitigated lung pathology. Along with a favorable pharmacokinetic profile, these results highlight DZIF-10c as a novel human SARS-CoV-2 neutralizing antibody with high in vitro and in vivo antiviral potency. The successful intranasal application of DZIF-10c paves the way for clinical trials investigating topical delivery of anti-SARS-CoV-2 antibodies. Significance StatementMonoclonal neutralizing antibodies are important in the global fight against the SARS-CoV-2 pandemic due to their ability to convey immediate protection. However, their intravenous application might lead to suboptimal bioavailability in the lung. We here precisely characterize a new monoclonal neutralizing antibody (DZIF-10c) that binds to the receptor binding domain of the spike protein of SARS-CoV-2. DZIF-10c neutralizes SARS-CoV-2 with exceptionally high potency and maintains activity against circulating variants of concern. The antibody has a favorable pharmacokinetic profile and protects mice from SARS-CoV-2 infection. Importantly, we show that intranasal administration of DZIF-10c generates protective efficacy. These results not only identify DZIF-10c as a novel highly potent neutralizing antibody, but further pave the way for a topical application of anti-SARS-CoV-2 antibodies.

Immunogenicity and efficacy of the COVID-19 candidate vector vaccine MVA SARS 2 S in preclinical vaccination

The severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) has emerged as the infectious agent causing the pandemic coronavirus disease 2019 (COVID-19) with dramatic consequences for global human health and economics. Previously, we reached clinical evaluation with our vector vaccine based on vaccinia virus MVA against the Middle East respiratory syndrome coronavirus (MERS-CoV), which causes an infection in humans similar to SARS and COVID-19. Here, we describe the construction and preclinical characterization of a recombinant MVA expressing full-length SARS-CoV-2 spike (S) protein (MVA-SARS-2-S). Genetic stability and growth characteristics of MVA-SARS-2-S, plus its robust synthesis of S antigen, make it a suitable candidate vaccine for industrial scale production. Vaccinated mice produced S antigen-specific CD8+ T cells and serum antibodies binding to S glycoprotein that neutralized SARS-CoV-2. Prime-boost vaccination with MVA-SARS-2-S protected mice sensitized with a human ACE2-expressing adenovirus from SARS-CoV-2 infection. MVA-SARS-2-S is currently being investigated in a phase I clinical trial as aspirant for developing a safe and efficacious vaccine against COVID-19. Significance StatementThe highly attenuated vaccinia virus MVA is licensed as smallpox vaccine, and as vector it is a component of the approved Adenovirus-MVA-based prime-boost vaccine against Ebola virus disease. Here we provide results from testing the COVID-19 candidate vaccine MVA-SARS-2-S, a poxvirus-based vector vaccine that proceeded to clinical evaluation. When administered by intramuscular inoculation, MVA-SARS-2-S expresses and safely delivers the full-length SARS-CoV-2 spike (S) protein, inducing balanced SARS-CoV-2-specific cellular and humoral immunity, and protective efficacy in vaccinated mice. Substantial clinical experience has already been gained with MVA vectors using homologous and heterologous prime-boost applications, including the immunization of children and immunocompromised individuals. Thus, MVA-SARS-2-S represents an important resource for developing further optimized COVID-19 vaccines.

Inhibiting coronavirus replication in cultured cells by chemical ER stress

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

Longitudinal isolation of potent near-germline SARS-CoV-2-neutralizing antibodies from COVID-19 patients

The SARS-CoV-2 pandemic has unprecedented implications for public health, social life, and world economy. Since approved drugs and vaccines are not available, new options for COVID-19 treatment and prevention are highly demanded. To identify SARS-CoV-2 neutralizing antibodies, we analysed the antibody response of 12 COVID-19 patients from 8 to 69 days post diagnosis. By screening 4,313 SARS-CoV-2-reactive B cells, we isolated 255 antibodies from different time points as early as 8 days post diagnosis. Among these, 28 potently neutralized authentic SARS-CoV-2 (IC100 as low as 0.04 g/ml), showing a broad spectrum of V genes and low levels of somatic mutations. Interestingly, potential precursors were identified in naive B cell repertoires from 48 healthy individuals that were sampled before the COVID-19 pandemic. Our results demonstrate that SARS-CoV-2 neutralizing antibodies are readily generated from a diverse pool of precursors, fostering the hope of rapid induction of a protective immune response upon vaccination.

Epidemiological study to detect active SARS-CoV-2 infections and seropositive persons in a selected cohort of employees in the Frankfurt am Main metropolitan area

So far, 170,000 Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections have been confirmed in Germany, of which more than 5,000 have been detected in the Frankfurt am Main metropolitan region. When examining 1,000 nasopharyngeal swabs and serum samples from healthy volunteers from this region, one RT-PCR-positive and five antibody-positive persons were identified. The five positive serum samples were confirmed to be specific. Four of the five positive sera cross-neutralized SARS-CoV.

TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets

In December 2019, a novel coronavirus named SARS-CoV-2 first reported in Wuhan, China, emerged and rapidly spread to numerous other countries globally, causing the current pandemic. SARS-CoV-2 causes acute infection of the respiratory tract (COVID-19) that can result in severe disease and lethality. Currently, there is no approved antiviral drug for treating COVID-19 patients and there is an urgent need for specific antiviral therapies and vaccines. In order for SARS-CoV-2 to enter cells, its surface glycoprotein spike (S) must be cleaved at two different sites by host cell proteases, which therefore represent potential drug targets. In the present study we investigated which host cell proteases activate the SARS-CoV-2 S protein in Calu-3 human airway epithelial cells. We show that S can be cleaved by both the proprotein convertase furin at the S1/S2 site and the transmembrane serine protease 2 (TMPRSS2) at the S2 site. We demonstrate that TMPRSS2 is essential for activation of SARS-CoV-2 S in Calu-3 cells through antisense-mediated knockdown of TMPRSS2 expression. Further, we show that SARS-CoV-2 replication can be efficiently inhibited by two synthetic inhibitors of TMPRSS2 and also by the broad range serine protease inhibitor aprotinin. Additionally, SARS-CoV-2 replication was also strongly inhibited by the synthetic furin inhibitor MI-1851. Combining various TMPRSS2 inhibitors with MI-1851 produced more potent antiviral activity against SARS-CoV-2 than an equimolar amount of any single serine protease inhibitor. In contrast, inhibition of endosomal cathepsins by E64d did not affect virus replication. Our data demonstrate that both TMPRSS2 and furin are essential for SARS-CoV-2 activation in human airway cells and are promising drug targets for the treatment of COVID-19 either by targeting one of these proteases alone or by a combination of furin and TMPRSS2 inhibitors. Therefore, this approach has a high therapeutic potential for treatment of COVID-19.