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INTRODUCTIONThe pandemic readiness toolbox needs to be extended, providing diagnostic tools that target different biomolecules, using orthogonal experimental setups and fit-for-purpose specification of detection. Here we build on a previous Cov-MS effort that used liquid chromatography-mass spectrometry (LC-MS) and describe a method that allows accurate, high throughput measurement of SARS-CoV-2 nucleocapsid (N) protein. MATERIALS and METHODSWe used Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) technology to enrich and quantify proteotypic peptides of the N protein from trypsin-digested samples from COVID-19 patients. RESULTSThe Cov2MS assay was shown to be compatible with a variety of sample matrices including nasopharyngeal swabs, saliva and blood plasma and increased the sensitivity into the attomole range, up to a 1000-fold increase compared to direct detection in matrix. In addition, a strong positive correlation was observed between the SISCAPA antigen assay and qPCR detection beyond a quantification cycle (Cq) of 30-31, the level where no live virus can be cultured from patients. The automatable "addition only" sample preparation, digestion protocol, peptide enrichment and subsequent reduced dependency upon LC allow analysis of up to 500 samples per day per MS instrument. Importantly, peptide enrichment allowed detection of N protein in a pooled sample containing a single PCR positive sample mixed with 31 PCR negative samples, without loss in sensitivity. MS can easily be multiplexed and we also propose target peptides for Influenza A and B virus detection. CONCLUSIONSThe Cov2MS assay described is agnostic with respect to the sample matrix or pooling strategy used for increasing throughput and can be easily multiplexed. Additionally, the assay eliminates interferences due to protein-protein interactions including those caused by anti-virus antibodies. The assay can be adapted to test for many different pathogens and could provide a tool enabling longitudinal epidemiological monitoring of large numbers of pathogens within a population, applied as an early warning system.
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The ongoing COVID-19 pandemic, caused by SARS-CoV-2, constitutes a tremendous global health issue. Continuous monitoring of the virus has become a cornerstone to make rational decisions on implementing societal and sanitary measures to curtail the virus spread. Additionally, emerging SARS-CoV-2 variants have increased the need for genomic surveillance to detect particular strains because of their potentially increased transmissibility, pathogenicity and immune escape. Targeted SARS-CoV-2 sequencing of wastewater has been explored as an epidemiological surveillance method for the competent authorities. Few quality criteria are however available when sequencing wastewater samples, and those available typically only pertain to constructing the consensus genome sequence. Multiple variants circulating in the population can however be simultaneously present in wastewater samples. The performance, including detection and quantification of low-abundant variants, of whole genome sequencing (WGS) of SARS-CoV-2 in wastewater samples remains largely unknown. Here, we evaluated the detection and quantification of mutations present at low abundances using the SARS-CoV-2 lineage B.1.1.7 (alpha variant) defining mutations as a case study. Real sequencing data were in silico modified by introducing mutations of interest into raw wild-type sequencing data, or by mixing wild-type and mutant raw sequencing data, to mimic wastewater samples subjected to WGS using a tiling amplicon-based targeted metagenomics approach and Illumina sequencing. As anticipated, higher variation, lower sensitivity and more false negatives, were observed at lower coverages and allelic frequencies. We found that detection of all low-frequency variants at an abundance of 10%, 5%, 3% and 1%, requires at least a sequencing coverage of 250X, 500X, 1500X and 10,000X, respectively. Although increasing variability of estimated allelic frequencies at decreasing coverages and lower allelic frequencies was observed, its impact on reliable quantification was limited. This study provides a highly sensitive low-frequency variant detection approach, which is publicly available at https://galaxy.sciensano.be, and specific recommendations for minimum sequencing coverages to detect clade-defining mutations at specific allelic frequencies.
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We have identified camelid single-domain antibodies (VHHs) that cross-neutralize SARS-CoV-1 and -2, such as VHH72, which binds to a unique highly conserved epitope in the viral receptor-binding domain (RBD) that is difficult to access for human antibodies. Here, we establish a protein engineering path for how a stable, long-acting drug candidate can be generated out of such a VHH building block. When fused to human IgG1-Fc, the prototype VHH72 molecule prophylactically protects hamsters from SARS-CoV-2. In addition, we demonstrate that both systemic and intranasal application protects hACE-2-transgenic mice from SARS-CoV-2 induced lethal disease progression. To boost potency of the lead, we used structure-guided molecular modeling combined with rapid yeast-based Fc-fusion prototyping, resulting in the affinity-matured VHH72_S56A-Fc, with subnanomolar SARS-CoV-1 and -2 neutralizing potency. Upon humanization, VHH72_S56A was fused to a human IgG1 Fc with optimized manufacturing homogeneity and silenced effector functions for enhanced safety, and its stability as well as lack of off-target binding was extensively characterized. Therapeutic systemic administration of a low dose of VHH72_S56A-Fc antibodies strongly restricted replication of both original and D614G mutant variants of SARS-CoV-2 virus in hamsters, and minimized the development of lung damage. This work led to the selection of XVR011 for clinical development, a highly stable anti-COVID-19 biologic with excellent manufacturability. Additionally, we show that XVR011 is unaffected in its neutralizing capacity of currently rapidly spreading SARS-CoV-2 variants, and demonstrate its unique, wide scope of binding across the Sarbecovirus clades.
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Introductory paragraphSince the emergence of SARS-CoV-2 causing COVID-19, the world is being shaken to its core with numerous hospitalizations and hundreds of thousands of deaths. In search for key targets of effective therapeutics, robust animal models mimicking COVID-19 in humans are urgently needed. Here, we show that productive SARS-CoV-2 infection in the lungs of mice is limited and restricted by early type I interferon responses. In contrast, we show that Syrian hamsters are highly permissive to SARS- CoV-2 and develop bronchopneumonia and a strong inflammatory response in the lungs with neutrophil infiltration and edema. Moreover, we identify an exuberant innate immune response as a key player in pathogenesis, in which STAT2 signaling plays a dual role, driving severe lung injury on the one hand, yet restricting systemic virus dissemination on the other. Finally, we assess SARS-CoV- 2-induced lung pathology in hamsters by micro-CT alike used in clinical practice. Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients.
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The pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV-1) and COVID-19 coronavirus (SARS-CoV-2) have all emerged into the human population with devastating consequences. These viruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics to combat these highly pathogenic coronaviruses. Here, we describe the isolation and characterization of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs are capable of potently neutralizing MERS-CoV or SARS-CoV-1 S pseudotyped viruses. The crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs block receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S, and demonstrate that this cross-reactive VHH is capable of neutralizing SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.
