Project description:We developed a genomic surveillance program for real-time monitoring of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) in Uruguay. We report on a PCR method for SARS-CoV-2 VOCs, the surveillance workflow, and multiple independent introductions and community transmission of the SARS-CoV-2 P.1 VOC in Uruguay.
Project description:The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike-RBD and efficiently neutralize pseudotyped and live-viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially-discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100- and 1000-fold increase in neutralization potency, respectively. Cryo-EM of the sybody-spike complex additionally reveals a novel up-out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally-circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.
Project description:With the emergence of SARS-CoV-2 variants that may increase transmissibility and/or cause escape from immune responses 1-3 , there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant first detected in the UK 4,5 could be serendipitously detected by the ThermoFisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern that lack spike Δ69-70, such as B.1.351 (also 501Y.V2) detected in South Africa 6 and P.1 (also 501Y.V3) recently detected in Brazil 7 . We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all three variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open source PCR assay to detect SARS-CoV-2 variants of concern 8 . Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence spread of B.1.1.7, B.1.351, and P.1.
Project description:With the emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants that may increase transmissibility and/or cause escape from immune responses, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant, first detected in the United Kingdom, could be serendipitously detected by the Thermo Fisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike Δ69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern (VOC) that lack spike Δ69-70, such as B.1.351 (also 501Y.V2), detected in South Africa, and P.1 (also 501Y.V3), recently detected in Brazil. We identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in all 3 variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, we designed and validated an open-source PCR assay to detect SARS-CoV-2 VOC. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence and spread of B.1.1.7, B.1.351, and P.1.
Project description:The spread of the current Sars-Cov-2 pandemics leads to the development of mutations that are constantly monitored because they could affect the efficacy of vaccines. Three recently identified mutated strains, known as variants of concern, are rapidly spreading worldwide. Here, we study possible effects of these mutations on the immune response to Sars-Cov-2 infection using NetTepi a computational method based on artificial neural networks that considers binding and stability of peptides obtained by proteasome degradation for widely represented HLA class I alleles present in human populations as well as the T-cell propensity of viral peptides that measures their immune response. Our results show variations in the number of potential highly ranked peptides ranging between 0 and 20% depending on the specific HLA allele. The results can be useful to design more specific vaccines.
Project description:The emergence and rapid spread of novel variants of concern (VOC) of the coronavirus 2 constitute a major challenge for spatial disease surveillance. We explore the possibility to use close to real-time crowdsourced data on reported VOC cases (mainly the Alpha variant) at the local area level in Germany. The aim is to use these data for early-stage estimates of the statistical association between VOC reporting and the overall COVID-19 epidemiological development. For the first weeks in 2021 after international importation of VOC to Germany, our findings point to significant increases of up to 35-40% in the 7-day incidence rate and the hospitalization rate in regions with confirmed VOC cases compared to those without such cases. This is in line with simultaneously produced international evidence. We evaluate the sensitivity of our estimates to sampling errors associated with the collection of crowdsourced data. Overall, we find no statistical evidence for an over- or underestimation of effects once we account for differences in data representativeness at the regional level. This points to the potential use of crowdsourced data for spatial disease surveillance, local outbreak monitoring and public health decisions if no other data on new virus developments are available.
Project description:The global spread of SARS-CoV-2 lead to the most challenging pandemic in this century, posing major economic and health challenges worldwide. Revealing host genes essential for infection by multiple variants of SARS-CoV-2 can provide insights into the virus pathogenesis, and facilitates the development of novel broad-spectrum host-directed therapeutics. Here, employing genome-scale CRISPR screens, we provide a comprehensive data-set of cellular factors that are exploited by WT-SARS-CoV-2 as well as two additional recently emerged variants of concerns (VOCs), Alpha and Beta. These screens identified known and novel host factors critical for SARS-CoV-2 infection, including various components belonging to the Clathrin-dependent transport pathway, ubiquitination and Heparan sulfate biogenesis. In addition, the host phosphatidylglycerol biosynthesis processes appeared to have major anti-viral functions. Comparative analysis of the different VOCs revealed the host factors KREMEN2 and SETDB1 as potential unique candidates required only to the Alpha variant, providing a possible explanation for the increased infectivity of this variant. Furthermore, the analysis identified GATA6, a zinc finger transcription factor, as an essential pro-viral gene for all variants inspected. We revealed that GATA6 directly regulates ACE2 transcription and accordingly, is critical for SARS-CoV-2 cell entry. Analysis of clinical samples collected from SARS-CoV-2 infected individuals showed an elevated level of GATA6, indicating the important role GATA6 may be playing in COVID-19 pathogenesis. Finally, pharmacological inhibition of GATA6 resulted in down-modulation of ACE2 and consequently to inhibition of the viral infectivity. Overall, we show GATA6 represents a target for the development of anti-SARS-CoV-2 therapeutic strategies and reaffirm the value of the CRISPR loss-of-function screens in providing a list of potential new targets for therapeutic interventions.
Project description:The high sequence identity of the first SARS-CoV-2 samples collected in December 2019 at Wuhan did not foretell the emergence of novel variants in the United Kingdom, North and South America, India, or South Africa that drive the current waves of the pandemic. The viral spike receptor possesses two surface areas of high mutagenic plasticity: the supersite in its N-terminal domain (NTD) that is recognised by all anti-NTD antibodies and its receptor binding domain (RBD) where 17 residues make contact with the human Ace2 protein (angiotensin I converting enzyme 2) and many neutralising antibodies bind. While NTD mutations appear at first glance very diverse, they converge on the structure of the supersite. The mutations within the RBD, on the other hand, hone in on only a small number of key sites (K417, L452, E484, N501) that are allosteric control points enabling spike to escape neutralising antibodies while maintaining or even gaining Ace2-binding activity. The D614G mutation is the hallmark of all variants, as it promotes viral spread by increasing the number of open spike protomers in the homo-trimeric receptor complex. This review discusses the recent spike mutations as well as their evolution.
Project description:Escape variants of SARS-CoV-2 are threatening to prolong the COVID-19 pandemic. To address this challenge, we developed multivalent protein-based minibinders as potential prophylactic and therapeutic agents. Homotrimers of single minibinders and fusions of three distinct minibinders were designed to geometrically match the SARS-CoV-2 spike (S) trimer architecture and were optimized by cell-free expression and found to exhibit virtually no measurable dissociation upon binding. Cryo-electron microscopy (cryoEM) showed that these trivalent minibinders engage all three receptor binding domains on a single S trimer. The top candidates neutralize SARS-CoV-2 variants of concern with IC 50 values in the low pM range, resist viral escape, and provide protection in highly vulnerable human ACE2-expressing transgenic mice, both prophylactically and therapeutically. Our integrated workflow promises to accelerate the design of mutationally resilient therapeutics for pandemic preparedness.One-sentence summaryWe designed, developed, and characterized potent, trivalent miniprotein binders that provide prophylactic and therapeutic protection against emerging SARS-CoV-2 variants of concern.
Project description:In view of the rapid development of the COVID-19 pandemic and SARS-CoV-2 mutation, we characterized the emerging SARS-CoV-2 variants of concern (VOCs) by both bioinformatics methods and experiments. The representative genomic sequences of SARS-CoV-2 VOCs were first downloaded from NCBI, including the prototypic strain, Alpha (B.1.1.7) strain, Beta (B.1.351) strain, Delta (B.1.617.2), and Omicron (B1.1.529) strain. Bioinformatics analysis revealed that the D614G mutation led to formation of a protruding spike (S) in the tertiary structure of spike protein, which could be responsible for the enhanced binding to angiotensin-converting enzyme 2 (ACE2) receptor. The epitope analysis further showed that the S protein antigenicity of the Omicron variant changed dramatically, which was possibly associated with its enhanced ability of immune escape. To verify the bioinformatics results, we performed experiments of pseudovirus infection and protein affinity assay. Notably, we found that the spike protein of Omicron variant showed the weakest infectivity and binding ability among all tested strains. Finally, we also proved this through virus infection experiments, and found that the cytotoxicity of Omicron seems to be not strong enough. The results in this study provide guidelines for prevention and control of COVID-19.