31 December, 2021

Subgenomic SARS-CoV-2 replicon and reporter replicon cell lines enable ultrahigh throughput antiviral screening and mechanistic studies with antivirals, viral mutations or host factors that affect COVID-19 replication -

Replicon-based technologies were used to develop reagents and assays for advanced drug discovery efforts against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and for examining all facets of the SARS-CoV-2 replication cycle at reduced biocontainment level. Specifically: a) 21 replicons were cloned in bacterial artificial chromosomes (BACs) and delivered as transfectable plasmid DNA or transcribed RNA in various cell types. Replicons carrying mutations that affect the activity or antiviral susceptibility of SARS-CoV-2 enzymes were used to establish utility for mechanistic studies while reducing the community risks associated with gain-of-function studies in fully infectious virus. b) A BHK-21 stable cell line harboring SARS-CoV-2 replicon was generated and characterized in robust high/ultra- high throughput assays of antiviral efficacy with orthogonal SARS-CoV-2 replication reporter genes (Nano luciferase and enhanced green fluorescent protein-eGFP); the estimated antiviral potencies in the fully infectious SARS-CoV-2 system and in the transient or stable replicon systems were similar. HEK293 and Calu1 stable cell lines expressing SARS-CoV-2 replicon have also been prepared. Finally, c) we generated trans-encapsidated replicons by co-expression with SARS-CoV-2 structural proteins, thus producing single-round infectious SARS-CoV-2 virus-like particles able to transduce susceptible cell types, thus expanding utility to enable study of virion assembly and entry into target cells. Hence, these SARS-CoV-2 replicon-based reagents include a novel approach to replicon-harboring cell line generation and are valuable tools that can be used at lower biosafety level (BSL2) for drug discovery efforts, characterization of SARS- CoV-2 and variant evolution in the COVID-19 pandemic, mechanisms of inhibition and resistance, and studies on the role of SARS-CoV-2 genes and host dependency factors.

🖺 Full Text HTML: Subgenomic SARS-CoV-2 replicon and reporter replicon cell lines enable ultrahigh throughput antiviral screening and mechanistic studies with antivirals, viral mutations or host factors that affect COVID-19 replication

SARS-CoV-2 diverges from other betacoronaviruses in only partially activating the IRE1α/XBP1 ER stress pathway in human lung-derived cells -

Despite the efficacy of vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 5 million individuals worldwide and continues to spread in countries where the vaccines are not yet widely available or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed essential for viral replication. We examined the activation status and requirement of the master UPR sensor IRE1 kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and the murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1 as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1 whereby it autophosphorylates, but its RNase fails to splice XBP1. Moreover, IRE1 was dispensable for optimal replication in human cells for all coronaviruses tested. Our findings demonstrate that IRE1 activation status differs upon infection with distinct betacoronaviruses and is not essential for efficient replication of any of them. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1 through an unknown mechanism, perhaps as a strategy to eliminate detection by the host immune system.

🖺 Full Text HTML: SARS-CoV-2 diverges from other betacoronaviruses in only partially activating the IRE1α/XBP1 ER stress pathway in human lung-derived cells

Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron -

Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccines, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring the vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS- CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.

🖺 Full Text HTML: Molecular probes of spike ectodomain and its subdomains for SARS-CoV-2 variants, Alpha through Omicron

Preserved T cell reactivity to the SARS-CoV-2 Omicron variant indicates continued protection in vaccinated individuals. -

Importance: The emergence of the highly contagious Omicron variant of SARS-CoV-2 and the findings of a significantly reduced neutralizing potency of sera from convalescent or vaccinated individuals imposes the study of cellular immunity to predict the degree of immune protection to the yet again new coronavirus. Design: Prospective monocentric observational study. Setting: Conducted between December 20-21 at the Santa Lucia Foundation IRCCS. Participants: 61 volunteers (Mean age 41.62, range 21-62; 38F/23M) with different vaccination and SARS-CoV-2 infection backgrounds donated 15 ml of blood. Of these donors, one had recently completed chemotherapy, and one was undergoing treatment with monoclonal antibodies; the others reported no known health issue. Main Outcome(s) and Measure(s): The outcomes were the measurement of T cell reactivity to the mutated regions of the Spike protein of the Omicron SARS-CoV-2 variant and the assessment of remaining T cell immunity to the spike protein by stimulation with peptide libraries. Results: Lymphocytes from freshly drawn blood samples were isolated and immediately tested for reactivity to the Spike protein of SARS-CoV-2. T cell responses to peptides covering the mutated regions in the Omicron variant were decreased by over 47% compared to the same regions of the ancestral vaccine strain. However, overall reactivity to the peptide library of the full-length protein was largely maintained (estimated 83%). No significant differences in loss of immune recognition were identified between groups of donors with different vaccination and/or infection histories. Conclusions and Relevance: We conclude that despite the mutations in the Spike protein, the SARS-CoV-2 Omicron variant is nonetheless recognized by the cellular component of the immune system. It is reasonable to assume that protection from hospitalization and severe disease is maintained.

🖺 Full Text HTML: Preserved T cell reactivity to the SARS-CoV-2 Omicron variant indicates continued protection in vaccinated individuals.

Viral population genomics reveals host and infectivity impact on SARS-CoV-2 adaptive landscape -

Public health surveillance, drug treatment development, and optimization of immunological interventions all depend on understanding pathogen adaptation, which differ for specific pathogens. SARS-CoV-2 is an exceptionally successful human pathogen, yet complete understanding of the forces driving its evolution is lacking. Here, we leveraged almost four million SARS-CoV-2 sequences originating mostly from non-vaccinated naive patients to investigate the impact of functional constraints and natural immune pressures on the sequence diversity of the SARS-CoV-2 genome. Overall, we showed that the SARS-CoV-2 genome is under strong and intensifying levels of purifying selection with a minority of sites under diversifying pressure. With a particular focus on the spike protein, we showed that sites under selection were critical for protein stability and virus fitness related to increased infectivity and/or reduced neutralization by convalescent sera. We investigated the genetic diversity of SARS-CoV-2 B and T cell epitopes and determined that the currently known T cell epitope sequences were highly conserved. Outside of the spike protein, we observed that mutations under selection in variants of concern can be associated to beneficial outcomes for the virus. Altogether, the results yielded a comprehensive map of all sites under selection across the entirety of SARS-CoV-2 genome, highlighting targets for future studies to better understand the virus spread, evolution and success.

🖺 Full Text HTML: Viral population genomics reveals host and infectivity impact on SARS-CoV-2 adaptive landscape

Unsupervised genome-wide cluster analysis: nucleotide sequences of the omicron variant of SARS-CoV-2 are similar to sequences from early 2020 -

The GISAID database contains more than 100,000 SARS-CoV-2 genomes, including sequences of the recently discovered SARS-CoV-2 omicron variant and of prior SARS-CoV-2 strains that have been collected from patients around the world since the beginning of the pandemic. We applied unsupervised cluster analysis to the SARS-CoV-2 genomes, assessing their similarity at a genome-wide level based on the Jaccard index and principal component analysis. Our analysis results show that the omicron variant sequences are most similar to sequences that have been submitted early in the pandemic around January 2020. Furthermore, the omicron variants in GISAID are spread across the entire range of the first principal component, suggesting that the strain has been in circulation for some time. This observation supports a long-term infection hypothesis as the omicron strain origin.

🖺 Full Text HTML: Unsupervised genome-wide cluster analysis: nucleotide sequences of the omicron variant of SARS-CoV-2 are similar to sequences from early 2020

Increased resistance of SARS-CoV-2 Omicron Variant to Neutralization by Vaccine-Elicited and Therapeutic Antibodies -

Currently authorized vaccines for SARS-CoV-2 have been highly successful in preventing infection and lessening disease severity. The vaccines maintain effectiveness against SARS-CoV-2 Variants of Concern but the heavily mutated, highly transmissible Omicron variant poses an obstacle both to vaccine protection and monoclonal antibody therapies. Analysis of the neutralization of Omicron spike protein-pseudotyped lentiviruses showed a 26-fold relative resistance (compared to D614G) to neutralization by convalescent sera and 26-34-fold resistance to Pfizer BNT162b2 and Moderna vaccine-elicited antibodies following two immunizations. A booster immunization increased neutralizing titers against Omicron by 6-8-fold. Previous SARS-CoV-2 infection followed by vaccination resulted in the highest neutralizing titers against Omicron. Regeneron REGN10933 and REGN10987, and Lilly LY-CoV555 and LY-CoV016 monoclonal antibodies were ineffective against Omicron, while Sotrovimab was partially effective. The results highlight the benefit of a booster immunization in providing protection against Omicron but demonstrate the challenge to monoclonal antibody therapies.

🖺 Full Text HTML: Increased resistance of SARS-CoV-2 Omicron Variant to Neutralization by Vaccine-Elicited and Therapeutic Antibodies

Pipeline for retrieval of COVID-19 immune signatures -

Objective: The accelerating pace of biomedical publication has made retrieving papers and extracting specific comprehensive scientific information a key challenge. A timely example of such a challenge is to retrieve the subset of papers that report on immune signatures (coherent sets of biomarkers) to understand the immune response mechanisms which drive differential SARS-CoV-2 infection outcomes. A systematic and scalable approach is needed to identify and extract COVID-19 immune signatures in a structured and machine-readable format. Materials and Methods: We used SPECTER embeddings with SVM classifiers to automatically identify papers containing immune signatures. A generic web platform was used to manually screen papers and allow anonymous submission. Results: We demonstrate a classifier that retrieves papers with human COVID-19 immune signatures with a positive predictive value of 86%. Semi-automated queries to the corresponding authors of these publications requesting signature information achieved a 31% response rate. This demonstrates the efficacy of using a SVM classifier with document embeddings of the abstract and title, to retrieve papers with scientifically salient information, even when that information is rarely present in the abstract. Additionally, classification based on the embeddings identified the type of immune signature (e.g., gene expression vs. other types of profiling) with a positive predictive value of 74%. Conclusions: Coupling a classifier based on document embeddings with direct author engagement offers a promising pathway to build a semi-structured representation of scientifically relevant information. Through this approach, partially automated literature mining can help rapidly create semi-structured knowledge repositories for automatic analysis of emerging health threats.

🖺 Full Text HTML: Pipeline for retrieval of COVID-19 immune signatures

Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody -

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies has been limited by the continuous emergence of viral variants, and by the restricted diffusion of antibodies from circulation into the sites of respiratory virus infection. Here, we report the identification of two highly conserved regions on Omicron variant RBD recognized by broadly neutralizing antibodies. Based on this finding, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant RBD as revealed by Cryo-EM structures. This inhalable antibody exhibited exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. The structures also deciphered an uncommon cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics.

🖺 Full Text HTML: Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody

Structural and functional characterizations of altered infectivity and immune evasion of SARS-CoV-2 Omicron variant -

The SARS-CoV-2 Omicron with increased fitness is spreading rapidly worldwide. Analysis of cryo-EM structures of the Spike (S) from Omicron reveals amino acid substitutions forging new interactions that stably maintain an active conformation for receptor recognition. The relatively more compact domain organization confers improved stability and enhances attachment but compromises the efficiency of viral fusion step. Alterations in local conformation, charge and hydrophobic microenvironments underpin the modulation of the epitopes such that they are not recognized by most NTD- and RBD-antibodies, facilitating viral immune escape. Apart from already existing mutations, we have identified three new immune escape sites: 1) Q493R, 2) G446S and 3) S371L/S373P/S375F that confers greater resistance to five of the six classes of RBD-antibodies. Structure of the Omicron S bound with human ACE2, together with analysis of sequence conservation in ACE2 binding region of 25 sarbecovirus members as well as heatmaps of the immunogenic sites and their corresponding mutational frequencies sheds light on conserved and structurally restrained regions that can be used for the development of broad-spectrum vaccines and therapeutics.

🖺 Full Text HTML: Structural and functional characterizations of altered infectivity and immune evasion of SARS-CoV-2 Omicron variant

The Origin of the SARS‑CoV‑2 in the United States as a Biological Weapon -

Introduction: The SARS-CoV-2 virus was first reported in Wuhan China in Dec 2019, since then 279 million have been infected and 5.4 million have died. This has raised the question where did the SARs-CoV-2 virus originate? Methodology: In this study, the literature was reviewed, and the scientific and intelligence evidence assessed. Interviews were made with scientists and victims involved in the creation of the virus in both the United States and China. Results: The evidence suggest that the SARs-CoV-2 virus began as bat virus which was then manipulated in the lab via gain of function research in the United States funded by the National Institutes of Health under Dr. Fauci. This proto-biological weapon was then given to the Chinese and passed through Uighur prisoners. It is hypothesized that the modest common Altaic ancestry between American Native Americans and the Uighur from North Central Asia may in part account for the increased death rates of Native Americans in the United States. Conclusion: The SARS- CoV-2 virus with near scientific certainty originated in the United States as a proto-biological weapon which was further clinically developed in China in a collaborative effort as a biological weapon to target ethnic and racial minorities by both China and the United States.

🖺 Full Text HTML: The Origin of the SARS‑CoV‑2 in the United States as a Biological Weapon

Structures of the Omicron spike trimer with ACE2 and an anti-Omicron antibody: mechanisms for the high infectivity, immune evasion and antibody drug discovery -

The Omicron variant (B.1.1.529) of SARS-CoV-2 has rapidly become the dominant infective strain and the focus efforts against the ongoing COVID-19 pandemic. Here we report an extensive set of structures of the Omicron spike trimer by its own or in complex with ACE2 and an anti-Omicron antibody. These structures reveal that most Omicron mutations are located on the surface of the spike protein, which confer stronger ACE2 binding but become inactive epitopes resistant to many therapeutic antibodies. Importantly, both RBD and the closed conformation of the Omicron spike trimer are thermodynamically unstable, making the spiker trimer prone to random open conformations. An unusual RBD-RBD interaction in the ACE2-spike complex unique to Omicron is observed to support the open conformation and ACE2 binding, serving the basis for the higher infectivity of Omicron. A broad spectrum therapeutic antibody, which has completed Phase 1 clinical trial, is found to interact with the same two RBDs to inhibit ACE2 binding, in a mode that is distinguished from all previous antibodies, thus providing the structural basis for the potent inhibition of Omicron by this antibody. Together with biochemical data, our structures provide crucial insights into higher infectivity, antibody evasion and inhibition of Omicron.

🖺 Full Text HTML: Structures of the Omicron spike trimer with ACE2 and an anti-Omicron antibody: mechanisms for the high infectivity, immune evasion and antibody drug discovery

SARS-CoV-2 Omicron-B.1.1.529 Variant leads to less severe disease than Pango B and Delta variants strains in a mouse model of severe COVID-19 -

COVID-19 is a spectrum of clinical symptoms in humans caused by infection with SARS-CoV-2. The B.1.1.529 Omicron variant is rapidly emerging and has been designated a Variant of Concern (VOC). The variant is highly transmissible and partially or fully evades a spectrum of neutralising antibodies due to a high number of substitutions in the spike glycoprotein. A major question is the relative severity of disease caused by the Omicron variant compared with previous and currently circulating variants of SARS-CoV-2. To address this, a mouse model of infection that recapitulates severe disease in humans, K18-hACE2 mice, were infected with either a Pango B, Delta or Omicron variant of SARS-CoV-2 and their relative pathogenesis compared. In contrast to mice infected with Pango B and Delta variant viruses, those infected with the Omicron variant had less severe clinical signs (weight loss), showed recovery and had a lower virus load in both the lower and upper respiratory tract. This is also reflected by less extensive inflammatory processes in the lungs. Although T cell epitopes may be conserved, the antigenic diversity of Omicron from previous variants would suggest that a change in vaccine may be required to mitigate against the higher transmissibility and global disease burden. However, the lead time to develop such a response may be too late to mitigate the spread and effects of Omicron. These animal model data suggest the clinical consequences of infection with the Omicron variant may be less severe but the higher transmissibility could still place huge burden upon healthcare systems even if a lower proportion of infected patients are hospitalised.

🖺 Full Text HTML: SARS-CoV-2 Omicron-B.1.1.529 Variant leads to less severe disease than Pango B and Delta variants strains in a mouse model of severe COVID-19

Forecasting COVID-19 infection trends and new hospital admissions in England due to SARS-CoV-2 Variant of Concern Omicron -

Objectives: On November 26, 2021, WHO designated the variant B.1.1.529 as a new SARS-CoV-2 variant of concern (VoC), named Omicron, originally identified in South Africa. Several mutations in Omicron indicate that it may have an impact on how it spreads, resistance to vaccination, or the severity of illness it causes. We used our previous modelling algorithms to forecast the spread of Omicron in England. Design: We followed EQUATOR TRIPOD guidance for multivariable prediction models. Setting: England. Participants: Not applicable. Interventions: Non-interventional, observational study with a predicted forecast of outcomes. Main outcome measures: Trends in daily COVID-19 cases with a 7-day moving average and of new hospital admissions. Methods: Modelling included a third-degree polynomial curve in existing epidemiological trends on the spread of Omicron and a new Gaussian curve to estimate a downward trend after a peak in England. Results: Up to February 15, 2022, we estimated a projection of 250,000 COVID-19 daily cases of Omicron spread in the worse scenario, and 170,000 in the best scenario. Omicron might represent a relative increase from the background daily rates of COVID-19 infection in England of mid December 2021 of 1.9 to 2.8-fold. With a 5-day lag-time, daily new hospital admissions would peak at around 5,063 on January 23, 2022 in the worse scenario. Conclusion: This warning of pandemic surge of COVID-19 due to Omicron is calling for further reinforcing in England and elsewhere of universal hygiene interventions (indoor ventilation, social distance, and face masks), and anticipating the need of new total or partial lockdowns in England.

🖺 Full Text HTML: Forecasting COVID-19 infection trends and new hospital admissions in England due to SARS-CoV-2 Variant of Concern Omicron

Short-term Projections based on Early Omicron Variant Dynamics in England. -

Throughout the ongoing COVID-19 pandemic, the worldwide transmission and replication of SARS- COV-2, the causative agent of COVID-19 disease, has resulted in the opportunity for multiple mutations to occur that may alter the virus transmission characteristics, the effectiveness of vaccines and the severity of disease upon infection. The Omicron variant (B.1.1.529) was first reported to the WHO by South Africa on 24 November 2021 and was declared a variant of concern by the WHO on 26 November 2021. The variant was first detected in the UK on 27 November 2021 and has since been reported in a number of countries globally where it is frequently associated with rapid increase in cases. Here we present analyses of UK data showing the earliest signatures of the Omicron variant and mathematical modelling that uses the UK data to simulate the potential impact of this variant in the UK. In order to account for the uncertainty in transmission advantage, vaccine escape and severity at the time of writing, we carry out a sensitivity analysis to assess the impact of these variant characteristics on future risk.

🖺 Full Text HTML: Short- term Projections based on Early Omicron Variant Dynamics in England.

E6以及人肺腺癌细胞Calu‑3，检测肌醇六磷酸酯钠水合物的抗SARS‑CoV‑2活性。实验结果显示，肌醇六磷酸酯钠水合物能有效抑制SARS‑CoV‑2对上述易感细胞的感染，且细胞毒性较小，有希望作为有效抗SARS‑CoV‑2感染的药物，具有应用前景。 - link

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