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1.
Nat Biotechnol ; 2022 Jul 21.
Article in English | MEDLINE | ID: covidwho-1947381

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with potential resistance to existing drugs emphasizes the need for new therapeutic modalities with broad variant activity. Here we show that ensovibep, a trispecific DARPin (designed ankyrin repeat protein) clinical candidate, can engage the three units of the spike protein trimer of SARS-CoV-2 and inhibit ACE2 binding with high potency, as revealed by cryo-electron microscopy analysis. The cooperative binding together with the complementarity of the three DARPin modules enable ensovibep to inhibit frequent SARS-CoV-2 variants, including Omicron sublineages BA.1 and BA.2. In Roborovski dwarf hamsters infected with SARS-CoV-2, ensovibep reduced fatality similarly to a standard-of-care monoclonal antibody (mAb) cocktail. When used as a single agent in viral passaging experiments in vitro, ensovibep reduced the emergence of escape mutations in a similar fashion to the same mAb cocktail. These results support further clinical evaluation of ensovibep as a broad variant alternative to existing targeted therapies for Coronavirus Disease 2019 (COVID-19).

2.
Nucleic Acids Res ; 2022 Jun 17.
Article in English | MEDLINE | ID: covidwho-1901218

ABSTRACT

Antiviral innate immunity represents the first defense against invading viruses and is key to control viral infections, including SARS-CoV-2. Body temperature is an omnipresent variable but was neglected when addressing host defense mechanisms and susceptibility to SARS-CoV-2 infection. Here, we show that increasing temperature in a 1.5°C window, between 36.5 and 38°C, strongly increases the expression of genes in two branches of antiviral immunity, nitric oxide production and type I interferon response. We show that alternative splicing coupled to nonsense-mediated decay decreases STAT2 expression in colder conditions and suggest that increased STAT2 expression at elevated temperature induces the expression of diverse antiviral genes and SARS-CoV-2 restriction factors. This cascade is activated in a remarkably narrow temperature range below febrile temperature, which reflects individual, circadian and age-dependent variation. We suggest that decreased body temperature with aging contributes to reduced expression of antiviral genes in older individuals. Using cell culture and in vivo models, we show that higher body temperature correlates with reduced SARS-CoV-2 replication, which may affect the different vulnerability of children versus seniors toward severe SARS-CoV-2 infection. Altogether, our data connect body temperature and pre-mRNA processing to provide new mechanistic insight into the regulation of antiviral innate immunity.

3.
Mol Ther ; 30(5): 1952-1965, 2022 05 04.
Article in English | MEDLINE | ID: covidwho-1783847

ABSTRACT

For coronavirus disease 2019 (COVID-19), effective and well-understood treatment options are still scarce. Since vaccine efficacy is challenged by novel variants, short-lasting immunity, and vaccine hesitancy, understanding and optimizing therapeutic options remains essential. We aimed at better understanding the effects of two standard-of-care drugs, dexamethasone and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies, on infection and host responses. By using two COVID-19 hamster models, pulmonary immune responses were analyzed to characterize effects of single or combinatorial treatments. Pulmonary viral burden was reduced by anti-SARS-CoV-2 antibody treatment and unaltered or increased by dexamethasone alone. Dexamethasone exhibited strong anti-inflammatory effects and prevented fulminant disease in a severe disease model. Combination therapy showed additive benefits with both anti-viral and anti-inflammatory potency. Bulk and single-cell transcriptomic analyses confirmed dampened inflammatory cell recruitment into lungs upon dexamethasone treatment and identified a specifically responsive subpopulation of neutrophils, thereby indicating a potential mechanism of action. Our analyses confirm the anti-inflammatory properties of dexamethasone and suggest possible mechanisms, validate anti-viral effects of anti-SARS-CoV-2 antibody treatment, and reveal synergistic effects of a combination therapy, thus informing more effective COVID-19 therapies.


Subject(s)
COVID-19 , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Antibodies, Viral , Antiviral Agents , COVID-19/drug therapy , Cricetinae , Dexamethasone/pharmacology , SARS-CoV-2 , Transcriptome
4.
J Virol ; 96(5): e0218621, 2022 03 09.
Article in English | MEDLINE | ID: covidwho-1736028

ABSTRACT

Recent emergence of SARS-CoV-1 variants demonstrates the potential of this virus for targeted evolution, despite its overall genomic stability. Here we show the dynamics and the mechanisms behind the rapid adaptation of SARS-CoV-2 to growth in Vero E6 cells. The selective advantage for growth in Vero E6 cells is due to increased cleavage efficiency by cathepsins at the mutated S1/S2 site. S1/S2 site also constitutes a heparan sulfate (HS) binding motif that influenced virus growth in Vero E6 cells, but HS antagonist did not inhibit virus adaptation in these cells. The entry of Vero E6-adapted virus into human cells is defective because the mutated spike variants are poorly processed by furin or TMPRSS2. Minor subpopulation that lack the furin cleavage motif in the spike protein rapidly become dominant upon passaging through Vero E6 cells, but wild type sequences are maintained at low percentage in the virus swarm and mediate a rapid reverse adaptation if the virus is passaged again on TMPRSS2+ human cells. Our data show that the spike protein of SARS-CoV-2 can rapidly adapt itself to available proteases and argue for deep sequence surveillance to identify the emergence of novel variants. IMPORTANCE Recently emerging SARS-CoV-2 variants B.1.1.7 (alpha variant), B.1.617.2 (delta variant), and B.1.1.529 (omicron variant) harbor spike mutations and have been linked to increased virus pathogenesis. The emergence of these novel variants highlights coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt to selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genomes with original genotype are maintained in the virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.


Subject(s)
COVID-19/metabolism , Furin/metabolism , SARS-CoV-2/physiology , Serine Endopeptidases/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Adaptation, Physiological , Animals , COVID-19/genetics , COVID-19/virology , Chlorocebus aethiops , Cytopathogenic Effect, Viral , Furin/genetics , HEK293 Cells , High-Throughput Nucleotide Sequencing , Humans , Mutation , SARS-CoV-2/genetics , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/genetics , Vero Cells , Virus Replication
5.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-308569

ABSTRACT

Global pandemics by influenza or coronaviruses cause severe disruptions to the public health and lead to severe morbidity and mortality. Vaccines against these pathogens remain a medical need. CMV (cytomegalovirus) is a β-herpesvirus that induces uniquely robust immune responses, where outstandingly large populations of antigen-specific CD8+ T cells are maintained for a lifetime. Hence, CMV has been proposed and investigated as a novel vaccine vector expressing antigenic peptides or proteins to elicit protective cellular immune responses against numerous pathogens. We generated two recombinant murine CMV (MCMV) vaccine vectors expressing the hemagglutinin (HA) of influenza A virus (MCMVHA) or the spike protein of the severe acute respiratory syndrome coronavirus 2 (MCMVS). A single shot of MCMVs expressing either viral protein induced potent neutralizing antibody responses, which strengthened over time. Importantly, MCMVHA vaccinated mice were protected from illness following challenge with the influenza virus, and we excluded that this protection was due to effects of memory T cells. Conclusively, we show here that MCMV vectors do not only induce long-term cellular immunity, but also humoral responses that provide long-term immune protection against clinically relevant respiratory pathogens.

6.
Cell Mol Immunol ; 19(2): 234-244, 2022 02.
Article in English | MEDLINE | ID: covidwho-1612184

ABSTRACT

Global pandemics caused by influenza or coronaviruses cause severe disruptions to public health and lead to high morbidity and mortality. There remains a medical need for vaccines against these pathogens. CMV (cytomegalovirus) is a ß-herpesvirus that induces uniquely robust immune responses in which remarkably large populations of antigen-specific CD8+ T cells are maintained for a lifetime. Hence, CMV has been proposed and investigated as a novel vaccine vector for expressing antigenic peptides or proteins to elicit protective cellular immune responses against numerous pathogens. We generated two recombinant murine CMV (MCMV) vaccine vectors expressing hemagglutinin (HA) of influenza A virus (MCMVHA) or the spike protein of severe acute respiratory syndrome coronavirus 2 (MCMVS). A single injection of MCMVs expressing either viral protein induced potent neutralizing antibody responses, which strengthened over time. Importantly, MCMVHA-vaccinated mice were protected from illness following challenge with the influenza virus, and we excluded that this protection was due to the effects of memory T cells. Conclusively, we show here that MCMV vectors induce not only long-term cellular immunity but also humoral responses that provide long-term immune protection against clinically relevant respiratory pathogens.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Immunity, Humoral , Influenza A virus/immunology , Influenza Vaccines/immunology , Orthomyxoviridae Infections/prevention & control , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccination/methods , Animals , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/virology , Chlorocebus aethiops , Cytomegalovirus/immunology , Dogs , Female , HEK293 Cells , Humans , Immunity, Cellular , Madin Darby Canine Kidney Cells , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Orthomyxoviridae Infections/virology , Vero Cells
7.
Sci Adv ; 7(49): eabk0172, 2021 Dec 03.
Article in English | MEDLINE | ID: covidwho-1546430

ABSTRACT

Vaccines are instrumental and indispensable in the fight against the COVID-19 pandemic. Several recent SARS-CoV-2 variants are more transmissible and evade infection- or vaccine-induced protection. We constructed live attenuated vaccine candidates by large-scale recoding of the SARS-CoV-2 genome and showed that the lead candidate, designated sCPD9, protects Syrian hamsters from a challenge with ancestral virus. Here, we assessed immunogenicity and protective efficacy of sCPD9 in the Roborovski dwarf hamster, a nontransgenic rodent species that is highly susceptible to SARS-CoV-2 and severe COVID-19­like disease. We show that a single intranasal vaccination with sCPD9 elicited strong cross-neutralizing antibody responses against four current SARS-CoV-2 variants of concern, B.1.1.7 (Alpha), B.1.351 (Beta), B.1.1.28.1 (Gamma), and B.1.617.2 (Delta). The sCPD9 vaccine offered complete protection from COVID-19­like disease caused by the ancestral SARS-CoV-2 variant B.1 and the two variants of concern B.1.1.7 and B.1.351.

8.
PLoS One ; 16(8): e0255622, 2021.
Article in English | MEDLINE | ID: covidwho-1388954

ABSTRACT

The SARS-CoV-2 pandemic is a major global threat that sparked global research efforts. Pre-clinical and biochemical SARS-CoV-2 studies firstly rely on cell culture experiments where the importance of choosing an appropriate cell culture model is often underestimated. We here present a bottom-up approach to identify suitable permissive cancer cell lines for drug screening and virus research. Human cancer cell lines were screened for the SARS-CoV-2 cellular entry factors ACE2 and TMPRSS2 based on RNA-seq data of the Cancer Cell Line Encyclopedia (CCLE). However, experimentally testing permissiveness towards SARS-CoV-2 infection, we found limited correlation between receptor expression and permissiveness. This underlines that permissiveness of cells towards viral infection is determined not only by the presence of entry receptors but is defined by the availability of cellular resources, intrinsic immunity, and apoptosis. Aside from established cell culture infection models CACO-2 and CALU-3, three highly permissive human cell lines, colon cancer cell lines CL-14 and CL-40 and the breast cancer cell line CAL-51 and several low permissive cell lines were identified. Cell lines were characterised in more detail offering a broader choice of non-overexpression in vitro infection models to the scientific community. For some cell lines a truncated ACE2 mRNA and missense variants in TMPRSS2 might hint at disturbed host susceptibility towards viral entry.


Subject(s)
COVID-19/virology , Receptors, Virus , SARS-CoV-2/physiology , Virus Internalization , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Cell Line, Tumor , Humans , Receptors, Virus/genetics , Receptors, Virus/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism
9.
Cell Rep ; 36(4): 109433, 2021 07 27.
Article in English | MEDLINE | ID: covidwho-1300649

ABSTRACT

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/genetics , COVID-19/virology , Humans , Mutation/genetics , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Protein Domains/genetics , Spike Glycoprotein, Coronavirus/immunology
10.
Nat Commun ; 12(1): 1577, 2021 03 11.
Article in English | MEDLINE | ID: covidwho-1132068

ABSTRACT

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Antibodies, Neutralizing/genetics , Antibodies, Viral/genetics , COVID-19/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/chemistry , Animals , Antibodies, Neutralizing/isolation & purification , Antibodies, Viral/isolation & purification , Antibody Affinity , COVID-19/epidemiology , Cell Line , Chlorocebus aethiops , Gene Library , Healthy Volunteers , Host Microbial Interactions/immunology , Humans , Immunoglobulin G/genetics , Immunoglobulin G/isolation & purification , Models, Molecular , Mutation , Neutralization Tests , Pandemics , Peptide Library , Protein Interaction Domains and Motifs , Recombinant Proteins/genetics , Recombinant Proteins/immunology , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry , Vero Cells
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