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1.
Int J Mol Sci ; 23(15)2022 Jul 25.
Article in English | MEDLINE | ID: covidwho-1957347

ABSTRACT

Mutations in the spike protein of SARS-CoV-2 can lead to evasion from neutralizing antibodies and affect the efficacy of passive and active immunization strategies. Immunization of mice harboring an entire set of human immunoglobulin variable region gene segments allowed to identify nine neutralizing monoclonal antibodies, which either belong to a cluster of clonally related RBD or NTD binding antibodies. To better understand the genetic barrier to emergence of SARS-CoV-2 variants resistant to these antibodies, escape mutants were selected in cell culture to one antibody from each cluster and a combination of the two antibodies. Three independently derived escape mutants to the RBD antibody harbored mutations in the RBD at the position T478 or S477. These mutations impaired the binding of the RBD antibodies to the spike protein and conferred resistance in a pseudotype neutralization assay. Although the binding of the NTD cluster antibodies were not affected by the RBD mutations, the RBD mutations also reduced the neutralization efficacy of the NTD cluster antibodies. The mutations found in the escape variants to the NTD antibody conferred resistance to the NTD, but not to the RBD cluster antibodies. A variant resistant to both antibodies was more difficult to select and only emerged after longer passages and higher inoculation volumes. VOC carrying the same mutations as the ones identified in the escape variants were also resistant to neutralization. This study further underlines the rapid emergence of escape mutants to neutralizing monoclonal antibodies in cell culture and indicates the need for thorough investigation of escape mutations to select the most potent combination of monoclonal antibodies for clinical use.


Subject(s)
Antibodies, Neutralizing , COVID-19 , Animals , Antibodies, Monoclonal , Antibodies, Viral , Humans , Mice , Mutation , Neutralization Tests , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/chemistry
2.
EMBO J ; : e111608, 2022 Jul 14.
Article in English | MEDLINE | ID: covidwho-1934722

ABSTRACT

The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19.

3.
Cells ; 11(8)2022 04 08.
Article in English | MEDLINE | ID: covidwho-1785538

ABSTRACT

The COVID-19 pandemic caused by SARS-CoV-2 has lasted for more than two years. Despite the presence of very effective vaccines, the number of virus variants that escape neutralizing antibodies is growing. Thus, there is still a need for effective antiviral treatments that target virus replication independently of the circulating variant. Here, we show for the first time that deficiency or pharmacological inhibition of the cellular lysine-methyltransferase SMYD2 decreases TMPRSS2 expression on both mRNA and protein levels. SARS-CoV-2 uses TMPRSS2 for priming its spike protein to infect target cells. Treatment of cultured cells with the SMYD2 inhibitors AZ505 or BAY598 significantly inhibited viral replication. In contrast, treatment of Vero E6 cells, which do not express detectable amounts of TMPRSS2, had no effect on SARS-CoV-2 infection. Moreover, by generating a recombinant reporter virus that expresses the spike protein of the Delta variant of SARS-CoV-2, we demonstrate that BAY598 exhibits similar antiviral activity against this variant of concern. In summary, SMYD2 inhibition downregulates TMPRSS2 and blocks viral replication. Targeting cellular SMYD2 represents a promising tool to curtail SARS-CoV-2 infection.


Subject(s)
COVID-19 , Epithelial Cells , Histone-Lysine N-Methyltransferase , Serine Endopeptidases , Antiviral Agents/pharmacology , COVID-19/pathology , Epithelial Cells/metabolism , Epithelial Cells/virology , Histone-Lysine N-Methyltransferase/genetics , Humans , SARS-CoV-2/drug effects , Serine Endopeptidases/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
4.
mBio ; 13(2): e0040222, 2022 04 26.
Article in English | MEDLINE | ID: covidwho-1765083

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection triggers cytokine-mediated inflammation, leading to a myriad of clinical presentations in COVID-19. The SARS-CoV-2 open reading frame 8 (ORF8) is a secreted and rapidly evolving glycoprotein. Patients infected with SARS-CoV-2 variants with ORF8 deleted are associated with mild disease outcomes, but the molecular mechanism behind this is unknown. Here, we report that SARS-CoV-2 ORF8 is a viral cytokine that is similar to but distinct from interleukin 17A (IL-17A) as it induces stronger and broader human IL-17 receptor (hIL-17R) signaling than IL-17A. ORF8 primarily targeted blood monocytes and induced the heterodimerization of hIL-17RA and hIL-17RC, triggering a robust inflammatory response. Transcriptome analysis revealed that besides its activation of the hIL-17R pathway, ORF8 upregulated gene expression for fibrosis signaling and coagulation dysregulation. A naturally occurring ORF8 L84S variant that was highly associated with mild COVID-19 showed reduced hIL-17RA binding and attenuated inflammatory responses. This study reveals how SARS-CoV-2 ORF8 by a viral mimicry of the IL-17 cytokine contributes to COVID-19 severe inflammation. IMPORTANCE Patients infected with SARS-CoV-2 variants lacking open reading frame 8 (ORF8) have been associated with milder infection and disease outcome, but the molecular mechanism behind how this viral accessory protein mediates disease pathogenesis is not yet known. In our study, we revealed that secreted ORF8 protein mimics host IL-17 to activate IL-17 receptors A and C (IL-17RA/C) and induces a significantly stronger inflammatory response than host IL-17A, providing molecular insights into the role of ORF8 in COVID-19 pathogenesis and serving as a potential therapeutic target.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Inflammation/genetics , Interleukin-17/genetics , Open Reading Frames , SARS-CoV-2/genetics , Viral Proteins/metabolism
5.
Eur J Immunol ; 52(5): 770-783, 2022 05.
Article in English | MEDLINE | ID: covidwho-1589126

ABSTRACT

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Animals , Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , Humans , Mice , SARS-CoV-2
6.
Nat Commun ; 12(1): 6855, 2021 11 25.
Article in English | MEDLINE | ID: covidwho-1537312

ABSTRACT

The bat sarbecovirus RaTG13 is a close relative of SARS-CoV-2, the cause of the COVID-19 pandemic. However, this bat virus was most likely unable to directly infect humans since its Spike (S) protein does not interact efficiently with the human ACE2 receptor. Here, we show that a single T403R mutation increases binding of RaTG13 S to human ACE2 and allows VSV pseudoparticle infection of human lung cells and intestinal organoids. Conversely, mutation of R403T in the SARS-CoV-2 S reduces pseudoparticle infection and viral replication. The T403R RaTG13 S is neutralized by sera from individuals vaccinated against COVID-19 indicating that vaccination might protect against future zoonoses. Our data suggest that a positively charged amino acid at position 403 in the S protein is critical for efficient utilization of human ACE2 by S proteins of bat coronaviruses. This finding could help to better predict the zoonotic potential of animal coronaviruses.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Protein Binding , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Animals , COVID-19/virology , COVID-19 Vaccines , Caco-2 Cells , Cloning, Molecular , HEK293 Cells , Humans , Molecular Dynamics Simulation , Mutation , Replicon , Species Specificity , Stem Cells , Zoonoses
7.
Nat Commun ; 12(1): 6871, 2021 11 26.
Article in English | MEDLINE | ID: covidwho-1537309

ABSTRACT

Several effective SARS-CoV-2 vaccines are currently in use, but effective boosters are needed to maintain or increase immunity due to waning responses and the emergence of novel variants. Here we report that intranasal vaccinations with adenovirus 5 and 19a vectored vaccines following a systemic plasmid DNA or mRNA priming result in systemic and mucosal immunity in mice. In contrast to two intramuscular applications of an mRNA vaccine, intranasal boosts with adenoviral vectors induce high levels of mucosal IgA and lung-resident memory T cells (TRM); mucosal neutralization of virus variants of concern is also enhanced. The mRNA prime provokes a comprehensive T cell response consisting of circulating and lung TRM after the boost, while the plasmid DNA prime induces mostly mucosal T cells. Concomitantly, the intranasal boost strategies lead to complete protection against a SARS-CoV-2 infection in mice. Our data thus suggest that mucosal booster immunizations after mRNA priming is a promising approach to establish mucosal immunity in addition to systemic responses.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Immunity, Mucosal , Immunization, Secondary/methods , SARS-CoV-2/immunology , Adenoviridae/genetics , Administration, Intranasal , Animals , Antibodies, Viral/immunology , COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/genetics , Genetic Vectors , Immunization Schedule , Immunogenicity, Vaccine , Mice , Vaccines, DNA/administration & dosage , Vaccines, DNA/genetics , Vaccines, DNA/immunology , /immunology
8.
Int J Mol Sci ; 22(19)2021 Sep 22.
Article in English | MEDLINE | ID: covidwho-1438627

ABSTRACT

The ongoing pandemic coronavirus (CoV) disease 2019 (COVID-19) by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has already caused substantial morbidity, mortality, and economic devastation. Reverse genetic approaches to generate recombinant viruses are a powerful tool to characterize and understand newly emerging viruses. To contribute to the global efforts for countermeasures to control the spread of SARS-CoV-2, we developed a passage-free SARS-CoV-2 clone based on a bacterial artificial chromosome (BAC). Moreover, using a Lambda-based Red recombination, we successfully generated different reporter and marker viruses, which replicated similar to a clinical isolate in a cell culture. Moreover, we designed a full-length reporter virus encoding an additional artificial open reading frame with wild-type-like replication features. The virus-encoded reporters were successfully applied to ease antiviral testing in cell culture models. Furthermore, we designed a new marker virus encoding 3xFLAG-tagged nucleocapsid that allows the detection of incoming viral particles and, in combination with bio-orthogonal labeling for the visualization of viral RNA synthesis via click chemistry, the spatiotemporal tracking of viral replication on the single-cell level. In summary, by applying BAC-based Red recombination, we developed a powerful, reliable, and convenient platform that will facilitate studies answering numerous questions concerning the biology of SARS-CoV-2.


Subject(s)
COVID-19/virology , Cloning, Molecular/methods , Genome, Viral , SARS-CoV-2/genetics , Animals , Chlorocebus aethiops , HEK293 Cells , Humans , Mutagenesis , Plasmids/genetics , Recombination, Genetic , Vero Cells
9.
Pathogens ; 10(9)2021 Aug 25.
Article in English | MEDLINE | ID: covidwho-1374480

ABSTRACT

Currently, human infections with the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are accelerating the ongoing spread of the pandemic. Several innovative types of vaccines have already been developed, whereas effective options of antiviral treatments still await a scientific implementation. The development of novel anti-SARS-CoV-2 drug candidates demands skillful strategies and analysis systems. Promising results have been achieved with first generation direct-acting antivirals targeting the viral polymerase RdRp or the protease 3CLpro. Such recently approved or investigational drugs like remdesivir and GC376 represent a basis for further development and optimization. Here, we establish a multi-readout assay (MRA) system that enables the antiviral assessment and mechanistic characterization of novel test compounds, drug repurposing and combination treatments. Our SARS-CoV-2-specific MRA combines the quantitative measurement of several parameters of virus infection, such as the intracellular production of proteins and genomes, enzymatic activities and virion release, as well as the use of reporter systems. In this regard, the antiviral efficacy of remdesivir and GC376 has been investigated in human Caco-2 cells. The readouts included the use of spike- and double-strand RNA-specific monoclonal antibodies for in-cell fluorescence imaging, a newly generated recombinant SARS-CoV-2 reporter virus d6YFP, the novel 3CLpro-based FRET CFP::YFP and the previously reported FlipGFP reporter assays, as well as viral genome-specific RT-qPCR. The data produced by our MRA confirm the high antiviral potency of these two drugs in vitro. Combined, this MRA approach may be applied for broader analyses of SARS-CoV-2-specific antivirals, including compound screenings and the characterization of selected drug candidates.

10.
Clin Microbiol Infect ; 27(9): 1353.e1-1353.e5, 2021 Sep.
Article in English | MEDLINE | ID: covidwho-1240261

ABSTRACT

OBJECTIVES: Detection and surveillance of SARS-CoV-2 is of eminent importance, particularly due to the rapid emergence of variants of concern (VOCs). In this study we evaluated if a commercially available quantitative real-time PCR (qRT-PCR) assay can identify SARS-CoV-2 B.1.1.7 lineage samples by a specific N gene dropout or Ct value shift compared with the S or RdRp gene. METHODS: VOC B.1.1.7 and non-B.1.1.7 SARS-CoV-2-positive patient samples were identified via whole-genome sequencing and variant-specific PCR. Confirmed B.1.1.7 (n = 48) and non-B.1.1.7 samples (n = 58) were analysed using the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay for presence of SARS-CoV-2 S, RdRp and N genes. The N gene coding sequence of SARS-CoV-2 with and without the D3L mutation (specific for B.1.1.7) was cloned into pCR™II-TOPO™ vectors to validate polymorphism-dependent N gene dropout with the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay. RESULTS: All studied B.1.1.7-positive patient samples showed significantly higher Ct values in qRT-PCR (Δ6-10, N gene dropout on Ct values > 29) of N gene than the corresponding values of S (p ≤ 0.0001) and RdRp (p ≤ 0.0001) genes. The assay reliably discriminated B.1.1.7 and non-B.1.1.7 positive samples (area under the curve = 1) in a receiver operating characteristic curve analysis. Identical Ct value shifts (Δ7-10) were detected in reverse genetic experiments, using isolated plasmids containing N gene coding sequences corresponding to D3 or 3L variants. DISCUSSION: An N gene dropout or Ct value shift is shown for B.1.1.7-positive samples in the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay. This approach can be used as a rapid tool for B.1.1.7 detection in single assay high throughput diagnostics.


Subject(s)
COVID-19/diagnosis , Coronavirus Nucleocapsid Proteins/genetics , SARS-CoV-2/classification , Whole Genome Sequencing/methods , COVID-19 Nucleic Acid Testing , Genome, Viral , High-Throughput Nucleotide Sequencing , Humans , Multiplex Polymerase Chain Reaction , Mutation , ROC Curve , SARS-CoV-2/genetics , Sensitivity and Specificity
12.
Eur J Clin Microbiol Infect Dis ; 40(4): 751-759, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-880323

ABSTRACT

SARS-CoV-2 has emerged as a previously unknown zoonotic coronavirus that spread worldwide causing a serious pandemic. While reliable nucleic acid-based diagnostic assays were rapidly available, only a limited number of validated serological assays were available in the early phase of the pandemic. Here, we evaluated a novel flow cytometric approach to assess spike-specific antibody responses.HEK 293T cells expressing SARS-CoV-2 spike protein in its natural confirmation on the surface were used to detect specific IgG and IgM antibody responses in patient sera by flow cytometry. A soluble angiotensin-converting-enzyme 2 (ACE-2) variant was developed as external standard to quantify spike-specific antibody responses on different assay platforms. Analyses of 201 pre-COVID-19 sera proved a high assay specificity in comparison to commercially available CLIA and ELISA systems, while also revealing the highest sensitivity in specimens from PCR-confirmed SARS-CoV-2-infected patients. The external standard allowed robust quantification of antibody responses among different assay platforms. In conclusion, our newly established flow cytometric assay allows sensitive and quantitative detection of SARS-CoV-2-specific antibodies, which can be easily adopted in different laboratories and does not rely on external supply of assay kits. The flow cytometric assay also provides a blueprint for rapid development of serological tests to other emerging viral infections.


Subject(s)
Antibodies, Viral/immunology , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoglobulin G/immunology , Immunoglobulin M/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2 , Enzyme-Linked Immunosorbent Assay , Flow Cytometry/methods , HEK293 Cells , Humans , Reference Standards , Reproducibility of Results , Sensitivity and Specificity
13.
Euro Surveill ; 25(39)2020 10.
Article in English | MEDLINE | ID: covidwho-808986

ABSTRACT

We found that a single nucleotide polymorphism (SNP) in the nucleoprotein gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a patient interfered with detection in a widely used commercial assay. Some 0.2% of the isolates in the EpiCoV database contain this SNP. Although SARS-CoV-2 was still detected by the other probe in the assay, this underlines the necessity of targeting two independent essential regions of a pathogen for reliable detection.


Subject(s)
Betacoronavirus/genetics , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , Nucleoproteins/genetics , Pandemics , Pneumonia, Viral/diagnosis , Point Mutation , Polymorphism, Single Nucleotide , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , Viral Proteins/genetics , Base Sequence , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/methods , Contact Tracing , Coronavirus Infections/virology , DNA Primers , Diagnostic Errors , False Negative Reactions , Female , Genes, Viral , Humans , Middle Aged , Nasopharynx/virology , Nucleoproteins/analysis , Phylogeny , Pneumonia, Viral/virology , Real-Time Polymerase Chain Reaction/methods , Romania , SARS-CoV-2 , Travel-Related Illness , Viral Proteins/analysis
14.
Geburtshilfe Frauenheilkd ; 80(4): 380-390, 2020 Apr.
Article in English | MEDLINE | ID: covidwho-118977

ABSTRACT

In December 2019, cases of pneumonia of unknown cause first started to appear in Wuhan in China; subsequently, a new coronavirus was soon identified as the cause of the illness, now known as Coronavirus Disease 2019 (COVID-19). Since then, infections have been confirmed worldwide in numerous countries, with the number of cases steadily rising. The aim of the present review is to provide an overview of the new severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) and, in particular, to deduce from it potential risks and complications for pregnant patients. For this purpose, the available literature on cases of infection in pregnancy during the SARS epidemic of 2002/2003, the MERS (Middle East respiratory syndrome) epidemic ongoing since 2012, as well as recent publications on cases infected with SARS-CoV-2 in pregnancy are reviewed and reported. Based on the literature available at the moment, it can be assumed that the clinical course of COVID-19 disease may be complicated by pregnancy which could be associated with a higher mortality rate. It may also be assumed at the moment that transmission from mother to child in utero is unlikely. Breastfeeding is possible once infection has been excluded or the disease declared cured.

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