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1.
Front Immunol ; 13: 882918, 2022.
Article in English | MEDLINE | ID: covidwho-1993786

ABSTRACT

In light of the decreasing immune protection against symptomatic SARS-CoV-2 infection after initial vaccinations and the now dominant immune-evasive Omicron variants, 'booster' vaccinations are regularly performed to restore immune responses. Many individuals have received a primary heterologous prime-boost vaccination with long intervals between vaccinations, but the resulting long-term immunity and the effects of a subsequent 'booster', particularly against Omicron BA.1, have not been defined. We followed a cohort of 23 young adults, who received a primary heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination, over a 7-month period and analysed how they responded to a BNT162b2 'booster'. We show that already after the primary heterologous vaccination, neutralization titers against Omicron BA.1 are recognizable but that humoral and cellular immunity wanes over the course of half a year. Residual responsive memory T cells recognized spike epitopes of the early SARS-CoV-2 B.1 strain as well as the Delta and BA.1 variants of concern (VOCs). However, the remaining antibody titers hardly neutralized these VOCs. The 'booster' vaccination was well tolerated and elicited both high antibody titers and increased memory T cell responses against SARS-CoV-2 including BA.1. Strikingly, in this young heterologously vaccinated cohort the neutralizing activity after the 'booster' was almost as potent against BA.1 as against the early B.1 strain. Our results suggest that a 'booster' after heterologous vaccination results in effective immune maturation and potent protection against the Omicron BA.1 variant in young adults.


Subject(s)
Antibodies, Neutralizing , COVID-19 , Antibodies, Viral , BNT162 Vaccine , COVID-19/prevention & control , ChAdOx1 nCoV-19 , Humans , SARS-CoV-2 , Vaccination , Young Adult
2.
Commun Biol ; 5(1): 681, 2022 Jul 08.
Article in English | MEDLINE | ID: covidwho-1927105

ABSTRACT

The transmembrane serine protease 2 (TMPRSS2) primes the SARS-CoV-2 Spike (S) protein for host cell entry and represents a promising target for COVID-19 therapy. Here we describe the in silico development and in vitro characterization of peptidomimetic TMPRSS2 inhibitors. Molecular docking studies identified peptidomimetic binders of the TMPRSS2 catalytic site, which were synthesized and coupled to an electrophilic serine trap. The compounds inhibit TMPRSS2 while demonstrating good off-target selectivity against selected coagulation proteases. Lead candidates are stable in blood serum and plasma for at least ten days. Finally, we show that selected peptidomimetics inhibit SARS-CoV-2 Spike-driven pseudovirus entry and authentic SARS-CoV-2 infection with comparable efficacy as camostat mesylate. The peptidomimetic TMPRSS2 inhibitors also prevent entry of recent SARS-CoV-2 variants of concern Delta and Omicron BA.1. In sum, our study reports antivirally active and stable TMPRSS2 inhibitors with prospects for further preclinical and clinical development as antiviral agents against SARS-CoV-2 and other TMPRSS2-dependent viruses.


Subject(s)
COVID-19 , Peptidomimetics , COVID-19/drug therapy , Cell Culture Techniques , Humans , Molecular Docking Simulation , Peptidomimetics/pharmacology , SARS-CoV-2 , Serine Endopeptidases/genetics
3.
Viruses ; 14(6)2022 06 15.
Article in English | MEDLINE | ID: covidwho-1911637

ABSTRACT

Screening of a protein kinase inhibitor library identified SB431542, targeting activin receptor-like kinase 5 (ALK5), as a compound interfering with SARS-CoV-2 replication. Since ALK5 is implicated in transforming growth factor ß (TGF-ß) signaling and regulation of the cellular endoprotease furin, we pursued this research to clarify the role of this protein kinase for SARS-CoV-2 infection. We show that TGF-ß1 induces the expression of furin in a broad spectrum of cells including Huh-7 and Calu-3 that are permissive for SARS-CoV-2. The inhibition of ALK5 by incubation with SB431542 revealed a dose-dependent downregulation of both basal and TGF-ß1 induced furin expression. Furthermore, we demonstrate that the ALK5 inhibitors SB431542 and Vactosertib negatively affect the proteolytic processing of the SARS-CoV-2 Spike protein and significantly reduce spike-mediated cell-cell fusion. This correlated with an inhibitory effect of ALK5 inhibition on the production of infectious SARS-CoV-2. Altogether, our study shows that interference with ALK5 signaling attenuates SARS-CoV-2 infectivity and cell-cell spread via downregulation of furin which is most pronounced upon TGF-ß stimulation. Since a TGF-ß dominated cytokine storm is a hallmark of severe COVID-19, ALK5 inhibitors undergoing clinical trials might represent a potential therapy option for COVID-19.


Subject(s)
COVID-19 , Transforming Growth Factor beta1 , Cell Fusion , Furin , Humans , Protein Serine-Threonine Kinases , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/metabolism , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta1/metabolism
4.
Antiviral Res ; 203: 105343, 2022 07.
Article in English | MEDLINE | ID: covidwho-1850637

ABSTRACT

Besides pandemic SARS-CoV-2, also endemic seasonal human common cold coronaviruses (hCoVs) have a significant impact on human health and economy. Studies on hCoVs and the identification of antivirals are therefore crucial to improve human well-being. However, hCoVs have long been neglected and the methodology to study virus infection, replication and inhibition warrants being updated. We here evaluated the established plaque-based assays to determine viral titers and cell-to-cell spread and developed protocols for the immunodetection of the viral nucleocapsid protein by flow cytometry and in-cell ELISA to study infection rates at early time points. The developed protocols allow detection of hCoV-229E infection after 2, and hCoV-NL63 and -OC43 infection after 3 days at a single cell level or in a 96 well microtiter format, in large sample numbers without being laborious or expensive. Both assays can be applied to assess the susceptibility of cells to hCoV infection and replication, and to determine the efficacy of antiviral compounds as well as neutralizing antibodies in a sensitive and quantitative manner. Application revealed that clinically applied SARS-CoV-2 targeting monoclonal antibodies are inactive against hCoVs, but that the viral polymerase targeting antivirals remdesivir and molnupiravir are broadly active also against all three hCoVs. Further, the in-cell ELISA provided evidence that nirmatrelvir, previously shown to broadly inhibit coronavirus proteases, also prevents replication of authentic hCoVs. Importantly, the protocols described here can be easily adapted to other coronavirus strains and species as well as viruses of other families within a short time. This will facilitate future research on known and emerging (corona)viruses, support the identification of antivirals and increase the preparedness for future virus outbreaks.


Subject(s)
COVID-19 , Common Cold , Coronavirus NL63, Human , Antiviral Agents/pharmacology , COVID-19/diagnosis , COVID-19/drug therapy , Common Cold/diagnosis , Common Cold/drug therapy , Humans , SARS-CoV-2 , Seasons
5.
Adv Sci (Weinh) ; 9(20): e2201378, 2022 07.
Article in English | MEDLINE | ID: covidwho-1838136

ABSTRACT

Inhibitors of viral cell entry based on poly(styrene sulfonate) and its core-shell nanoformulations based on gold nanoparticles are investigated against a panel of viruses, including clinical isolates of SARS-CoV-2. Macromolecular inhibitors are shown to exhibit the highly sought-after broad-spectrum antiviral activity, which covers most analyzed enveloped viruses and all of the variants of concern for SARS-CoV-2 tested. The inhibitory activity is quantified in vitro in appropriate cell culture models and for respiratory viral pathogens (respiratory syncytial virus and SARS-CoV-2) in mice. Results of this study comprise a significant step along the translational path of macromolecular inhibitors of virus cell entry, specifically against enveloped respiratory viruses.


Subject(s)
COVID-19 , Metal Nanoparticles , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Gold , Mice , SARS-CoV-2 , Virus Internalization
6.
Clin Infect Dis ; 2022 Jan 25.
Article in English | MEDLINE | ID: covidwho-1774348

ABSTRACT

BACKGROUND: Most of the millions of people that are vaccinated against SARS-CoV-2, the causative agent of COVID-19, have previously been infected by related circulating human coronaviruses (hCoVs) causing common colds and will experience further encounters with these viruses in the future. Whether or not COVID-19 vaccinations impact neutralization of seasonal coronaviruses is largely unknown. METHODS: We analyzed the capacity of sera derived from 24 individuals before and after heterologous ChAdOx1 nCoV-19 BNT162b2 prime-boost vaccination to neutralize genuine OC43, NL63 and 229E hCoVs, as well as viral pseudoparticles carrying the SARS-CoV-1, SARS-CoV-2, MERS-CoV, hCoV-OC43, -NL63 and -229E spike proteins. Genuine hCoVs or spike containing pseudovirions were incubated with different concentrations of sera and neutralization efficiencies were determined by measuring viral RNA yields, intracellular viral nucleocapsid expression, or reporter gene expression in Huh-7 cells. RESULTS: All individuals showed strong preexisting immunity against hCoV-OC43. Neutralization of hCoV-NL63 was more variable and all sera showed only modest inhibitory activity against genuine hCoV-229E. SARS-CoV-2 vaccination resulted in efficient cross-neutralization of SARS-CoV-1 but not of MERS-CoV. On average, vaccination significantly increased the neutralizing activity against genuine hCoV-OC43, -NL63 and -229E. CONCLUSIONS: Heterologous COVID-19 vaccination may confer some cross-protection against endemic seasonal coronaviruses.

7.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-317546

ABSTRACT

Preexisting diabetes increases the risk of a severe course of the pandemic coronavirus disease 2019 (COVID-19). Vice versa, exacerbations of a preexisting diabetes as well as new-onset diabetes have been reported upon SARS-CoV-2 infection. Thus, there is an imperative need to clarify whether human pancreatic endocrine cells organized within an islet of Langerhans are permissive for and affected by SARS-CoV-2 infection, and to elucidate the mechanisms underlying the development of diabetes upon COVID-19. Here, we (i) defined ACE2 and TMPRSS2 expression patterns in human pancreatic endocrine and exocrine cell types, (ii) employed human pancreatic islet cultures to demonstrate susceptibility to SARS-CoV-2 infection and to viral replication in β-cells, (iii) showed that SARS-CoV-2 attenuates glucose-stimulated insulin secretion, and (iv) tested remdesivir as eventually effective to prevent β-cell failure. In addition, we (v) visualized viral particles replicating in endocrine pancreatic cells and define their subcellular localization patterns via transmission electron microscopy, and finally (vi) present examples of cell type specific pancreatic infection patterns of COVID-19 deceased patients. Overall, our data demonstrate that SARS-CoV-2 can infect both the exocrine and endocrine compartments of the pancreas and can perturb β-cell integrity, which might lead to an increased risk for diabetes.

8.
J Clin Virol ; 147: 105062, 2022 02.
Article in English | MEDLINE | ID: covidwho-1670705

ABSTRACT

Since diagnostic sampling material must be considered as infectious, we evaluated whether extraction buffers of SARS-CoV-2 rapid antigen test kits may inactivate SARS-CoV-2. Of concern, seven of nine tested buffers lacked potent virucidal activity. To reduce risk of infection during assay performance, virucidal antigen extraction buffers that efficiently inactivate virus should replace the extraction buffers in these commercially available point-of-care devices.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Immunologic Tests , Point-of-Care Systems
9.
Nat Commun ; 12(1): 4584, 2021 07 28.
Article in English | MEDLINE | ID: covidwho-1387354

ABSTRACT

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Antigens, Differentiation/genetics , Membrane Proteins/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/genetics , Amino Acid Sequence , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Neutralizing/pharmacology , Antigens, Differentiation/metabolism , Binding Sites , COVID-19/virology , Gene Expression Regulation , Host-Pathogen Interactions/drug effects , Host-Pathogen Interactions/genetics , Humans , Interferon-beta/pharmacology , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/antagonists & inhibitors , RNA-Binding Proteins/metabolism , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Spike Glycoprotein, Coronavirus/metabolism , Virus Attachment/drug effects
10.
Nat Commun ; 12(1): 1726, 2021 03 19.
Article in English | MEDLINE | ID: covidwho-1142436

ABSTRACT

SARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α1-antitrypsin (α1AT), a highly abundant circulating serine protease inhibitor. Here, we report that α1AT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that α1AT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein α1AT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of α1AT-containing drugs has prospects for the therapy of COVID-19.


Subject(s)
COVID-19/drug therapy , SARS-CoV-2/drug effects , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , alpha 1-Antitrypsin/pharmacology , Antibodies, Viral/blood , Antiviral Agents/pharmacology , COVID-19/blood , Caco-2 Cells , Humans , Immunoglobulin G/blood , Molecular Docking Simulation , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects , Virus Replication/drug effects
11.
Am J Physiol Lung Cell Mol Physiol ; 320(5): L750-L756, 2021 05 01.
Article in English | MEDLINE | ID: covidwho-1076012

ABSTRACT

Pharmaceutical interventions are urgently needed to prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and transmission. As SARS-CoV-2 infects and spreads via the nasopharyngeal airways, we analyzed the antiviral effect of selected nasal and oral sprays on virus infection in vitro. Two nose sprays showed virucidal activity but were cytotoxic precluding further analysis in cell culture. One nasal and one mouth spray suppressed SARS-CoV-2 infection of TMPRSS2-expressing Vero E6 cells and primary differentiated human airway epithelial cultures. The antiviral activity in both sprays could be attributed to polyanionic ι- and κ-carrageenans. Thus, application of carrageenan-containing nasal and mouth sprays may reduce the risk of acquiring SARS-CoV-2 infection and may limit viral spread, warranting further clinical evaluation.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , COVID-19/prevention & control , Carrageenan/pharmacology , SARS-CoV-2/drug effects , Adult , Animals , Cell Line , Chlorocebus aethiops , Epithelial Cells/drug effects , Epithelial Cells/virology , Female , Humans , Male , Middle Aged , Nasal Sprays , Oral Sprays , Serine Endopeptidases/metabolism , Vero Cells
12.
Nat Metab ; 3(2): 149-165, 2021 02.
Article in English | MEDLINE | ID: covidwho-1065968

ABSTRACT

Infection-related diabetes can arise as a result of virus-associated ß-cell destruction. Clinical data suggest that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19), impairs glucose homoeostasis, but experimental evidence that SARS-CoV-2 can infect pancreatic tissue has been lacking. In the present study, we show that SARS-CoV-2 infects cells of the human exocrine and endocrine pancreas ex vivo and in vivo. We demonstrate that human ß-cells express viral entry proteins, and SARS-CoV-2 infects and replicates in cultured human islets. Infection is associated with morphological, transcriptional and functional changes, including reduced numbers of insulin-secretory granules in ß-cells and impaired glucose-stimulated insulin secretion. In COVID-19 full-body postmortem examinations, we detected SARS-CoV-2 nucleocapsid protein in pancreatic exocrine cells, and in cells that stain positive for the ß-cell marker NKX6.1 and are in close proximity to the islets of Langerhans in all four patients investigated. Our data identify the human pancreas as a target of SARS-CoV-2 infection and suggest that ß-cell infection could contribute to the metabolic dysregulation observed in patients with COVID-19.


Subject(s)
Islets of Langerhans/virology , SARS-CoV-2/growth & development , Aged , Aged, 80 and over , Angiotensin-Converting Enzyme 2/biosynthesis , Angiotensin-Converting Enzyme 2/genetics , COVID-19/physiopathology , Cells, Cultured , Diabetes Mellitus , Female , Humans , Islets of Langerhans/cytology , Islets of Langerhans/physiopathology , Male , Pancreas, Exocrine/cytology , Pancreas, Exocrine/physiopathology , Pancreas, Exocrine/virology , Pancreatic Diseases/etiology , Pancreatic Diseases/virology , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/genetics , Virus Internalization , Virus Replication
13.
Cell Mol Gastroenterol Hepatol ; 11(4): 935-948, 2021.
Article in English | MEDLINE | ID: covidwho-917333

ABSTRACT

BACKGROUND AND AIMS: The COVID-19 pandemic has spread worldwide and poses a severe health risk. While most patients present mild symptoms, descending pneumonia can lead to severe respiratory insufficiency. Up to 50% of patients show gastrointestinal symptoms like diarrhea or nausea, intriguingly associating with prolonged symptoms and increased severity. Thus, models to understand and validate drug efficiency in the gut of COVID-19 patients are of urgent need. METHODS: Human intestinal organoids derived from pluripotent stem cells (PSC-HIOs) have led, due to their complexity in mimicking human intestinal architecture, to an unprecedented number of successful disease models including gastrointestinal infections. Here, we employed PSC-HIOs to dissect SARS-CoV-2 pathogenesis and its inhibition by remdesivir, one of the leading drugs investigated for treatment of COVID-19. RESULTS: Immunostaining for viral entry receptor ACE2 and SARS-CoV-2 spike protein priming protease TMPRSS2 showed broad expression in the gastrointestinal tract with highest levels in the intestine, the latter faithfully recapitulated by PSC-HIOs. Organoids could be readily infected with SARS-CoV-2 followed by viral spread across entire PSC-HIOs, subsequently leading to organoid deterioration. However, SARS-CoV-2 spared goblet cells lacking ACE2 expression. Importantly, we challenged PSC-HIOs for drug testing capacity. Specifically, remdesivir effectively inhibited SARS-CoV-2 infection dose-dependently at low micromolar concentration and rescued PSC-HIO morphology. CONCLUSIONS: Thus, PSC-HIOs are a valuable tool to study SARS-CoV-2 infection and to identify and validate drugs especially with potential action in the gut.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , COVID-19/drug therapy , COVID-19/metabolism , Human Embryonic Stem Cells , Intestinal Mucosa , Organoids , SARS-CoV-2/physiology , Virus Replication/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Caco-2 Cells , Human Embryonic Stem Cells/metabolism , Human Embryonic Stem Cells/pathology , Human Embryonic Stem Cells/virology , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Intestinal Mucosa/virology , Organoids/metabolism , Organoids/pathology , Organoids/virology
14.
J Am Chem Soc ; 142(40): 17024-17038, 2020 10 07.
Article in English | MEDLINE | ID: covidwho-772998

ABSTRACT

Broad-spectrum antivirals are powerful weapons against dangerous viruses where no specific therapy exists, as in the case of the ongoing SARS-CoV-2 pandemic. We discovered that a lysine- and arginine-specific supramolecular ligand (CLR01) destroys enveloped viruses, including HIV, Ebola, and Zika virus, and remodels amyloid fibrils in semen that promote viral infection. Yet, it is unknown how CLR01 exerts these two distinct therapeutic activities. Here, we delineate a novel mechanism of antiviral activity by studying the activity of tweezer variants: the "phosphate tweezer" CLR01, a "carboxylate tweezer" CLR05, and a "phosphate clip" PC. Lysine complexation inside the tweezer cavity is needed to antagonize amyloidogenesis and is only achieved by CLR01. Importantly, CLR01 and CLR05 but not PC form closed inclusion complexes with lipid head groups of viral membranes, thereby altering lipid orientation and increasing surface tension. This process disrupts viral envelopes and diminishes infectivity but leaves cellular membranes intact. Consequently, CLR01 and CLR05 display broad antiviral activity against all enveloped viruses tested, including herpesviruses, Measles virus, influenza, and SARS-CoV-2. Based on our mechanistic insights, we potentiated the antiviral, membrane-disrupting activity of CLR01 by introducing aliphatic ester arms into each phosphate group to act as lipid anchors that promote membrane targeting. The most potent ester modifications harbored unbranched C4 units, which engendered tweezers that were approximately one order of magnitude more effective than CLR01 and nontoxic. Thus, we establish the mechanistic basis of viral envelope disruption by specific tweezers and establish a new class of potential broad-spectrum antivirals with enhanced activity.


Subject(s)
Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Bridged-Ring Compounds/pharmacology , Organophosphates/pharmacology , Viral Envelope Proteins/drug effects , Acid Phosphatase/chemistry , Acid Phosphatase/metabolism , Amyloid/antagonists & inhibitors , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Arginine/chemistry , Betacoronavirus/drug effects , Bridged-Ring Compounds/chemistry , Cell Membrane/chemistry , Cell Membrane/drug effects , Cell Membrane/virology , HIV Infections/drug therapy , HIV-1/drug effects , Humans , Lipids/chemistry , Lysine/chemistry , Magnetic Resonance Spectroscopy , Organophosphates/chemistry , SARS-CoV-2 , Seminal Vesicle Secretory Proteins/chemistry , Seminal Vesicle Secretory Proteins/metabolism , Structure-Activity Relationship , Viral Envelope Proteins/metabolism , Zika Virus/drug effects
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