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1.
Sci Immunol ; 7(69): eabo2202, 2022 03 25.
Article in English | MEDLINE | ID: covidwho-1673343

ABSTRACT

The severe acute respiratory distress syndrome coronavirus 2 (SARS-CoV-2) Omicron variant is spreading rapidly, even in vaccinated individuals, raising concerns about immune escape. Here, we studied neutralizing antibodies and T cell responses targeting SARS-CoV-2 D614G [wild type (WT)] and the Beta, Delta, and Omicron variants of concern in a cohort of 60 health care workers after immunization with ChAdOx-1 S, Ad26.COV2.S, mRNA-1273, or BNT162b2. High binding antibody levels against WT SARS-CoV-2 spike (S) were detected 28 days after vaccination with both mRNA vaccines (mRNA-1273 or BNT162b2), which substantially decreased after 6 months. In contrast, antibody levels were lower after Ad26.COV2.S vaccination but did not wane. Neutralization assays showed consistent cross-neutralization of the Beta and Delta variants, but neutralization of Omicron was significantly lower or absent. BNT162b2 booster vaccination after either two mRNA-1273 immunizations or Ad26.COV2 priming partially restored neutralization of the Omicron variant, but responses were still up to 17-fold decreased compared with WT. SARS-CoV-2-specific T cells were detected up to 6 months after all vaccination regimens, with more consistent detection of specific CD4+ than CD8+ T cells. No significant differences were detected between WT- and variant-specific CD4+ or CD8+ T cell responses, including Omicron, indicating minimal escape at the T cell level. This study shows that vaccinated individuals retain T cell immunity to the SARS-CoV-2 Omicron variant, potentially balancing the lack of neutralizing antibodies in preventing or limiting severe COVID-19. Booster vaccinations are needed to further restore Omicron cross-neutralization by antibodies.


Subject(s)
COVID-19 , SARS-CoV-2 , CD8-Positive T-Lymphocytes , COVID-19/prevention & control , COVID-19 Vaccines , Humans
2.
J Clin Immunol ; 42(2): 232-239, 2022 02.
Article in English | MEDLINE | ID: covidwho-1669888

ABSTRACT

PURPOSE: To study the effect of interferon-α2 auto-antibodies (IFN-α2 Abs) on clinical and virological outcomes in critically ill COVID-19 patients and the risk of IFN-α2 Abs transfer during convalescent plasma treatment. METHODS: Sera from healthy controls, cases of COVID-19, and other respiratory illness were tested for IFN-α2 Abs by ELISA and a pseudo virus-based neutralization assay. The effects of disease severity, sex, and age on the risk of having neutralizing IFN-α2 Abs were determined. Longitudinal analyses were performed to determine association between IFN-α2 Abs and survival and viral load and whether serum IFN-α2 Abs appeared after convalescent plasma transfusion. RESULTS: IFN-α2 neutralizing sera were found only in COVID-19 patients, with proportions increasing with disease severity and age. In the acute stage of COVID-19, all sera from patients with ELISA-detected IFN-α2 Abs (13/164, 7.9%) neutralized levels of IFN-α2 exceeding physiological concentrations found in human plasma and this was associated with delayed viral clearance. Convalescent plasma donors that were anti-IFN-α2 ELISA positive (3/118, 2.5%) did not neutralize the same levels of IFN-α2. Neutralizing serum IFN-α2 Abs were associated with delayed viral clearance from the respiratory tract. CONCLUSIONS: IFN-α2 Abs were detected by ELISA and neutralization assay in COVID-19 patients, but not in ICU patients with other respiratory illnesses. The presence of neutralizing IFN-α2 Abs in critically ill COVID-19 is associated with delayed viral clearance. IFN-α2 Abs in COVID-19 convalescent plasma donors were not neutralizing in the conditions tested.


Subject(s)
Autoantibodies/immunology , COVID-19/immunology , COVID-19/therapy , Interferon alpha-2/immunology , Plasma/immunology , Adult , Aged , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antiviral Agents/immunology , Blood Component Transfusion/methods , Critical Illness , Female , Humans , Immunization, Passive/methods , Immunoglobulin G/immunology , Male , Middle Aged , SARS-CoV-2/immunology
3.
Nat Commun ; 12(1): 5498, 2021 09 17.
Article in English | MEDLINE | ID: covidwho-1428814

ABSTRACT

Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Biological Specimen Banks , CRISPR-Cas Systems , Coronavirus , Dipeptidyl Peptidase 4/genetics , Organoids/metabolism , Serine Endopeptidases/genetics , COVID-19 , Cell Line , Humans , Middle East Respiratory Syndrome Coronavirus , SARS-CoV-2 , Transcriptome , Virus Replication
4.
Elife ; 102021 04 09.
Article in English | MEDLINE | ID: covidwho-1389777

ABSTRACT

Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases - prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.


Subject(s)
Epithelial Cells , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Virus Cultivation/methods , Virus Internalization , Animals , Cell Line , Chlorocebus aethiops , Epithelial Cells/cytology , Epithelial Cells/metabolism , Epithelial Cells/virology , Humans , Proteolysis , Respiratory System/cytology , Respiratory System/virology , Serine Proteases/metabolism
5.
Elife ; 102021 01 04.
Article in English | MEDLINE | ID: covidwho-1063492

ABSTRACT

Coronavirus entry is mediated by the spike protein that binds the receptor and mediates fusion after cleavage by host proteases. The proteases that mediate entry differ between cell lines, and it is currently unclear which proteases are relevant in vivo. A remarkable feature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells and, more frequently, formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy.


Subject(s)
Organoids/virology , Respiratory System/virology , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/chemistry , Virus Internalization , Amino Acid Motifs , Animals , COVID-19/virology , Cell Fusion , Cell Line, Tumor , Chlorocebus aethiops , Humans , SARS Virus/chemistry , SARS Virus/physiology , SARS-CoV-2/chemistry , Serine Endopeptidases , Vero Cells
6.
Cell ; 184(5): 1188-1200.e19, 2021 03 04.
Article in English | MEDLINE | ID: covidwho-1046538

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication in the upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.


Subject(s)
COVID-19 Vaccines/administration & dosage , COVID-19 Vaccines/immunology , Macaca fascicularis , Spike Glycoprotein, Coronavirus/chemistry , Animals , Antibodies, Neutralizing , B-Lymphocytes/immunology , COVID-19/immunology , COVID-19/prevention & control , Mice , Mice, Inbred BALB C , Models, Animal , Nanoparticles/administration & dosage , Rabbits , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/blood , T-Lymphocytes/immunology , Viral Load
7.
Emerg Microbes Infect ; 10(1): 1-7, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-990475

ABSTRACT

Transmission of severe acute respiratory coronavirus-2 (SARS-CoV-2) between livestock and humans is a potential public health concern. We demonstrate the susceptibility of rabbits to SARS-CoV-2, which excrete infectious virus from the nose and throat upon experimental inoculation. Therefore, investigations on the presence of SARS-CoV-2 in farmed rabbits should be considered.


Subject(s)
COVID-19/transmission , Rabbits/virology , SARS-CoV-2/isolation & purification , Angiotensin-Converting Enzyme 2/physiology , Animals , COVID-19/etiology , COVID-19/veterinary , Disease Susceptibility/veterinary , Female , HEK293 Cells , Humans , Virus Shedding
8.
EMBO J ; 40(5): e105912, 2021 03 01.
Article in English | MEDLINE | ID: covidwho-962496

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air-liquid interface culture system which was characterized by confocal and electron microscopy and single-cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self-renewing fetal lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with mainly surfactant protein C-positive alveolar type II-like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens.


Subject(s)
Alveolar Epithelial Cells/metabolism , COVID-19/metabolism , Models, Biological , Organoids/metabolism , SARS-CoV-2/physiology , Virus Replication , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Animals , COVID-19/virology , Chlorocebus aethiops , Gene Expression Regulation , Humans , Interferon Type I/biosynthesis , Interferons/biosynthesis , Organoids/pathology , Organoids/virology , Vero Cells
10.
Science ; 369(6499): 50-54, 2020 07 03.
Article in English | MEDLINE | ID: covidwho-154670

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause coronavirus disease 2019 (COVID-19), an influenza-like disease that is primarily thought to infect the lungs with transmission through the respiratory route. However, clinical evidence suggests that the intestine may present another viral target organ. Indeed, the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on differentiated enterocytes. In human small intestinal organoids (hSIOs), enterocytes were readily infected by SARS-CoV and SARS-CoV-2, as demonstrated by confocal and electron microscopy. Enterocytes produced infectious viral particles, whereas messenger RNA expression analysis of hSIOs revealed induction of a generic viral response program. Therefore, the intestinal epithelium supports SARS-CoV-2 replication, and hSIOs serve as an experimental model for coronavirus infection and biology.


Subject(s)
Betacoronavirus/physiology , Enterocytes/virology , Ileum/virology , Virus Replication , Angiotensin-Converting Enzyme 2 , Betacoronavirus/ultrastructure , Cell Culture Techniques , Cell Differentiation , Cell Lineage , Cell Proliferation , Culture Media , Enterocytes/metabolism , Enterocytes/ultrastructure , Gene Expression , Humans , Ileum/metabolism , Ileum/ultrastructure , Lung/virology , Male , Organoids , Peptidyl-Dipeptidase A/genetics , Peptidyl-Dipeptidase A/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Virus/genetics , Receptors, Virus/metabolism , Respiratory Mucosa/virology , SARS Virus/physiology , SARS-CoV-2
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