Your browser doesn't support javascript.
Show: 20 | 50 | 100
Results 1 - 14 de 14
Filter
1.
PLoS Biol ; 20(3): e3001592, 2022 03.
Article in English | MEDLINE | ID: covidwho-1770633

ABSTRACT

Gastrointestinal effects associated with Coronavirus Disease 2019 (COVID-19) are highly variable for reasons that are not understood. In this study, we used intestinal organoid-derived cultures differentiated from primary human specimens as a model to examine interindividual variability. Infection of intestinal organoids derived from different donors with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) resulted in orders of magnitude differences in virus replication in small intestinal and colonic organoid-derived monolayers. Susceptibility to infection correlated with angiotensin I converting enzyme 2 (ACE2) expression level and was independent of donor demographic or clinical features. ACE2 transcript levels in cell culture matched the amount of ACE2 in primary tissue, indicating that this feature of the intestinal epithelium is retained in the organoids. Longitudinal transcriptomics of organoid-derived monolayers identified a delayed yet robust interferon signature, the magnitude of which corresponded to the degree of SARS-CoV-2 infection. Interestingly, virus with the Omicron variant spike (S) protein infected the organoids with the highest infectivity, suggesting increased tropism of the virus for intestinal tissue. These results suggest that heterogeneity in SARS-CoV-2 replication in intestinal tissues results from differences in ACE2 levels, which may underlie variable patient outcomes.


Subject(s)
COVID-19 , Angiotensin-Converting Enzyme 2/genetics , Humans , Organoids , SARS-CoV-2
2.
Front Immunol ; 12: 719077, 2021.
Article in English | MEDLINE | ID: covidwho-1575525

ABSTRACT

The COVID-19 pandemic caused by the coronavirus SARS-CoV-2 is a major global public threat. Currently, a worldwide effort has been mounted to generate billions of effective SARS-CoV-2 vaccine doses to immunize the world's population at record speeds. However, there is still a demand for alternative effective vaccines that rapidly confer long-term protection and rely upon cost-effective, easily scaled-up manufacturing. Here, we present a Sindbis alphavirus vector (SV), transiently expressing the SARS-CoV-2 spike protein (SV.Spike), combined with the OX40 immunostimulatory antibody (αOX40) as a novel, highly effective vaccine approach. We show that SV.Spike plus αOX40 elicits long-lasting neutralizing antibodies and a vigorous T-cell response in mice. Protein binding, immunohistochemical, and cellular infection assays all show that vaccinated mice sera inhibits spike functions. Immunophenotyping, RNA Seq transcriptome profiles, and metabolic analysis indicate a reprogramming of T cells in vaccinated mice. Activated T cells were found to mobilize to lung tissue. Most importantly, SV.Spike plus αOX40 provided robust immune protection against infection with authentic coronavirus in transgenic mice expressing the human ACE2 receptor (hACE2-Tg). Finally, our immunization strategy induced strong effector memory response, potentiating protective immunity against re-exposure to SARS-CoV-2 spike protein. Our results show the potential of a new Sindbis virus-based vaccine platform to counteract waning immune response, which can be used as a new candidate to combat SARS-CoV-2. Given the T-cell responses elicited, our vaccine is likely to be effective against variants that are proving challenging, as well as serve as a platform to develop a broader spectrum pancoronavirus vaccine. Similarly, the vaccine approach is likely to be applicable to other pathogens.


Subject(s)
Antigens, Differentiation/immunology , COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Sindbis Virus/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Cricetinae , Female , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Sindbis Virus/genetics , T-Lymphocytes/immunology , Vaccination
3.
Viruses ; 13(12)2021 12 04.
Article in English | MEDLINE | ID: covidwho-1554971

ABSTRACT

Epidemic RNA viruses seem to arise year after year leading to countless infections and devastating disease. SARS-CoV-2 is the most recent of these viruses, but there will undoubtedly be more to come. While effective SARS-CoV-2 vaccines are being deployed, one approach that is still missing is effective antivirals that can be used at the onset of infections and therefore prevent pandemics. Here, we screened FDA-approved compounds against SARS-CoV-2. We found that atovaquone, a pyrimidine biosynthesis inhibitor, is able to reduce SARS-CoV-2 infection in human lung cells. In addition, we found that berberine chloride, a plant-based compound used in holistic medicine, was able to inhibit SARS-CoV-2 infection in cells through direct interaction with the virion. Taken together, these studies highlight potential avenues of antiviral development to block emerging viruses. Such proactive approaches, conducted well before the next pandemic, will be essential to have drugs ready for when the next emerging virus hits.


Subject(s)
Antiviral Agents/pharmacology , Atovaquone/pharmacology , Berberine/pharmacology , SARS-CoV-2/drug effects , Virus Replication/drug effects , Alveolar Epithelial Cells , Animals , Berberine/chemistry , Cell Proliferation/drug effects , Chlorides/chemistry , Chlorides/pharmacology , Chlorocebus aethiops , Drug Synergism , Humans , Proguanil/pharmacology , Vero Cells , Virion/drug effects
4.
J Thromb Haemost ; 19(12): 3139-3153, 2021 12.
Article in English | MEDLINE | ID: covidwho-1526388

ABSTRACT

OBJECTIVE: Heightened inflammation, dysregulated immunity, and thrombotic events are characteristic of hospitalized COVID-19 patients. Given that platelets are key regulators of thrombosis, inflammation, and immunity they represent prime candidates as mediators of COVID-19-associated pathogenesis. The objective of this study was to understand the contribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the platelet phenotype via phenotypic (activation, aggregation) and transcriptomic characterization. APPROACH AND RESULTS: In a cohort of 3915 hospitalized COVID-19 patients, we analyzed blood platelet indices collected at hospital admission. Following adjustment for demographics, clinical risk factors, medication, and biomarkers of inflammation and thrombosis, we find platelet count, size, and immaturity are associated with increased critical illness and all-cause mortality. Bone marrow, lung tissue, and blood from COVID-19 patients revealed the presence of SARS-CoV-2 virions in megakaryocytes and platelets. Characterization of COVID-19 platelets found them to be hyperreactive (increased aggregation, and expression of P-selectin and CD40) and to have a distinct transcriptomic profile characteristic of prothrombotic large and immature platelets. In vitro mechanistic studies highlight that the interaction of SARS-CoV-2 with megakaryocytes alters the platelet transcriptome, and its effects are distinct from the coronavirus responsible for the common cold (CoV-OC43). CONCLUSIONS: Platelet count, size, and maturity associate with increased critical illness and all-cause mortality among hospitalized COVID-19 patients. Profiling tissues and blood from COVID-19 patients revealed that SARS-CoV-2 virions enter megakaryocytes and platelets and associate with alterations to the platelet transcriptome and activation profile.


Subject(s)
COVID-19 , Thrombosis , Blood Platelets , Humans , SARS-CoV-2 , Severity of Illness Index
5.
J Thromb Haemost ; 19(12): 3139-3153, 2021 12.
Article in English | MEDLINE | ID: covidwho-1429991

ABSTRACT

OBJECTIVE: Heightened inflammation, dysregulated immunity, and thrombotic events are characteristic of hospitalized COVID-19 patients. Given that platelets are key regulators of thrombosis, inflammation, and immunity they represent prime candidates as mediators of COVID-19-associated pathogenesis. The objective of this study was to understand the contribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to the platelet phenotype via phenotypic (activation, aggregation) and transcriptomic characterization. APPROACH AND RESULTS: In a cohort of 3915 hospitalized COVID-19 patients, we analyzed blood platelet indices collected at hospital admission. Following adjustment for demographics, clinical risk factors, medication, and biomarkers of inflammation and thrombosis, we find platelet count, size, and immaturity are associated with increased critical illness and all-cause mortality. Bone marrow, lung tissue, and blood from COVID-19 patients revealed the presence of SARS-CoV-2 virions in megakaryocytes and platelets. Characterization of COVID-19 platelets found them to be hyperreactive (increased aggregation, and expression of P-selectin and CD40) and to have a distinct transcriptomic profile characteristic of prothrombotic large and immature platelets. In vitro mechanistic studies highlight that the interaction of SARS-CoV-2 with megakaryocytes alters the platelet transcriptome, and its effects are distinct from the coronavirus responsible for the common cold (CoV-OC43). CONCLUSIONS: Platelet count, size, and maturity associate with increased critical illness and all-cause mortality among hospitalized COVID-19 patients. Profiling tissues and blood from COVID-19 patients revealed that SARS-CoV-2 virions enter megakaryocytes and platelets and associate with alterations to the platelet transcriptome and activation profile.


Subject(s)
COVID-19 , Thrombosis , Blood Platelets , Humans , SARS-CoV-2 , Severity of Illness Index
6.
Sci Adv ; 7(37): eabh2434, 2021 Sep 10.
Article in English | MEDLINE | ID: covidwho-1405214

ABSTRACT

Given the evidence for a hyperactive platelet phenotype in COVID-19, we investigated effector cell properties of COVID-19 platelets on endothelial cells (ECs). Integration of EC and platelet RNA sequencing revealed that platelet-released factors in COVID-19 promote an inflammatory hypercoagulable endotheliopathy. We identified S100A8 and S100A9 as transcripts enriched in COVID-19 platelets and were induced by megakaryocyte infection with SARS-CoV-2. Consistent with increased gene expression, the heterodimer protein product of S100A8/A9, myeloid-related protein (MRP) 8/14, was released to a greater extent by platelets from COVID-19 patients relative to controls. We demonstrate that platelet-derived MRP8/14 activates ECs, promotes an inflammatory hypercoagulable phenotype, and is a significant contributor to poor clinical outcomes in COVID-19 patients. Last, we present evidence that targeting platelet P2Y12 represents a promising candidate to reduce proinflammatory platelet-endothelial interactions. Together, these findings demonstrate a previously unappreciated role for platelets and their activation-induced endotheliopathy in COVID-19.

8.
Nat Microbiol ; 6(10): 1245-1258, 2021 10.
Article in English | MEDLINE | ID: covidwho-1380902

ABSTRACT

Respiratory failure is associated with increased mortality in COVID-19 patients. There are no validated lower airway biomarkers to predict clinical outcome. We investigated whether bacterial respiratory infections were associated with poor clinical outcome of COVID-19 in a prospective, observational cohort of 589 critically ill adults, all of whom required mechanical ventilation. For a subset of 142 patients who underwent bronchoscopy, we quantified SARS-CoV-2 viral load, analysed the lower respiratory tract microbiome using metagenomics and metatranscriptomics and profiled the host immune response. Acquisition of a hospital-acquired respiratory pathogen was not associated with fatal outcome. Poor clinical outcome was associated with lower airway enrichment with an oral commensal (Mycoplasma salivarium). Increased SARS-CoV-2 abundance, low anti-SARS-CoV-2 antibody response and a distinct host transcriptome profile of the lower airways were most predictive of mortality. Our data provide evidence that secondary respiratory infections do not drive mortality in COVID-19 and clinical management strategies should prioritize reducing viral replication and maximizing host responses to SARS-CoV-2.


Subject(s)
Bronchoalveolar Lavage Fluid/microbiology , COVID-19/therapy , Respiration, Artificial , SARS-CoV-2/pathogenicity , Adaptive Immunity , Adult , Aged , Bacteria/classification , Bacteria/genetics , Bacteria/isolation & purification , Bacterial Load , Bronchoalveolar Lavage Fluid/immunology , Bronchoalveolar Lavage Fluid/virology , COVID-19/immunology , COVID-19/microbiology , COVID-19/mortality , Critical Illness , Female , Hospitalization , Humans , Immunity, Innate , Male , Microbiota , Middle Aged , Odds Ratio , Prognosis , Prospective Studies , Respiratory System/immunology , Respiratory System/microbiology , Respiratory System/virology , SARS-CoV-2/immunology , Viral Load
9.
Genes Dev ; 35(13-14): 1005-1019, 2021 07 01.
Article in English | MEDLINE | ID: covidwho-1334329

ABSTRACT

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.


Subject(s)
Coronavirus OC43, Human/physiology , RNA Processing, Post-Transcriptional/genetics , SARS-CoV-2/physiology , Virus Replication/genetics , Adenosine/analogs & derivatives , Adenosine/genetics , Adenosine/metabolism , Cell Line , Coronavirus Infections/metabolism , Coronavirus Infections/virology , Gene Expression Regulation/drug effects , Host-Pathogen Interactions/drug effects , Humans , Methyltransferases/antagonists & inhibitors , Methyltransferases/metabolism , Nucleocapsid Proteins , RNA, Viral/metabolism , RNA-Binding Proteins/metabolism , Virus Replication/drug effects
10.
Immunity ; 54(6): 1304-1319.e9, 2021 06 08.
Article in English | MEDLINE | ID: covidwho-1246001

ABSTRACT

Despite mounting evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engagement with immune cells, most express little, if any, of the canonical receptor of SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Here, using a myeloid cell receptor-focused ectopic expression screen, we identified several C-type lectins (DC-SIGN, L-SIGN, LSECtin, ASGR1, and CLEC10A) and Tweety family member 2 (TTYH2) as glycan-dependent binding partners of the SARS-CoV-2 spike. Except for TTYH2, these molecules primarily interacted with spike via regions outside of the receptor-binding domain. Single-cell RNA sequencing analysis of pulmonary cells from individuals with coronavirus disease 2019 (COVID-19) indicated predominant expression of these molecules on myeloid cells. Although these receptors do not support active replication of SARS-CoV-2, their engagement with the virus induced robust proinflammatory responses in myeloid cells that correlated with COVID-19 severity. We also generated a bispecific anti-spike nanobody that not only blocked ACE2-mediated infection but also the myeloid receptor-mediated proinflammatory responses. Our findings suggest that SARS-CoV-2-myeloid receptor interactions promote immune hyperactivation, which represents potential targets for COVID-19 therapy.


Subject(s)
COVID-19/metabolism , COVID-19/virology , Host-Pathogen Interactions , Lectins, C-Type/metabolism , Membrane Proteins/metabolism , Myeloid Cells/immunology , Myeloid Cells/metabolism , Neoplasm Proteins/metabolism , SARS-CoV-2/physiology , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , COVID-19/genetics , Cell Line , Cytokines , Gene Expression Regulation , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Inflammation Mediators/metabolism , Lectins, C-Type/chemistry , Membrane Proteins/chemistry , Models, Molecular , Neoplasm Proteins/chemistry , Protein Binding , Protein Conformation , Single-Domain Antibodies/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , Structure-Activity Relationship
11.
Sci Rep ; 11(1): 5538, 2021 03 10.
Article in English | MEDLINE | ID: covidwho-1125909

ABSTRACT

Understanding antibody responses to SARS-CoV-2 is indispensable for the development of containment measures to overcome the current COVID-19 pandemic. Recent studies showed that serum from convalescent patients can display variable neutralization capacities. Still, it remains unclear whether there are specific signatures that can be used to predict neutralization. Here, we performed a detailed analysis of sera from a cohort of 101 recovered healthcare workers and we addressed their SARS-CoV-2 antibody response by ELISA against SARS-CoV-2 Spike receptor binding domain and nucleoprotein. Both ELISA methods detected sustained levels of serum IgG against both antigens. Yet, the majority of individuals from our cohort generated antibodies with low neutralization capacity and only 6% showed high neutralizing titers against both authentic SARS-CoV-2 virus and the Spike pseudotyped virus. Interestingly, higher neutralizing sera correlate with detection of -IgG, IgM and IgA antibodies against both antigens, while individuals with positive IgG alone showed poor neutralization response. These results suggest that having a broader repertoire of antibodies may contribute to more potent SARS-CoV-2 neutralization. Altogether, our work provides a cross sectional snapshot of the SARS-CoV-2 neutralizing antibody response in recovered healthcare workers and provides preliminary evidence that possessing multiple antibody isotypes can play an important role in predicting SARS-CoV-2 neutralization.


Subject(s)
Antibodies, Neutralizing/immunology , COVID-19/immunology , SARS-CoV-2/immunology , Adult , Antibodies, Viral/immunology , COVID-19/therapy , Cohort Studies , Cross-Sectional Studies , Enzyme-Linked Immunosorbent Assay/methods , Epitopes/immunology , Female , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Male , Neutralization Tests/methods , Pandemics , SARS-CoV-2/pathogenicity , Serum/immunology , Spike Glycoprotein, Coronavirus/immunology
12.
Cell Rep ; 33(12): 108528, 2020 12 22.
Article in English | MEDLINE | ID: covidwho-978234

ABSTRACT

Soluble forms of angiotensin-converting enzyme 2 (ACE2) have recently been shown to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We report on an improved soluble ACE2, termed a "microbody," in which the ACE2 ectodomain is fused to Fc domain 3 of the immunoglobulin (Ig) heavy chain. The protein is smaller than previously described ACE2-Ig Fc fusion proteins and contains an H345A mutation in the ACE2 catalytic active site that inactivates the enzyme without reducing its affinity for the SARS-CoV-2 spike. The disulfide-bonded ACE2 microbody protein inhibits entry of SARS-CoV-2 spike protein pseudotyped virus and replication of live SARS-CoV-2 in vitro and in a mouse model. Its potency is 10-fold higher than soluble ACE2, and it can act after virus bound to the cell. The microbody inhibits the entry of ß coronaviruses and virus with the variant D614G spike. The ACE2 microbody may be a valuable therapeutic for coronavirus disease 2019 (COVID-19) that is active against viral variants and future coronaviruses.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , Immunoglobulin Fc Fragments/metabolism , Microbodies/metabolism , SARS-CoV-2/drug effects , Amino Acid Sequence , Animals , COVID-19/prevention & control , COVID-19/virology , Disease Models, Animal , Disulfides/metabolism , Female , HEK293 Cells , Humans , Male , Mice, Transgenic , Protein Domains , Protein Multimerization , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Virion/metabolism , Virus Internalization/drug effects
13.
J Mol Biol ; 433(3): 166748, 2021 02 05.
Article in English | MEDLINE | ID: covidwho-974282

ABSTRACT

The COVID-19 pandemic remains a global threat, and host immunity remains the main mechanism of protection against the disease. The spike protein on the surface of SARS-CoV-2 is a major antigen and its engagement with human ACE2 receptor plays an essential role in viral entry into host cells. Consequently, antibodies targeting the ACE2-interacting surface (ACE2IS) located in the receptor-binding domain (RBD) of the spike protein can neutralize the virus. However, the understanding of immune responses to SARS-CoV-2 is still limited, and it is unclear how the virus protects this surface from recognition by antibodies. Here, we designed an RBD mutant that disrupts the ACE2IS and used it to characterize the prevalence of antibodies directed to the ACE2IS from convalescent sera of 94 COVID-19-positive patients. We found that only a small fraction of RBD-binding antibodies targeted the ACE2IS. To assess the immunogenicity of different parts of the spike protein, we performed in vitro antibody selection for the spike and the RBD proteins using both unbiased and biased selection strategies. Intriguingly, unbiased selection yielded antibodies that predominantly targeted regions outside the ACE2IS, whereas ACE2IS-binding antibodies were readily identified from biased selection designed to enrich such antibodies. Furthermore, antibodies from an unbiased selection using the RBD preferentially bound to the surfaces that are inaccessible in the context of whole spike protein. These results suggest that the ACE2IS has evolved less immunogenic than the other regions of the spike protein, which has important implications in the development of vaccines against SARS-CoV-2.


Subject(s)
Angiotensin-Converting Enzyme 2/immunology , Angiotensin-Converting Enzyme 2/metabolism , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/metabolism , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Binding Sites , Chlorocebus aethiops , Epitopes/immunology , Host-Pathogen Interactions , Humans , Immune Sera , Immunoglobulin G/metabolism , Mutation , Spike Glycoprotein, Coronavirus/genetics , Vero Cells
SELECTION OF CITATIONS
SEARCH DETAIL