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1.
Immunity ; 55(6): 998-1012.e8, 2022 Jun 14.
Article in English | MEDLINE | ID: covidwho-1778212

ABSTRACT

SARS-CoV-2 infection or vaccination produces neutralizing antibody responses that contribute to better clinical outcomes. The receptor-binding domain (RBD) and the N-terminal domain (NTD) of the spike trimer (S) constitute the two major neutralizing targets for antibodies. Here, we use NTD-specific probes to capture anti-NTD memory B cells in a longitudinal cohort of infected individuals, some of whom were vaccinated. We found 6 complementation groups of neutralizing antibodies. 58% targeted epitopes outside the NTD supersite, 58% neutralized either Gamma or Omicron, and 14% were broad neutralizers that also neutralized Omicron. Structural characterization revealed that broadly active antibodies targeted three epitopes outside the NTD supersite including a class that recognized both the NTD and SD2 domain. Rapid recruitment of memory B cells producing these antibodies into the plasma cell compartment upon re-infection likely contributes to the relatively benign course of subsequent infections with SARS-CoV-2 variants, including Omicron.


Subject(s)
COVID-19 , Spike Glycoprotein, Coronavirus , Antibodies, Monoclonal , Antibodies, Neutralizing , Antibodies, Viral , Epitopes , Humans , SARS-CoV-2
2.
J Virol ; 96(7): e0151621, 2022 04 13.
Article in English | MEDLINE | ID: covidwho-1744134

ABSTRACT

ADP-ribosylation is a highly dynamic posttranslational modification frequently studied in stress response pathways with recent attention given to its role in response to viral infection. Notably, the alphaviruses encode catalytically active macrodomains capable of ADP-ribosylhydrolase (ARH) activities, implying a role in remodeling the cellular ADP-ribosylome. This report decouples mono- and poly-ARH contributions to macrodomain function using a newly engineered Sindbis virus (SINV) mutant with attenuated poly-ARH activity. Our findings indicate that viral poly-ARH activity is uniquely required for high titer replication in mammalian systems. Despite translating incoming genomic RNA as efficiently as WT virus, mutant viruses have a reduced capacity to establish productive infection, offering a more complete understanding of the kinetics and role of the alphavirus macrodomain with important implications for broader ADP-ribosyltransferase biology. IMPORTANCE Viral macrodomains have drawn attention in recent years due to their high degree of conservation in several virus families (e.g., coronaviruses and alphaviruses) and their potential druggability. These domains erase mono- or poly-ADP-ribose, posttranslational modifications written by host poly-ADP-ribose polymerase (PARP) proteins, from undetermined host or viral proteins to enhance replication. Prior work determined that efficient alphavirus replication requires catalytically active macrodomains; however, which form of the modification requires removal and from which protein(s) had not been determined. Here, we present evidence for the specific requirement of poly-ARH activity to ensure efficient productive infection and virus replication.


Subject(s)
Coronavirus , Hydrolases , RNA, Viral , Sindbis Virus , Animals , Coronavirus/genetics , Hydrolases/metabolism , Mammals/genetics , Poly Adenosine Diphosphate Ribose/metabolism , RNA, Viral/genetics , Sindbis Virus/enzymology , Sindbis Virus/genetics , Virus Replication
3.
Cell Rep Med ; 3(2): 100522, 2022 02 15.
Article in English | MEDLINE | ID: covidwho-1650891

ABSTRACT

The molecular mechanisms underlying the clinical manifestations of coronavirus disease 2019 (COVID-19), and what distinguishes them from common seasonal influenza virus and other lung injury states such as acute respiratory distress syndrome, remain poorly understood. To address these challenges, we combine transcriptional profiling of 646 clinical nasopharyngeal swabs and 39 patient autopsy tissues to define body-wide transcriptome changes in response to COVID-19. We then match these data with spatial protein and expression profiling across 357 tissue sections from 16 representative patient lung samples and identify tissue-compartment-specific damage wrought by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, evident as a function of varying viral loads during the clinical course of infection and tissue-type-specific expression states. Overall, our findings reveal a systemic disruption of canonical cellular and transcriptional pathways across all tissues, which can inform subsequent studies to combat the mortality of COVID-19 and to better understand the molecular dynamics of lethal SARS-CoV-2 and other respiratory infections.


Subject(s)
COVID-19/genetics , COVID-19/pathology , Lung/pathology , SARS-CoV-2 , Transcriptome/genetics , Adult , Aged , Aged, 80 and over , COVID-19/metabolism , COVID-19/virology , Case-Control Studies , Cohort Studies , Female , Gene Expression Regulation , Humans , Influenza, Human/genetics , Influenza, Human/pathology , Influenza, Human/virology , Lung/metabolism , Male , Middle Aged , Orthomyxoviridae , RNA-Seq/methods , Respiratory Distress Syndrome/genetics , Respiratory Distress Syndrome/microbiology , Respiratory Distress Syndrome/pathology , Viral Load
4.
mSphere ; 6(6): e0071121, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1546463

ABSTRACT

The COVID-19 pandemic has highlighted the need to identify additional antiviral small molecules to complement existing therapies. Although increasing evidence suggests that metabolites produced by the human microbiome have diverse biological activities, their antiviral properties remain poorly explored. Using a cell-based SARS-CoV-2 infection assay, we screened culture broth extracts from a collection of phylogenetically diverse human-associated bacteria for the production of small molecules with antiviral activity. Bioassay-guided fractionation uncovered three bacterial metabolites capable of inhibiting SARS-CoV-2 infection. This included the nucleoside analogue N6-(Δ2-isopentenyl)adenosine, the 5-hydroxytryptamine receptor agonist tryptamine, and the pyrazine 2,5-bis(3-indolylmethyl)pyrazine. The most potent of these, N6-(Δ2-isopentenyl)adenosine, had a 50% inhibitory concentration (IC50) of 2 µM. These natural antiviral compounds exhibit structural and functional similarities to synthetic drugs that have been clinically examined for use against COVID-19. Our discovery of structurally diverse metabolites with anti-SARS-CoV-2 activity from screening a small fraction of the bacteria reported to be associated with the human microbiome suggests that continued exploration of phylogenetically diverse human-associated bacteria is likely to uncover additional small molecules that inhibit SARS-CoV-2 as well as other viral infections. IMPORTANCE The continued prevalence of COVID-19 and the emergence of new variants has once again put the spotlight on the need for the identification of SARS-CoV-2 antivirals. The human microbiome produces an array of small molecules with bioactivities (e.g., host receptor ligands), but its ability to produce antiviral small molecules is relatively underexplored. Here, using a cell-based screening platform, we describe the isolation of three microbiome-derived metabolites that are able to prevent SARS-CoV-2 infection in vitro. These molecules display structural similarities to synthetic drugs that have been explored for the treatment of COVID-19, and these results suggest that the microbiome may be a fruitful source of the discovery of small molecules with antiviral activities.


Subject(s)
Antiviral Agents/pharmacology , Bacteria/metabolism , Culture Media/chemistry , Metabolic Networks and Pathways , Microbiota/physiology , SARS-CoV-2/drug effects , Symbiosis/physiology , Bacteria/chemistry , Bacteria/classification , Bacteria/growth & development , Biological Assay , Cell Line, Tumor , Culture Media/pharmacology , Humans , Molecular Docking Simulation , Protease Inhibitors/pharmacology , Protein Binding
5.
Science ; 374(6571): 1099-1106, 2021 Nov 26.
Article in English | MEDLINE | ID: covidwho-1467657

ABSTRACT

Molecular virology tools are critical for basic studies of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and for developing new therapeutics. Experimental systems that do not rely on viruses capable of spread are needed for potential use in lower-containment settings. In this work, we use a yeast-based reverse genetics system to develop spike-deleted SARS-CoV-2 self-replicating RNAs. These noninfectious self-replicating RNAs, or replicons, can be trans-complemented with viral glycoproteins to generate replicon delivery particles for single-cycle delivery into a range of cell types. This SARS-CoV-2 replicon system represents a convenient and versatile platform for antiviral drug screening, neutralization assays, host factor validation, and viral variant characterization.


Subject(s)
RNA, Viral/genetics , Replicon/physiology , SARS-CoV-2/genetics , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Antiviral Agents/pharmacology , Cell Line , Humans , Interferons/pharmacology , Microbial Sensitivity Tests , Mutation , Plasmids , RNA, Viral/metabolism , Replicon/genetics , Reverse Genetics , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Saccharomyces cerevisiae/genetics , Spike Glycoprotein, Coronavirus/genetics , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virion/genetics , Virion/physiology , Virus Replication
7.
Nature ; 599(7885): 465-470, 2021 11.
Article in English | MEDLINE | ID: covidwho-1428880

ABSTRACT

Monoclonal antibodies with neutralizing activity against SARS-CoV-2 have demonstrated clinical benefits in cases of mild-to-moderate SARS-CoV-2 infection, substantially reducing the risk for hospitalization and severe disease1-4. Treatment generally requires the administration of high doses of these monoclonal antibodies and has limited efficacy in preventing disease complications or mortality among hospitalized patients with COVID-195. Here we report the development and evaluation of anti-SARS-CoV-2 monoclonal antibodies with optimized Fc domains that show superior potency for prevention or treatment of COVID-19. Using several animal disease models of COVID-196,7, we demonstrate that selective engagement of activating Fcγ receptors results in improved efficacy in both preventing and treating disease-induced weight loss and mortality, significantly reducing the dose required to confer full protection against SARS-CoV-2 challenge and for treatment of pre-infected animals. Our results highlight the importance of Fcγ receptor pathways in driving antibody-mediated antiviral immunity and exclude the possibility of pathogenic or disease-enhancing effects of Fcγ receptor engagement of anti-SARS-CoV-2 antibodies upon infection. These findings have important implications for the development of Fc-engineered monoclonal antibodies with optimal Fc-effector function and improved clinical efficacy against COVID-19 disease.


Subject(s)
Antibodies, Monoclonal/therapeutic use , COVID-19/drug therapy , COVID-19/immunology , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Fc Fragments/therapeutic use , SARS-CoV-2/drug effects , SARS-CoV-2/immunology , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/pharmacology , Antibodies, Neutralizing/therapeutic use , Cricetinae , Disease Models, Animal , Female , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/pharmacology , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Male , Mice , Pre-Exposure Prophylaxis , Receptors, IgG/chemistry , Receptors, IgG/immunology , Treatment Outcome
8.
PLoS One ; 16(6): e0252949, 2021.
Article in English | MEDLINE | ID: covidwho-1282295

ABSTRACT

To address the need for simple, safe, sensitive, and scalable SARS-CoV-2 tests, we validated and implemented a PCR test that uses a saliva collection kit use at home. Individuals self-collected 300 µl saliva in vials containing Darnell Rockefeller University Laboratory (DRUL) buffer and extracted RNA was assayed by RT-PCR (the DRUL saliva assay). The limit of detection was confirmed to be 1 viral copy/µl in 20 of 20 replicate extractions. Viral RNA was stable in DRUL buffer at room temperature up to seven days after sample collection, and safety studies demonstrated that DRUL buffer immediately inactivated virus at concentrations up to 2.75x106 PFU/ml. Results from SARS-CoV-2 positive nasopharyngeal (NP) swab samples collected in viral transport media and assayed with a standard FDA Emergency Use Authorization (EUA) test were highly correlated with samples placed in DRUL buffer. Direct comparison of results from 162 individuals tested by FDA EUA oropharyngeal (OP) or NP swabs with co-collected saliva samples identified four otherwise unidentified positive cases in DRUL buffer. Over six months, we collected 3,724 samples from individuals ranging from 3 months to 92 years of age. This included collecting weekly samples over 10 weeks from teachers, children, and parents from a pre-school program, which allowed its safe reopening while at-risk pods were quarantined. In sum, we validated a simple, sensitive, stable, and safe PCR-based test using a self-collected saliva sample as a valuable tool for clinical diagnosis and screening at workplaces and schools.


Subject(s)
COVID-19 Nucleic Acid Testing , COVID-19 , SARS-CoV-2 , Saliva/virology , Schools , Specimen Handling , COVID-19/diagnosis , COVID-19/genetics , Child , Female , Humans , Male
9.
Nature ; 595(7867): 426-431, 2021 07.
Article in English | MEDLINE | ID: covidwho-1267998

ABSTRACT

More than one year after its inception, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains difficult to control despite the availability of several working vaccines. Progress in controlling the pandemic is slowed by the emergence of variants that appear to be more transmissible and more resistant to antibodies1,2. Here we report on a cohort of 63 individuals who have recovered from COVID-19 assessed at 1.3, 6.2 and 12 months after SARS-CoV-2 infection, 41% of whom also received mRNA vaccines3,4. In the absence of vaccination, antibody reactivity to the receptor binding domain (RBD) of SARS-CoV-2, neutralizing activity and the number of RBD-specific memory B cells remain relatively stable between 6 and 12 months after infection. Vaccination increases all components of the humoral response and, as expected, results in serum neutralizing activities against variants of concern similar to or greater than the neutralizing activity against the original Wuhan Hu-1 strain achieved by vaccination of naive individuals2,5-8. The mechanism underlying these broad-based responses involves ongoing antibody somatic mutation, memory B cell clonal turnover and development of monoclonal antibodies that are exceptionally resistant to SARS-CoV-2 RBD mutations, including those found in the variants of concern4,9. In addition, B cell clones expressing broad and potent antibodies are selectively retained in the repertoire over time and expand markedly after vaccination. The data suggest that immunity in convalescent individuals will be very long lasting and that convalescent individuals who receive available mRNA vaccines will produce antibodies and memory B cells that should be protective against circulating SARS-CoV-2 variants.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/immunology , SARS-CoV-2/immunology , Adult , Aged , Antibodies, Monoclonal/immunology , B-Lymphocytes/immunology , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Female , Humans , Immunologic Memory/immunology , Male , Middle Aged , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Time Factors
10.
Cell ; 184(15): 3962-3980.e17, 2021 07 22.
Article in English | MEDLINE | ID: covidwho-1252549

ABSTRACT

T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.


Subject(s)
Epitopes, T-Lymphocyte/immunology , Histocompatibility Antigens Class I/immunology , Open Reading Frames/genetics , Peptides/immunology , Proteome/immunology , SARS-CoV-2/immunology , A549 Cells , Alleles , Amino Acid Sequence , Animals , Antigen Presentation/immunology , COVID-19/immunology , COVID-19/virology , Female , HEK293 Cells , Humans , Kinetics , Male , Mice , Peptides/chemistry , T-Lymphocytes/immunology
11.
J Exp Med ; 218(4)2021 04 05.
Article in English | MEDLINE | ID: covidwho-1066211

ABSTRACT

Yellow fever virus (YFV) live attenuated vaccine can, in rare cases, cause life-threatening disease, typically in patients with no previous history of severe viral illness. Autosomal recessive (AR) complete IFNAR1 deficiency was reported in one 12-yr-old patient. Here, we studied seven other previously healthy patients aged 13 to 80 yr with unexplained life-threatening YFV vaccine-associated disease. One 13-yr-old patient had AR complete IFNAR2 deficiency. Three other patients vaccinated at the ages of 47, 57, and 64 yr had high titers of circulating auto-Abs against at least 14 of the 17 individual type I IFNs. These antibodies were recently shown to underlie at least 10% of cases of life-threatening COVID-19 pneumonia. The auto-Abs were neutralizing in vitro, blocking the protective effect of IFN-α2 against YFV vaccine strains. AR IFNAR1 or IFNAR2 deficiency and neutralizing auto-Abs against type I IFNs thus accounted for more than half the cases of life-threatening YFV vaccine-associated disease studied here. Previously healthy subjects could be tested for both predispositions before anti-YFV vaccination.


Subject(s)
Antibodies, Neutralizing/immunology , Autoantibodies/immunology , Autoimmune Diseases , COVID-19 , Genetic Diseases, Inborn , Interferon-alpha , Receptor, Interferon alpha-beta , SARS-CoV-2 , Yellow Fever Vaccine , Yellow fever virus , Adolescent , Adult , Aged , Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , COVID-19/genetics , COVID-19/immunology , Female , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/immunology , HEK293 Cells , Humans , Interferon-alpha/genetics , Interferon-alpha/immunology , Male , Middle Aged , Receptor, Interferon alpha-beta/deficiency , Receptor, Interferon alpha-beta/immunology , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Vaccines, Attenuated/genetics , Vaccines, Attenuated/immunology , Yellow Fever Vaccine/adverse effects , Yellow Fever Vaccine/genetics , Yellow Fever Vaccine/immunology , Yellow fever virus/genetics , Yellow fever virus/immunology
12.
Cell ; 184(1): 120-132.e14, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-1064914

ABSTRACT

The coronavirus disease 2019 (COVID-19) pandemic has claimed the lives of over one million people worldwide. The causative agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a member of the Coronaviridae family of viruses that can cause respiratory infections of varying severity. The cellular host factors and pathways co-opted during SARS-CoV-2 and related coronavirus life cycles remain ill defined. To address this gap, we performed genome-scale CRISPR knockout screens during infection by SARS-CoV-2 and three seasonal coronaviruses (HCoV-OC43, HCoV-NL63, and HCoV-229E). These screens uncovered host factors and pathways with pan-coronavirus and virus-specific functional roles, including major dependency on glycosaminoglycan biosynthesis, sterol regulatory element-binding protein (SREBP) signaling, bone morphogenetic protein (BMP) signaling, and glycosylphosphatidylinositol biosynthesis, as well as a requirement for several poorly characterized proteins. We identified an absolute requirement for the VMP1, TMEM41, and TMEM64 (VTT) domain-containing protein transmembrane protein 41B (TMEM41B) for infection by SARS-CoV-2 and three seasonal coronaviruses. This human coronavirus host factor compendium represents a rich resource to develop new therapeutic strategies for acute COVID-19 and potential future coronavirus pandemics.


Subject(s)
Coronavirus Infections/genetics , Genome-Wide Association Study , SARS-CoV-2/physiology , A549 Cells , Cell Line , Clustered Regularly Interspaced Short Palindromic Repeats , Coronavirus 229E, Human/physiology , Coronavirus Infections/virology , Coronavirus NL63, Human/physiology , Coronavirus OC43, Human/physiology , Gene Knockout Techniques , HEK293 Cells , Host-Pathogen Interactions/drug effects , Humans , Membrane Proteins/metabolism , Metabolic Networks and Pathways/drug effects , Protein Interaction Mapping
13.
Cell ; 184(1): 133-148.e20, 2021 01 07.
Article in English | MEDLINE | ID: covidwho-987228

ABSTRACT

Flaviviruses pose a constant threat to human health. These RNA viruses are transmitted by the bite of infected mosquitoes and ticks and regularly cause outbreaks. To identify host factors required for flavivirus infection, we performed full-genome loss of function CRISPR-Cas9 screens. Based on these results, we focused our efforts on characterizing the roles that TMEM41B and VMP1 play in the virus replication cycle. Our mechanistic studies on TMEM41B revealed that all members of the Flaviviridae family that we tested require TMEM41B. We tested 12 additional virus families and found that SARS-CoV-2 of the Coronaviridae also required TMEM41B for infection. Remarkably, single nucleotide polymorphisms present at nearly 20% in East Asian populations reduce flavivirus infection. Based on our mechanistic studies, we propose that TMEM41B is recruited to flavivirus RNA replication complexes to facilitate membrane curvature, which creates a protected environment for viral genome replication.


Subject(s)
Flavivirus Infections/genetics , Flavivirus/physiology , Membrane Proteins/metabolism , Animals , Autophagy , COVID-19/genetics , COVID-19/metabolism , COVID-19/virology , CRISPR-Cas Systems , Cell Line , Flavivirus Infections/immunology , Flavivirus Infections/metabolism , Flavivirus Infections/virology , Gene Knockout Techniques , Genome-Wide Association Study , Host-Pathogen Interactions , Humans , Immunity, Innate , Membrane Proteins/genetics , Polymorphism, Single Nucleotide , SARS-CoV-2/physiology , Virus Replication , Yellow fever virus/physiology , Zika Virus/physiology
14.
Cell Host Microbe ; 29(2): 267-280.e5, 2021 02 10.
Article in English | MEDLINE | ID: covidwho-978239

ABSTRACT

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks.


Subject(s)
COVID-19/virology , SARS-CoV-2/metabolism , CRISPR-Cas Systems , Coronavirus 229E, Human/genetics , Coronavirus 229E, Human/metabolism , Coronavirus NL63, Human/genetics , Coronavirus NL63, Human/metabolism , Coronavirus OC43, Human , Genes, Viral , Host-Pathogen Interactions , Humans , SARS-CoV-2/genetics , Viral Proteins/genetics , Viral Proteins/metabolism
15.
Sci Transl Med ; 13(577)2021 01 20.
Article in English | MEDLINE | ID: covidwho-963896

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), primarily infects cells at mucosal surfaces. Serum neutralizing antibody responses are variable and generally low in individuals that suffer mild forms of COVID-19. Although potent immunoglobulin G (IgG) antibodies can neutralize the virus, less is known about secretory antibodies such as IgA that might affect the initial viral spread and transmissibility from the mucosa. Here, we characterize the IgA response to SARS-CoV-2 in a cohort of 149 convalescent individuals after diagnosis with COVID-19. IgA responses in plasma generally correlated with IgG responses. Furthermore, clones of IgM-, IgG-, and IgA-producing B cells were derived from common progenitor cells. Plasma IgA monomers specific to SARS-CoV-2 proteins were demonstrated to be twofold less potent than IgG equivalents. However, IgA dimers, the primary form of antibody in the nasopharynx, were, on average, 15 times more potent than IgA monomers against the same target. Thus, dimeric IgA responses may be particularly valuable for protection against SARS-CoV-2 and for vaccine efficacy.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19/diagnosis , Immunoglobulin A/blood , SARS-CoV-2/immunology , Animals , Biomarkers/blood , COVID-19/blood , COVID-19/immunology , COVID-19/virology , Cell Line, Tumor , Chlorocebus aethiops , Convalescence , HEK293 Cells , Host-Pathogen Interactions , Humans , Protein Multimerization , Vero Cells
16.
Elife ; 92020 10 28.
Article in English | MEDLINE | ID: covidwho-895692

ABSTRACT

Neutralizing antibodies elicited by prior infection or vaccination are likely to be key for future protection of individuals and populations against SARS-CoV-2. Moreover, passively administered antibodies are among the most promising therapeutic and prophylactic anti-SARS-CoV-2 agents. However, the degree to which SARS-CoV-2 will adapt to evade neutralizing antibodies is unclear. Using a recombinant chimeric VSV/SARS-CoV-2 reporter virus, we show that functional SARS-CoV-2 S protein variants with mutations in the receptor-binding domain (RBD) and N-terminal domain that confer resistance to monoclonal antibodies or convalescent plasma can be readily selected. Notably, SARS-CoV-2 S variants that resist commonly elicited neutralizing antibodies are now present at low frequencies in circulating SARS-CoV-2 populations. Finally, the emergence of antibody-resistant SARS-CoV-2 variants that might limit the therapeutic usefulness of monoclonal antibodies can be mitigated by the use of antibody combinations that target distinct neutralizing epitopes.


The new coronavirus, SARS-CoV-2, which causes the disease COVID-19, has had a serious worldwide impact on human health. The virus was virtually unknown at the beginning of 2020. Since then, intense research efforts have resulted in sequencing the coronavirus genome, identifying the structures of its proteins, and creating a wide range of tools to search for effective vaccines and therapies. Antibodies, which are immune molecules produced by the body that target specific segments of viral proteins can neutralize virus particles and trigger the immune system to kill cells infected with the virus. Several technologies are currently under development to exploit antibodies, including vaccines, blood plasma from patients who were previously infected, manufactured antibodies and more. The spike proteins on the surface of SARS-CoV-2 are considered to be prime antibody targets as they are accessible and have an essential role in allowing the virus to attach to and infect host cells. Antibodies bind to spike proteins and some can block the virus' ability to infect new cells. But some viruses, such as HIV and influenza, are able to mutate their equivalent of the spike protein to evade antibodies. It is unknown whether SARS-CoV-2 is able to efficiently evolve to evade antibodies in the same way. Weisblum, Schmidt et al. addressed this question using an artificial system that mimics natural infection in human populations. Human cells grown in the laboratory were infected with a hybrid virus created by modifying an innocuous animal virus to contain the SARS-CoV-2 spike protein, and treated with either manufactured antibodies or antibodies present in the blood of recovered COVID-19 patients. In this situation, only viruses that had mutated in a way that allowed them to escape the antibodies were able to survive. Several of the virus mutants that emerged had evolved spike proteins in which the segments targeted by the antibodies had changed, allowing these mutant viruses to remain undetected. An analysis of more than 50,000 real-life SARS-CoV-2 genomes isolated from patient samples further showed that most of these virus mutations were already circulating, albeit at very low levels in the infected human populations. These results show that SARS-CoV-2 can mutate its spike proteins to evade antibodies, and that these mutations are already present in some virus mutants circulating in the human population. This suggests that any vaccines that are deployed on a large scale should be designed to activate the strongest possible immune response against more than one target region on the spike protein. Additionally, antibody-based therapies that use two antibodies in combination should prevent the rise of viruses that are resistant to the antibodies and maintain the long-term effectiveness of vaccines and therapies.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/therapy , Mutation , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Monoclonal/immunology , Base Sequence , COVID-19/virology , Epitopes/genetics , Epitopes/immunology , Genes, Reporter , Humans , Immunization, Passive , Neutralization Tests , Protein Domains , Protein Isoforms/immunology , Reassortant Viruses/immunology , Receptors, Virus/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Selection, Genetic , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Vesiculovirus/genetics , Virus Replication
17.
bioRxiv ; 2020 Oct 26.
Article in English | MEDLINE | ID: covidwho-835256

ABSTRACT

T cell-mediated immunity may play a critical role in controlling and establishing protective immunity against SARS-CoV-2 infection; yet the repertoire of viral epitopes responsible for T cell response activation remains mostly unknown. Identification of viral peptides presented on class I human leukocyte antigen (HLA-I) can reveal epitopes for recognition by cytotoxic T cells and potential incorporation into vaccines. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two human cell lines at different times post-infection using mass spectrometry. We found HLA-I peptides derived not only from canonical ORFs, but also from internal out-of-frame ORFs in Spike and Nucleoprotein not captured by current vaccines. Proteomics analyses of infected cells revealed that SARS-CoV-2 may interfere with antigen processing and immune signaling pathways. Based on the endogenously processed and presented viral peptides that we identified, we estimate that a pool of 24 peptides would provide one or more peptides for presentation by at least one HLA allele in 99% of the human population. These biological insights and the list of naturally presented SARS-CoV-2 peptides will facilitate data-driven selection of peptides for immune monitoring and vaccine development.

18.
J Cell Biol ; 219(10)2020 10 05.
Article in English | MEDLINE | ID: covidwho-713813

ABSTRACT

With the rapid global spread of SARS-CoV-2, we have become acutely aware of the inadequacies of our ability to respond to viral epidemics. Although disrupting the viral life cycle is critical for limiting viral spread and disease, it has proven challenging to develop targeted and selective therapeutics. Synthetic lethality offers a promising but largely unexploited strategy against infectious viral disease; as viruses infect cells, they abnormally alter the cell state, unwittingly exposing new vulnerabilities in the infected cell. Therefore, we propose that effective therapies can be developed to selectively target the virally reconfigured host cell networks that accompany altered cellular states to cripple the host cell that has been converted into a virus factory, thus disrupting the viral life cycle.


Subject(s)
Antiviral Agents/pharmacology , Host Microbial Interactions/drug effects , Virus Diseases/drug therapy , Virus Replication/drug effects , Drug Discovery , Humans , Immunologic Factors/pharmacology , Metabolic Networks and Pathways/drug effects , Protein Interaction Maps , Proteolysis , RNA Viruses/drug effects , RNA Viruses/physiology , Virus Diseases/genetics
19.
J Exp Med ; 217(11)2020 11 02.
Article in English | MEDLINE | ID: covidwho-697830

ABSTRACT

The emergence of SARS-CoV-2 and the ensuing explosive epidemic of COVID-19 disease has generated a need for assays to rapidly and conveniently measure the antiviral activity of SARS-CoV-2-specific antibodies. Here, we describe a collection of approaches based on SARS-CoV-2 spike-pseudotyped, single-cycle, replication-defective human immunodeficiency virus type-1 (HIV-1), and vesicular stomatitis virus (VSV), as well as a replication-competent VSV/SARS-CoV-2 chimeric virus. While each surrogate virus exhibited subtle differences in the sensitivity with which neutralizing activity was detected, the neutralizing activity of both convalescent plasma and human monoclonal antibodies measured using each virus correlated quantitatively with neutralizing activity measured using an authentic SARS-CoV-2 neutralization assay. The assays described herein are adaptable to high throughput and are useful tools in the evaluation of serologic immunity conferred by vaccination or prior SARS-CoV-2 infection, as well as the potency of convalescent plasma or human monoclonal antibodies.


Subject(s)
Antibodies, Neutralizing/analysis , Antibodies, Viral/analysis , Betacoronavirus/immunology , Coronavirus Infections/immunology , Immunoassay/methods , Pneumonia, Viral/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/genetics , COVID-19 , Cell Line , Chimera/genetics , Chimera/immunology , Chlorocebus aethiops , Coronavirus Infections/virology , HEK293 Cells , HIV-1/genetics , HIV-1/immunology , Humans , Neutralization Tests/methods , Pandemics , Pneumonia, Viral/virology , Recombination, Genetic , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Vero Cells , Vesicular stomatitis Indiana virus/genetics , Vesicular stomatitis Indiana virus/immunology
20.
Nat Microbiol ; 5(11): 1330-1339, 2020 11.
Article in English | MEDLINE | ID: covidwho-676586

ABSTRACT

Zoonotic coronaviruses (CoVs) are substantial threats to global health, as exemplified by the emergence of two severe acute respiratory syndrome CoVs (SARS-CoV and SARS-CoV-2) and Middle East respiratory syndrome CoV (MERS-CoV) within two decades1-3. Host immune responses to CoVs are complex and regulated in part through antiviral interferons. However, interferon-stimulated gene products that inhibit CoVs are not well characterized4. Here, we show that lymphocyte antigen 6 complex, locus E (LY6E) potently restricts infection by multiple CoVs, including SARS-CoV, SARS-CoV-2 and MERS-CoV. Mechanistic studies revealed that LY6E inhibits CoV entry into cells by interfering with spike protein-mediated membrane fusion. Importantly, mice lacking Ly6e in immune cells were highly susceptible to a murine CoV-mouse hepatitis virus. Exacerbated viral pathogenesis in Ly6e knockout mice was accompanied by loss of hepatic immune cells, higher splenic viral burden and reduction in global antiviral gene pathways. Accordingly, we found that constitutive Ly6e directly protects primary B cells from murine CoV infection. Our results show that LY6E is a critical antiviral immune effector that controls CoV infection and pathogenesis. These findings advance our understanding of immune-mediated control of CoV in vitro and in vivo-knowledge that could help inform strategies to combat infection by emerging CoVs.


Subject(s)
Antigens, Surface/metabolism , Coronavirus Infections/immunology , Coronavirus Infections/virology , Coronavirus/physiology , GPI-Linked Proteins/metabolism , Angiotensin-Converting Enzyme 2 , Animals , Antigens, Surface/genetics , Antigens, Surface/immunology , Betacoronavirus/immunology , Betacoronavirus/physiology , COVID-19 , Coronavirus/immunology , Female , GPI-Linked Proteins/genetics , GPI-Linked Proteins/immunology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Middle East Respiratory Syndrome Coronavirus/immunology , Middle East Respiratory Syndrome Coronavirus/physiology , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , SARS Virus/immunology , SARS Virus/physiology , SARS-CoV-2 , Virus Internalization
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