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1.
Nature ; 601(7891): 110-117, 2022 01.
Article in English | MEDLINE | ID: covidwho-1510600

ABSTRACT

Individuals with potential exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) do not necessarily develop PCR or antibody positivity, suggesting that some individuals may clear subclinical infection before seroconversion. T cells can contribute to the rapid clearance of SARS-CoV-2 and other coronavirus infections1-3. Here we hypothesize that pre-existing memory T cell responses, with cross-protective potential against SARS-CoV-2 (refs. 4-11), would expand in vivo to support rapid viral control, aborting infection. We measured SARS-CoV-2-reactive T cells, including those against the early transcribed replication-transcription complex (RTC)12,13, in intensively monitored healthcare workers (HCWs) who tested repeatedly negative according to PCR, antibody binding and neutralization assays (seronegative HCWs (SN-HCWs)). SN-HCWs had stronger, more multispecific memory T cells compared with a cohort of unexposed individuals from before the pandemic (prepandemic cohort), and these cells were more frequently directed against the RTC than the structural-protein-dominated responses observed after detectable infection (matched concurrent cohort). SN-HCWs with the strongest RTC-specific T cells had an increase in IFI27, a robust early innate signature of SARS-CoV-2 (ref. 14), suggesting abortive infection. RNA polymerase within RTC was the largest region of high sequence conservation across human seasonal coronaviruses (HCoV) and SARS-CoV-2 clades. RNA polymerase was preferentially targeted (among the regions tested) by T cells from prepandemic cohorts and SN-HCWs. RTC-epitope-specific T cells that cross-recognized HCoV variants were identified in SN-HCWs. Enriched pre-existing RNA-polymerase-specific T cells expanded in vivo to preferentially accumulate in the memory response after putative abortive compared to overt SARS-CoV-2 infection. Our data highlight RTC-specific T cells as targets for vaccines against endemic and emerging Coronaviridae.


Subject(s)
Asymptomatic Infections , COVID-19/immunology , COVID-19/virology , DNA-Directed RNA Polymerases/immunology , SARS-CoV-2/immunology , Seroconversion , Cell Proliferation , Cohort Studies , DNA-Directed RNA Polymerases/metabolism , Evolution, Molecular , Female , Health Personnel , Humans , Male , Membrane Proteins/immunology , Multienzyme Complexes/immunology , SARS-CoV-2/enzymology , SARS-CoV-2/growth & development , Transcription, Genetic/immunology
2.
J Virol ; 95(20): e0059221, 2021 09 27.
Article in English | MEDLINE | ID: covidwho-1440799

ABSTRACT

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , SARS-CoV-2/immunology , Vesicular stomatitis Indiana virus/genetics , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Brain/virology , COVID-19/immunology , Cell Line , Cytokine Release Syndrome/prevention & control , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Humans , Immunogenicity, Vaccine , Lung/immunology , Lung/pathology , Lung/virology , Mesocricetus , Methyltransferases/genetics , Methyltransferases/metabolism , Mice , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology , Th1 Cells/immunology , Vaccines, Synthetic/immunology , Vesicular stomatitis Indiana virus/enzymology , Vesicular stomatitis Indiana virus/physiology , Viral Proteins/genetics , Viral Proteins/metabolism , Virus Replication
4.
Biophys J ; 120(6): 1085-1096, 2021 03 16.
Article in English | MEDLINE | ID: covidwho-1033766

ABSTRACT

This work builds upon the record-breaking speed and generous immediate release of new experimental three-dimensional structures of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and complexes, which are crucial to downstream vaccine and drug development. We have surveyed those structures to catch the occasional errors that could be significant for those important uses and for which we were able to provide demonstrably higher-accuracy corrections. This process relied on new validation and correction methods such as CaBLAM and ISOLDE, which are not yet in routine use. We found such important and correctable problems in seven early SARS-CoV-2 structures. Two of the structures were soon superseded by new higher-resolution data, confirming our proposed changes. For the other five, we emailed the depositors a documented and illustrated report and encouraged them to make the model corrections themselves and use the new option at the worldwide Protein Data Bank for depositors to re-version their coordinates without changing the Protein Data Bank code. This quickly and easily makes the better-accuracy coordinates available to anyone who examines or downloads their structure, even before formal publication. The changes have involved sequence misalignments, incorrect RNA conformations near a bound inhibitor, incorrect metal ligands, and cis-trans or peptide flips that prevent good contact at interaction sites. These improvements have propagated into nearly all related structures done afterward. This process constitutes a new form of highly rigorous peer review, which is actually faster and more strict than standard publication review because it has access to coordinates and maps; journal peer review would also be strengthened by such access.


Subject(s)
Peer Review , SARS-CoV-2/chemistry , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/chemistry , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/chemistry , Alanine/pharmacology , Antibodies, Viral , Catalytic Domain , DNA-Directed RNA Polymerases/metabolism , Humans , Models, Molecular , Nucleocapsid/chemistry , Phosphoproteins/chemistry , RNA-Binding Proteins/chemistry , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Zinc/metabolism
5.
Cell Cycle ; 19(24): 3399-3405, 2020 12.
Article in English | MEDLINE | ID: covidwho-972502

ABSTRACT

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. Until now, diverse drugs have been used for the treatment of COVID-19. These drugs are associated with severe side effects, e.g. induction of erythrocyte death, named eryptosis. This massively affects the oxygen (O2) supply of the organism. Therefore, three elementary aspects should be considered simultaneously: (1) a potential drug should directly attack the virus, (2) eliminate virus-infected host cells and (3) preserve erythrocyte survival and functionality. It is known that PKC-α inhibition enhances the vitality of human erythrocytes, while it dose-dependently activates the apoptosis machinery in nucleated cells. Thus, the use of chelerythrine as a specific PKC-alpha and -beta (PKC-α/-ß) inhibitor should be a promising approach to treat people infected with SARS-CoV-2.


Subject(s)
Antiviral Agents/pharmacology , Benzophenanthridines/pharmacology , COVID-19/drug therapy , Erythrocytes/immunology , Protein Kinase C beta/antagonists & inhibitors , Protein Kinase C-alpha/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Respiratory Tract Diseases/virology , Antiviral Agents/adverse effects , Antiviral Agents/therapeutic use , Apoptosis/drug effects , Benzophenanthridines/adverse effects , Benzophenanthridines/therapeutic use , COVID-19/immunology , COVID-19/metabolism , DNA-Directed RNA Polymerases/metabolism , Erythrocytes/drug effects , Erythrocytes/metabolism , Humans , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 2/metabolism , Protein Biosynthesis/drug effects , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/therapeutic use , RNA Viruses/genetics , RNA Viruses/metabolism , Respiratory Tract Diseases/enzymology , Respiratory Tract Diseases/metabolism
6.
Nat Biomed Eng ; 4(12): 1168-1179, 2020 12.
Article in English | MEDLINE | ID: covidwho-780008

ABSTRACT

The control of viral outbreaks requires nucleic acid diagnostic tests that are sensitive, simple and fast. Here, we report a highly sensitive and specific one-pot assay for the fluorescence-based detection of RNA from pathogens. The assay, which can be performed within 30-50 min of incubation time and can reach a limit of detection of 0.1-attomolar RNA concentration, relies on a sustained isothermal reaction cascade producing an RNA aptamer that binds to a fluorogenic dye. The RNA aptamer is transcribed by the T7 RNA polymerase from the ligation product of a promoter DNA probe and a reporter DNA probe that hybridize with the target single-stranded RNA sequence via the SplintR ligase (a Chlorella virus DNA ligase). In 40 nasopharyngeal SARS-CoV-2 samples, the assay reached positive and negative predictive values of 95 and 100%, respectively. We also show that the assay can rapidly detect a range of viral and bacterial RNAs.


Subject(s)
COVID-19/diagnosis , Molecular Diagnostic Techniques/methods , RNA, Viral/genetics , SARS-CoV-2/genetics , Transcription, Genetic/genetics , COVID-19/virology , Chlorella/metabolism , DNA/genetics , DNA Ligases/metabolism , DNA-Directed RNA Polymerases/metabolism , Diagnostic Tests, Routine/methods , Fluorescence , Humans , Nucleic Acid Amplification Techniques , Pandemics/prevention & control , Sensitivity and Specificity , Viral Proteins/metabolism
8.
Cells ; 9(5)2020 05 20.
Article in English | MEDLINE | ID: covidwho-324261

ABSTRACT

The current coronavirus disease-2019 (COVID-19) pandemic is due to the novel coronavirus SARS-CoV-2. The scientific community has mounted a strong response by accelerating research and innovation, and has quickly set the foundation for understanding the molecular determinants of the disease for the development of targeted therapeutic interventions. The replication of the viral genome within the infected cells is a key stage of the SARS-CoV-2 life cycle. It is a complex process involving the action of several viral and host proteins in order to perform RNA polymerization, proofreading and final capping. This review provides an update of the structural and functional data on the key actors of the replicatory machinery of SARS-CoV-2, to fill the gaps in the currently available structural data, which is mainly obtained through homology modeling. Moreover, learning from similar viruses, we collect data from the literature to reconstruct the pattern of interactions among the protein actors of the SARS-CoV-2 RNA polymerase machinery. Here, an important role is played by co-factors such as Nsp8 and Nsp10, not only as allosteric activators but also as molecular connectors that hold the entire machinery together to enhance the efficiency of RNA replication.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Pneumonia, Viral/virology , RNA, Viral/metabolism , Virus Replication/physiology , Animals , COVID-19 , Catalytic Domain , DNA-Directed RNA Polymerases/metabolism , Exoribonucleases/chemistry , Exoribonucleases/metabolism , Genome, Viral/genetics , Humans , Methyltransferases/chemistry , Methyltransferases/metabolism , Pandemics , Protein Conformation, alpha-Helical , RNA Helicases/chemistry , RNA Helicases/metabolism , RNA, Messenger/metabolism , SARS-CoV-2 , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/chemistry , Viral Regulatory and Accessory Proteins/metabolism
9.
Nature ; 582(7813): 561-565, 2020 06.
Article in English | MEDLINE | ID: covidwho-164589

ABSTRACT

Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.


Subject(s)
Betacoronavirus/genetics , Cloning, Molecular/methods , Coronavirus Infections/virology , Genome, Viral/genetics , Genomics/methods , Pneumonia, Viral/virology , Reverse Genetics/methods , Synthetic Biology/methods , Animals , COVID-19 , China/epidemiology , Chlorocebus aethiops , Chromosomes, Artificial, Yeast/metabolism , Coronavirus Infections/epidemiology , DNA-Directed RNA Polymerases/metabolism , Evolution, Molecular , Humans , Mutation , Pandemics/statistics & numerical data , Pneumonia, Viral/epidemiology , Respiratory Syncytial Viruses/genetics , SARS-CoV-2 , Saccharomyces cerevisiae/genetics , Vero Cells , Viral Proteins/metabolism , Zika Virus/genetics
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