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Virus Res ; 304: 198508, 2021 10 15.
Article in English | MEDLINE | ID: covidwho-1331289


The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to public health. An explicit investigation of COVID-19 immune responses, particularly the host immunity in recovered subjects, will lay a foundation for the rational design of therapeutics and/or vaccines against future coronaviral outbreaks. Here, we examined virus-specific T cell responses and identified T cell epitopes using peptides spanning SARS-CoV-2 structural proteins. These peptides were used to stimulate peripheral blood mononuclear cells (PBMCs) derived from COVID-19-recovered subjects, followed by an analysis of IFN-γ-secreting T cells by enzyme-linked immunosorbent spot (ELISpot). We also evaluated virus-specific CD4 or CD8 T cell activation by flow cytometry assay. By screening 52 matrix pools (comprised of 315 peptides) of the spike (S) glycoprotein and 21 matrix pools (comprised of 102 peptides) spanning the nucleocapsid (N) protein, we identified 28 peptides from S protein and 5 peptides from N protein as immunodominant epitopes. The immunogenicity of these epitopes was confirmed by a second ELISpot using single peptide stimulation in memory T cells, and they were mapped by HLA restrictions. Notably, SARS-CoV-2 specific T cell responses positively correlated with B cell IgG and neutralizing antibody responses to the receptor-binding domain (RBD) of the S protein. Our results demonstrate that defined levels of SARS-CoV-2 specific T cell responses are generated in some, but not all, COVID-19-recovered subjects, fostering hope for the protection of a proportion of COVID-19-exposed individuals against reinfection. These results also suggest that these virus-specific T cell responses may induce protective immunity in unexposed individuals upon vaccination, using vaccines generated based on the immune epitopes identified in this study. However, SARS-CoV-2 S and N peptides are not potently immunogenic, and none of the single peptides could universally induce robust T cell responses, suggesting the necessity of using a multi-epitope strategy for COVID-19 vaccine design.

CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , Epitopes, T-Lymphocyte/immunology , Pandemics , Spike Glycoprotein, Coronavirus/immunology , Adult , CD8-Positive T-Lymphocytes/cytology , COVID-19/epidemiology , Female , Humans , Immunodominant Epitopes/immunology , Male , Middle Aged , SARS-CoV-2/immunology , Young Adult
Sci Rep ; 11(1): 5558, 2021 03 10.
Article in English | MEDLINE | ID: covidwho-1125779


The recent COVID-19 pandemic poses a serious threat to global public health, thus there is an urgent need to define the molecular mechanisms involved in SARS-CoV-2 spike (S) protein-mediated virus entry that is essential for preventing and/or treating this emerging infectious disease. In this study, we examined the blocking activity of human COVID-19 convalescent plasma by cell-cell fusion assays using SARS-CoV-2-S-transfected 293 T as effector cells and ACE2-expressing 293 T as target cells. We demonstrate that the SARS-CoV-2 S protein exhibits a very high capacity for membrane fusion and is efficient in mediating virus fusion and entry into target cells. Importantly, we find that COVID-19 convalescent plasma with high titers of IgG neutralizing antibodies can block cell-cell fusion and virus entry by interfering with the SARS-CoV-2-S/ACE2 or SARS-CoV-S/ACE2 interactions. These findings suggest that COVID-19 convalescent plasma may not only inhibit SARS-CoV-2-S but also cross-neutralize SARS-CoV-S-mediated membrane fusion and virus entry, supporting its potential as a preventive and/or therapeutic agent against SARS-CoV-2 as well as other SARS-CoV infections.

COVID-19/immunology , COVID-19/therapy , Spike Glycoprotein, Coronavirus/immunology , Adult , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/prevention & control , Cell Fusion/methods , Female , Humans , Immunization, Passive/methods , Male , Membrane Fusion/drug effects , Middle Aged , Pandemics/prevention & control , Plasma/chemistry , Receptors, Virus/metabolism , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization/drug effects
Bioconjug Chem ; 31(11): 2553-2563, 2020 11 18.
Article in English | MEDLINE | ID: covidwho-872629


As a large enveloped RNA virus, coronavirus is of considerable medical and veterinary significance, and anticoronavirus treatment is challenging due to its biodiversity and rapid variability. In this study, Au@Ag nanorods (Au@AgNRs) were successfully synthesized by coating AuNRs with silver and were shown for the first time to have activity against the replication of porcine epidemic diarrhea virus (PEDV). Viral titer analysis demonstrated that Au@AgNRs could inhibit PEDV infection by 4 orders of magnitude at 12 h post-infection, which was verified by viral protein expression analysis. The potential mechanism of action showed that Au@AgNRs could inhibit the entry of PEDV and decrease the mitochondrial membrane potential and caspase-3 activity. Additionally, we demonstrated that a large amount of virus proliferation can cause the generation of reactive oxygen species in cells, and the released Ag+ and exposed AuNRs by Au@AgNRs after the stimulation of reactive oxygen species has superior antiviral activity to ensure long-term inhibition of the PEDV replication cycle. The integrated results support that Au@AgNRs can serve as a potential therapeutic strategy to prevent the replication of coronavirus.

Gold/chemistry , Gold/pharmacology , Metal Nanoparticles/chemistry , Porcine epidemic diarrhea virus/drug effects , Porcine epidemic diarrhea virus/physiology , Silver/chemistry , Virus Replication/drug effects , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Antiviral Agents/toxicity , Chlorocebus aethiops , Dose-Response Relationship, Drug , Gold/toxicity , Nanotubes/chemistry , Vero Cells