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1.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-333124

ABSTRACT

Viral CD8 + epitopes are generated by the cellular turnover of viral proteins, predominantly by the proteasome. Mutations located within viral epitopes can result in escape from memory T cells but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two of the most dominant SARS-CoV-2 nucleoprotein CD8 + epitopes, we identified mutations in epitope flanking regions and investigated the contribution of these mutations to antigen processing and T cell activation using SARS-CoV-2 nucleoprotein transduced B cell lines and in vitro proteasomal processing of peptides. We found that decreased NP 9-17 -B*27:05 CD8 + T cell responses to the NP-Q7K mutation correlated with lower epitope surface expression, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103N/Y mutations flanking the NP 9-17 -B*27:05 and NP 105-113 -B*07:02 epitopes, respectively, increased CD8 + T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on antigen processing and presentation, thereby contributing to escape from immunodominant T cell responses. Alternatively, mutations could enhance antigen processing and efficacy of T cell recognition, opening new avenues for improving future vaccine designs. One Sentence Summary Natural mutations in the flanking regions of known immunodominant SARS-CoV-2 nucleoprotein epitopes can decrease CD8 + T cell responses leading to partial escape.

2.
Lancet Microbe ; 3(5): e348-e356, 2022 05.
Article in English | MEDLINE | ID: covidwho-1758021

ABSTRACT

Background: The memory immune response is crucial for preventing reinfection or reducing disease severity. However, the robustness and functionality of the humoral and T-cell response to SARS-CoV-2 remains unknown 12 months after initial infection. The aim of this study is to investigate the durability and functionality of the humoral and T-cell response to the original SARS-CoV-2 strain and variants in recovered patients 12 months after infection. Methods: In this longitudinal cohort study, we recruited participants who had recovered from COVID-19 and who were discharged from the Wuhan Research Center for Communicable Disease Diagnosis and Treatment at the Chinese Academy of Medical Sciences, Wuhan, China, between Jan 7 and May 29, 2020. Patients received a follow-up visit between Dec 16, 2020, and Jan 27, 2021. We evaluated the presence of IgM, IgA, and IgG antibodies against the SARS-CoV-2 nucleoprotein, Spike protein, and the receptor-binding domain 12 months after initial infection, using ELISA. Neutralising antibodies against the original SARS-CoV-2 strain, and the D614G, beta (B.1.351), and delta (B.1.617.2) variants were analysed using a microneutralisation assay in a subset of plasma samples. We analysed the magnitude and breadth of the SARS-CoV-2-specific memory T-cell responses using the interferon γ (IFNγ) enzyme-linked immune absorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) assay. The antibody response and T-cell response (ie, IFN-γ, interleukin-2 [IL-2], and tumour necrosis factor α [TNFα]) were analysed by age and disease severity. Antibody titres were also analysed according to sequelae symptoms. Findings: We enrolled 1096 patients, including 289 (26·4%) patients with moderate initial disease, 734 (67·0%) with severe initial disease, and 73 (6·7%) with critical initial disease. Paired plasma samples were collected from 141 patients during the follow-up visits for the microneutralisation assay. PBMCs were collected from 92 of 141 individuals at the 12-month follow-up visit, of which 80 were analysed by ELISpot and 92 by ICS assay to detect the SARS-CoV-2-specific memory T-cell responses. N-IgG (899 [82·0%]), S-IgG (1043 [95·2%]), RBD-IgG (1032 [94·2%]), and neutralising (115 [81·6%] of 141) antibodies were detectable 12 months after initial infection in most individuals. Neutralising antibodies remained stable 6 and 12 months after initial infection in most individuals younger than 60 years. Multifunctional T-cell responses were detected for all SARS-CoV-2 viral proteins tested. There was no difference in the magnitude of T-cell responses or cytokine profiles in individuals with different symptom severity. Moreover, we evaluated both antibody and T-cell responses to the D614G, beta, and delta viral strains. The degree of reduced in-vitro neutralising antibody responses to the D614G and delta variants, but not to the beta variant, was associated with the neutralising antibody titres after SARS-CoV-2 infection. We also found poor neutralising antibody responses to the beta variant; 83 (72·2%) of 115 patients showed no response at all. Moreover, the neutralising antibody titre reduction of the recovered patient plasma against the delta variant was similar to that of the D614G variant and lower than that of the beta variant. By contrast, T-cell responses were cross-reactive to the beta variant in most individuals. Importantly, T-cell responses could be detected in all individuals who had lost the neutralising antibody response to SARS-CoV-2 12 months after the initial infection. Interpretation: SARS-CoV-2-specific neutralising antibody and T-cell responses were retained 12 months after initial infection. Neutralising antibodies to the D614G, beta, and delta viral strains were reduced compared with those for the original strain, and were diminished in general. Memory T-cell responses to the original strain were not disrupted by new variants. This study suggests that cross-reactive SARS-CoV-2-specific T-cell responses could be particularly important in the protection against severe disease caused by variants of concern whereas neutralising antibody responses seem to reduce over time. Funding: Chinese Academy of Medical Sciences, National Natural Science Foundation, and UK Medical Research Council.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , COVID-19/epidemiology , Cohort Studies , Cytokines , Humans , Immunoglobulin G , Longitudinal Studies , T-Lymphocytes
3.
EuropePMC; 2021.
Preprint in English | EuropePMC | ID: ppcovidwho-309642

ABSTRACT

NP 105-113 -B*07:02 specific CD8 + T-cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP 105-113 -B*07:02 specific T-cell clones and single cell sequencing were performed concurrently, with functional avidity and anti-viral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with TCR usage, transcriptome signature, and disease severity (acute N=77, convalescent N=52). We demonstrated a beneficial association of NP 105-113 -B*07:02 specific T-cells in COVID-19 disease progression, linked with expansion of T-cell precursors, high functional avidity and anti-viral effector function. Broad immune memory pools were narrowed post-infection but NP 105-113 -B*07:02 specific T-cells were maintained 6 months after infection with preserved anti-viral efficacy to the SARS-CoV-2 Victoria strain, as well as new Alpha, Beta and Gamma variants. Our data shows that NP 105-113 -B*07:02 specific T-cell responses associate with mild disease and high anti-viral efficacy, pointing to inclusion for future vaccine design.

4.
EuropePMC;
Preprint in English | EuropePMC | ID: ppcovidwho-328783

ABSTRACT

Little is known of the role of cytotoxic CD4 + T-cells in the control of viral replication. Here, we investigate CD4 + T-cell responses to three dominant SARS-CoV-2 epitopes and evaluate antiviral activity, including cytotoxicity and antiviral cytokine production. Diverse T cell receptor (TCR) usage including public TCRs were identified;surprisingly, cytotoxic CD4 + T-cells were found to have signalling and cytotoxic pathways distinct from classical CD8 + T-cells, with increased expression of chemokines and tissue homing receptors promoting migration. We show the presence of cytolytic CD4 + T-cells during primary infection associates with COVID-19 disease severity. Robust immune memory 6-9 months post-infection or vaccination provides CD4 + T-cells with potent antiviral activity. Our data support a model where CD4 + killer cells drive immunopathogenesis during primary infection and CD4 + memory responses are protective during secondary infection. Our study highlights the unique features of cytotoxic CD4 + T-cells that use distinct functional pathways, providing preventative and therapeutic opportunities.

5.
Nat Immunol ; 23(1): 50-61, 2022 01.
Article in English | MEDLINE | ID: covidwho-1545628

ABSTRACT

NP105-113-B*07:02-specific CD8+ T cell responses are considered among the most dominant in SARS-CoV-2-infected individuals. We found strong association of this response with mild disease. Analysis of NP105-113-B*07:02-specific T cell clones and single-cell sequencing were performed concurrently, with functional avidity and antiviral efficacy assessed using an in vitro SARS-CoV-2 infection system, and were correlated with T cell receptor usage, transcriptome signature and disease severity (acute n = 77, convalescent n = 52). We demonstrated a beneficial association of NP105-113-B*07:02-specific T cells in COVID-19 disease progression, linked with expansion of T cell precursors, high functional avidity and antiviral effector function. Broad immune memory pools were narrowed postinfection but NP105-113-B*07:02-specific T cells were maintained 6 months after infection with preserved antiviral efficacy to the SARS-CoV-2 Victoria strain, as well as Alpha, Beta, Gamma and Delta variants. Our data show that NP105-113-B*07:02-specific T cell responses associate with mild disease and high antiviral efficacy, pointing to inclusion for future vaccine design.


Subject(s)
HLA-B7 Antigen/immunology , Immunodominant Epitopes/immunology , Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , T-Lymphocytes, Cytotoxic/immunology , Aged , Amino Acid Sequence , Antibodies, Viral/immunology , Antibody Affinity/immunology , COVID-19/immunology , COVID-19/pathology , Cell Line, Transformed , Female , Gene Expression Profiling , Humans , Immunologic Memory/immunology , Male , Middle Aged , Receptors, Antigen, T-Cell/immunology , Severity of Illness Index , Vaccinia virus/genetics , Vaccinia virus/immunology , Vaccinia virus/metabolism
6.
iScience ; 24(11): 103353, 2021 Nov 19.
Article in English | MEDLINE | ID: covidwho-1509904

ABSTRACT

We identify amino acid variants within dominant SARS-CoV-2 T cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T cell responsiveness was seen due to Q213K in the A∗01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215; due to P13L, P13S, and P13T in the B∗27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17; and due to T362I and P365S in the A∗03:01/A∗11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T cell lines unable to recognize variant epitopes have diverse T cell receptor repertoires. These data demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.

7.
Cell ; 184(8): 2183-2200.e22, 2021 04 15.
Article in English | MEDLINE | ID: covidwho-1086819

ABSTRACT

Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 µg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public V-genes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Binding Sites, Antibody , CHO Cells , Chlorocebus aethiops , Cricetulus , Epitopes , Female , HEK293 Cells , Humans , Male , Mice , Mice, Transgenic , Models, Molecular , Protein Binding , Protein Structure, Tertiary , SARS-CoV-2/immunology , Vero Cells
8.
Nat Immunol ; 21(11): 1336-1345, 2020 11.
Article in English | MEDLINE | ID: covidwho-889210

ABSTRACT

The development of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines and therapeutics will depend on understanding viral immunity. We studied T cell memory in 42 patients following recovery from COVID-19 (28 with mild disease and 14 with severe disease) and 16 unexposed donors, using interferon-γ-based assays with peptides spanning SARS-CoV-2 except ORF1. The breadth and magnitude of T cell responses were significantly higher in severe as compared with mild cases. Total and spike-specific T cell responses correlated with spike-specific antibody responses. We identified 41 peptides containing CD4+ and/or CD8+ epitopes, including six immunodominant regions. Six optimized CD8+ epitopes were defined, with peptide-MHC pentamer-positive cells displaying the central and effector memory phenotype. In mild cases, higher proportions of SARS-CoV-2-specific CD8+ T cells were observed. The identification of T cell responses associated with milder disease will support an understanding of protective immunity and highlights the potential of including non-spike proteins within future COVID-19 vaccine design.


Subject(s)
Antigens, Viral/immunology , Betacoronavirus/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Immunologic Memory/immunology , COVID-19 , COVID-19 Vaccines , Coronavirus Infections/immunology , Coronavirus Infections/pathology , Coronavirus Infections/prevention & control , Epitopes, T-Lymphocyte/immunology , Humans , Immunodominant Epitopes/immunology , Pandemics , Pneumonia, Viral/immunology , Pneumonia, Viral/pathology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/immunology , United Kingdom , Viral Vaccines/immunology
10.
JCI Insight ; 5(13)2020 07 09.
Article in English | MEDLINE | ID: covidwho-541270

ABSTRACT

BACKGROUND: Identifying immune correlates of COVID-19 disease severity is an urgent need for clinical management, vaccine evaluation, and drug development. Here, we present a temporal analysis of key immune mediators, cytokines, and chemokines in blood of hospitalized COVID-19 patients from serial sampling and follow-up over 4 weeks. METHODS: A total of 71 patients with laboratory-confirmed COVID-19 admitted to Beijing You'an Hospital in China with either mild (53 patients) or severe (18 patients) disease were enrolled with 18 healthy volunteers. We measured 34 immune mediators, cytokines, and chemokines in peripheral blood every 4-7 days over 1 month per patient using a bioplex multiplex immunoassay. RESULTS: We found that the chemokine RANTES (CCL5) was significantly elevated, from an early stage of the infection, in patients with mild but not severe disease. We also found that early production of inhibitory mediators including IL-10 and IL-1RA were significantly associated with disease severity, and a combination of CCL5, IL-1 receptor antagonist (IL-1RA), and IL-10 at week 1 may predict patient outcomes. The majority of cytokines that are known to be associated with the cytokine storm in virus infections such as IL-6 and IFN-γ were only significantly elevated in the late stage of severe COVID-19 illness. TNF-α and GM-CSF showed no significant differences between severe and mild cases. CONCLUSION: Together, our data suggest that early intervention to increase expression of CCL5 may prevent patients from developing severe illness. Our data also suggest that measurement of levels of CCL5, as well as IL-1RA and IL-10 in blood individually and in combination, might be useful prognostic biomarkers to guide treatment strategies.


Subject(s)
Chemokine CCL5/immunology , Coronavirus Infections/immunology , Interleukin 1 Receptor Antagonist Protein/immunology , Interleukin-10/immunology , Pneumonia, Viral/immunology , Adult , Aged , Betacoronavirus , COVID-19 , Case-Control Studies , Coronavirus Infections/physiopathology , Cytokine Release Syndrome/immunology , Female , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Hospitalization , Humans , Immunoassay , Interferon-gamma/immunology , Interleukin-6/immunology , Longitudinal Studies , Male , Middle Aged , Pandemics , Pneumonia, Viral/physiopathology , SARS-CoV-2 , Severity of Illness Index , Tumor Necrosis Factor-alpha/immunology
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