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1.
Lancet Microbe ; 2021 Nov 09.
Article in English | MEDLINE | ID: covidwho-1510521

ABSTRACT

Background: Previous infection with SARS-CoV-2 affects the immune response to the first dose of the SARS-CoV-2 vaccine. We aimed to compare SARS-CoV-2-specific T-cell and antibody responses in health-care workers with and without previous SARS-CoV-2 infection following a single dose of the BNT162b2 (tozinameran; Pfizer-BioNTech) mRNA vaccine. Methods: We sampled health-care workers enrolled in the PITCH study across four hospital sites in the UK (Oxford, Liverpool, Newcastle, and Sheffield). All health-care workers aged 18 years or older consenting to participate in this prospective cohort study were included, with no exclusion criteria applied. Blood samples were collected where possible before vaccination and 28 (±7) days following one or two doses (given 3-4 weeks apart) of the BNT162b2 vaccine. Previous infection was determined by a documented SARS-CoV-2-positive RT-PCR result or the presence of positive anti-SARS-CoV-2 nucleocapsid antibodies. We measured spike-specific IgG antibodies and quantified T-cell responses by interferon-γ enzyme-linked immunospot assay in all participants where samples were available at the time of analysis, comparing SARS-CoV-2-naive individuals to those with previous infection. Findings: Between Dec 9, 2020, and Feb 9, 2021, 119 SARS-CoV-2-naive and 145 previously infected health-care workers received one dose, and 25 SARS-CoV-2-naive health-care workers received two doses, of the BNT162b2 vaccine. In previously infected health-care workers, the median time from previous infection to vaccination was 268 days (IQR 232-285). At 28 days (IQR 27-33) after a single dose, the spike-specific T-cell response measured in fresh peripheral blood mononuclear cells (PBMCs) was higher in previously infected (n=76) than in infection-naive (n=45) health-care workers (median 284 [IQR 150-461] vs 55 [IQR 24-132] spot-forming units [SFUs] per 106 PBMCs; p<0·0001). With cryopreserved PBMCs, the T-cell response in previously infected individuals (n=52) after one vaccine dose was equivalent to that of infection-naive individuals (n=19) after receiving two vaccine doses (median 152 [IQR 119-275] vs 162 [104-258] SFUs/106 PBMCs; p=1·00). Anti-spike IgG antibody responses following a single dose in 142 previously infected health-care workers (median 270 373 [IQR 203 461-535 188] antibody units [AU] per mL) were higher than in 111 infection-naive health-care workers following one dose (35 001 [17 099-55 341] AU/mL; p<0·0001) and higher than in 25 infection-naive individuals given two doses (180 904 [108 221-242 467] AU/mL; p<0·0001). Interpretation: A single dose of the BNT162b2 vaccine is likely to provide greater protection against SARS-CoV-2 infection in individuals with previous SARS-CoV-2 infection, than in SARS-CoV-2-naive individuals, including against variants of concern. Future studies should determine the additional benefit of a second dose on the magnitude and durability of immune responses in individuals vaccinated following infection, alongside evaluation of the impact of extending the interval between vaccine doses. Funding: UK Department of Health and Social Care, and UK Coronavirus Immunology Consortium.

2.
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.

3.
BMJ Open ; 11(10): e055435, 2021 10 22.
Article in English | MEDLINE | ID: covidwho-1480255

ABSTRACT

OBJECTIVES: The steroid hormone vitamin D has roles in immunomodulation and bone health. Insufficiency is associated with susceptibility to respiratory infections. We report 25-hydroxy vitamin D (25(OH)D) measurements in hospitalised people with COVID-19 and influenza A and in survivors of critical illness to test the hypotheses that vitamin D insufficiency scales with illness severity and persists in survivors. DESIGN: Cross-sectional study. SETTING AND PARTICIPANTS: Plasma was obtained from 295 hospitalised people with COVID-19 (International Severe Acute Respiratory and emerging Infections Consortium (ISARIC)/WHO Clinical Characterization Protocol for Severe Emerging Infections UK study), 93 with influenza A (Mechanisms of Severe Acute Influenza Consortium (MOSAIC) study, during the 2009-2010 H1N1 pandemic) and 139 survivors of non-selected critical illness (prior to the COVID-19 pandemic). Total 25(OH)D was measured by liquid chromatography-tandem mass spectrometry. Free 25(OH)D was measured by ELISA in COVID-19 samples. OUTCOME MEASURES: Receipt of invasive mechanical ventilation (IMV) and in-hospital mortality. RESULTS: Vitamin D insufficiency (total 25(OH)D 25-50 nmol/L) and deficiency (<25 nmol/L) were prevalent in COVID-19 (29.3% and 44.4%, respectively), influenza A (47.3% and 37.6%) and critical illness survivors (30.2% and 56.8%). In COVID-19 and influenza A, total 25(OH)D measured early in illness was lower in patients who received IMV (19.6 vs 31.9 nmol/L (p<0.0001) and 22.9 vs 31.1 nmol/L (p=0.0009), respectively). In COVID-19, biologically active free 25(OH)D correlated with total 25(OH)D and was lower in patients who received IMV, but was not associated with selected circulating inflammatory mediators. CONCLUSIONS: Vitamin D deficiency/insufficiency was present in majority of hospitalised patients with COVID-19 or influenza A and correlated with severity and persisted in critical illness survivors at concentrations expected to disrupt bone metabolism. These findings support early supplementation trials to determine if insufficiency is causal in progression to severe disease, and investigation of longer-term bone health outcomes.


Subject(s)
COVID-19 , Influenza A Virus, H1N1 Subtype , Influenza, Human , Vitamin D Deficiency , Critical Illness , Cross-Sectional Studies , Humans , Influenza, Human/complications , Influenza, Human/epidemiology , Pandemics , SARS-CoV-2 , Survivors , Vitamin D , Vitamin D Deficiency/complications , Vitamin D Deficiency/epidemiology
4.
Cell ; 184(23): 5699-5714.e11, 2021 11 11.
Article in English | MEDLINE | ID: covidwho-1466093

ABSTRACT

Extension of the interval between vaccine doses for the BNT162b2 mRNA vaccine was introduced in the United Kingdom to accelerate population coverage with a single dose. At this time, trial data were lacking, and we addressed this in a study of United Kingdom healthcare workers. The first vaccine dose induced protection from infection from the circulating alpha (B.1.1.7) variant over several weeks. In a substudy of 589 individuals, we show that this single dose induces severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibody (NAb) responses and a sustained B and T cell response to the spike protein. NAb levels were higher after the extended dosing interval (6-14 weeks) compared with the conventional 3- to 4-week regimen, accompanied by enrichment of CD4+ T cells expressing interleukin-2 (IL-2). Prior SARS-CoV-2 infection amplified and accelerated the response. These data on dynamic cellular and humoral responses indicate that extension of the dosing interval is an effective immunogenic protocol.


Subject(s)
COVID-19 Vaccines/immunology , Vaccines, Synthetic/immunology , Adult , Aged , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/immunology , COVID-19/virology , Cross-Priming/immunology , Dose-Response Relationship, Immunologic , Female , Humans , Immunity , Immunoglobulin G/immunology , Linear Models , Male , Middle Aged , Reference Standards , SARS-CoV-2/immunology , T-Lymphocytes/immunology , Treatment Outcome , Young Adult
5.
Nat Commun ; 12(1): 2055, 2021 04 06.
Article in English | MEDLINE | ID: covidwho-1171493

ABSTRACT

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/immunology , COVID-19/virology , Cross Reactions/immunology , Immunoassay/methods , SARS-CoV-2/physiology , T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/epidemiology , Cell Proliferation , Cytokines/metabolism , HEK293 Cells , Health Personnel , Humans , Immunoglobulin G/immunology , Immunologic Memory , Interferon-gamma/metabolism , Pandemics , Peptides/metabolism , SARS-CoV-2/drug effects
6.
Nat Commun ; 12(1): 1951, 2021 03 29.
Article in English | MEDLINE | ID: covidwho-1157905

ABSTRACT

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests do not require special equipment, are read by eye, have short development times, low cost and can be applied at the Point of Care. Here we describe a quantitative Haemagglutination test (HAT) for the detection of antibodies to the receptor binding domain of the SARS-CoV-2 spike protein. The HAT has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. We will supply aliquots of the test reagent sufficient for ten thousand test wells free of charge to qualified research groups anywhere in the world.


Subject(s)
Antibodies, Viral/analysis , COVID-19 Testing/methods , COVID-19/diagnosis , Hemagglutination Tests/methods , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/immunology , Agglutination Tests/methods , Antibodies, Monoclonal/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/immunology , COVID-19/virology , Enzyme-Linked Immunosorbent Assay/methods , Humans , Point-of-Care Systems , Polymerase Chain Reaction , SARS-CoV-2/immunology , Sensitivity and Specificity , Seroconversion
7.
Wellcome Open Res ; 5: 139, 2020.
Article in English | MEDLINE | ID: covidwho-1140800

ABSTRACT

Background: The COVID-19 pandemic caused >1 million infections during January-March 2020. There is an urgent need for reliable antibody detection approaches to support diagnosis, vaccine development, safe release of individuals from quarantine, and population lock-down exit strategies. We set out to evaluate the performance of ELISA and lateral flow immunoassay (LFIA) devices. Methods: We tested plasma for COVID (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) IgM and IgG antibodies by ELISA and using nine different LFIA devices. We used a panel of plasma samples from individuals who have had confirmed COVID infection based on a PCR result (n=40), and pre-pandemic negative control samples banked in the UK prior to December-2019 (n=142). Results: ELISA detected IgM or IgG in 34/40 individuals with a confirmed history of COVID infection (sensitivity 85%, 95%CI 70-94%), vs. 0/50 pre-pandemic controls (specificity 100% [95%CI 93-100%]). IgG levels were detected in 31/31 COVID-positive individuals tested ≥10 days after symptom onset (sensitivity 100%, 95%CI 89-100%). IgG titres rose during the 3 weeks post symptom onset and began to fall by 8 weeks, but remained above the detection threshold. Point estimates for the sensitivity of LFIA devices ranged from 55-70% versus RT-PCR and 65-85% versus ELISA, with specificity 95-100% and 93-100% respectively. Within the limits of the study size, the performance of most LFIA devices was similar. Conclusions: Currently available commercial LFIA devices do not perform sufficiently well for individual patient applications. However, ELISA can be calibrated to be specific for detecting and quantifying SARS-CoV-2 IgM and IgG and is highly sensitive for IgG from 10 days following first symptoms.

8.
Sci Immunol ; 6(57)2021 03 10.
Article in English | MEDLINE | ID: covidwho-1127535

ABSTRACT

While it is now widely accepted that host inflammatory responses contribute to lung injury, the pathways that drive severity and distinguish coronavirus disease 2019 (COVID-19) from other viral lung diseases remain poorly characterized. We analyzed plasma samples from 471 hospitalized patients recruited through the prospective multicenter ISARIC4C study and 39 outpatients with mild disease, enabling extensive characterization of responses across a full spectrum of COVID-19 severity. Progressive elevation of levels of numerous inflammatory cytokines and chemokines (including IL-6, CXCL10, and GM-CSF) were associated with severity and accompanied by elevated markers of endothelial injury and thrombosis. Principal component and network analyses demonstrated central roles for IL-6 and GM-CSF in COVID-19 pathogenesis. Comparing these profiles to archived samples from patients with fatal influenza, IL-6 was equally elevated in both conditions whereas GM-CSF was prominent only in COVID-19. These findings further identify the key inflammatory, thrombotic, and vascular factors that characterize and distinguish severe and fatal COVID-19.


Subject(s)
COVID-19/blood , Cytokines/blood , Adult , Aged , COVID-19/immunology , Cytokines/immunology , Female , Humans , Inflammation/blood , Inflammation/immunology , Influenza A Virus, H1N1 Subtype , Influenza, Human/blood , Influenza, Human/immunology , Male , Middle Aged , SARS-CoV-2 , Severity of Illness Index
9.
Nature ; 591(7848): 92-98, 2021 03.
Article in English | MEDLINE | ID: covidwho-971937

ABSTRACT

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice.


Subject(s)
COVID-19/genetics , COVID-19/physiopathology , Critical Illness , 2',5'-Oligoadenylate Synthetase/genetics , COVID-19/pathology , Chromosomes, Human, Pair 12/genetics , Chromosomes, Human, Pair 19/genetics , Chromosomes, Human, Pair 21/genetics , Critical Care , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Drug Repositioning , Female , Genome-Wide Association Study , Humans , Inflammation/genetics , Inflammation/pathology , Inflammation/physiopathology , Lung/pathology , Lung/physiopathology , Lung/virology , Male , Multigene Family/genetics , Receptor, Interferon alpha-beta/genetics , Receptors, CCR2/genetics , TYK2 Kinase/genetics , United Kingdom
10.
Viruses ; 12(10)2020 10 14.
Article in English | MEDLINE | ID: covidwho-905965

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/virology , Pneumonia, Viral/virology , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , COVID-19 Vaccines , Clinical Laboratory Techniques , Coronavirus Infections/diagnosis , DNA, Complementary/analysis , DNA, Complementary/genetics , DNA, Viral/analysis , DNA, Viral/genetics , Genome, Viral , High-Throughput Nucleotide Sequencing/methods , Humans , Molecular Epidemiology , Multiplex Polymerase Chain Reaction , Pandemics , Pneumonia, Viral/diagnosis , RNA, Viral/analysis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction , SARS-CoV-2 , Sequence Analysis
11.
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
12.
Viruses ; 12(10):1164, 2020.
Article in English | MDPI | ID: covidwho-855539

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.

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