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1.
PLoS One ; 17(2): e0262149, 2022.
Article in English | MEDLINE | ID: covidwho-1677576

ABSTRACT

There is an urgent need for better diagnostic and analytical methods for vaccine research and infection control in virology. This has been highlighted by recently emerging viral epidemics and pandemics (Zika, SARS-CoV-2), and recurring viral outbreaks like the yellow fever outbreaks in Angola and the Democratic Republic of Congo (2016) and in Brazil (2016-2018). Current assays to determine neutralising activity against viral infections in sera are costly in time and equipment and suffer from high variability. Therefore, both basic infection research and diagnostic population screenings would benefit from improved methods to determine virus-neutralising activity in patient samples. Here we describe a robust, objective, and scalable Fluorescence Reduction Neutralisation Test (FluoRNT) for yellow fever virus, relying on flow cytometric detection of cells infected with a fluorescent Venus reporter containing variant of the yellow fever vaccine strain 17D (YF-17D-Venus). It accurately measures neutralising antibody titres in human serum samples within as little as 24 h. Samples from 32 vaccinees immunised with YF-17D were tested for neutralising activity by both a conventional focus reduction neutralisation test (FRNT) and FluoRNT. Both types of tests proved to be equally reliable for the detection of neutralising activity, however, FluoRNT is significantly more precise and reproducible with a greater dynamic range than conventional FRNT. The FluoRNT assay protocol is substantially faster, easier to control, and cheaper in per-assay costs. FluoRNT additionally reduces handling time minimising exposure of personnel to patient samples. FluoRNT thus brings a range of desirable features that can accelerate and standardise the measurement of neutralising anti-yellow fever virus antibodies. It could be used in applications ranging from vaccine testing to large cohort studies in systems virology and vaccinology. We also anticipate the potential to translate the methodology and analysis of FluoRNT to other flaviviruses such as West Nile, Dengue and Zika or to RNA viruses more generally.


Subject(s)
Antibodies, Neutralizing/immunology , Yellow Fever/immunology , Yellow fever virus/immunology , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Antibodies, Viral/immunology , Chlorocebus aethiops , Fluorescence , Humans , Neutralization Tests/economics , Neutralization Tests/methods , Vero Cells , Yellow Fever/blood , Yellow Fever/virology
2.
J Extracell Vesicles ; 10(14): e12173, 2021 12.
Article in English | MEDLINE | ID: covidwho-1544291

ABSTRACT

Infection with SARS-CoV-2 is associated with thromboinflammation, involving thrombotic and inflammatory responses, in many COVID-19 patients. In addition, immune dysfunction occurs in patients characterised by T cell exhaustion and severe lymphopenia. We investigated the distribution of phosphatidylserine (PS), a marker of dying cells, activated platelets and platelet-derived microparticles (PMP), during the clinical course of COVID-19. We found an unexpectedly high amount of blood cells loaded with PS+ PMPs for weeks after the initial COVID-19 diagnosis. Elevated frequencies of PS+ PMP+ PBMCs correlated strongly with increasing disease severity. As a marker, PS outperformed established laboratory markers for inflammation, leucocyte composition and coagulation, currently used for COVID-19 clinical scoring. PS+ PMPs preferentially bound to CD8+ T cells with gene expression signatures of proliferating effector rather than memory T cells. As PS+ PMPs carried programmed death-ligand 1 (PD-L1), they may affect T cell expansion or function. Our data provide a novel marker for disease severity and show that PS, which can trigger the blood coagulation cascade, the complement system, and inflammation, resides on activated immune cells. Therefore, PS may serve as a beacon to attract thromboinflammatory processes towards lymphocytes and cause immune dysfunction in COVID-19.


Subject(s)
COVID-19/blood , Leukocytes, Mononuclear/metabolism , Phosphatidylserines/blood , Adult , Blood Platelets/immunology , CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , COVID-19/physiopathology , Cell-Derived Microparticles/metabolism , Flow Cytometry , Humans , Platelet Membrane Glycoprotein IIb , Severity of Illness Index , Transcriptome
3.
PLoS Pathog ; 17(10): e1009742, 2021 10.
Article in English | MEDLINE | ID: covidwho-1456098

ABSTRACT

Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DCs) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute illness to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage-HLADR+ cells lacking DC markers. Increased frequency of CD163+ CD14+ cells within the recently discovered DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of programmed death-ligand 1 (PD-L1) in conventional DCs (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naïve CD4+ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.


Subject(s)
COVID-19/immunology , Dendritic Cells/immunology , Regeneration/immunology , SARS-CoV-2/immunology , Adult , Antigens, CD/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , COVID-19/pathology , Dendritic Cells/pathology , Female , Humans , Male , Middle Aged , Monocytes/immunology , Monocytes/pathology , Programmed Cell Death 1 Receptor/immunology
4.
Front Immunol ; 12: 601080, 2021.
Article in English | MEDLINE | ID: covidwho-1403468

ABSTRACT

COVID-19, the disease caused by SARS-CoV-2 infection, can assume a highly variable disease course, ranging from asymptomatic infection, which constitutes the majority of cases, to severe respiratory failure. This implies a diverse host immune response to SARS-CoV-2. However, the immunological underpinnings underlying these divergent disease courses remain elusive. We therefore set out to longitudinally characterize immune signatures of convalescent COVID-19 patients stratified according to their disease severity. Our unique convalescent COVID-19 cohort consists of 74 patients not confounded by comorbidities. This is the first study of which we are aware that excludes immune abrogations associated with non-SARS-CoV-2 related risk factors of disease severity. Patients were followed up and analyzed longitudinally (2, 4 and 6 weeks after infection) by high-dimensional flow cytometric profiling of peripheral blood mononuclear cells (PBMCs), in-depth serum analytics, and transcriptomics. Immune phenotypes were correlated to disease severity. Convalescence was overall associated with uniform immune signatures, but distinct immune signatures for mildly versus severely affected patients were detectable within a 2-week time window after infection.


Subject(s)
COVID-19/immunology , SARS-CoV-2/immunology , Adult , Aged , Aged, 80 and over , Convalescence , Female , Humans , Immunophenotyping , Male , Middle Aged , Severity of Illness Index , Young Adult
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