Development of a novel medium throughput flow-cytometry based micro-neutralisation test for SARS-CoV-2 with applications in clinical vaccine trials and antibody screening.

Autor: O'Reilly S; Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin, Ireland.; School of Medicine, University College Dublin, Belfield, Dublin, Ireland., Kenny G; Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin, Ireland.; School of Medicine, University College Dublin, Belfield, Dublin, Ireland.; Department of Infectious Diseases, St Vincent's University Hospital, Elm Park, Dublin, Ireland., Alrawahneh T; Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin, Ireland.; School of Medicine, University College Dublin, Belfield, Dublin, Ireland., Francois N; Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin, Ireland.; School of Medicine, University College Dublin, Belfield, Dublin, Ireland., Gu L; Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin, Ireland.; School of Medicine, University College Dublin, Belfield, Dublin, Ireland., Angeliadis M; Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin, Ireland.; School of Medicine, University College Dublin, Belfield, Dublin, Ireland., de Masson d'Autume V; Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin, Ireland.; School of Medicine, University College Dublin, Belfield, Dublin, Ireland., Garcia Leon A; Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin, Ireland.; School of Medicine, University College Dublin, Belfield, Dublin, Ireland., Feeney ER; Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin, Ireland.; School of Medicine, University College Dublin, Belfield, Dublin, Ireland.; Department of Infectious Diseases, St Vincent's University Hospital, Elm Park, Dublin, Ireland., Yousif O; Endocrinology Department, Wexford General Hospital, Carricklawn, Wexford, Ireland., Cotter A; Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin, Ireland.; School of Medicine, University College Dublin, Belfield, Dublin, Ireland.; Department of Infectious Diseases, Mater Misericordiae University Hospital, Eccles St, Dublin, Ireland., de Barra E; Department of Infectious Diseases, Beaumont Hospital, Beaumont, Dublin, Ireland.; Department of International Health and Tropical Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland., Horgan M; Department of Infectious Diseases, Cork University Hospital, Wilton, Cork, Ireland., Mallon PWG; Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin, Ireland.; School of Medicine, University College Dublin, Belfield, Dublin, Ireland.; Department of Infectious Diseases, St Vincent's University Hospital, Elm Park, Dublin, Ireland., Gautier V; Centre for Experimental Pathogen Host Research (CEPHR), University College Dublin, Belfield, Dublin, Ireland.; School of Medicine, University College Dublin, Belfield, Dublin, Ireland.; Conway Institute of Biomedical and Biomolecular Research, University College Dublin, Belfield, Dublin 4, Ireland.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2023 Nov 30; Vol. 18 (11), pp. e0294262. Date of Electronic Publication: 2023 Nov 30 (Print Publication: 2023).
DOI: 10.1371/journal.pone.0294262
Abstrakt: Quantifying neutralising capacity of circulating SARS-COV-2 antibodies is critical in evaluating protective humoral immune responses generated post-infection/post-vaccination. Here we describe a novel medium-throughput flow cytometry-based micro-neutralisation test to evaluate Neutralising Antibody (NAb) responses against live SARS-CoV-2 Wild Type and Variants of Concern (VOC) in convalescent/vaccinated populations. Flow Cytometry-Based Micro-Neutralisation Test (Micro-NT) was performed in 96-well plates using clinical isolates WT-B, WT-B.1.177.18 and/or VOCs Beta and Omicron. Plasma samples (All Ireland Infectious Diseases (AIID) Cohort) were serially diluted (8 points, half-log) from 1:20 and pre-incubated with SARS-CoV-2 (1h, 37°C). Virus-plasma mixture were added onto Vero E6 or Vero E6/TMPRSS2 cells for 18h. Percentage infected cells was analysed by automated flow cytometry following trypsinisation, fixation and SARS-CoV-2 Nucleoprotein intracellular staining. Half-maximal Neutralisation Titres (NT50) were determined using non-linear regression. Our assay was compared to Plaque Reduction Neutralisation Test (PRNT) and validated against the First WHO International Standard for anti-SARS-CoV-2 immunoglobulin. Both Micro-NT and PRNT achieved comparable NT50 values. Further validation showed adequate correlation with PRNT using a panel of secondary standards of clinical convalescent and vaccinated plasma samples. We found the assay to be reproducible through measuring both repeatability and intermediate precision. Screening 190 convalescent samples and 11 COVID-19 naive controls (AIID cohort) we demonstrated that Micro-NT has broad dynamic range differentiating NT50s <1/20 to >1/5000. We could also characterise immune-escape VOC Beta and Omicron BA.5, achieving fold-reductions in neutralising capacity similar to those published. Our flow cytometry-based Micro-NT is a robust and reliable assay to quantify NAb titres, and has been selected as an endpoint in clinical trials.
Competing Interests: E.F. has received consulting fees from Gilead, ViiV and Vidacare Ireland, and has been awarded a grant from Science Foundation Ireland outside the submitted work. E.d.B. has received consulting fees from Sanofi Pasteur and an honoraria/travel grant from Pfizer. P.M. has received honoraria and/or travel grants from Gilead Sciences, MSD, Astrazeneca, and ViiV Healthcare, and has been awarded grants by Science Foundation Ireland outside the submitted work. The remaining authors declare that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.
(Copyright: © 2023 O’Reilly et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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