Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS-CoV-2 spike protein

A correct identification of seropositive individuals for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T-cell line that stably expresses the full-length native spike “S” protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double-staining with the test sera and anti-EGFR. The method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. As examples of its use, we show that as much as 28% of the personnel working at the CBMSO in Madrid is already immune. Additionally, we show that anti-S antibodies with protective neutralizing activity are long-lasting and can be detected in sera 8 months after infectionThis work was funded by intramural grant CSIC-COVID19-004: 202020E081 (to B.A.) and CSIC-COVID19-004: 202020E165 (to MF). L.H has been supported by an FPI fellowship from the Spanish Ministry of Science and Innovation. I.B. has been supported by an H2020-MSCA-ITN-2016 training network grant of the European Union (GA 721358

Decreased breadth after boost

The rapid development of vaccines to prevent infection by SARS-CoV-2 virus causing COVID-19 makes necessary to compare the capacity of the different vaccines in terms of development of a protective humoral response. Here, we have used a highly sensitive and reliable flow cytometry method to measure the titers of antibodies of the IgG1 isotype in blood of volunteers after receiving one or two doses of the vaccines being administered in Spain. We took advantage of the multiplexed capacity of the method to measure simultaneously the reactivity of antibodies with the S protein of the original strain Wuhan-1 and the variant B.1.1.7 (Alpha). We found significant differences in the titer of anti-S antibodies produced after a first dose of the vaccines ChAdOx1 nCov-19/AstraZeneca, mRNA-1273/Moderna, BNT162b2/Pfizer-BioNTech and Ad26.COV.S/Janssen. Most important, we found a relative reduction in the reactivity of the sera with the B.1.1.7 versus the Wuhan-1 variant after the second boosting immunization. These data allow to make a comparison of different vaccines in terms of anti-S antibody generation and cast doubts about the convenience of repeatedly immunizing with the same S protein sequence.This work was funded by intramural grant CSIC-COVID19-004: 202020E081 (to B.A.) and CSIC-COVID19-004: 202020E165 (to MF). L.H has been supported by an FPI fellowship from the Spanish Ministry of Science and Innovation. I.B. has been supported by an H2020-MSCA-ITN2016 training network grant of the European Union (GA 721358)Peer reviewe

Decreased breadth of the antibody response to the spike protein of SARS-CoV-2 after repeated vaccination

IntroductionThe rapid development of vaccines to prevent COVID-19 has raised the need to compare the capacity of different vaccines in terms of developing a protective humoral response. Previous studies have shown inconsistent results in this area, highlighting the importance of further research to evaluate the efficacy of different vaccines.MethodsThis study utilized a highly sensitive and reliable flow cytometry method to measure the titers of IgG1 isotype antibodies in the blood of healthy volunteers after receiving one or two doses of various vaccines administered in Spain. The method was also used to simultaneously measure the reactivity of antibodies to the S protein of the original Wuhan strain and variants B.1.1.7 (Alpha), B.1.617.2 (Delta), and B.1.617.1 (Kappa).ResultsSignificant differences were observed in the titer of anti-S antibodies produced after a first dose of the vaccines ChAdOx1 nCov-19/AstraZeneca, mRNA-1273/Moderna, BNT162b2/Pfizer-BioNTech, and Ad26.COV.S/Janssen. Furthermore, a relative reduction in the reactivity of the sera with the Alpha, Delta, and Kappa variants, compared to the Wuhan strain, was observed after the second boosting immunization.DiscussionThe findings of this study provide a comparison of different vaccines in terms of anti-S antibody generation and cast doubts on the convenience of repeated immunization with the same S protein sequence. The multiplexed capacity of the flow cytometry method utilized in this study allowed for a comprehensive evaluation of the efficacy of various vaccines in generating a protective humoral response. Future research could focus on the implications of these findings for the development of effective COVID-19 vaccination strategies

Procédé multiplexe par cytométrie en flux de détection d'anticorps anti-SARS-COV-2

[EN] The present invention relates to a first method for in vitro detection of anti-S protein antibodies against SARS-CoV2 in a subject and a second method for diagnostic of the exposure to SARS-CoV2 virus of a subject, based both in flow cytometry, which comprising stable expressing either by transfection or viral transduction of a vector comprising the S protein of SARS-CoV2 in a cell line in suspension. Further, the present invention relates to a kit for in vitro detection of anti-S protein antibodies against SARS-CoV2 and a kit for diagnostic of the exposure to SARS-CoV2, as well as its uses.[FR] La présente invention concerne un premier procédé de détection in vitro d'anticorps anti-protéine S contre le SARS-CoV2 chez un sujet et un second procédé de diagnostic de l'exposition au virus SRAS-CoV2 d'un sujet, les deux par cytométrie de flux, qui comprennent une expression stable par transfection ou par transduction virale d'un vecteur comprenant la protéine S du SARS-CoV2 dans une lignée cellulaire en suspension. La présente invention concerne en outre un kit pour la détection in vitro d'anticorps anti-protéine S contre le SARS-CoV2 et un kit pour le diagnostic de l'exposition au SARS-CoV2, ainsi que leurs utilisations.NoConsejo Superior de Investigaciones Científicas (CSIC)A1 Solicitud de patente con informe sobre el estado de la técnic

Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS-COV-2 spike protein

A correct identification of seropositive individuals for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T cell line that stably expresses the full-length native spike S protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a ratio of the mean fluorescence intensities obtained by double- staining with the sera and a monoclonal antibody specific for EGFR. We show that the method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. It can also be employed to detect neutralizing activity in the sera of individuals. Finally, the method can be used in a multiplexed format to simultaneously measure all anti-S human immunoglobulin isotypes in blood and mucosal fluids including total saliva.Peer reviewe