Suchergebnisse

Currently, there is a need for reliable tests that allow identification of individuals that have been infected with SARS-CoV-2 even if the infection was asymptomatic. To date, the vast majority of the serological tests for SARS-CoV-2 specific antibodies are based on serum detection of antibodies to either the viral spike glycoprotein (the major target for neutralising antibodies) or the viral nucleocapsid protein that are known to be highly immunogenic in other coronaviruses. Conceivably, exposure of antigens released from infected cells could stimulate antibody responses that might correlate with tissue damage and, hence, they may have some value as a prognostic indicator. We addressed whether other non-structural viral proteins, not incorporated into the infectious viral particle, specifically the viral cysteine-like protease, might also be potent immunogens. Using ELISA tests, coating several SARS-CoV-2 proteins produced in vitro, we describe that COVID-19 patients make high titre IgG, IgM and IgA antibody responses to the Cys-like protease from SARS-CoV-2, also known as 3CLpro or Mpro, and it can be used to identify individuals with positive serology against the coronavirus. Higher antibody titres in these assays associated with more severe disease and no cross-reactive antibodies against prior betacoronavirus were found. Remarkably, IgG antibodies specific for Mpro and other SARS-CoV-2 antigens can also be detected in saliva. In conclusion, Mpro is a potent antigen in infected patients that can be used in serological tests and its detection in saliva could be the basis for a rapid, non-invasive test for COVID-19 seropositivity. ; This work was supported by the Spanish National Research Council (CSIC, project number 202020E079) and grants from Madrid Regional Government IMMUNOTHERCAN [S2017/BMD-3733-2 (MVG)]; the Spanish Ministry of Science and Innovation [(MCIU/AEI/FEDER, EU): RTI2018-093569-B-I00 (MVG), SAF2017-82940-R (JMRF), SAF2017-83265-R (HTR); SAF2017-82886-R (FSM)]; RETICS Program of ISCIII [RD16/0012/0006; RIER (JMRF); RD16/0011/0012, PI18/0371 (IGA), PI19/00549 (AA)]. The study was also funded by La Caixa Banking Foundation (HR17-00016 to FSM) and Fondo Supera COVID (CRUE-Banco de Santander) to FSM ; No

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19. ; This work was supported by: Spanish National Research Council (CSIC-202020E079, CSIC-COVID19-028); Madrid Regional Government "IMMUNOTHERCAN" [S2017/BMD-3733-2 (MVG)]; Spanish Ministry of Science and Innovation [(MCIU/AEI/FEDER, EU, RTI2018-093569-B-I00 (MVG), SAF2017-82940-R (JMRF), SAF2017-83265-R (HTR); SAF2017-82886-R (FSM)]; Health Institute Carlos III (ISCIII) [RETICS Program RD16/0012/0006; RIER (EMGC); PI19/00549 (AA)]; "La Caixa Bank Foundation" (HR17-00016), Fondo Supera COVID (CRUE-Banco de Santander), both to FSM. ; Peer reviewed

Many new aspects of COVID-19 disease, including different clinical manifestations, have been identified during the pandemic. The wide array of symptoms and variation in disease severity after SARS-CoV-2 infection might be related to heterogeneity in the immune responses of different patients. Here we describe a new method for a simple multi-antigen serological test that generates a full picture of seroconversion in a single reaction. The assay is based on the detection by flow cytometry of multiple immunoglobulin classes (isotypes) specific for four SARS-CoV-2 antigens: the Spike glycoprotein (one of the highly immunogenic proteins), its RBD fragment (the major target for neutralising antibodies), the nucleocapsid protein and the main cysteine-like protease. Until now, most diagnostic serological tests measured antibodies to only one antigen and some patients seemed to not make any antibody response. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. Combining all the four antigens and using machine learning techniques, it was possible to clearly discriminate between patients and healthy controls with 100% confidence. Further, combination of antigens and different immunoglobulin isotypes in this multi-antigen assay improved the classification of patients with mild and severe disease. Introduction of this method will facilitate massive screenings of patients to evaluate their immune response. It could also support vaccination campaigns both to select non-immune individuals and to distinguish infected patients from vaccine responders. ; This work was supported by the Spanish National Research Council (CSIC, project numbers 202020E079 and CSIC-COVID19-028) and grants from Madrid Regional Government "IMMUNOTHERCAN" [S2017/BMD-3733-2 (MVG)]; the Spanish Ministry of Science and Innovation [(MCIU/AEI/FEDER, EU): RTI2018-093569-B-I00 (MVG), SAF2017-82940-R (JMRF), SAF2017-83265-R (HTR); SAF2017-82886-R (FSM)]; Health Institute Carlos III (ISCIII) [RETICS Program RD16/0012/0006; RIER (JMRF); PI19/00549 (AA)]. The study was also funded by "La Caixa Banking Foundation" (HR17-00016 to FSM) and Fondo Supera COVID (CRUE-Banco de Santander) to FSM. ; No