Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS‐CoV‐2 spike protein
2021; Springer Nature; Volume: 13; Issue: 3 Linguagem: Inglês
10.15252/emmm.202013549
ISSN1757-4684
AutoresLydia Horndler, Pilar Delgado, David Abia, Ivaylo Balabanov, Pedro Martínez‐Fleta, Georgina H. Cornish, Miguel Ángel Llamas, Sergio Serrano‐Villar, Francisco Sánchez‐Madrid, Manuel Fresno, Hisse M. van Santen, Balbino Alarcón,
Tópico(s)Single-cell and spatial transcriptomics
ResumoArticle17 February 2021Open Access Transparent process Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS-CoV-2 spike protein Lydia Horndler Lydia Horndler Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Pilar Delgado Pilar Delgado Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author David Abia David Abia Bioinformatics Facility, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Ivaylo Balabanov Ivaylo Balabanov Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Pedro Martínez-Fleta Pedro Martínez-Fleta Immunology Department, Hospital Universitario La Princesa, HS-IP, Madrid, Spain Search for more papers by this author Georgina Cornish Georgina Cornish The Francis Crick Institute, London, UK Search for more papers by this author Miguel A Llamas Miguel A Llamas orcid.org/0000-0002-0075-9020 EMPIREO Diagnóstico Molecular SL, Madrid, Spain Search for more papers by this author Sergio Serrano-Villar Sergio Serrano-Villar orcid.org/0000-0002-5447-3554 Hospital Universitario Ramón y Cajal, Universidad de Alcalá, IRYCIS, Madrid, Spain Search for more papers by this author Francisco Sánchez-Madrid Francisco Sánchez-Madrid Immunology Department, Hospital Universitario La Princesa, HS-IP, Madrid, Spain Search for more papers by this author Manuel Fresno Manuel Fresno Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Hisse M van Santen Hisse M van Santen orcid.org/0000-0003-0769-4511 Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Balbino Alarcón Corresponding Author Balbino Alarcón [email protected] orcid.org/0000-0001-7820-1070 Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Lydia Horndler Lydia Horndler Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Pilar Delgado Pilar Delgado Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author David Abia David Abia Bioinformatics Facility, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Ivaylo Balabanov Ivaylo Balabanov Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Pedro Martínez-Fleta Pedro Martínez-Fleta Immunology Department, Hospital Universitario La Princesa, HS-IP, Madrid, Spain Search for more papers by this author Georgina Cornish Georgina Cornish The Francis Crick Institute, London, UK Search for more papers by this author Miguel A Llamas Miguel A Llamas orcid.org/0000-0002-0075-9020 EMPIREO Diagnóstico Molecular SL, Madrid, Spain Search for more papers by this author Sergio Serrano-Villar Sergio Serrano-Villar orcid.org/0000-0002-5447-3554 Hospital Universitario Ramón y Cajal, Universidad de Alcalá, IRYCIS, Madrid, Spain Search for more papers by this author Francisco Sánchez-Madrid Francisco Sánchez-Madrid Immunology Department, Hospital Universitario La Princesa, HS-IP, Madrid, Spain Search for more papers by this author Manuel Fresno Manuel Fresno Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Hisse M van Santen Hisse M van Santen orcid.org/0000-0003-0769-4511 Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Balbino Alarcón Corresponding Author Balbino Alarcón [email protected] orcid.org/0000-0001-7820-1070 Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain Search for more papers by this author Author Information Lydia Horndler1, Pilar Delgado1, David Abia2, Ivaylo Balabanov1, Pedro Martínez-Fleta3, Georgina Cornish4, Miguel A Llamas5, Sergio Serrano-Villar6, Francisco Sánchez-Madrid3, Manuel Fresno1, Hisse M van Santen1 and Balbino Alarcón *,1 1Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain 2Bioinformatics Facility, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas (CSIC), Universidad Autónoma de Madrid, Madrid, Spain 3Immunology Department, Hospital Universitario La Princesa, HS-IP, Madrid, Spain 4The Francis Crick Institute, London, UK 5EMPIREO Diagnóstico Molecular SL, Madrid, Spain 6Hospital Universitario Ramón y Cajal, Universidad de Alcalá, IRYCIS, Madrid, Spain *Corresponding author. Tel: +34 911964555; Fax: +34 911964420; E-mail: [email protected] EMBO Mol Med (2021)13:e13549https://doi.org/10.15252/emmm.202013549 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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 infection. Synopsis This study shows the development of a new method for the classification of human blood donors according to the presence of anti-Spike (S) antibodies of SARS-CoV-2 that bind to the native S protein expressed on the surface of the human Jurkat human T cell line. The flow cytometry method provides quantitative data over a wide range of fluorescence intensities. The co-expression of the hEGFRt marker allows to set a clear positive/negative threshold according to the slope of fluorescence intensities. The method is more sensitive and reliable than serological methods based on the expression of recombinant proteins. The method correlates better with the presence of protective neutralizing antibodies than ELISA methods. The use of the method allows to conclude that the humoral response anti-SARS-CoV-2 is long-lasting and that the percentage of the population already immune is higher than suspected. The paper explained Problem Current serological tests for detection of antibodies to SARS-CoV-2 are based on the use of recombinant fragments of proteins, including the spike S protein, that do not reproduce the native form of the protein in the context of the full virus or infected cells. This may lead to missing important antibodies that recognize conformational epitopes present in the native forms. Results By expressing in a stable manner the S protein of SARS-CoV-2 and a EGFR reporter construct on the surface of the human T lymphoblastic leukemia cell line Jurkat, we have created a flow cytometry-based method of detection of antibodies against the S protein that results more sensitive, more specific, and more relevant for the detection of functional neutralizing antibodies than those using recombinant proteins. Impact The method will allow to assess in a precise manner the degree of humoral immunity in human populations before and after vaccination, allowing to evaluate how close we are to the sought herd immunity. Introduction Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the global pandemic COVID-191. Phylogenetic analysis of the full genome classifies SARS-CoV-2 as a Betacoronavirus subgenus Sarbecovirus, lineage B and is related to bat isolates of SARS-CoV and is 79% identical to the SARS virus causing a viral epidemic in 2002 (Lu et al, 2020). Like other coronaviruses, SARS-CoV-2 encodes for 16 non-structural proteins at the 5′ end of the genomic RNA and structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N) at the 3′ end (Su et al, 2016). The spike S protein is responsible for binding to ACE2 in host cells which seems to be the main cellular receptor for the virus (Hoffmann et al, 2020). In native state, the S protein forms homotrimers and is composed of two fragments S1 and S2 that result from proteolytic cleavage of S upon ACE2 binding (Walls et al, 2020). The S1 fragment contains a central RBD sequence that is the actual ACE2-binding sequence and the target of neutralizing antibodies such as those described to neutralize SARS-CoV-1. Diagnosis of active infection is currently carried out by PCR amplification of viral RNA comprising fragments of the N, E, S, and RdRP genes from biological samples taken from bronchoalveolar lavage (BAL), sputum, nasal swabs, pharyngeal swabs, and fibronchoscope brush biopsies, with the highest positive rate resulting from PCR of BAL samples (Venter & Richter, 2020). Positivity in the PCR test vanishes as the infection is resolved although viral RNA shedding and PCR positivity could take place even after there is no further production of infective virions and therefore possibility of transmission. Unlike PCR-based tests, serological tests are not highly valuable to determine which individual has an active infection with SARS-CoV-2 but are key in epidemiology and Public Health policies since it can provide an estimate of what segment of a population has been infected with the virus and is likely to have acquired total or partial immunity against ongoing and future resurgences of the pandemics. Herd immunity achieved either by natural infection or as a consequence of vaccination is a goal for all health authorities in the world. Seropositivity is usually established by detecting the presence of viral antigen-specific IgG or IgM in the serum of individuals using recombinant fragments of the S or N proteins and tests based on ELISA or lateral flow assay (Venter & Richter, 2020; Weissleder et al, 2020). A disadvantage of those tests is that neutralizing antibodies are not directed against the N protein and that recombinant fragments of S miss the quaternary structure of the S protein trimer, which is the native form of the spike protein in the viral envelope. Therefore, part of the neutralizing antibodies directed against the native S trimer could be missed in serological tests based on the expression of recombinant proteins. Flow cytometry of cells that are transfected with vectors that express the S protein represents an attractive alternative strategy for serological tests. This strategy allows detection of antibodies against the native form of the S protein. Such system has been used by transient transfection of HEK293T human cells to analyze different cohorts of COVID-19 patients (Grzelak et al, 2020). We have developed here a flow cytometry serological test using stably transfected Jurkat, a human leukemic T-cell line, that co-expresses the native S protein of SARS-CoV-2 and a truncated form of the human EGFR that serves as a normalizer. The Jurkat-S system is amenable to standardization and can be used for multiplexed detection of human immunoglobulins of all isotypes in a single assay. Finally, we show that the system is superior to ELISA-based methods to detect sera of donors containing neutralizing antibodies. Results We used a lentiviral vector to express the full spike "S" protein of SARS-CoV-2 followed by a truncated human EGFR protein (huEGFRt) linked by a T2A self-cleaving sequence in transduced cells (Fig 1A). This system allows expression of the two proteins from a monocistronic mRNA. We produced transducing supernatants to express the construct in the human leukemic cell line Jurkat. Taking advantage of the fact that after cleavage the T2A sequence remains attached to the N-terminal protein, we studied whether the S protein was expressed at the cell surface of the transduced Jurkat cells (from now on, Jurkat-S cells). To this end, cell surface membrane proteins of Jurkat-S cells were surface biotinylated followed by immunoprecipitation with anti-T2A, SDS–PAGE, and Western blot with streptavidin–peroxidase. This showed the presence of a polypeptide of ~ 120 kD when resolved by SDS–PAGE under reducing conditions in Jurkat-S cells but not in the parental cells (Fig 1B). Interestingly, larger sizes of the immune-reactive polypeptide were resolved in the absence of reducing agents under non-denaturing conditions (i.e., without boiling; Fig 1C). These data suggest that the S protein of SARS-CoV-2 could be expressed on Jurkat-S plasma membrane as native homotrimers. To determine whether the S protein expressed on Jurkat-S cells was functional, we analyzed if it could promote the formation of syncytia when Jurkat-S cells were co-incubated with the ACE2-expressing human hepatocarcinoma cell line HepG2. We labeled Jurkat-S cells with the green dye CFSE and HepG2 with the far red dye Cell Trace Far Red (CTFR). After overnight incubation, we detected a Jurkat-S dose-dependent formation of mixed cells that were not detected when HepG2 were incubated with parental Jurkat cells not expressing the S protein (Fig 1D). To determine whether the mixed cells labeled with CFSE and CTFR were syncytia and not just cell doublets, the dual color cell population was sorted and seeded on coverslips for confocal microscopy analysis. The sorted population was formed by large cells with multiple nuclei that were stained with CFSE and CTFR, demonstrating that they were syncytia formed by fusion of Jurkat-S and HepG2 cells (Fig 1E). These data indicated that the S protein expressed in Jurkat-S cells has fusogenic activity and therefore that it must be in a native conformation. Figure 1. Jurkat-S cells expressing native SARS-CoV-2 spike S protein allow the detection of anti-S protein antibodies in human sera Lentiviral construct used to permanently express S protein in Jurkat. The full-length mature S protein is preceded by a leader sequence of GM-CSF and followed by a T2A sequence which is followed by a tail-less truncated human EGFR construct. Expression of S protein on the plasma membrane of Jurkat-S cells assessed by surface biotinylation and followed by immunoprecipitation with anti-T2A, SDS–PAGE under reducing conditions, and Western blot with streptavidin–peroxidase. Biotinylation of the parental Jurkat cells was carried out in parallel as a negative control. The arrow indicates the position of the reduced S protein. Expression of the S protein in native form was assessed by surface biotinylation of Jurkat-S and Jurkat cells followed by immunoprecipitation with anti-T2A followed by SDS–PAGE under non-denaturing conditions (i.e., without reducing agents and without boiling). The nitrocellulose membrane was blotted with streptavidin–peroxidase. Arrows point at different oligomerization forms of the S protein. Formation of syncytia between the CTFR-labeled ACE2+ human cell line and the CFSE-labeled Jurkat-S cells was measured by flow cytometry by analyzing the percentage of cells that become double positive for CTFR and CFSE markers. The bar plot to the right shows the effect of different doses of HepG2 cells on the formation of syncytia with a fixed number of Jurkat. Parental Jurkat cells (not expressing S protein) are considered negative controls. Data represent the mean ± SD of triplicated datasets. Formation of syncytia between the CTFR-labeled ACE2+ human cell line and the CFSE-labeled Jurkat-S cells, in an experiment as in (D), was confirmed by confocal microscopy after sorting cells double positive for CTFR and CFSE. Nuclei were identified by DAPI staining. Red arrowhead in the DAPI image indicates a nucleus of Hep-G2 origin; the green arrowhead the nucleus of Jurkat-S origin. Overlay plot of Jurkat-S cells that were incubated with anti-EGFR mAb conjugated to Bv421 and serum from either donor #15 or from a pre-COVID-19 donor and followed by a secondary anti-human IgG1 antibody conjugated to PE. Download figure Download PowerPoint The Jurkat-S cells were analyzed using a flow cytometry (FC) assay, where staining with an anti-EGFR monoclonal antibody detects and quantitates expression of the huEGFRt construct alongside detection and quantitation of anti-S protein antibodies within sera from SARS-CoV-2-infected blood donors. Figure 1F shows Jurkat-S cells stained with anti-EGFR mAb and either a serum sample taken from an individual before the COVID-19 pandemics (pre-COVID-1 serum) or serum taken from an asymptomatic donor (donor #15) determined positive with multiple serological assays (Table EV1). Serum from donor #15 could strongly detect the S protein expressed on Jurkat-S cells, whereas pre-COVID-1 serum did not, proving this method to be valid for capture and detection of anti-spike antibodies present in sera from SARS-CoV-2-exposed individuals. This assay was highly sensitive and detected anti-S protein antibodies over a wide range of mean fluorescence intensities (MFI) when staining with sera from different blood donors (Fig 2A). In comparison with commercial serological assays, this Jurkat-S clone FC assay detected more cases of seropositive individuals (Table EV1) and facilitated verification of positive seroconversion in patient samples that had previously given uncertain results (Fig 2B, orange symbols). Figure 2. Flow cytometry of Jurkat-S cells allows to detect anti-S immunoglobulins poorly detected by commercial tests with a wide dynamic range A. Overlay histograms of Jurkat-S staining with different human sera diluted 1:50. A pre-COVID-19 serum sample is taken as negative control (gray histogram). B. Bar plots of flow cytometry data generated with Jurkat-S cells and the panel of serum samples of Table EV1 classified according to their result in the indicated commercial tests: green, positive for the test; orange, weak, or unclear; magenta, negative samples. Flow cytometry data are expressed as the ratio between the MFI of the antibody anti-S and the MFI for EGFR. Negative values for the flow cytometry test are those with a S/EGFR MFI ratio lower than 0.5. This ratio was set in order to fit most of the data negative for the other serological tests (pink triangles) under that threshold. Data represent the mean ± s.e.m. All datapoints are shown. Download figure Download PowerPoint In order to compare the sensitivity of our Jurkat-S assay with a conventional ELISA, we used recombinant Spike proteins, specifically the S1 and receptor binding domain (RBD) fragments, both kind gifts from Dr. Peter Cherepanov (CRICK, London). We coated the plates with 2 µg/ml of either S1 or RBD and incubated the coated plates with a 1:50 dilution of human sera. Binding of human IgG1 antibodies was detected using a peroxidase-labeled anti-human IgG1 monoclonal antibody. Comparing absorbance values from the ELISA with MFI values of the Jurkat-S FC assay across sera stratified by experienced COVID-19 symptoms (asymptomatic, mild, moderate, moderate–severe, and severe) showed the MFI values to be spread across a wider range of values than absorbance values (Fig 3A). Finally, the comparison of absorbance values in the two ELISA tests (anti-S1 and anti-RBD) produced a good-fitted straight line (R2 = 0.83; Fig 3B), whereas the comparison of the FC MFI with the absorbance values (against S1 and RBD) poorly adjust to a straight line (R2 = 0.55 and R2 = 0.32; Fig 3B), with samples that gave a poor signal in the ELISA giving a clear signal with the FC assay. This indicates that detecting S-specific IgG1 using the Jurkat-S FC assay increases sensitivity for detecting SARS-CoV-2-exposure in individuals testing negative by ELISA. These individuals may have generated antibodies against other fragments of the Spike, e.g. S2 (Ng et al, 2020), or against the native trimeric structure of the S protein, that are found on the surface of Jurkat-S cells but not in the ELISAs. For example, sera from donors #8, #46, #48, and #49 were apparently negative for anti-S IgG1 by ELISA but clearly positive by FC with Jurkat-S. By contrast, anti-S IgG1 was detected in sera from donors #15, #31, and #52 by both ELISA and FC, and serum from donor #58, appeared to be detected better by ELISA than by FC (Fig 3B). To determine whether those differences were maintained at different dilutions of the sera, a titration test was carried out in parallel using the FC Jurkat-S method and ELISA of the S1 fragment. The results showed that all sera, including that of donor #58, were clearly positive by FC, even at a 1:450 dilution, whereas by ELISA, sera #8, #46, #48, and #49 remained negative (Fig 3C). These results indicate that sera, which could have been considered negative by ELISA, are indeed positive for S-specific IgG1 by FC with Jurkat-S. Sample #49 was from an asymptomatic individual but sera #8, #46, and #48 were from individuals who had experienced symptoms that ranged from mild to severe (Table EV1). Figure 3. Flow cytometry of Jurkat-S cells detects anti-S antibodies in sera otherwise negative for ELISA using recombinant S1 and RBD proteins Box and whiskers plot of MFI and absorbance data in human sera according to their clinical classification of COVID-19 symptoms. Clinical classification was done according to the following parameters: Asymptomatic, no symptoms; mild, three or more of the following symptoms: non-productive cough, hyperthermia, headache, odynophagia, dyspnea, asthenia, myalgia, ageusia, anosmia, cutaneous involvement; moderate, three or more of the above symptoms plus gastrointestinal symptoms, or more than three of the above for seven or more days; and moderate–severe, three or more of the above symptoms plus pneumonia. Box and whiskers are shown to represent the minimum and maximum values as well as the median. All datapoints are shown. Comparison of MFI vs absorbance data generated by flow cytometry and ELISA for all human sera. A lineal regression curve was adjusted, with a 95% confidence interval, to all data with the R2 values indicated in the plots. Selected samples of outliers (#4, #5, #8, #31, #46, #48, #49, and #58) as well as samples close to a diagonal (#36, #52, and #15) are indicated. Titration of selected human sera by ELISA using the S1 protein and by flow cytometry with Jurkat-S cells. Samples with antibodies detected by flow cytometry and not by ELISA are indicated (#8, #46, #48, #49). Data represent the mean ± SD of duplicates. An unpaired two-tailed t-test was carried out to compare the results of 1:50, 1:150, and 1:450 dilutions of sera #8, #46, #48 and #49 by FC to the pre-COVID-1 sample as a negative control. *P < 0.05; **P < 0.005; ***P < 0.0005. Download figure Download PowerPoint To determine which of the two methods, ELISA or FC, was producing a more reliable picture of the immune status of the serum donors, we assayed sera testing positive by FC and negative by ELISA for the capacity to neutralize S protein function. We generated pseudotyped lentiviral reporter particles coated either with the S protein of SARS-CoV-2 or, as a control, with the G protein of VSV virus (Fig 4A). To test the functionality of the viral particles, they were used to transduce HEK293T cells stably transfected with ACE2 in the presence of different dilutions of serum from donor #66, that was identified as positive by the FC method (Table EV1), o from the pre-COVID-1 donor. Serum #66 neutralized the entry of the S protein-pseudotyped lentivirus in a dose-dependent manner, whereas the pre-COVID-1 serum did not (Fig 4B). The effect of the #66 serum was due to specifically neutralizing the S protein since this serum did not inhibit transduction of ACE2+ HEK293T cells by lentivirus pseudotyped with VSV G protein (Fig 4B). We then interrogated whether sera from donors #46 and #48, testing positive by FC but not by ELISA (Fig 3C), were also able to neutralize the S protein-pseudotyped lentivirus. They did inhibit (Fig 4C), suggesting that these serum samples contain neutralizing antibodies and therefore that the Jurkat-S FC assay can be superior to ELISA for detecting protective immunity to SARS-CoV-2. Figure 4. Sera not detected by ELISA but detected by flow cytometry with Jurkat-S contain neutralizing antibodies Cartoon of the strategy for generation of pseudotyped lentiviruses. HEK-293T cells were transfected with a lentiviral construct containing the EGFP marker gene and two more plasmids encoding for the gag and pol genes of HIV-1 and for the S protein of SARS-CoV-2. Alternatively, the latter plasmid was replaced by another one encoding the G protein of VSV. The cell supernatants collected after 48 h of transfection were used to transduce HEK-293T cells stably transfected with human ACE2. Validation of the neutralization method was carried out with cell HEK-293T cell culture supernatants containing lentiviral particles pseudotyped with either the S protein or with the VSV G protein. The supernatants were mixed with sera from donor #66 at the indicated dilutions or from a pre-COVID sample as a control before addition onto HEK-293T-ACE2+ cell cultures. No serum refers to a control containing no human sera. Data represent the mean ± SD of triplicates. The presence of neutralizing antibodies in sera from donors #46 and #48 found negative by ELISA and positive by flow cytometry (Fig 3C) was demonstrated as in panel B. Data represent the mean ± SD of triplicates. *P < 0.05; **P < 0.005; ****P < 0.00005 (paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample). Bar plot showing the S/EGFR MFI ratio determined by flow cytometry analysis with Jurkat-S of serum samples from 30 sanitary personnel repeatedly tested as PCR negative for SARS-CoV-2 at the Ramón y Cajal Hospital of Madrid. A negative result is considered for a ratio lower than 0.5. Two clear cases of sera positive for anti-S IgG1 are indicated (RyC52 and RyC65). A borderline sample just above the threshold line (RyC58) is also indicated. Data show the mean ± s.e.m. All datapoints are shown. Two-color dot plot overlay of anti-S protein fluorescence vs EGFR fluorescence for borderline serum sample RyC58 (blue) and the clearly negative RyC70 sample (red). The line plot to the right shows the MFI for EGFR taken at the three sectors indicated in the two-color plot and the corresponding MFI values for EGFR. Plot of the S/EGFR MFI ratio for the 30 RyC samples vs a Score generated according to the slope of S/EGFR and shape (see Materials and Methods and Fig EV1). The green line indicates the threshold point (0.024) for the Score above which a serum is considered positive. The presence of neutralizing antibodies in sera from donors RyC46, RyC52, RyC56, and RyC58 diluted 1/15 was demonstrated as in panel B. Data represent the mean ± SD of triplicates. *P < 0.05; **P < 0.005; ****P < 0.00005 (unpaired two-tailed t-test comparing each serum to the no serum sample). Download figure Download PowerPoint To further determine the capacity of the FC Jurkat-S method to determine seropositivity in samples scored as negative by other methods, we tested serum samples collected between March and May 2020, from 30 healthcare workers (Hospital Ramón y Cajal, Madrid) that were repeatedly tested by PCR and ELISA for SARS-CoV-2 and determined to be negative in both tests (Table EV2). These samples were re-screened using the Jurkat-S FC assay and two sera (RyC52 and RyC65) resulted clearly positive for S-specific IgG1 (Fig 4D). To increase the sensitivity for detecting S-specific IgG1 in sera giving S/EGFR MFI ratios close to the 0.5 threshold for positivity, we plotted the EGFR MFI vs S protein MFI (Fig 4E) such as it was done in Fig 1F. S protein expression in Jurkat-S is coupled to huEGFRt expression (Fig 1A), thus positive s
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