Rapid response flow cytometric assay for the detection of antibody responses to SARS-CoV-2

Lapuente, Dennis; Maier, Clara; Irrgang, Pascal; Hübner, Julian; Peter, Sophia Katharina; Hoffmann, Markus; Ensser, Armin; Ziegler, Katharina; Winkler, Thomas; Birkholz, Torsten; Kremer, Andreas E.; Steininger, Philipp; Korn, Klaus; Neipel, Frank; Überla, Klaus; Tenbusch, Matthias

doi:10.1101/2020.05.09.20091447

Cited by 23 publications

(26 citation statements)

References 17 publications

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“…Antibodies against S also decreased over time in these plasma samples, with the decrease being significant ~74 days post-symptom onset onwards (Figure 2A). This finding was corroborated using a previously characterized flow-cytometry method to quantify SARS-CoV-2 Spike-specific antibodies using 293T cells transiently transfected with a plasmid encoding the full-length Spike 10,11,[19][20][21] (Figure 2B), and the MFI obtained from both these methods correlated significantly (r = 0.9207, p<0.0001) ( Figure 2C). Results obtained with both flow cytometry assays, using transduced or transfected 293T cells, also positively correlated with the levels of RBD-specific antibodies as quantified by ELISA in the recently published study using the same cohort 18 (Figure 2C).…”

supporting

confidence: 74%

High-throughput detection of antibodies targeting the SARS-CoV-2 Spike in longitudinal convalescent plasma samples

Anand

Prévost

Richard

et al. 2020

Preprint

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Background: The SARS-CoV-2 virus is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic, infecting millions of people and causing more than a million deaths. The SARS-CoV-2 Spike glycoproteins mediate viral entry and represent the main target for antibody responses. Humoral responses were shown to be important for preventing and controlling infection by coronaviruses. A promising approach to reduce the severity of COVID-19 is the transfusion of convalescent plasma. However, longitudinal studies revealed that the level of antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 Spike declines rapidly after the resolution of the infection. Study Design and Methods: To extend this observation beyond the RBD domain, we performed a longitudinal analysis of the persistence of antibodies targeting the full-length SARS-CoV-2 Spike in the plasma from 15 convalescent donors. We generated a 293T cell line constitutively expressing the SARS-CoV-2 Spike and used it to develop a high-throughput flow cytometry-based assay to detect SARS-CoV-2 Spike specific antibodies in the plasma of convalescent donors. Results and Conclusion: We found that the level of antibodies targeting the full-length SARS-CoV-2 Spike declines gradually after the resolution of the infection. This decline was not related to the number of donations, but strongly correlated with the decline of RBD-specific antibodies and the number of days post-symptom onset. These findings help to better understand the decline of humoral responses against the SARS-CoV-2 Spike and provide important information on when to collect plasma after recovery from active infection for convalescent plasma transfusion.

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supporting

confidence: 74%

High-throughput detection of antibodies targeting the SARS-CoV-2 Spike in longitudinal convalescent plasma samples

Anand

Prévost

Richard

et al. 2020

Preprint

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“…IgG-and IgM-antibodies against the SARS-CoV-2-Spike protein were first determined by a flow cytometric antibody assay measuring antibodies against human embryonic kidney cells (HEK 293T cells) transfected with either 30µg pCG1_CoV_2019-S plus 15 µg fluorescent protein (BFP) or with the control plasmid 30µg pcDNA3.1 and 15µg fluorescent protein (dsRed) as described (18). In addition, a subgroup of patients was tested with commercially available assays such as the iFlash-SARS-CoV-2 CLIA (Yhlo Biotech Co., Shenzhen, China) or the SARS-CoV-2-ELISA (Euroimmun, Lübeck, Germany).…”

Section: Measurement Of Sars-cov-2-antibodiesmentioning

confidence: 99%

SARS-CoV-2-Seronegative Subjects Target CTL Epitopes in the SARS-CoV-2 Nucleoprotein Cross-Reactive to Common Cold Coronaviruses

et al. 2021

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The beta-coronavirus SARS-CoV-2 induces severe disease (COVID-19) mainly in elderly persons with risk factors, whereas the majority of patients experience a mild course of infection. As the circulating common cold coronaviruses OC43 and HKU1 share some homologous sequences with SARS-CoV-2, beta-coronavirus cross-reactive T-cell responses could influence the susceptibility to SARS-CoV-2 infection and the course of COVID-19. To investigate the role of beta-coronavirus cross-reactive T-cells, we analyzed the T-cell response against a 15 amino acid long peptide (SCoV-DP15: DLSPRWYFYYLGTGP) from the SARS-CoV-2 nucleoprotein sequence with a high homology to the corresponding sequence (QLLPRWYFYYLGTGP) in OC43 and HKU1. SCoV-DP15-specific T-cells were detected in 4 out of 23 (17.4%) SARS-CoV-2-seronegative healthy donors. As HIV-1 infection is a potential risk factor for COVID-19, we also studied a cohort of HIV-1-infected patients on antiretroviral therapy. 44 out of these 116 HIV-1-infected patients (37.9%) showed a specific recognition of the SCoV-DP15 peptide or of shorter peptides within SCoV-DP15 by CD4+ T-cells and/or by CD8+ T-cells. We could define several new cross-reactive HLA-I-restricted epitopes in the SARS-CoV-2 nucleoprotein such as SPRWYFYYL (HLA-B*07, HLA-B*35), DLSPRWYFYY (HLA-A*02), LSPRWYFYY (HLA-A*29), WYFYYLGTGP and WYFYYLGT. Epitope specific CD8+ T-cell lines recognized corresponding epitopes within OC43 and HKU1 to a similar degree or even at lower peptide concentrations suggesting that they were induced by infection with OC43 or HKU1. Our results confirm that SARS-CoV-2-seronegative subjects can target SARS-CoV-2 not only by beta-coronavirus cross-reactive CD4+ T-cells but also by cross-reactive CD8+ cytotoxic T-cells (CTL). The delineation of cross-reactive T-cell epitopes contributes to an efficient epitope-specific immunomonitoring of SARS-CoV-2-specific T-cells. Further prospective studies are needed to prove a protective role of cross-reactive T-cells and their restricting HLA alleles for control of SARS-CoV-2 infection. The frequent observation of SARS-CoV-2-reactive T-cells in HIV-1-infected subjects could be a reason that treated HIV-1 infection does not seem to be a strong risk factor for the development of severe COVID-19.

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“…In our flow cytometric assay 54 , spike-specific IgG, IgG1, and IgG2a could be easily detected in serum and BAL of animals treated with the prime-boost strategies, while antibodies in the BAL after a single dose of Ad19a or Ad5 were almost absent (Fig. 1 B-D).…”

Section: A Systemic Dna Prime Significantly Increases the Mucosal Immunogenicity Of An Intranasal Adenoviral Vector Vaccinementioning

confidence: 74%

Protective mucosal immunity against SARS-CoV-2 after heterologous systemic RNA-mucosal adenoviral vector immunization

Lapuente

Fuchs

Willar

et al. 2021

Preprint

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Several effective SARS-CoV-2 vaccines are currently in use, but in the light of waning immunity and the emergence of novel variants, effective boost modalities are needed in order to maintain or even increase immunity. Here we report that intranasal vaccinations with adenovirus 5 and 19a vectored vaccines following a systemic DNA or mRNA priming result in strong systemic and mucosal immunity in mice. In contrast to two intramuscular injections with an mRNA vaccine, the mucosal boost with adenoviral vectors induced high levels of IgA and tissue-resident memory T cells in the respiratory tract. Mucosal neutralization of virus variants of concern was also enhanced by the intranasal boosts. Importantly, priming with mRNA provoked a more comprehensive T cell response consisting of circulating and tissue-resident memory T cells after the boost, while a DNA priming induced mostly mucosal T cells. Concomitantly, the intranasal boost strategies provided protection against symptomatic disease. Therefore, a mucosal booster immunization after mRNA priming is a promising approach to establish mucosal immunity in addition to systemic responses.

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Rapid response flow cytometric assay for the detection of antibody responses to SARS-CoV-2

Cited by 23 publications

References 17 publications

High-throughput detection of antibodies targeting the SARS-CoV-2 Spike in longitudinal convalescent plasma samples

High-throughput detection of antibodies targeting the SARS-CoV-2 Spike in longitudinal convalescent plasma samples

SARS-CoV-2-Seronegative Subjects Target CTL Epitopes in the SARS-CoV-2 Nucleoprotein Cross-Reactive to Common Cold Coronaviruses

Protective mucosal immunity against SARS-CoV-2 after heterologous systemic RNA-mucosal adenoviral vector immunization

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