Image-based and machine learning-guided multiplexed serology test for SARS-CoV-2

Here, we describe a scalable and automated, high-content microscopy -based mini-immunofluorescence assay (mini-IFA) for serological testing i.e., detection of antibodies. Unlike conventional IFA, which often relies on the use of cells infected with the target pathogen, our assay employs transfected cells expressing individual viral antigens. The assay builds on a custom neural network-based image analysis pipeline for the automated and multiplexed detection of immunoglobulins (IgG, IgA, and IgM) in patient samples. As a proof-of-concept, we employed high-throughput equipment to set up the assay for measuring antibody response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection with spike (S), membrane (M), and nucleo (N) proteins, and the receptor-binding domain (R) as the antigens. We compared the automated mini-IFA results from hundreds of patient samples to the visual observations of human experts and to the results obtained with conventional ELISA. The comparisons demonstrated a high correlation to both, suggesting high sensitivity and specificity of the mini-IFA. By testing pre-pandemic samples and those collected from patients with RT-PCR confirmed SARS-CoV-2 infection, we found mini-IFA to be most suitable for IgG and IgA detection. The results demonstrated N and S proteins as the ideal antigens, and the use of these antigens can serve to distinguish between vaccinated and infected individuals. The assay principle described enables detection of antibodies against practically any pathogen, and none of the assay steps require high biosafety level environment. The simultaneous detection of multiple Ig classes allows for distinguishing between recent and past infection. Public abstractThe manuscript describes a miniaturized immunofluorescence assay (mini-IFA) for measuring antibody response in patient blood samples. The automated method builds on machine-learning -guided image analysis with SARS-CoV-2 as the model pathogen. The method enables simultaneous measurement of IgM, IgA, and IgG responses against different virus antigens in a high throughput manner. The assay relies on antigens expressed through transfection and allows for differentiation between vaccine-induced and infection-induced antibody responses. The transfection-based antigen expression enables performing the assay at a low biosafety level laboratory and allows fast adaptation of the assay to emerging pathogens. Our results provide proof-of-concept for the approach, demonstrating fast and accurate measurement of antibody responses in a clinical and research set-up.

A highly sensitive and specific SARS-CoV-2 spike- and nucleoprotein-based fluorescent multiplex immunoassay (FMIA) to measure IgG, IgA and IgM class antibodies

BackgroundValidation and standardization of accurate serological assays are crucial for the surveillance of the coronavirus disease 2019 (COVID-19) pandemic and population immunity. MethodsWe describe the analytical and clinical performance of an in-house fluorescent multiplex immunoassay (FMIA) for simultaneous quantification of antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleoprotein and spike glycoprotein. Furthermore, we calibrated IgG-FMIA against World Health Organisation (WHO) International Standard and compared FMIA results to an in-house enzyme immunoassay (EIA) and a microneutralisation test (MNT). We also compared the MNT results of two laboratories. ResultsIgG-FMIA displayed 100% specificity and sensitivity for samples collected 13-150 days post-onset of symptoms (DPO). For IgA- and IgM-FMIA 100% specificity and sensitivity were obtained for a shorter time window (13-36 and 13-28 DPO for IgA- and IgM-FMIA, respectively). FMIA and EIA results displayed moderate to strong correlation, but FMIA was overall more specific and sensitive. IgG-FMIA identified 100% of samples with neutralising antibodies (NAbs). Anti-spike IgG concentrations correlated strongly ({rho}=0.77-0.84, P<2.2x10-16) with NAb titers. The NAb titers of the two laboratories displayed a very strong correlation ({rho}=0.95, P<2.2x10-16). DiscussionOur results indicate good correlation and concordance of antibody concentrations measured with different types of in-house SARS-CoV-2 antibody assays. Calibration against WHO international standard did not, however, improve the comparability of FMIA and EIA results.

Synergistic interferon alpha-based drug combinations inhibit SARS-CoV-2 and other viral infections in vitro

There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. In the present study, we found that recombinant human interferon-alpha (IFNa) triggers intrinsic and extrinsic cellular antiviral responses, as well as reduces replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. Although IFNa alone was insufficient to completely abolish SARS-CoV-2 replication, combinations of IFNa with remdesivir or other antiviral agents (EIDD-2801, camostat, cycloheximide, or convalescent serum) showed strong synergy and effectively inhibited SARS-CoV-2 infection in human lung epithelial Calu-3 cells. Furthermore, we showed that the IFNa-remdesivir combination suppressed virus replication in human lung organoids, and that its single prophylactic dose attenuated SARS-CoV-2 infection in lungs of Syrian hamsters. Transcriptome and metabolomic analyses showed that the combination of IFNa-remdesivir suppressed virus-mediated changes in infected cells, although it affected the homeostasis of uninfected cells. We also demonstrated synergistic antiviral activity of IFNa2a-based combinations against other virus infections in vitro. Altogether, our results indicate that IFNa2a-based combination therapies can achieve higher efficacy while requiring lower dosage compared to monotherapies, making them attractive targets for further pre-clinical and clinical development.

Rapid homogeneous assay for detecting antibodies against SARS-CoV-2

Accurate and rapid diagnostic tools are needed for management of the ongoing coronavirus disease 2019 (COVID-19) pandemic. Antibody tests enable detection of individuals past the initial phase of infection and will help to examine possible vaccine responses. The major targets of human antibody response in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the spike glycoprotein (S) and nucleocapsid protein (N). We have developed a rapid homogenous approach for antibody detection termed LFRET (protein L-based time-resolved Forster resonance energy transfer immunoassay). In LFRET, fluorophore-labeled protein L and antigen are brought to close proximity by antigen-specific patient immunoglobulins of any isotype, resulting in TR-FRET signal generation. We set up LFRET assays for antibodies against S and N and evaluated their diagnostic performance using a panel of 77 serum/plasma samples from 44 individuals with COVID-19 and 52 negative controls. Moreover, using a previously described S construct and a novel N construct, we set up enzyme linked immunosorbent assays (ELISAs) for antibodies against SARS-CoV-2 S and N. We then compared the LFRET assays with these enzyme immunoassays and with a SARS-CoV-2 microneutralization test (MNT). We found the LFRET assays to parallel ELISAs in sensitivity (90-95% vs. 90-100%) and specificity (100% vs. 94-100%). In identifying individuals with or without a detectable neutralizing antibody response, LFRET outperformed ELISA in specificity (91-96% vs. 82-87%), while demonstrating an equal sensitivity (98%). In conclusion, this study demonstrates the applicability of LFRET, a 10-minute mix and read assay, to detection of SARS-CoV-2 antibodies.

Effects of environmental factors on severity and mortality of COVID-19

BackgroundMost respiratory viruses show pronounced seasonality, but for SARS-CoV-2 this still needs to be documented. MethodsWe examined the disease progression of COVID-19 in 6,914 patients admitted to hospitals in Europe and China. In addition, we evaluated progress of disease symptoms in 37,187 individuals reporting symptoms into the COVID Symptom Study application. FindingsMeta-analysis of the mortality risk in eight European hospitals estimated odds ratios per one day increase in the admission date to be 0.981 (0.973-0.988, p<0.001) and per increase in ambient temperature of one degree Celsius to be 0.854 (0.773-0.944, p=0.007). Statistically significant decreases of comparable magnitude in median hospital stay, probability of transfer to Intensive Care Unit and need for mechanical ventilation were also observed in most, but not all hospitals. The analysis of individually reported symptoms of 37,187 individuals in the UK also showed the decrease in symptom duration and disease severity with time. InterpretationSeverity of COVID-19 in Europe decreased significantly between March and May and the seasonality of COVID-19 is the most likely explanation. Mucosal barrier and mucociliary clearance can significantly decrease viral load and disease progression, and their inactivation by low relative humidity of indoor air might significantly contribute to severity of the disease.

Systems-level immunomonitoring from acute to recovery phase of severe COVID-19

The immune response to SARS-CoV2 is under intense investigation, but not fully understood att this moment. Severe disease is characterized by vigorous inflammatory responses in the lung, often with a sudden onset after 5-7 days of stable disease. Efforts to modulate this hyperinflammation and the associated acute respiratory distress syndrome, rely on the unraveling of the immune cell interactions and cytokines that drive such responses. Systems-level analyses are required to simultaneously capture all immune cell populations and the many protein mediators by which cells communicate. Since every patient analyzed will be captured at different stages of his or her infection, longitudinal monitoring of the immune response is critical. Here we report on a systems-level blood immunomonitoring study of 39 adult patients, hospitalized with severe COVID-19 and followed with up to 14 blood samples from acute to recovery phases of the disease. We describe an IFN{gamma} - Eosinophil axis activated prior to lung hyperinflammation and changes in cell-cell coregulation during different stages of the disease. We also map an immune trajectory during recovery that is shared among patients with severe COVID-19. HIGHLIGHTSSystems-level immunomonitoring from acute to recovery in severe COVID-19 An IFN{gamma} - Eosinophil axis involved in lung hyperinflammation Cell-cell coregulation differ during four disease stages Basophils and hyperinflammation modulate humoral responses A shared trajectory of immunological recovery in severe COVID-19