High-throughput detection of neutralizing antibodies to SARS-CoV-2 variants using flow cytometry (preprint)

Detecting neutralizing antibodies (NAbs) to SARS-CoV-2 variants is crucial for controlling the spread of COVID-19. In this work, we developed a high-throughput assay for the broad systematic examination of NAbs to eleven SARS-CoV variants of concern (VOCs), which include D614G, Alpha, Beta, Gamma, Delta, Kappa, and Omicron sub-lineages BA.1, BA.2, BA.3, BA.4, and BA.5. The assay is cost-effective, reliable, 35-fold more sensitive than Luminex technology, and can include the new variants during SARS-CoV-2 evolution. Importantly, our results highly correlated with a commercial IgG serological assay (R = 0.89) and cPass, a U.S. FDA-approved surrogate virus neutralization test (sVNT) assay (R = 0.93). With our high-throughput NAb platform, we constructed a comprehensive overview of the interactions between SARS-CoV-2 VOCs' Spike trimer proteins and ACE2 receptors, leading to the identification of a monoclonal Ab with broad neutralizing activity. Furthermore, when compared to the D614G variant, we found that the serum NAbs elicited by the third dose vaccine (administered after 28 days) demonstrated decreased inhibition to multiple SARS-CoV-2 variants, including Gamma (0.94x), Alpha (0.91x), Delta (0.91x), Beta (0.81x), Kappa (0.81x), BA.2 (0.44x), BA.1 (0.43x), BA.3 (0.41x), BA.5 (0.35x) and BA.4 (0.33x), in cohort of 56 vaccinated individuals. Altogether, our proteomics platform proves to be an effective tool to detect broad NAbs in the population and aid in the development of future COVID-19 vaccines and vaccination strategies.

IP-10 and MCP-1 as biomarkers associated with disease severity of COVID-19 (preprint)

Background: COVID-19 is a viral respiratory disease caused by the severe acute respiratory syndrome-Coronavirus type 2 (SARS-CoV-2). Patients with this disease may be more prone to venous or arterial thrombosis because of the activation of many factors involved in it, including inflammation, platelet activation and endothelial dysfunction. Interferon gamma inducible protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein 1-alpha (MIP1α) are cytokines related to thrombosis. Therefore, this study focused on these three indicators in COVID-19, with the hope to find biomarkers that are associated with patients’ outcome. Methods: This is a retrospective single-center study involving 74 severe and critically ill COVID-19 patients recruited from the ICU department of the Tongji Hospital in Wuhan, China. The patients were divided into two groups: severe patients and critically ill patients. The serum IP-10, MCP-1 and MIP1α level in both groups was detected using the enzyme-linked immunosorbent assay (ELISA) kit. The clinical symptoms, laboratory test results, and the outcome of COVID-19 patients were retrospectively analyzed. Results: The serum IP-10 and MCP-1 level in critically ill patients was significantly higher than that in severe patients ( P <0.001). However, no statistical difference in MIP1α between the two groups was found. The analysis of dynamic changes showed that these indicators remarkably increased in patients with poor prognosis. Since the selected patients were severe or critically ill, no significant difference was observed between survival and death. Conclusions: IP-10 and MCP-1 are biomarkers associated with the severity of COVID-19 disease and can be related to the risk of death in COVID-19 patients.

The change pattern and significance of IgM and IgG in the progress of COVID-19 disease (preprint)

Background: To investigate the significance of IgM and IgG in the progress of COVID-19. Method: A multicenter cross-sectional study conducted in suspected and confirmed patients from four hospitals of China and a cohort study to identify the change pattern and significance in the process of COVID-19 disease. Results: A total of 571 patients were enrolled in the cross-sectional study, including 235 confirmed SARS-CoV-2 infection with 91.9% patients IgG positive and 92.3% IgM positive. 30 patients diagnosed with SARS-CoV-2 infection were enrolled in the cohort study for flowing-up in 20 days. The peak of IgM and IgG reached in 10th and 20 th day separately after symptom onset. The relationship between clinical classification and serological antibodies were analysed. The positive rate of COVID-19 IgG and IgM increased along with the clinical classification and the delay of treatment time. Conclusion: We demonstrated the kinetics of IgM and IgG SARS-CoV-2 antibody in COVID-19 patients, which may contribute to explain the results of IgM and IgG SARS-CoV-2 antibody test and predict the prognosis of COVID-19.

Serum protein profiling reveals a landscape of inflammation and immune signaling in early-stage COVID-19 infection (preprint)

Coronavirus disease 2019 (COVID-19) is a highly contagious infection and threating the human lives in the world. The elevation of cytokines in blood is crucial to induce cytokine storm and immunosuppression in the transition of severity in COVID-19 patients. However, the comprehensive changes of serum proteins in COVID-19 patients throughout the SARS-CoV-2 infection is unknown. In this work, we developed a high-density antibody microarray and performed an in-depth proteomics analysis of serum samples collected from early COVID-19 (n=15) and influenza (n=13) patients. We identified a large set of differentially expressed proteins (n=125) that participate in a landscape of inflammation and immune signaling related to the SARS-CoV-2 infection. Furthermore, the significant correlations of neutrophil and lymphocyte with the CCL2 and CXCL10 mediated cytokine signaling pathways was identified. These information are valuable for the understanding of COVID-19 pathogenesis, identification of biomarkers and development of the optimal anti-inflammation therapy.

Proteome-wide analysis of differentially-expressed SARS-CoV-2 antibodies in early COVID-19 infection (preprint)

Rapid and accurate tests that detect IgM and IgG antibodies to SARS-CoV-2 proteins are essential in slowing the spread of COVID-19 by identifying patients who are infected with COVID-19. Using a SARS-CoV-2 proteome microarray developed in our lab, we comprehensively profiled both IgM and IgG antibodies in forty patients with early-stage COVID-19, influenza, or non-influenza who had similar symptoms. The results revealed that the SARS-CoV-2 N protein is not an ideal biomarker for COVID-19 diagnosis because of its low immunogenicity, thus tests that rely on this marker alone will have a high false negative rate. Our data further suggest that the S protein subunit 1 receptor binding domain (S1-RBD) might be the optimal antigen for IgM antibody detection, while the S protein extracellular domain (S1+S2ECD) would be the optimal antigen for both IgM and IgG antibody detection. Notably, the combination of all IgM and IgG biomarkers can identify 87% and 73.3% COVID-19 patients, respectively. Finally, the COVID-19-specific antibodies are significantly correlated with the clinical indices of viral infection and acute myocardial injury (p[≤]0.05). Our data may help understand the function of anti-SARS-CoV-2 antibodies and improve serology tests for rapid COVID-19 screening.

SARS-CoV-2 proteome microarray for mapping COVID-19 antibody interactions at amino acid resolution (preprint)

COVID-19 has quickly become a worldwide pandemic, which has significantly impacted the economy, education, and social interactions. Understanding the humoral antibody response to SARS-CoV-2 proteins may help identify biomarkers that can be used to detect and treat COVID-19 infection. However, no immuno-proteomics platform exists that can perform such proteome-wide analysis. To address this need, we created a SARS-CoV-2 proteome microarray to analyze antibody interactions at amino acid resolution by spotting peptides 15 amino acids long with 5-amino acid offsets representing full-length SARS-CoV-2 proteins. Moreover, the array processing time is short (1.5 hours), the dynamic range is ~2 orders of magnitude, and the lowest limit of detection is 94 pg/mL. Here, the SARS-CoV-2 proteome array reveals that antibodies commercially available for SARS-CoV-1 proteins can also target SARS-CoV-2 proteins. These readily available reagents could be used immediately in COVID-19 research. Second, IgM and IgG immunogenic epitopes of SARS-CoV-2 proteins were profiled in the serum of ten COVID-19 patients. Such epitope biomarkers provide insight into the immune response to COVID-19 and are potential targets for COVID-19 diagnosis and vaccine development. Finally, serological antibodies that may neutralize viral entry into host cells via the ACE2 receptor were identified. Further investigation into whether these antibodies can inhibit the propagation of SARS-CoV-2 is warranted. Antibody and epitope profiling in response to COVID-19 is possible with our peptide-based SARS-COV-2 proteome microarray. The data gleaned from the array could provide invaluable information to the scientific community to understand, detect, and treat COVID-19.