Correlation of patient serum IgG, IgA and IgM antigen binding with COVID-19 disease severity using multiplexed SARS-CoV-2 antigen microarray and maintained relative IgA and IgM antigen binding over time (preprint)

Zoonotic spillover of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to humans in December 2019 caused the coronavirus disease 2019 (COVID-19) pandemic. Serological monitoring is critical for detailed understanding of individual immune responses to infection and protection to guide clinical therapeutic and vaccine strategies. We developed a high throughput multiplexed SARS-CoV-2 antigen microarray incorporating spike (S) and nucleocapsid protein (NP) and fragments expressed in various hosts which allowed simultaneous assessment of serum IgG, IgA, and IgM responses. Antigen glycosylation influenced antibody binding, with S glycosylation generally increasing and NP glycosylation decreasing binding. Purified antibody isotypes demonstrated a binding pattern and intensity that differed from the same isotype in the presence of other isotypes in whole serum, probably due to competition. Using purified antibody isotypes from naive Irish COVID-19 patients, we correlated antibody isotype binding to different panels of antigens with disease severity, with significance for binding to the S region S1 expressed in insect cells (S1 Sf21) for all three antibody isotypes. Assessing longitudinal response for constant concentrations of antibody isotypes for a subset of patients demonstrated that while the relative proportion of antigen-specific IgGs decreased over time for severe disease, the relative proportion of antigen-specific IgA binding remained at the same magnitude at 5 and 9 months post-first symptom onset. Further, the relative proportion of IgM binding decreased for S antigens but remained the same for NP antigens. This may support antigen specific serum IgA and IgM playing a role in maintaining longer-term protection, of importance for developing and assessing vaccine strategies. Overall, these data demonstrate the multiplexed platform as a sensitive and useful platform for expanded humoral immunity studies, allowing detailed elucidation of antibody isotypes response against multiple antigens. This approach will be useful for monoclonal antibody therapeutic studies and screening of donor polyclonal antibodies for patient infusions.

Improved diagnosis of SARS-CoV-2 by using Nucleoprotein and Spike protein fragment 2 in quantitative dual ELISA tests (preprint)

SUMMARY The novel Coronavirus, SARS-CoV-2, is the causative agent of the 2020 worldwide coronavirus pandemic. Antibody testing is useful for diagnosing historic infections of a disease in a population. These tests are also a helpful epidemiological tool for predicting how the virus spreads in a community, relating antibody levels to immunity and for assessing herd immunity. In the present study, SARS-CoV-2 viral proteins were recombinantly produced and used to analyse serum from individuals previously exposed, or not, to SARS-CoV-2. The nucleocapsid (Npro) and Spike subunit 2 (S2Frag) proteins were identified as highly immunogenic, although responses to the former were generally greater. These two proteins were used to develop two quantitative ELISA assays that when used in combination resulted in a highly reliable diagnostic test. Npro and S2Frag-ELISAs could detect at least 10% more true positive COVID-19 cases than the commercially available ARCHITECT test (Abbott). Moreover, our quantitative ELISAs also show that specific antibodies to SARS-CoV-2 proteins tend to wane rapidly even in patients that had developed severe disease. As antibody tests complement COVID-19 diagnosis and determine population-level surveillance during this pandemic, the alternative diagnostic we present in this study could play a role in controlling the spread of the virus.