As the Coronavirus Disease 2019 (COVID-19), which is caused by the novel SARS-CoV-2, continues to spread rapidly around the world, there is a need for well validated serological assays that allow the detection of viral specific antibody responses in COVID-19 patients or recovered individuals. In this study, we established and used multiple indirect Enzyme Linked Immunosorbent Assay (ELISA)-based serological assays to study the antibody response in COVID-19 patients. In order to validate the assays we determined the cut off values, sensitivity and specificity of the assays using sera collected from pre-pandemic healthy controls, COVID-19 patients at different time points after disease-onset, and seropositive sera to other human coronaviruses (CoVs). The developed SARS-CoV-2 S1 subunit of the spike glycoprotein and nucleocapsid (N)-based ELISAs not only showed high specificity and sensitivity but also did not show any cross-reactivity with other CoVs. We also show that all RT-PCR confirmed COVID-19 patients tested in our study developed both virus specific IgM and IgG antibodies as early as week one after disease onset. Our data also suggest that the inclusion of both S1 and N in serological testing would capture as many potential SARS-CoV-2 positive cases as possible than using any of them alone. This is specifically important for tracing contacts and cases and conducting large-scale epidemiological studies to understand the true extent of virus spread in populations.
The Coronavirus Disease 2019 (COVID-19), caused by SARS-CoV-2, continues to spread globally with significantly high morbidity and mortality rates. Antigen-specific responses are of unquestionable value for clinical management of COVID-19 patients. Here, we investigated the kinetics of IgM, IgG against the spike (S) and nucleoproteins (N) proteins and their neutralizing capabilities in hospitalized COVID-19 patients with different disease presentations (i.e., mild, moderate or severe), need for intensive care units (ICU) admission or outcomes (i.e., survival vs death). We show that SARS-CoV-2 specific IgG, IgM and neutralizing antibodies (nAbs) were readily detectable in almost all COVID-19 patients with various clinical presentations. Interestingly, significantly higher levels of nAbs as well as anti-S1 and -N IgG and IgM antibodies were found in patients with more severe symptoms, patients requiring admission to ICU or those with fatal outcomes. More importantly, early after symptoms onset, we found that the levels of anti-N antibodies correlated strongly with disease severity. Collectively, these findings provide new insights into the kinetics of antibody responses in COVID-19 patients with different disease severity.
The Coronavirus Disease 2019 (COVID-19), caused by the novel SARS-CoV-2, continues to spread globally with significantly high morbidity and mortality rates. Immunological surrogate markers, in particular antigen-specific responses, are of unquestionable value for clinical management of patients with COVID-19. Here, we investigated the kinetics of IgM, IgG against the spike (S) and nucleoproteins (N) proteins and their neutralizing capabilities in hospitalized patients with RT-PCR confirmed COVID-19 infection. Our data show that SARS-CoV-2 specific IgG, IgM and neutralizing antibodies (nAbs) were readily detectable in almost all COVID-19 patients with various clinical presentations. Notably, anti-S and -N IgG, peaked 20-40 day after disease onset, and were still detectable for at least up to 70 days, with nAbs observed during the same time period. Moreover, nAbs titers were strongly correlated with IgG antibodies. Significantly higher levels of nAbs as well as anti-S1 and N IgG and IgM antibodies were found in patients with more severe clinical presentations, patients requiring admission to intensive care units (ICU) or those with fatal outcomes. Interestingly, lower levels of antibodies, particularly anti-N IgG and IgM in the first 15 days after symptoms onset, were found in survivors and those with mild clinical presentations. Collectively, these findings provide new insights into the characteristics and kinetics of antibody responses in COVID-19 patients with different disease severity.
The ongoing global pandemic of coronavirus disease 2019 (COVID-19) calls for an urgent development of effective and safe prophylactic and therapeutic measures. The spike (S) glycoprotein of severe acute respiratory syndrome-coronavirus (SARS-CoV-2) is a major immunogenic and protective protein and plays a crucial role in viral pathogenesis. In this study, we successfully constructed a synthetic codon-optimized DNA-based vaccine as a countermeasure against SARS-CoV-2, denoted VIU-1005. The design was based on a codon-optimized coding sequence of a consensus full-length S glycoprotein. The immunogenicity of the vaccine was tested in two mouse models (BALB/c and C57BL/6J). Th1-skewed systemic S-specific IgG antibodies and neutralizing antibodies (nAbs) were significantly induced in both models 4 weeks after three injections with 100 μg of the VIU-1005 vaccine via intramuscular needle injection but not intradermal or subcutaneous routes. Such immunization induced long-lasting IgG and memory T cell responses in mice that lasted for at least 6 months. Interestingly, using a needle-free system, we showed an enhanced immunogenicity of VIU-1005 in which lower or fewer doses were able to elicit significantly high levels of Th1-biased systemic S-specific immune responses, as demonstrated by the significant levels of binding IgG antibodies, nAbs and IFN-γ, TNF and IL-2 cytokine production from memory CD8+ and CD4+ T cells in BALB/c mice. Furthermore, compared to intradermal needle injection, which failed to induce any significant immune response, intradermal needle-free immunization elicited a robust Th1-biased humoral response similar to that observed with intramuscular immunization. Together, our results demonstrate that the synthetic VIU-1005 candidate DNA vaccine is highly immunogenic and capable of inducing long-lasting Th1-skewed humoral and cellular immunity in mice. Furthermore, we show that the use of a needle-free system could enhance the immunogenicity and minimize doses needed to induce protective immunity in mice, supporting further preclinical and clinical testing of this candidate vaccine.
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