In vitro, classical complement activation differs by disease severity and between SARS-CoV-2 antigens (preprint)

Antibodies specific for the spike glycoprotein (S) and nucleocapsid (N) SARS-CoV-2 proteins are typically present during severe COVID-19, and induced to S after vaccination. The binding of viral antigens by antibody can initiate the classical complement pathway. Since complement could play pathological or protective roles at distinct times during SARS-CoV-2 infection we determined levels of antibody-dependent complement activation along the complement cascade. Here, we used an ELISA assay to assess complement protein binding (C1q) and the deposition of C4b, C3b, and C5b to S and N antigens in the presence of anti-SARS-CoV-2 antibodies from different test groups: non-infected, single and double vaccinees, non-hospitalised convalescent (NHC) COVID-19 patients and convalescent hospitalised (ITU-CONV) COVID-19 patients. C1q binding correlates strongly with antibody responses, especially IgG1 levels. However, detection of downstream complement components, C4b, C3b and C5b shows some variability associated with the antigen and subjects studied. In the ITU-CONV, detection of C3b-C5b to S was observed consistently, but this was not the case in the NHC group. This is in contrast to responses to N, where median levels of complement deposition did not differ between the NHC and ITU-CONV groups. Moreover, for S but not N, downstream complement components were only detected in sera with higher IgG1 levels. Therefore, the classical pathway is activated by antibodies to multiple SARS-CoV-2 antigens, but the downstream effects of this activation may differ depending on the specific antigen targeted and the disease status of the subject. O_LISpike- and nucleocapsid-specific antibodies activate complement in vitro C_LIO_LIC1q binding correlates with IgG1 antibody levels C_LIO_LIGeneration of C4b, C3b and C5b relates to the antigen targeted and the patient group tested C_LI

Validation of a combined ELISA to detect IgG, IgA and IgM antibody responses to SARS-CoV-2 in mild or moderate non-hospitalised patients (preprint)

BackgroundFrequently SARS-CoV-2 results in mild or moderate disease with potentially lower concentrations of antibodies compared to those that are hospitalised. Here, we validated an ELISA using SARS-CoV-2 trimeric spike glycoprotein, with targeted detection of IgG, IgA and IgM (IgGAM) using serum and dried blood spots (DBS) from adults with mild or moderate disease. MethodsTargeting the SARS-CoV-2 trimeric spike, a combined anti-IgG, IgA and IgM serology ELISA assay was developed using 62 PCR-confirmed non-hospitalised, mild or moderate COVID-19 samples, [≥]14 days post symptom onset and 624 COVID-19 negative samples. The assay was validated using 73 PCR-confirmed non-hospitalised COVID-19 and 359 COVID-19 negative serum samples with an additional 81 DBSs, and further validated in 226 PCR-confirmed non-hospitalised COVID-19 and 426 COVID-19 negative clinical samples. ResultsA sensitivity and specificity of 98.6% (95% CI, 92.6-100.0), 98.3% (95% CI, 96.4-99.4), respectively, was observed following validation of the SARS-CoV-2 ELISA. No cross-reactivities with endemic coronaviruses or other human viruses were observed, and no change in results were recorded for interfering substances. The assay was stable at temperature extremes and components were stable for 15 days once opened. A matrix comparison showed DBS to correlate with serum results. Clinical validation of the assay reported a sensitivity of 94.7% (95% CI, 90.9-97.2%) and a specificity of 98.4% (95% CI, 96.6-99.3%). ConclusionsThe human anti-IgGAM SARS-CoV-2 ELISA provides accurate and sensitive detection of SARS-CoV-2 antibodies in non-hospitalised adults with mild or moderate disease. The use of dried blood spots makes the assay accessible to the wider community. Supplementary MaterialNo

Detection of antibodies to the SARS-CoV-2 spike glycoprotein in both serum and saliva enhances detection of infection (preprint)

Background: Detecting antibody responses during and after SARS-CoV-2 infection is essential in determining the seroepidemiology of the virus and the potential role of antibody in disease. Scalable, sensitive and specific serological assays are essential to this process. The detection of antibody in hospitalized patients with severe disease has proven straightforward; detecting responses in subjects with mild disease and asymptomatic infections has proven less reliable. We hypothesized that the suboptimal sensitivity of antibody assays and the compartmentalization of the antibody response may contribute to this effect. Methods: We systemically developed an ELISA assay, optimising different antigens and amplification steps, in serum and saliva from symptomatic and asymptomatic SARS-CoV-2-infected subjects. Results: Using trimeric spike glycoprotein, rather than nucleocapsid enabled detection of responses in individuals with low antibody responses. IgG1 and IgG3 predominate to both antigens, but more anti-spike IgG1 than IgG3 was detectable. All antigens were effective for detecting responses in hospitalized patients. Anti-spike, but not nucleocapsid, IgG, IgA and IgM antibody responses were readily detectable in saliva from non-hospitalized symptomatic and asymptomatic individuals. Antibody responses in saliva and serum were largely independent of each other and symptom reporting. Conclusions. Detecting antibody responses in both saliva and serum is optimal for determining virus exposure and understanding immune responses after SARS-CoV-2 infection. Funding. This work was funded by the University of Birmingham, the National Institute for Health Research (UK), the NIH National Institute for Allergy and Infectious Diseases, the Bill and Melinda Gates Foundation and the University of Southampton.

Serology confirms SARS-CoV-2 infection in PCR-negative children presenting with Paediatric Inflammatory Multi-System Syndrome (preprint)

Background. During the COVID-19 outbreak, reports have surfaced of children who present with features of a multisystem inflammatory syndrome with overlapping features of Kawasaki disease and toxic shock syndrome - Paediatric Inflammatory Multisystem Syndrome- temporally associated with SARS-CoV-2 pandemic (PIMS-TS). Initial reports find that many of the children are PCR-negative for SARS-CoV-2, so it is difficult to confirm whether this syndrome is a late complication of viral infection in an age group largely spared the worst consequences of this infection, or if this syndrome reflects enhanced surveillance. Methods. Children hospitalised for symptoms consistent with PIMS-TS between 28 April and 8 May 2020, and who were PCR-negative for SARS-CoV-2, were tested for antibodies to viral spike glycoprotein using an ELISA test. Results. Eight patients (age range 7-14 years, 63% male) fulfilled case-definition for PIMS-TS during the study period. Six of the eight patients required admission to intensive care. All patients exhibited significant IgG and IgA responses to viral spike glycoprotein. Further assessment showed that the IgG isotypes detected in children with PIMS-TS were of the IgG1 and IgG3 subclasses, a distribution similar to that observed in samples from hospitalised adult COVID-19 patients. In contrast, IgG2 and IgG4 were not detected in children or adults. IgM was not detected in children, which contrasts with adult hospitalised adult COVID-19 patients of whom all had positive IgM responses. Conclusions. Strong IgG antibody responses can be detected in PCR-negative children with PIMS-TS. The low detection rate of IgM in these patients is consistent with infection having occurred weeks previously and that the syndrome onset occurs well after the control of SARS-CoV-2 viral load. This implies that the disease is largely immune-mediated. Lastly, this indicates that serology can be an appropriate diagnostic tool in select patient groups.