Analysis of Serological Biomarkers of SARS-CoV-2 Infection in Convalescent Samples From Severe, Moderate and Mild COVID-19 Cases

Abstract

Precision monitoring of antibody responses during the COVID-19 pandemic is increasingly important during large scale vaccine rollout and rise in prevalence of Severe Acute Respiratory Syndrome-related Coronavirus-2 (SARS-CoV-2) variants of concern (VOC). Equally important is defining Correlates of Protection (CoP) for SARS-CoV-2 infection and COVID-19 disease. Data from epidemiological studies and vaccine trials identified virus neutralising antibodies (Nab) and SARS-CoV-2 antigen-specific (notably RBD and S) binding antibodies as candidate CoP. In this study, we used the World Health Organisation (WHO) international standard to benchmark neutralising antibody responses and a large panel of binding antibody assays to compare convalescent sera obtained from: a) COVID-19 patients; b) SARS-CoV-2 seropositive healthcare workers (HCW) and c) seronegative HCW. The ultimate aim of this study is to identify biomarkers of humoral immunity that could be used to differentiate severe from mild or asymptomatic SARS-CoV-2 infections. Some of these biomarkers could be used to define CoP in further serological studies using samples from vaccination breakthrough and/or re-infection cases. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (IU) for virus neutralisation assays or in Binding Antibody Units (BAU) for ELISA tests. In this work we used commercial and non-commercial antibody binding assays; a lateral flow test for detection of SARS-CoV-2-specific IgG/IgM; a high throughput multiplexed particle flow cytometry assay for SARS-CoV-2 Spike (S), Nucleocapsid (N) and Receptor Binding Domain (RBD) proteins); a multiplex antigen semi-automated immuno-blotting assay measuring IgM, IgA and IgG; a pseudotyped microneutralisation test (pMN) and an electroporation-dependent neutralisation assay (EDNA). Our results indicate that overall, severe COVID-19 patients showed statistically significantly higher levels of SARS-CoV-2-specific neutralising antibodies (average 1029 IU/ml) than those observed in seropositive HCW with mild or asymptomatic infections (379 IU/ml) and that clinical severity scoring, based on WHO guidelines was tightly correlated with neutralisation and RBD/S antibodies. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

Neutralisation Hierarchy of SARS-CoV-2 Variants of Concern Using Standardised, Quantitative Neutralisation Assays Reveals a Correlation With Disease Severity; Towards Deciphering Protective Antibody Thresholds

“…Here we report our findings in International Units through the use of the WHO International Standard for anti-SARS-CoV-2 Immunoglobulin (NIBSC code: 20/136) comprising a pool of 11 convalescent plasma sourced from the first wave of global infections, when the circulating SARS-CoV-2 sequences were relatively genetically homogeneous. These sera were also analysed in a previous study, that aimed to assess a multitude of different binding assays of which the results were then standardised using the WHO International Standard ( 60 ). One of the main questions regarding the antiviral neutralising antibody responses remains: what are the immune correlates of protection against SARS-CoV-2 infection?…”

“…Most notably, Khoury et al clearly stated that the 54 IU/mL estimate, the first of its kind for SARS-CoV-2, was based on aggregation of datasets using diverse neutralisation assays and vaccine clinical trial designs which did not use calibrated assays, and asserted that future standardisation was key to defining correlates of protection (23,67). Whilst studies have analysed the degree of correlation between different assays, it is difficult to account for inter-laboratory variation (60). Furthermore, our study only examines a single component of the immune response, and several other markers can be used as correlates of protection such as T cell or B cell responses, of which currently there is no estimated nor defined unitage that correlates with protection.…”

Standardization of two SARS-CoV-2 serology assays to the WHO 20/136 human standard reference material

“…The WHO IS has a single assigned value of 1000 binding antibody units (BAU/mL) per vial for serology assays of the same immunoglobulin class and specificity. Recent studies have calibrated their assays to the WHO IS ( Castillo-Olivares et al, 2021 ; Feng et al, 2021 ). One study compared results of several binding antibody assays on convalescent samples from severe, moderate, and mild COVID-19 cases and found a positive correlation between severity and RBD/S antibodies ( Castillo-Olivares et al, 2021 ).…”

“…Recent studies have calibrated their assays to the WHO IS ( Castillo-Olivares et al, 2021 ; Feng et al, 2021 ). One study compared results of several binding antibody assays on convalescent samples from severe, moderate, and mild COVID-19 cases and found a positive correlation between severity and RBD/S antibodies ( Castillo-Olivares et al, 2021 ). Here, we report the WHO IS calibration of two additional anti-S1 RBD Ig assays not previously reported in the literature.…”

Pseudotyped Bat Coronavirus RaTG13 is efficiently neutralised by convalescent sera from SARS-CoV-2 infected patients

“…Amendment 5). HCWs from RPH were recruited through staff email over the course of 2 months (20th April 2020–10th June 2020) as part of a prospective study to establish seroprevalence and immune correlates of protective immunity to SARS-CoV-2 42 . Patients were recruited in convalescence either pre-discharge or at the first post-discharge clinical review.…”