Direct detection of humoral marker corelates of COVID-19, glycated HSA and hyperglycosylated IgG3, by MALDI-ToF mass spectrometry.

The prefusion Spike protein of SARS-CoV2 binds advanced glycation end product (AGE) glycated human serum albumin (HSA) and a higher mass, hyperglycosylated/glycated, IgG3, as determined by matrix assisted laser desorption mass spectrometry (MALDI-ToF MS). We set out to investigate if the total blood plasma of patients who had recovered from acute respiratory distress as a result of COVID-19, contained more glycated HSA and higher mass (glycosylated/glycated) IgG3 than those with only clinically mild or asymptomatic infections. A direct dilution and disulphide bond reduction method was development and applied to plasma samples from SARS-CoV2 seronegative (N = 30) and seropositive (N = 31) healthcare workers and 38 convalescent plasma samples from patients who had been admitted with acute respiratory distress syndrome (ARDS) associated with COVID-19. Patients recovering from COVID-19 ARDS had significantly higher mass, AGE-glycated HSA and higher mass IgG3 levels. This would indicate that increased levels and/or ratios of hyper-glycosylation (probably terminal sialic acid) IgG3 and AGE glycated HSA may be predisposition markers for development of ARDS as a result of COVID-19 infection. Furthermore, rapid direct analysis of plasma samples by MALDI-ToF MS for such humoral immune correlates of COVID-19 presents a feasible screening technology for the most at risk; regardless of age or known health conditions. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/21260186v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@12bfa69org.highwire.dtl.DTLVardef@45344forg.highwire.dtl.DTLVardef@16d4f7forg.highwire.dtl.DTLVardef@17e5c34_HPS_FORMAT_FIGEXP M_FIG C_FIG

Determination of IgG1 and IgG3 SARS-CoV-2 spike protein and nucleocapsid binding. Who is binding who and why?

The involvement of IgG3 in the humoral immune response to SARS-CoV2 infection has been implicated in the pathogenesis of ARDS in COVID-19. The exact molecular mechanism is unknown but may be due to the differential ability of IgG3 Fc region to fix complement and stimulate cytokine release. We examined convalescent patients antibodies binding to immobilised nucleocapsid and spike protein by MALDI-ToF mass spectrometry. IgG3 was a major immunoglobulin found in all samples. Differential analysis of the spectral signatures found for nucleocapsid versus spike protein demonstrated that the predominant humoral immune response to nucleocapsid was IgG3, whilst against spike it was IgG1. However, the spike protein displayed a strong affinity for IgG3 itself which it would bind from control plasma samples as well as from those previously infected with SARS-CoV2, much in the way Protein-G binds IgG1. Furthermore, detailed spectral analysis indicated a mass shift consistent with hyper-glycosylation or glycation was a characteristic of the IgG3 captured by the spike protein. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/21259077v2_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@181773eorg.highwire.dtl.DTLVardef@bb8d58org.highwire.dtl.DTLVardef@13cbe05org.highwire.dtl.DTLVardef@df579e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Towards Internationally standardised humoral Immune Correlates of Protection from SARS CoV 2 infection and COVID-19 disease

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, was to identify biomarkers of humoral immunity that could be used as candidate CoP in internationally accepted unitage. Whenever suitable, the antibody levels of the samples studied were expressed in International Units (INU) for virus neutralisation assays or International 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 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 binding assays. In addition, there was a positive correlation between severity, N-antibody assays and intracellular virus neutralisation.

Critical care workers have lower seroprevalence of SARS-CoV-2 IgG compared with non-patient facing staff in first wave of COVID19.

With the first 2020 surge of the COVID-19 pandemic, many health care workers (HCW) were re-deployed to critical care environments to support intensive care teams to look after high numbers of patients with severe COVID-19. There was considerable anxiety of increased risk of COVID19 for staff working in these environments. Using a multiplex platform to assess serum IgG responses to SARS-CoV-2 N, S and RBD proteins, and detailed symptom reporting, we screened over 500 HCW (25% of the total workforce) in a quaternary level hospital to explore the relationship between workplace and evidence of exposure to SARS-CoV-2. Whilst 45% of the cohort reported symptoms that they consider may have represented COVID-19, overall seroprevalence was 14% with anosmia and fever being the most discriminating symptoms for seropositive status. There was a significant difference in seropositive status between staff working in clinical and non-clinical roles (9% patient facing critical care, 15% patient facing non-critical care, 22% nonpatient facing). In the seropositive cohort, symptom severity increased with age for men and not for women. In contrast, there was no relationship between symptom severity and age or sex in the seronegative cohort reporting possible COVID-19 symptoms. Of the 12 staff screened PCR positive (10 symptomatic), 3 showed no evidence of seroconversion in convalescence. ConclusionThe current approach to Personal Protective Equipment (PPE) appears highly effective in protecting staff from patient acquired infection in the critical care environment including protecting staff managing interhospital transfers of COVID-19 patients. The relationship between seroconversion and disease severity in different demographics warrants further investigation. Longitudinally paired virological and serological surveillance, with symptom reporting are urgently required to better understand the role of antibody in the outcome of HCW exposure during subsequent waves of COVID-19 in health care environments.