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

SARS-CoV-2 Spike protein binding of glycated serum albumin - its potential role in the pathogenesis of the COVID-19 clinical syndromes and bias towards individuals with pre-diabetes/type 2 diabetes & metabolic diseases.

Since the immune response to SARS-CoV2 infection requires antibody recognition of the Spike protein, we used MagMix, a semi-automated magnetic rack to reproducibly isolate patient plasma proteins bound to a pre-fusion stabilised Spike and nucleocapsid proteins conjugated to magnetic beads. Once eluted, MALDI-ToF mass spectrometry identified a range of immunoglobulins, but also in Spike protein magnetic beads we found a high affinity for human serum albumin. Careful mass comparison revealed a preferential capture of advanced glycation end product (AGE) glycated human serum albumin by the pre-fusion Spike protein. The ability of bacteria and viruses to surround themselves with serum proteins is a recognised process of immune evasion. A lower serum albumin concentration is a reported feature of COVID-19 patients with severe symptoms and high probability of death. This binding preference of the Spike protein for AGE glycated serum albumin may contribute to immune evasion and influence the severity & pathology of SARS-COV2 towards acute respiratory distress. Thus, it can be hypothesised, contributing to the symptom severity bias and mortality risk for the elderly and those with (pre)diabetic and atherosclerotic/metabolic diseases who contract SARS-CoV2 infections. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/21258871v3_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@2d9e7org.highwire.dtl.DTLVardef@1300c4corg.highwire.dtl.DTLVardef@1776193org.highwire.dtl.DTLVardef@a6ffc6_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

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

BackgroundDetecting 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. MethodsWe systemically developed an ELISA assay, optimising different antigens and amplification steps, in serum and saliva from symptomatic and asymptomatic SARS-CoV-2-infected subjects. ResultsUsing 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. ConclusionsDetecting antibody responses in both saliva and serum is optimal for determining virus exposure and understanding immune responses after SARS-CoV-2 infection. FundingThis 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.