key: cord-0952576-c88yemy8
authors: Iles, J. K.; Zmuidinaite, R.; Saddee, C.; Gardiner, A.; Lacy, J.; Harding, S.; Ule, J.; Roblett, D.; Heeney, J. L.; Baxendale, D. H.; Iles, R. K.
title: 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.
date: 2021-06-15
journal: nan
DOI: 10.1101/2021.06.14.21258871
sha: 5e4a92f2f90958aa4a1f429813b162e640c57d91
doc_id: 952576
cord_uid: c88yemy8

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 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 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.

The disease syndrome COVID-19 is caused by the enveloped corona virus SARS-CoV2.

However following infection its clinical manifestation can be very mild, even asymptomatic, to life threatening. Antibodies to SARS-CoV2 envelope proteins are seen in those infected and are particularly elevated in those with severe illness from the infection [1] .

The initial infection of the nasopharyngeal epithelia can be asymptomatic and the local immune response, such as mucosal IgA, in many, particularly the young, will be sufficient for the virus not to progress. However, if it does progress to involvement of the upper airways, the disease manifests with symptoms of fever, malaise and dry cough. There is a greater immune response during this phase involving the release of C-X-C motif chemokine ligand 10 (CXCL-10) and interferons (IFN-β and IFN-λ) from the virus-infected cells [2] .

The majority of patients do not progress beyond this phase as the mounted immune response is sufficient to contain the spread of infection.

About one-fifth of all infected patients progress to involvement of the lower respiratory tract and progression to Acute Respiratory Distress Syndrome (ARDS). The virus-laden pneumocytes release many different cytokines and inflammatory markers such as interleukins, tumour necrosis factor-α, Interferons, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1α. This 'COVID-19 cytokine storm' acts as a chemoattractant for neutrophils, CD4 helper T cells and CD8 cytotoxic T cells. Sequestered into the lung tissue, these cells are responsible for the subsequent inflammation and lung injury, culminating in an acute respiratory distress syndrome. [3, 4] .

At admission, the majority of patients had lymphopenia and platelet abnormalities, elevated neutrophils, aspartate aminotransferase (AST), lactate dehydrogenase (LDH) along with these inflammatory biomarkers [5] . Medical imaging (CT or X-ray) frequently shows patients have bilateral pneumonia and pleural effusion occurs in only 10% of the patients [6] . Compared to patients in general, refractory patients have a higher level of neutrophils, AST, LDH, and Creactive protein (CRP) but lower level of platelets [7, 8] and albumin [9, 10] .

The Spike complex is the principal target for neutralising antibodies as this viral envelope surface exposed structure is functionally responsible for the virus target and entry into cells via the ACE 2 receptor expressed on the target cells. The spike complex is a trimer of the large S protein which, after binding to the ACE-2 receptor, undergoes activation via a twostep protease cleavage: the first one for S protein priming at the S1/S2 cleavage site and the second cleavage for activation at a position adjacent to a fusion peptide within the S2 subunit [11, 12, 13, 14] . The initial cleavage stabilises the S2 subunit at the attachment site and the subsequent cleavage activates the S protein causing conformational changes leading to viral and host cell membrane fusion [15] .

As part of an investigation of serum/plasma binding proteins to SARS CoV2 antigenic proteins albumin was found to bind strongly to stabilised complete spike protein but not nucleocapsid protein nor control Protein-G. Characterised by MALDI-ToF mass spectrometry the albumin bound was found to vary in molecular mass consistent with advanced glycation end products (AGE).

Serum and plasma samples were obtained from Health care workers (HCWs) and patients referred to the Royal Papworth Hospital for critical care. COVID-19 patients hospitalised during the first wave and as well as NHS healthcare workers working at the Royal Papworth Hospital in Cambridge, UK served as the exposed HCW cohort (Study approved by Research Ethics Committee Wales, IRAS: 96194 12/WA/0148. Amendment 5). NHS HCW participants from the Royal Papworth Hospital were recruited through staff email over the course of 2 months (20 th April 2020-10 th June 2020) as part of a prospective study to establish seroprevalence and immune correlates of protective immunity to SARS-CoV-2.

Patients were recruited in convalescence either pre-discharge or at the first post-discharge clinical review. All participants provided written, informed consent prior to enrolment in the study. Sera from NHS HCW and patients were collected between July and September 2020, approximately 3 months after they were enrolled in the study [16] .

. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 15, 2021. ;  Clinical assessment and WHO criteria scoring of severity for both patients and NHS HCW was conducted following the 'COVID-19 Clinical Management: living guidance (https://www.who.int/publications/i/item/WHO-2019-nCoV-clinical-2021-1).

For cross-sectional comparison, representative convalescent serum and plasma samples from seronegative HCWs, seropositive HCW and convalescent PCR-positive COVID-19 patients were obtained. The serological screening to classify convalescent HCW as positive or negative was done according to the results provided by a CE-validated Luminex assay detecting N-, RBD-and S-specific IgG, a lateral flow diagnostic test (IgG/IgM) and an Electro-chemiluminescence assay (ECLIA) detecting N-and S-specific IgG. Any sample that produced a positive result by any of these assays was classified as positive. The clinical signs of the individuals from which the sample was obtained ranged from 0 to 7 according to the 

The viral Spike protein (S-protein) is present on virions as a pre-fusion trimers with the receptor binding domain of the S1 region stochastically open or closed, an intermediary where the S1 region is cleaved and discarded, and the S2 undergoes major confirmation changes to expose and then retract its fusion peptide domain [17] . Here the S-protein was modified to disable the S1/S2 cleavage site and maintain the pre-fusion stochastic Isolation of bound proteins with MagMix, a semi-automated magnetic rack Consistency in processing of magnetic bead-coupled antigen-captured binding-proteins from plasma sample was achieved using the semi-automated magnetic rack produced by the Crick Institute, London, UK (see figure 1 , panel 1) further details at bitomix.com. This instrument . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted June 15, 2021. ;  has a series of magnets on rotating discs set either side of the tube containing samples and magnetic beads. Rotation of the magnets not only pulls the antigen magnetic beads to one side, as is done by the commercially available static magnets, but also has a series of other automated functions. These include:

1. Engage/Disengage: Beads are immobilized allowing liquid to be aspirated or disengaged to transfer beads in solution to a new test tube. To prepare the beads, empty 1.5µl microcentrifuge tubes were loaded into the automated magnetic rack. The rack was turned on and set to the 'Disengage' setting (Figure 1c ). Protein G (GE), purified nucleocapsid or purified stabilized complete spike magnetic beads (Bindingsite, Birmingham UK) in their buffer solutions were vortexed to ensure an even distribution of beads within the solution, and 10µl of the appropriate magnetic beads were pipetted into each tube. 100µl of wash buffer, 0.1% Tween 20 in Dulbecco's phosphate, buffered saline (DPBS) was then added into each tube before the rack was set to the 'Resuspend' setting. During this period, the magnets continuously circulated for several rotations before stopping. Once the rack had finished resuspending, the settings were changed to 'Mix' for several minutes, and then changed back to the 'Resuspend' setting to mix once more. The rack was set to 'Engage' allowing the wash buffer to be carefully discarded using a pipette, while taking care not to disturb the beads. The wash cycle was repeated three times.

After 3 wash cycles, all of the remaining wash buffer in the tubes was discarded without disturbing the beads. The rack was set into the 'Disengage' setting.

To process the samples and elute the binding fraction from magnetic beads, 45µl of 10X DPBS was pipetted into each of the tubes containing the washed magnetic beads. 5µl of vortexed 1:10 diluted plasma was pipetted and pump mixed into a tube containing the beads, repeating for each plasma sample. The rack was set to the 'Resuspend' setting to ensure a . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ;  thorough mix of the beads with the diluted plasma. After the resuspension, the rack was set to 'Mix' for 20 minutes. After the mix, the rack was set to 'Engage' and the remaining solution was carefully discarded as to not disturb the beads. A further 3 wash cycles were conducted using 0.1% DPBS. Subsequently, another 3 wash cycles were conducted after this, using ultra-pure water, discarding the water after the last cycle. 15µl of recovery solution (20mM tris(2-carboxyethyl)phosphine (TCEP) (Sigma-Aldrich, UK) + 5% acetic acid + ultra-pure water) was pipetted into the tubes. The tubes were removed from the rack and mixed by flicking the tube to fully disturb the beads. All of the liquid was sent to the bottom of the tube by firmly tapping the tube on a surface to bring the droplets down. The tubes were securely placed back into the rack and were run alternatively between the 'Resuspend' setting and the 'Mix' setting for 5 minutes. The rack was set to 'Engage' and the recovery solution was carefully removed using a pipette and placed into a clean, labelled 0.6µl microcentrifuge tube. This recovery solution was the eluant from the beads and contained the desired proteins.

Mass spectra were generated using a 15mg/ml concentration of sinapinic acid (SA) matrix.

The elute from the beads was used to plate with no further processing. 1µl of the eluted samples were taken and plated on a 96 well stainless-steel target plate using a sandwich technique. The MALDI-ToF mass spectrometer (microflex® LT/SH, Bruker, Coventry, UK) was calibrated using a 2-point calibration of 2mg/ml bovine serum albumin (33,200 m/z and 66,400 m/z) (Pierce TM , ThermoFisher Scientific). Mass spectral data were generated in a positive linear mode. The laser power was set at 65% and the spectra was generated at a mass range between 10,000 to 200,000 m/z; pulsed extraction set to 1400ns.

A square raster pattern consisting of 15 shots and 500 positions per sample was used to give 7500 total profiles per sample. An average of these profiles was generated for each sample, giving a reliable and accurate representation of the sample across the well. The raw, averaged spectral data was then exported in a text file format to undergo further mathematical analysis.

Mass spectral data generated by the MALDI-ToF instrument were uploaded to an opensource mass spectrometry analysis software mMass™ [19] , where it was processed by using; a single cycle, Gaussian smoothing method with a window size of 300 m/z, and baseline correction with applicable precision and relative offset depending on the baseline of each . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ;  individual spectra. In software, an automated peak-picking was applied to produce peak list which was then tabulated and used in subsequent statistical analysis.

Peak mass and peak intensities were tabulated in excel and plotted in graphic comparisons of distributions for each antigen capture and patient sample group. Means and Medians were calculated and, given the asymmetric distributions found, non-parametric statistics were applied, such as Man Whitney U test, when comparing differences in group distributions.

This study relies on use of antigen-coupled magnetic beads to capture human serum proteins, which requires robust and reproducible processes of magnetic bead capture, washing, agitation and target binding protein elution (Figure 1c -d). If this is performed manually, the result can vary dramatically due to variations in timings between steps, in the intensity of mixing, while insufficient washing can result in large amounts of non-specific binding proteins being recovered and lead to variable efficiency of target binding proteins recoveries.

To overcome these problems, and to minimise individual operator variability, we employed a newly-developed automated magnetic rack system. By enabling automation, this system leads to consistent timing and intensity of binding, mixing and elution steps, and ensures gentle but thorough washing by enabling longer mixing with more repetitions.

Post elution from respective antigen coupled magnetic beads MALDI-ToF mass spectra was Although immunoglobulin light chains and heavy chains were recovered as expected (and subject to detailed analysis reports elsewhere) there was a significant recovery of human serum albumin by the magnetic beads to which stabilised complete Spike was conjugated ( Figure 2 ). Indeed the recovery was conspicuous in this one magnetic bead coupled SAR-S-Cov2 protein series recovered levels of HSA to a level similar to that seen for Protein G . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ;  binding and recovery of human IgG1, to which it has a known specificity. The same samples failed to bind albumin to the same degree despite its vast abundance in the samples to Protein-G or Nucleocapsid (see figure 2 ). Human serum albumin was detected in 72% of the samples of the Spike protein conjugated magnetic beads (compared to 33% for nucleocapsid and 13% Protein-G), and at an intensity level over 10x higher than that found for residual non-specific binding of HSA for nucleocapsid and Protein-G ( mean values; 1923 AU Spike protein, 159 AU N-protein and 100 AU Protein-G).

Closer examination of the bound and eluted HSA masses revealed that the average mass of the albumin recovered by the stabilised complete Spike protein was higher than that found for the low level non-specific absorption to nucleocapsid and Protein G (Δ152m/z, p<0.05, see figure 3 panel A). The binding of this higher molecular mass albumin was not a specific unique feature of plasma from patients who had recovered from COVID19 ARDS; but was also being bound from sero-negative and sero-positive HCW samples (see figure 3 panel B).

The average increase in HSA mass was 152m/z but ranged from ranged from 50 up to 500+m/z and Spike protein affinity was greatest for albumin with increased mass of about 150m/z (see figure 3 panel B).

Strong and specific human serum albumin binding to magnetic beads to which stabilised prefusion Spike protein is conjugated was found to occur with all plasma samples; be it from sero-negative & sero-positive medical staff (who had no symptoms or mild symptoms) or COVID-19 patients who had been treated on for ARDS on COVID-19 ITU wards. Albumin in these same plasma samples was not found to bind strongly to nucleocapsid or Protein-G coated magnetic beads, which acted as a control. This is despite the fact that significant human serum albumin binding has been reported as a feature of Protein-G [25].

Use of the semi-automated rack was essential to obtain reproducible data in the current study.

We had initially attempted to use a static magnetic rack and very gently vibrationally resuspend the particles during washing. Time consuming and labour intensive, when processing more than 6 samples, we also found that inconsistent and variability in intensity of vibrational mixing during the multiple washing steps would often result in no recovery of antibodies from the positive control Protein-G magnetic beads. Thus, we believe our study . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ;  serves as a proof-of-concept for the general utility of the semi-automated rack in any system where reproducible isolation of biomolecules with magnetic beads in 1.5-2ml tubes is required.

Many microorganisms that cause seriously harmful infections have developed mechanisms to evade the immune system. One such mechanism includes the specific binding of serum proteins to mask antigenic sites and thus evade antibody neutralisation and other immune processes. The spike protein is an extremely large antigenic target projecting and exposed from the virion Envelope. It is the focus of neutralising antibodies [26] . Thus, the ability to evade neutralising antibodies by coating with abundant serum protein(s), until such time as target cell receptor binding has occurred, is an ideal strategy to evolve, particularly when entering the blood stream. Bacteria express families of genes coding for surface proteins that bind serum proteins, in particular those that bind immunoglobulins, i.e. IgG by Protein-G (human group C and G streptococci) and Protein-A (Staphylococcus aureus). An analogous gene to that which codes for Protein-G has been found to code for a protein, termed PAB, which binds human serum albumin. PAB is highly expressed by the anaerobic bacterium Since detection of the eluted bound albumin was via MALDI-ToF mass spectrometry a more precise molecular mass of the bound albumin could be determined. It was found that the molecular mass of the elevated levels of albumin binding to the stabilised spike protein was higher than expected; and higher in molecular mass than that of the low levels of non-specific bound albumin which could be detected in some elution samples from nucleocapsid and Protein-G coated magnetic beads (Figure 3 ).

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The copyright holder for this preprint this version posted June 15, 2021. ;  Viruses such as influenza bind to specific glycan as part of their attachment and invasion mechanisms e.g. the viral envelope protein haemagglutinin recognises and binds sialic acid glycan residues [31]. Albumin is not a glycosylated protein [32] ; however, the increases in molecular mass seen here correspond to known advanced glycation end products (AGE) which form from blood saccharide reaction with long lived and abundant serum proteins. The AGE reaction occurs on specific lysine and arginine amino acid side chains of human serum albumin (see figure 4) . Glycation of serum proteins is more rapid and abundant than glycation of haemoglobin and has been used as biomarker of both diabetes and arthrosclerosis [33]. [34] demonstrated that Advanced Glycation End-product (AGE) modification of serum albumin could be detected by direct MALDI-ToF mass spectrometry via an increase in the averaged albumin peak molecular mass. This was reflected in a ratio consistent increase in mass of the albumin spectral singly and doubly charged peaks. We also detected similarly increased average mass of, MALDI-ToF MS determined singly and doubly charged, HSA peaks in our samples and the results are consistent with AGE glycation forms of HSA being preferentially captured by Spike protein.

By far the greatest at-risk group from dying following infection with SARS-CoV2 are those over 60 who developed COVID-19 symptoms; accounting for nearly 95% of all such deaths.

The next most dominate associations with severe symptoms and COVID-19 mortality are hypertension, obesity and type II diabetes [20,21,22,23]. The underlying pathological mechanism of these associations is unknown, but is likely to be multifactorial rather than a single determinant/marker [24] . Given our findings, it is likely that Spike protein variants with a stronger affinity to glycated/AGE'd serum albumin would enhance the pathogenic effects of SARS-CoV2 infections.

An unusual feature of the COVID-19 infection disease is micro thrombosis and localised disruption of the osmotic potential with pulmonary micro vascular dilation. Pulmonary thrombosis is common in sepsis induced ARDS. Coagulation dysfunction appears to be . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. common in COVID 19, and is detected by elevated D dimer levels. In fatal cases there is diffuse microvascular thrombosis, suggesting a thrombotic microangiopathy, and most deaths from COVID 19 ARDS have evidence of thrombotic disseminated intravascular coagulation [35] . This may explain some of the atypical or unexpected manifestations seen in the lung, such as dilated pulmonary vessels on chest CT, and episodes of pleuritic pain. Vascular enlargement is rarely reported in typical ARDS, yet was seen in most cases of COVID 19 ARDS.

Low circulating serum albumin is associated with pleural effusion, oedema and vascular constriction, as a result of serum albumin being sequested within the interstitial tissues.

However, in COVID-19 ARDS we see a lower overall serum albumin and yet pulmonary vascular dilation. Thus, where is the serum albumin being lost to?

The binding of albumin by SARS-CoV-2 has been postulated by Johnson et al [37] as a molecular contributor to fluid tissue-vascular imbalance giving rise to sceptic shock in COVID-19 cases. The possibility is that it is being caught locally within the pulmonary vasculature and contributing to the pathologic mechanism of local vascular dilation e.g.

within micro-thrombolytic clots found in the pulmonary blood vessels [38, 39] . Furthermore, deposition of glycated/AGE modified albumin has also been correlated with hyperinflammatory process seen in cardiovascular disease [34] .

In conclusion the stabilised/prefusion spike protein has a strong binding affinity for glycated/AGE'd albumin. The correlation of type 2 diabetes, obesity and age with greater probability of suffering from ARDS as a result of SARS-CoV2 infection that persist and progress; may in part be due to the Spike complex's affinity for glycated/AGE'd serum albumin. The ability to evade effective immune responses by hiding behind a coating of serum proteins, much like an octopus grabs shells and stones to hide from predatory sharks (Blue planet II 2017) [40] ; but in so doing it may cause tissue-vascular fluid physiological changes seen in COVID-19 ARDS even in those with subclinical pre-diabetes and metabolic diseases.

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The copyright holder for this preprint this version posted June 15, 2021. ;  17. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 15, 2021. ; Figure 1 A schematic of the capture and elution of binding proteins such, as immunoglobulins, is illustrated in panel 1. Plasma samples are mixed with magnetic beads coupled with Nucleocapsid (N-Protein), stabilised complete Spike (Spike protein) or G-protein. The magnetic particle processing rack can simultaneously process sixteen samples in Eppendorf tubes (1A). Using rotating disk containing magnets (1B) either side of the sample allows capture, removal of denuded sample, mixing and efficient wash off of non-specific absorption and final elution of the capture binder proteins (1C and D) in a consistent standardised process.

The eluted sample are spotted onto a MALDI-ToF mass spectrometry plate along with MALDI-matrix and subjected to mass spectral analysis following a standardised protocol and settings. Raw data is exported and analysed to give precise measurement of molecular mass of the eluted binder/complex proteins (e.g. Immunoglobulin light and heavy chains) along with intensity measurement for relative quantification purposes (Panel 2).

Example mass spectra of eluted sample from protein G bead captured proteins overlaid with Spike protein captured plasma proteins (panel A) and relative intensities of IgG1 heavy chains (IgG 1 Hc) recovered from the same samples by Protein G, nucleocapsid and stabilised spike protein (panel B); versus HSA (single: 1+ and doubly charged: 2+) recovered from the same samples by Protein G, nucleocapsid and stabilised spike protein (panel C).

Distribution of mass determined for captured and eluted human serum albumin (HSA) and the relative intensity of the increased mass albumins being bound: Panel A illustrates the mass variance of Protein G, Nucleocapsid (N-Protein) and stabilised complete Spike Protein along with average intensity. Panel B illustrates a lack of differences between patient groups plasma samples HSA binding by protein G, Nucleocapsid and stabilised complete Spike protein; whilst a clear preference of the S-protein to bind a higher molecular weight albumin from all samples. Blue represents data from SARS-CoV2 sero negative HCW, Orange from SARS-CoV2 HCW sero-positive and having recovered from with mild symptoms and Red sample data from convalescent patients recovering from COVID-19 ARDS.

Illustration of advanced glycation end product (AGE) as a result of human serum albumin Schiff base/ Amadori/Maillard reactions with blood circulating reducing monosaccharides (glucose and fructose), resulting in modification to exposed Lysine and Arginine amino acid side chains. The molecular mass effects of these AGE reactions on specific HSA residues is also detailed. 

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This study was undertaken by the Humoral Immune Correlates to COVID-19 (HICC) consortium, funded by the UKRI and NIHR; grant number G107217 (COV0170 -HICC: Humoral Immune Correlates for COVID-19). RKI is also funded by NISAD Ideell Förening (charitable association) Organisationsnummer 802528-6157We grateful acknowledge the loan of a prototype magnetic bead processing rack developed at the Francis Crick Institute London, UK, Bruker UK Ltd, Coventry for the loan of Microflex MALDI-Tof mass spectrometer and Dr Erika Tranfield and Dr Julie Green for technical support with running and data export from the Bruker Microflex.