Page 92 - Haematologica May 2022

J. Yu et al.
Figure 4. The Bb level is increased in COVID-19 patient serum and associated with disease severity. Serum Bb level was significantly elevated in COVID-19 patients who required minimal oxygen support, high flow nasal cannula oxygen therapy (Hi-Flow oxygen) and mechanical ventilation (Vent) as compared to pooled normal human serum (NHS) and healthy controls (healthy ctrl). Patients requiring mechanical ventilation also had significantly higher serum Bb level than patients with min- imal oxygen support (P<0.05), suggesting greater alternative pathway of complement dysregulation. All experiments were run in duplicate. CVF: cobra venom factor (used as a positive control).
Increased alternative pathway of complement activation is associated with COVID-19 disease severity
We utilized standard enzyme-linked immunosorbant assay (ELISA) to measure the factor Bb level in COVID-19 patient serum. Factor Bb, which results from cleavage of factor B by factor D, is a biomarker of APC activation. Regardless of disease severity, the serum level of Bb was significantly higher in COVID-19 patients compared to the healthy controls. We also found that COVID-19 patients requiring intubation (WHO score 7) had significantly high- er Bb levels than those requiring minimal oxygen support (WHO score 4) (Figure 4). These observations suggested that increased APC activation is associated with disease severity in COVID-19.
SARS-CoV-2 spike proteins compete with complement factor H for cell surface heparan sulfate binding
We previously demonstrated that SARS-CoV-2 spike proteins (both subunit 1 and 2) bind heparan sulfate on the cell surface.17 Heparan sulfate also serves as a necessary co- factor for binding of SARS-CoV-2 spike proteins to the angiotensin receptor 2 (ACE2).23 CFH, a negative regulator of APC, also utilizes glycosaminoglycans, such as heparan sulfate, and a2,3 N-linked sialic acid residues for binding to nucleated cells; thus, we hypothesized that SARS-CoV-2 spike protein competes with CFH for binding to heparan sulfate and its tissue specific, more highly-sulfated variant, heparin. In order to evaluate whether SARS-CoV-2 spike proteins block CFH from binding to heparan sulfate, we compared the heparin-binding activity of CFH in the pres- ence and absence of the SARS-CoV-2 spike proteins using heparin-linked beads. CFH alone bound to the heparin- beads with high affinity (Figure 5A, lane 1). In the presence of SARS-CoV-2 S1 and S2, binding of CFH to the heparin- beads was markedly reduced (Figure 5A, lanes 2 and 3).
We also compared the heparin-binding ability of SARS- CoV-2 spike proteins in the presence and absence of CFH. S1 alone showed strong binding to heparin-beads and retained high heparin-binding activity in the presence of CFH (Figure 5B). S2 bound to heparin-beads with similarly high efficiency under both conditions (Figure 5C). These results indicated the SARS-CoV-2 spike proteins have high-
er binding affinity for heparin than CFH and interfere with the binding of CFH to heparin.
SARS-CoV-2 mRNA vaccine does not markedly increase complement activity in healthy individuals
The mRNA COVID-19 vaccines employ the SARS-CoV- 2 spike protein as an immunogenic target. Given that SARS-CoV-2 spike proteins activate complement in vitro, concern arises whether the COVID-19 vaccine could also trigger transient complement dysregulation in vivo through generation of the spike protein.
In order to test this, we obtained serum samples from five healthy individuals who received both doses of the Pfizer-BioNTech (BTN162b2) COVID-19 vaccine at three time points: before the vaccine (baseline), 24 to 48 hours after receiving the first vaccine dose, and 24 to 48 hours after the second dose. We measured the serum level of fac- tor Bb in these individuals pre- and post-COVID-19 vacci- nation. Two of five individuals showed significantly higher serum Bb levels from their baseline after receiving the COVID-19 vaccine (Figure 6). Notably, these two individ- uals experienced side effects post vaccination including fever, headache and fatigue. We next performed functional assays to assess for cell surface complement amplification post-vaccination. On average, serum collected after the first vaccine dose did not lead to increased C5b-9 deposi- tion on the surface of TF1PIGAnull cells compared to the individual’s serum pre-vaccination. The second dose of the vaccine led to an 11% increase in the C5b-9 deposition. All individuals had negative mHam results at baseline, which remained negative after receiving the COVID-19 vaccine (data not shown). These results from the functional assays demonstrate that the Pfizer-BioNTech SARS-CoV-2 mRNA vaccine does not sufficiently alter complement regulation in healthy individuals.
Discussion
Previously, we showed that the SARS-CoV-2 spike pro- tein dysregulates the alternative complement pathway in vitro by interfering with CFH function on the cell.17 Here,
1100
haematologica | 2022; 107(5)