Page 88 - Haematologica May 2022

J. Yu et al.
system consists of three major pathways – the classical, lectin and alternative pathways (APC). The three path- ways converge on the cleavage of C3 and subsequently C5, which produces anaphylatoxins (C3a and C5a, respec- tively) that are associated with the acute inflammatory response and thrombosis.16 The membrane attack complex (C5b-9) is the terminal product of complement activation.15
Evidence for the role of complement in COVID-19 con- tinues to emerge, but understanding of the underlying mechanisms remains incomplete. Recently, we demon- strated that the SARS-CoV-2 spike protein (subunits 1 and 2) converts inactivator surfaces into activator surfaces through interference with the function of complement fac- tor H (CFH), a critical negative regulator of APC on host cells.17 These studies were performed by adding recombi- nant spike protein to normal human serum (NHS) and measuring complement-mediated killing on the surface of nucleated cells. Complement inhibition with C5 and factor D inhibitors effectively prevented C5b-9 accumulation induced by the SARS-CoV-2 spike proteins.17 In order to confirm that complement is a rational target for treating COVID-19, it is important to show that serum from patients infected with SARS-CoV-2 also displays comple- ment dysregulation. In this study, we evaluated cell surface C5b-9 deposition and complement-mediated cell killing induced by COVID-19 patient sera in the modified Ham (mHam) test5,18,19 and showed that impaired complement regulation correlates with COVID-19 disease severity and can be mitigated with complement inhibitors.
Methods
Patients and samples collection
Serum samples and a limited clinical data set were obtained for 58 COVID-19 patients from the Clinical Characterization Protocol for Severe Infectious Diseases repository (clinicaltrials gov. Identifier: NCT04496466), approved by the Johns Hopkins Institutional Review Board. Samples were collected between April and May 2020. One sample collected outside of the patient’s hospitalization for COVID-19 was excluded. We also recruited one COVID-19 patient requiring mechanical ventilation and five healthy individuals who received the Pfizer-BioNTech (BTN162b2) COVID-19 vaccine between November and December 2020. Informed written consent for sample use from the Complement Associated Disorders Registry study was obtained. Blood was collected by venipuncture in serum separator tubes.
Date of COVID-19 diagnosis was based on the earliest known positive SARS-CoV-2 nucleic acid amplification test from a nasopharyngeal swab or time an infection flag was placed on the patient’s chart. Severity of COVID-19 was graded based on World Health Organization (WHO) eight-point ordinal outcome scale.20
The modified Ham test
The mHam test was used to assess complement-mediated cell killing of TF1PIGAnull cells induced by patient serum as previous- ly described.5,17–19,21 The assay is detailed in the Online Supplementary Appendix.
Detection of complement activity by flow cytometry
C5b-9 and C3c deposition after incubation of TF1PIGAnull cells with COVID-19 patient serum was measured by flow cytometry as previously described.5,17,21 The assay is detailed in Online Supplementary Appendix.
Complement inhibition in patient serum
Patient serum was incubated with 1 μM factor D inhibitor (ACH145951, Achillion Pharmaceuticals) or 50 μg anti-C5 mono- clonal antibody (anti-C5Ab, Alexion pharmaceuticals) and then added to cells. C5b-9 and C3c deposition was measured by flow cytometry.
Quantification of serum factor Bb by enzyme-linked immunosorbant assay
Factor Bb level in patient serum was measured using MicroVue Bb Plus EIA kit (Quidel). NHS preincubated with 20 μg/mL cobra venom factor (Complement Technology) served as a positive con- trol.
Competitive heparin binding assay
Immunoprecipitation of His-tagged SARS-CoV-2 spike protein subunit 1 (S1, RayBiotech) and subunit 2 (S2, RayBiotech) was performed using Heparin-Sepharose beads (BioVision). We incu- bated 0.2 M S1 or S2 overnight at 4°C with 80 μL beads in the presence and absence of 0.2 M CFH protein (Complement Technology) in phosphate buffered saline. After washing, the beads were denatured with LDS buffer (Invitrogen) and reducing agent (Invitrogen) at 70°C for 15 minutes and then centrifuged. The supernatants were collected for western blotting. We used 10 μL of input protein solution for western blotting.
We loaded 10 μL reduced proteins to mini-PROTEIN TGX Gels (Bio-Rad Laboratories) and then transferred to PVDF membranes. The membrane was probed with anti-CFH antibody (1:1,500, Cat. Sc-53073, Santa Cruz Biotechnology) or anti-His antibody (1:2,000, Cat. Sc-53073, Santa Cruz Biotechnology). The blot was incubated with HRP-linked anti-mouse immunoglobulin (Ig) G (1:10,000, Cat. 7076S, Cell Signaling Technology) and imaged with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher).
Statisticalanalysis
Data was summarized as mean ± standard error (SE). c2 test was used to compare the rates of mHam positivity across WHO groups. Student’s t-test was used to evaluate differences between unpaired groups. P<0.05 was considered statistically significant.
Results
Patient characteristics
All 58 patients studied required hospitalization for their COVID-19 illness (WHO score 4-8). Thirty-two patients required minimal oxygen support (WHO score 4), seven required high flow nasal cannula oxygen therapy (WHO score 5), 17 received mechanical ventilation and additional organ support (WHO score 7), and two died (WHO score 8) (Table 1). Five healthy individuals were recruited at the same hospital system and their serum samples post COVID-19 vaccination were tested for complement activa- tion.
COVID-19 patient serum induced complement-mediated cell killing
The mHam measures the ability of a nucleated cell to protect itself from complement-mediated cell killing in vitro in the absence of downstream cell-surface complement regulators, CD55 and CD59.18,21 Thus, mHam exposes defects in complement regulation in patient serum. This assay has been validated in complement-mediated disor- ders such as aHUS, HELLP and CAPS.5,18,21,22 The mHam
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