key: cord-0721346-t39gkw0t
authors: Gerber, Gloria F.; Yuan, Xuan; Yu, Jia; Cher, Benjamin A.Y.; Braunstein, Evan M.; Chaturvedi, Shruti; Brodsky, Robert A.
title: COVID-19 Vaccines Induce Severe Hemolysis in Paroxysmal Nocturnal Hemoglobinuria
date: 2021-05-06
journal: Blood
DOI: 10.1182/blood.2021011548
sha: d50352bb71f01ff4e04e984fd7585feaf73f8cf4
doc_id: 721346
cord_uid: t39gkw0t

nan

Complement has emerged as a likely driver of the immune response and end-organ damage in coronavirus-19 . In patients with severe disease, deposition of terminal complement and microthrombosis have been observed in the lung, skin, kidney and heart. [1] [2] [3] [4] Recently, we demonstrated that the severe acute respiratory coronavirus 2 (SARS-CoV-2) spike protein leads to alternative pathway of complement amplification on cell surfaces through competition with complement factor H (CFH) for binding heparan sulfate. 5 Thus, in vitro, the SARS-CoV-2 spike protein can convert an inactivator surface to an activator surface on nucleated cells.

Two mRNA-based vaccines that lead to transient expression of the SARS-CoV-2 spike protein are highly efficacious in preventing severe infection. 6, 7 Reactions to these vaccines are generally mild; however, increased complement amplification could theoretically lead to more severe effects in diseases like paroxysmal nocturnal hemoglobinuria (PNH), where blood cells lack complement regulatory proteins. 8 Here, we describe significant adverse reactions to COVID-19 vaccines in four patients with PNH. We also present two PNH patients who received the vaccine without significant adverse effects or hemolysis.

Patient characteristics and reactions to the COVID-19 vaccines are shown in Table 1 . Patients were identified based on self-report of receiving the vaccine. Patients were aged 25-63 years, had PNH granulocyte clones of ≥80%, and had not received transfusions in the past year.

Reactions occurred from the day of administration to five days later and lasted one to six days. He also developed hemoglobinuria on post-vaccination days 1 and 2, associated with 2.7 g/dL hemoglobin decrease. Patient 3 is a 32-year-old woman with a 10-year history of PNH on ravulizumab with last dose four weeks prior to vaccination. She was also part of a clinical trial with danicopan, an oral complement factor D inhibitor. She missed two doses of danicopan immediately after her second vaccination due to concerns that the drug may interfere with vaccine potency; therefore, danicopan was not at therapeutic levels at the time of her reaction. Notably, she took danicopan throughout her first vaccination and did not experience breakthrough hemolysis. Approximately 12 hours after receiving the second dose of the Moderna mRNA-1273 (Moderna) COVID-19 vaccine, she had a fever (39ºC) and rigors. She presented to a local hospital, where she was noted to have a 3 g/dL hemoglobin decrease and received two units packed red blood cells.

Patient 4 is a 63-year-old man diagnosed with PNH 30 years ago, currently treated with ravulizumab. He experienced fatigue and darkening of his urine following his first dose of the Moderna vaccine. He had an approximately 1 g/dL hemoglobin decrease on labs three days following his first vaccination. Additionally, his total bilirubin rose to 7.1 mg/dL from baseline 2.4 mg/dL. Following his second dose, he noted fevers, diarrhea, vomiting, severe fatigue and dark urine. Labs one week later, as symptoms were resolving, showed >4 g/dL hemoglobin decrease from his baseline.

Based on these observations, we sought to evaluate whether the SARS-CoV-2 spike protein directly leads to increased hemolysis. Recent data suggests the SARS-CoV-2 spike protein binds heparan sulfate on nucleated cells 9 and amplifies the alternative pathway of complement through interference with the binding of CFH, an alternative pathway inhibitor. 5 However, CFH primarily binds sialic acid on human erythrocytes, 10 and mature erythrocytes express little heparan sulfate. 11 To test the effect of the SARS-CoV-2 spike protein on hemolysis, we performed erythrocyte lysis using PNH patient erythrocytes and acidified normal human serum (aNHS) with addition of the SARS-CoV-2 spike protein subunit 1 (S1) (see Supplemental Methods). Briefly, type O positive red blood cells from one PNH and control patient were collected. aNHS pre-incubated with and without S1 was added to the erythrocytes. Following incubation at 37°C for 1 hour, absorbance at 405 nm was measured in the cell-free supernatants. Hemolysis in each sample was compared to total water-induced lysis of the erythrocytes. S1 did not increase hemolysis of PNH erythrocytes as compared to aNHS alone ( Figure 1 ). Further, S1 does not appear to bind erythrocytes (data not shown). This suggests that post-vaccination hemolysis is not mediated by the direct effect of the spike protein. We postulate that strong complement amplification as a byproduct of the inflammatory response is responsible for the clinically observed hemolysis, as has been reported with other vaccines, infections, and surgeries.

In PNH patients on complement inhibition, strong complement amplifying conditions such as infection, surgery or pregnancy may trigger pharmacodynamic breakthrough (as previously defined by Risitano et al.) 12 Complement amplifying conditions lead to C3b accumulation on the cell surface; at high densities of C3b, C5 can assume a conformational change, disrupting the ability of eculizumab to inhibit terminal complement. 13, 14 Hemolysis following COVID-19 vaccination, which occurred in three patients on ravulizumab, suggests pharmacodynamic breakthrough. While many vaccines can lead to hemolysis and thrombosis in PNH, this effect is mitigated in most patients on complement inhibitors. 15 Ravulizumab, a new C5 inhibitor with a half-life four times longer than eculizumab, is reported to have significantly fewer instances of pharmacokinetic breakthrough hemolysis. 16 Further, three instances of breakthrough hemolysis occurred four weeks from the last ravulizumab infusion, making suboptimal C5 inhibition unlikely. As seen in patients 3 and 6, a proximal complement inhibitor, such as danicopan, may prevent breakthrough hemolysis precipitated by the vaccine; however, it is equally possible that the stronger immune response after the second vaccine dose was primarily responsible for the breakthrough hemolysis in patient 3.

This study provides insight into the mechanism of pharmacodynamic breakthrough precipitated by COVID-19 vaccination in PNH patients on ravulizumab. Similar disease flares may be anticipated in other complement-mediated disorders such as complement-mediated hemolytic uremic syndrome, cold agglutinin disease, catastrophic antiphospholipid syndrome, and HELLP syndrome. 17 As SARS-CoV-2 leads to a severe inflammatory state, the benefits of vaccinating patients with PNH likely outweigh the risks; however, clinicians and patients should be aware of this serious adverse effect, and patients should be educated to report any symptoms postvaccination. We recommend vaccination within 4 weeks of the last ravulizumab infusion and 1

week of eculizumab infusion and that patients maintain optimal hydration. Adverse reactions appear time-limited and can be managed with supportive care and transfusions as needed. Hemolysis is not increased with addition of the SARS-CoV-2 spike protein subunit 1 (S1) to PNH erythrocytes. Addition of 40% acidified normal human serum (NHS) with various concentrations of the spike protein to type O positive erythrocytes from a PNH patient (49% PNH red blood cell clone; 25% type III and 24% type II cells) and type O positive erythrocytes from a healthy control, incubated at 37º C for 1 hour. Water (H 2 O) serves as a positive control.

EDTA added to serum serves as an inhibitor of hemolysis. Data shown as mean ± SEM of duplicate wells.

Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: A report of five cases

Is the COVID-19 thrombotic catastrophe complementconnected?

Pulmonary Vascular Endothelialitis, Thrombosis, and Angiogenesis in Covid-19

Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition

Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine

How I treat paroxysmal nocturnal hemoglobinuria

SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2

Disturbed sialic acid recognition on endothelial cells and platelets in complement attack causes atypical hemolytic uremic syndrome

Heparan sulphate identified on human erythrocytes: a Plasmodium falciparum receptor

Anti-complement Treatment for Paroxysmal Nocturnal Hemoglobinuria: Time for Proximal Complement Inhibition? A Position Paper From the SAAWP of the EBMT

Complement inhibition at the level of C3 or C5: mechanistic reasons for ongoing terminal pathway activity

Incomplete inhibition by eculizumab: mechanistic evidence for residual C5 activity during strong complement activation

Thrombotic Events with Neisseria Meningitidis Vaccination in Patients with Paroxysmal Nocturnal Hemoglobinuria, UK Experience

Characterization of breakthrough hemolysis events observed in the phase 3 randomized studies of ravulizumab versus eculizumab in adults with paroxysmal nocturnal hemoglobinuria

Complementopathies and precision medicine

COVID-19 denotes coronavirus disease 2019, PNH paroxysmal nocturnal hemoglobinuria, LDH lactate dehydrogenase, AST aspartate amino transferase, RBC red blood cells

Additionally on danicopan, however two doses were missed immediately following vaccination