Page 95 - Haematologica May 2022

Complement dysregulation & COVID-19
COVID-19, at a time when the SARS-CoV-2 viral load is decreasing,3,31 as opposed to measuring cell surface C5b-9 deposition within minutes after adding spike protein to normal human serum. We also demonstrated that mHam positivity (measuring the activation of all complement pathways) from SARS-CoV-2 infected patients is associat- ed with the need for mechanical ventilation; however, we did not find as strong a correlation between cell surface C5b-9 and the mHam as we did after supplementing nor- mal human serum with the spike protein.
Virtually all patients had elevated levels of Bb in their serum, even two of the healthy subjects after COVID-19 mRNA vaccination, suggesting that APC activation is an early event in the pathogenesis of SARS-CoV-2 infection. This is in agreement with the prominent role for APC found in proteomics studies, which showed increased complement factor B (CFB) levels in serum from severe COVID-19 patients.32 CFB deposition was also observed in the lung tissue of COVID-19 patients, and a CFB inhibitor blocked the C3a generated by infection of respi- ratory epithelial cells with SARS-CoV-2.33,34 Pekayvaz et al.35 further showed upregulation of complement factor D (CFD), produced mainly in adipocytes, in monocytes of severe COVID-19 patients. Ma et al.36 demonstrated that enhanced activation of the APC is associated with mark- ers of endothelial injury and hypercoagulability in severe COVID-19 patients as compared to other non-COVID-19 patients admitted to the intensive care unit with acute res- piratory failure.
C5b-9 deposition induced by COVID-19 patient serum is blocked by both a terminal complement inhibitor (anti- C5 antibody) and an alternative pathway specific inhibitor (factor D inhibitor, ACH145951). The factor D inhibitor was more effective in blocking C3c deposition induced by COVID-19 patient serum as compared to the anti-C5 anti- body. These results are supported by observations from case series of eculizumab, a monoclonal anti-C5 antibody, in which treated COVID-19 patients showed significant improvements in clinical parameters.12,13 However, the phase III trial of eculizumab (clinicaltrials gov. Identifier: NCT04355494) in COVID-19 patients on mechanical ven- tilation was paused due to interim analysis of 122 patients showing that the drug did not meet its prespecified effica- cy outcome of survival on day 29. Final results from this trial are eagerly anticipated as are those for COVID-19 patients who are hospitalized but not on mechanical ven- tilation. COVID-19 patients may derive benefit from com- plement inhibition early in their disease course or from more proximal complement inhibition.
A comparative study of eculizumab versus AMY-101, an upstream C3 inhibitor, in a small number of patients showed that both decreased inflammatory markers and led to improvements in lung functions. The three patients who received AMY-101 demonstrated greater reduction in plasma levels of C3a, sC5b-9 and CFB as compared to patients who received eculizumab.37 This limited clinical data in addition to our in vitro results suggests that proxi- mal complement inhibitors may be more effective than terminal inhibitors in reducing COVID-19 disease severi- ty. Notably, treatment of six severely ill COVID-19 patients with Narsoplimab, a monoclonal antibody against MASP-2 inhibiting lectin-pathway activation, showed rapid reduction in serum inflammatory markers and survival in all patients.38 In addition to the COVID-19 infection itself, there are likely multifactorial contributions
from tissue damage, secondary infections, and thrombo- sis, leading to complement activation from all pathways. Further studies comparing different complement inhibitors would be valuable to identify the most appro- priate therapeutic targets.
The SARS-CoV-2 spike protein interferes with the func- tion of CFH and this is likely an early event in the patho- genesis of COVID-19. In our prior work, we found that addition of purified CFH protein to serum treated with the SARS-CoV-2 spike protein decreased C3c and C5b-9 dep- osition on the cell surface.17 Here, we used competitive immunoprecipitation experiments to show that the SARS- CoV-2 spike protein directly blocks CFH from binding to heparin, which may explain the APC dysregulation observed in COVID-19 infection (Figure 5). When binding to glycosaminoglycans on cell surface, including heparan sulfate, and a2-3 N-linked sialic acid residues, CFH achieves a more active conformation that allows for C3b binding.39 Interestingly, genetic variants in CFH, that occur in the same region where factor H binds heparan sulfate, have been identified as an important risk factor for mor- bidity and mortality from COVID-19.27
COVID-19 vaccines lead to the transient expression of the SARS-CoV-2 spike protein and are effective in pre- venting severe infection.40,41 Our vaccine studies (Figure 6) are reassuring that mRNA vaccines should not induce clin- ically significant complement amplification in healthy individuals, as suggested by the negative results from our functional assays; however, more data is necessary in patients with disorders of complement regulation, such as paroxysmal nocturnal hemoglobinuria (PNH), aHUS, CAPS, HELLP and cold agglutinin disease.42 Relapse of aHUS has been reported in patients with COVID-19 infec- tion,43 and PNH patients have experienced adverse reac- tions to COVID-19 vaccines including severe hemolysis and need for blood transfusions even while on a C5 inhibitor.44 This evidence suggests that although comple- ment activation induced by COVID-19 vaccines is well- controlled in healthy individuals, patients with disorders of complement regulation could be at higher risk for adverse reactions to vaccine.
Limitations of our study are that we received a limited amounts of patient serum for experiments and were unable to test other complement markers such as C4d deposition on the cell surface. We also had limited access to clinical information from which to draw robust conclu- sions regarding the association of complement activation with clinical parameters. Further, serum sample collection was not standardized and occurred at different time points from the initial diagnosis of COVID-19 and hospital admission. For example, in one of the two patients who died due to multiorgan failure from COVID-19, serum was collected near the end of his clinical course, at which point peak amplification of complement may have passed. Finally, we do not have serial samples from patients to estimate the persistence of complement activation over time. In future studies, it will be important to do serial mHam, Bb, and surface C5b-9 deposition studies starting soon after infection and correlating with SARS-CoV-2 viral load.
In summary, we showed that COVID-19 patient serum can induce complement dysregulation on cell surfaces that tracks with disease severity. Our previous data showed that the SARS-CoV-2 spike proteins convert inactivator surfaces to activator surfaces. Taken together, we postu-
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