key: cord-0283600-8hlhp8i7
authors: Lo, Martin W.; Amarilla, Alberto A.; Lee, John D.; Albornoz, Eduardo A.; Modhiran, Naphak; Clark, Richard J.; Ferro, Vito; Chhabra, Mohit; Khromykh, Alexander A.; Watterson, Daniel; Woodruff, Trent M.
title: SARS-CoV-2 Triggers Complement Activation through Interactions with Heparan Sulfate
date: 2022-01-11
journal: bioRxiv
DOI: 10.1101/2022.01.11.475820
sha: 6a38a37eca6e3d35995ae3d878123723e0fdfb00
doc_id: 283600
cord_uid: 8hlhp8i7

The complement system has been heavily implicated in severe COVID-19 with clinical studies revealing widespread gene induction, deposition, and activation. However, the mechanism by which complement is activated in this disease remains incompletely understood. Herein we examined the relationship between SARS-CoV-2 and complement by inoculating the virus in lepirudin-anticoagulated human blood. This caused progressive C5a production after 30 minutes and 24 hours, which was blocked entirely by inhibitors for factor B, C3, C5, and heparan sulfate. However, this phenomenon could not be replicated in cell-free plasma, highlighting the requirement for cell surface deposition of complement and interactions with heparan sulfate. Additional functional analysis revealed that complement-dependent granulocyte and monocyte activation was delayed. Indeed, C5aR1 internalisation and CD11b upregulation on these cells only occurred after 24 hours. Thus, SARS-CoV-2 is a non-canonical complement activator that triggers the alternative pathway through interactions with heparan sulfate.

COVID-19 is a highly contagious respiratory infection caused by the severe acute 53 respiratory syndrome coronavirus 2 (SARS-CoV-2). In the last two years, this disease 54 has affected over 300 million individuals and caused over 5.4 million deaths (1). Thus, 55 unprecedented efforts have been put towards vaccine and drug development, but with 56 the possibility of new variants and the inevitability of future pandemics, a fundamental 57 understanding of severe COVID-19 is still needed. In this context, SARS-CoV-2 58

replicates in an unchecked manner and evades the immune system by exploiting 59 several inborn and acquired weaknesses (2, 3). At a critical mass, these virions then 60 trigger a hyperinflammatory response that results in acute respiratory distress 61 syndrome (ARDS) (4). Emerging evidence suggests that the complement system 62 plays a key role in this process (5-7). Indeed, complement activation has been 63 correlated with disease severity (8) and small case studies have shown that 64 complement inhibition can be effective in critical patients, prompting at least six anti-65 complement drugs to be taken to clinical trials (5). However, whilst in vitro mechanistic 66 studies have demonstrated that specific viral proteins can activate complement, the 67 relationship between SARS-CoV-2 and complement activation remains incompletely 68 understood. 69 70 Complement-mediated disease in COVID-19 appears to be confined to severely ill 71 patients who are unable to bring the virus under immunological control. In these 72 patients, SARS-CoV-2 exploits defects in the type 1 interferon system and replicates 73 in an unchecked manner, which at a critical mass, is believed to drive a form of 74 complement-mediated hyperinflammation (3). Indeed, evidence of complement 75 activation has been correlated with disease severity and includes serum C5a and C5b-76 9 concentrations (8), monocyte and granulocyte CD11b expression (9) which can be 77 due to C5aR1 activation (10, 11), and post-mortem immunochemistry (7, 12). These 78 features occur on the background of airway and intravascular complement synthesis 79 (13, 14) and are particularly prominent in individuals who are genetically prone to C5 80 cleavage (15), who have elevated mannose binding protein levels (16) However, SARS-CoV-2 S-protein did not generate complement activation products in 93 human serum without such cells or after heparan sulfate or factor H supplementation. 94

In addition, a more recent study found that the SARS-CoV-2 S and N proteins are able 95 to activate the lectin pathway via MASP-2 (22). Thus, early molecular studies using 96 viral proteins suggest that SARS-CoV-2 can directly activate the complement system, 97 but conclusive evidence for this with live virus is still outstanding. 98 99 Therefore, here we inoculated SARS-CoV-2 into lepirudin-anticoagulated human 100 blood and used ELISAs and flow cytometry to measure complement activation and 101 functionality respectively. We show that SARS-CoV-2 activates complement via the 102 alternative pathway by interacting with heparan sulfate, and in doing so causes 103 delayed leukocyte activation through C5a-C5aR1 signalling. inhibitor, 100g/mL; Ichorbio, Wantage, United Kingdom, #ICH4005)), EGCG 153 (heparan sulfate inhibitor, 100M; Sigma-Aldrich E4143-50MG), or pixatimod/PG545 154 (heparan sulfate mimetic, 100g/mL; synthesized in house (24)). This assay was 155 repeated with plasma isolated from whole blood after centrifugation at 2000g for 10 156 minutes at room temperature. For the flow cytometry experiments, whole blood was 157 mixed 1:1 with RPMI1640 (Gibco, Waltham, Massachusetts, #42401-018) and 158 inoculated with SARS-CoV-2 at a MOI of 0.1 or 1.0 or a mock solution (as above) and 159 incubated for 3 or 24 hours at 37°C with 5% CO2. Certain samples were pre-treated 160 with PMX205 (10M; synthesized as previously described (25)) or eculizumab (as 161 above) for 30 minutes at 37°C with 5% CO2. 

Next, we sought to determine if SARS-CoV-2-mediated complement activation was 258 sufficient to induce a functional response. As C5a stimulation of myeloid cells causes 259

C5aR1 internalisation and CD11b upregulation at the cell surface (10, 11), we 260 CD11b responses between SARS-CoV-2 inoculated blood at 3 (n = 4) and 24h (n = 6) expressed as a percentage change from mock inoculation (B, D, F). Boxes depict medians and inter-quartile ranges and have Tukey whiskers. MOI = multiplicity of infection; * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 using a one-way ANOVA and Dunnett's post-test or unpaired t test with two stage step-up 1% false discovery rate correction. Flow cytometry gating strategy and incidental findings are provided (Figure 3figure supplement 1 and 2) .

To investigate the functional role of the terminal complement pathway in mediating 311 leukocyte activation in response to SARS-CoV-2, we next pre-incubated whole blood 312 with a C5 inhibitor (eculizumab) and a C5aR1 antagonist (PMX205). At 24 hours post 313 inoculation, both drugs inhibited SARS-CoV-2-dependent C5aR1 internalisation and 314

CD11b upregulation in neutrophils and eosinophils with similar efficacy (figure 4a-b) . 315

By contrast, on monocytes, these drugs were only able to partially inhibit C5aR1 316 internalisation and did not lower CD11b upregulation (figure 4b). This latter finding 317 suggests that complement at the level of C5 is not involved in this process in Anti-C5/C5aR1 inhibitors eculizumab (100μg/mL) and PMX205 (10μM) were administered to lepirudin-anticoagulated whole blood (n = 4) prior to SARS-CoV-2 inoculation at MOI 1.0 for 24 hours and analysed with flow cytometry. Activation markers C5aR1 and CD11b were measured as MFI. Boxes depict medians and interquartile ranges and have Tukey whiskers. The dashed line represents the mock infection baseline. MOI = multiplicity of infection; MFI = median fluorescence intensity; * P<0.05, ** P<0.01, using a paired one-tailed t test. release (5), genetic susceptibility to complement activation (15-17), and local 361 complement synthesis (13). But in this regard, our participants were seronegative for 362 anti-SARS-CoV-2 antibodies, which would imply that the classical pathway was not 363 activated in this study. Additionally, given that C5a-mediated immune activation 364 typically occurs within 60 minutes (11, 26), the delayed response requiring 24 hours 365 in our model is consistent with the gradual progression (4) and upregulation of CD11b 366 on leukocytes in severe cases (9). Thus, these results strongly support the emerging 367 paradigm of heparan sulfate-and alternative pathway-mediated disease in severe 368

370

When placed in the context of in vivo viral titres, our results also suggest that SARS-372

CoV-2-mediated complement activation is localized to organs that can support 373 replication. On average COVID-19 patients have median viral titres of ~10 6 RNA 374 copies/ml in their airways; as determined from nasopharyngeal swaps, sputum, saliva, 375 and bronchoalveolar lavage fluid; and ~10 3 RNA copies/ml in their serum, in which the 376 former is at least 10 times higher in severe cases compared to that in mild cases (27, 377 28). By comparison, in this study whole blood inoculated with SARS-CoV-2 at MOI 0.1 378 and 1.0 was exposed to virion concentrations of ~3.5-5.5 x 10 5 and ~3.5-5.5 x 10 6 379 FFU/ml respectively. Thus, complement activation in severe COVID-19 is most likely 380 localised to tissues that support replication (e.g., lung parenchyma) with changes in 381 plasma complement occurring as a secondary phenomenon. This implies that anti- Mounting evidence suggests that complement plays a key role in its most severe form 406 and here we show that SARS-CoV-2 interacts with heparan sulfate to activate the 407 alternative pathway, which ultimately drives innate leukocyte activation through C5a-408 C5aR1 signalling. In doing so, these findings support the use of targeted anti-409 complement treatments in severe COVID-19. Participants were recruited from the local Brisbane area and had no history of COVID-19, no history of acute illness or vaccination in the last 2 weeks, no immunodeficiencies or autoinflammatory/autoimmune conditions, and were not on any immunomodulatory medications (e.g., corticosteroids). No significant sex or age differences were found between the cohorts at different time points for the ELISA and flow cytometry experiments. Upregulation of (A) CD16 and HLA-DR on monocytes and (B) CD11b on neutrophils exposed to SARS-CoV-2. SARS-CoV-2 inoculated lepirudin-anticoagulated whole blood was analysed with flow cytometry at 24 (n = 5-6) and 3 hours post-inoculation (n = 4) respectively. Surface markers were quantified as MFI. MOI = multiplicity of infection; MFI = median fluorescence intensity; * P<0.05, *** P<0.001 using a oneway ANOVA and Dunnett's post-test.

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Representative plots of SARS-CoV-2 inoculated whole blood stained with fluorophore-antibodies and a viability dye for flow cytometry (A-I). For comparison, representative plots of virus-naïve whole blood stained in the same fashion are also provided for the monocyte gates (J-L)

sulfuric acid stop solution were then added prior to absorbance analysis

/130A is a human covalence serum obtained from National Institute for Biological 817 Standards and Control

Initially, a sample size of 3 was determined to be most appropriate for this study. This 794 was decided in reference to previous ex vivo complement experiments that have been 795 conducted with the lepirudin whole blood system (34). However, we also generated 796 additional biological replicates according to donor availability and achieved final 797 sample sizes of between 3 and 6. 798 799Each experiment was performed on one occasion and all data reflects biological 800replication except for that in supplementary figure 3, in which data points reflect 801 technical replication. Biological and technical replication was defined as the replication 802 of an assay in a blood sample from a distinct or the same donor respectively. One 803 outlier was excluded in supplementary figure 2a as its mock data point was more than 804 3 standard deviations from the mean. No other data was excluded. 805 806Supplementary Methods 5: SARS-CoV-2 Serology Testing 807Trimeric SARS-CoV-2 spike protein was coated at 2 μg/ml on an ELISA plate overnight 808(35). Plates were blocked for 1 hour at room temperature with a blocking buffer (PBS 809 containing 0.05% Tween-20 and milk sera diluent/blocking solution (Seracare, Milford, 810Massachusetts)). Plasma from the whole blood used for the C5a ELISA study, a 811 positive plasma control NIBSC 20/130 and a pre-COVID-19 serum negative 812 control were serially diluted in blocking buffer and added to the plate for 1 hour at 37 813 °C. Plates were washed and probed by goat anti-human HRP antibody (1:2500) in 814blocking buffer for 1 hour in 37 °C. Tetramethylbenzidine substrate solution and 815