A Significant Contribution of the Classical Pathway of Complement in SARS-CoV-2 Neutralization of Convalescent and Vaccinee Sera

In December 2019, a novel beta-coronavirus, SARS-CoV-2, was identified in Wuhan, China, representing the etiological agent that caused the COVID-19 pandemic. Symptoms of the disease included fever, dry cough, dyspnea, fatigue, and lymphopenia in most patients, with viral pneumonia and severe acute respiratory syndrome in more severe cases (1, 2). SARS-CoV-2–infected individuals were shown to produce anti-nucleocapsid Ab and anti-spike Abs, with IgM being the dominant isotype peaking at 9 d after symptom onset and quickly class switching to IgG and IgA around day 14 and plateauing 6 d later (3–7). Convalescent individuals were found to neutralize SARS-CoV-2 at serum dilutions of 1:40 to 1:80 in live virus neutralization assays, indicating that Abs offer putative protection (3). Infected individuals at 4 d after symptom onset had undetectable IgM and IgG titers, but by 9 and 20 d after symptom onset, IgG neutralization titers reached 1:80 and 1:1280 whereas the IgM neutralization titers reached 1:80 and 1:320, respectively (8). Thus, SARS-CoV-2 neutralization in serum appears to peak at ∼20 d after symptom onset, after which it stabilizes and is maintained for at least 6 mo (8–11). As more studies were reported, most convalescent serum samples showed 50% neutralizing titers of two to three orders of magnitude against wild-type ancestral SARS-CoV-2 (Wuhan isolate) (12).

Neutralization assays against SARS-CoV-2 are emerging as a reliable correlate of protection to infection, or reinfection with homologous virus strains. Challenge studies in rhesus macaques showed that passively immunizing naive macaques requires an infusion of a neutralizing Ab titer of at least 1:50 for protection (13, 14). Recent work suggests that a neutralizing Ab titer of 20 and 3% of convalescent serum will provide 50% protection against symptomatic and severe disease, respectively (15). A separate study suggested that after mRNA-1273 vaccination IC₅₀ neutralization titers of 10 will provide 78% protection against ancestral SARS-CoV-2, and IC₅₀ of 100 will provide 91% protection from infection using a pseudovirus neutralization assay (16).

Human and animal immune sera have been used for decades as a surrogate marker of protection against virus infection by showing that sera from immunized animals/humans can inactivate viruses and bacteriophages in vitro (17–23). Immunity was indicated by demonstration of reduction in virus plaque formation, where immune sera was cocultured with viral stocks, which was then used to infect susceptible cell lines, in what is known as a plaque reduction neutralization test (18). In these studies, there was considerable variability in the methods employed in their protocols: naive and immune sera were used (17, 18, 20, 21), heat-inactivated (HI) sera were used (18, 19, 22), or HI sera reconstituted with complement proteins (23) were used in neutralization assays.

Early on, Eduard Buchner, Jules Bordet, and Paul Ehrlich recognized that a heat-labile factor existed in immune sera, which was a nonspecific effector of Ab-mediated lysis of bacteria and viruses (24–29). This factor was coined with the term “complement” by Ehrlich, who wanted to indicate that the heat-labile factor “complemented” the activities of specific Abs. However, these studies did not explore the importance of complement in terms of enhancing neutralizing activity in the context of various virus infections. Various neutralization protocols show a common trend to use HI sera. However, a thorough review of the published literature does not document the rationale or the purpose of heat inactivation (HI). HI may reduce potential adverse effects on target cell lines, but may also inactivate viral particles (30). It has been suggested that HI may result in denaturation and aggregation of Igs, with loss of Ag binding reported in some studies but not in others (31–35). As a result, neutralization assays that rely on Ab structure and binding have the potential to be negatively impacted by HI and thus their neutralizing capabilities could be underestimated.

Historically, HI was done at 56°C for 30 min to primarily inactivate the complement system by denaturing the heat-liable proteins C2 and factor B, which can cause interference with experimental assays (36, 37). Although there is speculation that HI serves to reduce contaminants from sources such as bacteria, viruses, and cytokines, there is little literature to support theses suppositions. HI nevertheless has now become a standard operating procedure to test serum neutralization of pathogens across laboratories, and it was adopted by the World Health Organization laboratory manual for influenza serological assays (38).

A major mechanism by which Abs neutralize viruses is by blocking virus entry through their cellular receptors by steric interference (39–41). Abs, however, can also have effector functions after binding to the virus through the complement pathway. The importance of complement has been demonstrated for West Nile, Nipah, vaccinia, hepatitis C, as well as other viruses, where Ab-dependent complement activation significantly increased the neutralization by sera of the virus (42–47). Complement activation is induced specifically through the presence of a glycosylated Asn²⁹⁷ in the constant H chain 2 region of the Ab, which is bound by C1q (the first component of the classical complement cascade) (48). Removal of this oligosaccharide was shown to render Abs, mainly IgG, sensitive to proteases, alter Ab conformation, and prevent proper C1q binding (48, 49). A single pathogen-bound IgM or several pathogen-bound IgG Abs in close proximity are required to trigger the complement cascade. The Ab binding induces a conformational change revealing the complement binding region on the constant H chain 2, leading to the neutralization of the pathogen via activation of the classical pathway (50–52). Virus-bound Abs serve as the scaffold for complement activation, which enhances viral neutralization (51, 53). The mannose-binding pathway, instead, works as part of the innate immune system, where mannose-binding lectins directly bind mannose on the pathogen, which then trigger complement activation. Lastly, the alternative pathway relies on the spontaneous C3 activation and buildup on target cells (51). Viral neutralization by complement can occur through opsonization and aggregation as well as by viral lysis through the membrane attack complex (MAC) (52). Importantly, Ab-mediated viral lysis is theoretically independent of the specific recognition of epitopes on the spike protein that are involved in recognizing the angiotensin converting enzyme 2 receptor.

In the current study, we investigated the neutralization activity of SARS-CoV-2 of convalescent and vaccinee sera and explored the relative contribution of the complement pathway arms are to the neutralizing activity found in ex vivo serum.

Healthy subjects and individuals with COVID-19 infection as diagnosed by positive nasopharyngeal SARS-CoV-2 PCR were recruited. Informed consent was obtained for blood draws and/or leukapheresis using Institutional Research Ethics Board–approved protocols (St Michael’s Hospital/Unity Health Toronto REB20-044, University of Toronto HRP no. 00028628, University of Toronto RIS Human Protocol no. 23901). COVID-19–negative controls had no history of viral infection and had negative serology (IgG) for SARS-CoV-2 spike glycoprotein receptor-binding domain (RBD) and nucleocapsid phosphoprotein by ELISA as previously described (54). All research on human subjects was done in compliance with the Declaration of Helsinki.

The following clinical definitions were used: unexposed, samples taken and stored prior to the SARS-CoV-2 pandemic in 2018 or earlier; convalescent asymptomatic, subjects who reported no symptoms but had a previously positive nasopharyngeal SARS-CoV-2 PCR (samples were obtained a minimum of 14 d after a negative PCR test); convalescent mild, subjects who recovered from COVID19 and had a SARS-CoV-2 PCR-positive test with symptoms that allowed recovery at home (samples were obtained a minimum of 14 d after a negative PCR test); convalescent moderate, subjects who recovered from COVID19 and had a nasopharyngeal SARS-CoV-2 PCR-positive test with symptoms that required hospitalization but no intensive care unit (ICU) admission (samples were obtained a minimum of 14 d after a negative PCR test); convalescent severe, subjects who recovered from COVID-19 and had a nasopharyngeal SARS-CoV-2 PCR-positive test with symptoms that required hospitalization and ICU admission (samples were obtained a minimum of 14 d after a negative PCR test).

Serum samples were incubated in a water bath set to 56°C for 30 min, then allowed to cool to room temperature before use in experiments.

Samples was treated with 15 U/ml cobra venom factor (CVF; Quidel, A600) for 90 min in a water bath set to 37°C.

Vero E6 cells (American Type Culture Collection, CRL-1586) were cultured in D-10 (DMEM supplemented with 10% HI FBS, 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM l-glutamine), after which 0.06 × 10⁶ Vero E6 cells per well were seeded into a 96-well flat-bottom culture plate and rested overnight. Serum samples were serially diluted in serum-free DMEM and incubated with 100 median tissue culture infectious dose U of SARS-CoV-2-SB2-PB clone 1 for 1 h at 37°C with 5% CO₂. The Vero E6 cells were then inoculated with the sample/virus coculture for 1 h at 37°C with 5% CO₂. After 1 h, the inoculum was removed and D-2 (DMEM supplemented with 2% HI FBS, 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM l-glutamine) was added to the wells. The cells were incubated for 5 d at 37°C with 5% CO₂ and cytopathic events were evaluated by visually examining the cells under a phase-contrast microscope. For each experiment samples were assayed in quadruplicates. Results were analyzed using the Prism software package (GraphPad Software, La Jolla, Ca) with the four-parameter logistic nonlinear regression analysis to determine IC₅₀ values. IC₅₀ was defined as the serum dilution at which 50% of the wells were negative for cytopathic events. All manipulations with live SARS-CoV-2 were performed in the Combined Containment Level 3 Unit at the Temerty Faculty of Medicine, University of Toronto.

Sera were incubated with 100 mM mannose (Sigma-Aldrich) for 30 min at 37°C to sequester free mannose-binding lectins as previously described (55).

Neutralizing sera were incubated with protein G–Sepharose beads (Thermo Fisher Scientific) to deplete the sera samples of IgG Abs for 1 h at 4°C. The sera were then centrifuged at 500 × g for 10 min to pellet the beads, and the supernatant was used for subsequent experiments.

Neutralizing sera were incubated with 10 μg/ml of an eculizumab biosimilar anti-C5 Ab (clone h5G1.1, Bio-Rad) for 1 h at 4°C.

Neutralizing sera were incubated with 10 μg/ml anti-human complement factor B AF2739 (R&D Systems) for 1 h at 4°C (56).

Total C3 and C4, along with their activation products, were measured in serum using a commercial immunoturbidimetric assay (Roche Diagnostics C3C-2 kit 3001938322c501V9.0). In this method, 10 μl of serum was diluted and loaded into the COBAS c311 analyzer according to the manufacturer’s protocol. The proteins of interest form a precipitate when mixed with a goat anti-human C3 or C4 Ab in the reaction. The formed complex is determined turbidimetrically at 340 nm. These assays do not distinguish intact C3 and C4 from cleaved C3 and C4.

Fifty percent complement hemolytic (CH₅₀) was measured in serum using an automated homogeneous liposome-based assay (Fujifilm Wako Pure Chemical). For this assay, 10 μl of serum was diluted with 250 μl of buffer R1 and loaded into the Hitachi 717 analyzer, and the automated CH₅₀ protocol was run following the manufacturer’s protocol. Briefly, liposomes containing entrapped glucose-6-phosphate (G6P) dehydrogenase react with Abs to form a complex that activates the classical pathway of the complement system. When activated, the MAC breaks the liposome membrane, releasing G6P dehydrogenase, which reacts with NAD and G6P in the assay reagent to create a product that is measured at 340 nm spectrophotometrically. An increase in absorbance is proportional to the complement activity in the sample.

All statistical tests were performed using GraphPad Prism version 9.3.1 for Windows (GraphPad Software, San Diego, CA). Data are presented as mean ± SD.

Because serum complement can be inactivated by heat treatment, we compared live SARS-CoV-2 virus microneutralization assays using HI and non-HI convalescent sera from a cohort of subjects who recovered from SARS-CoV-2 infection and who had varying levels of disease severity, ranging from asymptomatic infection to ICU admission. The loss of complement function after HI was verified using the CH₅₀ test (Fig. 1G). For HI serum, which is what standard neutralization assays use, convalescent sera of severe cases displayed significantly higher mean neutralization titers compared with others with less severe disease (Fig. 1A, 1B). Mean serum IC₅₀ titers in individuals who convalesced after severe illness were 275.5 ± 209.2 versus 33.35 ± 26.34 for asymptomatic, 21.80 ± 30.01 for mild, and 14.09 ± 18.71 for moderate infection. Thus, individuals who recovered from severe disease had significantly higher neutralization titers than did those with mild and moderate infection (p = 0.0001 and 0.0028 respectively), whereas those with nonsevere illness had similar levels of neutralization.

Using the identical samples without prior HI of serum, we found that the neutralization titers from all participants were significantly greater when compared with HI serum (mean IC₅₀ 75.92 ± 52.52 for asymptomatic, 79.86 ± 47.76 for mild, 72.92 ± 46.89 for moderate, and 654.7 ± 488.2 for severe) (Fig. 1C, 1D), with the maintenance of the trend of severe sera showing greatest neutralization. Of note, one of the five unexposed control sera collected in 2018 showed a low-level SARS-CoV-2 neutralization titer with an IC₅₀ of 20, which was lost when the sample was HI. Thus, HI inhibits some aspects of in vitro live virus microneutralization.

To determine that non-HI sera had no artifactual effect on target cells used in the assay, human sera from five participants were split into two aliquots, one of which was HI, and subsequently cultured with Vero E6 cells, with the highest concentration of sera being 1:20, matching the lower dilutions used in our neutralization assay dilution series. We found no toxic effects of ex vivo sera on Vero E6 cells, which maintained 100% confluency and proper morphology in 5-d cultures (Fig. 1E). The effect of HI on Ab aggregation/denaturation was also evaluated using a published SARS-CoV-2 spike-reactive mAb, clone C23 (57). Serial dilutions of C23 in three different SARS-CoV-2 nonimmune participant sera with or without prior HI were used in neutralization assays (57). The untreated and the HI Ab showed comparable IC₅₀ titers (Fig. 1F). Thus, untreated human sera had no artifactual effect on assay target cells, and heating a monoclonal neutralizing Ab at 56°C did not affect its neutralizing capacity, consistent with previous literature (34, 58).

To assess whether the heat-sensitive contribution to the neutralization was due to the complement system, sera were treated with CVF, which structurally mimics the complement protein C3b and binds factor B, thus triggering the complement cascade downstream from C3 convertase (59). This causes the subsequent proteins to activate irreversibly, resulting in complement-depleted serum (60–62). The loss of complement function after CVF treatment was verified using the CH₅₀ test (Fig. 1G). To determine the role of complement in mediating virus neutralization preheat inactivation, we compared live virus neutralization of non-HI serum, non-HI CVF-treated serum, and HI serum (Fig. 2A, 2B). In all samples, CVF treatment significantly reduced neutralization titers to levels approaching HI sera. The neutralization in the SARS-CoV-2 unexposed samples dropped to undetectable levels (IC₅₀ 3.217 ± 7.879 to 0.00 ± 0.00). CVF treatment of COVID-19 asymptomatic, mild, moderate, and severe sera reduced IC₅₀ neutralization by 43% to 46.61 ± 39.65 (p = 0.0319), 72% to 27.92 ± 36.93 (p = < 0.0001), 62% to 27.16 ± 15.11 (p = 0.0261), and 40% to 385.5 ± 259.5 (p = 0.0083), respectively (Fig. 2C). Overall, complement contributed, on average, 57% of the neutralizing activity of ex vivo SARS-CoV-2 sera.

Across all disease states, complement played a significant role in increasing the neutralization potential of convalescent serum. These results indicate that the effector function of SARS-CoV-2–specific Abs can also be mediated via complement rather than direct blocking of virus entry. Complement activation requires the binding of a single IgM Ab or several IgG class-switched Abs, thus opening the C1q binding site (42). It has recently been shown that neutralization by Abs that bind to the RBD is not enhanced with the addition of complement for SARS-CoV-2 (63). It was also recently observed that non-RBD binding, but SARS-CoV-2 spike-specific Abs, which are more abundant in serum than anti-RBD Abs, can neutralize the virus via complement activity (63). In this regard, SARS-CoV-1 Abs, which are capable of neutralizing SARS-CoV-1 and can cross-react with SARS-CoV-2 spike, but do not neutralize SARS-CoV-2, can become SARS-CoV-2 neutralizing Abs in the added context of the complement system (64–66). In our studies, we tested a serum sample from a convalescent SARS-CoV-1 participant (OM8085), which had undetectable live virus neutralization when using HI serum; however, when using non-HI serum, one obtains a detectable live virus neutralization with an IC₅₀ of 20.6 (Fig. 2D). This finding supports the existing literature that SARS-CoV-1 Abs that are capable of cross-reacting to SARS-CoV-2 in a nonneutralizing manner can become neutralizing when the complement system remains intact.

We analyzed the contribution of the complement initiation pathways to complement-mediated neutralization of SARS-CoV-2, specifically the classical, lectin, and alternate pathways. To assess the classical pathway mediated by IgG, convalescent sera were preincubated with protein G–Sepharose beads, then analyzed with a live virus neutralization assay to quantify the contribution of the Ab-mediated classical pathway activation. IgG depletion did not impact complement activity of the sera (Fig. 1G) while resulting in near complete abrogation of SARS-CoV-2 neutralization (Fig. 3A). Thus, a substantial contribution of complement-mediated virus neutralization can be demonstrated via the classical pathway. The mannose-binding lectin pathway was inactivated by adding 100 mM mannose to the samples as previously described (55). Inhibition of the lectin pathway did not result in changes in the neutralizing capabilities of the sera (Fig. 3B). Finally, the alternative pathway was examined, which is initiated by the spontaneous hydrolysis of a labile thioester bond, and converts C3 to a bioactive form C3b-like C3(H₂O) in the fluid phase (67). SARS-CoV-2 unexposed and unvaccinated sera should thus spontaneously react with SARS-CoV-2 viral particles in this arm of the complement system, thus granting a detectable level of neutralization. In our neutralization assay, unexposed and unvaccinated individuals did not show detectable levels of neutralization, indicating that the virus does not directly activate though the alternative pathway (Fig. 1A, 1C). The second aspect of the alternative pathway is the amplification loop, a positive feedback loop initiated after C3 cleavage and formation of C3b, regardless of the initiation pathway that triggered C3 cleavage. The C3b associates with factor B and forms a C3bBb complex that mimics the traditional C3 convertase, initiating a positive feedback loop that promotes additional C3 cleavage. This loop can be inhibited by addition of anti–factor B Abs. Inhibition of the C3 amplification loop resulted in a reduction of complement-mediated neutralization levels by 31% (p = 0.0459), showing the involvement of the amplification loop as previously described (56). The amplification loop, however, also plays a role in amplifying the classical and lectin pathways, making factor B a central player in complement activation (68). Taken together, we demonstrate that the Ab-mediated classical pathway is the main contributor to complement-mediated neutralization in SARS-CoV-2 sera, with the amplification loop significantly increasing complement-mediated neutralization by 31% (Fig. 3D).

Eculizumab is a mAb that targets the complement protein C5, thus blocking the cleavage of C5 convertases, and is used to treat disorders with hyperactivation of the complement system by inhibiting the formation of the MAC (69). Ravulizumab is a modified version of eculizumab, optimized to extend its therapeutic half-life (70). We tested the contribution of complement in the neutralization activity of two COVID-19 convalescent individuals, one receiving eculizumab for atypical hemolytic uremic syndrome, and the other receiving ravulizumab for paroxysmal nocturnal hemoglobinuria. The virus-neutralizing activity of these samples was unaffected by either HI or CVF treatment (Fig. 4A, 4B). These findings suggest that the mechanism of action of complement-dependent neutralization is through the formation of the MAC, as eculizumab and ravulizumab block this step of complement activation. To further validate our findings, an eculizumab biosimilar anti-C5 Ab, clone h5G1.1, was used for preincubation with neutralizing sera samples. A 48% drop in neutralization was observed (p = 0.0024), comparable to the reduction following HI of the sera (Fig. 4C). Blocking the C5 convertase prevents the formation of the downstream MAC. This finding strengthens the hypothesis that the main mechanism of action is through the MAC.

We determined the role of complement in sera of donors who were vaccinated with homologous SARS-CoV-2 vaccines, that is, ChAdOx1-S, BNT162b2, or mRNA1273 (data from all subjects are combined in Fig. 5). Two weeks after their first dose, we observed mean serum IC₅₀ of 111.0 ± 82.73, 36.52 ± 28.47, and 33.48 ± 27.21 for untreated, CVF-treated, and HI serum, respectively. Two weeks after their second dose of vaccine we observed mean IC₅₀ of 1183 ± 857.1, 659.0 ± 411.7, and 557.4 ± 353.1 for untreated, CVF-treated, and HI serum, respectively (mean ± SD). Neutralization after the first dose significantly dropped 65% after CVF treatment (p = 0.0015), and after the second dose it significantly dropped 43% after CVF treatment (p = 0.0031). There was no significant difference between CVF-treated neutralization and HI neutralization in the first dose of vaccinations. This suggests that the main mechanism of increasing neutralization in the untreated serum samples was due to complement. Of note, two donors (Fig. 5, red data points) displayed readily observable neutralization titers 2 wk after their first dose in untreated serum that was abolished after CVF and HI treatment, suggesting that protection may exist due to complement effects in the absence of detectable neutralization by standard assays.

A subset of convalescent sera was available for total C3 and C4 levels as well as complement activity through the CH₅₀ assay. We observed an increasing trend for total C3 and C4 levels across all SARS-CoV-2 convalescent samples, consistent with increasing disease severity (Fig. 6). CH₅₀ assays showed intact complement activity in all samples. Thus, convalescent sera contained normal to increased levels of complement and competent complement activity. This is in keeping with recent literature showing higher C3 and C4 levels during COVID-19 illness (71).

In this study, we report that individuals who survived severe COVID-19 developed the highest levels of neutralizing Abs (IC₅₀ > 100) compared with asymptomatic or mild infections, suggesting that severe disease will be highly protective against reinfection by a homologous virus. Because all cases we studied, including those with severe disease, had survived their illness, we hypothesize that prolonged Ag exposure from prolonged illness and inflammation allowed the adaptive immune system to impact the affinity maturation of Abs, thus allowing higher neutralization titers in severe participants to be observed during convalescence.

Individuals with asymptomatic to moderate illness displayed lower neutralization titers during convalescence (IC₅₀ of 14–26) and are possibly not being fully protected against reinfection. Our analysis uncovered another potential mechanism of Ab-mediated neutralization/protection that is not routinely measured in current neutralization assays: complement-mediated neutralization. In this regard, complement-mediated neutralization, on average, accounted for 57% of the neutralization activity found in sera, and it increased levels of neutralization in all convalescent individuals by at least 2-fold. We observed that the Ab-mediated classical pathway is responsible for initiating the complement-mediated neutralization in these individuals, as experimental inhibition of either the mannose-binding lectin pathway or the alternative pathway had nonexistent neutralization. In contrast, the C3 amplification loop significantly contributes to the positive feedback loop following complement activation through the classical pathway, being responsible for 31% of the complement-mediated neutralization, which is consistent with previously reported literature (56). Importantly, note that complement can function in additional ways such as opsonization as well as internalization through phagocytosis, which we did not assess in our assays and will likely require future work.

The effects of serum complement were further confirmed by examining convalescent sera from individuals treated with eculizumab or ravulizumab in vitro, where the protective effect of complement in sera is neutralized. This raises concerns for individuals being treated with eculizumab or ravulizumab, as they might be at an increased risk for infection and serious disease as reported in some cases, but these studies are few and other studies have found no significant association (72–76). Increased complement activation has also been associated with more severe COVID-19 disease, which initiated a clinical trial of eculizumab as a treatment against severe disease, but the results are inconclusive (69, 77–80). Importantly, we also observed a substantial level of complement-mediated neutralization of the virus after vaccination. Although standard HI neutralization levels are low after one dose of vaccination, we find complement-mediated neutralization to be readily detectable. Other studies have also shown enhanced Ab-mediated function when neutralization titers were low after one dose of vaccination. The antiviral effects of serum through Fc-mediated Ab-dependent direct cytotoxicity against SARS-CoV-2 has been shown (81, 82). Interestingly, when neutralizing titers were high, then the Fc domain of neutralizing Abs appeared to be redundant when given as a prophylaxis (83). In addition, animal models using SARS-CoV-2 Abs for postexposure treatment demonstrated that a functional Fc domain correlated with decreased viral burden, lung pathology, immune activation, levels of inflammation, as well as improved respiratory mechanics, suggesting the importance of the Fc domain in cell-mediated virus clearance (83). Although RBD binding Abs are the best correlate of neutralization by blocking spike interactions with ACE-2, complement-mediated neutralization can use other spike-binding Abs recognizing epitopes outside the RBD domain for its effect (84). Indeed, extensive analysis of the IgG repertoire in convalescent individuals showed that >80% of spike Abs target outside of the RBD domain (63). While various enveloped viruses, such as pox viruses and HIV, have evolved to escape the effects of human complement activity, current SARS-CoV-2 virus strains appear to be susceptible to complement-mediated neutralization (85, 86). Our study demonstrates the robust contribution of complement-mediated virus neutralization as Ab-mediated effector function independent of interference with spike binding to angiotensin converting enzyme 2. This mechanism may contribute to the high frequency of asymptomatic cases and cases with a mild disease course following SARS-CoV-2 infection.

C.J.P. has received Advisory Board Honoraria (Alexion, Apellis, BioCryst, Sanofi, Sobi, and Takeda) and Speaking Honoraria (Alexion, Sanofi, and Sobi), and has been a Site Investigator for clinical trials sponsored by Alexion, Apellis, BioCryst, Regeneron, and Roche. The other authors have no financial conflicts of interest.

Training and support for high-containment research activities were provided by the technical staff of the University of Toronto Combined CL3 Unit in the Temerty Faculty of Medicine.