SARS-CoV-2 Spike- and Nucleoprotein-Specific Antibodies Induced After Vaccination or Infection Promote Classical Complement Activation
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Abstract
Antibodies specific for the spike glycoprotein (S) and nucleocapsid (N) SARS-CoV-2 proteins are typically present during severe COVID-19, and induced to S after vaccination. The binding of viral antigens by antibody can initiate the classical complement pathway. Since complement could play pathological or protective roles at distinct times during SARS-CoV-2 infection we determined levels of antibody-dependent complement activation along the complement cascade. Here, we used an ELISA assay to assess complement protein binding (C1q) and the deposition of C4b, C3b, and C5b to S and N antigens in the presence of antibodies to SARS-CoV-2 from different test groups: non-infected, single and double vaccinees, non-hospitalised convalescent (NHC) COVID-19 patients and convalescent hospitalised (ITU-CONV) COVID-19 patients. C1q binding correlates strongly with antibody responses, especially IgG1 levels. However, detection of downstream complement components, C4b, C3b and C5b shows some variability associated with the subject group from whom the sera were obtained. In the ITU-CONV, detection of C3b-C5b to S was observed consistently, but this was not the case in the NHC group. This is in contrast to responses to N, where median levels of complement deposition did not differ between the NHC and ITU-CONV groups. Moreover, for S but not N, downstream complement components were only detected in sera with higher IgG1 levels. Therefore, the classical pathway is activated by antibodies to multiple SARS-CoV-2 antigens, but the downstream effects of this activation may differ depending the disease status of the subject and on the specific antigen targeted.
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SciScore for 10.1101/2021.11.22.21266681: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Ethical approval for obtaining samples for groups 1 -4 was provided by the London – Camden and Kings Cross Research Ethics Committee reference 20/HRA/1817. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Group 3: Individuals without evidence of infection (absence of anti-N antibodies) who had received their second dose of BNT162b2 vaccine at least 28 days previously (DOUBLE VACC). anti-Nsuggested: NoneHRP-conjugated anti-human secondary antibodies were added for 1 hr at RT: anti-IgGAM, neat (EACONJ654, The Binding Site), anti-IgM clone … SciScore for 10.1101/2021.11.22.21266681: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Ethical approval for obtaining samples for groups 1 -4 was provided by the London – Camden and Kings Cross Research Ethics Committee reference 20/HRA/1817. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Group 3: Individuals without evidence of infection (absence of anti-N antibodies) who had received their second dose of BNT162b2 vaccine at least 28 days previously (DOUBLE VACC). anti-Nsuggested: NoneHRP-conjugated anti-human secondary antibodies were added for 1 hr at RT: anti-IgGAM, neat (EACONJ654, The Binding Site), anti-IgM clone AF6, 1:2000, anti-IgG1 clone MG6.41, 1:3000, anti-IgG3 clone MG5.161, 1:1000 (all produced at the University of Birmingham). HRP-conjugated anti-human secondary antibodies were added for 1 hr at RT: anti-IgGAM, neat (EACONJ654suggested: Noneanti-IgMsuggested: Noneanti-IgG1suggested: (Fitzgerald Industries International Cat# 61R-I161aPE, RRID:AB_1286147)anti-IgG3suggested: NoneAfter washing, 50 µl COVID negative normal human serum (same source used throughout all assays, containing no detectable S or N specific antibodies as measured by IgGAM ELISA) at a dilution of 1:40 (in 2% BSA plus 5 mM calcium chloride and 5 mM magnesium chloride) was added to each well for 1hr at RT. 100µl of rabbit anti-C1q FITC antibody (Invitrogen PA5-16601) at a 1:200 dilution in PBS-0.1% Tween 20 was added and incubated at 37□C for 1 hr. anti-C1q FITCsuggested: (Abcam Cat# ab4223, RRID:AB_304387)The following anti-human monoclonal complement antibodies (100ul, diluted in PBS-0.1% Tween 20) were added and incubated at 37□C for 1 hr: mouse anti-C4b, 1:22,500 (Invitrogen, LF-MA0198); mouse anti-C3b, 1:10,000 (Invitrogen MA1-70053); mouse anti-C5b, 1:10,000 (Invitrogen DIA 011-01-02). anti-human monoclonal complementsuggested: (Acris Antibodies GmbH Cat# LF-MA0198, RRID:AB_1874068)anti-C4bsuggested: Noneanti-C3bsuggested: Noneanti-C5bsuggested: NoneExperimental Models: Cell Lines Sentences Resources HEK293F cells were cultured in Freestyle 293 Expression medium (Fisher Scientific) and maintained at a density of 0.2 × 106 cells/mL at 37°C, 8% CO2 and 125 rpm shaking. HEK293Fsuggested: NoneSoftware and Algorithms Sentences Resources Statistics: Statistical analysis was carried out using GraphPad Prism 9.0. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:One caveat in this argument is that exogenous sources of complement in the form of sera from non-infected individuals were used in these studies and that patients own sera may differ in potency. This was not assessed here as the focus was on antibody-mediated activation of complement. Additionally, these assessments were made using sera from patients who were infected or immunized weeks previously and the antibodies present may not reflect the antibodies present at the time of infection. Certainly, it could be expected that the affinity of the antibodies would increase over time. One striking feature was the variability in the anti-C4b/C3b response detected in the VACC group. It is unclear why this is the case, but it could simply be that there is variability within the wider population in the ability to activate complement downstream. The complement cascade has been reported to be activated through multiple pathways after SARS-CoV-2 infection [19-21]. Amongst these, the engagement of the classical pathway is distinct to the non-antibody-dependent pathways due to the potential multiple roles antibody can play during the course of infection. If induced whilst an infection is ongoing, then the activation of the complement cascade by antibody could worsen disease, particularly as antibody responses become detectable concomitant with risk of severe disease. This could happen either through enhanced inflammation, such as observed during acute respiratory distress syndrome, or thro...
Results from TrialIdentifier: No clinical trial numbers were referenced.
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Results from scite Reference Check: We found no unreliable references.
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