BREAKING! SARS-CoV-2 Induced Antibody-Mediated Complement Activation And Subsequent Cellular Priming Exacerbates COVID-19 Severity!

A new study by researchers from University of Copenhagen and the Copenhagen University Hospital that explored the adverse effects of SARS-CoV-2 induced antibody responses has alarmingly found that SARS-CoV-2 induced antibody-mediated complement activation and subsequent cellular priming exacerbates COVID-19 severity!

 
The current COVID-19 pandemic caused by the SARS-CoV-2 coronavirus continues to constitute a serious public health threat worldwide. To date more than 260 million people globally have been infected with the SARS-CoV-2 coronavirus and more than 5.2 million people have died from the disease according to official data. In reality, the figures could be many folds and these figures are expected to rise in coming weeks and months catastrophically due to emerging SARS-CoV-2 variants and sub-variants and also due to contributions by viral priming due to exposure to spike proteins of earlier lineages.
 
Despite protective antibody-mediated viral neutralization in response to SARS-CoV-2 infection been firmly characterized and the effects of the antibody response generally considered to be beneficial, an important biological question regarding potential negative outcomes of a SARS-CoV-2 antibody response has yet to be answered.
 
The study team determined the distribution of IgG subclasses and complement activation levels in plasma from convalescent individuals using in-house developed ELISAs.
 
Interestingly, the IgG response towards SARS-CoV-2 receptor-binding domain (RBD) after natural infection appeared to be mainly driven by IgG1 and IgG3 subclasses, which are the main ligands for C1q mediated classical complement pathway activation. The deposition of the complement components C4b, C3bc, and TCC as a consequence of SARS-CoV-2 specific antibodies were depending primarily on the SARS-CoV-2 RBD and significantly correlated with both IgG levels and disease severity, indicating that individuals with high levels of IgG and/or severe disease, might have a more prominent complement activation during viral infection.
 
Furthermore, freshly isolated monocytes and a monocyte cell line (THP-1) were used to address the cellular mediated inflammatory response as a consequence of Fc-gamma receptor engagement by SARS-CoV-2 specific antibodies. Monocytic Fc gamma receptor charging resulted in a significant rise in the secretion of the pro-inflammatory cytokine TNF-α.

Detection of IgG1, IgG2, IgG3 and IgG4 in recovered SARS-CoV-2 individuals. Groups divided according to levels of total IgG; high, intermediate, low and negative healthy controls, n = 240. Levels were assessed by coating plates with 1 µg/nm RBD and detecting with HRP-conjugated antibodies against IgG1, 2, 3 and 4 (1 µg/ml). Samples were measured in a 1:50 dilution, except for IgG1 which was measured in a 1:150 dilution. The dynamic range is represented in signal-to-noise ratios (S/N). A p value < 0.05 was considered significant. ns, not significant, ***p < 0.001 using Tukey all-pair comparisons.

The study findings indicate that SARS-CoV-2 antibodies might drive significant inflammatory responses through the classical complement pathway and via cellular immune-complex activation that could have negative consequenc es during COVID-19 disease.
 
The study findings validate that increased classical complement activation was highly associated to COVID-19 disease severity. The combination of antibody-mediated complement activation and subsequent cellular priming could constitute a significant risk of exacerbating COVID-19 severity.
 
The study findings were published in the peer reviewed journal: Frontiers In Immunology. https://www.frontiersin.org/articles/10.3389/fimmu.2021.767981/full
 
Co-lead researcher Dr Peter Garred from the Laboratory of Molecular Medicine, Department of Clinical Immunology, Copenhagen University Hospital told Thailand Medical News, “Typically, infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggers an immune response driven by the immunoglobulin G (IgG) family of antibodies in the infected individual. This response against the SARS-CoV-2 receptor-binding domain (RBD) is driven primarily by IgG1 and IgG3 sub-classes. While several studies have established the positive outcomes of this antibody-mediated viral neutralization, its potential adverse effects are yet unknown.”
 
The study team used Sandwich ELISA tests to determine the distribution of IgG sub-classes and complement activation levels in plasma from convalescent individuals.
 
The research included a total of 180 people who recovered from coronavirus disease 2019 (COVID-19), as confirmed by their negative reverse transcription-polymerase chain reaction (RT-PCR) test. Plasma samples from these individuals were ethylenediaminetetraacetic acid (EDTA)-treated and kept frozen at −80°C until they were used.
 
All the RT-PCR positive participants comprised males and females in the age group 18–86, with the course of disease ranging from mild (few symptoms) to severe (needed hospitalization). Healthy blood donors were used as negative controls to collect a total of 60 EDTA plasma samples (from before 2020) having no SARS-CoV-2 antibodies.

Complement deposition in recovered SARS-CoV-2 individuals. (A) Measurements of complement components C4, C3 and TCC in convalescent EDTA plasma. Groups divided according to levels of total IgG; high, intermediate, low and negative healthy controls, n = 240. Levels were assessed by coating plates with 3 µg/nm RBD, followed by incubation with EDTA plasma diluted 1:60 (for high IgG samples) and 1:20 (for intermediate, low and negative IgG samples). A normal human serum pool was applied in a 1:50 dilution. Biotinylated monoclonal antibodies against C4b, C3bc and TCC (2 µg/ml) was used for detection. Dynamic range represented in arbitrary units (AU). A p value < 0.05 was considered significant. ***p < 0.001 using Tukey all-pair comparisons. (B) The correlation between deposition of either C4, C3 or TCC and IgG levels using Spearman rank correlation analysis.

IgG Sub-Classes And Complement Molecules Levels
 
It is known that during natural infection, IgG1 and IgG3 sub-classes effectively activate the classical complement pathway to eliminate the pathogen (here SARS-CoV-2) employing methods such as microbial lysis by the terminal complement complex (TCC).
 
Importantly, the complement system, an integral part of innate immunity, is initiated by three distinct pathways - the classical, the lectin, and the alternative pathway - differing in the modes of initiation but all generating complement factor (C3) and other central and terminal effector molecules.
 
The deposition of such effector molecules (C4b, C3bc, and TCC), as a consequence of SARS-CoV-2-specific antibodies, are significantly correlated with both IgG levels and disease severity, indicating that patients with high IgG levels might have more prominent complement activation during viral infection.
 
Complement Activation: A Critical Pathogenic Feature Of COVID-19
 
Although the activation of the complement system may effectively contribute to the control of infection, it may also contribute to several pathologies like acute respiratory distress syndrome (ARDS) in severely ill COVID-19 patients due to its pro-inflammatory effects.
 
The research findings suggest that antibody-mediated complement activation and subsequent cellular priming constitute a significant risk of increasing COVID-19 severity.
 
In addition, the results indicate a preferential generation of SARS-CoV-2-specific IgG3 (and not IgG1) during COVID-19 infection, while IgG2 and IgG4 are barely detected. This excess IgG3 could be responsible for excess inflammation rather than tissue repair, worsening the COVID-19 symptoms in patients.
 
Implications Of Depletion Of Antibodies Against RBD
 
The study findings demonstrated that once the RBD antibodies depleted in convalescent plasma samples, the deposition of C4, C3 and TCC complement molecules stopped.
 
However, while this proved that the activation of the complement system through deposition of C4, C3, and TCC is mediated by RBD antibody, the results could not decipher the exact residues of the RBD involved in triggering the complement activation.
 
Additionally, the levels of C4, C3, and TCC are positively correlated with both IgG levels and disease severity. Both C4 and C3 levels were significantly lower in patients with high disease severity than patients with low severity, indicating that C4 and C3 are readily absorbed in severely ill patients.
 
Importantly, an aggressive cellular mediated inflammatory response observed in SARS-CoV-2-infected individuals is a consequence of Fc-gamma receptor (FcγR) engagement by SARS-CoV-2 specific antibodies. Simultaneously, it releases heaps of pro-inflammatory cytokines, referred to as the "cytokine storm."  
 
Although numerous studies have investigated the role of antibodies and FcγRs during viral infections concerning virus neutralization and antibody-dependent enhancement of infection, data on FcγR-mediated cytokine responses in the context of viral infections is limited.
 
In order to fill this knowledge gap, the study team primed THP-1 monocytes and freshly isolated human monocytes with SARS-CoV-2 RBD immune complexes, the antigen being RBD. Upon evaluating the cytokine secretion of IL-6, IL-1β, and TNF-α by THP-1 cells, observed TNF-α levels were significantly high, but IL-6 and IL-1β levels were not. The monocytes and monocyte-derived macrophages are phagocytic cells contributing to inflammation, immunity, and anti-viral responses.
 
The study team concluded, “In conclusion, our study findings support the notion that antibodies against SARS-CoV-2 could represent a two-edged sword. It is known that particularly antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity can drive harmful and systemic proinflammatory responses that can have severe pathophysiological consequences. Our investigations indicate that SARS-CoV-2 antibodies might drive significant inflammatory responses through the classical complement pathway and cellular immune-complex activation that could have negative consequences during COVID-19 disease. However, it is important to note that the data presented does not give direct evidence of classical pathway activation, since a contribution from the lectin pathway cannot be excluded. The combination of antibody-mediated complement activation and subsequent cellular priming could constitute a significant risk of exacerbating COVID-19 severity.”
 
The research data provides an essential insight into the overall immunological response during a COVID-19 infection and emphasize the need for further studies to shed light on all the possible harmful effects of these antibodies and their interaction with the complement system.

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