The analysis of convalescent serum from individuals infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is rich in neutralizing antibodies in good responders. Among these, the immunoglobulin G (IgG) subtype IgG3 is the most important antibody.
Study: IgG3 and IgM Identified as Key to SARS-CoV-2 Neutralization in Convalescent Plasma Pools. Image Credit: Corona Borealis Studio/Shutterstock
A recent study examined this fact for its veracity, using large pools comprising approximately 250 to 570 convalescent donors. The researchers found that both IgG3 and IgM elicited by the spike S1 subunit and the receptor-binding domain (RBD) correlate with viral neutralization.
Background
Neutralizing antibodies disrupt SARS-CoV-2 entry into the host cell to cause infection by preventing the binding of the viral spike to the host cell surface receptor molecule, the angiotensin-converting enzyme 2 (ACE2). Earlier, various methods to assess specific antibodies to the viral spike have been described, based on which extensive seroprevalence studies have been carried out to identify and monitor those who have had infection with the virus.
The scientists in this study, published online on the PLOS ONE server, used pooled convalescent serum to examine the serologic response in a population at risk of infection. Using seven such pools, they found that each pooled specimen had higher neutralization titers for SARS-CoV-2 than those collected before the pandemic began.
Each pool also tested positive for S1-specific enzyme-linked immunosorbent assay (ELISA) testing. The most abundant antibody in any pool was IgG3 and then IgG1. IgA showed a high level of binding, but IgM was at baseline level.
The ELISA test was carried out using optimized recombinant nucleocapsid, S1, and the S1-RBD antigens to capture the antibodies to the virus, if present, with antibodies specific to the different Ig subtypes to detect the antigen-antibody capture complexes. This again confirmed the relative abundance of IgG3 in SARS-CoV-2 binding antibodies.
The highest correlation with neutralization titers for each binding antibody was with the S1-RBD-IgG3, IgM-S1, and IgM-S1-RBD antibodies, while IgA showed the least correlation with neutralizing activity.
When each of the Ig classes was depleted selectively from the convalescent plasma pool derived from 567 donors, it was shown that about 40% of the total neutralizing activity was due to the presence of IgG3 and IgM antibodies, making up 3% and 8% of the total Ig antibodies. Though IgG1 made up half the total Ig mass, it contributed only 16% of the neutralizing activity.
The contribution of IgA was half its percentage-wise share of the total mass, with IgG2 and IgG4 being negligible.
What are the implications?
The results demonstrate the importance of IgG3 and IgM to the neutralizing activity of convalescent plasma against SARS-CoV-2, with the presence of antibodies to the S1 and RBD being most closely correlated with overall neutralizing potency. The direct role of IgG3 in neutralization has been earlier shown with respect to the human immunodeficiency virus type 1.
Some scientists think this is due to the length of the hinge region in IgG3, which makes it more flexible in its rotational movements while enhancing its binding to multivalent antigens. An earlier study found that IgG-mediated inhibition of S1-RBD-ACE2 binding prevented viral entry. This was confirmed in this study by the finding that IgG3 targeting the S1-RBD plays a crucial role in neutralizing viral entry.
The major contribution of IgM in neutralization is also clear. This is all the more interesting given its relatively low abundance. The explanation may lie in its polyvalency and increased avidity for antigens compared to divalent IgG and IgA since avidity is an important determinant of the effectiveness of the antibody response to a viral infection.
With the high affinity of binding between the viral RBD and the ACE2 receptor on the host cell, only avid antibodies could neutralize this binding, which may account for the inhibitory effect of IgM even at low concentrations.
An earlier paper showed that IgM and IgG are key to SARS-CoV-2 neutralization, using a selective-depletion approach. The difference from the current study lies in the large number of sera pooled to provide a population-level understanding of which antibodies are important in viral neutralization. Secondly, this study found that IgG3 was the most important in terms of SARS-CoV-2 neutralization, in contrast to IgM in the earlier paper.
Some investigators showed that the earliest neutralizing antibodies were of the IgM subtype, with the titer decreasing over time. Conversely, IgG neutralizing antibodies were slow to appear. Still, as expected from earlier studies on the evolution of an antibody response to a viral infection as the infection progressed or was cleared, they did not fall significantly with time.
Some other studies have shown the IgG1 to be the key antibody in neutralizing the virus, sometimes associated with IgM. These were directed against the spike and RBD proteins, in contrast to the spike target of IgG3.
Others have identified IgG1 and IgG3 as the most reactive to the S1 and RBD antigens. Such differences may be due to the different time points when the donated plasma was collected relative to the course of the disease and the severity of disease at the time of collection. Differences in the methods used could also contribute to these disparities.
IgG4 has been demonstrated to be a mortality marker when targeting the RBD, showing the predictive nature of the antibody depends on the time of collection and the type of cohort tested. Moreover, while IgG1 and IgG3 bind to soluble and membrane proteins, but IgG2 and IgG4 bind to polysaccharide bacterial capsular antigens and allergenic molecules, respectively.
When the monoclonal antibody binding the spike protein was expressed against a background of human IgG1-4, the binding affinity for IgG3 went up 5-fold, while neutralization was enhanced 50-fold, compared to other subclasses.
The authors postulate that IgG3 elicits a superior binding and neutralization effect against SARS-CoV-2 in an avidity-dependent manner via cross-linking the spike protein on the viral surface.”
Despite the high binding affinity of IgA, it has low to moderate neutralizing activity. This opposes earlier studies suggesting that IgS1- IgA is correlated with neutralizing activity in non-critical COVID-19, but with a rapid decline at 3-4 weeks from discharge, accompanied by a waning of the RBD-IgA titer.
Other studies have also shown that IgA antibodies drive the early phase of the specific antibody response to this virus, with IgA plasmablast expansion occurring soon after symptoms begin and peaking at day 22 or so. Thus, IgA may be a major driver of the immune response in early infection.
The current study’s apparent contradiction of these findings may be due to the difference in the time points of plasma sampling, with these samples being collected at 28 or more days from symptom resolution. At this point, IgA and IgG are high, but the former has begun to wane while the latter remains stable.
This study suggests that enriching convalescent plasma for IgG3 concentrations, and IgG in general, may provide a more effective treatment for COVID-19, compared with the ordinary pooled samples used in many studies. Hyperimmune globulin use provides evidence of this effect, being manufactured to contain a standardized level of antibodies raised against this virus. Not only is it more selective and effective in its action, but it has a longer shelf life and a better safety profile, besides being easily scaled up for mass production.
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