Determination of IgG1 and IgG3 SARS-CoV-2 Spike Protein and Nucleocapsid Binding-Who Is Binding Who and Why?

Abstract

The involvement of immunoglobulin (Ig) G3 in the humoral immune response to SARS-CoV-2 infection has been implicated in the pathogenesis of acute respiratory distress syndrome (ARDS) in COVID-19. The exact molecular mechanism is unknown, but it is thought to involve this IgG subtype's differential ability to fix, complement and stimulate cytokine release. We examined the binding of convalescent patient antibodies to immobilized nucleocapsids and spike proteins by matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) mass spectrometry. IgG3 was a major immunoglobulin found in all samples. Differential analysis of the spectral signatures found for the nucleocapsid versus the spike protein demonstrated that the predominant humoral immune response to the nucleocapsid was IgG3, whilst for the spike protein it was IgG1. However, the spike protein displayed a strong affinity for IgG3 itself, as it would bind from control plasma samples, as well as from those previously infected with SARS-CoV-2, similar to the way protein G binds IgG1. Furthermore, detailed spectral analysis indicated that a mass shift consistent with hyper-glycosylation or glycation was a characteristic of the IgG3 captured by the spike protein.

Keywords: COVID-19; IgG1; IgG3; convalescent plasma; nucleocapsid; predictive profile; spike protein.

Figure 1

A schematic of the humoral response-immunoglobulin major class switching following SARS-CoV-2 infection and COVID-19 progress is illustrated in (A). IgG predominates 3–4 weeks after the onset of symptoms and the major structural and functional domains of IgG are also illustrated. The variation in the neck region of the 4 subclasses of human IgG are shown in (B), while (C) shows structure and functional interaction regions of immunoglobulin. IgG3 is the largest and its heavy chain (Hc) resolution in MALDI-ToF mass spectra (D) is indicated at 54,000 m/z (IgG3 Hc). Also indicated is the IgG1 heavy chain (IgG1 Hc), which resolves at 51,000 m/z. The position of the IgM heavy chain peak (IgM Hc) is indicated at 74,000 m/z. An Ig (IgX Hc), which is thought to be either IgG2 or IgG4 but has yet to be fully identified, was found in patient samples, and is shown at 49,000 m/z.

Figure 2

Relative intensities and variance in peak apex molecular mass of IgG1 heavy chains (IgG1 Hc) recovered from the same samples by protein G, nucleocapsid and stabilized spike protein. The non-parametric Kruskal-Wallis statistical test was carried out to test differences between study cohorts. The p values (alpha: 0.05) were as follows: intensity p < 0.0001 and molecular mass p < 0.0001.

Figure 3

Distribution of intensity for captured and eluted IgG1 heavy chains (IgG1 Hc) and the relative peak molecular mass being bound. The dot plots to the right are intensity versus molecular mass for individual samples; Blue represents data from SARS-CoV-2 sero-negative HCWs, orange from SARS-CoV-2 sero-positive HCWs having recovered from mild symptoms and red sample data from convalescent patients recovering from COVID-19 ARDS. The non-parametric Kruskal-Wallis statistical test was carried out to test differences between study cohorts. The p values (alpha: 0.05) were as follows: protein G capture intensity p = 0.66, molecular weight p = 0.038; protein N capture intensity p = 0.84, molecular weight p = 0.15; protein S capture intensity p = 0.0003, molecular weight p = 0.63.

Figure 4

Comparative table of IgG1 (A) and IgG3 (B) spectral analyses: Detection frequency, intensity and molecular mass for all samples captured by protein G , nucleocapsid and prefusion complete spike protein . Further delineation is done by sample infection status: blue represents data from SARS-CoV-2 sero-negative HCWs, orange from SARS-CoV-2 sero-positive HCWs having recovered from mild symptoms and red sample data from convalescent patients recovering from COVID-19 ARDS.

Figure 5

Relative intensities and variance in peak apex molecular mass of IgG3 heavy chains (IgG3 Hc) recovered from the same samples by protein G, nucleocapsid and stabilized spike protein. The non-parametric Kruskal-Wallis statistical test was carried out to test differences between study cohorts, the p values (alpha: 0.05) were as follows: intensity p < 0.0001, molecular mass p < 0.0001.

Figure 6

Distribution of intensity for captured and eluted IgG3 heavy chains (IgG3 Hc) and the relative peak molecular masses bound and separated into sample groups. The dot plots to the right are intensity versus molecular mass for individual samples. Blue represents data from SARS-CoV-2 sero-negative HCWs, orange from SARS-CoV-2 sero-positive HCWs having recovered from mild symptoms and red sample data from convalescent patients recovering from COVID-19 ARDS. The non-parametric Kruskal-Wallis statistical test was carried out to test differences between study cohorts; the p values (alpha: 0.05) were as follows: protein G capture intensity p = 0.61, molecular weight p = 0.006; protein N capture intensity p = 0.067, molecular weight p = 0.19; protein S capture intensity p = 0.75, molecular weight p = 0.97.