Quantitative, multiplexed, targeted proteomics for ascertaining variant specific SARS-CoV-2 antibody response

Abstract

Determining the protection an individual has to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VoCs) is crucial for future immune surveillance, vaccine development, and understanding of the changing immune response. We devised an informative assay to current ELISA-based serology using multiplexed, baited, targeted proteomics for direct detection of multiple proteins in the SARS-CoV-2 anti-spike antibody immunocomplex. Serum from individuals collected after infection or first- and second-dose vaccination demonstrates this approach and shows concordance with existing serology and neutralization. Our assays show altered responses of both immunoglobulins and complement to the Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.1) VoCs and a reduced response to Omicron (B1.1.1529). We were able to identify individuals who had prior infection, and observed that C1q is closely associated with IgG1 (r > 0.82) and may better reflect neutralization to VoCs. Analyzing additional immunoproteins beyond immunoglobulin (Ig) G, provides important information about our understanding of the response to infection and vaccination.

Trial registration: ClinicalTrials.gov NCT04318314.

Keywords: COVID-19; SARS-CoV-2; complement: immunoglobulin; delta variant; mass spectrometry; omicron variant; proteomics; vaccination; variant of concern.

Graphical abstract

Figure 1

Principle of the targeted LC-MS/MS immunocomplex assay (A) A summarized workflow and schematical representation of the bait-capture LC-MS/MS assay. (B) Composition of the immunocomplex at different vaccination and pre-infection stages. Mean values used for each protein. (C) Heatmap of all proteins measured in the multiplex as determined by normalized mean values of the protein ratioed to spike peptide against each S1 bait variant and vaccination group. MAC, membrane attack complex. Blue to red color scale indicates lowest to highest values.

Figure 2

Comparison of LC-MS/MS IgG1 levels with other serology method and live viral neutralization (A) Roche Elecsys Anti-S assay versus IgG1 LC-MS/MS. (B) Wuhan Hu-1 neutralizing antibodies versus Wuhan S1 IgG1 LC-MS/MS. (C) Alpha neutralizing antibodies versus Alpha and Wuhan S1 IgG1 LC-MS/MS. (D) Beta neutralizing antibodies versus Beta and Wuhan S1 IgG1 LC-MS/MS. (E) Delta neutralizing antibodies versus Delta and Wuhan S1 IgG1 LC-MS/MS. Significance determined by Spearman correlation. n = 141 in total for n = 24 infection-naive group, n = 23 previous infection group at pre, first, and second vaccination time points.

Figure 3

Application of immunocomplex assay to vaccinated participant cohort (A) The comparison of IgG1 levels binding to each VoC spike according to vaccine status in healthcare workers, who have had (+) or not had (−) a prior SARS CoV-2 infection. Results are shown for the wild-type Wuhan Hu-1, Alpha, Beta, and Delta spike variants. (B) Summaries of average percentage of the IgG1 response to the VoC compared with Wuhan Hu-1. Comparison of other proteins of immunocomplex according to vaccine status for (C) IgM, (D) IgG3, (E) IgA1, (F) complement C4, and (G) complement C9. Vaccination groups colored according to exposure status. Green, no exposure; yellow, first exposure (X1); orange, second exposure (X2), and red, third exposure(X3). Data normalized for VoC comparison. Significance determined by non-parametric ANOVA and Spearman correlation p values ∗ ≤0.05, ∗∗ ≤ 0.01, ∗∗∗ ≤ 0.001, ∗∗∗∗ ≤ 0.0001. Colored significance bars indicate change due to pre-infection. n = 24 infection-naive group, n = 23 previous infection group.

Figure 4

C1q response to variant S1 protein (A) The comparison of C1Q levels binding to each VoC spike according to vaccine status in healthcare workers, who have had (+) (n = 23) or not had (−) (n = 24) a prior SARS CoV-2 infection. Results are shown for the wild-type Wuhan Hu-1, Alpha, Beta, and Delta spike variants. Linear regression based on correlation showing changes in the slope/ratio for Delta for (B) C1Q versus IgG1, and no change in slope/ratio for Delta for (C) C1Q versus nAbs (log scale) analysis performed on all four vaccination groups combined (n = 94). (D) A summary of significant changes of the immunocomplex due to VoC compared with Wuhan Hu-1 response. Blue decreased and red increased. Vaccination groups colored according to exposure status. Green, no exposure; yellow, first exposure; orange, second exposure; and red, third exposure. Significance determined by non-parametric ANOVA and Spearman correlation p values ∗ ≤ 0.05, ∗∗ ≤ 0.01, ∗∗∗ ≤ 0.001, ∗∗∗∗ ≤ 0.0001.

Figure 5

Response of triple-vaccinated HCWs to the Omicron BA.1 VoC Comparison of triple vaccinated with or without prior infection against S1 protein from Wuhan Hu-1 or Omicron BA.1 infection; naive n = 25 (blue), prior infected n = 36 (red). Mean ± 1 standard deviation are indicated on all plots. (A) IgG1. (B) IgA. (C) C1q. (D) C4. (E) C9. (F) Comparison of ratio of C1q/IgG1 between VoCs for double (n = 47) and triple (n = 76) vaccinated. Significance determined by non-parametric Kruskal-Wallis test.p-values ∗ ≤ 0.05, ∗∗ ≤ 0.01, ∗∗∗ ≤ 0.001, ∗∗∗∗ ≤ 0.0001