Humoral immune responses during SARS-CoV-2 mRNA vaccine administration in seropositive and seronegative individuals

Abstract

Background: The global pandemic of coronavirus disease 2019 (COVID-19) is caused by infection with the SARS-CoV-2 virus. Currently, there are three approved vaccines against SARS-CoV-2 in the USA, including two based on messenger RNA (mRNA) technology that has demonstrated high vaccine efficacy. We sought to characterize humoral immune responses, at high resolution, during immunization with the BNT162b2 (Pfizer-BioNTech) vaccine in individuals with or without prior history of natural SARS-CoV-2 infection.

Methods: We determined antibody responses after each dose of the BNT162b2 SARS-CoV-2 vaccine in individuals who had no prior history of SARS-CoV-2 infection (seronegative) and individuals that had previous viral infection 30-60 days prior to first vaccination (seropositive). To do this, we used both an antibody isotype-specific multiplexed bead-based binding assays targeting multiple SARS-CoV-2 viral protein antigens and an assay that identified potential SARS-CoV-2 neutralizing antibody levels. Moreover, we mapped antibody epitope specificity after immunization using SARS-CoV-2 spike protein peptide arrays.

Results: Antibody levels were significantly higher after a single dose in seropositive individuals compared to seronegative individuals and were comparable to levels observed in seronegative individuals after two doses. While IgG was boosted by vaccination for both seronegative and seropositive individuals, only seronegative individuals had increased IgA or IgM antibody titers after primary immunization. We identified immunodominant peptides targeted on both SARS-CoV-2 spike S1 and S2 subunits after vaccination.

Conclusion: These findings demonstrated the antibody responses to SARS-CoV-2 immunization in seropositive and seronegative individuals and provide support for the concept of using prior infection history as a guide for the consideration of future vaccination regimens. Moreover, we identified key epitopes on the SARS-CoV-2 spike protein that are targeted by antibodies after vaccination that could guide future vaccine and immune correlate development.

Keywords: Antibody response; SARS-CoV-2; mRNA vaccine.

Fig. 1

Antibody response to SARS-CoV-2 mRNA vaccine. A Multiplex bead-based antibody binding assay that measures the IgG antibody response to 4 SARS-CoV-2 viral antigens (S1, S2, RBD, and NP). Median Fluorescent Intensity (MFI) is shown and background well subtraction has been used to remove nonspecific signal. Each dot represents an individual at baseline before vaccine (week 0), 3 weeks after the first dose of vaccine (week 3) or 4 weeks after the second dose (week 7). Bars represent the group median. The number of individuals in each group are shown below the graphs. Individuals with a previous history of SARS-CoV-2 infection (seropositive; blue), no previous history of infection (seronegative; red), and individuals with possible undiagnosed infection (green). The dashed line indicates a threshold determined by the sum of the mean and standard deviation for the negative control (beads without antigen). B Neutralization antibody proxy assay that determines the level of antibodies that block the RBD-ACE2 receptor-binding expressed as the percentage of binding that was blocked relative to control with no plasma (representing maximum binding). The assay threshold for positivity was 30%. Each point represents an individual at baseline before the vaccine, 3 weeks after the first dose of vaccine (week 3) or 4 weeks after the second dose (week 7). Bars represent the group median. The number of individuals in each group are shown below the graphs. Individuals with a previous history of SARS-CoV-2 infection (seropositive; blue), no previous history of infection (seronegative; red), and individuals with possible undiagnosed infection (green). Statistical tests for significant differences between groups were unpaired, two-tailed Wilcoxon-Mann-Whitney test with a significant threshold of P < 0.05

Fig. 2

Antibody response to primary immunization based on sex and age. A Results of the neutralization proxy blocking assay after primary immunization at week 3 with individuals stratified by sex (male, female) or age (18–49 years old, ≥ 50 years old). Each point represents an individual 3 weeks after the first dose of vaccine (week 3). Bars represent the group median. Statistical tests for significant differences between groups were unpaired, two-tailed Wilcoxon-Mann-Whitney test with a significant threshold of P < 0.05

Fig. 3

IgM and IgA antibody isotype responses to SARS-CoV-2 after immunization. A, B Multiplex bead-based antibody binding assay that measures the IgM (A) or IgA-specific (B) antibody response to 4 SARS-CoV-2 viral antigens (S1, S2, RBD, and NP). Median Fluorescent Intensity (MFI) is shown and background well subtraction has been used to remove the nonspecific signal. Each dot represents an individual at baseline before vaccine (week 0), 3 weeks after the first dose of BNT162b2 COVID-19 vaccine (week 3) or 4 weeks after the second dose of BNT162b2 COVID-19 vaccine (week 7). Bars represent the group median. The number of individuals in each group are shown below the graphs in Fig. 1. Individuals with a previous history of SARS-CoV-2 infection (seropositive; blue), no previous history of infection (seronegative; red), and individuals with possible undiagnosed infection (green). The dashed line indicates a threshold determined by the sum of the mean and standard deviation for the negative control (beads without antigen). Statistical tests for significant differences between groups were unpaired, two-tailed Wilcoxon-Mann-Whitney test with a significant threshold of P < 0.05

Fig. 4

IgG subclass responses to SARS-CoV-2. A, B Dot plots of ELISA End Point titers calculated as the highest plasma dilution where the O.D. is 3× background. The bar represents the group median, and the points display individual data. Secondary antibodies specific for each IgG subclass (IgG1, IgG2, IgG3, IgG4) were used to determine subclass-specific responses. A Subclass responses to SARS-CoV-2 spike subunits (S1, S2) and nucleocapsid protein (NP) in SARS-CoV-2 infected individuals (n = 24) at baseline before the vaccine. B Subclass responses to SARS-CoV-2 receptor-binding domain (RBD) in SARS-CoV-2 infected individuals at baseline before vaccine (seropositive; n = 24) and three weeks after the first dose of BNT162b2 COVID-19 vaccine in seronegative individuals (vaccine; n = 24)

Fig. 5

High-resolution mapping of SARS-CoV-2 antibody responses after COVID-19 vaccine. A, B Plasma IgG binding to SARS-CoV-2 peptides spanning the SARS-CoV-2 spike protein (A S1 and B S2 subunits separated;12-mer overlapping peptides) in 14 SARS-CoV-2 seronegative adults after primary COVID-19 immunization (week 3). Each peptide was printed in triplicate and the log2 of the average median fluorescent intensity (F635) for each peptide graphed. Row color corresponds to the minimum and maximum intensity for all peptides for each individual. Known regions of the spike protein are annotated above heatmaps. Orange section in RBD depicts critical ACE2 contact residues. Line graphs displaying the mean group Z-score for each peptide in S1 and S2 from the vaccine group (blue; n = 14)