Isolation of antigen-specific IgA+ plasma cells from mice that were intranasally immunized with SpikWuhan.

(A) Mice were inoculated via the intranasal route with 10 μg of SpikWuhan or PBS with 1 μg CT as an adjuvant. Nasal lavage fluid and serum were collected from the mice at one week after the last immunization, and antibody responses were evaluated using ELISA.

(B) FACS gating strategy for the isolation of S1-specific plasma cells from mice.

Cells from nasal mucosa or spleen were stained with anti-CD138, ER-tracker, anti-mouse IgA, and S1. Plots (I)-(III) represent the sequential gating strategy. (I) FSC vs. SSC with gate R1 representing lymphocytes. (II) The CD138+ ER-TrackerHigh fraction was defined as plasma cells. (R2). (III) The S1-specific plasma cells were defined as IgA+, ER-TrackerHigh and S1+ (R3 gate). The numbers indicate the percentages of cells in the gated area. A total of 100,000 events were recorded. Representative data from the No. 1 mouse are shown.

Intranasal immunization induces functionally diverse antibodies in the nasal mucosa and spleen.

(A) Characterization of S1-specific monoclonal antibodies obtained from No. 1 mouse. The heatmap represents the relative intensity of antibody binding to RBDs and blocking of the RBD-ACE2 interaction. Blue (0–25%), green (25–50), orange (50–75%) and red (>75%). NTD binding was considered positive (+) when the OD at 405 nm was >0.3 after the background was subtracted. Neutralizing activity was considered positive (+) when the antibody suppressed Wuhan pseudotyped virus infection by 50%. The figure reports values from a single experiment. UN, antibody type not determined. ND, antibody activity not determined.

(B) Maximum-likelihood phylogenetic tree of the VH and VL chains of the S1-specific antibodies.

Different colored fonts indicate antibodies obtained from the nose (red), spleen (blue), and lung (green). Antibody groups are indicated by bands on the outer ring. The color of the band indicates antibody types: Type 1 (red), Type 2 (orange), Type 3 (green), Type 4 (blue) and Type 5 (gray). The antibody group is defined as clones using the same V-(D)-J usage and having an overall sequence identity of at least 95% from the signal peptide to framework 4 (FR4). The prefixes N, S and L in the antibody clone numbers refer to antibodies derived from the nose, spleen and lung, respectively. The suffixes A, G and K in the antibody clone numbers refer to alpha, gamma and kappa chain, respectively.

(C) Nucleotide sequence arraignment of VH and VL genes in the G2 and G3 antibodies form No. 1 mouse. The VH and VL sequences from the beginning of the signal peptide through the end of FR4 are shown as horizontal lines. Nucleotide changes relative to S632A and N109A are depicted as vertical bars across the horizontal lines. Different colored fonts indicate antibodies derived from the nose (red) and spleen (blue). Antibody phylogenetic trees based on VH/VK paired sequences are depicted. Gray circles represent the hypothetical germline configuration. White circles represent hypothetical ancestors. Colors indicate nasal (red) and splenic (blue) antibodies. Circles and squares indicate IgA and IgG, respectively.

Characterization of S1-specific monoclonal antibodies obtained from No. 2 mouse.

(A) A total of 51 S1-reactive antibodies were analyzed for their properties as in Fig 2A. UN, antibody type not determined. ND, antibody activity not determined.

(B) Maximum-likelihood phylogenetic tree of the VH and VL chains of the S1-specific antibodies. Different colored fonts indicate antibodies obtained from the nose (red), spleen (blue), lung (green) and blood (magenta). Antibody groups are indicated by bands on the outer ring. The color of the band indicates antibody types: Type 1 (red), Type 2 (orange), Type 3 (green), Type 4 (blue) and Type 5 (gray). The prefixes N, S and L in the antibody clone numbers refer to antibodies derived from the nose, spleen and lung, respectively. The suffixes A, G and K in the antibody clone numbers refer to alpha, gamma and kappa chain, respectively.

(C) Nucleotide sequence arraignment of VH and VL genes in the G6 and G8 antibodies from No. 2 mouse. The. nucleotide changes relative to N5120A and N5105A are depicted as vertical bars across the horizontal lines. Different colored fonts indicate antibodies derived from nose (red), spleen (blue), lung (green) and blood (magenta). Antibody phylogenetic trees based on VH/VK paired sequences are depicted. Gray circles represent the hypothetical germline configuration. White circles represent hypothetical ancestors. Colors indicate nasal (red), splenic (blue), lung (green) and blood (magenta) antibodies.

Characterization of S1-specific monoclonal antibodies obtained from No. 2 mouse.

(A) A total of 51 S1-reactive antibodies were analyzed for their properties as in Fig 2A. UN, antibody type not determined. ND, antibody activity not determined.

(B) Maximum-likelihood phylogenetic tree of the VH and VL chains of the S1-specific antibodies. Different colored fonts indicate antibodies obtained from the nose (red), spleen (blue), lung (green) and blood (magenta). Antibody groups are indicated by bands on the outer ring. The color of the band indicates antibody types: Type 1 (red), Type 2 (orange), Type 3 (green), Type 4 (blue) and Type 5 (gray). The prefixes N, S and L in the antibody clone numbers refer to antibodies derived from the nose, spleen and lung, respectively. The suffixes A, G and K in the antibody clone numbers refer to alpha, gamma and kappa chain, respectively.

(C) Nucleotide sequence arraignment of VH and VL genes in the G6 and G8 antibodies from No. 2 mouse. The. nucleotide changes relative to N5120A and N5105A are depicted as vertical bars across the horizontal lines. Different colored fonts indicate antibodies derived from nose (red), spleen (blue), lung (green) and blood (magenta). Antibody phylogenetic trees based on VH/VK paired sequences are depicted. Gray circles represent the hypothetical germline configuration. White circles represent hypothetical ancestors. Colors indicate nasal (red), splenic (blue), lung (green) and blood (magenta) antibodies.

Multimerization facilitates the neutralization activity of nonneutralizing M-IgAs.

(A) Graphs of the competitive ELISA results showing the binding of biotinylated ACE2 to the immobilized Wuhan, Delta or Omicron RBD in the presence of antibodies. The results are expressed as the mean ± SD of three technical replicates. The IC50 values of the indicated antibodies that inhibit the RBD-ACE2 interaction are shown in the diagrams.

(B) Comparison of neutralization activity between M-IgaA and S-IgA against SARS-CoV-2 pseudotyped viruses. Neutralization curves of the indicated antibody against pseudotyped viruses bearing spike proteins of Wuhan, Delta or Omicron are shown. Pseudotyped viruses preincubated with antibodies at the indicated concentrations were used to infect VeroE6 cells, and luciferase activities in cell lysates were measured at 20 h post transduction to calculate infection (%) relative to nonantibody-treated controls. The results are expressed as the mean ± SD of three technical replicates. The NT50 values of the indicated antibodies are shown in the diagrams. Antibodies that did not reach >70% inhibition at the highest concentration tested were listed as data not determined (ND).

(C) Comparison of neutralization potential between M-IgA and S-IgA against authentic SARS-CoV-2 BA.1. The neutralizing potential of the antibody was determined using an RT-PCR-based SARS-CoV-2 neutralization assay. VeroE6 cells preincubated with authentic SARS-CoV-2 BA.1 virus were incubated with the indicated antibodies at various concentrations. The virus in the cell culture medium was measured at 48 h post transduction to calculate infection (%) relative to nonantibody-treated controls. The results are expressed as the mean ± SD of three technical replicates. The NT50 values of the indicated antibodies are shown in the diagrams. Antibodies that did not reach >50% inhibition at the highest concentration tested are listed as ND. **p<0.01.