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

(A) Mice were inoculated intranasally with 10 μg of SpikeWuhan and 1 μg of Cholera toxin as an adjuvant in 10 μl of PBS, delivering the fluid dropwise into the nostril a total of three times at three-week intervals. Nasal lavage fluid and serum were collected from the mice one week after the last immunization, and antibody responses were evaluated using ELISA. The antibody titers are expressed as optical density (OD450) value per total protein in nasal lavage fluids or serum.

(B) FACS gating strategy for the isolation of S1-specific plasma cells from mice. Plots represent the sequential gating strategy. Lymphocytes (R1 gate) were stained with anti-CD138 and ER-Tracker to enrich plasma cells (CD138+ ER-TrackerHigh fraction, R2 gate). IgA+ plasma cells gated in R3 were selected from the R2-gated plasma cell fraction by staining with anti-IgA antibody. The antigen-specific plasma cells gated in R4 were further selected from the R3-gated Ig A+ plasma cell fraction by staining with S1 domain of Wuhan SARS-CoV-2 spike protein (S1). 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 ring 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 the 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.

Comparison of reactivity between monomeric and multimeric IgAs.

(A) Production of recombinant M-IgAs and S-IgAs. Recombinant M-IgA and S-IgA purified from the culture supernatant of CHO cells were subjected to SDS-PAGE and Blue native-PAGE analysis. Bands corresponding to a monomer (M), dimer (Di), trimer (Ti) and tetramer (Te) are shown. H, α heavy chain; L, light chain; J, J chain; SC, secretory component; M, M-IgA; S, S-IgA

(B) Binding dynamics of M-IgAs and S-IgAs to Wuhan, Delta or Omicron spike protein by SPR. The S-IgAs used are a mixture of dimers, trimers, and tetramers. The observed values refrect the average affinity of the S-IgAs. The curves shown are representative of two or three determinations. RU, resonance units. The table shows the association (ka) (M–1s–1), dissociation (kd) (s–1) rate constants and apparent equilibrium dissociation constants (KD) expressed as the mean of two or three determinations (lower panel).

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 non-antibody-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

Intranasal administration of S-IgA suppresses SARS-CoV-2 infection in Syrian hamsters.

(A) Experimental schedule. Three groups of hamsters received a single intranasal dose of 1.0 mg/kg of S-IgA three hours before infection (-3) for pre-exposure prophylaxis and at 24 hours (24) and 48 hours post-infection for early treatment, respectively. Control hamsters (n=3) received PBS at the same dose. On day 0, each hamster was intranasally challenged with the Wuhan SARS-CoV-2 virus (6 x 105 Median Tissue Culture Infectious Dose).

(B) Hamster body weights were recorded hourly (0, 24, 48 and 72 hours), and weight loss was defined as the percentage loss from 0 hours. Data represent the mean value ± SD at the indicated time points (n=3) at the indicated time points and were analyzed using a Kruskal–Wallis one-way ANOVA.

(C) Animals were euthanized 72 hours post-infection, and RNA was extracted from nasal turbinates and lungs. The SARS-CoV-2 viral load was analyzed using qRT-PCR targeting the SARS-CoV-2 nucleoprotein. Assays were normalized relative to total RNA levels. Data represent the mean value (n=3).

The affinity, ACE2 inhibitory activity, and the in vitro neutralizing activity of the indicated antibodies are illustrated in a 3D scatter plot.

N5203 M-IgA, categorized as Type 1, binds to the RBDWuhan and RBDDelta, inhibits ACE2-binding to these RBDs, and effectively neutralizes Wuhan and Delta pseudotyped viruses. The multimeric form of this antibody had a minimal effect on ACE-blocking and neutralization breadth despite the increased affinity for SpikeWuhan. This suggests that the increase in valency does not necessarily correlate with an increase in functionality. N142 M-IgA, categorized as Type 2, binds to the RBDWuhan and RBDDelta, inhibiting ACE2-binding to these RBDs, yet fails to neutralize the viruses. Interestingly, its S-IgA exhibited neutralization activity against both pseudotyped viruses without enhancing spike binding or ACE-blocking activity. These results suggest that the neutralizing activity of N142 S-IgA partially depends on the ACE2-blocking activity of the corresponding M-IgA, but its mechanism differs from that of N5203 S-IgA, whose monomeric form shows cross-neutralizing activity. Notably, two types of non-ACE2-blocking, non-neutralizing M-Abs (N114 and N217), expressed as S-IgA, gained the ability to neutralize the Wuhan but not the Delta pseudotyped viruses. These findings indicate that the two S-IgAs exert their neutralizing activity through mechanisms apart from the general ACE2-RBD axis.