Immunoglobulin A antibody composition is sculpted to bind the self gut microbiome

Abstract

Despite being the most abundantly secreted immunoglobulin isotype, the pattern of reactivity of immunoglobulin A (IgA) antibodies toward each individual's own gut commensal bacteria still remains elusive. By colonizing germ-free mice with defined commensal bacteria, we found that the binding specificity of bulk fecal and serum IgA toward resident gut bacteria resolves well at the species level and has modest strain-level specificity. IgA hybridomas generated from lamina propria B cells of gnotobiotic mice showed that most IgA clones recognized a single bacterial species, whereas a small portion displayed cross-reactivity. Orally administered hybridoma-produced IgAs still retained bacterial antigen binding capability, implying the potential for a new class of therapeutic antibodies. Species-specific IgAs had a range of strain specificities. Given the distinctive bacterial species and strain composition found in each individual's gut, our findings suggest the IgA antibody repertoire is shaped uniquely to bind "self" gut bacteria.

Fig. 1.. Fecal IgAs bind better towards bacteria that colonized them.

(A and B) Cross-reactivity of stool and serum IgA towards each bacterial species (A) or B. ovatus strains (B). (C and D) Stool and serum IgA binding capability towards each bacterial species of a cocktail of 8 bacterial species (8M Mix) or another cocktail of 8 bacterial species, which belong to the same species as 8M Mix but from different strains (8M* Mix). Stool and serum samples were harvested from gnotobiotic mice that were colonized with indicated bacterial strains or consortia for three weeks. The average value from 5 to 7 mice was used in heat map plotting. Data in A to D were normalized by row. Detailed information of bacterial strains is listed in table S1. p values were calculated by unpaired, nonparametric Mann-Whitney test: *p < 0.05, **p < 0.01).

Fig. 2.. Majority of monoclonal IgA antibodies produced by gut lamina propria IgA⁺ B cells bind towards bacterial antigens.

(A) Binding capability of 29 monoclonal IgA antibodies towards bacteria that colonized the gut of gnotobiotic mice. Germ-free mice were colonized with all 8 bacterial species for three weeks, then immune cells isolated from intestinal lamina propria were used for hybridoma fusion. A OD₄₅₀ > 0.25 at antibody concentration of 10 μg/ml is regarded as positive signal (dark gray square). (B) Representative results of hybridoma-produced IgA antibodies binding towards B. ovatus (I), B. caccae (II), R. gnavus (III), both B. ovatus and R. gnavus (IV) and all 8 bacterial species (V). The relative binding capability of IgA clones was analyzed by ELISA and the average value of two technical replicates is displayed. (C) Representative flow cytometry plots of hybridoma-produced IgA antibodies (clones B11, C2 and F2) binding towards all 8 bacterial species. Detailed information of bacterial strains is listed in table S1. OD₄₅₀: optical density at 450 nm.

Fig. 3.. Monoclonal IgAs isolated from lamina propria bind bacteria at species and strain levels.

(A to C) Reactivity of monoclonal IgA antibodies towards different B. ovatus strains (A), B. caccae strains (B) and R. gnavus strains (C) that were not used to colonize mice. Strains denoted by the white/open bar belong to the 8M community that colonized gnotobiotic mice. (D to F) Correlation of genomic similarity (X-axis) and binding similarity (Y-axis) of IgA clones that bind to B. ovatus (D), B. caccae (E) and R. gnavus (F). The genomic similarity was calculated as the genome k-mer similarity between each strain and the particular strain that was used to colonize gnotobiotic mice. The binding similarity was calculated as the area under curve of each strain over the area under curve of the particular strain that was used to colonize gnotobiotic mice. Results are the average value of two technical replicates (A to C). Dotted lines denote A.U. in negative control. Detailed information of bacterial strains is listed in table S1. A.U.: arbitrary units.

Fig. 4.. Hybridoma-produced monoclonal IgA antibodies retain binding capability towards bacterial antigen after passing through intestinal tract.

(A) Schematic representation of antibody gavage in Rag1^−/− mice (100 μg IgA per gavage). (B) IgA quantification in the stool of Rag1^−/− mice at different time points after IgA clone F2 antibody gavage. (C) The binding capability of IgA clone F2 in the stool of Rag1^−/− mice at hour 9 after initial gavage. sup.: supernatant. (D) Correlation of IgA clone F2 concentration in drinking water (X-axis) and in stool (Y-axis). Mice were given water containing different concentration of IgA clone F2 at 7 pm. The next day at 7 am, stool samples were collected and IgA level was quantified by ELISA. (E) IgA clone F2 concentration in the stool of mice at different time points. Mice were given water containing 0.2 mg/ml IgA clone F2 at 7 pm. Stool samples were collected every 12 hours in the following 36 hours. Data are shown as means ± SEM.