Destabilization of exotoxin A diminishes serum antibody titer and affinity maturation by reducing peptide-MHCII abundance

Abstract

Abstract Potent antibody responses depend on the proteolytic processing of protein antigens and the presentation of MHC Class II bound peptides to CD4+ T cells. We have previously reported a single amino acid substitution (R494A) made to pseudomonas exotoxin A domain III (PE-III) that alters processing and antibody responses with only minor changes to CD4+ T-cell priming. These findings could not explain how the R494A substitution causes a substantial reduction in antibody titer. Previous work by our laboratory and others has shown that the stability of protein antigens affects T-cell priming through effects on antigen processing. However, there is no direct mechanism for how antigen processing alters antibody immunogenicity. We hypothesized that destabilization of PE-III by the R494A substitution reduces the abundance of PE-III peptide-containing MHCII complexes on B cells and thereby reduces their capacity to solicit T cell help for affinity maturation. Here we have observed that antibodies raised against PE-III R494A exhibit lower avidity than those raised against wild type PE-III. Furthermore, R494A PE-III was a poorer source of T-cell epitopes when provided to splenocytes from mice immunized with either wild type or R494A. Currently, PE-III peptide abundance is being analyzed by mass spectrometry, and antigen specific B cells are being characterized by single cell RNA sequencing.