Abstract by Susanne Hedwig Dorothea Ständer

The characterization of the immune response induced by infection, vaccination or in autoimmunity is key for understanding the molecular mechanisms that guide immunological processes in health and disease. A key aspect to determine is which parts of an antigen or autoantigen (epitopes) are recognized by an antibody. Mapping of the epitopes is thus crucial for studying the basics of immunity, but also for elucidating autoantigenicity, as well as therapeutic antibody and vaccine mode of action.
In the presented PhD thesis, the first project aimed to structurally investigate the human neuroendocrine enzyme glutamate decarboxylase (GAD), since only one of the two existing isoforms, namely GAD65 but not GAD67, constitutes a predominant autoantigen in type 1 diabetes (T1D) and other autoimmune disorders. With this aim bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to investigate the solution phase conformational dynamics of GAD65 in the presence and absence of cofactor and an autoimmune polyendocrine syndrome type 2 (APS-2) patient-derived autoantibody (b96.11). HDX data were additionally compared with molecular dynamics (MD) simulations and Gaussian Network Model (GNM) analysis. Further structural details on GAD65 and its interaction with the corresponding autoantibody were obtained through cryogenic electron microscopy (cryo-EM) and X-ray crystallography. Lastly, Surface Plasmon Resonance Imaging (SPRi) was performed to assess the binding kinetics of GAD65-b96.11 interaction. In summary, our experimental findings together with the applied computational tools shed light on the structure-function relationship of GAD65 and gave new insights into its autoantigenicity on a molecular level.
The second part of the current PhD thesis was dedicated to characterize the immune response post vaccination. Today, IgG or B cell receptor (BCR) sequencing, as well as cloning of human B cells and the subsequent production of corresponding recombinant monoclonal antibodies (mAbs) have fueled investigations on the protective human immune response upon vaccination or infection. With this aim, a longitudinal study on the VDJ recombination and mutations occurring in BCRs of clonally expanded antigen-specific B cells was performed. Therefore BCR heavy chain sequences isolated from antigen-specific memory B cells (MBC) and plasmablasts (PBs) from four 4CMenB (Bexsero) vaccinated subjects were analysed.

Factor H binding protein (fHbp) is included in the Bexsero vaccine and represents one of the widely studied Neisseria meningitidis antigens, which elicits a robust and protective immune response in humans. In all four studied vaccinees BCRs of fHbp-specific B cells isolated at different time points post vaccination revealed a distinct CDR H3 length of 45 bp with a characteristic and highly conserved amino acid sequence motif. HDX-MS based epitope mapping of those mAbs sharing the same sequence motif in the CDR H3 revealed a common epitope in the N-terminal domain of fHbp which is recognized by each of the mAbs. Additional work was focused on the evaluation of the bactericidal capability of those mAbs via serum bactericidal assays (SBAs). With this aim each of the selected mAbs were tested alone or in combination with another mAb recognizing an epitope located at the C-terminal domain of fHbp.
However, it is not guaranteed that isolated analyses of multiple recombinantly produced mAbs deriving from B cells would lead to a comprehensive understanding of the simultaneous and competitive binding of a polyclonal antibody (pAb) population circulating in the blood. Thus, to pinpoint immuno-dominant epitopes, the entire population of elicited antibodies should also be evaluated. In the last part of the PhD project, a methodology for epitope mapping with HDX-MS was developed to characterize immuno-dominant epitopes recognized by the total of circulating pAbs deriving from the blood. The presented methodology is based on monitoring the HDX of the antigen in absence or presence of varied amounts of pAbs, as well as dissociating additives. The HDX-MS workflow was developed using pAb samples isolated from rabbits immunized with factor H-binding protein (fHbp). As a result, multiple well-defined immuno-dominant epitopes in the N-and C-terminal domain of the antigen could be identified by using the presented HDX-MS based approach.
As next step, the previously developed HDX-MS workflow was applied to pAb samples deriving from two human Bexsero vaccinees. Also in this study distinct immuno-dominant epitopes could be pinpointed, revealing that the developed HDX-MS approach is of great utility for fast epitope screening using human pAbs elicited post-vaccination. Further, in both studies using either rabbit or human pAb sample, we could show that the HDX-MS workflow is suitable for epitope mapping using the total set of pAbs without the need for additional antigen-specific antibody enrichment. Therefore only a very low amount (μL range) of serum is required for HDX-MS based epitope mapping using pAbs.

In summary, all obtained data from epitope mapping with anti-fHbp mAbs and pAbs deliver a package of structural and functional information on antibody-mediated immune protection upon vaccination, which pushes the boundaries in structural serology and its implementation in vaccine- and therapeutic mAb design.