Characterization of antibody-antigen interactions in human pathogens

Antibodies play an ever increasing role in biotechnology and basic research sectors due to their ability to recognize their target antigen with high affinity and selectivity. When their target is a pathogen, they can often neutralize it. Studying antibodies is also relevant for vaccine design, since most current vaccines aim to generate an antibody response. This project aims at investigating the interaction of antibodies with human pathogens, trying to improve our understanding of the molecular determinants for efficient neutralization. The main focus of our research group is the structural, biophysical and immunological characterization of antibody-antigen complexes by a multidisciplinary approach ranging from experimentally validated structural bio-computing to in cell infectivity assays. The long term goal is to improve our understanding of the determinants of efficient neutralization and selectivity by antibodies by knowing 1) which are the properties that render an antibody efficient and/or capable of recognizing pathogens; 2) the effect of antibody binding on the antigen, including dynamic effects; 3) whether with this information the antibodies can be manipulated to achieve better function. In this proposal, antibodies against Prion Protein and Diphtheria Toxin will be investigated. These molecules are not only responsible for biomedical relevant diseases but they are also good models for basic research studies on protein toxins and aggregation processes. Prion diseases are fatal neurodegenerative disorders affecting humans and animals for which no cure is available. Cellular prion protein, the causative agent, can convert into a toxic form ultimately leading to the accumulation of aggregates of prion protein in the brain. Recently, antibodies against the globular domain of prion protein have been shown to increase its toxicity. However, our group was able to show that single mutations in the antigen binding site render the molecule either toxic, non-toxic or protective, even when administered 20 days post prion infection. This has implications for therapy, since antibody treatment is considered a valid therapeutic strategy against prion diseases. However, this novel and exciting result also offers an opportunity to investigate the mechanism leading to toxicity and protection from toxicity, hopefully paving the way for future therapeutic approaches. In this proposal we aim to determine and compare the molecular properties of toxic, non-toxic and protective antibody mutants. Differences are likely to point out plausible determinants for toxicity and protection. Diphtheria epidemics are re-emerging in part of the world with insufficient vaccination coverage. Current therapy involves use of an “antitoxin” produced from horses known to cause “serum sickness”, a problem that would be circumvented by treatment with human antibodies such as those studied in this project. Despite decades of studies on Diphtheria toxin, no structural information is available on antibodies that can neutralize it. Thus in this proposal we aim to study the binding site and mechanism of action of a panel of human monoclonal antibodies. We are particularly interested in antibody D2.2, a potent neutralizer with the peculiarity of recognizing the toxin receptor binding domain and yet not inhibiting diphtheria binding to its host cellular receptor. The same multidisciplinary approaches and techniques will be used to study both objectives, which will be pursued in parallel by members of our research group.