The phagocytosis and destruction of microorganisms is an essential immunological function of macrophages and other phagocytes that protects us from invading pathogens. However current understanding of this process is incomplete because we have shown that bacteria are able to replicate and survive within macrophages that lack Perforin-2 (PRF2); until recently a largely uncharacterized protein. As expected from cell based studies, when challenged with pathogens PRF2 knockout mice succumb to infectious doses that the majority of their wild-type littermates survive. This is accompanied by replication and dissemination of bacteria to deeper tissues. Further underscoring the importance of PRF2 in host defense is the fact that some pathogens deploy effectors to block the delivery of PRF2 to phagosomes. Recent high resolution structures of PRF2 have shown that it is a pore-forming protein and that the transition from pre-pore-to-pore is driven by low pH; consistent with its role within acidifying phagosomes. This is accompanied by substantial intra- and inter-domain conformational changes. Although the data is not yet extensive, a clinical picture is beginning to emerge that certain mutations within PRF2 correlate with chronic bacterial infections. Further research in this area may well reveal that PRF2 haploinsufficiency is the underlying cause of recurring or persistent infections. These and others studies have established that PRF2 underpins one of the most basic functions of macrophages and other phagocytes. Although considerable advances have been made within this new area of research, progress is hindered by a critical lack of antibodies that are the mainstay of immunological investigations. Thus, the objective of this proposal is to isolate and evaluate heavy chain antibodies (VHHs) against PRF2. We further propose to develop conformation specific VHHs that are able to discriminate between the monomeric and, oligomeric pre-pore and pore conformations.
In Aim1 we will capitalize on the recent discovery that pH controls PRF2 conformations to isolate VHHs to both mouse and human pre-pore and pore complexes from a synthetic VHH library.
In Aim2 we will produce, rank, and evaluate VHHs in a variety of immunological assays. As an indicator of initial momentum we already have 18 unique clones from a screen against the P2 domain of PRF2; some of which are expected to be specific for PRF2 monomers. We anticipate that anti-PRF2 VHHs will remove current roadblocks that hinder progress to further elucidate the central role of PRF2 in innate immune defense. PRF2 research is highly significant to human health because our preliminary data, recent publications, and characterization of haploinsufficient individuals demonstrate that it underpins an essential function of phagocytes.
The phagocytosis and destruction of microorganisms is an essential function of phagocytes that protects us from invading pathogens. This application will further our understanding of this process through the development of antibodies to track and probe the conformational states of a pore-forming protein, Perforin-2, that underpins this critical protective function.
