Molecular interactions of the malarial vaccine candidate AMA1 from Plasmodium falciparum

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Molecular Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

Apical membrane antigen 1 (AMA1) is a malarial protein thought to be critical for parasite invasion of host red blood cells. AMA1 is an important vaccine candidate, however its precise function remains unclear. Investigations by Alexander et al (2006) revealed that AMA1 interacted with a rhoptry neck protein, RON4, in Plasmodium. An aim of this project was to dissect the molecular interactions of the two proteins. Data produced in this study reveals a lack of direct binding between the two proteins and suggests that the interaction may be through a complex involving other malarial proteins. These findings were supported by subsequent publications that identified a complex of RON proteins that interact with AMA1. Further publications revealed that RON2, another rhoptry neck protein, was also interacting with AMA1. In late 2011, two publications identified the Cterminus of PfRON2 as interacting with the hydrophobic trough of AMA1. PfRON2 became of particular interest as other studies showed it was the only protein found to directly bind to AMA1 through its C-terminus. In this study, the central conserved region within PfRON2 is investigated for its ability to bind to AMA1. A stretch of amino acids rich in hydrophobic residues was identified as possibly binding to AMA1. Data produced suggests that these PfRON2 peptides can compete with well-characterized AMA1 binding reagents. These peptides also compete with the PfRON2 C-terminus for binding to AMA1. The data is supported by other studies (published and unpublished) and provides a foundation for further investigation into this region. During this project a panel of novel inhibitory monoclonal antibodies that bind to AMA1 were characterized. These antibodies were raised against several strains of AMA1 and appear to recognize conserved epitopes on the surface of the antigen. Despite the observation that all monoclonals tested could inhibit merozoite invasion, only some of them could block RON2 binding to AMA1, therefore suggesting that inhibitory mAbs may have different mechanisms of inhibiting parasite invasion.