The first aim is to understand the molecular basis of malaria sporozoite (SPZ) infectivity in the liver. We have used SPZ invasion assays of HepG2 cells, and binding assays, to demonstrate that: 1. SPZ bind to HepG2 cells via a conserved 5 amino acid region of the vaccine candidate CS Protein; 2. The SPZ protein TRAP binds to hepatocytes through its RGD sequence to the fibronectin receptor a5b1 integrin which we have shown is expressed on HepG2 cells; 3. SPZ also bind to amino terminus of fibronectin independently of RGD; 4. SPZ bind to the C-terminus of thrombospondin (TSP) via a defined region of the SPZ micronemal protein TRAP, that TSP forms a molecular bridge between SPZ and hepatocyte CD36, and we have identified a CD36 sequence that acts as a low affinity receptor; 5. SPZ TRAP contains a sequence homologous with the A domain of b2 integrins that functions as CD11a (LFA-1) confirmed by binding to CD11a receptors; 6. SPZ express a mucin-like protein that is involved in binding to hepatic cells probably through O-linked N-acetylglucosamine using lectin inhibition assays; 7. SPZ contain a novel micronemal protein distinct from TRAP that may also mediate infectivity. 8. Identified a HepG2 receptor and used peptides from R. Boykins to define the active binding site. 9. Determined that SPZ invade many cell types in vitro and correlate this with expression of receptor in (8). 10. Further characterized the tissue and cell distribution of a novel hepatocyte carboxylesterase receptor We can now propose a more complete understanding of SPZ infectivity in which localization in the sinusoid is the most specific step, followed by passage across endothelium using receptors also involved in lymphocyte extravasation, and a relatively non-specific molecular interaction mediating actual hepatocyte invasion.
The second aim i s to investigate the relationship between CD4+ responses to defined peptides of the candidate vaccine LSA-1 and malaria morbidity in children in Papua New Guinea. Using peptides made by R. Boykins we have demonstrated that 95% of clinically immune adults gave CD4+ responses defined by cytokine release. We have now enrolled 420 children in a 1 year prospective study which will use these peptides, as well as two others now being made by the core facility. This work has resulted in 5 manuscripts in press, 1 submitted, and 9 ready for submission.
