Structure of the Plasmodium falciparum Circumsporozoite Protein, a Leading Malaria Vaccine Candidate

Abstract

The Plasmodium falciparum circumsporozoite protein (CSP) is critical for sporozoite function and invasion of hepatocytes. Given its critical nature, a phase III human CSP malaria vaccine trial is ongoing. The CSP is composed of three regions as follows: an N terminus that binds heparin sulfate proteoglycans, a four amino acid repeat region (NANP), and a C terminus that contains a thrombospondin-like type I repeat (TSR) domain. Despite the importance of CSP, little is known about its structure. Therefore, recombinant forms of CSP were produced by expression in both Escherichia coli (Ec) and then refolded (EcCSP) or in the methylotrophic yeast Pichia pastoris (PpCSP) for structural analyses. To analyze the TSR domain of recombinant CSP, conformation-dependent monoclonal antibodies that recognized unfixed P. falciparum sporozoites and inhibited sporozoite invasion of HepG2 cells in vitro were identified. These monoclonal antibodies recognized all recombinant CSPs, indicating the recombinant CSPs contain a properly folded TSR domain structure. Characterization of both EcCSP and PpCSP by dynamic light scattering and velocity sedimentation demonstrated that both forms of CSP appeared as highly extended proteins (R h 4.2 and 4.58 nm, respectively). Furthermore, high resolution atomic force microscopy revealed flexible, rod-like structures with a ribbon-like appearance. Using this information, we modeled the NANP repeat and TSR domain of CSP. Consistent with the biochemical and biophysical results, the repeat region formed a rod-like structure about 21-25 nm in length and 1.5 nm in width. Thus native CSP appears as a glycosylphosphatidylinositol-anchored, flexible rod-like protein on the sporozoite surface.

Reversible Conformational Change in the Plasmodium falciparum Circumsporozoite Protein Masks Its Adhesion Domains

“…The two recombinant forms of EcCSP and PpCSP were fermented in 5-liter bioreactors, purified using standard column chromatography, and fully biochemically and biophysically characterized as reported previously (11) and as detailed in the supplemental material.…”

“…Only a recombinant AMA-1 forming a stable complex with a constrained synthetic rhoptry neck protein-2 peptide induced protective antibodies in vivo against a lethal blood-stage challenge malaria parasite infection (8). When developing a novel CSP vaccine, these more recent developments need to be considered with regard to the potential for changes within the CSP, such as through in vivo processing or conformational changes (9,10) in a protein with a known extended rod-like structure (11), that could mask the adhesion domains located at the N-and C-terminal domains (9).…”

Overcoming Allelic Specificity by Immunization with Five Allelic Forms of Plasmodium falciparum Apical Membrane Antigen 1

“…The panel of recombinant AMA1 proteins was characterized essentially as described by Plassmeyer et al (23) and Uchime et al (24). In brief, the following analytical procedures were included here: Edman degradation, reversedphase high-performance liquid chromatography (HPLC), analytical SEC with in-line multiangle light scattering (MALS)-HPLC, matrix-assisted laser desorption ionization (MALDI) mass spectrometry, endotoxin concentration determination, immunoblotting with rat monoclonal antibody (MAb) 4G2dc1 (25) and anti-His 6 (Qiagen, Inc.), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).…”

Advances and challenges in malaria vaccine development

“…RTS,S also includes the thrombospondin domain, which binds receptors on liver cells (113). Monoclonal antibodies to the thrombospondin domain also block sporozoite invasion of liver cells, but to a lesser degree than antibodies to the repeat region (112). The CSP contains three known T cell epitopes: a highly variable CD4 + T cell epitope before the thrombospondin domain (114), a highly variable CD8 + T cell epitope within the thrombospondin domain (115), and a conserved "universal" CD4 + T cell epitope at the C-terminus (116).…”

“…HBsAg particles serve as the matrix carrier for RTS,S, 25% of which is fused to the CSP segment. The central repeat region contains the immunodominant B cell epitope, which induces antibodies that block sporozoite infection of liver cells in vitro (111,112). RTS,S immunization induces antibodies to the central repeat region that correlate with protection from P. falciparum infection (46, 47) but not clinical disease (41,45,46).…”