Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes. The P. vivax Duffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includes Plasmodium falciparum sialic acid binding protein and Plasmodium knowlesi Duffy binding protein. The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins. Here, we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli. Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies. We have developed methods to refold and purify recombinant PvRII in its functional conformation. Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Duffy-positive human erythrocytes with specificity. Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P. vivax Duffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate for P. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development.The invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions between host receptors and parasite ligands (1). Plasmodium vivax and the related simian malaria parasite Plasmodium knowlesi require interaction with the Duffy blood group antigen to invade human erythrocytes (2, 3). P. knowlesi can also invade rhesus erythrocytes using alternative Duffy-independent receptors (4). P. falciparum commonly uses sialic acid residues of glycophorin A as invasion receptors (5-9). Like P. knowlesi, P. falciparum also invades erythrocytes by multiple pathways and is not completely dependent on sialic acid residues of glycophorin A (8,10,12,13).Parasite ligands that bind host receptors to mediate erythrocyte invasion include P. vivax and P. knowlesi Duffy-binding proteins, P. knowlesi ␤ and ␥ proteins, which bind Duffy-independent receptors on rhesus erythrocytes, and P. falciparum sialic acid-binding protein (also known as EBA-175), which binds sialic acid residues on glycophorin A (4, 14 -18). These parasite ligands share similar features and belong to a family of erythrocyte-binding proteins (19). The extracellular domain of each erythrocyte-binding protein contains two conserved cysteine-rich regions, regions II and VI, at the amino and carboxyl ends, respectively. P. falciparum EBA-175 contains a tandem duplication (F1 and F2) of the N-terminal, conserved, cysteine-rich region. The functional receptor binding domain of each erythrocyte-binding protein lies in region II (20, 21). In the case of EBA-175, region F2 was found to have receptor b...

Athrombospondin-related anonymous protein (TRAP) of the human malaria parasite Plasmodium falciparum shares highly conserved amino acid sequence motifs with the circumsporozoite protein of all plasmodia sequenced so far, as well as with unrelated proteins like thrombospondin and properdin. Although it was first described as an asexual blood stages protein, there has been some controversy about its expression in these stages. Pursuant to our interest in the conserved sequences within the malaria antigens, we synthesized an 18-residue peptide (18-mer) representing a conserved motif of TRAP and raised polyclonal antibodies against it. In an immunoblot assay in which we probed proteins from the asexual blood stages of the parasite, we found that this antibody recognized predominantly a 78-kDa protein in the whole parasite lysate. Furthermore, in another immunoblot, the recombinant TRAP constructs containing the conserved-motif sequence were distinctly recognized by the antipeptide antibodies, whereas a construct lacking the motif sequence was not, suggesting that the antibodies specifically cross-reacted with a protein which might be a TRAP-like protein present in the asexual blood stages of the parasite. Also, in an immunofluorescence assay, this antibody brightly stained the acetone-fixed trophozoites of the parasite. Most significantly, anti-18-mer immunoglobulin G, as well as antipeptide antibody against a smaller (nonamer) construct representing the most conserved motif within the 18-mer, inhibited the merozoite invasion of erythrocytes in a dose-dependent manner. These results provide evidence of the expression of TRAP or a TRAP-like protein in the asexual blood stages of the parasite and of a possible role of the conserved motifs in the parasite-host cell interaction during the process of invasion.

Liver-stage antigen 1 (LSA-1) is a potential vaccine candidate against preerythrocytic stages of malaria. We report here the immunogenicity of linear synthetic constructs delineated as T H -cell determinants from the nonrepeat regions of Plasmodium falciparum LSA-1 in murine models and human subjects from areas where malaria is endemic in Rajasthan State, India. Seven peptide constructs (LS1.1 to LS1.7) corresponding to predicted T-cell sites from both the N-and C-terminal regions and peptide LS1R from a repeat region of PfLSA-1 were synthesized to analyze the cellular immune responses. These linear peptides were also tested for humoral responses in order to determine if there were any overlapping B-cell epitopes in the predicted T-cell sites. Most peptides induced cellular responses in peptide-immunized BALB/c and C57BL/6 mice as measured by proliferation and cytokine analysis. Cross-reactive T-cell recognition of P. falciparum-based peptides in Plasmodium berghei-immune animals was evaluated, but only one peptide, LS1.2 (amino acids 1742 to 1760) triggered T-cell proliferation and interleukin-2 and gamma interferon secretion in P. berghei-immune splenocytes of BALB/c and C57BL/6 mice as well as in Thamnomys gazellae (natural host of P. berghei ANKA). In an enzyme-linked immunosorbent assay with the peptides, only one peptide, LS1.1, was recognized by anti-P. berghei liver-stage serum. Three peptides (LS1.1, LS1.2, and LS1.3) of the eight peptides tested in this study were recognized by a relatively large percentage of P. falciparum-exposed human subjects; the reactivities ranged from ϳ45% for LS1.3 to ϳ60% for LS1.1 and LS1.2. Interestingly, all of the eight putative T-cell determinants were also recognized by the sera collected from malaria patients, although the response was variable in nature. These T H -and B-cell epitopes may be of potential value for preerythrocytic antigen-based malaria subunit vaccine formulations.

A heptapeptide containing two dehydrophenylalanine residues, Boc-Gly-AzPhe-Ala-Phe-Leu-AzPhe-Ala-NH Me (Boc = t-butoxycarbonyl) has been synthesised and its solution conformation investigated using 500 MHz 'H NMR and IR spectroscopy. 'H NMR studies of the solvent accessibility of NH resonances and observation of successive N,H ++ N,+, H nuclear Overhauser effects (NOEs) suggest the presence of a significant population of folded, helical structures in CDCI,. A 5-+1 intramolecular hydrogen bonding pattern provides evidence in favour of an a-helix conformation. In (CD,),SO, although the peptide largely favours the helical conformation, observation of a few C;H -N,+,H NOEs gives an indication of some conformational heterogeneity. IR studies in chloroform have provided supporting evidence in favour of these conclusions. Dehydrophenylalanine residues may be of potential use in designing peptides with preferred secondary structures.

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