Antibodies to a conserved-motif peptide sequence of the Plasmodium falciparum thrombospondin-related anonymous protein and circumsporozoite protein recognize a 78-kilodalton protein in the asexual blood stages of the parasite and inhibit merozoite invasion in vitro

Abstract

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.

Induction of Protective Immune Responses by Immunization with Linear Multiepitope Peptides Based on Conserved Sequences fromPlasmodium falciparumAntigens

“…Immunoassays. We had found earlier that anti-region II peptide Abs immunoreacted with a TRAP-like molecule in the P. falciparum blood lysate (38). Since both P60 and P32 contained the conserved region II sequences which are present in both CSP and TRAP and which are also found conserved in different plasmodium species (32), we wondered if anti-P60 Abs would also recognize antigens from the blood stages of P. falciparum and P. yoelii.…”

“…(i) Affinity purification of immunoglobulins. Peptide-specific Abs were purified from the rabbit anti-P60 sera by immunoaffinity chromatography essentially as described in our earlier work (38). Briefly, gamma globulin fraction from rabbit serum was obtained by ammonium sulfate precipitation followed by ion-exchange chromatography on an Econo-Pac immunoglobulin G (IgG) purification column (Bio-Rad Laboratories, Richmond, Calif.) according to the manufacturer's instructions.…”

“…The peptide-specific IgG Abs were eluted with glycine-HCl buffer (0.1 M; pH 2.5), and the fractions were neutralized by the addition of Tris base (2.0 M). The fractions were pooled and characterized as described previously (38).…”

“…The role of TRAP and, indeed, its expression and location at the blood stages, is not yet known, although TRAP-specific mRNA has been detected in infected erythrocytes (32). We have recently shown that synthetic peptides representing the conserved motif sequences and the antisera raised against these peptides inhibited the merozoite invasion of erythrocytes (38). Further, the anti-peptide Abs recognized a TRAP-like molecule in the blood stage lysate of P. falciparum (38).…”

“…We have recently shown that synthetic peptides representing the conserved motif sequences and the antisera raised against these peptides inhibited the merozoite invasion of erythrocytes (38). Further, the anti-peptide Abs recognized a TRAP-like molecule in the blood stage lysate of P. falciparum (38). A better understanding of the structure of the region II peptide sequences and immune responses against them may provide the basis for their inclusion in a subunit malaria vaccine.…”