Characterization of Protective Epitopes in a Highly ConservedPlasmodium falciparumAntigenic Protein Containing Repeats of Acidic and Basic Residues

Abstract

The delineation of putatively protective and immunogenic epitopes in vaccine candidate proteins constitutes a major research effort towards the development of an effective malaria vaccine. By virtue of its role in the formation of the immune clusters of merozoites, its location on the surface of merozoites, and its highly conserved nature both at the nucleotide sequence level and the amino acid sequence level, the antigen which contains repeats of acidic and basic residues (ABRA) of the human malaria parasitePlasmodium falciparum represents such an antigen. Based upon the predicted amino acid sequence of ABRA, we synthesized eight peptides, with six of these (AB-1 to AB-6) ranging from 12 to 18 residues covering the most hydrophilic regions of the protein, and two more peptides (AB-7 and AB-8) representing its repetitive sequences. We found that all eight constructs bound an appreciable amount of antibody in sera from a large proportion of P. falciparummalaria patients; two of these peptides (AB-1 and AB-3) also elicited a strong proliferation response in peripheral blood mononuclear cells from all 11 human subjects recovering from malaria. When used as carrier-free immunogens, six peptides induced a strong, boostable, immunoglobulin G-type antibody response in rabbits, indicating the presence of both B-cell determinants and T-helper-cell epitopes in these six constructs. These antibodies specifically cross-reacted with the parasite protein(s) in an immunoblot and in an immunofluorescence assay. In another immunoblot, rabbit antipeptide sera also recognized recombinant fragments of ABRA expressed in bacteria. More significantly, rabbit antibodies against two constructs (AB-1 and AB-5) inhibited the merozoite reinvasion of human erythrocytes in vitro up to ∼90%. These results favor further studies so as to determine possible inclusion of these two constructs in a multicomponent subunit vaccine against asexual blood stages of P. falciparum.

Immunogenicity of recombinant fragments of Plasmodium falciparum acidic basic repeat antigen produced in Escherichia coli

“…For example, in the CS protein of P. falciparum, the central repeat region forms the immunodominant epitope, resulting in masking of the highly conserved and functional epitope lying in region II, leading to poor immune response to this epitope (39). The observed poor immune response to ABRA (N) is also in agreement with our earlier findings which showed that peptides based on the N-terminal region of ABRA were poorly immunogenic in animals (25). However, when the N-terminal was expressed along with the middle portion of the protein, the resulting construct, ABRA (P), was highly immunogenic in mice.…”

“…ABRA constructs used for this study were as follows: (a) ABRA (N) representing the N-terminal portion of the protein including the TNDEED repeats; (b) ABRA (M) containing the middle fragment of the protein without any repeats; (c) ABRA (C) which represents the highly charged C-terminal tail of the protein containing the acidic and basic KEKEEK repeats; and (d) ABRA (P) representing two-thirds of the protein from the N-terminal ( Figure 1a). The constructs were designed in such a way that they contained at least one epitope sequence that had been identified in our earlier study with ABRA peptides (25). ABRA (M) and ABRA (C) were expressed as fusion proteins with MBP at their N-terminii whereas ABRA (N) and ABRA (P) were expressed with N-terminal 6X histidine tag (Figure 1a).…”

“…Our earlier studies showed that the peptides based on ABRA sequence elicit a strong antibody response in rabbits and some of these antibodies exert a strong inhibitory effect on the merozoite invasion of erythrocytes (25). Some of these peptide sequences were found to be the targets of immune responses generated during natural infection, and they also stimulated the peripheral blood mononuclear cells from convalescent patients living in endemic areas (25). Although our attempts to obtain full ABRA protein have been unsuccessful, we were able to express and characterize several large fragments of this protein and show that the antibodies raised against these fragments recognized the native parasite protein (21).…”

Antigenic Diversity of Plasmodium falciparum and Antibody‐Mediated Parasite Neutralization

“…Both SERA and ABRA are found in the immune clusters formed by antibodies inhibiting merozoite dispersal in parasite cultures [39]. Antibodies to SERA have, furthermore, been shown to inhibit P. falciparum growth in vitro and antibodies to ABRA are ef®cient inhibitors of merozoite invasion [55]. parasite-neutralizing antibodies, inhibiting the intraerythrocytic growth of the parasite [8].…”

Protective Immunity Induced by Virus-Like Particle Containing Merozoite Surface Protein 9 of Plasmodium berghei

“…There is experimental evidence that MSP-9 participates in merozoite release from infected erythrocytes and binds to the human erythrocyte surface via N-terminal cysteine-rich regions or high binding activity peptides (HBAPs) [8,9]. MSP-9 has been suggested to be an important protein in vaccine development due to its role in erythrocyte invasion and immunogenicity in animal models [10][11][12][13].…”