Sharma P, Kumar A, Singh B, Bharadwaj A, Sailaja V N, Adak T, Kushwaha A, Malhotra P, Chauhan V S
International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, India.
Infect Immun. 1998 Jun;66(6):2895-904. doi: 10.1128/IAI.66.6.2895-2904.1998.
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 parasite Plasmodium 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. falciparum malaria 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 approximately 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.
确定候选疫苗蛋白中假定具有保护性和免疫原性的表位是开发有效疟疾疫苗的一项主要研究工作。由于其在裂殖子免疫簇形成中的作用、位于裂殖子表面以及在核苷酸序列水平和氨基酸序列水平上的高度保守性，恶性疟原虫中含有酸性和碱性残基重复序列的抗原（ABRA）就代表了这样一种抗原。基于ABRA的预测氨基酸序列，我们合成了8种肽，其中6种（AB - 1至AB - 6）长度为12至18个残基，覆盖该蛋白最亲水区域，另外两种肽（AB - 7和AB - 8）代表其重复序列。我们发现，所有这8种构建体都能与很大一部分恶性疟原虫疟疾患者血清中的相当数量抗体结合；其中两种肽（AB - 1和AB - 3）还在11名从疟疾中康复的人类受试者的外周血单核细胞中引发了强烈的增殖反应。当用作无载体免疫原时，6种肽在兔子中诱导了强烈的、可增强的免疫球蛋白G型抗体反应，表明这6种构建体中存在B细胞决定簇和T辅助细胞表位。这些抗体在免疫印迹和免疫荧光测定中与寄生虫蛋白发生特异性交叉反应。在另一次免疫印迹中，兔抗肽血清也识别在细菌中表达的ABRA重组片段。更重要的是，针对两种构建体（AB - 1和AB - 5）的兔抗体在体外可将裂殖子对人红细胞的再入侵抑制高达约90%。这些结果有利于进一步研究，以确定在针对恶性疟原虫无性血液阶段的多组分亚单位疫苗中是否可能包含这两种构建体。
