Posttranslational modification of recombinant Plasmodium falciparum apical membrane antigen 1:: Impact on functional immune responses to a malaria vaccine candidate
Posttranslational modification of recombinant Plasmodium falciparum apical membrane antigen 1:: Impact on functional immune responses to a malaria vaccine candidate
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DOI：
10.1128/iai.73.7.3963-3970.2005
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发表时间：
2005-07-01
影响因子：
3.1
通讯作者：
Singh, S
中科院分区：
文献类型：
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作者：
Giersing, B;Miura, K;Singh, S
重组顶端膜抗原1（AMA 1）是恶性疟原虫疟疾的主要候选疫苗，因为针对重组恶性疟原虫AMA 1（PfAMA 1）的抗体中断裂殖子侵入红细胞。为了研究翻译后修饰在调节对重组AMA 1的功能性免疫应答中的作用，使用大肠杆菌和巴斯德毕赤酵母表达系统产生了PfAMA 1的两个单独的等位基因（FVO和3D 7），其中天然N-糖基化位点已被突变。重组巴斯德毕赤酵母AMA 1-FVO（PpAMA 1-FVO）和PpAMA 1 - 3D 7是O-连接的糖基化的，45%的PpAMA 1 - 3D 7是切口的，尽管所有四种重组分子都与构象特异性单克隆抗体反应。为了探讨O-糖基化的免疫学效应，我们比较了E. coli AMA 1-FVO（EcAMA 1-FVO）和PpAMA 1-FVO抗原，因为两种分子都是完整的。然后通过比较EcAMA 1 - 3D 7和PpAMA 1 - 3D 7的免疫原性来研究抗原切口的作用。我们的数据表明，针对EcAMA 1-FVO和PpAMA 1-FVO或针对EcAMA 1 - 3D 7和PpAMA 1 - 3D 7引发的兔抗体滴度没有显著差异。此外，我们已经证明，重组AMA 1（FVO或3D 7），无论是表达和复性从E。大肠杆菌中表达的或由毕赤酵母表达系统产生的蛋白质是等效的，并且在体外生长抑制测定实验中模拟天然蛋白质的功能。我们的结论是，在重组AMA 1的情况下，E.大肠杆菌和巴斯德毕赤酵母衍生的抗原在免疫学和功能上是等同的，并且不受这两种系统中表达所导致的翻译后修饰的影响。
Recombinant apical membrane antigen 1 (AMA1) is a leading vaccine candidate for Plasmodium falciparum malaria, as antibodies against recombinant P. falciparum AMA1 (PfAMA1) interrupt merozoite invasion into erythrocytes. In order to investigate the role of posttranslational modification in modulating the functional immune response to recombinant AMA1, two separate alleles of PfAMA1 (FVO and 3D7), in which native N-glycosylation sites have been mutated, were produced using Escherichia coli and a Pichia pastoris expression system. Recombinant Pichia pastoris AMA1-FVO (PpAMA1-FVO) and PpAMA1-3D7 are O-linked glycosylated, and 45 % of PpAMA1-3D7 is nicked, though all four recombinant molecules react with conformation-specific monoclonal antibodies. To address the immunological effect of O-linked glycosylation, we compared the immunogenicity of E. coli AMA1-FVO (EcAMA1-FVO) and PpAMA1-FVO antigens, since both molecules are intact. The effect of antigen nicking was then investigated by comparing the immunogenicity of EcAMA1-3D7 and PpAMA1-3D7. Our data demonstrate that there is no significant difference in the rabbit antibody titer elicited towards EcAMA1-FVO and PpAMA1-FVO or to EcAMA1-3D7 and PpAMA1-3D7. Furthermore, we have demonstrated that recombinant AMA1 (FVO or 3D7), whether expressed and refolded from E. coli or produced from the Pichia expression system, is equivalent and mimics the functionality of the native protein in in vitro growth inhibition assay experiments. We conclude that in the case of recombinant AMA1, the E. coli and P. pastoris-derived antigens are immunologically and functionally equivalent and are unaffected by the posttranslational modification resulting from expression in these two systems.