Studies on attenuated malaria parasites for the development of vaccine.

从现场观察中众所周知，疟疾感染不会产生对再次感染的强大保护性免疫力。这一事实给疟疾疫苗的开发投下了悲观的阴影。我们发现，减毒的突变体可以产生更强的免疫力，从而产生持久的影响。伯氏疟原虫XAT是伯氏疟原虫NK65辐射诱导的减毒变异体。野生型NK65对小鼠免疫功能低下，致死率达100%。而伯氏假单胞菌在Balb/c小鼠中引起适度的自限性感染，单次接种变异株可诱导小鼠对原始毒力NK65株的挑战免疫产生持久的免疫力。因此，NK65和XAT寄生虫的联合实验将是发展长效疫苗研究的良好模式。在本研究中，利用单抗在分子水平上研究了原始NK65毒株和衍生XAT毒株之间的差异。研制了抗XAT株的单抗，其中一株命名为D3-1A12，在裂殖体阶段仅与XAT反应。该单抗从来自XAT的代谢标记的寄生虫抗原中沉淀出一个240kD的分子。另一方面，分别制备了针对强毒株NK65和弱毒株XAT的多克隆抗体。每种抗体的Western印迹结果显示，在减毒寄生虫中，30kD的蛋白存在缺陷。经O‘Farrell双向凝胶电泳法检测到两个明显的多肽斑点。用Yoelii黑敏系统验证了X射线辐射衰减的重复性。

It is widely known from field observation that malaria infections do not create strong protective immunity to reinfections. The fact casts a pessimistic shadow over the vaccine development against malaria. We have found that an attenuated mutant can produce much stronger immunity in terms of long lasting effect. Plasmodium (P) berghei XAT is an irradiation-induced attenuated variant of P. berghei NK65. The wild NK65 induces poor immunity in mice and causes 100% lethality in mice. While, P. berghei-XAT causes modest self-limiting infections in Balb/c mice and a single inoculation with the variant strain induces a long lasting immunity in mice against a challenge inoculation with the original virulent NK65 strain. Hence, combination experiments of NK65 and XAT parasites will be a good model to develop long lasting and effective vaccine studies. In this study, differences between the original NK65 strain and the derivative XAT were studied at the molecular level using monoclonal antibodies. Monoclonal antibodies to XAT strain were developed and one designated D3-1A12 was reactive only with XAT at the stage of schizont. The monoclonal antibody precipitated a molecule of 240 Kd from metabolically labeled parasite antigens derived from XAT. On the other hand, polyclonal antibodies to virulent NK65 and to attenuated XAT were prepared respectively. Western blot using each antibody showed that 30 Kd protein was defective in attenuated parasite. By O'Farrell's two dementional electrophoresis two distinctive polypeptide spots were detected. Reproducibility of X-ray irradiation attenuation were confirmed with P. yoelii nigeriensismice system.