一种新型间日疟疫苗候选抗原：受感染红细胞表膜相关分子PvTRAg-26/ 29免疫原性及免疫效果研究

The widespread transmission and high motality rate of malaria as well as increasing antimalaria drug resistence urge the need for the development of malaria vaccines. However, current vaccines development have been limited to parasitic organism related antigens polymorphism, low and/or short immunogenicity and so on. The development of a successful multivalent malaria vaccine therefore requires identification of novel potential vaccine antigens. It has been reported that Tryptophan-rich antigens (TRAgs) in Plasmodium falciparum are proved to have protection for merozoite invasion. While, Plasmodium vivax trags (Pvtrags) have been found to include more family members than other malarial species. PvTRAgs contain positionally conserved tryptophan residues in a TR domain. Several of these proteins have erythrocyte binding activity. Based on our previously data, PvTRAg-26/29, which were proved as infected RBC membrane associated antigens, have high, stable and longlasting antigenicity among South Eastern Asian patients that strongly suggest PvTRAg-26/29 as potential vaccine candidates. Thus, In the present project, to adequately evaluate the immunogenicity and its vaccine potential, we assessed its immunogenicity by stimulating patients’ PBMCs and immunizing BALB/c mice using PvTRAg-26/29 as well as PvTRAg-29/rPvCSP-C recombinant protein. So far, no relevant approach has been available. This study will further firm up the basis for understanding the immune mechanism of PvTRAgs and may provide vaccine candidate(s) for new malaria vaccine design.

疟疾的广泛流行，高致死率以及日益严重的原虫耐药性，使得其疫苗研制成为当今疟疾防治亟待解决的问题。然而现有虫体来源的抗原多态性、弱免疫原性及短效性等成为疫苗发展的瓶颈。发现高效特异、长效稳定的分子疫苗已成为疟疾疫苗研发的关键。近年发现，恶性疟原虫色氨酸富集蛋白(TRAgs)具有一定的免疫保护价值。而间日疟含有更多的TRAgs，具有高度保守性，且能粘附正常红细胞。我们前期研究发现，PvTRAg-26/29为受染RBC膜相关蛋白，在东南亚间日疟患者中具有强力而稳定的抗原性，强烈提示其具有作为疫苗候选抗原的价值。本课题运用免疫芯片筛选，体内外免疫原性比较，包括诱导既往感染和现症感染患者抗体和CKs生成能力、动物体内接种后抗体和CKs产生水平和持续时间、记忆B细胞测定等，以评价疫苗的效果。本课题为深入探讨PvTRAgs免疫机制和作为候选疫苗的价值提供依据。此类研究国内外尚未见报道。

疟原虫色氨酸富集蛋白(TRAgs)具有潜在的药物和疫苗研究价值。相比于其他疟原虫，间日疟含有更多的TRAgs，且具有高度保守性。PvTRAg_26/29为间日疟TRAgs家族中的成员，在东南亚间日疟患者中具有强力而稳定的抗原性，强烈提示其具有作为疫苗候选抗原的价值。本研究采用大肠杆菌表达系统表达重组PvTRAg_26/29蛋白并经Ni-NTA琼脂糖亲和柱纯化后对小鼠进行免疫。为了探究PvTRAg_26/29的抗原性和免疫原性，通过酶联免疫吸附法（ELISA）在35例间日疟原虫感染的患者和一组免疫小鼠中检测到PvTRAg_26/29的IgG亚型的特异性抗体。随后，进行间接免疫荧光实验以探究PvTRAg_26/29在受感染红细胞上的定位，并在体外试验中确定了经PvTRAg_26/29免疫后小鼠脾细胞的抗原特异性免疫应答和Th1 / Th2型细胞因子模式。同时，初步探究PvTRAg_29与重组PvCSP组成融合蛋白的免疫原性。结果表明，PvTRAg_26/29蛋白的亚细胞定位与间日疟原虫早期感染阶段的感染性红细胞膜上的薛氏点结构有关；各组中间日疟原虫感染患者的IgG1和IgG3水平显著高于未感染健康人对照组。在经PvTRAg_26/29免疫的小鼠中，观察到抗原特异性IgG抗体产生，其中IgG1是主要的IgG亚型，Th1型细胞因子的水平相对高于Th2型细胞因子。而此外，融合表达/混合表达组，免疫原性优于单个抗原，特别是体液免疫水平有所提升。研究结果显示PvTRAg_26/29在疟原虫入侵早期就被运送到受感染红细胞膜上，体外实验及动物实验结果表明其具有良好的抗原性及免疫原性，且与PvCSP组合形成的复合抗原在免疫原性上有所提升，为PvTRAg_26/29及其与PvCSP组合形成的多期复合疫苗可能成为间日疟原虫疫苗开发的潜在候选抗原提供了实验基础。