Identification of Transmission blocking epitopes on P. vivax 48/45 protein

描述（由申请方提供）：Pfs 48/45是一种对雄性配子生育力很重要的蛋白质，作为恶性疟原虫传播阻断疫苗正处于开发的晚期阶段。间日疟原虫对应物Pvs 48/45同样预期是重要的候选疫苗。尽管有显著的序列鉴定和假定的保守生物学功能，但对于针对Pvs 48/45的抗体的传递阻断功效以及其是否与针对Pfs 48/45的功能性抗体相似知之甚少，因为没有观察到交叉阻断反应性。开发间日疟原虫疟疾传播阻断疫苗的几个障碍推迟了这一长期目标的进展。这些问题包括构象上合适的重组蛋白的可用性、缺乏体内激发模型以及无法在实验室中产生间日疟原虫配子体以测试抗体的生物学功能。在本申请中，我们建议开发某些急需的试剂，以利用合作研究者实验室中Pfs 48/45的成功推动该领域的发展。在目标1中，我们提出产生针对Pvs 48/45的单克隆抗体，并通过体外膜饲养测定来表征其结合参数和功能。目的二是检测Pvs 48/45基因在自然分离株中的多态性，并鉴定自然感染产生的抗体所识别的表位。第三个目标将决定功能 使用蛋白质片段鉴定参与形成与传递相关的构象表位的蛋白质结构域。抗Pvs 48/45单克隆抗体试剂的开发将极大地有助于理解该蛋白的生物学，并有助于开发基于Pvs 48/45的间日疟原虫疟疾传播阻断疫苗。虽然所提出的工作的目的可能看起来是常规的，但是这些试剂的产生和表位的表征对于如上所述的更显著的收获以及具有传播阻断潜力的mAb在开发和建立基于转基因寄生虫的鼠模型（其在本申请的范围之外）中的使用是至关重要的。

DESCRIPTION (provided by applicant): Pfs48/45, a protein important for male gamete fertility, is in advanced stages of development as a Plasmodium falciparum transmission blocking vaccine. The Plasmodium vivax counterpart Pvs 48/45 is likewise expected to be an important vaccine candidate. In spite of significant sequence identify and presumed conserved biological function not much is known about the transmission blocking efficacy of antibodies against Pvs48/45 and if it will be similar to functional antibodies against Pfs48/45 as no cross-blocking reactivity has been observed. Several impediments to developing a transmission blocking vaccine for P. vivax malaria have delayed progress for this long term goal. These have included availability of conformationally suitable recombinant protein, lack of an in vivo challenge model and the inability to produce P. vivax gametocytes in the lab to test biological function of antibodies. In this application, we propose to develop certain much needed reagents to move the field forward capitalizing on success with Pfs48/45 in the co-investigator's lab. In Aim 1 we propose to generate monoclonal antibodies directed against Pvs48/45 and characterize their binding parameters and functionality through in vitro membrane feeding assays. Aim 2 will focus on examining the polymorphisms of Pvs48/45 in natural isolates and identification of epitopes recognized by antibodies elicited by natural infection. The third aim will determine the functional domains in Pvs48/45 using fragments of the protein to identify protein domains involved in forming conformational epitopes of relevance to transmission. The development of the mAb reagents against Pvs48/45 will greatly aid in understanding the biology of the protein and assist in the development of a transmission blocking vaccine based on Pvs48/45 for P. vivax malaria. While the objectives of the proposed work may appear routine, the generation of these reagents and characterization of epitopes are critical for more significant gains as discussed above as well as use of mAbs with transmission blocking potential in developing and establishing a murine model based on transgenic parasites (which is outside the scope of this application).