High throughput approaches to strain-transcending malaria vaccine candidate selection

间日疟原虫--发现于南美洲和东南亚--是分布最广的一种疟原虫。感染人类，并日益被承认会造成严重的疾病负担。间日疟原虫研究中的一个特殊障碍(与恶性疟原虫不同)是无法培养这些寄生虫，因为它们偏爱入侵网织红细胞(年轻的红细胞)。在培养中，网织红细胞迅速成熟为正常细胞(完全形成的红细胞)，留下足够的种群供后续寄生虫世代入侵。2013年，间日疟原虫研究向前迈出了重要的一步，穆恩等人改造了密切相关的猿猴寄生虫诺氏疟原虫，使其在人类红细胞中生长。这项技术的明显好处是有助于诺氏疟原虫的研究，因为这种寄生虫是亚洲新出现的人畜共患病的健康威胁，而且众所周知，它被显微镜误诊了。令人兴奋的是，这些寄生虫也被证明使用CRISPR/Cas9系统进行了有效的基因改造。间日疟原虫和诺氏间日疟原虫具有密切的亲缘关系，并利用相似的侵袭配体进行红细胞内复制，包括使用两个关键的蛋白质超家族，网织红细胞结合样(RBL)配体和Duffy结合蛋白(DBPs)来选择和入侵宿主红细胞。在这个模型中，用PvDBP同源替换诺氏P.nowlesi DBPA-两者都识别人类红细胞上的同一受体DARC-允许对以PvDBP-RII为基础的疫苗免疫的志愿者产生的单抗进行体外评估，从而允许在需要CHMI之前进行合理的疫苗设计。该实验室以前的一位成员最近的工作还发现，在诺氏疟原虫中，必需的RBL和DBP蛋白在红细胞入侵过程中依次起作用，分别介导宿主细胞变形和裂殖子重定向。这就提出了一个问题，即这两个蛋白质家族是否应该被评估为疫苗靶标--可能同时使用以提高疫苗效力。该项目重点解决四个关键问题。首先，我将使用野生型和表达PvDBP的诺氏拟青霉菌株来研究DBP-DARC相互作用是如何介导宿主细胞趋向性的，重点是宿主细胞DARC突变如何影响PvDBP和PkDBPA结合亲和力的异同。其次，我将探讨间日疟原虫RBL是否负责介导宿主细胞对网织红细胞的限制；我将特别关注PvRBP2b和PvRBP1a，因为这两种蛋白最近发现了宿主细胞受体，与正常细胞相比，这两种受体在网织红细胞中都显著上调。我将在一个可诱导的PkNBPXa互补系中表达这些蛋白质，这将允许在体外有限的周期内研究潜在的网织红细胞限制入侵基因，而不会影响该系在正常细胞中长期生长的能力。第三，我将使用阻断PvDBP-DARC识别的单抗和识别PkNBPXa的新纯化抗体的组合，来研究当阻断RBL和DBP与宿主红细胞的结合时是否存在协同作用。如果是这样的话，在一种疫苗中针对这两个蛋白质家族可能会对间日疟原虫和诺氏疟原虫提供更强有力的保护。最后，在时间允许的情况下，这些研究可以进一步评估间日疟原虫和诺氏疟原虫中RBL和DBP基因的变异如何改变疫苗的效力。由于这些寄生虫蛋白具有免疫原性，它们容易受到寄生虫分离株之间的选择和显著差异的影响；有效的疫苗必须能够针对广泛的变种，以提供跨株免疫。通过表达PvRBPs和PkNBPXa的不同变体，可以在长期培养模型中评估不同抗体的交叉反应性。

P. vivax - found across South America and southeast Asia - is the most widespread of the Plasmodium spp. infecting humans and is increasingly acknowledged to cause a significant burden of disease. A particular hurdle in P. vivax research (unlike P. falciparum) has been the inability to culture these parasites, due to their preference for invading reticulocytes ('young' red blood cells, RBCs). In culture, reticulocytes mature quickly into normocytes (fully formed RBCs), leaving an insufficient population for subsequent parasite generations to invade. A major step forward in P. vivax research came in 2013 with the adaptation of the closely related simian parasite, P. knowlesi, to grow in human RBCs by Moon et al. The clear benefit of this technique is in aiding P. knowlesi research, as the parasite is an emerging zoonotic health threat in Asia and is notoriously misdiagnosed by microscopy. Excitingly, these parasites were also shown to be efficiently genetically modified using the CRISPR/Cas9 system. P. vivax and P. knowlesi are closely related and utilise similar invasion ligands to undergo intraerythrocytic replication, including the use of two key protein superfamilies, the reticulocyte binding-like (RBL) ligands and the Duffy-binding proteins (DBPs), to select and invade host erythrocytes. Orthologue replacement of P. knowlesi DBPa with PvDBP - both of which recognise the same receptor on human erythrocytes, DARC - in this model has allowed in vitro assessment of monoclonal antibodies generated by volunteers immunised with PvDBP-RII-based vaccines, permitting rational vaccine design prior to necessitating CHMI. Recent work by a previous member of the lab additionally identified that, in P. knowlesi, the essential RBL and DBP proteins act sequentially during erythrocytic invasion, mediating host cell deformation and merozoite reorientation, respectively. This raises the question as to whether both protein families should be assessed as vaccine targets - potentially used in tandem to improve vaccine efficacy. This project focuses on four key questions. Firstly, I will use both wild-type and PvDBP-expressing P. knowlesi lines to investigate how the DBP-DARC interaction mediates host cell tropism, with specific focus on the similarities and differences of how binding affinity of PvDBP and PkDBPa is affected by mutations in host cell DARC. Secondly, I will explore whether P. vivax RBLs are responsible for mediating host cell restriction to reticulocytes; my particular focus will be on PvRBP2b and PvRBP1a, as these proteins have recently identified host cell receptors which are both significantly upregulated in reticulocytes compared to normocytes. I will be expressing these proteins in an inducible PkNBPXa complementation line, which will permit investigation of potential reticulocyte-restricting invasion genes over a limited number of cycles in vitro without compromising the ability of the line to be grown in normocytes long-term. Thirdly, I will be using a combination of monoclonal antibodies blocking PvDBP-DARC recognition, alongside newly purified antibodies recognising PkNBPXa, to investigate whether there is synergy when blocking both RBL and DBP binding to host erythrocytes. If so, targeting both families of proteins in a single vaccine might provide more robust protection against P. vivax and P. knowlesi malaria. Finally, time permitting, these investigations can be taken one step further to assess how variation in RBL and DBP genes in P. vivax and P. knowlesi may alter the efficacy of vaccines. As these parasite proteins are immunogenic, they are susceptible to selection and significant variability between parasite isolates; an effective vaccine must be able to target a wide range of variants to provide strain-transcending immunity. By expressing different variants of PvRBPs and PkNBPXa, the cross-reactivity of different antibodies can be assessed in a long-term culture model.