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-是最广泛的疟原虫属。感染人类，并且越来越多地被认为会造成重大的疾病负担。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，因为它们偏好入侵网织红细胞（“年轻”红细胞，RBC）。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），从而为随后的寄生虫世代入侵留下不足的群体。2013年，Moon等人对与间日疟原虫密切相关的猿类寄生虫诺氏疟原虫进行了改造，使其在人类红细胞中生长，这一技术的明显好处是有助于诺氏疟原虫研究，因为该寄生虫是亚洲新兴的人畜共患病健康威胁，并且众所周知被显微镜误诊。令人兴奋的是，这些寄生虫也被证明可以使用CRISPR/Cas9系统进行有效的遗传修饰。间日疟原虫和诺氏疟原虫密切相关，并利用类似的侵入配体进行红细胞内复制，包括使用两个关键蛋白超家族，网织红细胞结合样（RBL）配体和达菲结合蛋白（DBP），以选择和侵入宿主红细胞。在该模型中，用PvDBP（两者均识别人红细胞上的相同受体DARC）替代诺氏毕赤酵母DBPa的直系同源物，允许对用基于PvDBP-RII的疫苗免疫的志愿者产生的单克隆抗体进行体外评估，从而允许在需要CHMI之前进行合理的疫苗设计。实验室前成员最近的工作还发现，在诺氏疟原虫中，必需的RBL和DBP蛋白在红细胞侵入过程中依次起作用，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点--可能串联使用以提高疫苗效力。该项目侧重于四个关键问题。首先，我将使用野生型和表达PvDBP的诺氏毕赤酵母细胞系来研究DBP-DARC相互作用如何介导宿主细胞嗜性，特别关注宿主细胞DARC中突变如何影响PvDBP和PkDBPa的结合亲和力的相似性和差异。其次，我将探索间日疟原虫RBL是否负责介导宿主细胞对网织红细胞的限制;我的特别重点将是PvRBP 2b和PvRBP 1a，因为这些蛋白质最近已经鉴定出与正常细胞相比在网织红细胞中均显著上调的宿主细胞受体。我将在诱导型PkNBPXa互补系中表达这些蛋白质，这将允许在体外有限数量的循环中研究潜在的网织红细胞限制性侵袭基因，而不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识别的单克隆抗体与新纯化的识别PkNBPXa的抗体的组合，以研究当阻断RBL和DBP与宿主红细胞结合时是否存在协同作用。如果是这样的话，在单一疫苗中靶向两个蛋白质家族可能会提供更强大的抗间日疟原虫和诺氏疟原虫疟疾的保护。最后，如果时间允许，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，因此它们容易受到选择的影响，并且寄生虫分离株之间存在显著的变异性;有效的疫苗必须能够靶向广泛的变体，以提供超越菌株的免疫力。通过表达PvRBP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.