US Military Malaria Vaccine Program, Naval Medical Research Center, Silver Spring, Maryland, USA.
Malar J. 2013 Nov 5;12:394. doi: 10.1186/1475-2875-12-394.
Experimental vaccines targeting Plasmodium falciparum have had some success in recent years. These vaccines use attenuated parasites, recombinant sporozoite proteins, or DNA and virus combinations to induce cell-mediated immune responses and/or antibodies targeting sporozoite surface proteins. To capitalize on the success of these vaccines and understand the mechanisms by which these vaccines function, it is important to develop assays that measure correlates of protection in volunteers. The inhibition of liver stage development assay (ILSDA) tests antibodies for the ability to block sporozoite development in hepatocytes. As such the ILSDA is an excellent candidate assay to identify correlates of humoral protection, particularly against the liver stage of malaria infection. In addition, the ILSDA can be used as a tool to evaluate novel sporozoite antigens for future vaccine development. Historically the ILSDA has suffered from low sporozoite infection rates, absence of standardized reagents, and the subjectivity associated with the traditional primary outcome measures, which depend on microscopy of stained hepatocyte cultures. This study worked to significantly improve sporozoite infection rates in hepatocytes, modify key steps in the assay protocol to reduce experimental variability, and demonstrate the utility of the ILSDA in testing antibodies targeting the circumsporozoite protein.
Cryopreserved primary human hepatocytes, Plasmodium falciparum sporozoites, and circumsporozoite antibodies were used to optimize the ILSDA.
Inoculation of cryopreserved primary human hepatocytes with Plasmodium falciparum sporozoites improved liver stage development in the ILSDA compared to HCO4 cells. In the ILSDA, circumsporozoite antibodies suppressed liver stage development in cryopreserved primary human hepatocytes in a concentration-dependent manner. Antibody-mediated suppression of parasite development in the ILSDA at a 96-hour endpoint was more robust than the 24-hour endpoint.
ILSDA performance is improved by the use of cryopreserved primary human hepatocytes, expediting interactions between sporozoites and hepatocytes, and extending the assay endpoint.
近年来，针对恶性疟原虫的实验性疫苗取得了一定的成功。这些疫苗使用减毒寄生虫、重组子孢子蛋白或 DNA 和病毒组合来诱导细胞介导的免疫反应和/或针对子孢子表面蛋白的抗体。为了利用这些疫苗的成功，并了解这些疫苗的作用机制，开发测量志愿者保护相关性的检测方法非常重要。抑制肝期发育检测（ILSDA）检测抗体阻断子孢子在肝细胞中发育的能力。因此，ILSDA 是识别体液保护相关性的极好候选检测方法，特别是针对疟疾感染的肝期。此外，ILSDA 可用于评估新型子孢子抗原用于未来疫苗的开发。历史上，ILSDA 受到低子孢子感染率、缺乏标准化试剂以及与传统主要终点测量相关的主观性的困扰，这些主观性依赖于染色肝细胞培养物的显微镜检查。本研究旨在显著提高肝细胞中的子孢子感染率，修改检测方案中的关键步骤以减少实验变异性，并展示 ILSDA 在测试针对环子孢子蛋白的抗体中的效用。
使用冷冻保存的原代人肝细胞、恶性疟原虫子孢子和环子孢子抗体来优化 ILSDA。
与 HCO4 细胞相比，用恶性疟原虫子孢子接种冷冻保存的原代人肝细胞可改善 ILSDA 中的肝期发育。在 ILSDA 中，环子孢子抗体以浓度依赖的方式抑制冷冻保存的原代人肝细胞中的肝期发育。在 96 小时终点时，抗体介导的对子孢子发育的抑制作用比 24 小时终点更为显著。
使用冷冻保存的原代人肝细胞可提高 ILSDA 的性能，加速子孢子与肝细胞之间的相互作用，并延长检测终点。
