Predicted B and T cell epitopes of PE antigens.

Plasmodium vivax pre-erythrocytic (PE) vaccine research has lagged far behind efforts to develop Plasmodium falciparum vaccines. There is a critical gap in our knowledge of PE antigen targets that can induce functionally inhibitory neutralizing antibody responses. To overcome this gap and guide the selection of potential PE vaccine candidates, we considered key characteristics such as surface exposure, essentiality to infectivity and liver stage development, expression as recombinant proteins, and functional immunogenicity. Selected P. vivax sporozoite antigens were surface sporozoite protein 3 (SSP3), sporozoite microneme protein essential for cell traversal (SPECT1), sporozoite surface protein essential for liver-stage development (SPELD), and M2 domain of MAEBL. Sequence analysis revealed little variation occurred in putative B-cell and T-cell epitopes of the PE candidates. Each antigen was tested for expression as refolded recombinant proteins using an established bacterial expression platform and only SPELD failed. The successfully expressed antigens were immunogenic in vaccinated laboratory mice and were positively reactive with serum antibodies of P. vivax-exposed residents living in an endemic region in Thailand. Vaccine immune antisera were tested for reactivity to native sporozoite proteins and for their potential vaccine efficacy using an in vitro inhibition of liver stage development assay in primary human hepatocytes quantified on day 6 post-infection by high content imaging analysis. The anti-PE sera produced significant inhibition of P. vivax sporozoite invasion and liver stage development. This report provides an initial characterization of potential new PE candidates for a future P. vivax vaccine.

间日疟原虫（Plasmodium vivax）红细胞前期（pre-erythrocytic, PE）疫苗研发工作，长期以来远远落后于恶性疟原虫（Plasmodium falciparum）疫苗的开发进程。当前学界对于可诱导功能性抑制性中和抗体应答的PE抗原靶点，仍存在关键认知空白。为填补这一空白并指导潜在PE疫苗候选抗原的筛选，本研究综合考量了多项核心特性：抗原的表面暴露水平、对寄生虫感染性及肝期发育的必需性、可作为重组蛋白表达的可行性，以及功能性免疫原性。
本研究选取的间日疟原虫子孢子抗原包括：子孢子表面蛋白3（surface sporozoite protein 3, SSP3）、细胞穿越必需的子孢子微线蛋白（sporozoite microneme protein essential for cell traversal, SPECT1）、肝期发育必需的子孢子表面蛋白（sporozoite surface protein essential for liver-stage development, SPELD），以及MAEBL的M2结构域。序列分析结果显示，上述PE候选抗原的推定B细胞与T细胞表位几乎未发生序列变异。
研究采用成熟的细菌表达平台，对各候选抗原进行复性重组蛋白表达测试，结果仅SPELD未能成功表达。成功表达的抗原在免疫后的实验小鼠体内均表现出良好免疫原性，且可与泰国某疟疾流行区曾暴露于间日疟原虫的居民血清抗体呈阳性反应。
本研究进一步检测了疫苗免疫抗血清对天然子孢子蛋白的反应性，并通过原代人肝细胞体外肝期发育抑制实验评估其潜在疫苗效力：于感染后第6天采用高内涵成像分析对肝期发育情况进行定量检测。实验结果表明，抗PE血清可显著抑制间日疟原虫子孢子入侵宿主细胞及肝期发育进程。
本报告首次对用于未来间日疟原虫疫苗研发的潜在新型PE候选抗原进行了系统初步表征，可为后续相关疫苗开发提供关键理论与实验依据。