Plasmodium vivax 48/45 gametocyte protein: functional characterization and vaccine potential assessment in preclinical studies

DESCRIPTION（由申请人提供）：在流行地区持续暴露于疟疾感染的个体会产生免疫反应，这种免疫反应已被证明可以减少或阻止寄生虫从患者向蚊子媒介的传播，即所谓的传播阻断（TB）免疫。在这种情况下，针对配子体、受精卵和卵母细胞阶段表达的特定疟原虫抗原的抗体（Abs）可以诱导这种阻断，为开发结核病疫苗提供基础。其中一种抗原P48/45是一种在所有疟原虫中表达的保守蛋白，是寄生虫受精所必需的，目前正在开发作为恶性疟原虫的候选疫苗。间日疟原虫同源基因在大肠杆菌中克隆表达，重组产物约60kDa，具有较高的抗原性和免疫原性。暴露于间日疟原虫的个体产生对Pvs48/45的抗体，并呈年龄依赖性增加，α-Pvs48/45抗体滴度较高的个体也表现出较高的体外TB活性。初步研究表明，rPvs48/45蛋白在小鼠和猴子中都具有高度的免疫原性，并且诱导的抗体具有体外阻断寄生虫向蚊子传播的能力。总之，所有这些数据使Pvs48/45成为一种非常有希望的结核病候选疫苗。该提案的目标是确定rPvs48/45用于人类的疫苗潜力。我们将利用来自流行地区的个体的血清和细胞进一步表征rPvs48/45的抗原性，并利用rPvs48/45结构域和合成肽来绘制相关的免疫和功能表位。此外，我们将确认并进一步表征Pvs48/45全长和选定片段在非人灵长类动物中的免疫原性。此外，蛋白质的三维结构数据将通过物理化学分析[圆二色性（CD）和核磁共振（NMR）光谱和质谱（MS）分析]，生物信息学和分子建模获得。在哥伦比亚、巴西和布基纳法索流行地区暴露于间日疟原虫和恶性疟原虫的个体中诱导的天然抗体将通过ELISA和流式细胞术的t细胞反应进行评估。将检测血清和亲和纯化的IgG在MFAs中的结核能力。此外，小鼠和猴子的免疫原性研究将解决寄生虫感染对免疫引起的Ab反应的潜在交叉启动/增强效应，反之亦然。此外，还将测试诱导的抗体对恶性疟原虫的潜在交叉反应性。所有所需的设施和技术都是可用的，并由参与小组定期进行。在研究结束时，我们预计将确定Pvs48/45在人类和动物中诱导有效结核病免疫的最佳条件，以及用于进一步临床开发的最佳疫苗配方。

DESCRIPTION (provided by applicant): Individuals continuously exposed to malaria infections in endemic areas develop immune responses that have been shown to reduce or block parasite transmission from patients to the mosquito vector in what is called transmission-blocking (TB) immunity. In this context, antibodies (Abs) targeting specific Plasmodium antigens expressed on gametocyte, zygote, and ookinete stages can induce this blockage, providing the bases for the development of TB vaccines. One of these antigens, P48/45 is a conserved protein expressed in all Plasmodium species, required for parasite fertilization, and currently being developed as a vaccine candidate for P. falciparum. The P. vivax orthologue, cloned and expressed in E. coli, as a ~60kDa recombinant product has been shown to be highly antigenic and immunogenic. Individuals exposed to P. vivax produce Abs to Pvs48/45 which increases in an age dependent manner, and those individuals with higher α-Pvs48/45 Ab titers also display high ex vivo TB activity. Pilot studies have shown that the rPvs48/45 protein is highly immunogenic both in mice and monkeys and Abs elicited have the capacity to ex vivo block parasite transmission to mosquitoes. Together, all these data make Pvs48/45 a very promising TB vaccine candidate. The goal of this proposal is to determine the vaccine potential of rPvs48/45 for human use. We will further characterize the antigenicity of rPvs48/45 using sera and cells of individuals from endemic areas, and rPvs48/45 domains and synthetic peptides to map the relevant immune and functional epitopes. Additionally, we will confirm and further characterize the immunogenicity of full length Pvs48/45 and selected fragments in non-human primates. Furthermore, the 3D structure data of the protein(s) will be obtained by physico-chemical analyses [circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies and mass spectrometry (MS) analysis], and bioinformatics and molecular modeling. Natural Abs induced in individuals exposed to P. vivax and P. falciparum in endemic areas of Colombia, Brazil and Burkina Faso will be assessed by ELISA and T-cell responses by flow cytometry. Sera and affinity purified IgG will be tested for TB capacity in MFAs. Furthermore, immunogenicity studies in mice and monkeys will address the potential cross prime/boost effect of parasite infection on Ab response elicited by immunization and vice versa. Potential cross-reactivity of elicited Abs against P. falciparum will be also tested. All facilities and techniques required are available and routinely conducted by the participant groups. At the end of the study we expect to have identified the optimal conditions for Pvs48/45 to induce effective TB immunity in humans and animals and the best vaccine formulations for further clinical development.