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

描述（由申请人提供）：在流行地区持续暴露于疟疾感染的个体产生免疫应答，已显示这种免疫应答可减少或阻断寄生虫从患者向蚊媒的传播，即所谓的传播阻断（TB）免疫。在这种情况下，针对配子体、受精卵和动合子阶段表达的特异性疟原虫抗原的抗体（Ab）可以诱导这种阻断，为TB疫苗的开发提供基础。这些抗原之一，P48/45是在所有疟原虫物种中表达的保守蛋白，是寄生虫受精所需的，并且目前正在开发作为恶性疟原虫的候选疫苗。克隆了间日疟原虫同源基因，并在大肠杆菌中表达。大肠杆菌中的约60 kDa重组产物已被证明具有高度抗原性和免疫原性。暴露于间日疟原虫的个体产生针对Pvs 48/45的抗体，该抗体以年龄依赖性方式增加，并且具有较高α-Pvs 48/45抗体滴度的个体也显示出较高的离体TB活性。初步研究表明，rPvs 48/45蛋白在小鼠和猴中均具有高度免疫原性，并且所引发的Ab具有离体阻断寄生虫向蚊子传播的能力。总之，所有这些数据使Pvs 48/45成为非常有希望的结核病候选疫苗。本提案的目的是确定rPvs 48/45的人用疫苗潜力。我们将进一步表征rPvs 48/45的抗原性，使用来自流行地区的个体的血清和细胞，以及rPvs 48/45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全长Pvs 48/45和选定片段在非人灵长类动物中的免疫原性。此外，将通过理化分析[圆二色谱（CD）和核磁共振（NMR）光谱和质谱（MS）分析]以及生物信息学和分子建模获得蛋白质的3D结构数据。将通过ELISA和流式细胞术评估在哥伦比亚、巴西和布基纳法索流行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和T细胞应答。将检测血清和亲和纯化IgG在MFA中的TB能力。此外，在小鼠和猴中进行的免疫原性研究将解决寄生虫感染对免疫引起的Ab应答的潜在交叉初免/加强效应，反之亦然。还将检测引发的抗恶性疟原虫抗体的潜在交叉反应性。所需的所有设施和技术都是可用的，并由参与者团体定期进行。在研究结束时，我们期望已经确定了Pvs 48/45在人类和动物中诱导有效TB免疫的最佳条件以及用于进一步临床开发的最佳疫苗制剂。

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.