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

描述(申请人提供)：在疟疾流行地区持续暴露于疟疾感染的个人会产生免疫反应，这种免疫反应已被证明可以减少或阻止患者向蚊子媒介传播寄生虫，即所谓的传播阻断(TB)免疫。在这种背景下，针对配子体、受精卵和动粒体期表达的特定疟原虫抗原的抗体可以诱导这种阻断，为结核病疫苗的开发提供了基础。P48/45是这些抗原之一，是一种在所有疟原虫物种中表达的保守蛋白，是寄生虫受精所必需的，目前正被开发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和表达，作为~60 kDa的重组产物，已被证明具有高度的抗原性和免疫原性。暴露于间日疟原虫的个体产生α-Pvs48/45抗体，抗体滴度随年龄增加而增加，而抗体滴度较高的个体也表现出较高的体外结核病活性。初步研究表明，rPvs48/45蛋白对小鼠和猴子都具有高度的免疫原性，所诱导的抗体具有体外阻止寄生虫传播到蚊子的能力。所有这些数据使Pvs48/45成为非常有希望的结核病疫苗候选疫苗。这项提案的目标是确定rPvs48/45用于人类的疫苗潜力。我们将利用流行区个体的血清和细胞，以及rPvs48/45结构域和合成肽来进一步表征rPvs48/45的抗原性，以绘制相关的免疫和功能表位。此外，我们将确认和进一步鉴定全长Pvs48/45和部分片段在非人类灵长类动物中的免疫原性。此外，蛋白质(S)的三维结构数据将通过物理化学分析[圆二色谱(CD)、核磁共振(NMRs)光谱和质谱分析(MS)]以及生物信息学和分子建模获得。在哥伦比亚、巴西和布基纳法索的流行地区，暴露于间日疟原虫和恶性疟原虫的个体诱导的天然抗体将通过ELISA进行评估，T细胞反应将通过流式细胞仪进行评估。血清和亲和纯化的免疫球蛋白将在MFA中进行结核能力测试。此外，在小鼠和猴子身上的免疫原性研究将解决寄生虫感染对免疫引发的抗体反应的潜在交叉启动/增强效应，反之亦然。还将测试诱导的抗体对恶性疟原虫的潜在交叉反应。所有所需的设施和技术都是可用的，并由参与者小组进行常规操作。在研究结束时，我们预计已经确定了Pvs48/45在人和动物中诱导有效结核病免疫的最佳条件，以及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.