Evaluation of Novel Preerythrocytic Anti-infection Malaria Vaccine Candidates

从我们今年的合作出版物中，我们报告了以下进展： 我们有兴趣开发动物模型，以更好地预测正在考虑进行人体试验的疟疾疫苗的疗效。在FDA同事领导的这项研究中（Chawla B等人，Infection and Immunity），我们提供了来自约氏疟原虫的重组CSP（rPyCSP），当在Montanide伊萨51佐剂中递送时，在C57 BL/6和BALB B/c小鼠品系中诱导针对子孢子攻击的杀菌免疫。对子孢子攻击的保护与表达IL-2、IL-4和TNF-α的CD 4 + T细胞的较高频率相关。在RTS，S疫苗（一种基于CSP的疫苗）的人体研究中，临床免疫与较高的IgG水平和较高的产生IL-2和TNF的CD 4 + T细胞频率相关。这些结果提供了一个很好的模型系统，以评估新的佐剂的效力，疫苗剂量和确定免疫相关性，以寻找上级候选疟疾疫苗。 18 S rRNA是一种生物标志物，在控制人类疟疾感染（CHMI）试验中提供了厚血涂片的替代方法。与华盛顿大学领导的多个机构的同事一起，（Seilie A等AJTMH），我们验证了疟原虫18 S rRNA的多重定量逆转录-聚合酶链反应（qRT-PCR），前瞻性地比较了三项试验的血涂片和qRT-PCR，并模拟了不同生物标志物定义的寄生虫密度下的治疗效果，以评估对感染检测、症状减轻、并测量干预效果。我们确定疟原虫18 S rRNA是一种敏感和特异的生物标志物，可以替代血液涂片在非流行性环境中的CHMI试验中进行感染检测。本研究通过美国食品药品监督管理局对生物标志物进行了认证，以用于非流行性研究中心的CHMI研究，这将促进生物标志物在药物和疫苗试验的合格使用背景下的使用。 我们在本报告所述期间未公布的进展包括以下进展： 在小鼠和人类中，如果RAS保持足够的活力以侵入肝细胞，则可以通过多次暴露于辐射减毒子孢子（RAS）寄生虫形式来诱导针对疟疾感染的无菌保护。我们对PfSPZ为基础的RAS疫苗进行的临床试验一致表明，对CSP的抗体应答与保护自然获得的恶性疟原虫感染有关。因此，需要新的亚单位疫苗策略。CSP耐受性转基因小鼠在RAS免疫后也受到保护，这表明另外的红细胞前抗原作为无菌免疫的靶标。RTS，S基于Pf环子孢子蛋白（PfCSP），主要的子孢子表面抗原，是最先进的亚基候选者，但在III期试验中仅显示出对疟疾发作的部分功效。我们已经试图鉴定新的候选红细胞前疫苗抗原（PEVA），其可以增加单独使用CSP免疫原实现的保护水平。我们假设PEVA候选者在肝期（LS）发育过程中转录，并使用我们实验室开发的转录组学数据来鉴定此类候选抗原。LMIV已经评估了这些免疫原中的一些通过DNA疫苗接种在啮齿动物疟疾模型中的保护效力。 于2019财政年度，我们向下选择了2个PyPEVA候选物以研究PfPEVA直向同源物。选择的两个候选人是SHMT和305 w。我们还研究了PyPEVA和PfPEVA在E.而不是昆虫细胞。通过修饰N-末端氨基酸以增加蛋白质在E.结果，观察到PySHMT、PfSHMT和Pf 305 w的蛋白表达。已经确定了获得可溶性蛋白质的一些初始复性条件。 于2019财政年度，我们开发了新试剂，以测试全生物PfSPZ疫苗试验中患者的PfCSP菌株特异性和结构域特异性反应性。从具有筛分证据的PfCSP中的不同结构域设计肽。形成结构的CSP（TSR）的C-末端肽作为SUMO融合物重组产生，其具有位点特异性生物素标签以特异性连接至Bio-Biological珠。我们产生了毫克量的切割的和生物素化的肽。我们通过测试与小鼠单克隆抗体的反应性来测试这些肽的正确结构，所述小鼠单克隆抗体与折叠的TSR结构反应并具有活性。首先，我们确定NF 54 TSR肽识别肽，表明我们的肽正确折叠。其次，我们观察到菌株特异性肽减少了与单克隆抗体的结合。

From our collaborative publications this year, we report the following advances: We are interested in developing animal models that can better predict the efficacy of malaria vaccines being considered for human trials. In this study led by colleagues at the FDA (Chawla B, et al, Infection and Immunity) we contributed recombinant CSP from Plasmodium yoelli (rPyCSP), which when delivered in Montanide ISA 51 adjuvant, induced sterilizing immunity against sporozoite challenge in C57BL/6 and BALB/c strains of mice. Protection against sporozoite challenge correlated with the higher frequency of CD4+ T cells that express IL-2, IL-4 and TNF-. In human studies of the RTS,S vaccine (a CSP-based vaccine), clinical immunity was associated with higher IgG levels and greater frequency of IL-2 and TNF- producing CD4+ T cells. These results provide an excellent model system to evaluate the efficacy of novel adjuvants, vaccine dosage and determine the correlates of immunity in search for superior candidate malaria vaccines. 18S rRNA is a biomarker that provides an alternative to thick blood smears in controlled human malaria infection (CHMI) trials. Together with colleagues at multiple institutions led from the University of Washington (Seilie A, et al. AJTMH), we validated a multiplex quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for Plasmodium 18S rRNA, prospectively compared blood smears and qRT-PCR for three trials, and modeled treatment effects at different biomarker-defined parasite densities to assess the impact on infection detection, symptom reduction, and measured intervention efficacy. We determined that Plasmodium 18S rRNA is a sensitive and specific biomarker that can justifiably replace blood smears for infection detection in CHMI trials in non-endemic settings. This study led to biomarker qualification through the U.S. Food and Drug Administration for use in CHMI studies at non-endemic sites, which will facilitate biomarker use for the qualified context of use in drug and vaccine trials. Our unpublished progress during this reporting period includes the following advances: Sterile protection against malaria infection can be induced by multiple exposures to radiation-attenuated sporozoite (RAS) parasite forms in mice and humans, if the RAS remain sufficiently viable to invade hepatocytes. Our clinical trials of PfSPZ-based RAS vaccines have consistently shown that antibody responses to CSP are associated with protection of naturally acquired P. falciparum infection. Thus, new subunit vaccine strategies are needed. CSP tolerant transgenic mice are also protected after RAS immunization, implicating additional pre- erythrocytic antigens as targets of sterile immunity. RTS,S based on Pf circumsporozoite protein (PfCSP), the predominant sporozoite surface antigen, is the most advanced subunit candidate, but has shown only partial efficacy against malaria episodes in Phase III testing. We have sought to identify novel candidate pre-erythrocytic vaccine antigens (PEVA) that could add to the level of protection achieved with CSP immunogens alone. We assume that PEVA candidates are transcribed during liver stage (LS) development and have used transcriptomic data developed in our lab to identify such candidate antigens. LMIV has assessed the protective efficacy of some of these immunogens by DNA vaccination in rodent models of malaria. In FY2019, we down-selected to 2 PyPEVA candidates to pursue the PfPEVA orthologues. The two candidates selected were SHMT and 305w. We also pursued expression of both PyPEVA and PfPEVA in E. coli instead of insect cells. Stability of the protein was increased by modifying the N-terminal amino acid to increase the half-life of the protein in E. coli and as a result, protein expression was observed for PySHMT, PfSHMT and Pf305w. Some initial refolding conditions to achieve soluble protein have been identified. In FY2019, we developed new reagents to test strain-specific and domain-specific reactivity of PfCSP from patients in the whole organism PfSPZ vaccine trial. Peptides were designed from different domains in PfCSP that had evidence of sieving. C-terminal peptides of CSP (TSR) that form structure were generated recombinantly as a SUMO fusion with a site-specific biotin tag for specific attachment to Bio-Plex beads. We generated milligram quantities of cleaved and biotinylated peptides. We tested these peptides for correct structure by testing reactivity to a mouse monoclonal antibody that reacts to folded TSR structure and has activity. First, we determined that the NF54 TSR peptide recognizes the peptide, suggesting our peptide is correctly folded. Second, we observed that strain-specific peptides reduced binding to the monoclonal antibody.