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： 我们有兴趣开发能够更好地预测考虑用于人体试验的疟疾疫苗效力的动物模型。在FDA的同事(Chawla B，等人，感染和免疫)领导的这项研究中，我们贡献了来自约氏疟原虫的重组CSP(RPyCSP)，当它在Montanide ISA 51佐剂中投放时，在C57BL/6和BALB/c品系的小鼠中诱导了对子孢子攻击的灭菌免疫。对子孢子攻击的保护与表达IL-2、IL-4和TNF-的CD4+T细胞的频率较高有关。在对RTS的人体研究中，S疫苗(一种以CSP为基础的疫苗)的临床免疫与较高的免疫球蛋白水平以及较高的IL-2和产生肿瘤坏死因子的CD4+T细胞的频率有关。这些结果为评估新型佐剂的有效性、疫苗剂量和确定免疫相关因素以寻找更好的候选疟疾疫苗提供了一个很好的模型系统。 18S rRNA是一种生物标志物，在受控人类疟疾感染(CHMI)试验中提供了厚血涂片的替代。与华盛顿大学领导的多个机构的同事一起(Seilie A等人)。我们验证了针对疟原虫18S rRNA的多重定量逆转录-聚合酶链式反应(qRT-PCR)，前瞻性地比较了三个试验的血液涂片和qRT-PCR，并模拟了不同生物标记物定义的寄生虫密度下的治疗效果，以评估对感染检测、症状减轻和测量的干预效果的影响。我们确定疟原虫18S rRNA是一种敏感和特异的生物标志物，可以合理地取代血液涂片在非流行环境下的CHMI试验中用于感染检测。这项研究通过美国食品和药物管理局获得了用于非流行地点CHMI研究的生物标记物资格，这将促进在药物和疫苗试验中合格使用生物标记物。 在本报告所述期间，我们未公布的进展包括以下进展： 对疟疾感染的无菌保护可以通过多次暴露于辐射减弱子孢子(RAS)寄生虫形式在小鼠和人类中诱导，如果RAS仍然足够存活以入侵肝细胞的话。我们基于PfSPZ的RAS疫苗的临床试验一直表明，对CSP的抗体反应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。耐受CSP的转基因小鼠在RAS免疫后也受到保护，这意味着额外的前红细胞抗原是无菌免疫的目标。S的RTS基于Pf环子孢子蛋白(PfCSP)，PfCSP是主要的子孢子表面抗原，是最先进的候选亚基，但在第三阶段试验中仅显示出对疟疾发作的部分疗效。我们试图确定新的候选前红细胞疫苗抗原(PEVA)，以增加单独使用CSP免疫原所达到的保护水平。我们假设PEVA候选抗原在肝脏发育阶段(LS)被转录，并使用我们实验室开发的转录数据来识别这些候选抗原。LMIV通过在疟疾啮齿动物模型中接种DNA疫苗，评估了其中一些免疫原的保护效果。 在2019财年，我们从2个PfPEVA候选者中选出了PfPEVA的直写词。入选的两名候选人是SHMT和305W。我们还寻求在大肠杆菌中表达pYPEVA和pfPEVA，而不是在昆虫细胞中。通过修饰N-末端氨基酸来延长该蛋白在大肠杆菌中的半衰期，从而提高了该蛋白的稳定性，并观察到了该蛋白在pYSHMT、PfSHMT和Pf305w中的表达。初步确定了获得可溶性蛋白质的复性条件。 在2019财年，我们开发了新的试剂来测试全生物PfSPZ疫苗试验中患者的PfCSP的菌株特异性和区域特异性反应性。从PfCSP的不同结构域中设计了具有筛选证据的多肽。形成结构的CSP(TSR)的C末端多肽被重组生成相扑与位点特异性生物素标记的融合，以特异性地附着在Bio-Plex珠子上。我们产生了毫克量的裂解和生物素化的多肽。我们通过测试对一种与折叠的TSR结构发生反应并具有活性的鼠单抗的反应性来测试这些多肽的正确结构。首先，我们确定NF54 TSR肽识别该肽，表明我们的肽正确折叠。其次，我们观察到菌株特异性多肽减少了与单抗的结合。

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.