Screening Complete TB Proteome for Protective Antigens

描述（由申请人提供）：该项目将完成对结核分枝杆菌（Mtb）基因组编码的所有蛋白质的鉴定和免疫原性分析，产生一组保护性抗原，这些抗原将立即作为结核病亚单位疫苗进入开发阶段。在我们之前的SBIR I期授权（R43 AI053636-01）中，我们开发了一种平台技术，用于高通量，基于蛋白质组的抗原鉴定，这些抗原在配制和作为疫苗使用时具有很高的诱导保护性细胞免疫的可能性。开发的高通量过程结合了高效的基因扩增和蛋白质表达方法，以及基于血清和脾细胞的检测，以确定抗体和t细胞对每种蛋白质的特异性反应性水平。利用I期奖金提供的资金，并与公认的Mtb疫苗领导者合作，我们已经验证了这一专有抗原发现系统，并成功完成了Mtb基因组384个基因（约10%）的免疫原性分析。我们现在寻求支持，将这一过程应用于其余约90%的结核分枝杆菌基因组，以产生用于疫苗（和适当的诊断）开发的候选抗原组合，并根据我们在Aeras全球结核疫苗基金会的合作者确定的抗原完成结核病候选疫苗的初步开发、生产和临床前测试。我们快速抗原发现过程的核心是我们的专利基因扩增技术，称为转录活性PCR （TAPR)，这是一种根据I期SBIR拨款（R43 AI47641-01）开发的无克隆方法，可产生转录活性PCR片段，可1）用于在培养细胞中表达蛋白质，2)用于直接接种动物（遗传疫苗）。3)作为模板，指导体外无细胞转录和翻译反应，产生大量用于免疫分析的蛋白质。由于它是无克隆的，TAP是快速合成和扩增基因组和相应蛋白质组的强大工具，并与B细胞和t细胞免疫测定相结合，作为高通量抗原发现平台，称为疫苗组学（TM/SM）。迄今为止，疫苗组学（TM/SM）已被用于快速扩增、表达和分析大约10%的结核分枝杆菌蛋白质组的免疫原性。我们现在建议对整个结核分枝杆菌蛋白质组完成这一过程：1)应用生物信息学分析来预测每个开放阅读框（ORF）的免疫原性，并相应地对基因组进行排序，然后2)合成和纯化蛋白质以创建蛋白质阵列，用于随后使用结核分枝杆菌感染小鼠、豚鼠和人类结核患者的材料进行免疫筛选，以选择疫苗候选抗原。通过T细胞试验确定的候选疫苗将在包括mtb感染小鼠和豚鼠在内的动物模型中系统地评估其免疫原性和保护性免疫。首先，用候选疫苗免疫C57BL/6小鼠，检测其免疫原性。其次，进一步检测抗原对小鼠气溶胶结核感染的保护性免疫。在小鼠研究中确定的主要候选疫苗将在更严格的豚鼠模型中进行进一步检验，采用启动和增强策略，并直接与卡介苗（BCG）的保护效果进行比较。最后，在符合临床研究要求的条件下，通过重组卡介苗载体表达一些极有希望的候选疫苗，并在豚鼠体内测试其保护性免疫。基于这些研究，有希望的候选疫苗将在Aeras进入后期开发，预计将加快临床试验。我们还建议，这种方法可能有助于开发针对其他自然出现的和基因工程生物的合理疫苗和诊断方法，并且在需要对新型传染病（ID）和耐药ID（包括结核病）做出快速反应时也可能特别有用。

DESCRIPTION (provided by applicant): This project will complete the identification and immunogenicity analyses of all proteins encoded by the Mycobacterium tuberculosis (Mtb) genome, yielding a pool of protective antigens that will immediately enter development as subunit vaccines for tuberculosis. In our prior SBIR Phase I grant (R43 AI053636-01), we developed a platform technology for high throughput, proteome-based identification of antigens that have a high probability of inducing protective cellular immunity when formulated and administered as a vaccine. The high throughput process that was developed combines efficient gene amplification and protein expression methods with serum- and splenocyte-based assays to determine the level of antibody- and T-cell specific reactivity against each individualprotein. Using funding provided by our Phase I award, and in collaboration with recognized Mtb vaccine leaders, we have validated this proprietary antigen discovery system, and have successfully completed immunogenicity analysis of 384 genes (approximately 10%) of the Mtb genome. We now seek support to apply this process to the remaining approximately 90% of the Mtb genome to generate a portfolio of candidate antigens for use in vaccine (and diagnostics where appropriate) development, and to complete initial development, production and pre-clinical testing of tuberculosis vaccine candidates based on the identified antigens by our collaborators at Aeras Global TB Vaccine Foundation. Central to our rapid antigen discovery process is our patented gene amplification technology, called Transciptionally Active PCR (TAPR), a cloning-free method developed under a Phase I SBIR grant (R43 AI47641-01) that generates transcriptionally active PCR fragments that can 1) be used to express proteins in cultured cells, 2) be used to directly vaccinate animals (genetic vaccination), and 3) serve as templates to direct cell-free in vitro transcription and translation reactions that yield large amounts of proteins for use in immunoassays. Because it is cloning-free, TAP is a powerful tool for rapid synthesis and amplification of both genomes and the corresponding proteomes, and coupled with B- and T-cell immunoassays serve as a high-throughput antigen discovery platform called Vaccinomics(TM/SM). To date, Vaccinomics(TM/SM) has been used to rapidly amplify, express, and analyze the immunogenicity of approximately 10% of the Mtb proteome. We now propose to complete this process for the entire Mtb proteome by 1) applying bioinformatic analyses to predict the immunogenicity of each open reading frame (ORF) and rank the genome accordingly, then 2) synthesizing and purifying the proteins to create protein arrays for subsequent immunological screening using material from Mtb-infected mice, guinea pigs, and human TB patients to select vaccine antigen candidates: Vaccine candidates identified by the T cell assays will be systematically evaluated for their immunogenicity and protective immunity in animal models including Mtb-infected mice and guinea pigs. First, C57BL/6 mice will be immunized by vaccine candidates to examine their immunogenicity. Second, the antigens will be further examined for their protective immunity against aerosol TB infection of mice. The leading vaccine candidates identified in mouse studies will be further examined in the more stringent guinea pig model with a prime and boost strategy, and the protective efficacy is directly compared to that of Bacillus Calmette Guerin (BCG). Finally, a selected number of highly promising vaccine candidates will be expressed by recombinant BCG vectors and tested for their protective immunity in guinea pigs under conditions in compliance with clinical study requirements. Based on these studies, promising candidate vaccines will enter advanced development at Aeras in anticipation of expedited clinical testing. We also suggest that this approach will likely be useful for the development of rational vaccines and diagnostics against other naturally emerging and genetically engineered organisms, and may also be particularly useful when rapid responses to novel infectious diseases (ID) and drug-resistant IDs including TB are required.