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，产生一个保护性抗原池，将立即作为结核病亚单位疫苗进行开发。在我们之前的SBIR第一阶段拨款(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。开发的高通量方法将有效的基因扩增和蛋白质表达方法与基于血清和脾细胞的分析相结合，以确定针对每种单独蛋白质的抗体和T细胞特异性反应性水平。利用我们的第一阶段奖励提供的资金，并与公认的结核分枝杆菌疫苗领导者合作，我们已经验证了这一专利抗原发现系统，并成功地完成了对结核分枝杆菌基因组的384个基因(约10%)的免疫原性分析。我们现在寻求支持，将这一过程应用于结核分枝杆菌剩余约90%的基因组，以生成用于疫苗(和适当时的诊断)开发的候选抗原组合，并根据我们在AERAS全球结核病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Transcipally Active PCR(TPAR)，这是一种在SBIR I期拨款(R43 AI47641-01)下开发的免克隆方法，它可以产生转录活性的PCR片段，这些片段可以1)用于在培养细胞中表达蛋白质，2)用于直接接种动物(基因疫苗)，以及3)作为模板指导无细胞的体外转录和翻译反应，从而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细胞免疫分析相结合，成为一个高通量的抗原发现平台，称为疫苗组学(Vaccinology，TM/SM)。到目前为止，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。我们现在建议通过以下方式完成整个Mtb蛋白质组的这一过程：1)应用生物信息学分析来预测每个开放阅读框架(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，用于后续的免疫学筛选，使用来自Mtb感染小鼠、豚鼠和人类结核病患者的材料来选择疫苗候选抗原：通过T细胞检测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进行系统的免疫原性和保护性免疫评估。首先，C57BL/6小鼠将由候选疫苗免疫，以检查其免疫原性。其次，将进一步检测这些抗原对小鼠气雾性结核病感染的保护性免疫力。在小鼠研究中确定的领先候选疫苗将在更严格的豚鼠模型中进行进一步检查，采用PRIME和BOOST策略，其保护效果直接与卡介苗(BCG)进行比较。最后，选择一些极有希望的候选疫苗将通过重组卡介苗载体表达，并在符合临床研究要求的条件下测试其在豚鼠中的保护性免疫。基于这些研究，有希望的候选疫苗将在AERS进入高级开发阶段，预计将加快临床测试。我们还认为，这种方法可能对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有用，当需要对包括结核病在内的新型传染病(ID)和抗药性ID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.