Screening Complete TB Proteome for Protective Antigens

筛选完整的结核病蛋白质组以寻找保护性抗原

• 批准号: 7493092
• 负责人: Alan Greener
• 金额: $ 87.93万
• 依托单位: GENE THERAPY SYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7493092
• 关键词: Advanced Development; Aerosols; Animal Genetics; Animal Model; Animals; Antibodies; Antigens; Award; Bioinformatics; Biological Assay; C57BL/6 Mouse; CD4 Positive T Lymphocytes; Calmette-Guerin Bacillus; Cavia; Cells; Cellular Immunity; Clinical Research; Collaborations; Communicable Diseases; Condition; Coupled; Cultured Cells; DNA; Development; Diagnostic; Dose; Drug resistance; Engineering; Foundations; Funding; Gene Amplification; Genes; Genetic Transcription; Genome; Harvest; Histidine; Human; Immune response; Immunity; Immunoassay; In Vitro; Infection; Lead; Legal patent; Lung; Measures; Methods; Modeling; Mus; Mycobacterium tuberculosis; Numbers; Oligonucleotides; Open Reading Frames; Organism; Patients; Peripheral Blood Mononuclear Cell; Phase; Phase I Clinical Trials; Polymerase Chain Reaction; Probability; Process; Production; Protein Array; Proteins; Proteome; R43 grant; Reaction; Recombinants; Scanning; Screening procedure; Serum; Small Business Funding Mechanisms; Small Business Innovation Research Grant; Splenocyte; Staining method; Stains; Subunit Vaccines; System; T-Lymphocyte; Technology; Testing; Translations; Tuberculosis Vaccines; Vaccinated; Vaccination; Vaccine Antigen; Vaccines; Virulent; Work; base; cytokine; design; gene synthesis; genetic vaccine; high throughput technology; immunogenicity; immunoreactivity; magnetic beads; mouse model; novel; novel vaccines; pre-clinical; promoter; protein expression; research clinical testing; response; tool; vaccine development; vector

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.

描述（由申请人提供）：该项目将完成由结核分枝杆菌（MTB）基因组编码的所有蛋白质的鉴定和免疫原性分析，从而产生一系列保护性抗原，这些抗原将立即作为结核病的亚基疫苗进入发育。在我们先前的SBIR I期赠款（R43 AI053636-01）中，我们开发了一种平台技术，用于对抗原的高通量，基于蛋白质组的鉴定，这些抗原具有很高的诱导性细胞免疫力并作为疫苗施用时诱导保护性细胞免疫的可能性。开发的高吞吐过程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测定法相结合，以确定针对每个单个蛋白质的抗体和T细胞特异性反应性的水平。使用我们阶段奖提供的资金，并与公认的MTB疫苗领导者合作，我们验证了该专有的抗原发现系统，并成功地完成了对MTB基因组的384个基因（约10％）的免疫原性分析。 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.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，称为跨越活性PCR（TAPR），这是一种在I期SBIR Grant（R43 AI47641-01）下开发的无克隆方法模板直接无细胞的体外转录和翻译反应，这些反应产生了大量蛋白质，可用于免疫测定。由于它是无克隆的，因此TAP是一种强大的工具，用于快速合成和扩增基因组和相应的蛋白质组，并与B和T细胞免疫测定相结合，用作称为疫苗（TM/SM）的高通量抗原发现平台。迄今为止，疫苗（TM/SM）已用于快速扩大，表达和分析MTB蛋白质组约10％的免疫原性。现在，我们建议通过1）通过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（ORF），并相应地对基因组进行排名，然后对2）进行纯化并纯化蛋白质以创建蛋白质群体，以便使用MTB造成的材料来创建蛋白质群体，并在蛋白质群体中进行疫苗，并在人类的疫苗中进行疫苗，并构成人类疫苗，并构成对蛋白质群体，并构成蛋白质，并构成蛋白质，并将其构成人类的幼体，并构成对蛋白质群体，并创建蛋白质群体，并将其构成。候选者：在包括MTB感染的小鼠和豚鼠在内的动物模型中，将系统地评估由T细胞测定的疫苗候选物的免疫原性和保护性免疫。首先，C57BL/6小鼠将通过候选疫苗进行免疫以检查其免疫原性。其次，将进一步检查抗原的保护性免疫，以抗小鼠气溶胶结核病感染。在更严格的豚鼠模型中，将进一步研究了在小鼠研究中鉴定出的领先疫苗候选物，并将保护性疗效直接与芽孢杆菌Calmette Guerin（BCG）直接进行比较。最后，在符合临床研究要求的情况下，重组BCG载体将以重组BCG载体的形式表达选定数量的高度有希望的疫苗候选物，并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AERAS的高级发育中预期进行加急临床测试。我们还建议，这种方法可能对于针对其他自然出现和基因工程生物的理性疫苗和诊断的开发可能很有用，并且当需要快速对新型感染性疾病（ID）和耐药ID的快速反应时，也可能特别有用。