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，这些抗原将立即进入开发阶段，作为结核病亚单位疫苗。在我们之前的 SBIR 第一阶段资助 (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施用时很有可能诱导保护性细胞免疫。开发的高通量工艺将高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相结合，以确定针对每种蛋白质的抗体和 T 细胞特异性反应水平。利用我们第一阶段奖项提供的资金，并与公认的 Mtb 疫苗领导者合作，我们验证了这一专有的抗原发现系统，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，将此过程应用于剩余约 90% 的 Mtb 基因组，以生成用于疫苗（和适当的诊断）开发的候选抗原组合，并完成疫苗的初步开发、生产和临床前测试。我们在 Aeras 全球结核疫苗基金会的合作者根据已确定的抗原开发了候选结核疫苗。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)，这是一种在 I 期 SBIR 资助 (R43 AI47641-01) 下开发的无克隆方法，可生成转录活性 PCR 片段，该片段可以：用于在培养细胞中表达蛋白质，2) 用于直接对动物进行疫苗接种（基因疫苗接种），3) 作为模板指导无细胞体外转录和翻译反应，产生大量蛋白质用于免疫测定。由于无需克隆，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，成为称为疫苗组学 (TM/SM) 的高通量抗原发现平台）。迄今为止，Vaccinomics(TM/SM) 已被用于快速扩增、表达和分析大约 10% 的 Mtb 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结核分枝杆菌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（ORF）的免疫原性并对基因组进行相应的排名，然后2）合成和纯化蛋白质以创建蛋白质阵列以用于后续分析使用 Mtb 感染小鼠、豚鼠和人类结核病患者的材料进行免疫学筛选，以选择候选疫苗抗原：将系统评估通过 T 细胞测定确定的候选疫苗它们在动物模型（包括 Mtb 感染的小鼠和豚鼠）中的免疫原性和保护性免疫力。首先，用候选疫苗对 C57BL/6 小鼠进行免疫，以检查其免疫原性。其次，将进一步检查这些抗原对小鼠气溶胶结核感染的保护性免疫力。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通过初免和加强策略进行进一步检查，并将保护功效直接与卡介苗（BCG）进行比较。最后，将通过重组卡介苗载体表达选定的一些极具前景的候选疫苗，并在符合临床研究要求的条件下在豚鼠中测试其保护性免疫力。基于这些研究，有前景的候选疫苗将在 Aeras 进入高级开发阶段，以期加快临床测试。我们还建议，这种方法可能有助于开发针对其他自然出现和基因工程生物体的合理疫苗和诊断方法，并且在快速应对新型传染病（ID）和包括结核病在内的耐药性ID时也可能特别有用是必需的。