Delivery of passive immunity against HIV using commensal vaginal Lactobacillus bi

使用共生阴道双乳杆菌提供针对 HIV 的被动免疫

基本信息

批准号：
8329194
负责人：
Laurel A. Lagenaur
金额：
$ 29.99万
依托单位：
OSEL, INC.
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-04-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8329194
关键词：
AIDS/HIV problem Acquired Immunodeficiency Syndrome Address Affinity Chromatography Animals Antibodies Antibody Formation Ascaridil Bacterial Vaginosis Binding Biological Biological Assay Biological Products Biomedical Engineering Cell Line Cervical Characteristics Chromosomes Culture Media Cyanovirin-N Development Drug Formulations Engineering Enzyme-Linked Immunosorbent Assay Epidemic Epidemiologic Studies Exhibits Female Female of child bearing age Generations Genes Goals HIV HIV Antibodies HIV Entry Inhibitors HIV Envelope Protein gp120 HIV Infections HIV-1 Heterosexuals Human Immunoprecipitation In Situ In Vitro Infection Lactobacillus Lead Length Life Local Microbicides Macaca Macaca mulatta Modeling Mucous Membrane Neutralization Tests Passive Immunity Peripheral Blood Mononuclear Cell Phase Phase I Clinical Trials Plasmids Prevention Property Protective Agents Proteins Recombinants Recurrence Risk Safety Sexually Transmitted Diseases Site Surface Technology Testing Therapeutic Urinary tract infection Vagina Viral Viral Load result Virus Virus Diseases Western Blotting Woman base cost effective experience expression vector gel electrophoresis genetic strain homologous recombination in vivo inhibitor/antagonist manufacturing process development microbial microbicide nanobodies neutralizing antibody nonhuman primate novel pandemic disease pathogen pre-clinical prevent rectal safety testing simian human immunodeficiency virus social success transmission process vaginal lactobacilli vector

项目摘要

DESCRIPTION (provided by applicant): Women bear the brunt of the global AIDS epidemic due in part to their inherent social and biological vulnerability. Biologically, the cervical and vaginal mucosae are vulnerable targets for HIV transmission. In healthy women of childbearing age, mucosal antibodies and microflora, including Lactobacillus jensenii, help protect these surfaces and serve to block the colonization and infection by intruding pathogens. Epidemiological studies suggest that loss of vaginal lactobacilli is associated with an increased risk of heterosexual HIV-1 transmission and other sexually transmitted infections. Osel, Inc. and Profectus BioSciences are collaborating to exploit the properties of these natural protective agents by engineering a native human vaginal Lactobacillus, L. jensenii 1153, to express potent and broadly neutralizing single domain antibodies/nanobodies directed against HIV. Osel recently showed proof-of-concept for this recombinant Lactobacillus approach, by expressing an HIV-1 entry inhibitor from L. jensenii that reduced SHIV virus transmission in macaques by 63% (Lagenaur et al., Mucosal Immunol, (Jul 6, 2011)). The initial goal of this approach is to develop a Lactobacillus strain capable of delivering an antibody locally to prevent HIV infection at the vaginal or rectal mucosa. This strain can be formulated into a cost-effective product capable of persistent protection that is coitally-independent and controlled by women. We intend to evaluate this approach in two phases, and propose the following aims in Phase I: PHASE I PROPOSAL 1. Express secreted neutralizing single domain antibodies in the human vaginal Lactobacillus isolate, L. jensenii 1153. The genes encoding single domain antibodies will be subcloned into expression vector pOSEL175 and transformed into L. jensenii 1153. The resulting bacterial strains will be examined for antibody expression. The secreted proteins will be characterized using immunoprecipitation, gel electrophoresis, and gp120 ELISA. Full-length antibodies that bind to gp120 will be selected to for further characterization. 2. Characterize HI neutralizing activity of Lactobacillus-expressed single domain antibodies. The secreted antibodies in culture media will be purified using affinity chromatography and tested for neutralization of primary HIV-1 isolates in cell line-based and PBMC-based assays. The single domain antibodies that exhibit the best combination of HIV neutralization, (based on neutralization of isolates in the Profectus Virus Panel, scored by the number of isolates neutralized at >90% at 10 ¿g/ml concentration) will be selected for optimization of expression in L. jensenii 1153. 3. Optimize of plasmid-based expression of single domain antibodies by L. jensenii 1153. The expression cassettes of the leading single domain antibody candidates will be optimized for high level expression in L. jensenii 1153 and the expression levels compared by Western blot analysis. The candidate with the best combination of HIV neutralization potency and expression level will be selected for further strain development. 4. Construct integration vectors and integrate single domain expression cassettes into the L. jensenii 1153 chromosome. We will construct integration vectors and integrate the optimal antibody expression cassettes into the pox1 gene of L. jensenii 1153 via homologous recombination. The strains generated will be evaluated for antibody expression level, bioactivity against HIV, phenotypic characteristics compared to the parental strain and genetic stability. Technical feasibility in Phase I will be achieved by expressing intact and functionally active single domain antibodies in L. jensenii at concentrations high enough to block HIV-infectivity in vitro. In Phase II, we would complete development of the chromosomally integrated strain and test its ability to vaginally colonize rhesus macaques and produce sufficient levels of single domain antibody in vivo to protect against viral challenge. A proof of concept SHIV challenge study would then be conducted in animals colonized with the antibody- secreting strain to demonstrate protection from virus infection. Studies would also be conducted on formulation and manufacturing process development, and safety testing. If successful, this approach could provide a safe, yet inexpensive means to deliver passive immunity against HIV to the vaginal mucosa and to address the urgent need for female-controlled approaches to prevent sexually transmitted viral infection. PUBLIC HEALTH RELEVANCE: Our goal is to develop a novel female-controlled preventative against HIV by harnessing the vaginal microflora. We have developed technologies to bioengineer human vaginal Lactobacillus to express potent single domain antibodies in situ, thus creating a live microbicide. This project has the potential to reduce HIV acquisition in women of childbearing age in a coital-independent way.

描述（由适用提供）：妇女首当其冲，部分是由于其固有的社会和生物学脆弱性。从生物学上讲，宫颈粘膜和阴道粘膜是艾滋病毒传播的脆弱靶标。在育龄的健康女性中，包括乳杆菌的粘膜抗体和菌群（包括Jensenii）有助于保护这些表面，并通过侵入病原体来阻止定殖和感染。流行病学研究表明，阴道乳酸杆菌的丧失与异性恋HIV-1传播和其他性传播感染的风险增加有关。 Osel，Inc。和Profectus Biosciences正在协作，通过工程设计人类的人类阴道乳酸杆菌（L. jensenii 1153）来探索这些天然受保护剂的特性，以表达针对HIV的潜在和广泛中和单一域抗体/纳米生物的潜在中和单一域抗体。 OSEL最近通过表达L. jensenii的HIV-1进入抑制剂的概念证明，从L. jensenii表达了HIV-1进入抑制剂，该抑制剂将猕猴中的SHIV病毒传播降低了63％（Lagenaur等人，Mucosal Immunol，（Jul 6，2011））。这种方法的最初目标是开发能够在局部输送抗体以防止阴道或直肠粘膜的HIV感染的乳杆菌菌株。该菌株可以被配制为具有持久保护的具有成本效益的产品，该产品是与女性无关并控制的。我们打算分为两个阶段评估这种方法，并提出以下目的：第一阶段提案1。Express在人类阴道乳酸乳杆菌分离株中中和中和单个领域抗体，L。Jensenii1153。编码单个域抗体的基因将被列为表达量的1175和Transfore 175和transfore 175和tromensie。将检查菌株的抗体表达。分泌的蛋白质将使用免疫沉淀，凝胶电泳和GP120 ELISA进行表征。将选择与GP120结合的全长抗体以进行进一步表征。 2。表征乳杆菌表达的单域抗体的HI中和活性。培养基中的分泌抗体将使用亲和力色谱法纯化，并在基于细胞系和基于PBMC的测定中对原代HIV-1分离株进行神经化测试。单个结构域抗体表现出最佳组合HIV神经化的结合（基于Profectus病毒小组中分离株的神经化，由10％的分离株数量在10»g/mL浓度下中和以> 90％的中和数量进行评分），以优化在L. jensenii 1153. 3. l. jensenii1153。L.l. jensenii 1153。 1153。主要单域抗体的表达盒将优化用于高水平的Jensenii 1153中的高水平表达，并且通过Western blot分析比较了表达水平。将选择具有HIV神经化效力和表达水平最佳组合的候选人以进一步发展。 4。构造积分向量和集成的单域表达盒塞入L. jensenii 1153染色体中。我们将通过同源重组构建整合载体，并将最佳抗体表达盒整合到Jensenii 1153的Pox1基因中。与亲本菌株和遗传稳定性相比，将评估产生的菌株的抗体表达水平，对HIV的生物活性，表型特征。通过表达完整且具有功能活跃的单个结构域抗体的浓度以足够高的浓度以阻止体外HIV感染性的完整且具有功能活跃的单个结构域抗体，可以实现I阶段I的技术可行性。在阶段 ii，我们将完成染色体整合菌株的发展，并测试其在体内进行恒河猕猴和产生足够水平的单域抗体的能力，以防止病毒挑战。然后将在用抗体分泌菌株定植的动物中进行概念证明SHIV挑战研究，以证明病毒感染的保护。还将对公式和制造过程开发以及安全测试进行研究。如果成功，这种方法可以提供一种安全但廉价的方法，以向阴道粘膜提供针对艾滋病毒的被动免疫学，并解决迫切需要对女性控制的方法，以防止性传播的病毒感染。公共卫生相关性：我们的目标是通过利用阴道菌群来开发一种新型的女性控制的预防性预防剂。我们已经开发了生物工程师人类阴道乳酸杆菌的技术，以原位表达潜在的单个结构域抗体，从而产生活生生。该项目有可能以核心独立的方式减少育龄妇女的艾滋病毒获取。