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

• 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
• 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

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（由申请人提供）：妇女承受着全球艾滋病流行的冲击，部分原因是她们固有的社会和生物脆弱性。从生物学上讲，宫颈和阴道粘膜是艾滋病毒传播的易受攻击目标。在育龄期的健康女性中，粘膜抗体和微生物群落，包括詹氏乳杆菌，有助于保护这些表面，并阻止入侵病原体的定植和感染。流行病学研究表明，阴道乳酸杆菌的损失与异性间HIV-1传播和其他性传播感染的风险增加有关。奥塞尔公司和Profectus生物科学公司正在合作开发这些天然保护剂的特性，通过工程改造一种天然的人类阴道乳酸杆菌，L。jensenii 1153，以表达针对HIV的有效和广泛中和的单结构域抗体/纳米抗体。Osel最近展示了这种重组乳酸杆菌方法的概念验证，通过表达来自乳酸杆菌的HIV-1进入抑制剂。jensenii，其将猕猴中的SHIV病毒传播降低了63%（Lagenaur等人，Mucobacterium Immunol，（2011年7月6日））。这种方法的最初目标是开发一种能够局部递送抗体以预防阴道或直肠粘膜处的HIV感染的乳杆菌菌株。这种菌株可以配制成一种具有成本效益的产品，能够持久保护，不依赖于性交，并由女性控制。我们打算分两个阶段评估这一方法，并在第一阶段提出以下目标：在人阴道乳杆菌分离株中表达分泌的中和性单域抗体。1153. jensenii将编码单域抗体的基因亚克隆到表达载体pOSEL 175中，转化L. 1153. jensenii将检查所得细菌菌株的抗体表达。分泌的蛋白质将使用免疫沉淀、凝胶电泳和gp 120 ELISA进行表征。将选择与gp 120结合的全长抗体用于进一步表征。 2.表征乳酸杆菌表达的单结构域抗体的HI中和活性。将使用亲和色谱法纯化培养基中分泌的抗体，并在基于细胞系和基于PBMC的试验中检测原代HIV-1分离株的中和作用。将选择表现出HIV中和的最佳组合的单结构域抗体（基于Profectus Virus Panel中分离株的中和，通过在10 μ g/ml浓度下中和>90%的分离株数量进行评分）用于在L. 1153. jensenii 3.单结构域抗体的L. 1153. jensenii主要单结构域抗体候选物的表达盒将被优化用于在L. jensenii 1153的表达水平，并通过Western印迹分析比较表达水平。将选择具有HIV中和效力和表达水平的最佳组合的候选物用于进一步菌株开发。 4.构建整合载体并将单结构域表达盒整合到L. jensenii 1153染色体。我们将构建整合载体，将优化的抗体表达盒整合到L. Jensenii 1153同源重组。将评价产生的菌株的抗体表达水平、抗HIV生物活性、与亲本菌株相比的表型特征和遗传稳定性。 I期的技术可行性将通过在L.浓度高到足以在体外阻断HIV感染。同相 II，我们将完成染色体整合菌株的开发，并测试其阴道定殖恒河猴的能力，并在体内产生足够水平的单域抗体以保护免受病毒攻击。然后在用抗体分泌菌株定殖的动物中进行概念验证SHIV攻击研究，以证明对病毒感染的保护。此外，亦会就配方和制造工序的发展，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而廉价的手段，将针对HIV的被动免疫传递到阴道粘膜，并解决对女性控制方法的迫切需求，以预防性传播病毒感染。 公共卫生相关性：我们的目标是通过利用阴道微生物菌群来开发一种新型的女性控制的艾滋病毒预防剂。我们已经开发了生物工程技术，人类阴道乳酸杆菌表达有效的单域抗体原位，从而创造了一个活的杀微生物剂。该项目有可能以不依赖性交的方式减少育龄妇女感染艾滋病毒。