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.

说明（由申请人提供）：妇女在全球艾滋病流行中首当其冲，部分原因是她们固有的社会和生理脆弱性。在生物学上，宫颈和阴道粘膜是艾滋病毒传播的脆弱目标。在育龄健康妇女中，粘膜抗体和微生物群，包括延seni乳杆菌，有助于保护这些表面，并有助于阻止入侵病原体的定植和感染。流行病学研究表明，阴道乳酸菌的缺失与异性传播HIV-1和其他性传播感染的风险增加有关。Osel, Inc.和Profectus BioSciences正在合作开发这些天然保护剂的特性，通过设计一种天然的人类阴道乳杆菌，L. jensenii 1153，来表达有效的和广泛中和的针对HIV的单域抗体/纳米体。Osel最近通过表达一种来自延森乳杆菌的HIV-1进入抑制剂，证明了这种重组乳酸菌方法的概念证明（Lagenaur等人，Mucosal immuno1,（2011年7月6日）），该抑制剂将SHIV病毒在猕猴中的传播减少了63%。该方法的最初目标是开发一种能够在阴道或直肠粘膜局部递送抗体的乳杆菌菌株，以防止HIV感染。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，不依赖于女性的生殖并由女性控制。我们打算分两个阶段对这一方法进行评估，并在第一阶段提出以下目标：在人阴道分离乳杆菌简氏乳杆菌1153中表达分泌的中和性单域抗体。将编码单域抗体的基因亚克隆到表达载体pOSEL175中，转化为大肠杆菌1153。所产生的菌株将被检测抗体表达。分泌蛋白将使用免疫沉淀、凝胶电泳和gp120 ELISA进行表征。将选择与gp120结合的全长抗体进行进一步表征。2. 鉴定乳酸杆菌表达的单域抗体的HI中和活性。培养基中分泌的抗体将使用亲和层析纯化，并在基于细胞系和基于pbmc的检测中检测HIV-1原代分离物的中和作用。将选择表现出最佳HIV中和组合的单域抗体（基于Profectus病毒面板中分离物的中和，以10¿g/ml浓度下以> / 90%中和的分离物数量评分）用于优化在L. jensenii 1153中的表达。3. 简氏乳杆菌1153质粒表达单域抗体的优化主要的单域候选抗体的表达盒将被优化，以便在L. jensenii 1153中高水平表达，并通过Western blot分析比较表达水平。将选择具有最佳HIV中和效力和表达水平组合的候选株进行进一步的毒株开发。4. 构建整合载体，将单域表达盒整合到简氏乳杆菌1153染色体上。我们将构建整合载体，并通过同源重组将最佳抗体表达盒整合到简氏乳杆菌1153的pox1基因中。产生的菌株将被评估抗体表达水平、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大肠杆菌中表达完整且功能活跃的单域抗体来实现，其浓度足以在体外阻断hiv感染。在阶段