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物脆弱性。从生物学角度来说，宫颈和阴道粘膜是艾滋病毒传播的脆弱目标。在健康的育龄妇女中，粘膜抗体和微生物群（包括詹氏乳杆菌）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，阴道乳酸杆菌的丧失与异性传播 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，通过改造天然人阴道乳杆菌 L. jensenii 1153，以表达针对 HIV 的有效且广泛中和的单域抗体/纳米抗体，从而利用这些天然保护剂的特性。 Osel 最近展示了这种重组乳杆菌方法的概念验证，通过表达来自詹氏乳杆菌的 HIV-1 进入抑制剂，可将猕猴中的 SHIV 病毒传播减少 63%（Lagenaur 等人，Mucosal 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的艾滋病毒感染的乳杆菌菌株。该菌株可以配制为具有成本效益的产品，能够持续保护性交独立且由女性控制。我们打算分两个阶段评估这种方法，并在第一阶段提出以下目标： 第一阶段建议 1. 在人阴道乳杆菌分离株 L. jensenii 1153 中表达分泌的中和单域抗体。编码单域抗体的基因将亚克隆到表达载体 pOSEL175 中并转化到 L. jensenii 1153 中。所得细菌 将检查菌株的抗体表达。将使用免疫沉淀、凝胶电泳和 gp120 ELISA 来表征分泌的蛋白质。将选择与 gp120 结合的全长抗体进行进一步表征。 2. 表征乳杆菌表达的单域抗体的 HI 中和活性。培养基中分泌的抗体将使用亲和层析进行纯化，并在基于细胞系和基于 PBMC 的测定中测试初级 HIV-1 分离株的中和作用。将选择表现出最佳 HIV 中和组合的单域抗体（基于 Profectus 病毒组中分离株的中和，通过在 10 µg/ml 浓度下中和 > 90% 的分离株数量进行评分），用于优化 L. jensenii 1153 中的表达。 3. 优化 L. jensenii 1153 中基于质粒的单域抗体表达。 主要单域抗体候选物的表达盒将针对在 L. jensenii 1153 中的高水平表达进行优化，并通过蛋白质印迹分析比较表达水平。将选择具有最佳 HIV 中和效力和表达水平组合的候选菌株进行进一步的菌株开发。 4. 构建整合载体并将单域表达盒整合到 L. jensenii 1153 染色体中。我们将构建整合载体，并通过同源重组将最佳的抗体表达盒整合到L. jensenii 1153的pox1基因中。将评估产生的菌株的抗体表达水平、针对 HIV 的生物活性、与亲本菌株相比的表型特征以及遗传稳定性。 第一阶段的技术可行性将通过在詹氏乳杆菌中表达完整且具有功能活性的单域抗体来实现，其浓度足以在体外阻断 HIV 感染。同相 II，我们将完成染色体整合菌株的开发，并测试其在恒河猴阴道定植并在体内产生足够水平的单域抗体以防止病毒攻击的能力。然后，将在感染了抗体分泌菌株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作用。还将对配方和制造工艺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，为阴道粘膜提供针对艾滋病毒的被动免疫，并满足对女性控制方法预防性传播病毒感染的迫切需求。 公共卫生相关性：我们的目标是通过利用阴道微生物群开发一种新型女性控制的艾滋病毒预防剂。我们开发了对人类阴道乳杆菌进行生物工程改造的技术，使其能够原位表达有效的单域抗体，从而创造出一种活的杀微生物剂。该项目有可能以独立于性交的方式减少育龄妇女感染艾滋病毒的可能性。