Delivery of passive immunity against HIV using commensal vaginal Lactobacillus bi

说明(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。从生物学上讲，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，包括詹森乳杆菌，有助于保护这些表面，并帮助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公司和Profectus BioSciences公司正在合作开发这些天然保护剂的特性，方法是设计一种天然的人阴道乳杆菌L.jensenii 1153，以表达针对HIV的有效和广泛中和的单域抗体/纳米抗体。Osel最近通过表达来自简氏乳杆菌的HIV-1进入抑制剂，将SIV病毒在猕猴中的传播减少了63%，从而展示了这种重组乳杆菌方法的概念验证(Lagenaur等人，Mucosal Invol，(2011年7月6日))。这种方法的最初目标是开发一种能够在局部传递抗体的乳杆菌菌株，以防止艾滋病毒在阴道或直肠粘膜上的感染。这种菌株可以配制成一种成本效益高的产品，能够提供持续的保护，而且是性交独立的，由妇女控制。我们打算分两个阶段对这种方法进行评估，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中表达分泌型中和单域抗体。将编码单域抗体的基因亚克隆到原核表达载体pOSEL175中，转化简氏乳杆菌1153。由此产生的细菌菌株将被检查抗体的表达。分泌的蛋白质将用免疫沉淀、凝胶电泳法和gp120-EL ISA进行鉴定。将选择与gp120结合的全长抗体进行进一步鉴定。2.鉴定乳杆菌表达的单域抗体的HI中和活性。培养上清液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的检测中测试初级HIV-1分离株的中和性。在简氏乳杆菌1153中，将选择表现出最佳的HIV中和组合的单域抗体(基于Productus Virus Panel中的分离物的中和，根据在10？g/ml浓度下中和的分离物的数量进行评分)，以优化表达。3.简氏乳杆菌1153表达单域抗体的优化主要单域抗体候选者的表达盒将被优化以在简氏乳杆菌1153中高效表达，并通过Western印迹分析比较其表达水平。将选出HIV中和能力和表达水平最佳组合的候选菌株进行进一步的毒株开发。4.构建整合载体，将单域表达框整合到简氏乳杆菌1153染色体上。我们将构建整合载体，并通过同源重组将优化的抗体表达盒整合到jensenii 1153的pox1基因中。将对产生的菌株进行抗体表达水平、抗艾滋病毒生物活性、与亲本菌株比较的表型特征和遗传稳定性的评估。第一阶段的技术可行性将通过在简氏乳杆菌中以足够高的浓度在体外表达完整的和具有功能活性的单域抗体来实现，以阻断艾滋病毒的传染性。同相 第二，我们将完成染色体整合株的开发，并测试其经阴道定植恒河猴的能力，并在体内产生足够水平的单域抗体，以保护其免受病毒攻击。然后，将在用抗体分泌菌株定居的动物中进行概念验证SHIV挑战研究，以证明对病毒感染的保护。还将就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而廉价的手段，向阴道粘膜提供对艾滋病毒的被动免疫，并满足对女性控制方法的迫切需要，以防止性传播病毒感染。 公共卫生相关性：我们的目标是通过利用阴道微生物群来开发一种新的女性控制的艾滋病毒预防药物。我们已经开发出生物工程人类阴道乳杆菌的技术，以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。该项目有可能以一种独立于性交的方式减少育龄妇女感染艾滋病毒的机会。

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.