Delivery of passive immunity against HIV using commensal vaginal Lactobacillus bioengineered to secrete broadly neutralizing domain antibodies

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易受攻击目标。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。奥塞尔公司已经利用这些天然保护剂的特性，通过工程改造天然的人类阴道乳杆菌，L。jensenii 1153，以表达针对HIV的有效和广泛中和的单结构域抗体，称为MucoCept-Ab。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L. jensenii，其使猕猴中的SHIV病毒传播减少63%（Lagenaur等人，Mucobacterium Immunol，（2011年7月6日））。II期研究将通过临床前开发推进MucoCept-Ab。这些研究将侧重于开发安全、有效和稳定的MucoCept-Ab产品，并将包括菌株优化、处方、工艺开发、生产、临床前安全性试验和非人灵长类动物（NHP）病毒攻毒模型中的有效性试验。第二阶段的目标是开发一种能够局部递送抗体的乳杆菌菌株，以预防阴道或直肠粘膜的HIV感染。这种菌株将被配制成一种具有成本效益的产品，能够持久保护，不依赖性交，并由女性控制。目标1：MucoCept-Ab API优化和表征：A）完整的MucoCept-Ab菌株优化：最大化启动子强度，优化L. jensenii，去除表位标签，并测试两个基因组整合位点; B）MucoCept-Ab菌株的完整微生物学表征;遗传稳定性，表达盒的序列染色体插入位点，生物化学谱，抗生素敏感性/抗性谱; AP-m36.4表达水平; C）建立MucoCept-Ab菌株阴道定殖雌性恒河猴的能力。目标二：MucoCept-Ab方法开发：A）开发抗体和ELISA以检测和测量AP-m36.4水平; B）开发qPCR测定以测量MucoCept-Ab菌株的水平。目标3：MucoCept-Ab制剂、工艺开发和生产：A）开发用于生产稳定MucoCept-Ab产品的制剂和冻干工艺; B）将MucoCept-Ab菌株和技术转移至GMP生产商，用于生产MC B和WCB，扩大工艺开发，并生产MucoCept-Ab用于临床前安全性研究。目标4：MucoCept-Ab临床前安全性和有效性：A）评价小鼠阴道给药后MucoCept-Ab菌株和纯化的AP-m36.4蛋白的急性毒性和全身暴露; B）评价配制的MucoCept-Ab在兔阴道刺激模型中的急性毒性; C）在雌性恒河猴中阴道施用MucoCept-Ab产品后评价亚慢性毒性、阴道定殖和AP-m36.4水平; D）使用重复阴道SHIV激发方案评估MucoCept-Ab产品在雌性恒河猴中的功效。 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。

DESCRIPTION: 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 microbiota, including Lactobacillus jensenii, help protect these surfaces and serve to block the colonization and infection by intruding pathogens. Epidemiological studies suggest that the loss of vaginal lactobacilli is associated with an increased risk of heterosexual HIV-1 transmission and other sexually transmitted infections. Osel, Inc. has used 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 directed against HIV, termed MucoCept-Ab. Osel recently showed proof-of-concept for this recombinant Lactobacillus approach, by expressing an HIV-1 entry inhibitor mCV-N, from L. jensenii, which reduced SHIV virus transmission in macaques by 63% (Lagenaur et al., Mucosal Immunol, (Jul 6, 2011)). Phase II studies will advance MucoCept-Ab through preclinical development. These studies will focus on developing a safe, efficacious, and stable MucoCept-Ab product, and will include strain optimization, formulation, process development, manufacturing, preclinical safety testing, and efficacy testing in a nonhuman primate (NHP) viral challenge model. The goal of this Phase II is to develop a Lactobacillus strain capable of delivering an antibody locally to prevent HIV infection at the vaginal or rectal mucosa. This strain will be formulated into a cost-effective product capable of persistent protection that is coitally-independent and controlled by women. Aim 1: MucoCept-Ab API optimization and characterization: A) Complete MucoCept-Ab strain optimization: maximize promoter strength, optimize codons for L. jensenii, remove epitope tag, and test two genomic integration sites; B) Complete microbiological characterization of the MucoCept-Ab strain; genetic stability, sequence chromosomal insertion site of expression cassette, biochemical profiling, antibiotic susceptibility/resistant profiling; AP-m36.4 expressio levels; C) Establish ability of the MucoCept-Ab strain to vaginally colonize female rhesus macaques. Aim 2: MucoCept-Ab method development: A) Develop antibodies and ELISA to detect and measure AP-m36.4 levels; B) Develop qPCR assay to measure levels of the MucoCept-Ab strain. Aim 3: MucoCept-Ab formulation, process development, and manufacturing: A) Develop formulation and lyophilization process for production of a stable MucoCept-Ab product; B) Transfer MucoCept-Ab strain and technology to GMP manufacturer for production of MCB and WCB, scale up process development, and production of MucoCept-Ab for preclinical safety studies. Aim 4: MucoCept-Ab preclinical safety and efficacy: A) Evaluate acute toxicity and systemic exposure of the MucoCept-Ab strain and purified AP-m36.4 protein following vaginal administration in mice; B) Evaluate acute toxicity of formulated MucoCept-Ab in the rabbit vaginal irritation model; C) Evaluate subchronic toxicity, vaginal colonization, and AP-m36.4 levels following vaginal administration of the MucoCept-Ab product in female rhesus macaques; D) Evaluate efficacy of the MucoCept-Ab product in female rhesus macaques using a repeated vaginal SHIV challenge protocol. 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.