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公司利用这些天然保护剂的特性，设计了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，称为MucoCept-Ab。Osel最近通过表达一种来自延森乳杆菌的HIV-1进入抑制剂mCV-N，证明了这种重组乳酸菌方法的概念验证，该方法将SHIV病毒在猕猴中的传播减少了63% （Lagenaur等人，muc粘膜免疫，（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一种安全、有效和稳定的MucoCept-Ab产品，包括菌株优化、配方、工艺开发、制造、临床前安全性测试和非人灵长类动物（NHP）病毒攻击模型的有效性测试。该II期的目标是开发一种能够在阴道或直肠粘膜局部递送抗体以预防HIV感染的乳酸杆菌菌株。这种菌株将被配制成一种具有成本效益的产品，能够持久地提供保护，这种产品不依赖于女性的生殖能力，并由女性控制。目的1:MucoCept-Ab API优化与表征：A) MucoCept-Ab菌株完整优化：最大化启动子强度，优化L. jensen的密码子，去除表位标签，测试两个基因组整合位点；B) MucoCept-Ab菌株的完整微生物学特征；遗传稳定性，序列染色体插入位点表达盒，生化分析，抗生素敏感性/耐药性分析；AP-m36.4表达水平；C)建立MucoCept-Ab菌株阴道定植雌性恒河猴的能力。目的2:MucoCept-Ab方法开发：A)开发抗体和ELISA检测和测量AP-m36.4水平；B)建立qPCR检测MucoCept-Ab菌株的水平。目标3:MucoCept-Ab制剂、工艺开发和生产：A)开发稳定MucoCept-Ab产品的制剂和冻干工艺；B)将MucoCept-Ab菌株和技术转让给GMP制造商，用于生产MCB和WCB，扩大工艺开发规模，并生产MucoCept-Ab用于临床前安全性研究。目的4:MucoCept-Ab临床前安全性和有效性：A)评估小鼠阴道给药后MucoCept-Ab菌株和纯化AP-m36.4蛋白的急性毒性和全身暴露；B)评价复方MucoCept-Ab在兔阴道刺激模型中的急性毒性；C)评估雌性恒河猴阴道给药MucoCept-Ab产品后的亚慢性毒性、阴道定植和AP-m36.4水平；D)评估MucoCept-Ab产品在雌性恒河猴中使用阴道SHIV重复攻击方案的功效。如果成功的话，这种方法可以提供一种安全而廉价的方法，向阴道粘膜提供抗艾滋病毒的被动免疫，并解决对女性控制方法的迫切需要，以防止性传播病毒感染。

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.