Optimizing AAV delivery of bNAbs for HIV prevention

项目摘要 HIV-1广泛中和抗体（bNAb）目前正在进行临床评估，以评估其预防HIV-1感染的能力。 传输在实现常规疫苗之前，需要重复给予bNAb， 维持保护性抗体浓度。被动输注bNAb的替代方案是使用腺病毒介导的细胞内注射。 相关病毒（AAV）载体，可以把肌肉变成抗体生产工厂。此一次性 治疗将具有明显的成本优势，超过连续生产，纯化和管理， 重组单克隆抗体。然而，宿主免疫应答限制了AAV载体的功效。 AAV转导的肌细胞的CD 8 + T细胞清除限制了产生抗体的细胞的总数， 并且预先存在的对AAV衣壳的免疫性限制了可以接受AAV的可能个体的数量 向量。我们和其他人先前已经表明，宿主免疫反应对AAV递送的免疫应答是有害的。 HIV-1抗体导致血清浓度低至不可检测。因此，克服宿主免疫 对AAV载体和表达的转基因的应答对于将来评估AAV递送的抗体是至关重要的 非人类灵长类动物的研究。限制宿主免疫应答的一个研究领域是 利用免疫检查点调节免疫系统途径。为此，在试点研究中，我们 在恒河猴中观察到HIV-1抗体浓度增加约21倍， 用编码恒河猴PD-L1的AAV载体共接种恒河猴。PD-L1功能 结合T细胞表达的PD-1，抑制细胞溶解和脱颗粒功能。它还有助于 调节性T细胞的发育。因此，我们假设肌细胞上PD-L1的表达被转导， 通过AAV载体表达抗体将避免T细胞清除并维持抗体的表达。 在此，我们试图证明共接种编码PD-L1的载体将导致血清浓度 bNAb将保护恒河猴免受重复的低剂量SHIV攻击。此外，我们将 通过评估减少AAV载体剂量的策略来开发该系统。此外，我们将设计 AAV转基因盒和评估用于增加肌内表达的新型AAV衣壳 接种。总之，这些研究的结果将为AAV基因治疗研究提供基础。 非人灵长类动物的研究，并导致用于表达HIV-1 bNAb的新型AAV载体的开发。

PROJECT SUMMARY HIV-1 broadly neutralizing antibodies (bNAbs) are currently under clinical evaluation for their ability to prevent transmission. Until a conventional vaccine is realized, repetitive dosing of a bNAb would be necessary to maintain protective antibody concentrations. An alternative to passive infusion of bNAbs is to use adeno- associated virus (AAV) vectors that can turn muscles into antibody production factories. This one-time treatment would have clear cost advantages over the continuous production, purification, and administration of recombinant monoclonal antibodies. However, host immune responses limit the efficacy of AAV vectors. CD8+ T cell clearance of AAV transduced muscle cells limits the total number of cells producing the antibody, and pre-existing immunity to AAV capsids limit the number of possible individuals that can receive AAV vectors. We and others have previously shown that host immune responses are detrimental to AAV-delivered HIV-1 antibodies resulting in low to no detectable serum concentrations. Thus, overcoming the host immune response to the AAV vector and expressed transgene is critical for future evaluation of AAV-delivered antibody studies in non-human primates. One area of investigation for limiting a host immune response would be to utilize immune checkpoints that regulate immune system pathways. To this end, in a pilot study, we have observed about a 21-fold increase in concentrations of an HIV-1 antibody in rhesus macaques when macaques were co-inoculated with an AAV vector encoding rhesus macaque PD-L1. PD-L1 functions in binding T cell expressed PD-1 to inhibit the cytolytic and degranulation functions. It also helps in the development of regulatory T cells. Thus, we hypothesize that expression of PD-L1 on muscle cells transduced by AAV vectors to express antibodies will avoid T cell clearance and maintain expression of the antibody. Here we seek to demonstrate that co-inoculation of vectors encoding PD-L1 will result in serum concentrations of a bNAb that will protect rhesus macaques from repetitive, low-dose SHIV challenges. Additionally, we will develop this system by evaluating strategies to reduce the dose of AAV vector. Furthermore, we will engineer AAV transgene cassettes and assess novel a novel AAV capsid for increasing expression from intramuscular inoculation. Together, the results from these studies will provide a foundation for AAV gene therapy studies in non-human primates as well as lead to the development of novel AAV vectors for expressing HIV-1 bNAbs.