Optimizing AAV delivery of bNAbs for HIV prevention

项目总结 HIV-1广谱中和抗体(BNAbs)目前正在接受临床评估，以确定其预防 变速箱。在实现常规疫苗之前，重复注射bNAb将是必要的 保持保护性抗体浓度。被动输注bNAbs的另一种选择是使用腺病毒- 相关病毒(AAV)载体可以将肌肉转化为抗体生产工厂。这一次 与连续生产、提纯和给药相比，处理将具有明显的成本优势 重组单抗。然而，宿主免疫反应限制了AAV载体的效果。 AAV转导的肌肉细胞的CD8+T细胞清除限制了产生抗体的细胞总数， 预先存在的对AAV衣壳的免疫力限制了可能感染AAV的个人数量 向量。我们和其他人之前已经证明，宿主免疫反应对甲型肝炎病毒传播是有害的 HIV-1抗体导致血清浓度低或检测不到。因此，克服宿主的免疫力 对AAV载体和表达的转基因的反应是未来评估AAV递送抗体的关键 对非人类灵长类动物的研究。限制宿主免疫反应的一个研究领域是 利用免疫检查点来调节免疫系统的途径。为此，在一项初步研究中，我们有 在猕猴体内观察到HIV-1抗体浓度增加了约21倍 将编码恒河猴PD-L1基因的AAV载体与恒河猴共接种。中的PD-L1函数 结合T细胞表达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PD-L1在转导的肌肉细胞上的表达 通过AAV载体表达抗体可以避免T细胞的清除，维持抗体的表达。 在这里，我们试图证明，共接种编码PD-L1的载体将导致血清浓度 一种bNAb将保护恒河猴免受重复的、低剂量的SIV挑战。此外，我们还将 通过评估减少AAV载体剂量的策略来开发该系统。此外，我们还将设计 AAV转化盒和评估一种新的AAV衣壳，用于增加肌肉内表达 接种疫苗。总之，这些研究的结果将为AAV基因治疗研究提供基础。 并导致了用于表达HIV-1 bNAbs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.