Conjugate nanoparticle platform development for HIV-1 envelope immunogens

摘要--总体 HIV-1广谱中和抗体(BNAbs)在HIV-1感染的动物模型中具有保护作用，但不具有保护作用 由目前的疫苗方案在人类身上引发的。为了诱导bNAbs，B细胞系疫苗设计方法旨在 以特定顺序注射多种免疫原，通过免疫学方法引导bNab成熟 通常会阻碍bNab开发的障碍。我们最近发现的一个障碍是体细胞突变 编码抗体功能的关键氨基酸，但很少由体细胞突变酶产生 激活诱导型胞苷脱氨酶。我们的核心疫苗设计假设是抗体(Abs) 编码这些不太可能的突变将是罕见的；因此，疫苗免疫原将需要具有更高的亲和力 对于有这些所需氨基酸变化的抗体，要比没有氨基酸变化的抗体进行选择 他们。这一策略面临的问题是，HIV-1 bNAbs的唯一抗原是HIV-1包膜(Env)，它 免疫原性差，bNab前体对其亲和力一般较低。我们和其他人发现 这两个障碍可以通过设计对bNab前体具有高亲和力的环境蛋白和通过 在纳米颗粒(NPs)上对这些环境蛋白进行多聚体以提供亲和力并改善生发细胞的抗原转运 中锋。然而，环境三聚体NP可能具有低表达，并呈现错误折叠的环境三聚体，从而诱导 不受欢迎的非中和抗体。此应用程序具有重要意义，因为它将建立符合cGMP的 快速生产高质量HIV-1环境三聚体NP疫苗的疫苗平台，无需耗时 迭代免疫原设计。这个平台使用分解酶A酶来很好地连接位点-具体地说，共价连接- 将HIV-1Env三聚体折叠到完整的幽门螺杆菌铁蛋白NPs上。由此产生的HIV-1环境三聚体分解酶A- 共轭NPs(ScNPs)仅显示折叠良好的环境三聚体，并在初步研究中成功 人bNab前体敲入小鼠和恒河猴中CD4bNAb的起始结合位点bNab谱系 猕猴。SCNP平台是通用的，因为它可以整合不同的I型病毒融合蛋白 通过简单地在其C末端添加6-氨基酸分解酶A标签。在具体目标1中，我们将比较 单价和双价HIV-1 env scNPs在人源化中引导CD4结合部位bNAbs亲和力成熟的研究 老鼠和恒河猴。在特定的目标2中，我们将产生并组装两个CD4结合位点-bNab- 在cGMP下靶向HIV-1环境三聚体scNP(Ch505 Tf SCNP和第二序列环境三聚体SCNP) 条件。该计划将提供一个优化的cGMP过程来制造scNPs，两个cGMP产生的环境 三聚体scNPs，以及额外的铁蛋白和分类酶A成分，用于制造未来的免疫原。这个 Ch505 Tf Env三聚体scNPs将通过HIV疫苗试验网络用于I期试验。最终， 这个平台的影响是，它将使cGMP下快速制造多个环境三聚体scNPs，使 在针对bNAbs的完整连续纳米颗粒疫苗的临床试验中进行迭代测试是可行的。

ABSTRACT – OVERALL HIV-1 broadly neutralizing antibodies (bnAbs) are protective in animal models of HIV-1 infection, but are not elicited in humans by current vaccine regimens. To elicit bnAbs, the B cell lineage vaccine design approach aims to administer multiple immunogens in a specific sequence to shepherd bnAb maturation through immunologic roadblocks that typically halt bnAb development. One roadblock we recently identified are somatic mutations that encode key amino acids for antibody function but that are rarely made by the somatic mutation enzyme activation-induced cytidine deaminase. Our central vaccine design hypothesis is that antibodies (Abs) encoding these improbable mutations, will be rare; thus, vaccine immunogens will need to have higher affinity for Abs with these desired amino acid changes than Abs without the amino acid changes in order to select for them. The problem facing this strategy is that the only antigen for HIV-1 bnAbs is HIV-1 envelope (Env), which is poorly immunogenic and for which bnAb precursors generally have low affinity. We and others have found these two obstacles can be overcome by designing Envs with high affinity for bnAb precursors and by multimerizing these Envs on nanoparticles (NPs) to provide avidity and improved antigen trafficking to germinal centers. However, Env trimer NPs can have low expression and present misfolded Env trimers that elicit undesired non-neutralizing Abs. This application is significant because it will establish a cGMP-compliant vaccine platform that rapidly generates higher quality HIV-1 Env trimer NP vaccines without time-consuming iterative immunogen design. This platform uses the sortase A enzyme to site-specifically, covalently-link well- folded HIV-1 Env trimers to intact Helicobacter pylori ferritin NPs. The resultant HIV-1 Env trimer sortase A- conjugated NPs (scNPs) display only well-folded Env trimers, and in preliminary studies, have successfully initiated CD4 binding site bnAb lineages in human bnAb precursor knock-in mice and CD4bs nAbs in rhesus macaques. The scNP platform is universal in nature since it can incorporate diverse viral type I fusion proteins by simply adding a 6-amino acid sortase A tag to their C-terminus. In Specific Aim 1, we will compare the ability of monovalent and bivalent HIV-1 Env scNPs to guide affinity maturation of CD4 binding site bnAbs in humanized mice and rhesus macaques. In Specific Aim 2, we will produce and assemble two CD4 binding site-bnAb- targeting HIV-1 Env trimer scNPs (CH505 TF scNP and a second sequential Env trimer scNP) under cGMP conditions. This program will deliver an optimized cGMP process for making scNPs, two cGMP-produced Env trimer scNPs, and additional ferritin and sortase A components for the manufacture of future immunogens. The CH505 TF Env trimer scNPs will be used in a Phase I trial through the HIV Vaccine Trial Network. Ultimately, the impact of this platform is that it will enable multiple Env trimer scNPs to be made rapidly under cGMP, making it feasible to do iterative testing in clinical trials of complete sequential nanoparticle vaccines that target bnAbs.