Conjugate nanoparticle platform development for HIV-1 envelope immunogens

摘要-总体 HIV-1广泛中和抗体（bnAbs）在HIV-1感染的动物模型中具有保护作用，但在HIV-1感染的动物模型中， 目前的疫苗方案在人类中引起的。为了引出bnAb，B细胞谱系疫苗设计方法旨在 以特定顺序施用多种免疫原以引导bnAb通过免疫学途径成熟， 这些障碍通常会阻止bnAb的发展。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 活化诱导的胞苷脱氨酶。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，疫苗免疫原将需要有更高的亲和力， 对于具有这些所需氨基酸变化的Ab，比不具有氨基酸变化的Ab， 他们该策略面临的问题是HIV-1 bnAb的唯一抗原是HIV-1包膜（Env）， 免疫原性差，且bnAb前体通常对其具有低亲和力。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Env和通过 将这些Env在纳米颗粒（NP）上多聚化以提供亲合力和改善的抗原运输至表皮细胞 中心.然而，Env三聚体NP可以具有低表达，并呈现错误折叠的Env三聚体，其引起细胞凋亡。 不需要的非中和Ab。该申请意义重大，因为它将建立符合cGMP的 一个疫苗平台，可快速生成更高质量的HIV-1 Env三聚体NP疫苗， 迭代免疫原设计。该平台使用分选酶A酶位点特异性地共价连接良好- 将折叠的HIV-1 Env三聚体转化为完整的幽门螺杆菌铁蛋白NP。得到的HIV-1 Env三聚体分选酶A- 缀合的纳米颗粒（scNPs）仅显示良好折叠的Env三聚体，并且在初步研究中，已经成功地 在人bnAb前体敲入小鼠中启动CD 4结合位点bnAb谱系和在恒河猴中启动CD 4 bs nAb 猕猴scNP平台在自然界中是通用的，因为它可以掺入不同的病毒I型融合蛋白 只需在其C末端添加6个氨基酸的分选酶A标签即可。在具体目标1中，我们将比较 单价和二价HIV-I Env scNP以引导人源化细胞中CD 4结合位点bnAb的亲和力成熟 小鼠和恒河猴。在特异性目标2中，我们将产生并组装两个CD 4结合位点-bnAb- 在cGMP下靶向HIV-1 Env三聚体scNP（CH 505 TF scNP和第二个连续的Env三聚体scNP） 条件该计划将提供一个优化的cGMP过程，用于制造scNP，两个cGMP生产的Env 三聚体scNP，以及用于制造未来免疫原的额外铁蛋白和分选酶A组分。的 CH 505 TF Env三聚体scNP将通过HIV疫苗试验网络用于I期试验。最后， 该平台的影响是，它将使多个Env三聚体scNP能够在cGMP下快速制备， 在靶向bnAb的完全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.