Damm Dominik, Suleiman Ehsan, Theobald Hannah, Wagner Jannik T, Batzoni Mirjam, Ahlfeld Née Kohlhauser Bianca, Walkenfort Bernd, Albrecht Jens-Christian, Ingale Jidnyasa, Yang Lifei, Hasenberg Mike, Wyatt Richard T, Vorauer-Uhl Karola, Überla Klaus, Temchura Vladimir
Institute of Clinical and Molecular Virology, Friedrich-Alexander University Erlangen-Nürnberg, 91054 Erlangen, Germany.
Department of Biotechnology, University of Natural Resources and Life Sciences, 1190 Vienna, Austria.
Pharmaceutics. 2022 Jun 30;14(7):1385. doi: 10.3390/pharmaceutics14071385.
Functionalization of experimental HIV-1 virus-like particle vaccines with heterologous T helper epitopes (T helper VLPs) can modulate the humoral immune response via intrastructural help (ISH). Current advances in the conjugation of native-like HIV-1 envelope trimers (Env) onto liposomes and encapsulation of peptide epitopes into these nanoparticles renders this GMP-scalable liposomal platform a feasible alternative to VLP-based vaccines. In this study, we designed and analyzed customizable Env-conjugated T helper liposomes. First, we passively encapsulated T helper peptides into a well-characterized liposome formulation displaying a dense array of Env trimers on the surface. We confirmed the closed pre-fusion state of the coupled Env trimers by immunogold staining with conformation-specific antibodies. These peptide-loaded Env-liposome conjugates efficiently activated Env-specific B cells, which further induced proliferation of CD4+ T cells by presentation of liposome-derived peptides on MHC-II molecules. The peptide encapsulation process was then quantitatively improved by an electrostatically driven approach using an overall anionic lipid formulation. We demonstrated that peptides delivered by liposomes were presented by DCs in secondary lymphoid organs after intramuscular immunization of mice. UFO (uncleaved prefusion optimized) Env trimers were covalently coupled to peptide-loaded anionic liposomes by His-tag/NTA(Ni) interactions and EDC/Sulfo-NHS crosslinking. EM imaging revealed a moderately dense array of well-folded Env trimers on the liposomal surface. The conformation was verified by liposomal surface FACS. Furthermore, anionic Env-coupled T helper liposomes effectively induced Env-specific B cell activation and proliferation in a comparable range to T helper VLPs. Taken together, we demonstrated that T helper VLPs can be substituted with customizable and GMP-scalable liposomal nanoparticles as a perspective for future preclinical and clinical HIV vaccine applications. The functional nanoparticle characterization assays shown in this study can be applied to other systems of synthetic nanoparticles delivering antigens derived from various pathogens.
用异源T辅助表位（T辅助病毒样颗粒）对实验性HIV-1病毒样颗粒疫苗进行功能化修饰可通过结构内辅助（ISH）调节体液免疫反应。目前，将天然样HIV-1包膜三聚体（Env）偶联到脂质体上以及将肽表位封装到这些纳米颗粒中的进展，使这个符合药品生产质量管理规范（GMP）的可扩展脂质体平台成为基于病毒样颗粒疫苗的可行替代方案。在本研究中，我们设计并分析了可定制的Env偶联T辅助脂质体。首先，我们将T辅助肽被动封装到一种特性明确的脂质体制剂中，该脂质体表面展示有密集排列的Env三聚体。我们用构象特异性抗体进行免疫金染色，证实了偶联的Env三聚体处于封闭的预融合状态。这些负载肽的Env-脂质体偶联物有效激活了Env特异性B细胞，这些B细胞通过在MHC-II分子上呈递脂质体衍生的肽进一步诱导CD4+ T细胞增殖。然后，通过使用整体阴离子脂质制剂以静电驱动的方法对肽封装过程进行了定量改进。我们证明，在小鼠肌肉内免疫后，脂质体递送的肽由次级淋巴器官中的树突状细胞（DC）呈递。未切割的预融合优化（UFO）Env三聚体通过His标签/NTA（Ni）相互作用和EDC/磺基-NHS交联共价偶联到负载肽的阴离子脂质体上。电子显微镜成像显示脂质体表面有排列适度密集的折叠良好的Env三聚体。通过脂质体表面荧光激活细胞分选术（FACS）验证了其构象。此外，阴离子Env偶联T辅助脂质体在与T辅助病毒样颗粒相当的范围内有效诱导Env特异性B细胞活化和增殖。综上所述，我们证明T辅助病毒样颗粒可以被可定制且符合GMP的可扩展脂质体纳米颗粒替代，作为未来临床前和临床HIV疫苗应用的一个前景。本研究中所示的功能性纳米颗粒表征分析可应用于递送源自各种病原体的抗原的其他合成纳米颗粒系统。
