Wyatt Lab, Department of Immunology and Microbiology, Scripps Research, La Jolla, CA, United States.
Weissman Lab, Penn Institute for RNA Innovation, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.
Front Immunol. 2024 Jul 25;15:1426232. doi: 10.3389/fimmu.2024.1426232. eCollection 2024.
The HIV-1 envelope glycoprotein (Env) is the sole neutralizing determinant on the surface of the virus. The Env gp120 and gp41 subunits mediate receptor binding and membrane fusion and are generated from the gp160 precursor by cellular furins. This cleavage event is required for viral entry. One approach to generate HIV-1 neutralizing antibodies following immunization is to express membrane-bound Env anchored on the cell-surface by genetic means using the natural HIV gp41 transmembrane (TM) spanning domain. To simplify the process of Env trimer membrane expression we sought to remove the need for Env precursor cleavage while maintaining native-like conformation following genetic expression. To accomplish these objectives, we selected our previously developed 'native flexibly linked' (NFL) stabilized soluble trimers that are both near-native in conformation and cleavage-independent. We genetically fused the NFL construct to the HIV TM domain by using a short linker or by restoring the native membrane external proximal region, absent in soluble trimers, to express the full HIV Env ectodomain on the plasma membrane. Both forms of cell-surface NFL trimers, without and with the MPER, displayed favorable antigenic profiles by flow cytometry when expressed from plasmid DNA or mRNA. These results were consistent with the presence of well-ordered cell surface native-like trimeric Env, a necessary requirement to generate neutralizing antibodies by vaccination. Inoculation of rabbits with mRNA lipid nanoparticles (LNP) expressing membrane-bound stabilized HIV Env NFL trimers generated tier 2 neutralizing antibody serum titers in immunized animals. Multiple inoculations of mRNA LNPs generated similar neutralizing antibody titers compared to immunizations of matched NFL soluble proteins in adjuvant. Given the recent success of mRNA vaccines to prevent severe COVID, these are important developments for genetic expression of native-like HIV Env trimers in animals and potentially in humans.
HIV-1 包膜糖蛋白（Env）是病毒表面唯一的中和决定簇。Env 的 gp120 和 gp41 亚基介导受体结合和膜融合，由细胞弗林蛋白酶从 gp160 前体中产生。这种切割事件是病毒进入所必需的。免疫接种后产生 HIV-1 中和抗体的一种方法是通过遗传手段使用天然 HIV gp41 跨膜（TM）跨越结构域将膜结合的 Env 表达在细胞表面上。为了简化 Env 三聚体膜表达的过程，我们试图在保持遗传表达后保持天然样构象的同时，去除 Env 前体切割的需要。为了实现这些目标，我们选择了我们之前开发的“天然灵活连接”（NFL）稳定的可溶性三聚体，这些三聚体在构象上非常接近天然，并且不依赖于切割。我们通过使用短接头或恢复可溶性三聚体中缺失的天然膜外近区，将 NFL 结构与 HIV TM 结构域基因融合，在质膜上表达完整的 HIV Env 外结构域。当从质粒 DNA 或 mRNA 表达时，两种形式的细胞膜 NFL 三聚体，无论是带有还是不带有 MPER，在流式细胞术上都显示出有利的抗原谱。这些结果与存在有序的细胞膜上类似天然的三聚体 Env 一致，这是通过接种疫苗产生中和抗体的必要条件。用表达膜结合稳定的 HIV Env NFL 三聚体的 mRNA 脂质纳米颗粒（LNP）接种兔子，在免疫动物中产生了 2 级中和抗体血清滴度。与在佐剂中免疫匹配的 NFL 可溶性蛋白相比，多次接种 mRNA LNPs 产生了类似的中和抗体滴度。鉴于 mRNA 疫苗最近在预防严重 COVID 方面的成功，这些进展对于在动物中，以及在人类中，对天然样 HIV Env 三聚体进行基因表达是重要的。
