Bale Shridhar, Gustchina Elena, Guenaga Javier, Ayala Victor, Lee Wen-Hsin, Ozorowski Gabriel, Whitney Stephen, Wilson Richard, Baboo Sabyasachi, Diedrich Jolene K, Doyle Esmeralda D, Hudacik Lauren, Ben-Akiva Elana, Rodrigues Kristen A, Irvine Darrell J, Yates John R, Paulson James C, Ward Andrew B, Fouts Timothy, Wyatt Richard T
Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA.
ABL Inc., Rockville, MD, USA.
NPJ Vaccines. 2025 Aug 20;10(1):198. doi: 10.1038/s41541-025-01218-6.
Evaluation of recombinant HIV-1 surface glycoproteins (Env) as vaccine candidates for Phase I human experimental trials often requires production of cGMP-grade well-ordered Env trimers. Here, we report an accelerated cGMP compatible approach for expression and purification of a stabilized HIV clade C-derived trimer '16055 DG4 NFL' (for native flexibly linked). This recombinant trimer was expressed from CHO-S™ cells using a MaxCyte® VLX™ electroporation-based transient transfection process. The 16055 DG4 NFL was designed with multiple internal stabilizing mutations and, as well, deletion of four N-linked glycans (DG4) proximal to the CD4 binding site (CD4bs) engineered to improve B cell recognition of this conserved neutralizing determinant. The transient process circumvents the need to develop stable cell lines expressing the Env trimers that is often the most time-consuming step impacting vaccine development timelines. The 16055 DG4 NFL trimer was purified by immunoaffinity chromatography using the broadly neutralizing antibody (bNAb), PGT145. Following additional downstream processing steps, purified trimer was vialed, frozen and stored at -80 °C. Upon thaw and analysis, the trimer displayed homogeneity and a near-native conformation as determined by size-exclusion chromatography (SEC), negative stain and cryo-electron microscopy (EM), differential scanning calorimetry (DSC) and biolayer interferometry (BLI). The immunogenicity of the trimer was tested in rabbits with bolus, escalating dose and divided dose immunization regimens. Rabbits from all three regimens elicited tier 2 autologous neutralizing antibodies that targeted the exposed protein region at the CD4bs. The trimer is currently under investigation in a human clinical trial (NCT06332339) for safety, tolerability and as a priming candidate followed by heterologous boosting to potentially elicit cross-neutralizing antibodies.
评估重组HIV-1表面糖蛋白（Env）作为I期人体试验候选疫苗时，通常需要生产符合cGMP标准的有序Env三聚体。在此，我们报告了一种加速的cGMP兼容方法，用于表达和纯化稳定的源自HIV C亚型的三聚体“16055 DG4 NFL”（天然柔性连接）。该重组三聚体通过基于MaxCyte® VLX™电穿孔的瞬时转染过程从CHO-S™细胞中表达。16055 DG4 NFL设计有多个内部稳定突变，并且还删除了靠近CD4结合位点（CD4bs）的四个N-连接聚糖（DG4），旨在改善B细胞对这个保守中和决定簇的识别。瞬时过程避免了开发表达Env三聚体的稳定细胞系的需要，而这通常是影响疫苗开发时间表的最耗时步骤。16055 DG4 NFL三聚体通过使用广泛中和抗体（bNAb）PGT145的免疫亲和色谱法进行纯化。经过额外的下游处理步骤后，纯化的三聚体装入小瓶，冷冻并储存在-80°C。解冻和分析后，通过尺寸排阻色谱（SEC）、负染和冷冻电子显微镜（EM）、差示扫描量热法（DSC）和生物层干涉术（BLI）确定，该三聚体显示出均一性和接近天然的构象。在兔中采用大剂量、递增剂量和分剂量免疫方案测试了该三聚体的免疫原性。来自所有三种方案的兔都产生了靶向CD4bs处暴露蛋白区域的2级自体中和抗体。该三聚体目前正在一项人体临床试验（NCT06332339）中进行安全性、耐受性研究，并作为引发候选物，随后进行异源加强免疫，以潜在地引发交叉中和抗体。
