table_1_Cleavage-Independent HIV-1 Trimers From CHO Cell Lines Elicit Robust Autologous Tier 2 Neutralizing Antibodies.xlsx

Native flexibly linked (NFL) HIV-1 envelope glycoprotein (Env) trimers are cleavage-independent and display a native-like, well-folded conformation that preferentially displays broadly neutralizing determinants. The NFL platform simplifies large-scale production of Env by eliminating the need to co-transfect the precursor-cleaving protease, furin that is required by the cleavage-dependent SOSIP trimers. Here, we report the development of a CHO-M cell line that expressed BG505 NFL trimers at a high level of homogeneity and yields of ~1.8 g/l. BG505 NFL trimers purified by single-step lectin-affinity chromatography displayed a native-like closed structure, efficient recognition by trimer-preferring bNAbs, no recognition by non-neutralizing CD4 binding site-directed and V3-directed antibodies, long-term stability, and proper N-glycan processing. Following negative-selection, formulation in ISCOMATRIX adjuvant and inoculation into rabbits, the trimers rapidly elicited potent autologous tier 2 neutralizing antibodies. These antibodies targeted the N-glycan “hole” naturally present on the BG505 Env proximal to residues at positions 230, 241, and 289. The BG505 NFL trimers that did not expose V3 in vitro, elicited low-to-no tier 1 virus neutralization in vivo, indicating that they remained intact during the immunization process, not exposing V3. In addition, BG505 NFL and BG505 SOSIP trimers expressed from 293F cells, when formulated in Adjuplex adjuvant, elicited equivalent BG505 tier 2 autologous neutralizing titers. These titers were lower in potency when compared to the titers elicited by CHO-M cell derived trimers. In addition, increased neutralization of tier 1 viruses was detected. Taken together, these data indicate that both adjuvant and cell-type expression can affect the elicitation of tier 2 and tier 1 neutralizing responses in vivo.

天然柔性连接（Native flexibly linked, NFL）的HIV-1包膜糖蛋白（envelope glycoprotein, Env）三聚体不依赖裂解过程，且呈现类天然、折叠完好的构象，可优先展示广谱中和决定簇。NFL平台无需共转染裂解依赖型SOSIP三聚体所必需的前体裂解蛋白酶弗林蛋白酶（furin），从而简化了Env的大规模生产流程。本研究构建了一株CHO-M细胞系，可高效表达均一性优异的BG505 NFL三聚体，表达产量约为1.8 g/L。经单步凝集素亲和层析纯化的BG505 NFL三聚体，呈现类天然的闭合构象，可被偏好识别三聚体的广谱中和抗体（broadly neutralizing antibodies, bNAbs）有效识别，不被非中和性CD4结合位点靶向及V3靶向抗体识别，具备长期稳定性与正常的N-聚糖加工修饰能力。经阴性筛选、采用ISCOMATRIX佐剂配制制剂并免疫家兔后，该三聚体可快速诱导强效的同源2级中和抗体。此类抗体靶向BG505 Env上天然存在的、靠近230、241及289位氨基酸残基的N-聚糖“空洞”。体外未暴露V3区域的BG505 NFL三聚体，在体内仅能诱导低效甚至无法检测到的1级病毒中和活性，表明其在免疫过程中保持了完整构象，未暴露V3区域。此外，采用293F细胞表达的BG505 NFL与BG505 SOSIP三聚体，经Adjuplex佐剂制剂后，可诱导相当水平的BG505同源2级中和效价，但该效价的活性弱于CHO-M细胞来源三聚体所诱导的效价，且可检测到针对1级病毒的中和活性增强。综上，本研究数据表明，佐剂选择与细胞表达体系均可影响体内2级及1级中和抗体应答的诱导。