Morales Javier F, Yu Bin, Perez Gerardo, Mesa Kathryn A, Alexander David L, Berman Phillip W
Department of Biomolecular Engineering, Baskin School of Engineering, University of California, 1156 High Street, MS-SOE2, Santa Cruz, CA 95064, United States.
Department of Biomolecular Engineering, Baskin School of Engineering, University of California, 1156 High Street, MS-SOE2, Santa Cruz, CA 95064, United States.
Mol Immunol. 2016 Sep;77:14-25. doi: 10.1016/j.molimm.2016.07.003. Epub 2016 Jul 21.
The V1/V2 domain of the HIV-1 envelope protein gp120 possesses two important epitopes: a glycan-dependent epitope recognized by the prototypic broadly neutralizing monoclonal antibody (bN-mAb), PG9, as well as an epitope recognized by non-neutralizing antibodies that has been associated with protection from HIV infection in the RV144 HIV vaccine trial. Because both of these epitopes are poorly immunogenic in the context of full length envelope proteins, immunization with properly folded and glycosylated fragments (scaffolds) represents a potential way to enhance the immune response to these specific epitopes. Previous studies showed that V1/V2 domain scaffolds could be produced from a few selected isolates, but not from many of the isolates that would be advantageous in a multivalent vaccine. In this paper, we used a protein engineering approach to improve the conformational stability and antibody binding activity of V1/V2 domain scaffolds from multiple diverse isolates, including several that were initially unable to bind the prototypic PG9 bN-mAb. Significantly, this effort required replicating both the correct glycan structure as well as the β-sheet structure required for PG9 binding. Although scaffolds incorporating the glycans required for PG9 binding (e.g., mannose-5) can be produced using glycosylation inhibitors (e.g., swainsonine), or mutant cell lines (e.g. GnTI(-) 293 HEK), these are not practical for biopharmaceutical production of proteins intended for clinical trials. In this report, we describe engineered glycopeptide scaffolds from three different clades of HIV-1 that bind PG9 with high affinity when expressed in a wildtype cell line suitable for biopharmaceutical production. The mutations that improved PG9 binding to scaffolds produced in normal cells included amino acid positions outside of the antibody contact region designed to stabilize the β-sheet and turn structures. The scaffolds produced address three major problems in HIV vaccine development: (1) improving antibody responses to poorly immunogenic epitopes in the V1/V2 domain; (2) eliminating antibody responses to highly immunogenic (decoy) epitopes outside the V1/V2 domain; and (3) enabling the production of V1/V2 scaffolds in a cell line suitable for biopharmaceutical production.
HIV-1包膜蛋白gp120的V1/V2结构域有两个重要表位：一个是原型广泛中和单克隆抗体（bN-mAb）PG9识别的糖基依赖性表位，另一个是在RV144 HIV疫苗试验中与预防HIV感染相关的非中和抗体识别的表位。由于这两个表位在全长包膜蛋白背景下免疫原性较差，用正确折叠和糖基化的片段（支架）进行免疫是增强对这些特定表位免疫反应的一种潜在方法。先前的研究表明，V1/V2结构域支架可以从少数选定的分离株中产生，但不能从许多在多价疫苗中具有优势的分离株中产生。在本文中，我们采用蛋白质工程方法来提高来自多种不同分离株的V1/V2结构域支架的构象稳定性和抗体结合活性，包括一些最初无法结合原型PG9 bN-mAb的分离株。值得注意的是，这一努力需要复制正确的糖基结构以及PG9结合所需的β-折叠结构。虽然可以使用糖基化抑制剂（如苦马豆素）或突变细胞系（如GnTI(-) 293 HEK）来生产包含PG9结合所需聚糖（如甘露糖-5）的支架，但这些方法对于用于临床试验的蛋白质的生物制药生产并不实用。在本报告中，我们描述了来自HIV-1三个不同分支的工程化糖肽支架，当在适合生物制药生产的野生型细胞系中表达时，它们能以高亲和力结合PG9。改善PG9与在正常细胞中产生的支架结合的突变包括设计用于稳定β-折叠和转角结构的抗体接触区域之外的氨基酸位置。所产生的支架解决了HIV疫苗开发中的三个主要问题：（1）改善对V1/V2结构域中免疫原性较差表位的抗体反应；（2）消除对V1/V2结构域之外高免疫原性（诱饵）表位的抗体反应；（3）能够在适合生物制药生产的细胞系中生产V1/V2支架。
