Generation and Characterization of a Bivalent HIV-1 Subtype C gp120 Protein Boost for Proof-of-Concept HIV Vaccine Efficacy Trials in Southern Africa

The viral envelope glycoprotein (Env) is the major target for antibody (Ab)-mediated vaccine development against the Human Immunodeficiency Virus type 1 (HIV-1). Although several recombinant Env antigens have been evaluated in clinical trials, only the surface glycoprotein, gp120, (from HIV-1 subtype B, MN, and subtype CRF_01AE, A244) used in the ALVAC prime-AIDSVAX gp120 boost RV144 Phase III HIV vaccine trial was shown to contribute to protective efficacy, although modest and short-lived. Hence, for clinical trials in southern Africa, a bivalent protein boost of HIV-1 subtype C gp120 antigens composed of two complementary gp120s, from the TV1.C (chronic) and 1086.C (transmitted founder) HIV-1 strains, was selected. Stable Chinese Hamster Cell (CHO) cell lines expressing these gp120s were generated, scalable purification methods were developed, and a detailed analytical analysis of the purified proteins was conducted that showed differences and complementarity in the antigenicity, glycan occupancy, and glycan content of the two gp120 molecules. Moreover, mass spectrometry revealed some disulfide heterogeneity in the expressed proteins, particularly in V1V2-C1 region and most prominently in the TV1 gp120 dimers. These dimers not only lacked binding to certain key CD4 binding site (CD4bs) and V1V2 epitope-directed ligands but also elicited reduced Ab responses directed to those epitopes, in contrast to monomeric gp120, following immunization of rabbits. Both monomeric and dimeric gp120s elicited similarly high titer Tier 1 neutralizing Abs as measured in standard virus neutralization assays. These results provide support for clinical evaluations of bivalent preparations of purified monomeric TV1.C and 1086.C gp120 proteins.

病毒包膜糖蛋白（Env）是针对1型人类免疫缺陷病毒（HIV-1）的抗体（Ab）介导疫苗研发的主要靶标。尽管已有多种重组Env抗原在临床试验中得到评估，但仅在ALVAC初免-AIDSVAX gp120加强的RV144 III期HIV疫苗临床试验中使用的、源自HIV-1 B亚型MN株以及CRF_01AE、A244亚型的表面糖蛋白gp120，被证实可提供一定的保护效力，不过该效力较为有限且持续时间较短。因此，针对南非地区的临床试验，研究人员选择了由两种互补gp120构成的HIV-1 C亚型gp120抗原二价蛋白加强剂，这两种gp120分别源自TV1.C（慢性感染株）和1086.C（传播创始株）HIV-1毒株。本研究构建了可稳定表达上述gp120的中国仓鼠细胞（CHO）株，开发了可规模化的纯化工艺，并对纯化后的蛋白开展详细分析，结果显示两种gp120分子在抗原性、糖基化位点占有率及糖基组成上既存在差异，又具有互补性。此外，质谱分析显示，表达的蛋白存在一定程度的二硫键异质性，该异质性主要集中于V1V2-C1区域，其中以TV1 gp120二聚体的表现最为显著。与单体gp120相比，此类二聚体不仅无法结合部分关键的CD4结合位点（CD4bs）及V1V2表位靶向配体，在对家兔进行免疫接种后，还会引发针对这些表位的抗体应答水平下降。在标准病毒中和试验中，单体与二聚体gp120均可诱导滴度相当的高活性Tier 1中和抗体。上述研究结果为纯化的TV1.C与1086.C单体gp120蛋白二价制剂的临床试验评估提供了依据。