Pritchard Laura K, Harvey David J, Bonomelli Camille, Crispin Max, Doores Katie J
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, United Kingdom.
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, United Kingdom
J Virol. 2015 Sep;89(17):8932-44. doi: 10.1128/JVI.01190-15. Epub 2015 Jun 17.
The gp120/gp41 HIV-1 envelope glycoprotein (Env) is highly glycosylated, with up to 50% of its mass consisting of N-linked glycans. This dense carbohydrate coat has emerged as a promising vaccine target, with its glycans acting as epitopes for a number of potent and broadly neutralizing antibodies (bnAbs). Characterizing the glycan structures present on native HIV-1 Env is thus a critical goal for the design of Env immunogens. In this study, we used a complementary, multistep approach involving ion mobility mass spectrometry and high-performance liquid chromatography to comprehensively characterize the glycan structures present on HIV-1 gp120 produced in peripheral blood mononuclear cells (PBMCs). The capacity of different expression systems, including pseudoviral particles and recombinant cell surface trimers, to reproduce native-like glycosylation was then assessed. A population of oligomannose glycans on gp120 was reproduced across all expression systems, supporting this as an intrinsic property of Env that can be targeted for vaccine design. In contrast, Env produced in HEK 293T cells failed to accurately reproduce the highly processed complex-type glycan structures observed on PBMC-derived gp120, and in particular the precise linkage of sialic acid residues that cap these glycans. Finally, we show that unlike for gp120, the glycans decorating gp41 are mostly complex-type sugars, consistent with the glycan specificity of bnAbs that target this region. These findings provide insights into the glycosylation of native and recombinant HIV-1 Env and can be used to inform strategies for immunogen design and preparation.
Development of an HIV vaccine is desperately needed to control new infections, and elicitation of HIV bnAbs will likely be an important component of an effective vaccine. Increasingly, HIV bnAbs are being identified that bind to the N-linked glycans coating the HIV envelope glycoproteins gp120 and gp41, highlighting them as important targets for vaccine design. It is therefore important to characterize the glycan structures present on native, virion-associated gp120 and gp41 for development of vaccines that accurately mimic native-Env glycosylation. In this study, we used a number of analytical techniques to precisely study the structures of both the oligomannose and complex-type glycans present on native Env to provide a reference for determining the ability of potential HIV immunogens to accurately replicate the glycosylation pattern on these native structures.
HIV-1包膜糖蛋白（Env）的gp120/gp41高度糖基化，其质量的多达50%由N-连接聚糖组成。这种密集的碳水化合物外壳已成为一个有前景的疫苗靶点，其聚糖作为多种强效和广泛中和抗体（bnAbs）的表位。因此，表征天然HIV-1 Env上存在的聚糖结构是Env免疫原设计的关键目标。在本研究中，我们采用了一种互补的多步骤方法，涉及离子淌度质谱和高效液相色谱，以全面表征外周血单核细胞（PBMC）中产生的HIV-1 gp120上存在的聚糖结构。然后评估了包括假病毒颗粒和重组细胞表面三聚体在内的不同表达系统再现天然样糖基化的能力。gp120上的一群低聚甘露糖聚糖在所有表达系统中都得到了再现，支持这是Env的一种固有特性，可作为疫苗设计的靶点。相比之下，在HEK 293T细胞中产生的Env未能准确再现PBMC来源的gp120上观察到的高度加工的复合型聚糖结构，特别是覆盖这些聚糖的唾液酸残基的精确连接。最后，我们表明，与gp120不同，修饰gp41的聚糖大多是复合型糖，这与靶向该区域的bnAbs的聚糖特异性一致。这些发现为天然和重组HIV-1 Env的糖基化提供了见解，并可用于指导免疫原设计和制备策略。
迫切需要开发一种HIV疫苗来控制新感染，诱导HIV bnAbs可能是有效疫苗的一个重要组成部分。越来越多与HIV包膜糖蛋白gp120和gp41上的N-连接聚糖结合的HIV bnAbs被鉴定出来，突出了它们作为疫苗设计的重要靶点。因此，表征天然的、与病毒体相关的gp120和gp41上存在的聚糖结构对于开发准确模拟天然Env糖基化的疫苗很重要。在本研究中，我们使用了多种分析技术来精确研究天然Env上存在的低聚甘露糖和复合型聚糖的结构，为确定潜在HIV免疫原准确复制这些天然结构上的糖基化模式的能力提供参考。
