Abstract
Generation of a stable, soluble mimic of the HIV-1 envelope glycoprotein (Env) trimer on the virion surface has been considered an important first step for developing a successful HIV-1 vaccine. Recently, a soluble native-like Env trimer (BG505 SOSIP.664) has been described. This protein has facilitated major advances in the HIV-1 vaccine field, since it was the first Env immunogen that induced consistent neutralizing antibodies against a neutralization-resistant (tier 2) virus. Moreover, BG505 SOSIP.664 enabled elucidation of the atomic resolution structure of the Env trimer and facilitated the isolation and characterization of new broadly neutralizing antibodies against HIV-1. Here, we designed and characterized the BG505 SOSIP.664 trimer fused to fluorescent superfolder GFP (sfGFP), a GFP variant that allows efficient folding (BG505 SOSIP.664-sfGFP). Despite the presence of the sfGFP, the Env protein largely retained its morphology, antigenicity, glycan composition, and thermostability. In addition, we show that BG505 SOSIP.664-sfGFP can be used for fluorescence-based assays, such as flow cytometry.
Highlights
The acquired immune deficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV-1) is one of the leading deadly communicable diseases and an effective vaccine is elusive
The BG505 SOSIP.664 trimer design is compatible with the C-terminal fusion of small tags, such as a His-tag, small epitope tags and AviTag for direct biotinylation [6,19]
We first verified whether the three superfolder GFP (sfGFP) molecules would clash when attached to the C-terminus of the SOSIP trimer in silico (Figure 1A, top)
Summary
The acquired immune deficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV-1) is one of the leading deadly communicable diseases and an effective vaccine is elusive. The resulting BG505 SOSIP.664 trimer was highly thermostable and its antigenic profile correlated well to that of Env on the parental virus strain [6] It was the first HIV-1 Env trimer for which the high-resolution structure was solved, enabling structure-based vaccine design [10,11,12,13]. We report the engineering and extensive characterization of a fluorescent soluble native-like Env trimer composed of BG505 SOSIP.664 fused to superfolder green fluorescent protein (sfGFP). Both gels were subjected to Western blotting using monoclonal antibodies 2G12. SOSIP and SOSIP-sfGFP proteins were captured onto the solid phase by bNAb 2G12 and probed with biotinylated 2G12 or PGT145
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