Engineering and Characterization of a Fluorescent Native-Like HIV-1 Envelope Glycoprotein Trimer

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.

N-Glycolylneuraminic Acid as a Receptor for Influenza A Viruses

“…HA encoding cDNAs (Genscript, USA), were cloned into the pCD5 expression as described previously (de Vries et al, 2010). The pCD5 expression vector was adapted so that the HA-encoding cDNAs are cloned in frame with DNA sequences coding for a signal sequence, a GCN4 trimerization motif (RMKQIEDKIEEIESKQKKIENEIARIKK), a super folder GFP (Sliepen et al, 2015), and the Strep-tag II (WSHPQFEKGGGSGGGSWSHPQFEK); IBA, Germany).…”

Restriction of HIV-1 Escape by a Highly Broad and Potent Neutralizing Antibody

“…To identify antibodies that accounted for the potent neutralizing activity of IDC561, we performed single-cell sorting of Env-reactive B cells that bound to native-like BG505 SOSIP.664 (Sanders et al, 2013;Sliepen et al, 2015) (0.08% of IgG + B cells) or to YU2 gp140 (Scheid et al, 2009;Yang et al, 2000) (0.72% of IgG + B cells) ( Figure 1B). Using a new amplification strategy with primer sets optimized for precise detection of highly mutated IgG gene segments (OPT5/oPR; Kreer et al, 2019), we obtained and analyzed 812 IgG heavy-chain sequences (BG505 SOSIP.664 , n = 445; YU2 gp140 , n = 367) ( Figure 1B).…”

Drivers of recombinant soluble influenza A virus hemagglutinin and neuraminidase expression in mammalian cells

“…In the MUNANA assay we determined enzymatic activity by measuring methylumberriferyl, which is released from sialic acid upon digestion, as previously described. 21,26 The sfGFP-TB-N2 and GCN4-N2 had the highest enzymatic activity (Figure 3a), and although TB-N2 also displayed sialic acid cleavage, it was significantly less active. We also tested enzymatic activity of additional sfGFP-TB NAs, two additional N2s (NL91 and NL19), an N1 from the 2009 pandemic (CA04) and the 2013 H7N9 NA (Figure 3b).…”