To summarize, crystallographic and EM analyses validated the structural integrity of UFO-BG trimers derived from five subtypes, supporting the notion that UFO-BG is a general and effective strategy for trimer stabilization. == Fig. on HIV-1 virions as a metastable trimer of heterodimers each made up of a (co-)receptor-binding protein, gp120, and a transmembrane protein, gp41, which anchors the Env spike within the viral membrane and drives the fusion process during cell entry (3). Because of its labile nature and a dense layer of surface glycans (4), Env has long resisted structure determination and trimer-based vaccine design efforts. While the functional necessity of a metastable Env for HIV-1 contamination is well comprehended, the molecular source of metastability and how to eliminate it from the Env trimer remain unclear. It is perhaps not an overstatement that overcoming Env metastability is usually central to trimer-based HIV-1 vaccine design. Several strategies have been proposed to create stable and soluble gp140 trimers as potential vaccine immunogens. The early generation of gp140 trimers was based on intuitive designs to overcome metastability via deletion of the cleavage site between gp120 and the gp41 ectodomain (gp41ECTO) and the addition of trimerization motifs at the C terminus (57). A more rigorous and successful trimer design, designated SOSIP.664, stressed the importance of the gp120/gp41ECTOcleavage, trimer stability, and solubility (8). This format uses a disulfide bond to covalently link gp120 and gp41ECTO, an I559P mutation in the heptad repeat 1 (HR1) region, and truncation of gp41ECTOat position 664. When applied to clade A BG505, the SOSIP Diclofensine hydrochloride trimer was shown to be a close mimic of the native Env spike (9) that enabled Env structure determination for the first time by x-ray crystallography and electron microscopy (EM) (10,11). The BG505 SOSIP trimer has substantially advanced HIV-1 research by providing an antigen for bNAb isolation and structural characterization (1223), a strategy for stabilizing diverse Envs (2427), and a template for trimer optimization either to shield nonneutralizing antibody (non-NAb) epitopes or to target bNAb precursors (2831). As the SOSIP design continued to evolve, new versions (designated SOSIP.v5 and SOSIP.v6) that further improved trimer stability and immunogenicity were proposed (32). Removal of the cleavage site has proven successful in the forms of single-chain gp140 (sc-gp140) (33), native flexibly linked (NFL) (34,35), and uncleaved prefusion-optimized (UFO) (36) trimers. However, it was not until recently that the primary cause of Env metastabilityan HR1 bend (residues 547 to 569) in gp41ECTOwas identified and targeted directly by rational redesign (36). Notably, the I559P mutation that improved trimer stability in the SOSIP design is within this HR1 bend (9). Glycine substitution in this region was also found to improve trimer properties in the NFL form (37). While attempts to elicit tier 2 NAb response with BG505 Mouse monoclonal to IKBKE SOSIP trimers in wild-type (WT) mice were unsuccessful (38), both SOSIP and NFL trimers consistently induced autologous tier 2 NAbs in rabbits and nonhuman primates (NHPs), although only sporadic neutralization was observed for heterologous tier 2 isolates (26,3943). Therefore, despite recent advances in rational design and structural analysis of native-like trimers (44,45), crucial barriers still remain in the path to an effective HIV-1 vaccine. In this study, we set out Diclofensine hydrochloride to address the diverse challenges in HIV-1 vaccine development with a coherent strategy Diclofensine hydrochloride centered on Env metastability. We first examined the power of a transient Chinese hamster ovary (CHO) cell line (ExpiCHO) to express native-like trimers, which resulted in superior trimer yield, purity, and antigenicity while displaying subtle differences in glycosylation pattern and B cell response compared to human embryonic kidney (HEK) 293 Fproduced trimers. We then exhibited that gp41ECTOis the main source of metastability by replacing WT gp41ECTOwith BG505 gp41ECTOof the UFO design for 10 Envs across clades A, B, C, B/C, and A/E. The gp41ECTO-swapped trimers (termed UFO-BG) exhibited substantial yield and purity and were structurally validated by negative-stain EM. Analysis of UFO and Diclofensine hydrochloride UFO-BG trimers by biolayer interferometry (BLI) against a large panel of 19 antibodies provided comprehensive antigenic profiles for each of the 10 Envs tested. A crystal structure was decided for the H078.14 UFO-BG trimer (tier.