Characterization of a stable HIV-1 B/C recombinant, soluble, and trimeric envelope glycoprotein (Env) highly resistant to CD4-induced conformational changes

Abstract

The HIV-1 envelope (Env) is a glycoprotein consisting of a trimer of heterodimers containing gp120 and gp41 subunits that mediates virus entry and is a major target of broadly neutralizing antibodies (bnAbs) developed during infection in some individuals. The engagement of the HIV-1 gp120 glycoprotein to the host CD4 protein triggers conformational changes in gp120 that allow its binding to co-receptors and is necessary for virus entry to establish infection. Native-like HIV-1 Env immunogens representing distinct clades have been proposed to improve immunogenicity. In the present study, we examined the basis of resistance of an HIV-1 B/C recombinant Env (LT5.J4b12C) to non-neutralizing antibodies targeting CD4-induced Env epitopes in the presence of soluble CD4 (sCD4). Using native polyacrylamide gel shift assay and negative-stain EM, we found that the prefusion conformational state of LT5.J4b12C trimeric Env was largely unaffected in the presence of excess sCD4 with most Env trimers appearing to be in a ligand-free state. This resistance to CD4-induced conformational changes was associated with a lower affinity for CD4. Moreover, the LT5.J4b12C trimeric Env preferentially bound to the neutralizing antibodies compared with non-neutralizing antibodies. Taken together, we report on an HIV-1 B/C recombinant, native-like trimeric Env protein that is highly resistant to CD4-induced conformational changes but displays epitopes recognized by a diverse array of bnAbs. Such features make this B/C recombinant trimeric Env a useful addition to the pool of other recently identified native-like HIV-1 Env trimers suitable for use as antigenic bait for bnAb isolation, structural studies, and use as potential immunogens.

Elicitation of potent serum neutralizing antibody responses in rabbits by immunization with an HIV-1 clade C trimeric Env derived from an Indian elite neutralizer

“…As shown in Figure 4A, the Tm for 1PGE-THIVC was observed to be approximately 62°C with an onset of melting at approximately 55°C. The high Tm obtained for the 1PGE-THIVC was comparable with the double cysteine mutant reported stable Env trimers like BG505.SOSIP.664 (13) and LT5.J4b12C SOSIP.664 (40). The soluble 1PGE-THIVC was also found to demonstrate stability at 37°C as measured by its ability to bind to different bnAbs by D7324-ELISA ( Figure 4B).…”

“…First, we assessed the antigenicity of 1PGE-THIVC Env by measuring binding to neutralizing and non-neutralizing mAbs by ELISA (avidity). To facilitate binding with mAbs in trimeric state, D7324 epitope was introduced in the C-terminus in 1PGE-THIVC as described before (40). As shown in Figure 2A, 1PGE-THIVC preferentially bound to neutralizing mAbs over non-neutralizing mAbs.…”

“…1PGE-THIVC gene was codon-optimized by GeneArt (Thermo Fisher Inc.) and cloned into pcDNA3.1(+) with following modifications to the wild-type Env sequence: A501C, T605C, I559C (for trimer stabilization), and gp120 -gp41 cleavage motif REKR changed to RRRRRR. The D7324 epitope sequence (GSAPTKAKRRVVQREKR) was added after residue 664 in gp41 ectodomain (ECTO) and preceding the stop codon to facilitate examining Env SOSIP binding by ELISA following published protocol (40,51). 1PGE-THIVC was expressed by transient transfection of 293F or Expi293 cells, and the trimeric protein fraction was purified from culture supernatants first by using PGT145 mAb affinity column (36).…”

“…Purified trimeric SOSIP proteins were analyzed in a gradient 4-15% BN-PAGE (Mini-PROTEAN TGXTM, Bio-Rad). The degree of 1PGE-THIVC cleavage was examined by incubating the SOSIP protein with 0.1 M dithiothreitol (DTT) and analyzed by SDS-PAGE under reducing conditions as described earlier (40).…”

Enhancing glycan occupancy of soluble HIV-1 envelope trimers to mimic the native viral spike

“…A now widely used immunogen design platform that is being explored in preclinical and clinical phase tests is the SOSIP trimer, a concept that has evolved from the initial prototype, BG505 SOSIP.664, to improved versions that better resemble viral Env [47][48][49][50][51][52] . SOSIP trimers can now be based on most viral sequences from various clades 14,18,35,37,38,[53][54][55][56][57] . However, the SOSIP trimers evaluated to date do not fully mimic viral Env in respect of glycan occupancy; the resulting artificial holes in the glycan shield are immunogenic, but they induce antibodies of generally narrow-specificity that are not on the pathway towards neutralization breadth.…”