A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers

Abstract

SUMMARY The heavily glycosylated native-like envelope (Env) trimer of HIV-1 is expected to have low immunogenicity, whereas misfolded forms are often highly immunogenic. High-quality correctly folded Envs may therefore be critical for developing a vaccine that induces broadly neutralizing antibodies. Moreover, the high variability of Env may require immunizations with multiple Envs. Here, we report a universal strategy that provides for correctly folded Env trimers of high quality and yield through a repair-and-stabilize approach. In the repair stage, we utilized a consensus strategy that substituted rare strain-specific residues with more prevalent ones. The stabilization stage involved structure-based design and experimental assessment confirmed by crystallographic feedback. Regions important for the refolding of Env were targeted for stabilization. Notably, the α9-helix and an intersubunit β sheet proved to be critical for trimer stability. Our approach provides a means to produce prefusion-closed Env trimers from diverse HIV-1 strains, a substantial advance for vaccine development.

HIV-1 Envelope and MPER antibody structures in lipid assemblies

“…A multitude of broadly neutralizing antibodies (bNAbs) have now been characterized, targeting various sites on HIV-1 Env (Haynes and Mascola, 2017;Sok and Burton, 2018). Structures of these bNAbs in complex with Env have paved the way for structure-based vaccine design by facilitating the identification of stabilizing mutations that allow large scale expression and purification of the unliganded ectodomain (Dey et al, 2018;Guenaga et al, 2017;Joyce et al, 2017;Klein et al, 2013;Kulp et al, 2017;Kwon et al, 2015;Ringe et al, 2017;Rutten et al, 2018;Schoofs et al, 2019;Sliepen et al, 2019;Torrents de la Pena and Sanders, 2018;Ward and Wilson, 2017;Yuan et al, 2019). The epitopes of MPER-targeting bNAbs were however truncated along with TMD and CTD from almost all of these Envs to increase solubility and stability and, therefore, remains perhaps the least understood epitope.…”

Disruption of the HIV-1 Envelope allosteric network blocks CD4-induced rearrangements

“…Centroids for the vectors in the analysis included a K46-K490 Cα centroid, W571 and W596 c-αs, c-αs of gp120 excluding variable loops the V1/V1 region residues, and the N- and C-termni, and a V1/V2+V3 c-α centroid Vectors between these reference positions were generated and included a projection of the W596 to K46-K490 centroid vector on to the W596 to W571 vector. Angles, distances, and dihedrals between these vectors were then compiled for a set of available crystal and cryo-EM structures with PDB IDs 4TVP 71 , 4ZMJ 25 , 5ACO 72 , 5CEZ 37 , 5CJX 73 , 5D9Q 74 , 5FYJ 75 , 5FYK 75 , 5FYL 75 , 5T3X 76 , 5T3Z 76 , 5U7M 33 , 5U7O 33 , 5UTF 13 , 5V7J 77 , 5V8L 78 , 5V8M 78 , 6CDE 38 , 6CDI 38 , 6CH7 79 , 6CH8 79 , 6CUE 80 , 6CUF 81 , and 6DE7 12 , 5THR 26 , 5VN3 27 , 5VN8 73 , 6CM3 31 , 6EDU 31 , 5FUU 82 , 6DCQ 46 , 6MAR 83 , 5U1F 55 , 5C7K 84 , 5I8H 85 , 5UM8 86 , 5UTY 13 , 5VIY 87 , 5VJ6 87 , 5WDU 14 , 6B0N 88 , 6CCB 89 , 6CE0 11 , 6CH9 79 , 6CHB 79 , 6CK9 90 , 6DCQ 46 , 6DID 91 , 6E5P 92 , 6IEQ 93 , 6MCO 94 , 6MDT 94 , 6MN7 95 , 6MPG 96 , 6MPH 96 , 6MTJ 97 , 6MTN 97 , 6MU6 97 , 6MU7 97 , 6MU8 97 , 6MUF 97 , 6MUG 97 , 6N1V 96 , 6N1W 96 , 6NC2 98 , 6NC3 98 , 6NF2 96 , 6NIJ 47 , 6NM6 99 , 6NNF 99 , 6NNJ 99 , 6OHY 100 , 6OKP 101 , 6OLP 47 , 6ORN 39 , 6ORO 39 , 6OR...…”

Stabilizing HIV-1 envelope glycoprotein trimers to induce neutralizing antibodies

“…1 the trimer core (A204I, T320L, E381M, Q422L) or the trimer stem (D589V, K655I, K658V, E662A) using structure-based design and mammalian cell display, which resulted in increased Env packing and reduced flexibility ( Fig. 1, Table 1) [49][50][51]54].…”

“…In structure-based HIV-1 immunogen design, several groups used the same principle and introduced aromatic residues to reduce V3 exposure (A316W, A319Y), and to increase stability of the trimer apex (Y177W, N302Y, N302F), the trimer base (E647F, N651F) and the trimer interface (gp120-gp41 interface: A223W, T538F and I548F; gp41-gp41 interface: I573F) ( Fig. 1, Table 1) [10,[48][49][50][51]54]. Overall, the introduction of hydrophobic and aromatic residues accounts for ~ 45% of the total number of mutations that are described in the literature to increase Env trimer stability.…”

“…Similarly, the introduction of glycine or proline residues in the HR1 and HR2 (N554G, L556P, A558P, I559G, T569P, T569G and S636G) further stabilized soluble HIV-1 Env trimers ( Fig. 1, Table 1) [1,54,58]. Kong et al computationally modeled HR1 loops with low Gibbs free energy that resulted in increased numbers of proline residues and rigidification of the HR1 loop [64].…”