Chemical Cross-Linking Stabilizes Native-Like HIV-1 Envelope Glycoprotein Trimer Antigens

Abstract

Major neutralizing antibody immune evasion strategies of the HIV-1 envelope glycoprotein (Env) trimer include conformational and structural instability. Stabilized soluble trimers such as BG505 SOSIP.664 mimic the structure of virion-associated Env but nevertheless sample different conformational states. Here we demonstrate that treating BG505 SOSIP.664 trimers with glutaraldehyde or a heterobifunctional cross-linker introduces additional stability with relatively modest effects on antigenicity. Thus, most broadly neutralizing antibody (bNAb) epitopes were preserved after cross-linking, whereas the binding of most weakly or nonneutralizing antibodies (non-NAb) was reduced. Cross-linking stabilized all Env conformers present within a mixed population, and individual conformers could be isolated by bNAb affinity chromatography. Both positive selection of cross-linked conformers using the quaternary epitope-specific bNAbs PGT145, PGT151, and 3BC315 and negative selection with non-NAbs against the V3 region enriched for trimer populations with improved antigenicity for bNAbs. Similar results were obtained using the clade B B41 SOSIP.664 trimer. The cross-linking method may, therefore, be useful for countering the natural conformational heterogeneity of some HIV-1 Env proteins and, by extrapolation, also vaccine immunogens from other pathogens.IMPORTANCE The development of a vaccine to induce protective antibodies against HIV-1 is of primary public health importance. Recent advances in immunogen design have provided soluble recombinant envelope glycoprotein trimers with near-native morphology and antigenicity. However, these trimers are conformationally flexible, potentially reducing B-cell recognition of neutralizing antibody epitopes. Here we show that chemical cross-linking increases trimer stability, reducing binding of nonneutralizing antibodies while largely maintaining neutralizing antibody binding. Cross-linking followed by positive or negative antibody affinity selection of individual stable conformational variants further improved the antigenic and morphological characteristics of the trimers. This approach may be generally applicable to HIV-1 Env and also to other conformationally flexible pathogen antigens.

Native‐like Env trimers as a platform for HIV‐1 vaccine design

“…The concept originates in an observation made over 20 years ago that chemical cross-linking of virion-associated Env proteins by formalin was compatible with the preservation of NAb epitopes (134). Since then, various groups have reported on the cross-linking of cell surface-associated Env, gp120 monomers and gp140 pseudotrimers (135–138), before the originators of the cross-linking concept then applied their method to native-like trimers (139). Thus, treating BG505 or B41 SOSIP.664 trimers with glutaraldehyde or a hetero-bifunctional cross-linker further stabilized them with only minimal adverse effects on antigenicity.…”

“…All the Env conformers present within a mixed population were, of course, cross-linked. However, the desired native-like trimers could be purified by positive selection on PGT145, PGT151 or 3BC315 affinity columns or via negative selection with a non-NAb against the V3 region (139). Of note is that the chemical cross-linking of BG505 and B41 SOSIP.664 trimers improves their abilities to induce autologous Tier-2 NAbs in rabbits (Q. Sattentau, personal communication) (immunogenicity studies, in general, are discussed below).…”

An insight into the thermodynamic characteristics of human thrombopoietin complexation with TN1 antibody

“…Antigenicity exhibited by hTPO was also reported in clinical trials using human erythropoietin and insulin . Although the mechanisms underlying the antigenicity of these proteins are unknown, biophysical properties associated with protein structure and thermodynamics are hypothesized to be prime contributors …”

“…[10][11][12][13] Although the mechanisms underlying the antigenicity of these proteins are unknown, biophysical properties associated with protein structure and thermodynamics are hypothesized to be prime contributors. [14][15][16] Previously, the crystal structure of hTPO in complex with an antigen-binding fragment (Fab) derived from the hTPO-neutralizing murine monoclonal antibody TN1 17 was described; however, 18,19 the crystal structure of free hTPO has yet to be reported. The difficulties associated with the crystallographic analysis of free hTPO may relate to its physicochemical properties; possibly, hTPO is highly-flexible and structurally stabilized by complexation with TN1.…”

Innovations in HIV-1 Vaccine Design

“…66 Various strategies have been used to design stabilized recombinant Env gp140 trimers that mimic the conformation of native Env trimers. 61,67,68 One approach involves stabilizing the gp120-gp41 interactions with an intermolecular disulfide bond (SOS gp140), modified with an isoleucine to proline (I559P) substitution to improve trimerization (SOSIP gp140). 69e71 Researchers designed native-like trimers derived from a subtype A transmitted/founder virus isolated from an HIV-infected infant, BG505.…”