Abstract
ABSTRACTMajor 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.
Highlights
Major neutralizing antibody immune evasion strategies of the HIV-1 envelope glycoprotein (Env) trimer include conformational and structural instability
We showed that GLA cross-linking of the nonnative uncleaved gp140CN54 trimer reduced its conformational flexibility, improved its antigenicity, and steered antibody responses induced in rabbits toward tier 1 Neutralizing antibodies (NAbs) epitopes associated with the CD4 binding site (CD4bs) [26]
We have reported that cross-linking soluble, uncleaved, nonnative gp140 proteins with glutaraldehyde (GLA), a bifunctional aldehyde that cross-links amine groups primarily associated with lysine side chains [57], prevented dissociation of gp140 subunits under the harsh denaturing conditions that apply during reducing SDS-PAGE but preserved the epitopes of many NAbs [26] (Fig. 1A)
Summary
Major neutralizing antibody immune evasion strategies of the HIV-1 envelope glycoprotein (Env) trimer include conformational and structural instability. Recent advances in immunogen design have provided soluble recombinant envelope glycoprotein trimers with near-native morphology and antigenicity These trimers are conformationally flexible, potentially reducing B-cell recognition of neutralizing antibody epitopes. 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 to other conformationally flexible pathogen antigens. Soluble recombinant Env trimers such as BG505 SOSIP.664, here termed BG505 trimers, have been designed that are antigenically very similar to the native, membrane-anchored Env spike; these trimers bind most broadly neutralizing antibodies (bNAbs) but few non-NAbs [12, 13]. Major goals in HIV-1 Env vaccine design are to prepare soluble trimers that expose only bNAb and not non-NAb or tier 1 V3 epitopes and that are conformationally and structurally stable
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