Asymmetric recognition of the HIV-1 trimer by broadly neutralizing antibody PG9

Abstract

PG9 is the founder member of an expanding family of glycandependent human antibodies that preferentially bind the HIV (HIV-1) envelope (Env) glycoprotein (gp) trimer and broadly neutralize the virus. Here, we show that a soluble SOSIP.664 gp140 trimer constructed from the Clade A BG505 sequence binds PG9 with high affinity (∼11 nM), enabling structural and biophysical characterizations of the PG9:Env trimer complex. The BG505 SOSIP.664 gp140 trimer is remarkably stable as assessed by electron microscopy (EM) and differential scanning calorimetry. EM, small angle X-ray scattering, size exclusion chromatography with inline multiangle light scattering and isothermal titration calorimetry all indicate that only a single PG9 fragment antigen-binding (Fab) binds to the Env trimer. An ∼18 Å EM reconstruction demonstrates that PG9 recognizes the trimer asymmetrically at its apex via contact with two of the three gp120 protomers, possibly contributing to its reported preference for a quaternary epitope. Molecular modeling and isothermal titration calorimetry binding experiments with an engineered PG9 mutant suggest that, in addition to the N156 and N160 glycan interactions observed in crystal structures of PG9 with a scaffolded V1/V2 domain, PG9 makes secondary interactions with an N160 glycan from an adjacent gp120 protomer in the antibody-trimer complex. Together, these structural and biophysical findings should facilitate the design of HIV-1 immunogens that possess all elements of the quaternary PG9 epitope required to induce broadly neutralizing antibodies against this region. R ational immunogen design is an increasingly promising approach for development of an effective human immunodeficiency virus-1 (HIV-1) vaccine. The recent discovery of many new and potent broadly neutralizing antibodies (bnAbs) has helped define conserved sites of vulnerability on the HIV-1 envelope (Env) glycoprotein (gp) complex that mediates viral entry into cells (refs. 1-6 and reviewed in refs. 7-11). Passive immunization studies show that sterilizing immunity can be achieved if sufficient amounts of bnAbs are present before virus challenge in macaques (12-16). Hence, intensive efforts are ongoing to design immunogens capable of re-eliciting these types of bnAbs by vaccination.The major difficulty in mounting an effective antibody response against HIV-1 resides in the multiple evasion strategies that have evolved in Env. An error-prone reverse transcriptase drives a high degree of Env sequence diversity (17-19). The few conserved regions of Env are shielded by an extensive array of glycans (20-24) and are often occluded by more variable structures, such as the V1-V5 loops. However, because some HIV-1-infected individuals can develop bnAbs over the course of infection, these various evasion strategies are not insurmountable (1,(25)(26)(27). Although bnAbs do not seem to confer significant protection against disease progression in infected individuals (28, 29), their induction through vaccination might prevent the acquisition of infection. ...

Recognition of synthetic glycopeptides by HIV-1 broadly neutralizing antibodies and their unmutated ancestors

“…The sensitivity of BnAb binding to Cys mutation suggests that the N 160 and N 156 glycans are perhaps spatially positioned more favorably in a dimer, thereby allowing for higheravidity binding or recognition of glycans on two V1V2 units. Asymmetric binding to adjacent V1V2 elements has been proposed in a recent model (27) to explain the preferential binding of PG9 to Env trimers (28). We also found that the introduction of the Cysto-Ala mutation resulted in the loss of the secondary structure of the Man 3 glycopeptide.…”

Identification and specificity of broadly neutralizing antibodies against HIV

“…The pioneering examples of this class are PG9 and PG16,23 which we showed bind to a novel trimer‐preferring, glycan‐dependent bnAb epitope 63, 64, 65. The glycan site at residue 160 is typically critical for these bnAbs and a decrease in neutralization is seen when additional glycan sites are removed from the V1, V2, and V3 loops in a viral isolate‐dependent manner 66.…”

Tyrosine sulfation in the second variable loop (V2) of HIV-1 gp120 stabilizes V2–V3 interaction and modulates neutralization sensitivity

“…Despite their definition as variable loops, the V2 and V3 regions of gp120 contain highly conserved domains (33). Because several lines of evidence suggest that V3 establishes direct interaction with V2 (12)(13)(14)(15)(16)(17)(18)(19)(20)(21), and the conserved base of V3 is the binding site for the N-terminal region of the CCR5 coreceptor (34), we looked for potential structural homology between V2 and CCR5. We recognized that the conserved central region of V2 contains two tyrosine residues (Tyr173 and Tyr177) with spacing identical to that of two tyrosines (Tyr10 and Tyr14) present in the N-terminal region of CCR5 (Fig.…”

“…Cryo-EM studies have provided evidence that in the prefusion conformation V2 and V3 are spatially contiguous and account for most of the density at the apex of the trimeric envelope spike (12)(13)(14)(15)(16)(17). Although various fragments of V2 and V3 were crystallized separately using antibody-complexed synthetic peptides (28,29), scaffolded chimeric constructs of the first and second variable loops (V1V2) (30,31), or a V3-containing gp120 core monomer (8), the only study in which the two loops were visualized simultaneously is the recent report of the BG505 SOSIP.664 trimer crystal structure (18).…”

“…As a consequence, most of the available high-definition structures of gp120 have been obtained with deglycosylated, variable looptruncated core monomers in complex with stabilizing ligands such as soluble CD4 (sCD4) (6)(7)(8)(9)(10). Important information regarding the overall conformation and ligand interactions of the trimeric spike at intermediate resolution has emerged from the use of increasingly refined cryo-electron microscopy (cryo-EM) technologies (11)(12)(13)(14)(15)(16)(17). Moreover, the crystal structure of a stabilized, soluble, cleaved gp140 trimer (BG505 SOSIP.664) at 4.7-Å resolution was reported recently (18).…”