Changes in Structure and Antigenicity of HIV-1 Env Trimers Resulting from Removal of a Conserved CD4 Binding Site-Proximal Glycan

Abstract

The envelope glycoprotein (Env) is the major target for HIV-1 broadly neutralizing antibodies (bNAbs). One of the mechanisms that HIV has evolved to escape the host's immune response is to mask conserved epitopes on Env with dense glycosylation. Previous studies have shown that the removal of a particular conserved glycan at N197 increases the neutralization sensitivity of the virus to antibodies targeting the CD4 binding site (CD4bs), making it a site of significant interest from the perspective of vaccine design. At present, the structural consequences that result from the removal of the N197 glycan have not been characterized. Using native-like SOSIP trimers, we examine the effects on antigenicity and local structural dynamics resulting from the removal of this glycan. A large increase in the binding of CD4bs and V3-targeting antibodies is observed for the N197Q mutant in trimeric Env, while no changes are observed with monomeric gp120. While the overall structure and thermostability are not altered, a subtle increase in the flexibility of the variable loops at the trimeric interface of adjacent protomers is evident in the N197Q mutant by hydrogen-deuterium exchange mass spectrometry. Structural modeling of the glycan chains suggests that the spatial occupancy of the N197 glycan leads to steric clashes with CD4bs antibodies in the Env trimer but not monomeric gp120. Our results indicate that the removal of the N197 glycan enhances the exposure of relevant bNAb epitopes on Env with a minimal impact on the overall trimeric structure. These findings present a simple modification for enhancing trimeric Env immunogens in vaccines. IMPORTANCEThe HIV-1 Env glycoprotein presents a dense patchwork of host cell-derived N-linked glycans. This so-called glycan shield is considered to be a major protective mechanism against immune recognition. While the positions of many N-linked glycans are isolate specific, some are highly conserved and are believed to play key functional roles. In this study, we examine the conserved, CD4 binding site-proximal N197 glycan and demonstrate that its removal both facilitates neutralizing antibody access to the CD4 binding site and modestly impacts the structural dynamics at the trimer crown without drastically altering global Env trimer stability. This indicates that surgical glycosylation site modification may be an effective way of sculpting epitope presentation in Env-based vaccines.T he trimeric HIV-1 envelope glycoprotein (Env), a trimer composed of gp120/gp41 heterodimers, is the primary antigenic feature on the virus and the sole target for neutralizing antibodies (1). Despite the extensive genetic diversity that exists among circulating HIV-1 variants, broadly neutralizing antibodies (bNAbs) capable of neutralizing a diverse panel of viral isolates have been identified in rare HIV-infected individuals. Elicitation of such bNAbs is thought to be a critical requirement for an effective HIV-1 vaccine (2, 3), but to date, HIV vaccine efforts have resulted in limited, narrow neu...

An antigenic atlas of HIV-1 escape from broadly neutralizing antibodies

“…For instance, the strongest escape mutations for 3BNC117 alone are N197S and N197T, which shift the N197 glycan to N195 ( Figure 4B and Figure 6A). However, eliminating the N197 glycan increases the virus's susceptibility to neutralization by antibodies targeting the same epitope as 10-1074 (Liang et al, 2016;Townsley et al, 2016). Mutations at site N197 are not selected by the pool of antibodies in our mutational antigenic profiling, presumably because any benefit with respect to escaping 3BNC117 is canceled out by increased susceptibility to 10-1074.…”

Comprehensive Cross-Clade Characterization of Antibody-Mediated Recognition, Complement-Mediated Lysis, and Cell-Mediated Cytotoxicity of HIV-1 Envelope-Specific Antibodies toward Eradication of the HIV-1 Reservoir

“…Thus, it is plausible that Env on the surface of infected primary CD4 T cells occludes the epitopes for certain antibodies with specificities for the CD4 binding site. Indeed, the removal of a glycan residue on trimeric envelope proximal to the CD4 binding site enhanced VRC01 binding relative to the wild-type trimer (46). Another potential mechanism at play might be that the viral envelope on the surface of infected CD4 T cells is constitutively engaged with the CD4 receptor, excluding the CD4 binding site antibodies from their epitope.…”

Evolution of B cell analysis and Env trimer redesign

“…In a general sense, selective N-glycan deletion of Env immunogens has been attempted previously, 110 but this approach may fare better in the modern era of well-ordered trimers, as was recently reported at residue N197. 111 Targeted N-glycan deletion, in combination with better tools to assess initial B cell activation and subsequent affinity maturation processes, will aid in analyzing the initial B cell response.…”

“…In principle, subsequent boosting with ordered trimers possessing the restored N-glycans may then allow B cell to mature and account for the full glycan pattern, permitting shield penetration by a subset of novel CD4bs-directed B cells. In a general sense, selective N-glycan deletion of Env immunogens has been attempted previously (112), but this approach may fare better in the modern era of well-ordered trimers, as was recently reported at residue N197 (113). Targeted N-glycan deletion, in combination with better tools to assess initial B cell activation and subsequent affinity maturation processes, will aid in analyzing the initial B cell response.…”