Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM

Abstract

Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic.

Antibody responses induced by SHIV infection are more focused than those induced by soluble native HIV-1 envelope trimers in non-human primates

“…Extensive serological analyses and the isolation of monoclonal antibodies (mAbs) from BG505 SOSIP-immunized rabbits, guinea pigs, and NHPs demonstrated the immunodominance of this glycan hole after vaccination [ 18 , 20 , 21 , 24 ], explaining the narrow breadth of these antibody responses. Furthermore, the lack of the N465 PNGS resulted in another immunodominant area on the BG505 Env trimer [ 19 , 24 – 27 ]. This PNGS is located near the 10-residue stretch in the C3 region that was under selective pressure in the BG505 HIV-1-infected infant [ 11 ], hence referred to as the C3/V5 epitope.…”

“…This C3/V5 epitope appeared to be more immunogenic in NHPs compared to rabbits [ 19 , 20 , 22 ]. Other previously identified targets for autologous NAbs on BG505 SOSIP include the gp41/gp120 interface, a region surrounding the glycan at residue 611, and an epitope near the apex of the trimer, involving the V1 region [ 19 , 21 – 24 , 27 ].…”

Structural Biology Illuminates Molecular Determinants of Broad Ebolavirus Neutralization by Human Antibodies for Pan-Ebolavirus Therapeutic Development

“…EM-based polyclonal epitope mapping (EMPEM) is a valuable new tool for studying the full antibody repertoire in survivor serum at various stages of infection, or as a tool for evaluating vaccines (127)(128)(129). Mapping polyclonal serum antibody specificities by EMPEM paints a more complete picture of the human immune response to filoviruses, and high-resolution cryo-EM studies could complement these efforts to reveal the more specific and subtle epitopes that are being accessed during natural infection or vaccination but have been missed by mAb studies (130,131). Such studies will be helpful in tuning vaccine design and for selecting antibodies that result from protective humoral responses.…”

High-resolution structural analysis of enterovirus-reactive polyclonal antibodies in complex with whole virions

“…Structural homology to published mouse antibody structures (primarily 3i9g (Wojciak et al, 2009)) was used to assign antibody heavy (H) and light (L) chains and individual CDR loops. The principal factors for discrimination are described previously (Antanasijevic et al, 2021). MolProbity (Williams et al, 2018) and EMRinger (Barad et al, 2015) metrics, were used to assess model quality.…”