Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM

Abstract

In BriefHerein, we evaluated the immunogenicity of several BG505 SOSIP-based HIV Env immunogens in the rhesus macaque animal model using a combination of serology and biophysical approaches. We applied electron cryo-microscopy for high-resolution mapping of elicited polyclonal antibody responses, which provided detailed insights into the binding modes of the most common classes of antibodies elicited by BG505 SOSIP immunogens as well as the critical differences in immunogenicity that can occur as a consequence of engineered stabilizing mutations and partial glycan occupancy at different sites.SummaryEngineered 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.Graphical abstract

Structural mapping of antibody landscapes to human betacoronavirus spike proteins

“…Here, we elucidate the -HCoV spike epitopes targeted by preexisting serum antibodies and compare them to those elicited following SARS-CoV-2 infection using electron microscopy-based polyclonal epitope mapping (EMPEM) methodology (24,25).…”

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

“…To further assess the diversity of polyclonal antibodies and precisely map epitope contacts, we subjected CV-A21 immune complexes to high-resolution cryoEM-based polyclonal epitope mapping (cryoEMPEM; (Antanasijevic et al, 2021)) (Figure 2A, Figure 2 - figure supplement 1 and 2). Briefly, the vitrified immune complexes were imaged using cryoEM and particles were aligned with icosahedral symmetry restraints imposed to obtain the initial reconstruction of the entire immune complex.…”

“…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.…”

“…However, these studies have been impeded by the relatively low throughput (one sample can be analyzed at a time) and the need for mAb isolation. EM-based polyclonal epitope mapping (EMPEM) is a tool for rapid and comprehensive structural analysis of immune complexes featuring vaccine-elicited polyclonal antibodies (pAbs) purified from sera (Bianchi et al, 2018)(Antanasijevic et al, 2021; Bangaru et al, 2021; Nogal et al, 2020). Additionally, we have recently shown that structural information from the reconstructed high-resolution maps can be used to identify sequences of polyclonal antibody families (Antanasijevic et al, 2022).…”

Structural mapping of antibody landscapes to human betacoronavirus spike proteins

“…It is not clear if infection triggers a recall of preexisting HCoV-specific antibodies or preferentially elicits cross-reactive β-CoV antibodies targeting conserved epitopes. Here, we elucidate the β-HCoV spike epitopes targeted by pre-existing serum antibodies and compare them to those elicited following SARS-2 infection using EMPEM methodology (24,25) Soluble ectodomains of spike proteins for β-CoVs, HKU1, OC43, SARS, MERS and SARS-2 (4 stabilized constructs were used for SARS-2) were generated and characterized by negative stain electron microscopy (ns-EM) and shown to be homogeneous in their prefusion conformation (Fig. S1A).…”