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.

Fig. 1. Immunization experiments with the stabilized BG505 SOSIP trimers.

A Immunogen (left) and immunization schedule (right). B ELISA binding titers (midpoint) and C neutralizing antibody titers (ID₅₀) for plasma samples collected at weeks 10, 26, and 38 (time points indicated above each graph; open circles—Grp 1; closed circles—Grp 2). Horizontal lines represent the geometric mean values at each time point (n = 6 animals in each group). ELISA and pseudovirus neutralization experiments were performed in triplicates (n = 3) for each serum sample at every time point and the mean value is plotted. D Composite figures from nsEMPEM analysis of polyclonal responses at week 26. Animal IDs and neutralizing antibody titers (at week 26) for corresponding animals are shown above and below each composite figure, respectively. A color-coding scheme for antibodies targeting different epitope clusters is shown at the bottom. BG505 SOSIP antigen is represented in gray.

Fig. 2. CryoEMPEM analysis of polyclonal antibodies elicited by BG505 SOSIP trimer immunogens.

A Schematic illustration of the data processing workflow of cryoEMPEM. B CryoEMPEM analysis of immune complexes generated using polyclonal Fabs isolated from plasma samples from animals Rh.32034 (top left), Rh.33104 (top right), and Rh.33311 (bottom). High-resolution trimer-Fab complexes featuring structurally unique antibody specificities detected in cryoEM datasets are shown in the corresponding panels. BG505 SOSIP antigen is represented in gray and Fab densities are colored according to the legend shown at the bottom. In the center of each panel is a composite figure from nsEMPEM. The apparent resolution of each reconstructed cryoEM map is indicated.

Fig. 3. Structural analysis of neutralizing and potentially neutralizing antibody responses.

Structures of BG505 SOSIP antigens in complex with polyclonal antibodies targeting C3/V5 (A), N241/N289 glycan hole (B), fusion peptide (C), and N611 glycan epitopes (D). BG505 SOSIP trimers are shown in gray with N-linked glycans in golden yellow. Antibodies are colored using the same scheme as in Figs. 1 and 2. Inferred heavy and light chains for each antibody are represented in different shades and labeled in each panel (HC and LC, respectively). The most relevant epitope/paratope components are indicated in the supporting panels. Surface representation is used for the full immune complex (left side of each panel) while ribbon and stick representations are used for the display of epitope-paratope contacts (right side of each panel). In panel B, sphere representations are used for residues S241 and N289 to improve visibility.

Fig. 4. Structural analysis of non-neutralizing antibody responses.

Structures of BG505 SOSIP antigens in complex with polyclonal antibodies targeting gp120–gp120 interface (A, B) and base epitopes (C). BG505 SOSIP trimers are shown in gray with N-linked glycans in golden yellow. Antibodies are colored using the same scheme as in Figs. 1 and 2. Inferred heavy and light chains for each antibody are represented in different colors and labeled in each panel (HC and LC, respectively). The most relevant epitope/paratope components are indicated in the panels. Surface representation is used for the full immune complex (left side of each panel). Trimer residues in direct contact with the antibodies are colored blue (ribbon and stick representation). Engineered stabilizing residues are represented as sticks and colored orange. EM Map is displayed as transparent light gray surfaces in panels (A) and (B).

Fig. 5. Design and characterization of the nanoparticle immunogen.

A Models of the two antigen-bearing components (left) and BG505 SOSIP-T33-31 nanoparticle (right). B CryoEM analysis of the designed BG505 SOSIP-T33-31 nanoparticle. Representative raw micrograph and sample 2D class-averages of imaged particles are shown in the top left corner. The scale bar in the micrograph is 100 nm. Low-pass filtered map of the entire nanoparticle is shown in the top right corner (orange—T33-31A; blue—T33-31B; light gray—BG505 SOSIP (A); dark gray—BG505 SOSIP (B)). Final maps and relaxed models from subparticle analysis are shown in the middle/bottom panels. Fourier shell correlation (FSC) curves for each subparticle map are presented in the bottom left corner. The cryoEM experiments have been performed once.

Fig. 6. Immunization experiments with the octameric BG505 SOSIP-T33-31 nanoparticle.

A Immunization information and schedule. B ELISA binding titers (midpoint) and C neutralizing antibody titers (ID₅₀) for plasma samples collected at time points indicated above each graph. Horizontal lines represent the geometric mean values at each time point (n = 6 animals for week 10 and n = 5 animals for weeks 26 and 38; see Method section for details). ELISA and pseudovirus neutralization experiments were performed in triplicates (n = 3) for each serum sample at every time point and the mean value is plotted. C Composite figures from nsEMPEM analysis of polyclonal responses at weeks 8, 10, 26, and 38. Animal IDs are shown above each dataset. Neutralizing antibody titers for corresponding animals are shown below each composite figure. A color-coding scheme for antibodies targeting different epitope clusters is shown at the bottom. BG505 SOSIP antigen is represented in gray.

Fig. 7. CryoEMPEM analysis of polyclonal antibodies elicited by BG505 SOSIP-T33-31 nanoparticle immunogen.

A CryoEMPEM analysis of the immune complexes generated using polyclonal Fabs isolated from animal Rh.33172 (week 38 plasma samples). BG505 SOSIP antigen is represented in gray and Fab densities are colored according to the scheme used in Fig. 6. The apparent resolution of each reconstructed map is indicated. B Structures of four V1/V2/V3-targeting pAbs. Models (ribbon representation) and maps (light gray mesh) are shown on the left, and close-up views of each structure with the most relevant epitope/paratope components are indicated (V1 loop—blue, V2 loop—salmon, V3 loop—magenta). Inferred heavy and light chains for each antibody are labeled in each panel and represented in a dark and light green color, respectively.