Integrity of Glycosylation Processing of a Glycan-Depleted Trimeric HIV‑1 Immunogen Targeting Key B‑Cell Lineages

Broadly neutralizing antibodies (bNAbs) that target the trimeric HIV-1 envelope glycoprotein spike (Env) are tools that can guide the design of recombinant Env proteins intended to engage the predicted human germline precursors of bNAbs (gl-bNAbs). The protein components of gl-bNAb epitopes are often masked by glycans, while mature bNAbs can evolve to accommodate or bypass these shielding glycans. The design of germline-targeting Env immunogens therefore includes the targeted deletion of specific glycan sites. However, the processing of glycans on Env trimers can be influenced by the density with which they are packed together, a highly relevant point given the essential contributions under-processed glycans make to multiple bNAb epitopes. We sought to determine the impact of the removal of 15 potential N-glycan sites (5 per protomer) from the germline-targeting soluble trimer, BG505 SOSIP.v4.1-GT1, using quantitative, site-specific N-glycan mass spectrometry analysis. We find that, compared with SOSIP.664, there was little overall change in the glycan profile but only subtle increases in the extent of processing at sites immediately adjacent to where glycans had been deleted. We conclude that multiple glycans can be deleted from BG505 SOSIP trimers without perturbing the overall integrity of the glycan shield

Comprehensive Antigenic Map of a Cleaved Soluble HIV-1 Envelope Trimer

<div><p>The trimeric envelope (Env) spike is the focus of vaccine design efforts aimed at generating broadly neutralizing antibodies (bNAbs) to protect against HIV-1 infection. Three recent developments have facilitated a thorough investigation of the antigenic structure of the Env trimer: 1) the isolation of many bNAbs against multiple different epitopes; 2) the generation of a soluble trimer mimic, BG505 SOSIP.664 gp140, that expresses most bNAb epitopes; 3) facile binding assays involving the oriented immobilization of tagged trimers. Using these tools, we generated an antigenic map of the trimer by antibody cross-competition. Our analysis delineates three well-defined epitope clusters (CD4 binding site, quaternary V1V2 and Asn332-centered oligomannose patch) and new epitopes at the gp120-gp41 interface. It also identifies the relationships among these clusters. In addition to epitope overlap, we defined three more ways in which antibodies can cross-compete: steric competition from binding to proximal but non-overlapping epitopes (e.g., PGT151 inhibition of 8ANC195 binding); allosteric inhibition (e.g., PGT145 inhibition of 1NC9, 8ANC195, PGT151 and CD4 binding); and competition by reorientation of glycans (e.g., PGT135 inhibition of CD4bs bNAbs, and CD4bs bNAb inhibition of 8ANC195). We further demonstrate that bNAb binding can be complex, often affecting several other areas of the trimer surface beyond the epitope. This extensive analysis of the antigenic structure and the epitope interrelationships of the Env trimer should aid in design of both bNAb-based therapies and vaccines intended to induce bNAbs.</p></div

Cross-competition analysis for bNAb binding to BG505 SOSIP.664 trimer by SPR.

<p>(<b>A</b>) Competition between 8ANC195 (competitor) and PGT151 and 35O22. Association-dissociation curves of the individual binding experiments were overlaid with the second association phase to detect competition. 0 on the y axis is the baseline for the single comparator injection and for the same analyte as the second in the double injection. Thus, the strength of all three responses can be read on the same scale, although the value for the first analyte in the double injection will be negative. (<b>B</b>) Competition between PGT151 (competitor) and 8ANC195 and 35O22. (<b>C</b>) Competition between 35O22 (competitor) and 8ANC195 and PGT151.</p

3D modeling to explain bNAb competition patterns.

<p>(<b>A</b>) 3D models for the nonreciprocal competition between PG9 and PGT122. Side and top views of the bNAbs are shown together with the footprints. (<b>B</b>) Analysis of the Asn332 supersite depicting the angles of approach to the Asn332 glycan taken by various bNAb subfamilies. (<b>C</b>) 3D models to explain the bidirectional competition between PGT151 and 8ANC195. (<b>D</b>) 3D models to explain the unidirectional competition between OD-glycan and CD4bs bNAbs and (<b>E</b>) 3D models to explain the nonreciprocal competition between 8ANC195 and CD4bs bNAbs.</p

CD4 binding to the BG505 SOSIP.664 trimer and the induction of CD4-like conformational changes.

<p>(<b>A</b>) Use of sCD4 as a competitor. (<b>B</b>) Use of CD4-IgG2 as the analyte. (<b>C</b>) Induction of conformational changes measured by 17b binding. The extent of 17b binding in the presence of sCD4 was defined as 100%. *** indicates a significant difference between 17b binding without sCD4 and in the presence of a bNAb, as calculated using a Mann-Whitney 2-tailed test (P <0.05).</p

bNAb epitopes mapped onto the 3D structure of the BG505 SOSIP.664 trimer.

<p>(<b>A</b>) Various bNAbs (not labeled) to different epitope clusters are modeled onto each protomer of the trimer, according to fitting of various EM density maps (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004767#sec013" target="_blank">material and methods</a>). (<b>B</b>) bNAbs to different epitope clusters are modeled onto the same EM density map. Only one Fab fragment per trimer is shown for clarity. (<b>C</b>) Footprints of the different bNAb Fab fragment densities displayed in (<b>B</b>). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004767#sec013" target="_blank">methods</a> for further details.</p

Comparison of PGT135 and VRC01 binding to HIV-1 gp120.

<p><b>(A)</b> View down the trimer three-fold axis showing a superimposition of PGT135 Fab in complex with the clade B JRFL gp120 core (PDB ID: 4jm2; red), and of VRC01 Fab in complex with the clade A/E 93TH057 gp120 core (PDB ID: 3ngb; blue), each aligned onto the crystal structure of the BG505 SOSIP.664 trimer (PDB ID: 4tvp; white). In addition, the same BG505 SOSIP.664 crystal structure is fit into the negative-stain EM reconstruction of JR-FL Env trimer in complex with PGT151 (EMD-5919; white). The resulting model clearly shows that PGT135 and VRC01 do not sterically block binding of each other. <b>(B)</b> Side view of <b>(A)</b> with only one gp120 monomer displayed for clarity. <b>(C)</b> Detailed view of the key glycans (spheres) that were resolved in the PGT135-gp120 structure, and of the same glycans from the BG505 SOSIP.664 crystal structure. The presumed steric clash between glycan on Asn362 (and perhaps also Asn363) and the CDR H2 loop of VRC01 is marked with an asterisk. The gp120 subunit of the BG505 SOSIP.664 trimer is displayed as white ribbons. The glycans depicted were limited to the components resolved in the crystal structures (GlncNAc₁ for Asn362, GlncNAc₂Man₁ for Asn386 and GlncNAc₂Man₆ for Asn392 in the PGT135-gp120 structure; GlncNAc₂ for Asn362, GlncNAc₂ for Asn386 and GlncNAc₂ for Asn392 in the BG505 SOSIP.664 trimer structure), and hence their sizes are underestimated compared to trimers produced in 293F cells or present on viruses.</p

Autologous NAb responses to sequentially delivered clade A and clade B trimers.

<p>The neutralization titers (IC₅₀) against Tier-2 Env-pseudotyped viruses are plotted on the y-axis as a function of time after the first immunization on the x-axis (weeks). The scales are kept constant within each panel to facilitate comparisons among groups. The schedule is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005864#ppat.1005864.g001" target="_blank">Fig 1C</a> with immunizations at weeks 0, 4, 20, 24, 36, 48 and 60, and for rabbits #5734–5, #5735–5 and #5736–5 also at week 73. Blood was taken for analysis immediately before and 2 weeks after each immunization. The test viruses were as follows: <b>A</b> and <b>C</b>, BG505.T332N; <b>B</b> and <b>D</b>, B41. The curves connecting the reciprocal neutralization titers are color-coded for each rabbit as per the key associated with each group. Thus, changes in titers can be monitored over time both for individual animals and on a group-wide basis.</p

Four mechanisms of bNAb binding interference.

<p>(<b>1</b>) Direct overlap of epitopes; (<b>2</b>) Steric inhibition; (<b>3</b>) Allosteric inhibition; (<b>4</b>) Reorientation of glycans. See text for details.</p

Correlations between trimer-binding and neutralizing antibody titers.

<p>The scatterplots show neutralization titers on the y-axes and the trimer-binding antibody titers on the x-axes. Within each plot the symbols corresponding to neutralization of the BG505.T332N and B41 viruses are color-coded as indicated on the figure panels. Spearman correlation coefficients (r-values) and the corresponding significances (p-values) are color-coded analogously. Correlation analyses were performed for both BG505 and B41 NAb and binding antibody titers for sera from the early monovalent immunogen regimens during weeks 4–36 (top panel, group 1; lower panel, group 3).</p