Targeted N-glycan deletion at the receptor-binding site retains HIV Env NFL trimer integrity and accelerates the elicited antibody response.

Extensive shielding by N-glycans on the surface of the HIV envelope glycoproteins (Env) restricts B cell recognition of conserved neutralizing determinants. Elicitation of broadly neutralizing antibodies (bNAbs) in selected HIV-infected individuals reveals that Abs capable of penetrating the glycan shield can be generated by the B cell repertoire. Accordingly, we sought to determine if targeted N-glycan deletion might alter antibody responses to Env. We focused on the conserved CD4 binding site (CD4bs) since this is a known neutralizing determinant that is devoid of glycosylation to allow CD4 receptor engagement, but is ringed by surrounding N-glycans. We selectively deleted potential N-glycan sites (PNGS) proximal to the CD4bs on well-ordered clade C 16055 native flexibly linked (NFL) trimers to potentially increase recognition by naïve B cells in vivo. We generated glycan-deleted trimer variants that maintained native-like conformation and stability. Using a panel of CD4bs-directed bNAbs, we demonstrated improved accessibility of the CD4bs on the N-glycan-deleted trimer variants. We showed that pseudoviruses lacking these Env PNGSs were more sensitive to neutralization by CD4bs-specific bNAbs but remained resistant to non-neutralizing mAbs. We performed rabbit immunogenicity experiments using two approaches comparing glycan-deleted to fully glycosylated NFL trimers. The first was to delete 4 PNGS sites and then boost with fully glycosylated Env; the second was to delete 4 sites and gradually re-introduce these N-glycans in subsequent boosts. We demonstrated that the 16055 PNGS-deleted trimers more rapidly elicited serum antibodies that more potently neutralized the CD4bs-proximal-PNGS-deleted viruses in a statistically significant manner and strongly trended towards increased neutralization of fully glycosylated autologous virus. This approach elicited serum IgG capable of cross-neutralizing selected tier 2 viruses lacking N-glycans at residue N276 (natural or engineered), indicating that PNGS deletion of well-ordered trimers is a promising strategy to prime B cell responses to this conserved neutralizing determinant

CD4bs-specific antibody binding profiles to the N-glycan deleted trimers.

<p>(a) Schematic presentation of N-glycan composition around the trimer CD4bs in the selected N-glycan-deleted trimers. Filled blue triangle—the N-glycan is present; empty blue triangles—the N-glycan is genetically deleted. (b) Comparison of the <i>+N332</i> PT (dark blue) with <i>+N332</i> N301Q (yellow), <i>+N332</i> N276Q/N360Q/N463 (red) and <i>+N332</i> N276Q/N360Q/N463/N301Q (light blue) trimers. Recognition of His-captured trimers by the trimer-elicited rabbit serum were analyzed in duplicate at each antibody dilution. The error bars indicate variance of the mean binding values (OD450 nm) and a representative experiment of three independent repeats is shown.</p

Binding kinetics for glycan deleted trimers with the VRC03 Fab.

<p>Bio-layer interferometry (BLI) curves were generated with the PT and N276Q/N463 trimers (blue fitted curves) and <i>+N332</i> PT with <i>+N332</i> N301Q, <i>+N332</i> N276Q/N360Q/N463 and <i>+N332</i> N276Q/N360Q/N463/N301Q trimers (red fitted curves) immobilized on an anti-His sensor with serial dilutions of the VRC03 Fab at the concentrations indicated. A tabular summary of the K_d, k_on and k_off is shown.</p

Characterization of lectin affinity-purified 16055 glycan-deleted trimers with the 332 N-glycan restored.

<p>(a) SEC profiles and EM 2D class averages. A1 or B1, A2, A3 and AB indicate trimers with one, two, three and four N-glycan deletions, respectively. SEC profiles of N-glycan-deleted trimers (solid line) are shown in comparison with the <i>+N332</i> PT trimer (dotted line) and the expression level relative to expression level of <i>+N332</i> PT is shown on each SEC graph. Percentage of native-like trimers is indicated above the 2D class averages representative images. (b) DSC thermal transition curves and derived T_ms of glycan-deleted trimers (red solid line) compared to the backbone glycoprotein <i>+N332</i> PT (black dotted line).</p

Antibody sensitivity of glycan-deleted variants of 16055 pseudovirus.

<p>Neutralization IC₅₀ values of the panel of bNAbs and mAbs are shown and color-coded for concentrations (μg/ml) regarding potency as indicated. NN = No Neutralization. These experiments were performed two independent times for the antibodies shown.</p

Neutralizing ID₅₀ titers (reciprocal serum, fold-dilution) against 16055 N-glycan-deleted viruses.

<p>ID₅₀ values are indicated in bold. Those derived by extrapolation are shown in non-bolded text (a) ID₅₀ values for the viruses with the same N-glycan deletions proximal to the CD4bs as those in the trimer immunogens. Statistical differences were evaluated by the non-parametric Mann-Whitney test and, when detected at a level of significance, are indicated under the specific data set with * P<0.05 and ** P<0.01. (b) Serum neutralization curves for 16055wt virus derived from mean values for each data point of three independent TZM-bl-based neutralization assays. Error bars represent the standard deviation of the values from three independently performed experiments.</p

Immunogenicity of glycan-deleted trimers.

<p>(a) The immunogenicity regimen and respective immunogens for Groups 1, 2 and 3 are shown. In brief, rabbits were immunized at weeks 0, 4, 12 and 24. Test bleeds are indicated by the red arrows following each immunization. (b) Representative negative stain EM images of the liposomes coupled with the respective trimers. The white scale bar on the top wt trimer-liposomes image is equivalent to 100 nm. (c) Geometric mean IgG titers (GMT) as measured by His-capture ELISA to the wt autologous trimer immunogen following each inoculation. Immunizations are indicated by the vertical dashed gray lines. Six data points per time point per group were determined. Two independent ELISA experiments were performed and a representative experiment is shown.</p

Quantification analysis of the neutralization mapping assay.

<p>Quantification analysis of the neutralization mapping assay.</p

Neutralizing ID₅₀ values for the singly N-glycan-deleted viruses.

<p>ID₅₀ values are indicated in bold; those derived by extrapolation are shown in non-bolded text. Statistical differences were evaluated by Mann-Whitney test and, when detected, were indicated under each data set with * P<0.05.</p

Purified serum IgG cross-neutralization.

<p>(a) IgG neutralization curves derived from mean values for each data point of three independent TZM-bl-based neutralization assays. Error bars represent the standard deviation. The rabbits are designated by the Group number first (1, 2 or 3) followed by a dash and the animal index number as indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006614#ppat.1006614.g007" target="_blank">Fig 7</a> (i.e., #3–5). If specified otherwise, the serum was analyzed following the fourth immunization. The “control rabbit” was immunized four times with blank liposomes in adjuvant and IgG was purified similarly to the experimental rabbit IgGs; the mean values of two experimental replicates are shown for this negative specificity control (b) ID₅₀ values were derived from the curves described above and are color-coded as indicated. Weak neutralizing values were extrapolated based on the two highest IgG dilution data points and are indicated in italics. (c) Cross-neutralization of IAVIC22Δ276 and BG505Δ276 viruses analyzed by depletion with the 16055 gp120 TriMut protein. Purified IgG from the serum of rabbit #2–5 and rabbit #3–5 were titrated at the concentrations indicated (horizontal axis) in the absence or presence of the 16055 gp120 TriMut (two left panels). The 16055 gp120 TriMut protein was used at fixed concentration of 100 mg/ml. The mean values of two independent TZM-bl-based neutralization assays are shown with the bars at each dilution indicating the individual values. VRC13 IgG was used as a CD4bs-directed antibody positive control (two right panels) and in case of BG505Δ276 virus representative control experiment is shown.</p