Cleavage-Independent HIV-1 Env Trimers Engineered as Soluble Native Spike Mimetics for Vaccine Design

Viral glycoproteins mediate entry by pH-activated or receptor-engaged activation and exist in metastable pre-fusogenic states that may be stabilized by directed rational design. As recently reported, the conformationally fixed HIV-1 envelope glycoprotein (Env) trimers in the pre-fusion state (SOSIP) display molecular homogeneity and structural integrity at relatively high levels of resolution. However, the SOSIPs necessitate full Env precursor cleavage, which requires endogenous furin overexpression. Here, we developed an alternative strategy using flexible peptide covalent linkage of Env subdomains to produce soluble, homogeneous, and cleavage-independent Env mimics, called native flexibly linked (NFL) trimers, as vaccine candidates. This simplified design avoids the need for furin co-expression and, in one case, antibody affinity purification to accelerate trimer scale-up for preclinical and clinical applications. We have successfully translated the NFL design to multiple HIV-1 subtypes, establishing the potential to become a general method of producing native-like, well-ordered Env trimers for HIV-1 or other viruses

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

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

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

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

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

HIV Env trimer N-glycans and the CD4bs.

<p>Structure of soluble BG505 SOSIP.664 HIV trimer (PDB accession number 5FYL) with gp120 in gray, gp41 in brown and the CD4bs shown as a magenta surface. N-glycans are shown in shades of blue. (a) The Group A N-glycans proximal to the CD4bs are shown in dark blue as indicated in bold and the N332 N-glycan is shown in dark turquoise. (b) The Group B N-glycans proximal to the CD4bs are shown in dark blue and are indicated in bold. N332 N-glycan is shown in dark turquoise. (c) Trimer docking models of VRC01 (purple), b12 (red) and CH103 (yellow) Fabs, each approaching the CD4bs with different angles of access.</p