HIV-1 ENVELOPE. Effect of the cytoplasmic domain on antigenic characteristics of HIV-1 envelope glycoprotein

Abstract

A major goal for HIV-1 vaccine development is the production of an immunogen to mimic native, functional HIV-1 envelope trimeric spikes (Env) on the virion surface. We lack a reliable description of a native, functional trimer, however, because of inherent instability and heterogeneity in most preparations. We describe here two conformationally homogeneous Envs derived from difficult-to-neutralize primary isolates. All their non-neutralizing epitopes are fully concealed and independent of their proteolytic processing. Most broadly neutralizing antibodies (bnAbs) recognize these native trimers. Truncation of their cytoplasmic tail has little effect on membrane fusion, but it diminishes binding to trimer-specific bnAbs while exposing non-neutralizing epitopes. These results yield a more accurate antigenic picture than hitherto possible of a genuinely untriggered and functional HIV-1 Env; they can guide effective vaccine development.

Fig. 1. Antigenic characteristics of the 92UG037.8 Env trimer presented on cell surfaces

Plots of antibody binding to the Env trimer on the 92UG037.8 gp160 cell surfaces in the absence (red) or presence (blue) of soluble CD4. Fluorescent signal for bound CD4 is shown in the presence of CD4 (cyan) or in the absence of CD4 (orange). Antibodies and their epitopes are indicated. The median inhibitory concentration (IC50) values were determined in a luciferase-based virus neutralization assay using 92UG037.8 gp160 and purified antibodies. Unless specified, all antibodies used are Fab fragments. Original flow cytometry histograms are shown in fig. S4. Extensive control experiments were carried out to ensure the binding specificity, and the experiments were repeated at least twice with almost identical results.

Fig. 2. Antigenic properties of the 92UG037.8 gp160-ΔCT

Plots of antibody binding to the Env trimer on the 92UG037.8 gp160-ΔCT cell surfaces in absence (red) or presence (blue) of soluble CD4. Fluorescent signal for bound CD4 is shown in the presence of CD4 (cyan) or in the absence of CD4 (orange). Antibodies and their epitopes are indicated. Unless specified, all antibodies used are Fab fragments. Original flow cytometry histograms are shown in fig. S11. The experiments were repeated at least twice with almost identical results.

Fig. 3. Effect of the gp41 cytoplasmic tail on antigenic properties of the ectodomain of Env

A tabulated summary of antibody binding to various 92UG037.8 Env constructs, including gp160, gp160-CT120, gp160-CT90, gp160-CT60, gp160-CT30, gp160-ΔCT, gp140-TMfd, gp140FL20-TM, gp140FL20-TMfd, and gp140-GPI. Epitopes targeted by the antibodies include CD4bs, CD4 binding site; CD4i, CD4-induced; V1V2+G, the V1V2 loop and glycans; V3+G, the V loop and glycans; MPER, membrane proximal external region; and gp41, cluster I and cluster II. Binding index is normalized by VRC01 binding to each untriggered Env construct in the absence of CD4, and it is defined as the ratio between the maximum MFI of a given antibody binding and the maximum MFI of VRC01 binding to the same Env construct. Cell-cell fusion capacity of each Env construct was monitored by syncytium formation when mixing Env-expressing cells and TZM-bl cells.

Fig. 4. A proposed relationship among all HIV-1 isolates based on their Env properties

Our gp160 and gp160-ΔCT may represent Envs of the difficult-to-neutralize primary isolates that cannot induce autologous neutralizing antibody responses and the easy-to-neutralize, laboratory-adapted strains that induce strong autologous neutralizing responses, respectively. Other isolates (of intermediate susceptibility to neutralization) may adopt intermediate conformations, like those of our partially truncated gp160-CT constructs. A few mutations can easily convert one form into another, perhaps even within a single patient, and thus an effective vaccine may need to protect against the viruses described in blue.