Dense Array of Spikes on HIV-1 Virion Particles

Abstract

HIV-1 is rare among viruses for having a low number of envelope glycoprotein (Env) spikes per virion, i.e., ϳ7 to 14. This exceptional feature has been associated with avoidance of humoral immunity, i.e., B cell activation and antibody neutralization. Virus-like particles (VLPs) with increased density of Env are being pursued for vaccine development; however, these typically require protein engineering that alters Env structure. Here, we used instead a strategy that targets the producer cell. We employed fluorescence-activated cell sorting (FACS) to sort for cells that are recognized by trimer cross-reactive broadly neutralizing antibody (bnAb) and not by nonneutralizing antibodies. Following multiple iterations of FACS, cells and progeny virions were shown to display higher levels of antigenically correct Env in a manner that correlated between cells and cognate virions (P ϭ 0.027). High-Env VLPs, or hVLPs, were shown to be monodisperse and to display more than a 10-fold increase in spikes per particle by electron microscopy (average, 127 spikes; range, 90 to 214 spikes). Sequencing revealed a partial truncation in the C-terminal tail of Env that had emerged in the sort; however, iterative rounds of "cell factory" selection were required for the high-Env phenotype. hVLPs showed greater infectivity than standard pseudovirions but largely similar neutralization sensitivity. Importantly, hVLPs also showed superior activation of Env-specific B cells. Hence, high-Env HIV-1 virions, obtained through selection of producer cells, represent an adaptable platform for vaccine design and should aid in the study of native Env.IMPORTANCE The paucity of spikes on HIV is a unique feature that has been associated with evasion of the immune system, while increasing spike density has been a goal of vaccine design. Increasing the density of Env by modifying it in various ways has met with limited success. Here, we focused instead on the producer cell. Cells that stably express HIV spikes were screened on the basis of high binding by bnAbs and low binding by nonneutralizing antibodies. Levels of spikes on cells correlated well with those on progeny virions. Importantly, high-Env virus-like particles (hVLPs) were produced with a manifest array of well-defined spikes, and these were shown to be superior in activating desirable B cells. Our study describes HIV particles that are densely coated with functional spikes, which should facilitate the study of HIV spikes and their development as immunogens.KEYWORDS antigenicity, broadly neutralizing antibodies, envelope glycoprotein, fluorescence-activated cell sorting, HIV-1, vaccine design, virus-like particles, cellPACK, electron microscopy, viral infectivity H uman immunodeficiency virus type 1 (HIV-1) displays around 7 to 14 envelope (Env) spikes per virion (1-3). This low number of spikes is unusual for enveloped viruses in comparison to numbers for influenza virus (ϳ400 to 500 spikes), vesicular

Immunogenicity of HIV-1 Vaccines Expressing Chimeric Envelope Glycoproteins on the Surface of Pr55 Gag Virus-Like Particles

“…It was possible in our case, however, that the modifications made to the HIV gp150 envelope protein already significantly increased the levels of Env in Gag VLPs to a level that could not be further increased by the replacement of the TM + CT regions with that of the influenza hemagglutinin protein. Various studies have shown that truncation of the HIV Env from gp160 to gp150 increases the levels of expression on the surface of virus particles [10,32]. In addition, the replacement of the native HIV Env leader sequence with other leader sequences has also been shown to increase expression [10,33,34].…”

Functional Stability of HIV-1 Envelope Trimer Affects Accessibility to Broadly Neutralizing Antibodies at Its Apex

“…BN-PAGE analysis showed that Comb-mut-LK trimers were recognized more efficiently and by a greater number of V2 bnAbs than Comb-mut and that the bnAb-Env association was improved further by chemical cross-linking. We note, too, that MPER and gp41 interface bnAbs 10E8, PGT151, and 35O22 potently neutralize Comb-mut-LKQ with high MPNs similar to those for Comb-mut (70), suggesting that this Env is relatively homogeneous throughout the trimer interfaces.…”

Electron tomography visualization of HIV-1 fusion with target cells using fusion inhibitors to trap the pre-hairpin intermediate

“…Thus if HIV-1 virions contain a random assortment of a small number of Env trimers that diffuse slowly or not at all in the membrane, then only a small number of Envs would be available on the small contact surface formed by a roughly spherical virion and a flat cell membrane, whereas more Envs would be available for contacts with a larger contact surface formed by a virion interacting with a concave cell membrane (Figure 1c). Indeed, a recent ET study of murine leukemia virus (MLV) attached to target cells revealed a concave surface on the target cell with ~28 spokes per attached virion [64], a number that would be predicted to be higher than observed for HIV-1 even if HIV-1 were interacting with a concave portion of its target cell because MLV includes more spikes (at least 100 per virion) [65] compared with HIV-1 (7-14 per virion) [52][53][54][55][56].…”