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., ∼7 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 = 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: HIV-1; antigenicity; broadly neutralizing antibodies; cellPACK; electron microscopy; envelope glycoprotein; fluorescence-activated cell sorting; vaccine design; viral infectivity; virus-like particles.

FIG 1

Sorting cells using bnAb and FACS to express high levels of HIV-1 Env trimers. (A) Human cells (HEK293T) were transduced with a lentiviral vector bearing env, and FACS was used to acquire single cells having a bnAb^high non-nAb^low phenotype (i.e., VRC01^high b6^low). A total of four rounds of sorting were performed. (B) Env-bearing cells expanded from each round (V1 to V4) were probed using a panel of bnAbs and non-nAbs by flow cytometry. The antibody staining profile of Env-bearing cells is diagnostic of mature Env trimers and increases with each round of sorting. (C) Env content (gp160 equivalents) in membrane fractions prepared from cell lines V1 to V4 as determined by ELISA. (D) Correlation between cell surface staining by bnAb as shown in panel B and the quantity of membrane Env as shown in panel C. (E) V1 to V4 cells were normalized to 300 cells per sample and solubilized, and Env was separated and probed using SDS-PAGE and Western blotting; the blot is representative of at least two independent experiments. LTR, long terminal repeat; SV40, simian virus 40; prom, promoter; MCS, multiple cloning site; MFI, mean fluorescence intensity; rep origin, replication of origin. *, P < 0.05.

FIG 2

The CTT of gp41 is partially truncated and is not exposed on hVLPs. (A) Sequence alignment of the Env CTTs from V1 and V4 cells. The env transgenes from V1 and V4 cell lines were sequenced and aligned to full-length Comb-mut. Only the C-terminal tail (CTT) of gp41 is shown where the only mutation was found, i.e., L555*. Described sequence motifs in the CTT are marked with colored boxes above the corresponding sequence. LLP, lentivirus lytic peptide. (B) Kennedy epitope on the CTT of gp41 is occluded on intact hVLPs. Purified V1 and V4 VLPs were immobilized and probed in an ELISA using the anti-Kennedy epitope antibody Chessie 8. Bald VLPs and anti-dengue virus DEN3 antibody were included as negative virus controls and negative antibody controls, respectively. Solubilized VLPs are treated with detergent to liberate gp41 from virions and were used as positive controls.

FIG 3

hVLPs are homogeneous and display trimeric spikes. (A) Nanoparticle tracking analysis (NTA) of purified VLPs, including bald VLPs, normal pseudotyped virus (293T VLPs), and V1 to V4 hVLPs shows that particles are monodisperse with an average diameter of 137 to 143 nm. (B) Virus ELISA was used to probe V1 to V4 hVLPs immobilized on microwells with a panel of bnAbs and non-nAbs. (C) Correlation between bnAb staining of producer cells by flow cytometry and bnAb binding to hVLPs by virus ELISA. (D) V1 to V4 hVLPs were solubilized in nonionic detergent, and Env was separated and probed using BN-block and Western blotting. OD₄₅₀, optical density at 450 nm.

FIG 4

V4 hVLPs display antigenically correct trimeric spikes. (A) V4 hVLPs were immobilized and probed by ELISA using a panel of bnAbs and non-nAbs. (B) V4 hVLPs were captured on wheat germ agglutinin-coated biosensors and probed using a subset of bnAbs and non-nAbs by biolayer light interferometry (BLI).

FIG 5

Electron microscopy (EM) of hVLPs. (A) Representative VLPs, including bald particles, VLPs generated by transient transfection (293T), and (h)VLPs V1 to V4 visualized using negative stain EM. Bar, 100 nm. (B) Visible spikes or puncta were counted on individual virions (bald VLPs, n = 24; 293T VLPs, n = 19; V1 VLPs, n = 37; V2 VLPs, n = 29; V3 VLPs, n = 31; V4 VLPs, n = 46), and the total number of estimated trimers is plotted per virion. Each dot in the graph represents an individual virion. (C) Relative staining of producer cells by trimer-specific bnAb correlates with the average number of spikes per virion (P < 0.0001). *, P < 0.05; ***, P < 0.001.

FIG 6

Computational analyses and modeling of Env on VLPs. (A) Env spikes from EM images were counted using a semiautomated procedure. The hVLP spike count, diameter, and minimum spike-spike distances are tabulated. (B) Computational models of virions were generated using cellPACK. From left to right, the five models illustrate a random distribution of an increasing number of spikes: 10 (average number of spikes for HIV-1 virions), 49 (average number of spikes for V1 hVLPs), 127 (average number of spikes for V4 hVLPs), 214 (highest number of spikes estimated from an EM image of a single V4 hVLP), and 330 (theoretical maximum number of spikes determined to fit on the same size of particle).

FIG 7

Infectivity and neutralization characteristics of hVLPs. (A) hVLPs are more infectious than standard pseudovirions generated in 293T cells but show no significant difference in infectivity levels between hVLPs V1, V2, V3, and V4. (B) A comparison of the neutralization sensitivities of V1 and V4 hVLPs (left) as well as of standard (gp160) pseudovirions produced by transient transfection and V1 VLPs against a panel of bnAbs (right). Results are plotted as the fold change in IC₅₀s in each panel. A star indicates that antibodies did not reach an IC₅₀ at the highest or lowest concentration tested, as shown in Fig. 8. *, P < 0.05.

FIG 8

Neutralization of hVLPs by a panel of bnAbs. Results are shown for a comparison of neutralization sensitivities to bnAbs between V1 and V4 VLPs (A) as well as between V1 and standard pseudovirions generated in 293T cells (293T VLPs) (B). The estimated fold change in the IC₅₀ between 293T VLPs and V4 VLPs shown in panel B (*) was calculated using IC₅₀s from two independent experiments, both of which included V1 VLPs, as follows: fold change in IC₅₀ = (IC₅₀ 293T VLPs/IC₅₀ V1 VLPs_{experiment B})/(IC₅₀ V4 VLPs/IC₅₀ V1 VLPs_{experiment A}). na, not applicable (the fold change in IC₅₀ could not be calculated because the antibodies did not reach an IC₅₀ at the highest or lowest concentration tested); sCD4, soluble CD4.

FIG 9

CTT truncation (755*) does not increase infectivity or Env content of VLPs produced by transient transfection of 293T cells. Virions were pseudotyped with Comb-mut gp160 (full-length), CTT-truncated Comb-mut (L755stop), and a 10-fold excess of plasmid DNA of full-length gp160 and were compared to V4 hVLPs. Virions were normalized for particle concentration and then assayed for percent infectivity in TZM-bl reporter cells (A) as well as for Env incorporation (B). For the latter, virions were probed with anti-gp120 antibodies b12, 2G12, and 447-52D in an ELISA, and the optical density (OD) at 450 nm is shown. ns, not significant. ***, P < 0.001.

FIG 10

hVLPs efficiently activate Env-specific B cells. B cells expressing BCRs of the anti-gp120 CD4bs bnAb VRC01 were cultured with three different concentrations of bald VLPs, normal pseudotyped virus (293T VLPs), V1 VLPs, or V4 VLPs. Following 24 h, flow cytometry of B cells was used to determine levels of the activation marker CD69 (A and B) and the B cell receptor hCk, which was downregulated upon activation (C and D), while levels of proinflammatory cytokines IL-6 (E) and TNF-α (F) in cell culture supernatants were determined by ELISA. Data shown are an average of two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.