Development and Evaluation of an Ebola Virus Glycoprotein Mucin-Like Domain Replacement System as a New Dendritic Cell-Targeting Vaccine Approach against HIV-1

Abstract

The development of efficient vaccine approaches against HIV infection remains challenging in the vaccine field. Here, we developed an Ebola virus envelope glycoprotein (EboGP)-based chimeric fusion protein system and demonstrated that replacement of the mucin-like domain (MLD) of EboGP with HIV C2-V3-C3 (134 amino acids [aa]) or C2-V3-C3-V4-C4-V5-C5 (243 aa) polypeptides (EbGPΔM-V3 and EbGPΔM-V3-V5, respectively) still maintained the efficiency of EboGP-mediated viral entry into human macrophages and dendritic cells (DCs). Animal studies using mice revealed that immunization with virus-like particles (VLPs) containing the above chimeric proteins, especially EbGPΔM-V3, induced significantly more potent anti-HIV antibodies than HIV gp120 alone in mouse serum and vaginal fluid. Moreover, the splenocytes isolated from mice immunized with VLPs containing EbGPΔM-V3 produced significantly higher levels of gamma interferon (IFN-γ), interleukin 2 (IL-2), IL-4, IL-5, and macrophage inflammatory protein 1α (MIP-1α). Additionally, we demonstrated that coexpression of EbGPΔM-V3 and the HIV Env glycoprotein in a recombinant vesicular stomatitis virus (rVSV) vector elicited robust anti-HIV antibodies that may have specifically recognized epitopes outside or inside the C2-V3-C3 region of HIV-1 gp120 and cross-reacted with the gp120 from different HIV strains. Thus, this study has demonstrated the great potential of this DC-targeting vaccine platform as a new vaccine approach for improving immunogen delivery and increasing vaccine efficacy. IMPORTANCE Currently, there are more than 38.5 million reported cases of HIV globally. To date, there is no approved vaccine for HIV-1 infection. Thus, the development of an effective vaccine against HIV infection remains a global priority. This study revealed the efficacy of a novel dendritic cell (DC)-targeting vaccination approach against HIV-1. The results clearly show that the immunization of mice with virus-like particles (VLPs) and VSVs containing HIV Env and a fusion protein composed of a DC-targeting domain of Ebola virus GP with HIV C2-V3-C3 polypeptides (EbGPΔM-V3) could induce robust immune responses against HIV-1 Env and/or Gag in serum and vaginal mucosa. These findings provide a proof of concept of this novel and efficient DC-targeting vaccine approach in delivering various antigenic polypeptides of HIV-1 and/or other emergent infections to the host antigen-presenting cells to prevent HIV and other viral infections.

Keywords: Ebola virus glycoprotein; HIV-1; VSV vector; dendritic cells; vaccines; virus-like particles.

FIG 1

Construction and expression of EboGPΔM-V3 and EboGPΔM-V3-V5 chimeric protein. (A) Codon-optimized EboGP gene and MLD deletion-containing (deletion encompassing aa 306 to 483) EboGP gene sequences were inserted into mammalian cell expression vectors named EboGPwt and EboGPΔM, respectively. PCR-amplified gene sequences including C2-V3-C3 and C2-V3-C3-V4-C4-V5 sequences were inserted into the MLD deletion-containing EboGP gene, and the resulting constructs were designated EboGPΔM-V3 and EboGPΔM-V3-V5. (B) Amino acid sequence of the C2-V3-C3-V4-C4-V5 region of HIV Env. (C) WB detected the expression of EboGPwt, EboGPΔM-V3, and EboGPΔM-V3-V5 in 293T cells by using anti-EboGP (42/3.7) and anti-gp120-V3 (5F7) antibodies. (D) Cytotoxicity of EboGP-based chimeric fusion proteins, detected by the trypan blue exclusion method.

FIG 2

Characterization of the virus entry mediated by HIV Env or EboGP-based chimeric fusion proteins. (A) 293T cells were cotransfected with HIV-1ΔRI/ΔE/GLuc⁺, Δ8.2, and HIV Env (T or M tropic), EboGPwt, EboGPΔM, EboGPΔM-V3, or EboGPΔM-V3-V5 plasmids. The VLPs produced were purified and analyzed by WB with anti-HIV gp120 (ID6 or 5F7), anti-EboGP antibody, or anti-HIV p24 antibody. (B to H) TZM-b1 cells, Vero E6 cells, CD4⁺ C8166 T cells, THP-1 cells, human MDMs, and human MDDCs were infected with the above-described VLPs. At different time points of infection, the supernatants were collected and subjected to a GLuc activity assay or p24 ELISA. (I) The THP1–NF-κB sensor cell line was incubated with various amounts of EboGPwt- or EboGPΔM-V3-pseudotyped HIV VLPs for 6 h to 18 h, and the activation of NF-κB signaling was detected by measurement of the Luc activity of treated cells. TNF-α treatment was used as a positive control. Error bars represent variation between duplicate samples, and the data are representative of results obtained in three independent experiments.

FIG 3

Gene expression of proinflammatory cytokines and chemokines induced by HIV Env or EboGPΔM-V3 VLPs in human MDMs. Human MDMs were treated with HIV Env or EboGPΔM-V3 VLPs for 4 or 24 h. LPS treatment was included as a control. The intracellular levels of mRNA transcripts encoding IL-2, IL-6, IL-10, MIP-1α, IFN-γ, and TNF-α (A to F) were analyzed by quantitative real-time PCR. Error bars represent variation between samples from three donors. Statistical significance was determined using an unpaired t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

FIG 4

Higher levels of anti-HIV antibodies induced by EboGPΔM-V3-pseudotyped VLPs in BALB/c mice. (A) Detection of the presence of HIV Env, EboGPΔM-V3, or EboGPΔM-V3-V5 in the VLPs used for immunization. (B) BALB/c mice were injected subcutaneously with 100 ng (p24) of HIV Env, EboGPΔM-V3, or EboGPΔM-V3-V5 VLPs on day 0 as indicated. At 21 and 49 days postimmunization, mice were given boosters with the same amounts of VLPs. Sera were collected at 7, 27, and 56 days postimmunization. (C) Levels of anti-HIV gp120 IgG (left) and anti-gp120 V3 IgG (right) in mouse sera. (D) Anti-Gagp24 antibodies in mouse sera were detected via ELISA. Statistical significance was determined using an unpaired t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

FIG 5

Significantly higher levels of anti-HIV antibodies and cytokines/chemokines were induced by immunization with HIV Env/EboGPΔM-V3-pseudotyped VLPs in BALB/c mice. (A) WB detected the presence of HIV Env and EboGPΔM-V3 in the pseudotyped VLPs. (B) BALB/c mice were injected subcutaneously with 100 ng (p24) of HIV Env or HIV Env/EboGPΔM-V3 VLPs and given boosters with the same amounts of VLPs at day 29. Sera were collected at 7, 28, and 42 days postimmunization. The levels of anti-HIV gp120 (C) and anti-HIV p24 (D) antibodies in sera were detected via ELISA at the time points indicated. The levels of anti-HIV gp120 IgG and IgA (E) and anti-HIV p24 IgG and IgA in vaginal (F) were detected via ELISA. Error bars represent variation between samples of each immunized group. (G) Splenocytes isolated from immunized mice were stimulated with HIV Env peptides. After 72 h of stimulation, supernatants were collected, and the release of cytokines and chemokines in the supernatants was quantified with an MSD V-plex kit mouse cytokine kit and counted in the MESO Quickplex SQ120 instrument. Statistical significance between the two groups was determined using an unpaired t test. *, P ≤ 0.05; **, P ≤ 0.01.

FIG 6

Generation of rVSV coexpressing HIV Env and EboGPΔM-V3 and induction of potent anti-HIV immune responses in mice. (A) Schematic structures of the rVSV vector coexpressing HIV Env and VSV-G (rVSVΔG/Henv/VSVG) and rVSV coexpressing HIV Env and EboGPΔM-V3 (rVSVΔG/Henv/EboGPΔM-V3). (B) WB detected the expression of HIV Env and EboGPΔM-V3 in the pseudotyped rVSV by using anti-HIV gp120, anti-HIV gp120V3 (top), or anti-ZGP (bottom). (C) The anti-HIV gp120 antibody levels in the sera of immunized mice were measured by anti-HIV gp120 IgG ELISA. The value is the individual amount for each mouse of each group. (D) The mouse serum pool from the rVSVΔG/Henv/VSVG and rVSVΔG/Henv/EboGPΔM-V3 groups was analyzed by WB against lysates from cells transiently expressing T- or M-tropic HIV Env protein (left). Meanwhile, the monoclonal anti-gp120 antibody (ID6) was used to detect HIV Env protein as a control (right). (E) ELISA was also done with the HIV-1 consensus B V3 peptide or HIV Env 405–423 and 419–433 peptides. (F) Cross-clade reactivity of anti-HIV gp120 antibodies from immunized mice. HIV gp120 recombinant proteins derived from clades B (IIIB0), C (C.1086D7), and AE (AE.A244D11) were used for ELISA to detect the cross-reactivity of the mouse serum pool from each group. Error bars show variation between triplicate samples of each group. Statistical significance was determined using an unpaired t test. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.