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. 2020 Apr 16;94(9):e01884-19.
doi: 10.1128/JVI.01884-19. Print 2020 Apr 16.

A Bivalent, Spherical Virus-Like Particle Vaccine Enhances Breadth of Immune Responses against Pathogenic Ebola Viruses in Rhesus Macaques

Karnail Singh  1   2   3 , Bishal Marasini  4 , Xuemin Chen  3 , Lingmei Ding  4 , Jaang-Jiun Wang  4   2   3 , Peng Xiao  5 , Francois Villinger  5 , Paul Spearman  4   2   3
Affiliations

A Bivalent, Spherical Virus-Like Particle Vaccine Enhances Breadth of Immune Responses against Pathogenic Ebola Viruses in Rhesus Macaques

Karnail Singh et al. J Virol. .

Abstract

The 2013-2016 Ebola outbreak in West Africa led to accelerated efforts to develop vaccines against these highly virulent viruses. A live, recombinant vesicular stomatitis virus-based vaccine has been deployed in outbreak settings and appears highly effective. Vaccines based on replication-deficient adenovirus vectors either alone or in combination with a multivalent modified vaccinia Ankara (MVA) Ebola vaccine also appear promising and are progressing in clinical evaluation. However, the ability of current live vector-based approaches to protect against multiple pathogenic species of Ebola is not yet established, and eliciting durable responses may require additional booster vaccinations. Here, we report the development of a bivalent, spherical Ebola virus-like particle (VLP) vaccine that incorporates glycoproteins (GPs) from Zaire Ebola virus (EBOV) and Sudan Ebola virus (SUDV) and is designed to extend the breadth of immunity beyond EBOV. Immunization of rabbits with bivalent Ebola VLPs produced antibodies that neutralized all four pathogenic species of Ebola viruses and elicited antibody-dependent cell-mediated cytotoxicity (ADCC) responses against EBOV and SUDV. Vaccination of rhesus macaques with bivalent VLPs generated strong humoral immune responses, including high titers of binding, as well as neutralizing antibodies and ADCC responses. VLP vaccination led to a significant increase in the frequency of Ebola GP-specific CD4 and CD8 T cell responses. These results demonstrate that a novel bivalent Ebola VLP vaccine elicits strong humoral and cellular immune responses against pathogenic Ebola viruses and support further evaluation of this approach as a potential addition to Ebola vaccine development efforts.IMPORTANCE Ebola outbreaks result in significant morbidity and mortality in affected countries. Although several leading candidate Ebola vaccines have been developed and advanced in clinical testing, additional vaccine candidates may be needed to provide protection against different Ebola species and to extend the durability of protection. A novel approach demonstrated here is to express two genetically diverse glycoproteins on a spherical core, generating a vaccine that can broaden immune responses against known pathogenic Ebola viruses. This approach provides a new method to broaden and potentially extend protective immune responses against Ebola viruses.

Keywords: Bundibugyo virus; Ebola glycoprotein; Ebola vaccine; Ebola virus; Ebola virus disease; Marburg virus; Sudan virus; Tai Forest virus; virus-like particles.

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Figures

FIG 1
FIG 1
Generation of stable cell lines secreting EBOV GP-Gag, SUDV GP-Gag, and bivalent EBOV/SUDV GP-Gag VLPs. (A) Schematics of pcDNA5/TO-puro EBOV GP, pcDNA5/TO-puro SUDV GP, pcDNA5/TO-neo SUDV GP, and pcDNA4/TO zeo HIV-1 Gag plasmids generated to develop different stable cell lines used in this study. (B) 293F cells selected after stable transfections with EBOV GP, SUDV GP, or both and HIV-1 Gag plasmids were cultured as such or induced with doxycycline for 24 h, and cell lysates were probed by Western blotting for EBOV GP, SUDV GP, HIV-1 Gag, and actin using specific antibodies. A representative Western blot from EBOV/SUDV GP-Gag 293F cells is shown. Similar results were obtained with EBOV GP-Gag and SUDV GP-Gag 293F cells. (C to E) Stable cells were induced with doxycycline for 40 h, and cleared supernatants were loaded on 20% sucrose cushions and subjected to ultracentrifugation. VLP pellets so obtained and the corresponding cell lysates were probed by Western blotting using anti-EBOV GP-, anti-SUDV GP-, and anti-HIV-1 Gag-specific antibodies.
FIG 2
FIG 2
Characterization of Ebola VLPs by electron microscopy and buoyant density. Ebola VLPs harvested from culture supernatants were analyzed by negative stain electron microscopy. (A and B) The analysis showed spherical particles abundantly covered with spikes of glycoproteins on their surface. (C to E) Equilibrium sucrose density gradient analysis of monovalent and bivalent Ebola VLPs. VLPs harvested from culture supernatants were fractionated by ultracentrifugation on 20 to 60% sucrose gradient. Fractions were analyzed by Western blotting for the presence of EBOV GP, SUDV GP, and HIV-1 Gag using specific antibodies. The refractive index of each fraction was measured and their buoyance density calculated. Densities of the fractions with largest amount of EBOV GP, SUDV GP, and HIV-1 Gag are shown with arrows above each fraction.
FIG 3
FIG 3
Humoral immunogenicity of Ebola VLPs in rabbits. (A to D) Serum samples collected from rabbits immunized with monovalent or bivalent Ebola VLPs were tested for binding antibodies by ELISA using recombinant EBOV GP, SUDV GP, BDBV GP, and MARV GP as the coating antigens. Shown on the y axis are the average of optical density values from representative experiments repeated thrice. (E to H) Levels of anti-Ebola neutralizing antibodies in immune sera were measured by using an Ebola neutralization assay. HIV-1ΔEnv pseudovirions expressing EBOV GP, SUDV GP, BDBV GP, TAFV GP, or MARV GP were preincubated for 1 h with increasing dilutions of the sera before adding onto the TZM-bl cells expressing luciferase in an HIV-1 tat-dependent manner. Forty-eight hours later, luciferase activity was measured and the percent neutralization calculated. The broken gray line in each panel represents the IC50 value (50% neutralization). Results are shown as means ± standard deviations (SD) of triplicates from representative experiments repeated thrice. Symbols in panel E: *, P value between EBOV versus SUDV; ***, P value between EBOV versus MARV. Symbol in panel F: ***, P values between SUDV versus other pseudovirions. Symbols in panels G and H: ***, P values between MARV versus other pseudovirions. (I to L) ADCC-mediating antibodies in immune sera were measured against target cells expressing either EBOV GP or SUDV GP, with CD16 overexpressing NK-92 effector cells in a luciferase-based Ebola ADCC assay as described in Materials and Methods. Results are shown as means ± SD from three independent experiments. The broken gray line in each panel represents the cutoff of the Ebola ADCC assay used. *, **, and *** represent P values of <0.05, <0.01, and <0.001, respectively.
FIG 4
FIG 4
Humoral immunogenicity of bivalent EBOV/SUDV GP-Gag VLPs in rhesus macaques. (A to C) Rhesus macaques were immunized with bivalent EBOV/SUDV GP-Gag VLPs, and plasma samples collected on day 0 (A), day 42 (B), and day 98 (C) were tested for the presence of anti-EBOV GP, SUDV GP, BDBV GP, and MARV GP binding antibodies by ELISA. Results are expressed as means ± standard errors of the means (SEM) of data obtained from 4 animals run in duplicate. (D to F) Plasma samples collected on day 0, day 42, and day 98 were tested for the presence of anti-Ebola neutralizing antibodies using recombinant HIV-1△Env pseudovirions bearing different Ebola/MARV GPs. Results are expressed as means ± SEM of data obtained from 4 animals run in duplicate. Broken gray lines represent IC50 values (plasma dilution giving 50% virus neutralization). Symbols in panel E: **, lower, P values between EBOV versus MARV and SUDV versus MARV; **, upper, ** P value between EBOV versus TAFV. Symbols in panel F: ***, P values between MARV versus other pseudovirions; **, P values between EBOV versus TAFV. (G to I) Plasma samples collected on day 0, day 42, and day 98 were tested for the presence of ADCC killing anti-EBOV GP and anti-SUDV GP antibodies using EBOV GP- or SUDV GP-expressing target cells and natural killer cells overexpressing CD16 as effector cells. Target cells expressing MARV GP were used as controls. Results from a representative experiment are shown as means ± SEM of data obtained from 4 animals. The broken gray line in each panel represents the cutoff of the Ebola ADCC assay used. *, **, and *** represent P values of <0.05, <0.01, and <0.001, respectively.
FIG 5
FIG 5
Frequencies of plasmablasts in PBMCs collected from rhesus macaques immunized with bivalent EBOV/SUDV GP-Gag VLPs. Rhesus macaques were immunized with bivalent EBOV/SUDV GP Gag VLPs, and frequencies of IgM-, IgG-, and IgA-secreting polyclonal (A), anti-EBOV GP (B), anti-SUDV GP (C), anti-BDBV GP (D), and anti-MARV GP (E) plasmablasts were measured on day 0, day 33 (5 days after the first VLP booster), and day 89 (5 days after the second VLP booster). Results are displayed as plasmablasts per million PBMCs. M, G, and A stand for IgM, IgG, and IgA, respectively.
FIG 6
FIG 6
Frequencies of cytokine-secreting (IFN-γ and TNF-α) CD4 and CD8 T cells in PBMCs from rhesus macaques. Rhesus macaques were immunized with bivalent Ebola VLPs as described in the text, and PBMCs collected on day 0, day 42, and day 98 were frozen in liquid nitrogen. Cells were thawed, rested overnight, and then stimulated for 12 h with recombinant EBOV GP, SUDV GP, BDBV GP, MARV GP (all expressed in mammalian cells), or TAFV GP (expressed in bacterial cells) in the presence of secretion inhibitor. Following stimulation, cells were washed and stained for surface CD3, CD4, and CD8. Cells were fixed, permeabilized, and then stained for IFN-γ and TNF-α. Frequencies of CD4 and CD8 T cells positive for IFN-γ and TNF-α were determined by FlowJo analysis. (A and B) Representative dot blots of CD4 and CD8 T cells secreting IFN-γ or TNF-α upon restimulation with EBOV GP. (C and D) Summary of CD4 and CD8 T cells secreting IFN-γ and/or TNF-α upon restimulation with different filovirus GPs obtained from four animals.
FIG 7
FIG 7
Durability of Ebola-specific immune responses in rhesus macaques. Plasma and/or PBMC samples were collected from the rhesus macaques, every 4 weeks for 6 months, after the last VLP booster dose. Plasma samples were analyzed for the presence of binding (A), neutralizing (IC50 values) (B), and ADCC-mediating (C) anti-Ebola GPs antibodies and data plotted versus time. Results are expressed as means ± SEM of data obtained from 4 animals. Plasma samples from none of the animals neutralized >50% at 1:30 dilution (lowest dilution used in this assay) after week 22. Similarly, plasma samples from none of the animals showed ADCC killing higher than the background levels after week 26. PBMC samples were analyzed for the presence of CD4 and CD8 T cells secreting IFN-γ and TNF-α upon restimulation with EBOV GP or SUDV GP. Data were plotted as percent frequencies of cytokine-secreting (IFN-γ and/or TNF-α) CD4 (D) and CD8 (E) T cells versus time (weeks). Results are expressed as means ± SEM of data obtained from 4 animals. *, **, and *** represent P values of < 0.05, <0.01, and <0.001, respectively, and are placed at the first time point after the peak response at which the antibody titers or cell frequencies showed a statistically significant decline.
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