Preclinical Development of Inactivated Rabies Virus-Based Polyvalent Vaccine Against Rabies and Filoviruses

Abstract

We previously described the generation of a novel Ebola virus (EBOV) vaccine based on inactivated rabies virus (RABV) containing EBOV glycoprotein (GP) incorporated in the RABV virion. Our results demonstrated safety, immunogenicity, and protective efficacy in mice and nonhuman primates (NHPs). Protection against viral challenge depended largely on the quality of the humoral immune response against EBOV GP.Here we present the extension and improvement of this vaccine by increasing the amount of GP incorporation into virions via GP codon-optimization as well as the addition of Sudan virus (SUDV) and Marburg virus (MARV) GP containing virions. Immunogenicity studies in mice indicate similar immune responses for both SUDV GP and MARV GP compared to EBOV GP. Immunizing mice with multiple antigens resulted in immune responses similar to immunization with a single antigen. Moreover, immunization of NHP with the new inactivated RABV EBOV vaccine resulted in high titer neutralizing antibody levels and 100% protection against lethal EBOV challenge when applied with adjuvant.Our results indicate that an inactivated polyvalent vaccine against RABV filoviruses is achievable. Finally, the novel vaccines are produced on approved VERO cells and a clinical grade RABV/EBOV vaccine for human trials has been produced.

Keywords: Ebola virus; Marburg virus; Sudan virus; filovirus; polyvalent vaccine; rabies virus; vaccine.

Figure 1.

Schematic of vaccine constructs used in this study. The genomes for the rabies virus vaccine vector (BNSP333), expressing wild-type Ebola virus (EBOV; strain Mayinga) glycoprotein (GP) (BNSP333-ZGP), codon-optimized EBOV GP (BNSP333-coZGP), or codon-optimized Sudan virus (strain Gulu) GP (BNSP333-coSGP), are shown.

Figure 2.

Ebola virus (EBOV) glycoprotein (GP) incorporation into rabies virus (RABV) particles. Sucrose-purified particles of BNSP333, BNSP333-ZGP, or BNSP333-coZGP were analyzed via Western blotting with a monoclonal antibody (15H10) against EBOV GP2 (approximately 25 kDa) and a rabbit polycloncal serum samples directed against RABV M (25 kDa) (A) or were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and stained with SYPRO Ruby for total protein analysis and quantification of EBOV GP (B) 1. Lanes 1–8 in B depict a bovine serum albumin standard (2000, 1500, 1000, 750, 500, 250, 125, and 25 ng); lanes 9–12, purified EBOV GP (10, 50, 100, and 500 ng); lanes 13–14, recombinant virions of BNSP333-coZGP (1000 and 2000 ng). EBOV GP1 and RABV G are indicated.

Figure 3.

Murine humoral immune response to Zaire glycoprotein (GP). Serum samples were analyzed for total immunoglobulin G with an Ebola virus (EBOV) GP–specific enzyme-linked immunosorbent assay for days 7 (first boost) (A), 14 (B), 21 (C), 28 (second boost) (D), 35 (E), and 84 (F). Optical density readings at 490 nm (OD₄₉₀) were compared with an anti–EBOV GP monoclonal antibody. Serum samples obtained before immunization on day 0 were used as negative controls. All samples were diluted 1:50 and analyzed in a 3-fold serial dilution.

Figure 4.

Murine humoral immune response to Sudan virus (SUDV) glycoprotein (GP). Serum samples were analyzed for total immunoglobulin (Ig) G with an SUDV GP–specific enzyme-linked immunosorbent assay for days 7 (first boost) (A), 14 (B), 21 (C), 28 (second boost) (D), 35 (E), and 84 (F). Optical density readings at 490 nm (OD₄₉₀) were compared with an anti–Ebola virus GP monoclonal antibody 15H10. Serum samples obtained before immunization on day 0 were used as negative controls. All serum samples were diluted 1:50 and analyzed in a 3-fold serial dilution.

Figure 5.

Immunoglobulin (Ig) G isotype responses to Ebola virus (EBOV) and Sudan virus (SUDV) glycoprotein (GP). A, IgG2c and IgG1 antibody responses to EBOV GP (A) or SUDV GP (B) were determined with enzyme-linked immunosorbent assay at an antibody dilution of 1:1000 on days 7, 14, 21, 28, and 35. Abbreviation: OD₄₉₀, optical density at 490 nm.

Figure 6.

Neutralizing antibody response elicited in nonhuman primates (NHPs) vaccinated twice with BNSP333-coZGP with 2 adjuvants. Groups of 2 animals were vaccinated with BNSP333-coZGP plus adjuvant 1 (NHP identifier [ID] 20447 and 27837), BNSP333-coZGP plus adjuvant 2 (NHP ID 20410 and 27914), or phosphate-buffered saline (PBS) (NHP ID 17847 and BB212E). Animals vaccinated with the inactivated rabies virus vaccine BNSP333-coZGP were vaccinated on days 0 and 42, and animals vaccinated with PBS on days 0, 21, and 42. Serum samples collected on days 42 and 54 were evaluated using an Ebola virus (EBOV)–specific enzyme-linked immunosorbent assay (A), and serum samples from day 54 were also analyzed for the presence of VNA by pseudovirion neutralization assay (PsVNA), using both EBOV Z76 and EBOV Z95 pseudovirions. B, PsVNA 50% and 80% neutralization titers (PsVNA₅₀ and PsVNA₈₀) titers were determined. Geometric mean titers for replicate values are shown with 95% confidence intervals. The positive control was serum samples from rabbits vaccinated 3 times with a EBOV Z76 DNA vaccine plasmid, pWRG/EBOV-Z76(opt), delivered by a PharmaJet disposable syringe jet injection device. The limit of quantification for titers was 20 (dashed line). Dotted lines at 1000 and 10 000 are included for reference points. C, Animals were challenged on day 70 with 100 plaque-forming units of EBOV and viral loads were followed. Abbreviations: BLD, below limit of detection (<10 genome equivalents); HRP, horseradish peroxidase; ND, not determined; OD₄₉₀, optical density at 490 nm; VNA, virus neutralizing antibody .