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. 2017 Jan 3;91(2):e02040-16.
doi: 10.1128/JVI.02040-16. Print 2017 Jan 15.

One-Health: A Safe, Efficient, Dual-Use Vaccine for Humans and Animals Against Middle East Respiratory Syndrome Coronavirus and Rabies Virus

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Free PMC article

One-Health: A Safe, Efficient, Dual-Use Vaccine for Humans and Animals Against Middle East Respiratory Syndrome Coronavirus and Rabies Virus

Christoph Wirblich et al. J Virol. .
Free PMC article

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012 and is a highly pathogenic respiratory virus. There are no treatment options against MERS-CoV for humans or animals, and there are no large-scale clinical trials for therapies against MERS-CoV. To address this need, we developed an inactivated rabies virus (RABV) that contains the MERS-CoV spike (S) protein expressed on its surface. Our initial recombinant vaccine, BNSP333-S, expresses a full-length wild-type MERS-CoV S protein; however, it showed significantly reduced viral titers compared to those of the parental RABV strain and only low-level incorporation of full-length MERS-CoV S into RABV particles. Therefore, we developed a RABV-MERS vector that contained the MERS-CoV S1 domain of the MERS-CoV S protein fused to the RABV G protein C terminus (BNSP333-S1). BNSP333-S1 grew to titers similar to those of the parental vaccine vector BNSP333, and the RABV G-MERS-CoV S1 fusion protein was efficiently expressed and incorporated into RABV particles. When we vaccinated mice, chemically inactivated BNSP333-S1 induced high-titer neutralizing antibodies. Next, we challenged both vaccinated mice and control mice with MERS-CoV after adenovirus transduction of the human dipeptidyl peptidase 4 (hDPP4) receptor and then analyzed the ability of mice to control MERS-CoV infection. Our results demonstrated that vaccinated mice were fully protected from the MERS-CoV challenge, as indicated by the significantly lower MERS-CoV titers and MERS-CoV and mRNA levels in challenged mice than those in unvaccinated controls. These data establish that an inactivated RABV-MERS S-based vaccine may be effective for use in animals and humans in areas where MERS-CoV is endemic.

Importance: Rabies virus-based vectors have been proven to be efficient dual vaccines against rabies and emergent infectious diseases such as Ebola virus. Here we show that inactivated rabies virus particles containing the MERS-CoV S1 protein induce potent immune responses against MERS-CoV and RABV. This novel vaccine is easy to produce and may be useful to protect target animals, such as camels, as well as humans from deadly MERS-CoV and RABV infections. Our results indicate that this vaccine approach can prevent disease, and the RABV-based vaccine platform may be a valuable tool for timely vaccine development against emerging infectious diseases.

Keywords: MERS-CoV; coronavirus; immunization; rabies; rhabdovirus.

Figures

FIG 1
FIG 1
Schematic illustration of the MERS-CoV vaccine constructs used in this study. Spike protein cDNA was inserted between the N and P genes of the SAD-B19-derived RABV vaccine vector BNSP333, which contains a mutation in the glycoprotein gene that eliminates neurotropism of the parent SAD-B19 strain. BNSP333-S contains the wild-type coding sequence of the spike protein of MERS-CoV. The BNSP333-S1-G construct expresses a chimeric protein that contains the entire S1 domain fused to the C-terminal part of the RABV G glycoprotein (amino acids 428 to 524), which encompasses the entire cytoplasmic domain (CD), the TM domain, and 31 amino acids of the ectodomain (E31) of RABV G. Different structural elements of the spike protein are indicated in the full-length construct, including the signal peptide, the receptor-binding domain (RBD), the fusion peptide (FP), heptad repeat (HR) regions 1 and 2, the TM domain, and the CD. Numbers indicate amino acid positions in the spike protein.
FIG 2
FIG 2
Characterization of BNSP333-S expressing the full-length MERS-CoV spike protein. (A) One-step growth curves on Vero cells demonstrate decreased fitness of the full-length S protein-expressing virus. (B) Western blot analysis of the time course of viral protein expression in Vero cells infected with the parent vector BNSP333 or BNSP333-S demonstrating S protein expression. (C) Immunofluorescence staining of permeabilized Vero cells at 48 h postinfection. Staining of cells infected with BSNP333-S revealed the presence of fused multinucleated cells. (D) SDS-PAGE analysis of purified virions after sucrose gradient purification. Letters indicate the positions of the RABV L, G, N, P, and M proteins. Numbers to the left indicate the sizes of the molecular mass standards. The bottom panel shows Western blot analysis of purified particles probed with anti-S antibody and with monoclonal antibody against RABV G.
FIG 3
FIG 3
Characterization of BNSP333-S1 expressing a chimeric S1-G fusion protein. (A) One-step growth curves on Vero cells. (B) Time course of protein expression in infected Vero cells. (C) Immunofluorescence staining of permeabilized Vero cells at 48 h postinfection labeled for either the RABV G protein or the MERS-CoV S protein. (D) SDS-PAGE analysis of purified virions after sucrose gradient purification. Letters indicate the positions of the RABV L, G, N, P, and M proteins. Numbers to the left indicate the sizes of the molecular mass standards. The bottom panel shows Western blotting of purified particles probed with anti-S antibody.
FIG 4
FIG 4
The S1-G chimera is incorporated into virions. Particles collected from the supernatant of infected Vero E6 cells were centrifuged on a 15 to 65% linear sucrose gradient. Viral proteins were detected in the collected fractions by Western blotting with the indicated antibodies. MERS-CoV S peaked in fraction 17, as did the RABV G, N, and P proteins. The rightmost lane shows particles pelleted from the supernatant of infected Vero cells. Numbers at the bottom show the sucrose concentrations in the collected fractions.
FIG 5
FIG 5
Immunoelectron microscopy of Vero E6 cells infected with BNSP333-S1. Filled arrowheads pointing to small gold particles indicate labeling with monoclonal antibody specific for RABV G. Large gold particles indicate staining with anti-S polyclonal rabbit serum.
FIG 6
FIG 6
Analysis of the immune response to BNSP333-S1 in mice. BALB/c mice were immunized three times (days 0, 7, and 21) with 10 μg chemically inactivated particles of BNSP333-S1 (groups B and C) (n = 10) or BNSP333-GP (BNSP333 expressing Zaire ebolavirus GP) (group A) (n = 5) or mock immunized with PBS (group D, n = 5). Serum was collected from each mouse at day 35 for analysis by a MERS-CoV S-specific ELISA. All mice immunized with BNSP333-S1 developed a strong humoral immune response to the S1 subunit and RABV G. No response above background levels was detected in unvaccinated control mice (PBS group). OD, optical density.
FIG 7
FIG 7
Sera from BNSP333-S1-vaccinated mice neutralize MERS-CoV. Sera collected on day 35 were assayed in neutralization assays against MERS-CoV on Vero E6 cells. The dilution at which 50% of the cells showed cytopathic effect was averaged across 5 mice and graphed as the geometric mean titer for each vaccination group described in the legend of Fig. 6. * denotes a P value of <0.5. LOD, limit of detection.

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