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Comparative Study
. 2018 Apr 4;7(1):60.
doi: 10.1038/s41426-018-0056-7.

Enhanced protection in mice induced by immunization with inactivated whole viruses compare to spike protein of middle east respiratory syndrome coronavirus

Yao Deng  1 , Jiaming Lan  1   2 , Linlin Bao  3 , Baoying Huang  1 , Fei Ye  1 , Yingzhu Chen  1 , Yanfeng Yao  3 , Wenling Wang  1 , Chuan Qin  3 , Wenjie Tan  4
Affiliations
Comparative Study

Enhanced protection in mice induced by immunization with inactivated whole viruses compare to spike protein of middle east respiratory syndrome coronavirus

Yao Deng et al. Emerg Microbes Infect. .

Abstract

The persistent public health threat of infection with Middle East respiratory syndrome coronavirus (MERS-CoV) highlights the need for an effective and safe MERS-CoV vaccine. In this study, we prepared and vaccinated mice with either a Spike (S) protein or inactivated whole MERS-CoV (IV) with a combined adjuvant (alum+CpG) as a vaccine formulation. Similar levels of the anti-S protein IgG response and neutralizing activity were induced by both the S protein and IV vaccines. In addition, immune responses against three other structural proteins, the envelope (E), membrane (M), and nucleocapsid (N) proteins, were also detected in sera of mice that received IV. No antigen-specific T-cell immunity was detected after vaccination based on the interferon-γ ELISpot assay. Mice were transduced with Ad5-hDPP4 after the final immunization and were then challenged with MERS-CoV (1 × 105 plaque-forming units). Compared with the control group (adjuvant alone), mice immunized with the S protein or IV showed slightly lower pathological damage in the lung, as well as reduced antigen expression and lung virus titers. Mice that received IV formulations also showed increased protective immunity (almost no live virus was isolated from the lung). In conclusion, our data indicate that immunization with our IV formulation induced enhanced protection in mice compared to immunization with the S protein against MERS-CoV, which should be further tested in camels and clinical trials.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Vaccine candidates and immunization schedule.
Western blot analyses of Middle East respiratory syndrome coronavirus (MERS-CoV) S and inactivated whole MERS-CoV(IV) vaccines using mouse anti-S (a) and anti-NP monoclonal antibodies (mAbs) (b). Schematic of the study (c)
Fig. 2
Fig. 2. Antigen-specific IgG response after vaccination.
Anti-S (a) and anti-NP (b) specific IgGs were detected at 2, 6, and 10 weeks. S protein-specific antibody isotypes induced by vaccination after 10 weeks (c). Values are the means ± standard error of the mean (SEM). Significant values are defined by *P < 0.05, **P < 0.01 and ***P < 0.001
Fig. 3
Fig. 3. Envelope (E) and membrane (M)-specific immune responses following vaccination with IV vaccine as determined by Western blotting (WB) and indirect immunofluorescence assay (IFA).
Samples were collected from 293 T cells that were transiently transfected with the pCAGGS-E (E), pCAGGS-M (M) or control plasmid pCAGGS at 24 h post-transfection and subjected to WB (a) or IFA (b) using IV-immunized mouse serum at a 1:400 dilution as the primary antibody
Fig. 4
Fig. 4. Neutralizing antibodies induced by S protein and IV vaccine against MERS-CoV pseudovirus particles and MERS-CoV.
Neutralizing antibody titers against MERS-CoV pseudovirus particles. (a, b) and MERS-CoV (c, d) were determined by plaque reduction neutralization assays at 6 and 10 weeks. Representative results of the plaque reduction neutralization (PRNT) assay for the detection of neutralization activity in the sera of mice (e). Approximately 30 pfu of the virus stock (hCoV-EMC) was used to infect Vero cells in 12-well plates with or without heat-inactivated sera from immunized mice 2 weeks after the third immunization. PRNT50 was calculated after the plaques were counted. Significant values are defined by **p < 0.01.
Fig. 5
Fig. 5. The IV formulation provides enhanced protection in mice compared to the S formulation, indicating ameliorated lung pathology, reduced viral titers and expression of virus antigens in the lungs of mice with IV or S.
Representative results of hematoxylin-eosin (HE) staining (×400) in the lungs of mock-treated or immunized mice (a). Immunohistochemistry staining (×400) with anti-S and anti-NP mAbs (b). Lung virus titers 3 days after MERS-CoV challenge (c) as detected by virus isolation and titration at day 3 post-challenge. Values are the means ± SEM. Significant values are defined by ***P < 0.001
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