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. 2017 Jan 3;35(1):10-18.
doi: 10.1016/j.vaccine.2016.11.064. Epub 2016 Nov 26.

The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

Lan Jiaming  1 , Yao Yanfeng  2 , Deng Yao  3 , Hu Yawei  4 , Bao Linlin  2 , Huang Baoying  3 , Yan Jinghua  4 , George F Gao  5 , Qin Chuan  2 , Tan Wenjie  6
Affiliations

The recombinant N-terminal domain of spike proteins is a potential vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) infection

Lan Jiaming et al. Vaccine. .

Abstract

The persistent public health threat of infection with the Middle East respiratory syndrome coronavirus (MERS-CoV) highlights the need for an effective MERS-CoV vaccine. Previous studies have focused mainly on the receptor-binding domain (RBD) on the spike protein of MERS-CoV. Herein, we investigated the immunogenicity and protective potential of the recombinant N-terminal domain (rNTD) of spike proteins as a vaccine candidate. BALB/c mice vaccinated with 5 or 10μg of rNTD protein demonstrated a significant humoral immune response (serum IgG and neutralizing activity). Additionally, according to the enzyme-linked immunospot, intracellular cytokine staining, and cytometric bead array assays, significant and functional T-cell immunity was induced by 10μg of the rNTD vaccination with aluminum and CpG adjuvant. Furthermore, rNTD-immunized mice showed reduced lung abnormalities in a MERS-CoV-challenge mouse model transfected with an adenoviral vector expressing human DPP4, showing protection consistent with that found with rRBD vaccination. These data show that rNTD induced potent cellular immunity and antigen-specific neutralizing antibodies in mice and that it demonstrated protective capacity against a viral challenge, indicating that rNTD is a vaccine candidate against MERS-CoV infection.

Keywords: Animal model; MERS-CoV; Mice; NTD; RBD; Vaccine.

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Figures

Fig. 1
Fig. 1
Description of the N-terminal domain (NTD) immunogen and vaccination schedule. (A) The location of the NTD protein on the Middle East respiratory syndrome coronavirus MERS-CoV spike (S) protein. The recombinant (r)NTD protein consists of 336 amino acid (aa) residues (18–353) of S protein. A gp67 signal peptide (SP) was added to the N terminus for expression of the rNTD protein. (B) Purified rNTD protein detected by SDS-PAGE (left) and Western blot (right). The purified rNTD protein was separated by a 10% SDS-PAGE and stained with 0.25% Coomassie brilliant blue. Anti-NTD polyclonal antibody and infrared ray-labeled secondary antibody were used for the Western blot assay. Lane 1: protein molecular weight marker; lane 2: purified rNTD protein. (C). Vaccination schedule and detection. Mice received three vaccinations consisting of 5 or 10 μg of rNTD protein combined with adjuvants at 4-week intervals. Sera were collected at the indicated times to analyze the humoral immune response. Six mice from each group were sacrificed 2 weeks after the last immunization. The spleens were harvested for enzyme-linked immunospot (ELISpot), intracellular cytokine staining (ICS), and cytometric bead array (CBA) assays. In parallel experiments, the remaining mice were challenged with MERS-CoV to detect the protective effect elicited by the rNTD protein. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Humoral immunity response induced in rNTD-immunized mice. (A) NTD-specific IgG titers were determined by enzyme-linked immunosorbent assay (ELISA) at 6, 10, and 22 weeks (14 weeks after the last immunization). (B) The neutralization activity in the sera of mice was detected using the pseudovirus neutralization assay at 10 or 22 weeks. (C) Representative results of the plaque reduction neutralization (PRNT) assay for the detection of neutralization activity in the sera of mice. Approximately 30 pfu 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 (10W) after the third immunization. PRNT50 was calculated after the plaques were counted. Statistical significance was set at ∗P < 0.05 and ∗∗∗P < 0.001.
Fig. 3
Fig. 3
Cellular immune response induced by the rNTD-immunized mice detected by ELISpot and ICS. (A) ELISpot analysis of IFN-γ secretion cells in spleen. Data are expressed as spot-forming cells (SFCs) responding to peptide-specific IFN-γ secretion and presented as means with standard deviation. (B) ICS assay to detect specific IFN-γ and IL-2 expression in CD8+ T-cells of the splenocytes in mice. (C) ICS assay to detect specific IFN-γ and IL-2 expression in the CD4+ T-cells of the splenocytes in mice. The thresholds for statistically significant differences between groups were set at ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.
Fig. 4
Fig. 4
Cytokine production of splenocytes among immunized mice was determined by CBA 2 weeks after the third immunization. Splenocytes were stimulated for 24 h with pooled peptides consisting of the NTD, and the supernatants were harvested. Cytokine concentration was detected using a CBA kit. The cytokines of Th1, Th2, and Th17 are shown in the figures. (A) IL-2 and IFN-γ. (B) IL-6 and IL-10. (C) IL-17A. Statistical significance was set at ∗P < 0.05.
Fig. 5
Fig. 5
rNTD or rRBD vaccination reduced respiratory tract pathology in mice after MERS-CoV challenge. Representative results of hematoxylin-eosin (HE) staining in the lung (A–C) and trachea (D–F) of mock-treated or immunized mice. Severe lesions including the loss of pulmonary alveolus (represented by the white vacuole) and diffuse inflammatory cell infiltration (represented by the dark purple point) are shown (figure A). In contrast, milder lesions were observed among mice immunized with rRBD (figure B) or NTD (figure C), as the pulmonary alveolus was highly visible with less inflammatory cell infiltration. Inflammatory cell infiltration and impaired epithelium of the tunica mucosa bronchiorum were seen in the mock group (D). rRBD (E) or rNTD (F) alleviated the pathologic damage in the trachea of immunized mice. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 6
Fig. 6
IHC detection of virus antigen expression in mouse tissue after challenge with MERS-CoV. Lung (A–C) and trachea (D–F) sections were assessed using rabbit polyclonal antibody to MERS-CoV nucleoprotein (NP) 3 days after the MERS-CoV challenge. The dark purple spot marked the inflammatory cell infiltration, and the brown particle marked the antigen of MERS-CoV. The MERS-CoV was located mainly in the trachea. Additionally, the lung tissue showed MERS-CoV expression in all immunized groups.
Supplementary Fig. 1
Supplementary Fig. 1
Synthesized peptide pools spanned the entire S protein were consisted of 161 short peptides. Each peptide was 18-mer and overlapped by 10 amino acids. Splenocytes of mice immunized with the recombinant Ad5 vector-based vaccines expressing full-length MERS-CoV S protein were collected for the detection of T lymphocyte immunodominant epitopes by an ELISpot assay. The represented results were shown in this figure. The highest level of T cell responses was induced by the peptide pools of X15 and Y15. Both of the two peptide pools contained the peptide of 37 which sequence of aa was TIKYYSIIPHSIRSIQSD. Thus this peptide was one of the T lymphocyte immunodominant epitopes of S protein.
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