Systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector-based vaccines carrying the spike protein of Middle East respiratory syndrome coronavirus

Abstract

An ideal vaccine against mucosal pathogens such as Middle East respiratory syndrome coronavirus (MERS-CoV) should confer sustained, protective immunity at both systemic and mucosal levels. Here, we evaluated the in vivo systemic and mucosal antigen-specific immune responses induced by a single intramuscular or intragastric administration of recombinant adenoviral type 5 (Ad5) or type 41 (Ad41) -based vaccines expressing the MERS-CoV spike (S) protein. Intragastric administration of either Ad5-S or Ad41-S induced antigen-specific IgG and neutralizing antibody in serum; however, antigen-specific T-cell responses were not detected. In contrast, after a single intramuscular dose of Ad5-S or Ad41-S, functional antigen-specific T-cell responses were elicited in the spleen and pulmonary lymphocytes of the mice, which persisted for several months. Both rAd-based vaccines administered intramuscularly induced systemic humoral immune responses (neutralizing IgG antibodies). Our results show that a single dose of Ad5-S- or Ad41-S-based vaccines represents an appealing strategy for the control of MERS-CoV infection and transmission.

Keywords: Middle East respiratory syndrome coronavirus; adenoviral vector; immunity; spike protein; vaccine.

Figure 1

Construction and identification of recombinant adenovirus type 41–Middle East respiratory syndrome coronavirus spike protein (Ad41-MERS-S) or Ad5-MERS-S. (a) Schematic of recombinant Ad41-MERS-S and Ad5-MERS-S. E1 region-deleted Ad41 or Ad5 vectors were constructed, with target genes (GFP or MERS-S) inserted into the deleted E1 region. A cytomegalovirus promoter and simian virus 40 polyA tail were used to control target gene expression. (b) Detection of MERS-S by Western blotting. Exponentially growing HEK293 cells were infected by Ad41-MERS-S or Ad5-MERS-S for 48 hr. Total proteins were then extracted and resolved by SDS–PAGE. MERS-S expression was confirmed by Western blotting using polyclonal rabbit anti-emc antibodies; proteins from GFP-containing viruses were used as a negative control. (c) Detection of MERS-S by immunofluorescence. HEK293 cells were infected as described in (b). The cells were then fixed with cold methanol in situ and probed with polyclonal rabbit anti-emc antibodies and FITC-conjugated goat anti-rabbit antibodies. Uninfected HEK293 cells were used as a negative control.

Figure 2

Adenovirus- and Middle East respiratory syndrome coronavirus spike protein (MERS-S)-specific IgG responses in the immunized mice. BALB/c mice (n = 10 per group) were immunized with different immunogens or by different routes of exposure, as described in Table1. Four weeks post-immunization (short term), half of the mice in each group were killed; various tissues and organs were then analysed for antibody responses against adenovirus or MERS-S by ELISA. The remaining mice were killed 16 weeks post-immunization (long term) and analysed as for the short-term group. (a) short-term anti-recombinant receptor-binding domain (rRBD) IgG in serum; (b) long-term anti-rRBD IgG in serum. Statistically significant differences are indicated as follows: *P < 0.05, **P < 0.01, and ***P < 0.001

Figure 3

Neutralization antibody responses in immunized mice. Sera were collected 4 (a) and 16 weeks (b) post-vaccination, heat-inactivated, and examined for neutralizing antibodies using the Middle East respiratory syndrome coronavirus (MERS-CoV) pseudovirus system. Virus neutralizing titres were defined as the dilution at which the relative inhibition rate was 90%. The data are presented as means ± standard error of the mean (SEM). Statistically significant differences are indicated as follows: *P < 0·05, **P < 0·01, and ***P < 0·001.

Figure 4

ELISPOT analysis of interferon-γ (IFN-γ) secretion by splenocytes and pulmonary lymphocytes. Lymphocytes were isolated 4 and 16 weeks after immunization. The data are expressed as spot-forming cells (SFCs) responding to receptor-binding domain (RBD)-specific peptides and presented as means ± standard error of the mean (SEM). Statistically significant differences are indicated as follows: *P < 0·05 and ***P < 0·001. Lymphocytes were isolated from the spleens (a) of the 4-week post-immunization mice, and from the spleens (b) and lungs (c) of the 16-week post-immunization mice.

Figure 5

Cytometric bead assay to determine the in vitro cytokine production of splenocytes from immunized mice 16 weeks after immunization. (a) Interferon-γ (IFN-γ), (b) interleukin-2 (IL-2), (c) IL-10 and (d) tumour necrosis factor-α (TNF-α). Statistical significance was defined by **P < 0·01. Each group contained six mice.