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. 2019 Jul 22;14(7):e0220196.
doi: 10.1371/journal.pone.0220196. eCollection 2019.

Superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length Spike protein of Middle East respiratory syndrome coronavirus

Myung Hee Kim  1 , Hyun Jik Kim  2 , Jun Chang  1
Affiliations

Superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length Spike protein of Middle East respiratory syndrome coronavirus

Myung Hee Kim et al. PLoS One. .

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) causes an acute and severe lower respiratory illness as well as vomiting, diarrhea, and renal failure. Because no licensed MERS-CoV vaccines are currently available, preventive and therapeutic measures are urgently needed. The surface spike (S) glycoprotein of MERS-CoV, which binds to the cellular receptor dipeptidyl peptidase 4 (DPP4), is considered as a major target for MERS-CoV vaccine development. Here, we designed recombinant replication-deficient adenovirus-based vaccines expressing the N-terminal domain (rAd/NTD) and receptor-binding domain (rAd/RBD) of the MERS-CoV S1 subunit and full-length Spike protein (rAd/Spike). We found that immunization with candidate vaccines via intranasal route induced S1-specific IgG antibodies and neutralizing antibodies against MERS spike pseudotyped virus. Especially, rAd/Spike induced the highest neutralizing antibody titer and the strongest cytokine-induced T cell responses among the three candidate vaccines. To compare the immune responses induced by different administration routes, rAd/Spike was administered via intranasal, sublingual, or intramuscular route. All these administration routes exhibited neutralizing effects in the serum. MERS-CoV-specific neutralizing IgA antibodies in the bronchoalveolar lavage fluid were only induced by intranasal and sublingual administration but not by intramuscular administration. Intranasal administration with rAd/Spike also created resident memory CD8 T cells in the airway and lung parenchyma. Taken together, our results showed that both the humoral and cellular immune responses are highly induced by rAd/Spike administration, suggesting that rAd/Spike may confer protection against MERS-CoV infection.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Construction and characterization of rAd-based vaccines.
(A) Schematic representation of rAd/Spik, rAd/RBD, and rAd/NTD. A shuttle vector carrying the codon-optimized full-length MERS-CoV Spike gene, RBD gene, and NTD gene was constructed, as shown in the diagram. The vectors were used to generate recombinant replication-deficient adenovirus rAd/Spike, rAd/RBD, and rAd/NTD by homologous recombination with adenoviral genomic DNA. Numbers indicate amino acids. SP, signal peptide; NTD, N-terminal domain; RBD, receptor-binding domain; TM, transmembrane domain. (B-D) Spike, RBD, and NTD expressions in the lysate (L) and supernatant (S) of HEK293 cells infected with (B) rAd/Spike, (C) rAd/RBD, and (D) rAd/NTD, respectively. The expressions of Spike and RBD were confirmed via immunoblotting assay using MERS-CoV spike protein S1 (aa 1–725) rabbit polyclonal antibody. NTD expression were confirmed by immunoblotting assay with anti-rAd/Spike immune serum.
Fig 2
Fig 2. Immunization with rAd/NTD, rAd/RBD, or rAd/Spike elicits humoral and CD8 T cell immune responses.
Female BALB/c mice (4 mice/group) were immunized twice at week 0 and 2 with 1×107 PFUs of rAd/NTD, rAd/RBD, or rAd/Spike via the intranasal (IN) route, whereas control animals received 1×107 PFUs of rAd/mock. Sera and BALFs were collected for measuring antibody responses 7 days after the second immunization. (A) Systemic MERS S1-specific serum IgG antibody titers were determined by ELISA. (B) Mucosal MERS S1-specific IgA antibody titers in BALFs were measured by ELISA. (C) On day 7 after the last immunization, lung cells were harvested from mice and stained with Kd/S291 tetramer, anti-CD8, and anti-CD44 antibody. The frequency of Kd/S291 tetramer-positive and CD44-positive cells among the total lung CD8-positive cell population is indicated in the upper right quadrant. (D) The average percentages of Kd/S291 tetramer-specific CD8 T cells in the lungs. Data are representative of at least two independent experiments with similar results and average SEM value of four mice. ns, not significant; ***p<0.001; **p<0.01.
Fig 3
Fig 3. The neutralizing capacity directed against MERS Spike pseudotyped virus.
Groups of mice were immunized as indicated in Fig 2. On day 7 after the last immunization, sera and BALFs were collected from each group. Serially diluted sera and BALFs were evaluated for their neutralizing activity against MERS Spike pseudotyped virus in Huh 7.5 cells. The transduction percentage was calculated by measuring GFP expression in comparison with the expression in the control pseudovirus-infected cells. The dotted horizontal line indicates 50% neutralization. (A) Neutralization activities in serially diluted sera. (B) The IC50 (50% inhibitory concentration) neutralization titers in the sera. (C) The neutralization activities in serially diluted BALFs. Data are representative of at least two independent experiments with similar results and average SEM value of four mice. (D) NHNE cells (106 cells/well) were inoculated with 300 μl of virus solution (MERS-CoV 10μl/PBS 10 ml) and N2 gene level of MERS-CoV was assessed at 1 day post-infection using real-time PCR. To prove the effect of neutralizing antibody for MERS-CoV spike protein, targeted antibody of MERS-CoV spike protein was diluted to 1:10, 1:50, 1:250 and the diluted antibodies were mixed with the virus solution at a ratio of 1:1 and maintained for 1 hour before infection to NHNE cells. Then, N2 gene level of MERS-CoV was assessed at 1 day post-infection using real-time PCR. The same experiments were carried out using mock antibody and the result was compared with that of targeted antibody for MERS-CoV spike protein. ns, not significant; ***p<0.001; **p<0.01; *p<0.05.
Fig 4
Fig 4. Characterization of neutralizing antibody responses induced by rAd/Spike.
Groups of mice were immunized as indicated in Fig 2. On day 7 after the last immunization, sera were collected from immune mice. (A) The NTD-specific, (B) RBD-specific, and (C) S2-specific IgG antibody responses were measured by ELISA. NTD, RBD, and S2 proteins were used as coating antigens (200 ng/well), and goat anti-mouse IgG-HPR was used as the detection antibody. (D) Protein competition neutralization assay. Sera collected from rAd/Spike immune mice were diluted at a ratio of 1:640 and were assayed for neutralization of MERS Spike pseudotyped virus in the presence of soluble NTD, RBD, S1, or S2 proteins at a concentration of 10 μg. Data are represented as mean ± SEM results of four mice from at least two independent experiments. ns, not significant; ***p<0.001; **p<0.01; *p<0.05.
Fig 5
Fig 5. Detection of humoral immunity induced by rAd/spike via different immunization routes.
Female BALB/c mice were immunized twice at week 0 and 2 with 1×108 PFU of rAd/Spike via IN, sublingual (SL), or intramuscular (IM) route. Control mice were immunized intranasally with 1×108 PFU of rAd/mock. Sera were collected 2 weeks after each vaccination for measuring antibody responses. BALFs were harvested 2 weeks after the last immunization (A) Systemic RBD-specific IgG titers in sera were measured using ELISA. (B) Mucosal RBD-specific IgA titers in BALFs were measured using ELISA. The data are presented as mean (Log2 endpoint titers) ± SEM values of four mice per group. ns, not significant; ***p<0.001; **p<0.01; *p<0.05; ND, not detected.
Fig 6
Fig 6. Comparison of neutralizing activity induced by rAd/spike via different immunization routes.
Groups of mice were immunized as indicated in Fig 5. Two weeks after last immunization, sera and BALFs were collected from immune mice and heat-inactivated at 56°C for 30 min before detecting neutralizing antibodies. (A) The neutralizing antibody titers in serially diluted sera were measured via MERS-CoV pseudovirus neutralization assay in Huh 7.5 cells. (B) IC50 neutralization titer in the serum. (C) Neutralizing antibody titers in serially diluted BALFs. (D) IC50 neutralization titer in BALFs. The data are presented as mean ± SEM values of four mice per group. ns, not significant; ***p<0.001.
Fig 7
Fig 7. Generation of CD8 TRM cells in the airway and lung parenchyma following intranasal immunization with rAd/Spike but not following intramuscular immunization.
On day 30 after immunization with 1×108 PFUs of rAd/Spike via the IN or IM route, mice were injected intravenously with 2 μg of anti-CD45 antibody and sacrificed after 5 min. (A) Representative staining of Kd/S291 tetramer-specific CD8 T cells distributed in the airway, lung parenchyma, lung vasculature, and spleen of mice immunized via the IN or IM route. (B) The numbers of Kd/S291 tetramer-positive CD8 T cells in the airway, lung parenchyma, lung vasculature, and spleen 30 days after IN (white bar) or IM (black bar) immunization. (C) Representative staining of CD69 and CD103 expressed on Kd/S291 tetramer-positive CD8 T cells, which are distributed in the airway, lung parenchyma, lung vasculature, and spleen of mice on day 30 after IN or IM immunization. (D) The number of Kd/S291 tetramer-specific CD8 T cells expressing CD69 and CD103 in the airway, lung parenchyma, lung vasculature, and spleen 30 days after IN or IM immunization. Data are presented as mean ± SEM values of four mice per group. ***p<0.001; **p<0.01.
Fig 8
Fig 8. Detection of long-lasting humoral immune responses elicited rAd/Spike immunization.
Female Balb/c mice were immunized three times at 0, 3, and 11 weeks with 1×107 PFU of rAd/Spike via IN route. Sera were collected for measuring antibody responses at 2, 6, 15, and 30 weeks. Control mice were immunized intranasally with 1×108 PFU of rAd/mock. (A) Immunization scheme. (B) Systemic RBD-specific IgG antibody titers were measured by ELISA after each immunization. (C) Mucosal RBD-specific IgA antibody titers in BALFs were measured 19 weeks after the last immunization by ELISA. (D) Neutralization antibody responses in sera were measured via MERS-CoV pseudovirus neutralization assay at 15 and 30 weeks. (E) Neutralizing activities in BALFs at 19 weeks after the third immunization. Data are presented as mean ± SEM values of four mice per group. ***p<0.001.
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This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea [Grant number: HI15C2923].
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