key: cord-0759824-10ux0fut
authors: Yang, Ye Lin; Kim, Ju; Jeong, Yongsu; Jang, Yong-Suk
title: Intranasal immunization with a Middle East respiratory syndrome-coronavirus antigen conjugated to the M-cell targeting ligand Co4B enhances antigen-specific mucosal and systemic immunity and protects against infection
date: 2021-12-30
journal: Vaccine
DOI: 10.1016/j.vaccine.2021.12.057
sha: f21b373ee47214a0a5daa89d820b92b8b161e71c
doc_id: 759824
cord_uid: 10ux0fut

Middle East respiratory syndrome (MERS) is a threat to public health worldwide. A vaccine against the causative agent of MERS, MERS-coronavirus (MERS-CoV), is urgently needed. We previously identified a peptide ligand, Co4B, which can enhance antigen (Ag) delivery to the nasal mucosa and promote Ag-specific mucosal and systemic immune responses following intranasal immunization. MERS-CoV infects via the respiratory route; thus, we conjugated the Co4B ligand to the MERS-CoV spike protein receptor-binding domain (S-RBD), and used this to intranasally immunize C57BL/6 and human dipeptidyl peptidase 4-transgenic (hDPP4-Tg) mice. Ag-specific mucosal immunoglobulin (Ig) A and systemic IgG, together with virus-neutralizing activities, were highly induced in mice immunized with Co4B-conjugated S-RBD (S-RBD-Co4B) compared to those immunized with unconjugated S-RBD. Ag-specific T cell-mediated immunity was also induced in the spleen and lungs of mice intranasally immunized with S-RBD-Co4B. Intranasal immunization of hDPP4-Tg mice with S-RBD-Co4B reduced immune cell infiltration into the tissues of virus-challenged mice. Finally, S-RBD-Co4B-immunized mice exhibited were better protected against infection, more likely to survive, and exhibited less body weight loss. Collectively, our results suggest that S-RBD-Co4B could be used as an intranasal vaccine candidate against MERS-CoV infection.

14 284 Comparisons among groups regarding survival rate were analyzed by the log-rank test.

285 The significance of differences between the groups was tested at p < 0.05, p < 0.01, and 286 p < 0.001. (Fig. 3) . After 17 h of S-RBD stimulation, the 345 frequency of IFN-γ-producing CD4 + and CD8 + CTLs was significantly higher in the 346 splenocyte pool of mice immunized with S-RBD-Co4B compared to those immunized 347 with unconjugated S-RBD (p < 0.05; Fig. 3A ). The cytotoxic potential was relatively 348 high in splenocytes collected from S-RBD-Co4B-immunized mice, as shown by the 349 high intracellular expression levels of CD107a (Fig. 3B) . Furthermore, effector CD4 + 350 and CD8 + CTL stimulation was significantly higher in lung lymphocytes collected from 351 mice immunized with S-RBD-Co4B compared to those immunized with unconjugated 352 S-RBD (p < 0.01) (Fig. 3C) . Likewise, the cytotoxic potential was significantly higher

The reverse primers used to amplify S-RBD and S-RBD-Co4B were 5

BamHI restriction sites are underlined

The amplified recombinant S-RBD and S-RBD-Co4B genes were cloned into a pCold II

The identities of the recombinant Ags were 132 confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western 133 blotting using anti-6× His tag (Qiagen) and polyclonal anti-RBD antibodies (Abs), as 134 described previously

The final endotoxin content was < 0.5 EU/μg of protein, as determined by 137 the LAL chromogenic endotoxin quantitation kit

Fecal immunoglobulin (Ig) A and serum IgG 145 were collected from the mice 3 days after the fifth immunization. Fecal samples were 8 146 collected by placing each mouse in a metabolic or ordinary cage for up to 30 min, and 147 the weight of the collected feces was measured. To prepare fecal extracts, 1 mL PBS 148 with 0.01% sodium azide was added to 100 mg fecal samples, followed by vortexing for 149 10 min at room temperature. Extracts were collected by centrifugation for 10 min at 150 13,000 rpm at 4℃. Splenocytes and lung lymphocytes were obtained 10 days after the 151 fifth immunization

To prepare lung lymphocytes, the lungs 155 were carefully obtained from the mice and then minced into small fragments using 156 scissors. The minced tissues were digested by collagenase D, and then the lymphocytes 157 were collected via Percoll density gradient centrifugation. Finally, after filtering the 158 cells using a cell strainer

The hDPP4-Tg mice were inoculated intranasally with 10 4 162 or 10 5 plaque-forming units (PFUs) of MERS-CoV in a total volume of 60 μL after the 163 fifth immunization. To test the efficacy of booster, mice were immunized with their 164 cognate Ag (S-RBD or S-RBD-Co4B) 28 days after the fifth immunization

Ag-specific IgA and IgG from fecal and serum samples, respectively, were 171 measured using indirect ELISAs. Briefly, 96-well ELISA plates

) were coated with S-RBD 173 protein (100 ng/well) dissolved in 100 mM bicarbonate/carbonate buffer (pH 9.6) and 174 incubated overnight at 4°C, then blocked with 5% non-fat dry milk for 2 h at 37°C

After adding serially diluted sample to each well, the plates were incubated for 2 h at 176 37°C, then washed with PBS containing Tween 20. Then, the bound Abs were 177 incubated with alkaline phosphatase-conjugated anti-mouse IgA or IgG secondary Ab 178 for 2 h at 37°C, after which p-nitrophenyl phosphate substrate was added. The 179 absorbance at 405 nm was read using an ELISA plate reader

The Vero E6 cells used for the virus inhibition assay were cultured in Dulbecco's 186 modified Eagle's medium (Welgene, Gyungsan, Korea) supplemented with 10% heat-187 inactivated FBS and 5% CO 2 at 37°C. To measure Ag-specific Ab-mediated inhibition 188 of the hDPP4 receptor and MERS-CoV, virus was pre-incubated with Abs collected 189 from intranasally immunized mice for 30 min at room temperature

Briefly, total RNA was extracted using TRI 194 Reagent according to the manufacturer's instructions. RNA was converted into cDNA 195 using an M-MLV Reverse Transcription Kit

The forward and reverse primer sequences for 199 amplifying the upE gene were 5′

The forward and reverse primer sequences 201 used to amplify the internal control gene β-actin were 5′-CGT ACC ACA GGC ATT 202 GTG A-3′ and 5′

To measure the number of IgG-secreting cells (SCs) in splenocytes, an enzyme-207 linked immunosorbent spot assay (ELISPOT) assay was performed. The wells of Multi-208 Screen filter plates (Millipore, Billerica, MA, USA) were activated with 35% ethanol, 209 pre-coated with goat anti-mouse IgG (100 ng/well; Bethyl Laboratories

Retroviruses pseudotyped with MERS-CoV spike (MERS-CoV-S) and β

After 24 h of incubation, total RNA 227 was extracted and subjected to qRT-PCR using specific primers for β-galactosidase (5'-228 GGG TTG TTA CTC GCT CAC ATT-3' and 5'-CGG TTT ATG CAG CAA CGA G-229 3') and Vero E6 β-actin

β-actin, and calculated in terms of the fold difference using 234 CFX Maestro software

RBD-Co4B compared to unconjugated S-RBD (p < 0.05) (Fig. 3D)

Next, we characterized the effects of intranasal immunization with Co4B-356 conjugated Ag on immune stimulation, by analyzing Th1 cytokine expression in 357 splenocytes and lung lymphocytes that had been stimulated for 48 h with S-RBD

IFN-γ, and TNF-α were remarkably enhanced in 359 both splenocytes and lung lymphocytes harvested from S-RBD-Co4B-immunized mice 360 compared to mice immunized with unconjugated S-RBD. In particular, IFN-γ 361 expression in splenocytes and lung lymphocytes following S-RBD stimulation was 362 greatly enhanced in S-RBD-Co4B-immunized mice. Collectively, these results suggest 363 that intranasal immunization with Co4B

370 the induction of S-RBD-specific mucosal and systemic immune responses in hDPP4-Tg 371 mice, because mice expressing murine DPP4 only are not susceptible to MERS-CoV 372 infection [25]. We evaluated the induction of S-RBD-specific fecal IgA and serum IgG 373 in hDPP4-Tg mice that were intranasally immunized with S-RBD or S-RBD-Co4B 374 (Fig. 4A). The levels of S-RBD-specific fecal IgA (left) and serum IgG (right) were 375 higher in mice immunized with S-RBD-Co4B compared to S-RBD, and the difference 18 with S-RBD-Co4B induces higher mucosal and systemic immune 378 responses than immunization

Abs could inhibit MERS-CoV infections in hDPP4 receptor-expressing Vero E6 cells 380 (Fig. 4B). We pre-incubated 100-fold diluted fecal samples with MERS-CoV

PFUs) and used the samples to infect Vero E6 cells; MERS-CoV upE gene expression 382 was significantly lower in Vero E6 cells infected with fecal samples collected from S-383 RBD-Co4B-immunized mice compared to those immunized with PBS (p < 0.01) and 384 unconjugated S-RBD (p < 0.05) (Fig. 4B, left)

Similar to the results from the 393 experiments with C57BL/6 mice, the expression of IL-10, IFN-γ, and TNF-α was 394 significantly higher in both splenocytes (p < 0.05) and lung lymphocytes (p < 0.001) 395 collected hDPP4-Tg mice immunized with from S-RBD-Co4B compared to 396 unconjugated S-RBD-immunized hDPP4-Tg mice. Taken together, these results suggest 397 that intranasal immunization with Co4B-conjugated Ag efficiently induced Th1-type 398 immune stimulation capable of promoting CD4 + and CD8 + effector CTL

Intranasal immunization with S-RBD-Co4B elicits potent protective immunity and 402 prevents lung damage in hDPP4-Tg mice challenged with MERS-CoV 403

In our previous study, we found that hDPP4-Tg mice challenged with 10 4 or 10 5

CoV exhibited body weight loss and mortality of 60% and 100% at 12 406 days post infection, respectively [15]. To determine whether intranasal immunization 407 with S-RBD-Co4B could effectively protect hDPP4-Tg mice challenged with MERS

we monitored the body weight, survival, and tissue damage of the mice following 409 intranasal challenge infection with MERS-CoV (Fig. 5). Following inoculation of 410 hDPP4-Tg mice with 10 4 PFUs of MERS-CoV, there was no significant difference in 411 body weight between the mice immunized with conjugated and unconjugated Ags

5A, left); of MERS-CoV (Fig. 5B). Body weight loss and 416 death began to occur at 6 days after the challenge infection with 10 5 PFUs of MERS

CoV (Fig. 5B, left); the survival rate was significantly higher in mice immunized with

40%) compared to those immunized with PBS control and unconjugated 419 S-RBD

We collected mouse lung and spleen tissues from the mice 7 days after MERS

CoV infection for histological analysis. We found no prominent differences in the 20

Although the lungs of all mice challenged with MERS-CoV 428 displayed squamous metaplasia and epithelial hypertrophy, S-RBD-Co4B-immunized 429 mice exhibited reduced lung tissue lesion formation compared to PBS-and S-RBD-430 immunized mice (Fig. 5C, upper panels). White pulp thickening, as well as marginal 431 zone and red pulp border collapse, were observed in the spleens of MERS-CoV-432 challenged mice, while marginal zone expansion and white pulp widening were detected 433 in the spleens of S-RBD-Co4B-immunized mice. Moreover, the red pulp and marginal 434 zone borders were clearly identifiable in the spleens of S-RBD-Co4B

Several MERS-CoV outbreaks have occurred since the first one in Saudi Arabia 442 in 2012. It is crucial to develop prophylactic measures for MERS-CoV infection; in this 443 respect, vaccines play an important role. Four types of MERS-CoV vaccines are 444 currently being developed: inactivated virus, live-attenuated, DNA, and recombinant 21 445 vaccines. However, no vaccine for MERS-CoV has been licensed to date

The fragment of the MERS-CoV S-RBD containing residues with Fc and the adjuvant MF59 elicited strong humoral and cellular immune 453 responses at low doses

The role of mucosal immunity in the lungs has not been widely studied, despite

Typically, vaccinations contain specific Ags, and an adjuvant, to 22 468 increase the immunogenicity of the Ags and their ability to induce specific adaptive

The 12-mer Co4B ligand was previously identified by bio-panning a 472 phage display library against an in vitro M-like cell culture system, and its ability to 473 target conjugated Ags to nasal-associated lymphoid tissue M cells, to induce Ag-474 specific immune responses

Therefore, we 478 examined the ability of Co4B to act as an adjuvant, to induce mucosal and systemic 479 immune responses specific to MERS-CoV S-RBD, in C57BL/6 and hDPP4-Tg mice 480 (Fig. 2A and Fig. 4A). Additionally, we confirmed that the mucosal and systemic Abs 481 induced by S-RBD-Co4B inhibited the interaction of MERS-CoV with DPP4 to a 482 greater extent than unconjugated S-RBD in both C57BL/6 and hDPP4-Tg mice

Similar to cytotoxic CD8 + T cells, functional CD4 + T cells have cytolytic 485 characteristics and exert protective effect against influenza virus infection

Effector 486 cells that produce IFN-γ in the lung also play a protective role at the site of infection

The frequency of IFN-γ-and CD107a-expressing CD4 + and CD8 + T cells in 488 splenocytes and lung lymphocytes collected from S-RBD-Co4B-immunized mice was 489 higher than in those harvested from S-RBD-immunized mice, which suggests that Ag-490 specific effector CTL cells were stimulated by intranasal immunization

In addition, the efficient stimulation of Th1-type cytokines such as IL-492 10, IFN-γ, and TNF-α implies that effective cell-mediated immunity was induced by 493 intranasal immunization with S-RBD-Co4B in both C57BL/6 and hDPP4-Tg mice

Notably, IFN-γ, which is a type II IFN, plays essential roles in 495 controlling various viral infections and promoting viral clearance by adaptive immune 496 cells [26]. The induction of pro-inflammatory cytokines and other IFNs is delayed 497 following MERS-CoV infection

498 efficient induction of IFN-γ by intranasal immunization with S-RBD-Co4B may be 499 critical for protective immunity in MERS-CoV infection

Although further research is needed to confirm the correlation, the 506 immunopathologic response might be partly attributed to vaccination [39

The roles of Th17 cells have been documented in regulating B-cell Ab 512 generation, formation of the germinal center, and inducible bronchus-associated 513 lymphoid tissue. Therefore, mucosal vaccination can induce robust Th17 responses

514 which are considered useful targets for mucosal vaccination. However

granulocyte-macrophage colony-stimulating factor, downregulating regulatory T cells, 519 and promoting neutrophil recruitment, but simultaneously inducing Th2 immune 520 responses. However, our observation of the increase in IgG2a/IgG1 in the sera of 521 immunized mice (Fig. S2). along with the level of Th1 responses (Fig 3E and Fig. 4C) 522 suggest

The hDPP4 receptor, which is recognized and used for entry by MERS-CoV, is 524 not present in the tissues of mice, meaning that they are not susceptible to MERS-CoV 525 infection; this has hampered MERS-CoV research. Moreover, respiratory infection and 526 pathological lung damage are key features of severe human disease caused by MERS

Therefore, a transgenic animal model with consistent hDPP4 expression 528 and pulmonary pathology is needed. Previously, we generated hDPP4-Tg mice that 529 express hDPP4 at high levels, which enabled MERS-CoV to develop

MERS-CoV infection in the respiratory tract causes fibrotic lung disease 532 symptoms including alveolar cell damage, inflammation, fibroblast proliferation, and 533 extracellular matrix deposition [5]. In the present study, we detected histopathologic 534 changes in the lung (e.g., pulmonary fibrosis), which is the initial infection site, and in 535 the spleen, a representative tissue of systemic immunity, after MERS-CoV infection in 536 hDPP4-Tg mice (Fig. 5C). Importantly

Moreover, mice immunized intranasally with S-539 RBD-Co4B showed superior survival rates following infection with MERS-CoV 540 compared to the other groups (Fig. 5A and Fig. 5B). Collectively, our findings

2019R1A2C2004711) and the Strategic International Collaborative Research Program 554 (2020K1A4A7A02095058) through the National Research Foundation, which was 555 funded by the Korean Ministry of Science and ICT. Dr. Yong-Suk Jang was supported 556 by the Research Base Construction Fund Program funded by Jeonbuk National 557 University in 2021. Y. L. Yang was supported by the BK21 FOUR program in the 558 Department of Bioactive Material Sciences. Some experiments were performed using 559 instruments installed in the

designed the research; YLY, JK and YJ performed the 565 research

566 Y-SJ acquired funding and supervised the research. All authors have read and approved

Middle East respiratory syndrome

Middle East respiratory 574 syndrome

Dipeptidyl 577 peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. 578

Cryo-580 electron microscopy structure of a coronavirus spike glycoprotein trimer

Receptor 583 variation and susceptibility to Middle East respiratory syndrome coronavirus 584 infection

Host species restriction of Middle East respiratory syndrome 587 coronavirus through its receptor, dipeptidyl peptidase 4

Intranasal vaccination against 593 plague, tetanus and diphtheria

596 Prevention and control of seasonal influenza with vaccines: recommendations of the 597 Advisory Committee on Immunization Practices (ACIP)

Immune 601 response in humans to a nasal boost with Streptococcus mutans antigens

Antibody-mediated 605 protection and the mucosal immune system of the genital tract: relevance to vaccine 606 design

Potential of nasopharynx-associated lymphoid tissue for vaccine 608 responses in the airways

The M cell-targeting ligand promotes 611 antigen delivery and induces antigen-specific immune responses in mucosal 612 vaccination

Nasal immunization 29 615 with M cell-targeting ligand-conjugated ApxIIA toxin fragment induces protective 616 immunity against Actinobacillus pleuropneumoniae infection in a murine model

Middle East respiratory syndrome-619 coronavirus infection into established hDPP4-transgenic mice accelerates lung 620 damage via activation of the pro-inflammatory response and pulmonary fibrosis

Searching for an 623 ideal vaccine candidate among different MERS coronavirus receptor-binding 624 fragments-the importance of immunofocusing in subunit vaccine design

Middle East respiratory syndrome coronavirus-encoded 627 accessory proteins impair MDA5-and TBK1-mediated activation of NF-κB

CD8 + T cell responses induced by a live attenuated tetravalent dengue vaccine are 632 directed against highly conserved epitopes

Effective clearance of mouse hepatitis virus from the 635 central nervous system requires both CD4 + and CD8 + T cells

CD8 + T cell effector mechanisms in 30 638 resistance to infection

641 Sensitive and viable identification of antigen-specific CD8 + T cells by a flow 642 cytometric assay for degranulation

645 Characterization of CD4 + CTLs ex vivo

Emergence of a CD4 + CD28 -granzyme B + , 649 cytomegalovirus-specific T cell subset after recovery of primary cytomegalovirus 650 infection

CD4 CTL, a cytotoxic subset of CD4 + T cells, their 653 differentiation and function

Pre-656 and postexposure efficacy of fully human antibodies against Spike protein in a novel 657 humanized mouse model of MERS-CoV infection

The immune 660 response and immune evasion characteristics in SARS-CoV, MERS-CoV, and SARS-CoV-2: Vaccine design strategies

Intranasal vaccination 664 with recombinant receptor-binding domain of MERS-CoV spike protein induces 665 much stronger local mucosal immune responses than subcutaneous immunization: 666 Implication for designing novel mucosal MERS vaccines

Crystal 669 structure of the receptor-binding domain from newly emerged Middle East 670 respiratory syndrome coronavirus

Molecular basis of binding between 673 novel human coronavirus MERS-CoV and its receptor CD26

Optimization of 676 antigen dose for a receptor-binding domain-based subunit vaccine against MERS 677 coronavirus

Middle East 680 Respiratory Syndrome Coronavirus (MERS-CoV) infection: epidemiology, 681 pathogenesis and clinical characteristics

Recent advances of vaccine adjuvants for infectious diseases

Role of secretory antibodies in the defence against infections

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Multifunctional CD4 cells 690 expressing gamma interferon and perforin mediate protection against lethal 691 influenza virus infection

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Middle East respiratory syndrome coronavirus: implications for pathogenesis and 696 treatment

A systematic review of SARS-698

CoV-2 vaccine candidates

Isolation of 701 potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small 702 animal model

Prior 704 Immunization with severe acute respiratory syndrome (SARS)-associated 705 coronavirus (SARS-CoV) nucleocapsid protein causes severe pneumonia in mice 706 infected with SARS-CoV

Th17 cell based vaccines in mucosal immunity

Palmitate conditions macrophages for enhanced responses toward inflammatory 712 stimuli via JNK activation

For each 723 vaccination group, five mice were immunized at 7-day intervals (total of five times) . Ten days after the fifth immunization, splenocytes and lung lymphocytes 726 were collected from two of the five mice. Then, the remaining mice were challenged 727 with MERS-CoV (10 4 or 10 5 plaque-forming units [PFUs]) on day 30 and booster-728 immunized with cognate Ags on day 56. Similarly, fecal and serum samples, and 729 splenocytes and lung lymphocytes

Intranasal immunization with S-RBD-Co4B induces S-RBD-specific mucosal 733 and systemic antibody (Ab) responses, together with Ab-mediated virus inhibition, in 734 C57BL/6 mice. (A) Concentrations of fecal immunoglobulin (Ig) A (left) and serum

IgG (right) in samples collected from C57BL/6 mice 3 days after the fifth immunization 736 with indicated Ags, as determined by enzyme linked immunosorbent assay (ELISA)

Fecal and serum samples were pre-incubated with MERS-CoV (10 4 PFUs)

gene expression levels via quantitative real-time polymerase chain reaction (qRT-PCR)

C57BL/6 mice 3 days after booster immunization with indicated Ags, as determined by 744 ELISA. (D) Splenocytes were stimulated with 1 μg of S-RBD Ag, and the number of S

RBD-specific IgG-secreting cells per 10 4 splenocytes was determined by enzyme-linked

Intranasal immunization with S-RBD-Co4B enhanced S-RBD-specific effector 750 cytotoxic T lymphocyte (CTL) stimulation in splenocytes and lung lymphocytes in

Splenocytes and lung lymphocytes were collected 10 days after the fifth 752 immunization with the indicated Ag, and then stimulated with S-RBD (1 μg) for up to 753 48 h. (A) The expression of IFN-γ in CD4 + (left) and CD8 + (right) cells in splenocytes 754 after stimulation with S-RBD for 17 h was analyzed by flow cytometry

of CD107a in CD4 + (left) and CD8 + (right) cells in splenocytes after stimulation with S-756 RBD for 24 h was analyzed by flow cytometry. (C) Expression of IFN

right) cells in lung lymphocytes after stimulation with S-RBD for 17 h was 758 analyzed by flow cytometry. (D) Expression of CD107a in CD4 + (left) and CD8 + (right) 759 cells in lung lymphocytes after stimulation with S-RBD for 24 h was analyzed by flow 760 cytometry

splenocytes (left panels) and lung lymphocytes (right panels) that had been stimulated 762 with S-RBD (1 μg) for 48 h, as determined by cytometric bead array. Experiments were 763 repeated three times and representative results are presented

Intranasal immunization with S-RBD-Co4B induces S-RBD-specific mucosal 767 and systemic Ab responses, together with Ab-mediated virus inhibition, in hDPP4-Tg 768 mice. (A) Concentrations of fecal IgA (left) and serum IgG (right) in samples collected 769 from hDPP4-Tg mice 3 days after the fifth immunization with the indicated Ag, as 770 determined by ELISA. (B) Fecal and serum samples were pre

Ab-mediated inhibition of MERS-CoV infection in Vero E6 cells the relative expression. Data are expressed as relative quantitation with the 775 level of PBS set as 1. (C) Cytokine concentrations in culture supernatants collected 776 from splenocytes (left panels) and lung lymphocytes (right panels) following 777 stimulation with S-RBD (1 μg) for 48 h

Experiments were repeated three times and representative results are presented

Intranasal immunization with Co4B-conjugated S-RBD elicits potent protective 782 immunity and prevents lung and spleen damage in hDPP4-Tg mice following MERS

Mice were intranasally immunized with the indicated Ags and 37 every 2 days for 2 weeks. (C) Histological changes in the lung and spleen 787 tissues of hDPP4-Tg mice immunized with indicated Ags (10 μg) and challenged with 788 MERS-CoV (10 5 PFUs)

Arrows indicate inflammatory immune cell infiltration, and arrow heads indicate 790 airspace

Scale bars = 100 μm

☐ The authors declare the following financial interests/personal relationships which

We prepared Co4B-conjugated receptor binding domain of MERS-CoV spike protein

We intranasally immunized C57BL/6 and hDPP4-Tg mice with S-RBD-Co4B

• Intranasal immunization of S-RBD-Co4B induced efficient Ag-specific immune 815 response