Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8⁺ T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine.     

Significant Spike-Specific IgG and Neutralizing Antibodies in Mice Induced by a Novel Chimeric Virus-Like Particle Vaccine Candidate for Middle East Respiratory Syndrome Coronavirus

正Dear Editor,Middle East respiratory syndrome coronavirus (MERS-CoV), first isolated in 2012, has emerged zoonotically among humans (van Boheemen et al. 2012). Since then,MERS-CoV continues to be a public health concern, with a fatality rate of 35%. On-going MERS-CoV outbreaks highlight the urgent need for the development of inter-     

Calling for rapid development of a safe and effective MERS vaccine

The geographic spread and rapid increase in the cases of Middle East respiratory syndrome (MERS) caused by a novel coronavirus (MERS-CoV) during the past two months have raised concern about its pandemic potential. Here we call for the rapid development of an effective and safe MERS vaccine to control the spread of MERS-CoV.     

Identification of a Receptor-Binding Domain in the S Protein of the Novel Human Coronavirus Middle East Respiratory Syndrome Coronavirus as an Essential Target for Vaccine Development

A novel human Middle East respiratory syndrome coronavirus (MERS-CoV) caused outbreaks of severe acute respiratory syndrome (SARS)-like illness with a high mortality rate, raising concerns of its pandemic potential. Dipeptidyl peptidase-4 (DPP4) was recently identified as its receptor. Here we showed that residues 377 to 662 in the S protein of MERS-CoV specifically bound to DPP4-expressing cells and soluble DPP4 protein and induced significant neutralizing antibody responses, suggesting that this region contains the receptor-binding domain (RBD), which has a potential to be developed as a MERS-CoV vaccine.     

A Truncated Receptor-Binding Domain of MERS-CoV Spike Protein Potently Inhibits MERS-CoV Infection and Induces Strong Neutralizing Antibody Responses: Implication for Developing Therapeutics and Vaccines

An emerging respiratory infectious disease with high mortality, Middle East respiratory syndrome (MERS), is caused by a novel coronavirus (MERS-CoV). It was first reported in 2012 in Saudi Arabia and has now spread to eight countries. Development of effective therapeutics and vaccines is crucial to save lives and halt the spread of MERS-CoV. Here, we show that a recombinant protein containing a 212-amino acid fragment (residues 377-588) in the truncated receptor-binding domain (RBD: residues 367–606) of MERS-CoV spike (S) protein fused with human IgG Fc fragment (S377-588-Fc) is highly expressed in the culture supernatant of transfected 293T cells. The purified S377-588-Fc protein efficiently binds to dipeptidyl peptidase 4 (DPP4), the receptor of MERS-CoV, and potently inhibited MERS-CoV infection, suggesting its potential to be further developed as a therapeutic modality for treating MERS-CoV infection and saving the patients’ lives. The recombinant S377-588-Fc is able to induce in the vaccinated mice strong MERS-CoV S-specific antibodies, which blocks the binding of RBD to DPP4 receptor and effectively neutralizes MERS-CoV infection. These findings indicate that this truncated RBD protein shows promise for further development as an effective and safe vaccine for the prevention of MERS-CoV infection.     

Epitope-Based Vaccine Target Screening against Highly Pathogenic MERS-CoV: An In Silico Approach Applied to Emerging Infectious Diseases

Middle East respiratory syndrome coronavirus (MERS-CoV) with pandemic potential is a major worldwide threat to public health. However, vaccine development for this pathogen lags behind as immunity associated with protection is currently largely unknown. In this study, an immunoinformatics-driven genome-wide screening strategy of vaccine targets was performed to thoroughly screen the vital and effective dominant immunogens against MERS-CoV. Conservancy and population coverage analysis of the epitopes were done by the Immune Epitope Database. The results showed that the nucleocapsid (N) protein of MERS-CoV might be a better protective immunogen with high conservancy and potential eliciting both neutralizing antibodies and T-cell responses compared with spike (S) protein. Further, the B-cell, helper T-cell and cytotoxic T lymphocyte (CTL) epitopes were screened and mapped to the N protein. A total of 15 linear and 10 conformal B-cell epitopes that may induce protective neutralizing antibodies were obtained. Additionally, a total of 71 peptides with 9-mer core sequence were identified as helper T-cell epitopes, and 34 peptides were identified as CTL epitopes. Based on the maximum HLA binding alleles, top 10 helper T-cell epitopes and CTL epitopes that may elicit protective cellular immune responses against MERS-CoV were selected as MERS vaccine candidates. Population coverage analysis showed that the putative helper T-cell epitopes and CTL epitopes could cover the vast majority of the population in 15 geographic regions considered where vaccine would be employed. The B- and T-cell stimulation potentials of the screened epitopes is to be further validated for their efficient use as vaccines against MERS-CoV. Collectively, this study provides novel vaccine target candidates and may prompt further development of vaccines against MERS-CoV and other emerging infectious diseases.     

Tailoring Subunit Vaccine Immunity with Adjuvant Combinations and Delivery Routes Using the Middle East Respiratory Coronavirus (MERS-CoV) Receptor-Binding Domain as an Antigen

The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV) pandemic. Some studies have indicated the receptor-binding domain (RBD) protein of MERS-CoV spike (S) is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m.) with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly I:C) or alum and cysteine-phosphate-guanine (CpG) oligodeoxynucleotides (ODN). The immune responses of mice vaccinated with RBD, incomplete Freund’s adjuvant (IFA) and CpG ODN by a subcutaneous (s.c.) route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies) and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production). Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting effective humoral and cellular immune responses.     

Development of A MERS-CoV Replicon Cell Line for Antiviral Screening

Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease with a high mortality of ~ 35%. The lack of approved treatments for MERS-CoV infection underscores the need for a user-friendly system for rapid drug screening. In this study, we constructed a MERS-CoV replicon containing the Renilla luciferase (Rluc) reporter gene and a stable luciferase replicon-carrying cell line. Using this cell line, we showed that MERS-CoV replication was inhibited by combined application of lopinavir and ritonavir, indicating that this cell line can be used to screen inhibitors of MERS-CoV replication. Importantly, the MERS-replicon cell line can be used for high-throughput screening of antiviral drugs without the need for live virus handling, providing an effective and safe tool for the discovery of antiviral drugs against MERS-CoV.     

Development of human neutralizing monoclonal antibodies for prevention and therapy of MERS-CoV infections

The recent Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak poses a serious threat to public health. Here, we summarize recent advances in identifying human neutralizing monoclonal antibodies (mAbs) against MERS-CoV, describe their mechanisms of action, and analyze their potential for treatment of MERS-CoV infections.     

Asymptomatic Middle East Respiratory Syndrome Coronavirus Infection in Rabbits

The ability of Middle East respiratory syndrome coronavirus (MERS-CoV) to infect small animal species may be restricted given the fact that mice, ferrets, and hamsters were shown to resist MERS-CoV infection. We inoculated rabbits with MERS-CoV. Although virus was detected in the lungs, neither significant histopathological changes nor clinical symptoms were observed. Infectious virus, however, was excreted from the upper respiratory tract, indicating a potential route of MERS-CoV transmission in some animal species.     

Coronaviruses: Important Emerging Human Pathogens

The identification of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 reaffirmed the importance of understanding how coronaviruses emerge, infect, and cause disease. By comparing what is known about severe acute respiratory syndrome coronavirus (SARS-CoV) to what has recently been found for MERS-CoV, researchers are discovering similarities and differences that may be important for pathogenesis. Here we discuss what is known about each virus and what gaps remain in our understanding, especially concerning MERS-CoV.     

Modulation of the immune response by Middle East respiratory syndrome coronavirus

Coronavirus (CoV) infections are commonly associated with respiratory and enteric disease in humans and animals. In 2012, a new human disease called Middle East respiratory syndrome (MERS) emerged in the Middle East. MERS was caused by a virus that was originally called human coronavirus-Erasmus Medical Center/2012 but was later renamed as Middle East respiratory syndrome coronavirus (MERS-CoV). MERS-CoV causes high fever, cough, acute respiratory tract infection, and multiorgan dysfunction that may eventually lead to the death of the infected individuals. The exact origin of MERS-CoV remains unknown, but the transmission pattern and evidence from virological studies suggest that dromedary camels are the major reservoir host, from which human infections may sporadically occur through the zoonotic transmission. Human to human transmission also occurs in healthcare facilities and communities. Recent studies on Middle Eastern respiratory continue to highlight the need for further understanding the virus-host interactions that govern disease severity and infection outcome. In this review, we have highlighted the major mechanisms of immune evasion strategies of MERS-CoV. We have demonstrated that M, 4a, 4b proteins and Plppro of MERS-CoV inhibit the type I interferon (IFN) and nuclear factor-κB signaling pathways and therefore facilitate innate immune evasion. In addition, nonstructural protein 4a (NSP4a), NSP4b, and NSP15 inhibit double-stranded RNA sensors. Therefore, the mentioned proteins limit early induction of IFN and cause rapid apoptosis of macrophages. MERS-CoV strongly inhibits the activation of T cells with downregulation of antigen presentation. In addition, uncontrolled secretion of interferon ɣ-induced protein 10 and monocyte chemoattractant protein-1 can suppress proliferation of human myeloid progenitor cells.     

Crystal Structure of the Receptor-Binding Domain from Newly Emerged Middle East Respiratory Syndrome Coronavirus

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 77 people, with a fatality rate of more than 50%. Alarmingly, the virus demonstrates the capability of human-to-human transmission, raising the possibility of global spread and endangering world health and economy. Here we have identified the receptor-binding domain (RBD) from the MERS-CoV spike protein and determined its crystal structure. This study also presents a structural comparison of MERS-CoV RBD with other coronavirus RBDs, successfully positioning MERS-CoV on the landscape of coronavirus evolution and providing insights into receptor binding by MERS-CoV. Furthermore, we found that MERS-CoV RBD functions as an effective entry inhibitor of MERS-CoV. The identified MERS-CoV RBD may also serve as a potential candidate for MERS-CoV subunit vaccines. Overall, this study enhances our understanding of the evolution of coronavirus RBDs, provides insights into receptor recognition by MERS-CoV, and may help control the transmission of MERS-CoV in humans.     

Differential Expression of the Middle East Respiratory Syndrome Coronavirus Receptor in the Upper Respiratory Tracts of Humans and Dromedary Camels

Middle East respiratory syndrome coronavirus (MERS-CoV) is not efficiently transmitted between humans, but it is highly prevalent in dromedary camels. Here we report that the MERS-CoV receptor—dipeptidyl peptidase 4 (DPP4)—is expressed in the upper respiratory tract epithelium of camels but not in that of humans. Lack of DPP4 expression may be the primary cause of limited MERS-CoV replication in the human upper respiratory tract and hence restrict transmission.     

Assessing Activity and Inhibition of Middle East Respiratory Syndrome Coronavirus Papain-Like and 3C-Like Proteases Using Luciferase-Based Biosensors

Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with an outbreak of more than 90 cases of severe pneumonia with high mortality (greater than 50%). To date, there are no antiviral drugs or specific therapies to treat MERS-CoV. To rapidly identify potential inhibitors of MERS-CoV replication, we expressed the papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro) from MERS-CoV and developed luciferase-based biosensors to monitor protease activity in cells. We show that the expressed MERS-CoV PLpro recognizes and processes the canonical CoV-PLpro cleavage site RLKGG in the biosensor. However, existing CoV PLpro inhibitors were unable to block MERS-CoV PLpro activity, likely due to the divergence of the amino acid sequence in the drug binding site. To investigate MERS-CoV 3CLpro activity, we expressed the protease in context with flanking nonstructural protein 4 (nsp4) and the amino-terminal portion of nsp6 and detected processing of the luciferase-based biosensors containing the canonical 3CLpro cleavage site VRLQS. Importantly, we found that a small-molecule inhibitor that blocks replication of severe acute respiratory syndrome (SARS) CoV and murine CoV also inhibits the activity of MERS-CoV 3CLpro. Overall, the protease expression and biosensor assays developed here allow for rapid evaluation of viral protease activity and the identification of protease inhibitors. These biosensor assays can now be used to screen for MERS-CoV-specific or broad-spectrum coronavirus PLpro and 3CLpro inhibitors.     

Adenosine Deaminase Acts as a Natural Antagonist for Dipeptidyl Peptidase 4-Mediated Entry of the Middle East Respiratory Syndrome Coronavirus

Middle East respiratory syndrome coronavirus (MERS-CoV) replicates in cells of different species using dipeptidyl peptidase 4 (DPP4) as a functional receptor. Here we show the resistance of ferrets to MERS-CoV infection and inability of ferret DDP4 to bind MERS-CoV. Site-directed mutagenesis of amino acids variable in ferret DPP4 thus revealed the functional human DPP4 virus binding site. Adenosine deaminase (ADA), a DPP4 binding protein, competed for virus binding, acting as a natural antagonist for MERS-CoV infection.     

Glycosylation of Mouse DPP4 Plays a Role in Inhibiting Middle East Respiratory Syndrome Coronavirus Infection

Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes dipeptidyl peptidase 4 (DPP4) as an entry receptor. Mouse DPP4 (mDPP4) does not support MERS-CoV entry; however, changes at positions 288 and 330 can confer permissivity. Position 330 changes the charge and glycosylation state of mDPP4. We show that glycosylation is a major factor impacting DPP4 receptor function. These results provide insight into DPP4 species-specific differences impacting MERS-CoV host range and may inform MERS-CoV mouse model development.