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Vaccines against Middle East respiratory syndrome coronavirus for humans and camels

Summary Middle East respiratory syndrome coronavirus (MERS‐CoV) is caused by a novel betacoronavirus that was isolated in late 2012 in Saudi Arabia. The viral infections have been reported in more than 1700 humans, ranging from asymptomatic or mild cases to severe pneumonia with a mortality rate of 40%. It is well documented now that dromedary camels contract the infection and shed the virus without notable symptoms, and such animals had been infected by at least the early 1980s. The mechanism of camel to human transmission is still not clear, but several primary cases have been associated with camel contact. There is no approved antiviral drug or vaccine against MERS‐CoV despite the active research in this area. Vaccine candidates have been developed using various platforms and regimens and have been tested in several animal models. Here, this article reviews the published studies on MERS‐CoV vaccines with more focus on vaccines tested in large animals, including camels. It is foreseeable that the 1‐health approach could be the best way of tackling the MERS‐CoV endemic in the Arabian Peninsula, by using the mass vaccination of camels in the affected areas to block camel to human transmission. Camel vaccines can be developed in a faster time with fewer regulations and lower costs and could clear this virus from the Arabian Peninsula if accompanied by efficient public health measures.


Summary
Middle East respiratory syndrome coronavirus (MERS-CoV) is caused by a novel betacoronavirus that was isolated in late 2012 in Saudi Arabia. The viral infections have been reported in more than 1700 humans, ranging from asymptomatic or mild cases to severe pneumonia with a mortality rate of 40%. It is well documented now that dromedary camels contract the infection and shed the virus without notable symptoms, and such animals had been infected by at least the early 1980s. The mechanism of camel to human transmission is still not clear, but several primary cases have been associated with camel contact. There is no approved antiviral drug or vaccine against MERS-CoV despite the active research in this area. Vaccine candidates have been developed using various platforms and regimens and have been tested in several animal models. Here, this article reviews the published studies on MERS-CoV vaccines with more focus on vaccines tested in large animals, including camels. It is foreseeable that the 1-health approach could be the best way of tackling the MERS-CoV endemic in the Arabian Peninsula, by using the mass vaccination of camels in the affected areas to block camel to human transmission. Camel vaccines can be to 40% mortality rate, mainly in countries of the Arabian Peninsula. 3 Dromedary camels are strongly believed to be an intermediate host and an important source of infection although the exact transmission mechanism is still unclear. [2][3][4] Human-to-human transmission has been documented, although this requires a very close and lengthy contact with patients. 4 In the most part, cases are mild or asymptomatic, and most fatal cases are the result of comorbidities, such as diabetes mellitus type 2, which is again very prevalent in the Arabian Peninsula. 4 Asymptomatic cases are estimated to be 2.1 times higher than the reported cases 5 and together with mild cases may serve to spread the virus from camels to humans. Primary cases that had no contact with infected patients are more likely to have contacted camels directly or indirectly. 6 However, camels are believed to be an intermediate with speculation that bats could be the primary host. 4 Dromedary camels were 80% to 90% seropositive, from samples dated back to as early as 1983, in African countries, 7 Arabian Peninsula countries, 8 and the Spanish Canary Islands, 9 but 0% in Australian dromedary camels or Bactrian camels, 10,11 and several groups reported viral shedding from infected camels. [12][13][14][15][16][17][18] Nevertheless, despite the impressive and quick elucidation of many issues about MERS-CoV, there are gaps in our understanding of this emerging virus, such as the precise route of transmission of camel-to-human infections, the risk factors for contracting the virus, and the protective level of neutralizing antibodies (nAb). More importantly, to date, there is no specific antiviral or vaccine for treatment or prevention of MERS-CoV infection. This article reviews the ongoing efforts on vaccine development and sheds some light on the challenges that can hinder both the progress and the speed of developing a MERS-CoV vaccine.

| ANIMAL MODELS FOR VACCINE EVALUATION
MERS-CoV tropism in humans and camels is determined by the binding of its spike glycoprotein to dipeptidyl peptidase 4 (DPP4) on the surface of mammalian cells such as respiratory epithelial cells. However, the virus does not infect most small experimental animals such as mice, ferrets, rats, and hamsters, mainly because of the different DPP4 in these animals therefore posing a challenge for developing a vaccine by delaying scientific investigation on the virus infection, pathogenicity, and transmission. A rabbit model was reported to support MERS-CoV, 19 but more small animal models were required to accelerate MERS-CoV research; therefore, susceptible mouse models were developed by different methods. The first model was developed by transducing mice with adenoviral vector encoding human DPP4 (AdV-hDPP4) delivered intranasally to enable mouse cells in the respiratory tract to express hDPP4, allowing viral entry and infection. 20 This model is easy to use and can be readily applied to a broad range of mouse strains, especially in laboratories with no transgenic animal facility available. Other models were developed by generating transgenic mice that are either expressing human DPP4 globally 20 or replacing murine DPP4 with the human ortholog. 21  subjects is yet to be investigated and determined. MERS-CoV replicates in the upper respiratory tract in camels, unlike humans where MERS-CoV infection affects mainly lungs and lower respiratory tract, and results in mild clinical manifestation with rhinorrhea. In experimental camel models, infectious viruses were detected in nasal and oropharyngeal swabs and in tissues up to 5 days postinfection, whereas viral RNA persisted in these samples for over a month. 18 All these developments and studies of animal models were conducted in less than 3 years and have accelerated our understanding of and responses to MERS-CoV. Some vaccines have already been tested in mouse, NHP, and camel models, as discussed in this section and in Figure 1 and Table 1, although there seems a long way to go before establishing a robust animal model for assessing immunogenicity and efficacy of MERS-CoV vaccine candidates. FIGURE 1 Development of MERS-CoV vaccines for humans and camels. Three vaccines based on different vectors have been developed and tested (or planned for testing) in camels and in humans. Camel vaccination could block MERS-CoV transmission and prevent any potential outbreaks in humans, applying the one-health concept. Vaccinating humans, especially health care workers or individuals with comorbidity, could then further prevent MERS-CoV infections and outbreaks. Bats are included as a suspected primary host of MERS-CoV although this is not confirmed. MERS-CoV can spread from infected camels to (i) naive camels or (ii) humans who may or may not show symptoms but are able to spread the virus to more susceptible individuals, causing an outbreak Rhesus macaques (n = 6). Three different regimens: DDD (S), DDP (S,S,S1), and PP (S1) 4-wk intervals, 100 μg of S1 protein,  Both doses induced strong RBD-specific antibodies 2 weeks after each vaccination, and similar nAb titers, but significantly higher than mockvaccinated monkeys. When compared with mock-vaccinated control animals, both doses of vaccines reduced the interstitial infiltration and the gross pathology in lungs, after the animals were challenged with MERS-CoV (Table 1). 24 Vaccines reduced the viral RNA load in lung and trachea tissues, and in throat, nasal, and rectal swab samples.
However, this reduction in viral load was statistically significant only in throat swabs from high-dose vaccinated animals (summarized in Table 1). Both doses also reduced the infectious virus titers in the lung and trachea by around 2-fold. Moreover, it was speculated that nAb were the underlying mechanism for this partial protection because cellular responses, as tested by IFN-γ secreting peripheral blood monocytes, were detectable only in animals immunized with the highest dose vaccine.
A series of studies was also conducted to first define the minimum sequence of MERS-CoV RBD to focus the immune responses. 25 Subunit vaccines are more appealing because it was found that the inactivated whole SARS-CoV used as a vaccine resulted in enhanced lung immunopathology when vaccinated animals were challenged with SARS-CoV. [28][29][30] This was attributed to the presence of N-specific antibodies rather than S-specific antibodies 31 ; therefore, using the whole S antigen in a vaccine could be as safe as using minimum RBD. In fact, some studies reported that nAb titers were directed against other parts of the S, outside the RBD boundaries. 32 A linear synthesized peptide, corresponding to 736 to 761 residues of the S protein (in the S2 subunit away from the RBD), induced nAb titers in mice. These titers were lower than using rRBD but other synthesized peptides based on immunoepitopes from the *Cellular immunogenicity detection reagents are not available for camels. S1 subunit of the spike protein (S1) and RBD regions induced binding, but not nAb. 33 This supports using the full-length S antigen for vaccine development.
Nanoparticles of the trimerized S protein were produced as a vaccine by baculovirus expression system and used to immunize BALB/c mice with alum or matrix M adjuvants. The latter induced significantly higher nAb levels, 4-fold higher than with alum. Moreover, there was no difference between high or low doses, in prime boost regimens. 34 In a separate study, the S antigen was tested in mice in 3 different forms, the full-length S, the S without its transmembrane domain, or the S1 subunit. All forms were tested in homologous regimens in mice, that is, prime boost regimen of 3 sequential DNA vaccinations (DDD) or prime boost regimen of 2 sequential protein vaccinations (PP), or in a heterologous regimen, that is, prime boost regimen of 2 sequential DNA vaccinations followed by 1 protein vaccination (DDP). 32 The study concluded that the full-length S DNA and S1-based protein induced the highest nAb titers in DDP or PP regimens, but it should be noted that there was no full-length S-based protein vaccine in this study. 32 The nAb titers cross-protected mice against 8 distinct MERS-CoV isolates; this could be predicted as the RBD sequence is highly conserved between the 8 isolates.
This study also showed that vaccinating with full-length S DNA was important to achieve maximal neutralization activity; therefore, vaccines that are based on the full S or S1 antigens were then further tested in Rhesus macaques, using DDD, PP, or DDP regimens.
The study used alum (ALPO4) to adjuvant the protein-based vaccines. The DDP followed tightly by PP reduced the peak proportional volume of pulmonary consolidation, as measured by computed tomography (CT), after the animals were challenged with MERS-CoV (Table 1). Therefore, it seemed that regimens involving protein-based vaccine were superior to DNA-based vaccines in driving this protection.
Another encouraging study also reported a partial protection in NHPs using only DNA vaccine based on the consensus sequence of the S gene. 35

| Poxviral vectors
Recombinant MVA encoding the full-length S antigen was used to immunize BALB/c mice via intramuscular or subcutaneous routes in a homologous prime boost vaccination. 36

| Human adenoviral vectors
Vaccines based on adenoviral vectors were developed by 2 different groups using human adenovirus type 5 (Ad5) and Ad41. The first group used Ad5 vectors encoding either the full-length S antigen or the S1 antigen to immunize BALB/c mice in a prime boost vaccination (intramuscular route for prime and intranasal route for the boost). Levels of IgG1 and IgG2a, which were similar for both antigens, increased over time and then were boosted to their peak level as they plateaued for 3 weeks postboost. Similarly, the nAb levels doubled after the boost, and they plateaued for 3 weeks. 40 The second group used an Ad41

| Venezuelan equine encephalitis replicons (VRP)
Nearly all vaccines developed for MERS-CoV were based on the S protein or its versions to induce nAb. However, eliciting cellular immunity can be achieved by vaccines that encode internal antigens such as nucleocapsid protein (N) or immunoepitopes from the N protein.
VRP encoding CD4 + T-cell-specific epitope from SARS-CoV nucleocapsid protein (VRP-SARS-N) was developed and tested in BALB/c mouse models. VRP-SARS-N induced airway CD4 + T cells that were able to activate dendritic cell migration to lymph nodes, which in turn stimulated epitope-specific cytotoxic CD8 + T cells. This resulted in the complete protection of vaccinated mice, immunized in prime boost regimen. 48 The same researchers then developed VRP-MERS-N vaccine that is based on the N epitope analogue from MERS-CoV, and similar results were obtained. The depletion of airway CD4 + T cells or inhibition of airway IFN-γ significantly decreased the protection of VRP-MERS-N. 48 This, in addition to the fact that the N epitope is a weak CD8 + T-cell epitope, supports the role and importance of airway This study clearly supports the cellular immunity role in developing MERS-CoV vaccine and paves the way toward a pan-vaccine as a universal encounter against existing or potentially emerging human CoVs. isolates were also used for the challenge (summarized in Table 1). 32

| Funding and cost-effectiveness of vaccines
Developing a vaccine for human use is a very long process that may take a decade to take a vaccine from bench through clinics to licensure, given that there is a productive and coherent collaboration between clinicians, scientists, regulators, and pharmaceutical industry.

| CONCLUDING REMARKS
To date, none of the tested vaccines against MERS-CoV failed in inducing nAb, protecting mice, or partially protecting large animals ( Table 1).
This virus does not seem very polymorphic in the spike protein that is involved in tropism and cell entry and contains neutralizing epitopes. costs and time to a great extent and make the world more prepared to distribute vaccines against the next emerging pathogen.

ACKNOWLEDGMENTS
The author thanks Prof. Sarah Gilbert, University of Oxford, for her comments on this article and Mr. Sultan Alharbi (srab4d@gmail.com) for the infographic design in Figure 1.