Towards a MERS-CoV vaccine: the importance of S

For about two or so years Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and fatal acute respiratory illness in humans and no prophylactic and antiviral therapeutics specifically targeting MERS-CoV have been identified and applied in the field.

One strategy is to generate a MERS-CoV pseudovirus, which can be used to infect target cells and generate an immune response. Such a pseudovirus is generated by inserting the MERS-CoV genome or the protein of interest of into a lentiviral plasmid and transfect this plasmid into a packaging cell line, i.e. a cell line that stably expresses the proteins required for generating lentiviral plasmids. Viral supernatant is then harvested 24-72 hrs post transfection and can be used to transduce target cells. The recombinant virus however is capable of infect these cells but cannot replicate within these - in other words, the virus is replication incompetent. The infection of the target cell with a recombinant virus expressing the S protein of MERS-CoV for instance -in this case antigen presenting cells of the immune system such as T-Lymphocytes, dendritic cells or cells of the respiratory tract- would then allow to induce the production of antibodies which are directed against the viral protein (in this case MERS-CoV S protein). Whilst the concept is used in laboratories worldwide to generate stable cell lines expressing a protein of interest, this technology faces obstacles when used in animals and humans alike, namely the to infect the target cells and the generation of a high antibody titer. Any MERS-CoV pseudovirus therefore must be able to bind the MERS-CoV receptor, hDPP4, and therefore contain the MERS-CoV S protein on its surface, and also be able to generate a high titer of neutralizing antibodies. Indeed a replication incompetent MERS-CoV pseudovirus has been developed and shown to infect a wide variety of human and animal DPP4 expressing cell lines. MERS-CoV pseudoviruses are also used in infectivity assays and to study the contribution of individual viral proteins to viral replication and pathology.

One might ask why not use an attenuated virus strain of MERS-CoV akin to the Polio Vaccine? Indeed such a strain,  a recombinant strain in which the viral E protein has been deleted (rMERS-CoV-ΔE) has been created based on a replicon system developed by Luis Enjuanes from the University of Madrid in 2013.  Interestingly although in cells transfected with cDNA from rMERS-CoV-ΔE the virus replicates, viral particles cannot be detected in the supernatant. If however the E protein is provided in trans, viral infectivity is restored. This however might become a problem in animals and/or humans where different Coronavirus’ can be detected in the same host - in other words, the co-infection with an otherwise benign Coronavirus might lead to the generation of a viable MERS-CoV. Supplementing the E protein in trans however although resulting in the generation of viral particles, these viral particles were propagation defective. Again however studies are required to assess the infectivity and propagation in the presence of other Coronavirus species. These studies need time and one should not forgot that rMERS-CoV-ΔE was first described prior the availably of a transgenic mouse model expressing hDPP4 (which was first described in 2014). Also, no studies regarding the induction of a high titer of neutralizing antibodies have been described yet.

Another strategy involves the Coronavirus S Protein.

                       

                    MERS-CoV S protein as a potential target

The Coronavirus S protein is considered to be the main determinant of cellular tropism; indeed swapping the gene encoding the S protein is sufficient to alter cell tropism. In the case of MERS-CoV, the S gene encodes for a protein of 1353 amino acids in length, which is N glycosylated and assembled into trimers. These trimers constitute the peplomers on the surface of the viral particle that gave the Coronaviridae its name. The S protein combines two functions, binding the host receptor and membrane fusion, which are required for viral entry into the host cell. In the case of MERS-CoV, the former is attributed to the S1 subunit (AA1-751) and the latter to the S2 subunit (AA 752-1353) respectively.  During viral entry, the S protein is cleaved into both subunits by host cell derived proteases such as type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS) in the case of SARS-CoV.

S proteins from different Coronaviridae and their respective domains

Human monoclonal antibodies generated against the MERS-CoV S proteinbcould be used not only as a potential vaccine during an epidemic (in particular local outbreaks) but also upon exposure to MERS-CoV prior to the onset of symptoms. Moreover the identification of potent antibodies targeting the S protein can assist in the development of vaccines targeting specific regions of the S protein and thus aid in the development of specific immunogens. Following the identification of a putative receptor binding domain (RBD) of the viral S protein by sequence comparison with the RBD of SARS-CoV, three potent antibodies with a high affinity for an epitope overlapping with the RBD have been identified by screening an antibody library. One of three monoclonal antibodies identified, m336, neutralized live and pseudotyped MERS-CoV with an exceptional potency of ID₅₀ (half maximal inhibitory concentration) of 0.005 (pseudotyped MERS-CoV) and 0.07  (live MERS-CoV) μg/ml, respectively, by competing with the hDPP4 receptor. In a different study, two more antibodies, MERS-4 and MERS-27, were isolated from an antibody library with an ID₅₀ at nanomolar concentrations as well. Although both antibodies were effective in blocking entry of MERS-CoV, only MERS-4 also inhibited the formation of syncytia mediated by MERS-CoV S protein and DPP4 and thus cell-cell transmission of MERS-CoV.  Highly effective neutralizing antibodies against MERS-CoV S 1 fragment spanning 231 amino acids were also raised in rabbits and successfully tested in HEK-293T cells, further highlighting the importance of the S1 subunit of the MERS-CoV S protein.

Subdomains of MERS-CoV S1 and SARS-CoV S1

Using bioinformatics the information gained from these studies may allow the generation of potent antivirals targeting the RBD of MERS-CoV as well as related bat-CoV and thus not only prevent further outbreaks of MERS but hopefully also the transmission of bat-CoV into the human/animal population. So far however this is not possible although mutagenesis studies identified key residues in the receptor-binding subdomain of the S1 subdomain of MERS-CoV S protein required for binding DPP4 and viral entry. This analysis showed that MERS-CoV RBD consists of core and a receptor-binding subdomain that interacts with the N-terminal domain of DPP4. Accordingly, a truncated fragment of the MERS-CoV S1 containing the RBD fused with human IgG Fc fragment (S377-588-Fc) not only prevents MERS-CoV infection of cell lines but also elicits a high antibody titre in rabbits and mice infected with MERS-CoV akin to an fusion protein generated based on the SARS S1 subunit.

Fc fragments of SARS-CoV and MERS-CoV S1 domains

These studies also confirmed the previous notion that the enzymatic activity of the intracellular domain of the viral receptor is not required for viral entry (similar to SARS-CoV) and indeed inhibitors of DPP4 do not affect virus entry.

Again however, the potential effect of preventing the disease has not been shown in an animal model.

Lastly, we have to consider that the approval process complicates the introduction of vaccine for the use in humans. It might be easier to vaccinate animals such as dromedary camels with a novel vaccine. Such an approach has been undertaken with the introduction of a camel pox vaccine. Since this vaccine is approved for the use in camels, it might be feasible to adopt the vaccine backbone to vaccinate camels against MERS-CoV by inserting parts of the MERS-CoV into the vaccine strain. One of the problems is that MERS-CoV might be more ubiquitous in dromedary camels than we currently know - after all blood samples from camels taken 20 years ago have been shown to contain MERS-CoV antibodies. Secondly, although the transmission of MERS-CoV from camels to humans would be prevented other animals still might be able to transmit the virus into the human population.

What will be the solution in the end? First, we need to identify the mode of transmission and all natural hosts. Second, we need to improve the diagnostics and the isolation procedures. Third, we need to establish how common the infection is among the general population. And lastly, after all we need a vaccine which is not effective but also readily available in future local outbreaks. 

Further reading

Almazán F, DeDiego ML, Sola I, Zuñiga S, Nieto-Torres JL, Marquez-Jurado S, Andrés G, & Enjuanes L (2013). Engineering a replication-competent, propagation-defective Middle East respiratory syndrome coronavirus as a vaccine candidate. mBio, 4 (5) PMID: 24023385 

Zhao G, Du L, Ma C, Li Y, Li L, Poon VK, Wang L, Yu F, Zheng BJ, Jiang S, & Zhou Y (2013). A safe and convenient pseudovirus-based inhibition assay to detect neutralizing antibodies and screen for viral entry inhibitors against the novel human coronavirus MERS-CoV. Virology journal, 10 PMID: 23978242

Ying T, Du L, Ju TW, Prabakaran P, Lau CC, Lu L, Liu Q, Wang L, Feng Y, Wang Y, Zheng BJ, Yuen KY, Jiang S, & Dimitrov DS (2014). Exceptionally potent neutralization of MERS-CoV by human monoclonal antibodies. Journal of virology PMID: 24789777

Tang XC, Agnihothram SS, Jiao Y, Stanhope J, Graham RL, Peterson EC, Avnir Y, Tallarico AS, Sheehan J, Zhu Q, Baric RS, & Marasco WA (2014). Identification of human neutralizing antibodies against MERS-CoV and their role in virus adaptive evolution. Proceedings of the National Academy of Sciences of the United States of America, 111 (19) PMID: 24778221

Jiang L, Wang N, Zuo T, Shi X, Poon KM, Wu Y, Gao F, Li D, Wang R, Guo J, Fu L, Yuen KY, Zheng BJ, Wang X, & Zhang L (2014). Potent Neutralization of MERS-CoV by Human Neutralizing Monoclonal Antibodies to the Viral Spike Glycoprotein. Science translational medicine, 6 (234) PMID: 24778414

Wang N, Shi X, Jiang L, Zhang S, Wang D, Tong P, Guo D, Fu L, Cui Y, Liu X, Arledge KC, Chen YH, Zhang L, & Wang X (2013). Structure of MERS-CoV spike receptor-binding domain complexed with human receptor DPP4. Cell research, 23 (8), 986-93 PMID: 23835475

Mou H, Raj VS, van Kuppeveld FJ, Rottier PJ, Haagmans BL, & Bosch BJ (2013). The receptor binding domain of the new Middle East respiratory syndrome coronavirus maps to a 231-residue region in the spike protein that efficiently elicits neutralizing antibodies. Journal of virology, 87 (16), 9379-83 PMID: 23785207

Du L, Kou Z, Ma C, Tao X, Wang L, Zhao G, Chen Y, Yu F, Tseng CT, Zhou Y, & Jiang S (2013). 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. PloS one, 8 (12) PMID: 24324708

Wong SK, Li W, Moore MJ, Choe H, & Farzan M (2004). A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. The Journal of biological chemistry, 279 (5), 3197-201 PMID: 14670965

Raj, V., Mou, H., Smits, S., Dekkers, D., Müller, M., Dijkman, R., Muth, D., Demmers, J., Zaki, A., Fouchier, R., Thiel, V., Drosten, C., Rottier, P., Osterhaus, A., Bosch, B., & Haagmans, B. (2013). Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC Nature, 495 (7440), 251-254 DOI: 10.1038/nature12005

PAOLETTI, E. (1990). Poxvirus Recombinant Vaccines Annals of the New York Academy of Sciences, 590 (1 Rickettsiolog), 309-325 DOI: 10.1111/j.1749-6632.1990.tb42239.x

Pastoret, P., & Vanderplasschen, A. (2003). Poxviruses as vaccine vectors Comparative Immunology, Microbiology and Infectious Diseases, 26 (5-6), 343-355 DOI: 10.1016/S0147-9571(03)00019-5  

Jones GJ, Boles C, & Roper RL (2014). Raccoonpoxvirus safety in immunocompromised and pregnant mouse models. Vaccine PMID: 24837508