A highly conserved WDYPKCDRA epitope in the RNA directed RNA polymerase of human coronaviruses can be used as epitope-based universal vaccine design

Coronaviruses are the diverse group of virus which infects domestic animals, birds as well as human [1]. Coronaviruses are enveloped viruses which are the members of Coronaviridae family [2]. Coronaviruses have positive strand RNA genome which is approximately 26–32 kb long. The overall structures of all coronaviruses are composed of the spike (S), envelope (E), membrane (M) and nucleocapsid (N) protein. The other non-structural proteins like RNA directed RNA polymerase, helicase, 3CL like proteinases etc are produced by the cleavage of replicase polyprotein 1ab or ORF 1ab polyprotein [3].

The first coronavirus 229E was identified in 1960. Since then different types of coronaviruses have emerged. HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HCoV-HKU1 and MERS-CoV are the coronaviruses which infect human [4]. Most of the coronaviruses cause respiratory, enteric, hepatic, or neurological diseases with highly variable severity in their hosts [5]. The first two coronavirus HCoV-229E and HCoV-OC43 infects lower respiratory tract where the later SARS-CoV, HCoV-NL63, HCoV-HKU1 infect both lower and upper respiratory tract [4, 6–8]. But the newly emerged MERS-CoV infects both lung and kidney that reflects how these viruses are changing their cell tropism and becoming highly pathogenic [9].

Most infections caused by human coronaviruses were relatively mild. Among all the human coronaviruses, SARS-CoV and MERS-CoV are much more deadly. The severe acute respiratory syndrome (SARS) outbreak caused by SARS-CoV in 2002 to 2003. The SARS-CoV created outbreak resulted in a total of 916 deaths among more than 8000 confirmed cases in over 30 countries [10, 11]. The newly emerged MERS-CoV is now posing a great threat for human. According to WHO, 75 people have died among more than 178 confirmed cases caused by MERS-CoV [4]. Though this MERS-CoV virus was first found in Saudi Arabia 2012, now it has been emerged in UK, France, Tunisia, Spain and Italy that indicates it’s going to create another outbreak like SARS-CoV [12–14]. From 1960 to till now there is no recommended drug or vaccine for MERS-CoV infection and treatment relies on exclusively supportive care, which gives the high case-fatality rate, is not highly effective [15].

In 2003 after the discovery of SARS-CoV, there were a significant increase in research on coronavirus, but no definitive antiviral or therapeutic treatment for coronavirus infections came from these researches [16]. From the clinical experience of SARS-CoV found that a number of interventions including ribavirin with and without corticosteroids, ribavirin with protease inhibitors and interferon with corticosteroids may improve outcome. But a definitive treatment was not clearly established and the therapeutic interventions have not been evaluated in vivo[17].

The identification of therapeutics is a high priority and though there is currently no specific therapy or vaccine for human coronaviruses, this disease has been severe with a high case-fatality rate [18]. As these viruses are now becoming pathogenic and causing outbreaks, so steps have to be taken to prevent human death. Vaccination is one of the most efficient ways to prevent infectious disease [19]. Effective vaccines controlling virus spread and disease are available for a number of infections, such as smallpox, poliomyelitis, measles, mumps, rubella, influenza, hepatitis A, and hepatitis B [20]. For coronavirus, this vaccine approach is hindered by the fact that human coronavirus strains are not structurally related and they are changing rapidly by recombination [21]. Therefore, designing a universal vaccine against conserved regions for all human coronaviruses is a major challenge at present.

With the disclosure of huge sequence information, epitope based vaccine design now has become a most promising approach for viral vaccine preparation [22]. In order to prepare vaccines, computational prediction of epitopes and vaccine design can reliably aid this process to reduce time and cost. Although the epitope based vaccine design is now a familiar concept, not much work has been done in case of coronaviruses.

In this study, we design an epitope based universal vaccine which can be use to prevent all kind of human coronaviruses. For this, bioinformatics analyses of viral proteins were done for finding the conserved peptide region and for mapping the evolutionary conserved epitope. The 3D structure of RNA directed RNA polymerase was determined by threading modeling technique and a highly immunogenic, accessible and conserved epitope was identified. This epitope can be used as a universal vaccine against all human coronaviruses.

Coronaviruses are one of the most diverse groups of virus which are becoming a deadly virus day by day. Though the first two strains were not so much deadly but the other members were pathogenic. After SARS outbreak, a new coronavirus strain called MERS-CoV is now going to cause another outbreak [4]. The cell tropism and cellular receptor of the six types of coronaviruses are not similar (Additional file 7: Table S1). Though at first it was thought that SARS-CoV and MERS-CoV are structurally similar and tried to treat MERS-CoV infected patient with the SASR-CoV treatment. But it was found that they bind to two different receptors, namely ACE2 and DPP4 or CD26 respectively [36]. These viruses are actually zoonotic origin, undergo recombination, and may be in future another strain of this group of virus will come [37]. Therefore, it is important to take preventing measures not only to prevent this new strain of coronavirus, also against all the strain of coronavirus. There is no recommended vaccine for coronaviruses which is necessary to prevent. Most of the cases, vaccines were designed by targeting spike protein. Similarly, researchers also reported to design vaccine against SARS-CoV and MERS-CoV spike protein [38, 39]. Fernando et al. also designed lived-attenuated MERS coronavirus by mutating MERS-CoV envelope protein as a vaccine which will be for only MERS-CoV [40]. These vaccines would thus be effective for only those strains not for others. Giving a universal vaccine for all strain of viruses is much more promising solution rather than giving individual vaccine for individual strain.

The concept of prevention of viruses by designing a universal vaccine has also been reported previously, for example against Influenza virus. In case of influenza virus, universal vaccine against matrix 2 protein which was found to be conserved among all Influenza subtypes was reported [41]. An attempt to design universal vaccine against members of coronaviruses, like feline infectious peritonitis (FIPV), canine coronavirus (CCV), gastroenteritis coronavirus (TGEV), bovine coronavirus (BCV) targeting their spike protein in 1993 were observed [42]. But this concept was not applied to human coronaviruses.

Vaccine development has been one of the most important contributions of immunology to public health to date. Traditionally vaccines were based on the intact pathogen, either inactivated or live attenuated. These types of vaccines had some crucial drawbacks like safety consideration and the loss of efficacy due to the genetic variation of the pathogen. But now a day’s these vaccine concepts are greatly replaced by novel vaccine approaches like naked DNA vaccine, epitope based vaccine. The main benefit of immunization with an epitope-based vaccine is the ability to immunize with a minimal structure and it will stimulate an effective specific immune response, while avoiding potential undesirable effects [43].

In this study, we aimed to design an epitope-based universal vaccine for all human coronavirus strain. For this purpose we did multiple sequence alignment of the spike (S), envelope (E), membrane (M), nucleocapsid (N) protein and replicase polyprotein 1ab of all six human coronaviruses. Replicase polyprotein 1ab was taken to check whether there is any conservancy among the non structural protein as this replicase polyprotein cleaved into 15 non-structural proteins. In case of S, E, M and N protein, no putative conserved region was found. But conserved region was found in case of replicae polyprotein 1ab where the conserved region was in the RNA directed RNA polymerase. This indicates that this protein is less mutating than the S, E, M and N protein. This RNA directed RNA polymerase protein was targeted to determine antigenic sites based on immunogenicity and surface accessibility using different bioinformatics analyses. The consensus antigenic sites were the desired one and their conservancy was also determined. From the conservancy analysis it was found that Bepipred [29] and IEDB [28] analysis predicted consensus epitope YPKCDRA and the surface accessibility analysis [30] predicted epitope WDYPKC are 100% conserved. This 100% conserved WDYPKC and YPKCDRA are actually located in the same region of RNA directed RNA polymerase and it was then taken as the targeted epitope. This epitope was found to be accessible and hydrophilic which is one of the crucial requirements for an epitope to be used as a vaccine. This reflects a promising scope to use this conserved epitope as a universal vaccine both as preventive and therapeutic treatment. As this epitope remain long been conserved since 1960, it may be possible to use this vaccine in future for upcoming human coronavirus strains as well. To become an effective vaccine, it needs to be highly immunogenic, stable inside the body. If it is poorly immunogenic or unstable, it needs to be conjugated with adjuvant [44]. Though this epitope based vaccine is designed by in silico analyses, the actual immunogenicity, stability, efficacy and their delivery strategy inside the recipients body can’t be determined by this in silico analysis. To address these questions in vitro and in vivo experiments are essential.

This study shows that though the human coronaviruses are not structurally related but it is possible to design an epitope-based universal vaccine for all human coronavirus strains. Our results are based on sequence analysis and computational predictions show predicted epitope would be a candidate target for the universal vaccine; and to determine the actual effectiveness of the peptide for mounting an immune response both in vitro and in vivo studies can be performed.