Middle East Respiratory Syndrome (MERS) also known as camel flu, was first documented in Saudi Arabia. MERS can be manifested as an asymptomatic infection or in critical cases, be fatal. Its symptoms include fever, cough, shortage of breath, nausea and can also progress to pneumonia. It is spread through contact with infected dromedary. Even though the transmission rate of MERS is low, almost 35% of infected patients have died [8]. Till date, no approved drugs or vaccines exist for the disease. The causative agent of MERS is the MERS- coronavirus (MERS- CoV) which is a positive sense,single stranded RNA Betacoronavirus. Its viral genome is about 30kb in size.The genome codes for 4 structural proteins (Spike (S) protein, Envelope (E) protein, Membrane (M) protein and Nucleocapsid (N) protein),16 non-structural proteins (NSP1-NSP16), and 5 accessory proteins (ORF3, ORF4a, ORF4b, ORF5, and ORF8b)[5].

Traditional vaccine development techniques rely on injection of killed/ live attenuated/ sub-unit microorganism into humans to induce an immune response relying largely on the memory of the adaptive immune response. However, this process can be time consuming and is largely based on trial and error. In the case of MERS,vaccine development has largely focused on eliciting a strong immune response against the envelope protein (S) and the nucleocapsid protein (N). Sub-unit vaccines based on the Receptor Binding Domain of the S protein vaccine have been tested on mouse models and have shown promise. Other candidates include full length S vaccine, N terminal domain, delivery of live attenuated virus and inactivated virus using viral vectors for optimal delivery etc [5]. In spite of showing promising results in pre- clinical studies, questions regarding potency in the human population that is heterogeneous and can elicit a range of immune responses, remain unanswered[3].

Pangenomic Ranjini
                                                  Reverse Vaccinology

With the advances in genomics and amalgamation of in silico analyses, structural and functional genomics, and experimental techniques, novel strategies encompassing pan-genomics and epigenomics have been developed for a more rapid identification of vaccine targets giving rise to the field of reverse vaccinology. Scientists have been able to process high throughput genomic data which has significantly reduced the time for target identification. Genome mining has made it possible to predict potential genes encoding factors that promote pathogenesis. Moreover, the development of post-genomics approaches has accelerated the discovery of factors related to pathogenesis, which are key elements in the design of new vaccines [1][4]. Pan- genomic studies (involving analyses of the entire genome of all viral strains) have shed light on heterogeneity in viral evolution and identification of core antigens to be included in vaccines for resistance to all strains of the virus. Studies on modulation of the epigenetic landscape in host due to MERS-CoV have shown that the virus has the potential to antagonize antigen presentation. This could imply that the virus can down regulate the elicited immune response in spite of vaccine administration [2][6]. Thus, understanding advances in pan-genomic and epi-genomic techniques could be the key to novel vaccine development for MERS.

Methodology

The reverse vaccinology technique starts with the entire genome of the virus as the starting material. This enables identification of molecular motifs responsible for viral entry and eliciting virulence. This enables efficient identification of potential vaccine targets[1]. Comparative genome hybridization studies using microarray techniques have shown that genetic diversity within a specie can hinder the immune response generated by vaccines that target only a specific domain of a particular strain.The virus can

thus evolve to have resistance to the virus. Today, it is possible to develop a universal vaccine using computational algorithms targeting the core antigens common to every strain [7].

Chromatin modification due to epigenetic modulation provides cells with a mechanism to retain acquired transcriptional regulation throughout cell division. Post transcriptional and transcriptional studies include epigenetic and proteomic studies. Proteomics has enabled identification of surface proteins using techniques relying on LC/ MS (especially MALDI- TOF) [6][7][11][12]. Affinity enrichment methods relying on antibodies to particular epigenetic marks are commonly used for detecting changes in histone modifications and DNA methylation between two cell types, or different stages of differentiation of a single cell type. Characterizing the transcriptional programming of the effector T cells and memory T cells of mice immunized with identified vaccine candidates can elucidate if these T cells undergo any changes due to acute/ chronic infection with the MERS- CoV. Characterization of methylation state and histone modification can most easily be achieved usingCHIP- seq and DNase- seq analysis[9][11][12].

Conclusion

Starting with the entire pangenome of the MERS- CoV followed by in silico analyses can provide scientists an entire list of potential candidates, their function, and their localization. Thein-silicoanalysis is the first step for the selection of secreted virulent proteins or coat proteins or membrane-associated proteins that interact with the human immune system. This also allows identification of the ideal combination of the proteins to be included (from different strains) in the vaccine. The next step is functional characterization of the antigen and comparative experimental studies in different conditions. This enables selection of the candidate viable in heterogeneous conditions. Rational design of target epitopes for vaccine candidates largely depends on structural biology (virology in this case). Homology modelling enables visualization of ligand receptor binding in MERS infection[4]. Epigenomic studies here, enable scientists to understand how MERS- CoV modulates the immune landscape and the differentiation status of T cells. This sheds light upon the inclusion of possible booster doses in the vaccination regimen.

Therefore, we have shown how reverse vaccinology encompassing techniques from bioinformatics, in vitro and animal model studies, genomics, pan- genomics, comparative genomics, structural virology, proteomics, and epigenomics to name a few, can pave the way towards identification of novel candidates or in improving existing vaccine candidates for MERS infections.

Abbreviations

LC: Liquid Chromatography MS: Mass Spectrometry

MALDI TOF: Matrix-assisted laser desorption/ionization- time-of-flight CHIP seq: Chromatin Immunoprecipitation- sequencing

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