Structural basis of development of multi-epitope vaccine against Middle East respiratory syndrome using in silico approach

Srivastava, Sukrit; Kamthania, Mohit; Singh, Soni; Saxena, Ajay K; Sharma, Nishi

The proteome of MERS-CoV consists of several important vaccine candidate and drug target proteins. These proteins are involved in infection and pathogenesis of MERS-CoV to human host cells. Spike (S) glycoprotein, in particular, its receptor-binding domain, is involved in virus and host cell interaction. 8 Envelope (E) protein plays an important role in host cell recognition. 9 Nucleocapsid (N) protein is involved in RNA binding during ribonucleocapsid formation by MERS-CoV. 10 Membrane (M) protein has interferon (IFN)-antagonizing properties, thus reducing IFN levels in infected patients. 11 Open reading frame (ORF) proteins also play critical roles in viral infection and pathogenesis. A mutation study of ORFs (ORF3, ORF4a, ORF4b, and ORF5) indicated that ORFs have major implications in viral infection involving disruption of host cell processes, dysregulated IFN pathway activation, and abrupt inflammation. 12 Proteins ORF1a (4P16) and ORF1ab (4WUR) are papain-like proteases (PL(pro)) involved in viral infection and are potential targets for the development of antiviral drugs. 13 PL(pro) facilitate infection by their proteolytic, deubiquitinating, and deISGylating activities suppressing innate immune response from the host cell. Protein ORF1a (4RSP), a protease (3CLpro), facilitates proteolytic activity during viral infection and replication. 14, 15 Protein ORF1ab (5WWP) is a helicase protein of MERS-CoV, and it is one of the most conserved proteins among nidoviruses. Protein ORF8b is a highly conserved protein, and during infection, it induces various immune responses. 16 In the present study, we propose two multi-epitope vaccine (MEV) designs for MERS. The proposed MEVs are composed of cytotoxic T lymphocyte (CTL) and helper T lymphocyte (HTL) epitopes. Both CTL and HTL MEVs contain overlapping regions of linear B-cell epitopes. Both MEVs also contain human β-defensin-2 (hBD-2) and human β-defensin-3 (hBD-3) as adjuvants to enhance the immunogenic response. 17, 18 Methodology To design the MEVs, different in silico tools were used to screen potential CTL, HTL, and B-cell epitopes from 13 MERS-CoV proteins that are involved in virus-host interaction and viral proliferation. All three types of epitopes were studied for overlapping consensus regions. CTL and HTL epitopes showing partial or complete overlapping regions with all or any of the three kinds of epitopes and the epitopes with the highest number of human leukocyte antigen (HLA) allele binders were selected for detailed analysis. The selected CTL and HTL epitopes were further validated for their molecular interaction with their HLA allele binders. Furthermore, the selected CTL epitopes were validated for their molecular interaction with cavity of transporter associated with antigen processing (TAP) to analyze their smooth passage from the cytoplasm to the endoplasmic reticulum (ER) lumen. 19 Three-dimensional (3D) structure model for both MEVs was generated, refined, validated, and analyzed for different physiochemical properties. Both CTL and HTL MEV models were further screened to have several B-cell discontinuous epitopes and IFN-γ-inducing epitopes. Because the immune system broadly recognizes pathogens partially due to the involvement of toll-like receptor 3 (TLR-3) and its signaling cascade, both CTL and HTL MEVs were further studied for their molecular interaction with TLR-3. 20,21 Moreover, the cDNAs of both MEVs were analyzed and were predicted to be highly expressing i