Development of a Novel Multi-Epitope Vaccine Against Crimean-Congo Hemorrhagic Fever Virus: An Integrated Reverse Vaccinology, Vaccine Informatics and Biophysics Approach

Abstract

Crimean-Congo hemorrhagic fever (CCHF) is a highly severe and virulent viral disease of zoonotic origin, caused by a tick-born CCHF virus (CCHFV). The virus is endemic in many countries and has a mortality rate between 10% and 40%. As there is no licensed vaccine or therapeutic options available to treat CCHF, the present study was designed to focus on application of modern computational approaches to propose a multi-epitope vaccine (MEV) expressing antigenic determinants prioritized from the CCHFV genome. Integrated computational analyses revealed the presence of 9 immunodominant epitopes from Nucleoprotein (N), RNA dependent RNA polymerase (RdRp), Glycoprotein N (Gn/G2), and Glycoprotein C (Gc/G1). Together these epitopes were observed to cover 99.74% of the world populations. The epitopes demonstrated excellent binding affinity for the B- and T-cell reference set of alleles, the high antigenic potential, non-allergenic nature, excellent solubility, zero percent toxicity and interferon-gamma induction potential. The epitopes were engineered into an MEV through suitable linkers and adjuvating with an appropriate adjuvant molecule. The recombinant vaccine sequence revealed all favorable physicochemical properties allowing the ease of experimental analysis in vivo and in vitro. The vaccine 3D structure was established ab initio. Furthermore, the vaccine displayed excellent binding affinity for critical innate immune receptors: TLR2 (-14.33 kcal/mol) and TLR3 (-6.95 kcal/mol). Vaccine binding with these receptors was dynamically analyzed in terms of complex stability and interaction energetics. Finally, we speculate the vaccine sequence reported here has excellent potential to evoke protective and specific immune responses subject to evaluation of downstream experimental analysis.

Keywords: Crimean-Congo hemorrhagic fever; Crimean-Congo hemorrhagic fever virus; immunoinformatics; molecular dynamics simulation; vaccine.

Figure 1

In silico approaches implemented to design vaccine construct against CCHFV. Multi-Epitopes Vaccine Design.

Figure 2

Venn diagram presenting the number of epitopes filtered at different phases. SBTCE, Shared B and T cell epitopes; AE, Antigenic Epitopes; NAE, Non-Allergen Epitopes; SE, Soluble Epitopes; FENTIF, Final set of Epitopes Nontoxic and IFN gamma positive producer.

Figure 3

(A) Schematic representation of MEV construct for CCHFV, (B) Secondary structure of MEV construct, (C) Predicted 3D structure of designed MEV construct and (D) Ramachandran plot of MEV construct.

Figure 4

(A) 3D structure of the MEV after disulfide engineering with mutated residue are shown in cyan-green spheres, (B) Reverse translated primary DNA sequence of the MEV, (C) Closeup view of start-site of MEV RNA structure (full structure is given in Figure S1 ) and (D) In silico cloning of MEV (shown in red) in pET28a(+) vector.

Figure 5

Host immune system simulation in response to. The immune response (generation of Igs) by antigen exposure are shown in (A), and cytokines and interleukins production in different stated with Simpson index is shown in (B).

Figure 6

Molecular docking between MEV and TLR receptors. (A) Docked conformation and residues interaction map of MEV (shown in pink) to TLR2 (shown in purple), and (B) Docked conformation and residues interaction map of MEV (shown in red) to TLR3 (shown in cyan green).

Figure 7

Molecular dynamics simulation analysis of MEV and TLR receptors. (A) RMSD, (B) RMSF, (C) RoG and (D) Hydrogen bond analysis.

Figure 8

Radial distribution plots for close interactions of MEV and TLRs. (A) Plots for MEV and TLR2 and (B) Plots for MEV and TLR3.