Design of synthetic Peptides as Vaccine candidates against Crimean-Congo haemorrhagic fever virus

Abstract

Crimean-Congo hemorrhagic fever virus (CCHFV) is endemic in 30 nations of Asia, Europe, Middle East, and Africa, and has the widest geographic distribution among tick-borne virus diseases. Hyalomma marginatum ticks serves as primary vector of CCHFV. Three billion people are thought to be susceptible to CCHFV infection. In 1944, the first case of CCHFV was found in the Crimean area of Eastern Europe. Later in 1956, it was found in the Belgian Congo, and it was called as Crimean-Congo hemorrhagic fever (CCHF). CCHF has reported 140 cases in 52 countries around the world and resulted an endemic situation. In 2011, India witnessed first case of CCHF disease in Ahmedabad and in subsequent years cases in other regions of India have been documented (Rajasthan and Kerala). From 2011-2020, 125 cases and 53 deaths of CCHF infection have been documented in India. World health organization (WHO) has categorized CCHFV as high priority pathogen. No treatment or vaccines for CCHFV have been approved till date for human use. Therefore, there is a crucial need of CCHFV vaccination which can offer protection against all CCHFV strains. Vaccine candidates targeting conserved regions of CCHFV and capable of binding with diverse human leukocyte antigen (HLA) alleles could serve as potential candidates against CCHF disease. Considering these facts, this study is focused on selection of conserved CCHFV RNA segment aided mutation profiling, homology modelling and simulation study of L, M and S segment followed by prediction of conserved, antigenic peptides containing multiple B and T cells without any detrimental response (autoimmunity and allergenicity) using immunoinformatics approach. Identified peptides were evaluated for population coverage and binding affinity with HLA alleles and T cell receptor aided docking study. Best docked complexes were simulated for 50 ns to assess stability of HLA-peptide-TCR docked complexes using GROMACS v2021 software. Furthermore, identified peptides were commercially synthesized for evaluating the ability of peptides to induce immune response in peripheral blood mononuclear cells (PBMCs). In mutation profiling, 106 sequences (L segment), 51 sequences (M segment) and 50 sequences (S segment) were evaluated for mutations at each amino acid position w.r.t. NCBI reference sequences: YP_325663.1, NP_950235.1 and NP_950237.1 respectively. Total 38, 47 and 7 highly frequent mutations (0.81-1) in L, M and S segment respectively were recorded. These mutations were mapped in the important region of RNA segments were selected to evaluate effect of point mutation of the stability of respective protein using homology modelling and molecular dynamic (MD) simulation of 50 ns. Six mutations in catalytic site domain were recorded and it was found that all six mutants are unstable w.r.t. wild catalytic site domain. Seven mutations mapped in structural proteins of M segment (glycoprotein C, Gc and glycoprotein N, Gn) were evaluated and large deviation and fluctuation was observed in all mutant models. In S segment, seven mutant models were evaluated and among them one mutant model displayed large deviation and fluctuation during 50ns simulation run. Mutation profiling concludes that S segment is more conserved than L and M segment, as only seven highly frequent mutations were recorded in S segment and all mutant models (except H195R) displayed stability upon introduction of single amino acid mutation during 50 ns simulation run. Therefore, S segment of CCHFV was selected for identification of conserved peptides and three conserved, antigenic peptides containing multiple B and T cell epitopes without any detrimental response were predicted. Population coverage analysis displayed ≥99% population coverage of three identified peptides across six continents. Docking study represented the comparative binding energy of three peptides and its associated epitopes to respective native peptides with both class I and II HLA alleles. Docking of HLA-peptide complexes with T cell receptor (TCR) also represented good binding interactions. MD simulation of best docked HLA-peptide (HLA B*35:05-P2_3 and HLA DR15-P3) and HLA-peptide-TCR (HLA A2-P2_3-TCR and HLA DR11-P3-TCR) complexes displayed stability during 50 ns simulation run. Three peptides further selected for in vitro study (MTT assay and ELISA assay) revealed all peptides exhibits peptide specific proliferation and IFN‐γ release in PBMCs of 10 healthy volunteer blood samples. P1 peptide displayed significant difference in peptide specific cells proliferation in 8 out of 10 blood samples from unstimulated cells. P2 and P3 displayed significant difference in five and six blood samples respectively from unstimulated cells. All peptides displayed significant difference in IFN‐γ release in all 10 blood samples from unstimulated cells. These results suggest that identified peptides are conserved, antigenic, exhibits high population coverage, good binding with diverse HLA alleles and TCR and have ability to induce immune response. Therefore, three peptides might serve as potent vaccine candidate to prevent from CCHF infection.