Identification of peptides containing epitopes of Ebola virus eliciting immune response

Abstract

Last 5 years have witnessed two of the worst Ebola virus outbreaks in history which have resulted in significant morbidity and mortality. Rapid spread of the virus across the globe has raised concerns over the safety of world citizens. No licensed vaccine or drug protective against human infecting Ebola species is available as yet. In a panic driven step, incompletely tested vaccines have been approved along with antiviral prophylaxis to counter unforeseen outbreaks. However, production difficulties, safety concerns, high booster dosage requirement, inefficient delivery systems and pre-existing immunity are some of the challenges faced by current vaccine development approaches. Hence, there is a pressing need to develop a vaccine strategy which can offer universal or at least broad protection against current and future Ebola virus strains in populations distributed worldwide. Highly conserved peptide fragments belonging to critical viral proteins and containing multiple epitopes which have the capacity to interact with a wide array of HLA molecules are anticipated to serve as potent candidates for a universal or broadly reactive Ebola vaccine. In light of the above facts, the present study is oriented towards identifying highly conserved promiscuous peptides containing multiple overlapping T (CD8+ and CD4+) and B cell epitopes and devoid of undesirable responses (autoimmunity, toxicity and allergenicity) in glycoprotein (GP) and nucleoprotein (NP) of Ebola virus using immunoinformatics techniques such as epitope prediction tools, HLA and population coverage analysis and molecular docking. Further, peripheral blood mononuclear cells (PBMC) from healthy volunteers were subjected to repetitive stimulation by peptides that presented the best in silico results to assess their immunogenic response by measuring cell proliferation and IFN-γ production with the help of MTT and ELISA assays respectively. Fifteen GP and eighteen NP peptide fragments containing multiple CD8+ T cell (HLA class I) epitopes while thirteen GP and fifteen NP fragments containing multiple CD4+ T cell (HLA class II) epitopes were obtained on merging the overlapping epitopes. Six GP and twelve NP peptide fragments common to their respective peptides containing both, CD8+ and CD4+ T cell epitopes were generated. B cell epitope prediction indicated the presence of B cell epitopes in four GP and eight NP peptide fragments. After screening for undesirable responses, four GP and six NP peptide fragments containing both, T and B cell epitopes, were obtained. Majority of the identified peptides were found to be 100% conserved in Zaire ebolavirus, the most pathogenic Ebola species while Gp3 and Np3 were also found to be 100% in other human infecting Ebola virus species (Sudan, Bundibugyo and Taï forest). Rest of the peptides were found to be conserved with minor variations while in other Filoviridae members (Lloviuvirus and Margburgvirus), these peptides have minor variations or could not be located. HLA coverage analysis based on prediction tools revealed that majority of the identified peptides displayed interaction with several HLA alleles/supertypes. Population coverage analysis indicated that the all peptides covered 69-100% population base in Asian, American, African and European continents with an average population coverage of ≥85% for these continents. Docking results revealed that the binding energy (Autodock Vina) and RMSD (CABS-dock) values obtained for all peptides were mostly found to be in range of native peptide values. Interestingly, results for some peptides outshined those of native peptides for corresponding HLA molecules. Based on the performance of identified peptides during peptide-HLA interaction analysis, six peptides (Gp1, Gp3 and Gp4, Np2, Np3 and Np5) were selected for further evaluation via in vitro experimentation. Peptide stimulation assays in PBMC revealed that all six peptides resulted in a significant peptide induced proliferation in healthy blood samples with Np3, Gp4 and Gp1 generating a significant response in ≥ 7 out of ten samples. Also, all peptides (except Np5) displayed a significant response in inducing peptide specific IFN-γ release in ≥ 7 samples. The results suggested that the in silico approach applied in combination with in vitro experimentation successfully selected peptides of glycoprotein and nucleoprotein which are capable of eliciting immune response, thus, making them potential candidates for global Ebola vaccine development.