In-silico design of a multi-epitope vaccine candidate against onchocerciasis and related filarial diseases

Abstract

Onchocerciasis is a parasitic disease with high socio-economic burden particularly in sub-Saharan Africa. The elimination plan for this disease has faced numerous challenges. A multi-epitope prophylactic/therapeutic vaccine targeting the infective L3 and microfilaria stages of the parasite's life cycle would be invaluable to achieve the current elimination goal. There are several observations that make the possibility of developing a vaccine against this disease likely. For example, despite being exposed to high transmission rates of infection, 1 to 5% of people have no clinical manifestations of the disease and are thus considered as putatively immune individuals. An immuno-informatics approach was applied to design a filarial multi-epitope subunit vaccine peptide consisting of linear B-cell and T-cell epitopes of proteins reported to be potential novel vaccine candidates. Conservation of the selected proteins and predicted epitopes in other parasitic nematode species suggests that the generated chimera could be helpful for cross-protection. The 3D structure was predicted, refined, and validated using bioinformatics tools. Protein-protein docking of the chimeric vaccine peptide with the TLR4 protein predicted efficient binding. Immune simulation predicted significantly high levels of IgG₁, T-helper, T-cytotoxic cells, INF-γ, and IL-2. Overall, the constructed recombinant putative peptide demonstrated antigenicity superior to current vaccine candidates.

Figure 1

Schematic presentation of the final multi-epitope vaccine peptide. The 599-amino acid long peptide sequence containing an adjuvant (green) at the amino terminal end linked with the multi-epitope sequence through an EAAAK linker (cyan). B epitopes and HTL epitopes are linked using GPGPG linkers (yellow) while the CTL epitopes are linked with the help of AAY linkers (purple). A 6x-His tag is added at the Carboxy terminus for purification and identification purposes.

Figure 2

C-ImmSim simulation of the cytokine levels induced by three injections given 4 weeks apart. The main plot shows cytokine levels after the injections. The insert plot shows IL-2 level with the Simpson index, D indicated by the dotted line. D is a measure of diversity. Increase in D over time indicates emergence of different epitope-specific dominant clones of T-cells. The smaller the D value, the lower the diversity.

Figure 3

Graphical representation of secondary structure features of the final subunit vaccine sequence. (A) The protein is predicted to comprise alpha-helices (22.0%), beta strands (10.0%) and coils (67.0%), and (B) 48% of positions are predicted as disordered.

Figure 4

Protein modelling, refinement and validation. (A) The final 3D model of the multi-epitope vaccine obtained after homology modelling on I-TASSER. (B) Refinement: superimposition of the refined 3D structure (coloured) on the ‘crude model’ (gray) by GalaxyRefine. Validation of the refined model with (C) Ramachandran plot analysis showing 94.5%, 4.5% and 1.0% of protein residues in favoured, allowed, and disallowed (outlier) regions respectively and (D) ProSA-web, giving a Z-score of −3.45.

Figure 5

Molecular docking of subunit vaccine with immune receptor (TLR4). (A) Docked complexes for adjuvant-TLR4 complex with adjuvant colored light pink, chain B of TLR4 colored light blue and the interface colored hot pink and blue, and (B) chimeric protein-TLR4 complex with protein colored light pink, B chain of TLR4 colored light blue and the interface colored in blue and hot pink. (C) Interface active residues for adjuvant-TLR4 complex with adjuvant active residues colored hotpink and TLR4 active residues colored in blue, and (D) chimeric protein-adjuvant complex with protein active residues colored hotpink and TLR4 active residues colored blue.

Figure 6

In silico restriction cloning of the final vaccine sequence into the pET30a (+) expression vector where the red part represents the gene coding for the vaccine and the black circle represents the vector backbone. The His-tag is located at the Carboxy-terminal end.

Figure 7

C-ImmSim presentation of an in silico immune simulation with the chimeric peptide. (A) Immunoglobulin production in response to antigen injections (black vertical lines); specific subclasses are indicated as coloured peaks. (B) The evolution of B-cell populations after the three injections. (C) The evolution of T-helper, and (D) T-cytotoxic cell populations per state after the injections. The resting state represents cells not presented with the antigen while the anergic state represents tolerance of the T-cells to the antigen due to repeated exposures.