Rational design and computational evaluation of a multi-epitope vaccine for monkeypox virus: Insights into binding stability and immunological memory

Abstract

Multi-epitope vaccines strategically tackle rapidly mutating viruses by targeting diverse epitopes from different proteins, providing a comprehensive and adaptable immune protection approach for enhanced coverage against various viral variants. This research employs a comprehensive approach that includes the mapping of immune cells activating epitopes derived from the six structural glycoproteins (A29L, A30L, A35R, L1R, M1R, and E8L) of Monkeypox virus (Mpox). A total of 7 T-cells-specific epitopes, 13 B-cells-specific epitopes, and 5 IFN-γ activating epitopes were forecasted within these glycoproteins. The selection process focused on epitopes indicating high immunogenicity and favorable binding affinity with multiple MHC alleles. Following this, a vaccine has been formulated by incorporating the chosen epitopes, alongside adjuvants (PADRE peptide) and various linkers (EAAAK, GPGPG, and AAY). The physicochemical properties and 3D structure of the multi-epitope hybrid vaccine were analysed for characterization. MD simulations were employed to predict the binding stability between the vaccine and various pathogen recognition receptors such as TLRs (TLR1, TLR2, TLR4, and TLR6), as well as both class I and II MHC, achieved through hydrogen bonding and hydrophobic interactions. Through in silico cloning and immune simulation, it was observed that the multi-epitopes vaccine induced a robust memory immune response upon booster doses, forecasting protective immunity upon viral challenge. This protective immunity was characterized by the production of IgM + IgG antibodies, along with release of inflammatory cytokines like IFN-γ, and IL12, and the activation of various immune cells. This study offers valuable insights into the potential of a multi-epitope vaccine targeting the Mpox virus.

Keywords: Immune simulation; Immunoinformatics; MD simulation; Monkeypox virus; Multi-epitope hybrid vaccine.

Graphical abstract

Fig. 1

Population Coverage. The plot of HLA population coverage of designed vaccine across different regions.

Fig. 2

Secondary structure components of Mpox vaccine. The per residue β-strand, helix and coil content present in the vaccine is shown in yellow, pink and black color, respectively.

Fig. 3

3D structure representing the final configuration of the vaccine. In the visualization, distinct colors highlight the adjuvant, various epitopes, and linkers. The amino acid sequence of the vaccine is comprised of 534 residues, underscoring the intricacy of its molecular composition.

Fig. 4

MD simulation of Mpox vaccine. (a) Time-dependent graph displaying root mean square deviation (RMSD) of the vaccine. Four snapshots at distinct time points: 0 ns, 100 ns, 200 ns, and 300 ns are indicated in cartoon representation. (b) Probability distribution of RMSD of Vaccine (c) RMSD of simulation 2 & 3 of vaccine (d) Time dependent SASA of vaccine with its probability distribution graph in the inset (e) Time dependent graph of R_g with its probability distribution graph in the inset (f) RMSF (g) Superimposed structures of vaccine at various time points (0, 50, 100, 150, 200, 250 and 300 ns) and (h–j) the most-population microstates m1, m2 and m3 of vaccine simulation. The percentage indicates the population of that microstates w.r.t to total number of conformations.

Fig. 5

The molecular docking of vaccine with (a) TLR1 receptor (b) TLR2 receptor of human. These docking simulations provide insights into the molecular interactions between the vaccine and human TLR1 or TLR2, highlighting their potential roles in initiating immune responses.

Fig. 6

The molecular docking of multi-epitope vaccine with (a) TLR4 receptor (b) TLR6 receptor of human. Molecular docking of the vaccine with the TLR4 and TLR6 demonstrates specific and favorable noncovalent binding interactions, elucidating the potential activation of immune responses.

Fig. 7

The molecular docking showing binding of Mpox vaccine with (a) MHC-I (b) MHC-II of human. Molecular docking of the vaccine with MHC I and II reveals precise and favorable binding interactions, offering insights into potential immune response activation mechanisms. The two chains of MHC-II are denoted by chain A = (a); chain B= (b).

Fig. 8

MD simulation of Mpox vaccine with TLR1, TLR2, TLR4, TLR6, MHC-I and MHC-II. (a) RMSD (b) Rg (c) RMSF (d) Number of hydrogen bond between Mpox vaccine and various receptors (TLR1, TLR2, TLR4, TLR6, MHC-I and MHC-II) (e) SASA plot showing the changes in solvent-accessible surface area of the vaccine and immune receptors.

Fig. 9

Clustering. The three most populated conformations (m1, m2 and m3) of six MD simulation systems (a) TLR1 + Vaccine; (b) TLR2 + Vaccine; (c) TLR4 + Vaccine; (d) TLR6 + Vaccine; (e) MHC-I + Vaccine; (f) MHC-II + Vaccine. The percentage indicates the population of the microstates (m1, m2 and m3) w.r.t to total number of conformations.

Fig. 10

PCA analysis. The displacement of Cα atoms along the eigenvector 1 and 2 for all systems are shown in panel a. The 2D representation of motion, based on the first two eigenvectors obtained through principal component analysis (PCA), is depicted for Vaccine only and the Vaccine with TLRs and MHC receptors in panel b–h.

Fig. 11

In silico cloning for expression of recombinant protein. The Mpox vaccine construct carrying multiple epitopes was virtually cloned into the pET-28b(+) expression vector, with the inserted segment depicted in red and the remaining sections representing the vector genome.

Fig. 12

Comparative immune simulation analyses showing high immunogenicity of the multi-epitope Mpox vaccine. (a) Immune response of administration of Mpox vaccine on 1st and 31st day followed by addition of virus on 59th, 186th and 366th day. (b) Control experiment involving administration of only virus at on 59th, 186th and 366th day.

Fig. 13

Abundance of T-cells activating and proliferating cytokines in the immune simulation studies with Mpox vaccine. Concentrations of cytokines and interleukins (ILs) were assessed in the comparative experiment involving (a) Vaccine + Virus and (b) Only Virus. The inset plot illustrates the presence of danger signals alongside the leukocyte growth factor IL2.

Fig. 14

Activation and proliferation of B-cells. Activation of B-cells population (cells per mm³) in the comparative experiment of (a) Vaccine + Virus (b) Only Virus at different time points. Cell counts are shown in per mm³ human blood.

Fig. 15

Activation and proliferation of Th-cells. Augmentation of CD4⁺ Th-cells population per state (cells per mm³) in the comparative experiment of (a) Vaccine + Virus (b) Only Virus at different time points. Cell counts are shown in per mm³ human blood.

Fig. 16

Activation and proliferation of Tc-cells. Concentration of CD8⁺ Tc-cells population per state (cells per mm³) in the comparative experiment of (a) Vaccine + Virus (b) Only Virus at different time points. Cell counts are shown in per mm³ human blood.

Fig. 17

The workflow of development of multi-epitope monkeypox vaccine. Strategic flowchart of developing a multi-epitope monkeypox vaccine, showcasing the systematic design and construction process to enhance efficacy and immune response.