Designing a multi-epitope influenza vaccine: an immunoinformatics approach

Abstract

Influenza continues to be one of the top public health problems since it creates annual epidemics and can start a worldwide pandemic. The virus's rapid evolution allows the virus to evade the host defense, and then seasonal vaccines need to be reformulated nearly annually. However, it takes almost half a year for the influenza vaccine to become accessible. This delay is especially concerning in the event of a pandemic breakout. By producing the vaccine through reverse vaccinology and phage display vaccines, this time can be reduced. In this study, epitopes of B lymphocytes, cytotoxic T lymphocytes, and helper T lymphocytes of HA, NA, NP, and M2 proteins from two strains of Influenza A were anticipated. We found two proper epitopes (ASFIYNGRL and LHLILWITDRLFFKC) in Influenza virus proteins for CTL and HTL cells, respectively. Optimal epitopes and linkers in silico were cloned into the N-terminal end of M13 protein III (pIII) to create a multi-epitope-pIII construct, i.e., phage display vaccine. Also, prediction of tertiary structure, molecular docking, molecular dynamics simulation, and immune simulation were performed and showed that the designed multi-epitope vaccine can bind to the receptors and stimulate the immune system response.

Keywords: Immunoinformatics; Influenza; Molecular dynamics simulation; Multi-epitope; Phage display vaccine; Reverse vaccinology.

Figure 1

Procedure for creating the multi-epitope-pIII vaccine. The plot was created by drawio server (https://www.drawio.com/).

Figure 2

Construction of the multi-epitope. AAY (violet) linkers are inserted between the CTL epitopes. GPGPG (orange) linkers are inserted between the HTL epitopes. KK (green) linkers are inserted between the BCL epitopes.

Figure 3

Secondary structure. The multi-epitope was made up of 26.48% alpha-helix, 25.86% β-sheet, and 47.66% coil.

Figure 4

In silico cloning of the multi-epitope. The multi-epitope was inserted into the N-terminal end of M13 protein III in the FADL-1e phage vector using the SnapGene software to create the multi-epitope-pIII construct, i.e., phage display vaccine. The multi-epitope is represented in the cyan area, while M13 pIII is represented in the purple portion afterward.

Figure 5

An assessment of 3D model of the vaccine construct prior to and subsequent refinement. (a) The primary model’s Z-score was − 2.09, while (b) the refined model’s Z-score was − 2.74. (c) In the Ramachandran plot, the residues in the favored, additional allowed, generously allowed, and disallowed regions, respectively, were 49.5%, 40.2%, 6.9%, and 3.4% of the residues of the primary model (d), whereas in the refined model, these values changed to 69.2%, 24.7%, 2.7%, and 3.3%, respectively.

Figure 6

3D model of the multi-epitope-pIII. The 3D model of the multi-epitope-pIII created by I-TASSER after refinement and 100 ns MDS. The multi-epitope is represented in pink, and M13 pIII is represented in yellow.

Figure 7

Conformational epitopes. A-C: conformational epitopes in the 3D model of the multi-epitope-pIII. The majority of the protein is represented as gray sticks, while epitopes are displayed as yellow surfaces.

Figure 8

The backbone RMSD of all free receptors. The backbone RMSD of MHC I, II, and TLR9 dimer during 20 ns MDS.

Figure 9

Docking complexes and molecular interactions after 100 ns MDS. (a) Molecular docking of CTL epitope 1 (red) and MHC I (green). (b) Molecular docking of HTL epitope 4 (magenta) and MHC II (green). (c) DIMPLOT diagram shows residues involved in CTL epitope 1(A)-MHC I (B) interactions. (d) The DIMPLOT diagram shows residues involved in HTL epitope 4 (A)-MHC II (B) interactions. (e) Molecular docking of a portion of the phage DNA genome (pink) and TLR9 dimer (green). (f) The DIMPLOT diagram shows the nucleotides (A) and residues (B) involved in the DNA-TLR9 dimer interaction. The horizontal dashed line represents the interface. In all cases, docking complexes and the interaction diagrams are after 100 ns MDS and include hydrogen bonds and non-bonded contacts.

Figure 10

The backbone RMSD of all complexes (receptors-ligand) also multi-epitope-pIII. Complexes of CTL epitope 1-MHC I, HTL epitope 4-MHC II, and phage DNA-TLR9 dimer also free multi-epitope-pIII during 100 ns MDS. Com: complex.

Figure 11

MM/PBSA binding energies of complexes. MM/PBSA binding energies of CTL epitope 1, HTL epitope 4, and phage DNA with their receptors during 100 MDS.

Figure 12

Chimeric vaccine molecular simulations. (A) Injection of antigens that produce immunoglobulins and their subclasses. (B) Development of plasma cell population following a single dose of vaccine injection. (C) CTL population. (D) HTL population (i.e., active, resting, anergic, and duplicating). Anergy state indicates cells that are tolerant of antigen, suggesting a lack of immunological reactivity, while resting phase indicates cells that have not been exposed to the antigen.