Table 1.
CTL epitope screening results.
Table 2.
HTL epitope screening results.
Table 3.
LBL epitope screening results.
Fig 1.
Schematic illustration of BtHKU5-CoV-2 vaccine construction.
(A) The amino acid sequence of the vaccine construct. Each part is represented in a different color, with β-defensin II adjuvant, CTL epitope, HTL epitope, LBL epitope, pan-HTL epitope and TAT peptide shown in green, blue, red, yellow, purple and orange, respectively. (B) The predicted secondary structure of the vaccine construct. Distinct structural elements labeled with different colors are indicated in the annotations below.
Fig 2.
Refined tertiary structure of BtHKU5-CoV-2 vaccine construct.
(A) Original tertiary structure model of the vaccine construct. (B) Refined tertiary structure model of the vaccine construct. (C) Structural alignment of the original and refined models, generated and analyzed using PyMOL.
Fig 3.
Quality evaluation for refined tertiary structure of BtHKU5-CoV-2 vaccine.
(A) Refined tertiary structure model of the vaccine. (B) The stereochemical quality of the refined tertiary structure was assessed using a Ramachandran plot. In the presented results, red represent the most favoured regions, yellow represent allowed regions and white represent the disallowed regions. Square and triangular dots represent residues distributed in different regions of the plot. (C) Evaluation of the Z-score of vaccine’s refined tertiary structure was performed by ProSA-web server. (D) Energy map of the refined structure. Most regions of the structure show negative energy values. (E) In the ERRAT plot, yellow and red represent error values exceeding the 95% and 99% thresholds, respectively. The error values of most regions in the protein were lower than the 95% confidence limit.
Table 4.
Conformation B epitope screening results.
Fig 4.
The conformational B cell epitopes within BtHKU5-CoV-2 vaccine tertiary structure.
(A-D) The conformational B cell epitopes predicted to be presented on the surface model are colored in yellow. These epitopes are distributed at different positions on the vaccine’s tertiary structure.
Table 5.
Results of molecular docking.
Fig 5.
Docking studies of BtHKU5-CoV-2 vaccine-TLR2 complex.
(A) Docking complex of BtHKU5-CoV-2 vaccine and TLR2. The vaccine is shown in light turquoise, while TLR2 is shown in purple. The box highlights the interacting residues between the two molecules. A simplified diagram below the box depicts the various forces and bonds between the vaccine and TLR2. (B) The connections, including salt bridges, disulphide bonds, hydrogen bonds and non-bonded contacts, predicted between amino acids in the docking complex, are represented in red, yellow, blue and orange, respectively. (C) Deformability plot of the complex. The graph describes the flexibility of each atom in the modeled structure. (D) Covariance plot of the complex. The plot illustrates the correlated and anti-correlated motions among residues. Red, white and blue colors indicate correlated motion, non-correlated motion, anti-correlated motion, respectively.
Fig 6.
Docking studies of BtHKU5-CoV-2 vaccine-TLR4 complex.
(A) Docking complex of BtHKU5-CoV-2 vaccine and TLR4. The vaccine is shown in light turquoise, while TLR4 is shown in yellow. The box highlights the interacting residues between the two molecules. A simplified diagram below the box depicts the various forces and bonds between the vaccine and TLR4. (B) The connections, including salt bridges, disulphide bonds, hydrogen bonds and non-bonded contacts, predicted between amino acids in the docking complex, are represented in red, yellow, blue and orange, respectively. (C) Deformability plot of the complex. The graph describes the flexibility of each atom in the modeled structure. (D) Covariance plot of the complex. The plot illustrates the correlated and anti-correlated motions among residues. Red, white and blue colors indicate correlated motion, non-correlated motion, anti-correlated motion, respectively.
Fig 7.
Population coverage of BtHKU5-CoV-2 vaccine across the world.
This map depicts the proportion of the population covered by the designed vaccine in various countries and regions. Countries and regions that have available data are labeled with blue dots. The global population coverage was visualized with pyecharts, utilizing a base map layer from Natural Earth (Natural Earth, 1:110 million Cultural Vectors, available at https://www.naturalearthdata.com/downloads/110m-cultural-vectors/; data publicly available at https://www.naturalearthdata.com/ under the Public Domain license).
Fig 8.
Immunological simulation analysis following administration of three doses of the BtHKU5-CoV-2 vaccine.
The antibody titers (IgM, IgG, and IgG subclasses) (A) and the levels of cytokines (IFN-γ, TGF-β, and IL-10) (B) were increased following three-dose vaccine administration. (C) The number of total B cells, memory B cells, and isotype-specific B cells increased after vaccination. (D) Plasma B-cell population (PLB), including IgM + IgG, IgM, and IgG1 isotype, exhibited transient expansion. (E) The population of activated Th cells surged following vaccine injection. (F) The active Tc cells expanded rapidly after immunization, and then the T cell population gradually shifted to a predominantly resting state. The populations of effector Tc cells (G), total NK cells (H), and DCs in presenting or resting states (I) were maintained at a stable level throughout the simulation period.
Fig 9.
Docking analysis of BtHKU5-CoV-2 vaccine (mRNA)-TLR complexes.
(A) Docking models of the vaccine mRNA with TLR3. The box highlights the interacting residues between the mRNA and TLR3. The simplified diagram below the box illustrates the various forces and bonds involved. (B) Docking models of the vaccine mRNA with TLR7. The interacting residues between the mRNA and TLR7 are shown in the box. The corresponding interaction forces and bonds are summarized in the schematic below. (C) Docking models of the vaccine mRNA with TLR8. The boxed region shows the interacting residues between the mRNA and TLR8, with the simplified diagram below depicting the various forces and bonds.