Epitope screening and vaccine molecule design of PRRSV GP3 and GP5 protein based on immunoinformatics

Abstract

Porcine reproductive and respiratory syndrome (PRRS) is a respiratory disease in pigs that causes severe economic losses. Currently, live PRRSV vaccines are commonly used but fail to prevent PRRS outbreaks and reinfection. Inactivated PRRSV vaccines have poor immunogenicity, making PRRSV a significant threat to swine health globally. Therefore, there is an urgent need to develop an effective PRRSV vaccine. This study used immunoinformatics to predict, screen, design and construct a candidate vaccine that fused B-cell epitopes, CTL- and HTL-dominant protective epitopes of PRRSV strain's GP3 and GP5 proteins. The study identified 12 B-cell epitopes, 6 CTL epitopes and 5 HTL epitopes of GP3 and GP5 proteins. The candidate vaccine was constructed with 50S ribosomal protein L7/L1 molecular adjuvant, which has antigenicity, solubility, stability, non-allergenicity and a high affinity for its target receptor, TLR-3. The C-ImmSim immunostimulation results showed significant increases in cellular and humoral responses (B cells and T cells) and production of TGF-β, IL-2, IL-10, IFN-γ and IL-12. The constructed vaccine was stable and immunogenic, and it can effectively induce strong T-cell and B-cell immune responses against PRRSV. Therefore, it is a promising candidate vaccine for controlling and preventing PRRSV outbreaks.

Keywords: GP3; GP5; immunoinformatics; porcine reproductive and respiratory syndrome virus; vaccine candidate.

FIGURE 1

The schematic diagram of candidate vaccine construction.

FIGURE 2

The prediction and analysis of the secondary structure of candidate vaccines. Different colours represent corresponding information about the secondary structure of a protein.

FIGURE 3

Modelling, refinement and evaluation of the tertiary structure of candidate vaccines. (A) The Omega parameter of the two‐dimensional structure; (B) The Phi parameter of the two‐dimensional structure; (C) The Distance parameter of the two‐dimensional structure; (D) The Theta parameter of the two‐dimensional structure; (E) The Contact parameter of the two‐dimensional structure; (F) Within the tertiary structure of the candidate vaccine, the 50S ribosomal L7/L12 molecular adjuvant is coloured in red, while the B‐cell epitope is green, the HTL epitope is yellow, and the CTL epitope is purple. The TM‐score of the structure is 0.358; (G) To verify the rough model using Ramachandran drawing; (H) The structure comparison between the rough model and the refined model, the rough model and the fine model are green and red, respectively; (I) To verify the refined model using Ramachandran drawing; (J) The Z score of the refined model ProSA SEB map is −3.03; (K) Local model quality assessment.

FIGURE 4

B cell‐dominant epitope map and candidates Docking results of vaccines and TLR3 molecules. (A) The highest‐scoring linear epitope binding position; (B) the highest‐scoring discontinuous epitope‐binding position. (C) The candidate vaccine structure is shown in red and the TLR3 structure in green; (D) Partial zoom‐in shows amino acid residues; (E) PDBsum draws a two‐dimensional interaction diagram; (F) Schematic diagram of the interaction between protein chains.

FIGURE 5

The changes in the number of immune B cells and Th in simulated immune stimulation. (A) The total counts of B‐lymphocyte, memory cells and subtypes IgM, IgG1 and IgG2; (B) The plasma B‐lymphocyte count broken down by isotype (IgM, IgG1 and IgG2); (C) B‐lymphocyte populations per entity status; (D) the counts of CD4 helper T lymphocytes. The figure shows total and memory counts; (E) CD4 helper T lymphocyte counts per entity status; (F) the counts of CD4 T‐regulatory lymphocytes.

FIGURE 6

The number changes of immune CTL, NK, MA, DC and EP cells after simulated immune stimulation. (A) The counts of CD8 T cytotoxic lymphocytes. Total and memory are shown; (B) the counts of CD8 T cytotoxic lymphocyte per entity state; (C) the counts of natural killer cells; (D) the counts of internalized and presented macrophage; (E) Dendritic cells can present antigenic peptides on MHC class I and II molecules; (F) the total counts of epithelial cells can be disaggregated into active, viral infection and presentation on MHC class I molecules.

FIGURE 7

Trends of immunoglobulins and immune complexes and concentration trends of cytokines and interleukins. (A) Antigens and immunoglobulins. Antibodies were subdivided by isotype (with 50S ribosomal L7/L12 molecular adjuvant); (B) antigen and immunoglobulin. Antibodies were subdivided by isotype (without 50S ribosomal L7/L12 molecular adjuvants); (C) the concentrations of cytokines and interleukins.

FIGURE 8

Schematic diagram of the connection between candidate vaccine and pET‐32a(+) prokaryotic expression vector.