A Universal Multi-Epitope Vaccine Design Against Porcine Reproductive and Respiratory Syndrome Virus via Bioinformatics and Immunoinformatics Approaches

Abstract

Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive disorders in sows and severe pneumonia in piglets, alongside immunosuppressive effects on the host. It poses a significant global threat to the swine industry, with no effective control measures currently available due to its complex pathogenesis and high variability. Conventional inactivated and attenuated vaccines provide inadequate protection and carry biosafety risks. In this study, we designed a universal multi-epitope peptide vaccine against PRRSV using bioinformatics and immunoinformatics approaches to address these limitations. By selecting sequences from seven representative PRRSV strains, we predicted highly conserved and immunogenic T cell (Th and CTL) epitopes across all encoded proteins. These were rationally concatenated with reported B cell neutralizing epitopes into a multi-epitope vaccine construct. We performed comprehensive assessments of the construct's physicochemical and biochemical properties, along with predictions and refinements of its secondary and tertiary structures. Molecular docking simulations with TLR2 and TLR4 revealed strong potential binding interactions. Immune simulations indicated that the multi-epitope vaccine could induce robust humoral and cellular immune responses. This study provides a scientific foundation for the development of safe and effective PRRSV subunit vaccines and offers new perspectives for designing vaccines against other viral diseases.

Keywords: PRRSV; bioinformatics; immunoinformatics; multi-epitope; universal vaccine.

Figure 1

Phylogenetic analysis of seven representative PRRSV strains. A Neighbor-Joining (NJ) tree was constructed based on full-length genomic sequences using the p-distance model with 1000 bootstrap replicates to assess the phylogenetic relationships among these strains.

Figure 2

Design of the finalized multi-epitope vaccine THs-CTLs-NEs. (A) Epitope concatenation order and sequence information. (B) BepiPred 2.0 assessment of the final NEs, with yellow regions indicating predicted B cell epitopes, while green regions are likely not to correspond to B cell epitopes.

Figure 3

Predicted secondary structure of the vaccine construct. (A) Distribution of α-helices, β-strands, and coils among the amino acid residues. (B) Distribution of small non-polar, hydrophobic, polar, and aromatic residues.

Figure 4

Evaluation of the tertiary structure of THs-CTLs-NEs. (A) AlphaFold3-predicted tertiary structure, optimized by GalaxyREFINE. (B) Structural assessment parameters, focusing on Ramachandran Favored regions. (C) Ramachandran plot with red dots representing residues in Favored, outlier, and rotamer outlier regions. A higher concentration in dark regions indicates better structural quality. (D) ProSA-web model quality assessment, where the Z-score evaluates the sample within the typical range for proteins of a similar size.

Figure 5

Molecular docking of THs-CTLs-NEs with porcine TLR2 and TLR4 via the HDOCK server.

Figure 6

In silico cloning of the vaccine candidate into PET-28a(+) for prokaryotic expression (A) and pFastBac1 for eukaryotic expression (B).

Figure 7

Immune response simulation of THs-CTLs-NEs via the C-ImmSim server across three injections: (A) antibody titers, (B) cytokine levels, (C) total B cell population, (D) active B cell population, (E) total Th cell population, (F) active Th cell population, (G) total TC cell population, and (H) active TC cell population.