Antigenic Protein Screening and Design of Multi-Epitope Vaccine Against Lactococcus garvieri and Streptococcus iniae for Combating Lactococcosis and Streptococcosis in Fish

Abstract

The illness caused by Lactococcus garvieae and Streptococcus iniae is well acknowledged as a disease that results in significant economic losses since it affects a diverse array of fish species. The constraints of existing vaccinations and techniques have prompted the exploration of novel approaches to manage this ailment. Multi-epitope vaccines that use a diverse range of immunogenic proteins have considerable potential. The primary objective of the present research endeavour was to develop a very effective multi-epitope vaccine targeting Streptococcus iniae and Lactococcus garvieae infection in fish. The immunogenic components of Lactococcus garvieae and Streptococcus iniae were used for epitope prediction. A multi-epitope vaccine was constructed using the immunogenic proteins' most effective B cell epitopes and the GFFY adjuvant. Subsequently, an assessment was conducted on many aspects of the developed vaccine, including physicochemical characteristics, antigenicity, secondary structure and tertiary structure. Furthermore, the molecular docking technique was used to study the interaction between the proposed vaccine and its TLR-5 receptor. The nucleotide sequence of the vaccine was subsequently modified to facilitate its expression in Lactococcus lactis. The findings of the current investigation indicate that the vaccine developed exhibited stability, as shown by its molecular weight of 93989.19 Da and antigenicity value of 0.8547. In addition, the study of the vaccine's structure indicated that it consisted of 32.24% alpha helix, with 88.41% of its residues located in the preferred area. The proposed vaccine effectively docked to its TLR5 receptor was shown, resulting in the lowest energy of -995.4. According to the data obtained, the developed vaccine has the potential to effectively prevent infection in fish caused by Lactococcus garvieae and Streptococcus iniae. Our findings suggest that the peptide vaccine might be a favourable choice for prophylaxis against Lactococcus garvieae and Streptococcus iniae.

Keywords: Lactococcus garvieae; Streptococcus iniae; immunoinformatic; multi‐epitope vaccine.

FIGURE 1

(A) Prevalence of Lactococcus garvieae and Streptococcus iniae protein LBL epitopes. (B) Solubility of Lactococcus garvieae and Streptococcus iniae selected proteins.

FIGURE 2

Schematic sequence of the constructed vaccine.

FIGURE 3

Web‐based display of protein surface and pH‐dependent properties for assessing the developability of biotherapeutics. (A) The vaccine structure's energy heat map illustrates the accurate distribution of energy. (B) Energy of the vaccine structure against the fab set at 0.05 IS, 0.15 IS and 0.3 IS. (C) The vaccine structure's charge heat map illustrates the accurate distribution of energy. (D) Charges of the vaccine structure against the fab set at 0.05 IS, 0.15 IS and 0.3 IS.

FIGURE 4

(A) The whole three‐dimensional configuration of the vaccination construct. (B) A Ramachandran plot explicitly developed for the vaccine. According to the Ramachandran plot, it was observed that 88.41% of the residues were situated inside the favoured zone. (C) Discontinuous epitopes on the vaccine's intended surface. (D) Discontinuous B cell epitopes were expected to have a particular significance. (E) The predicted structure was validated with a Z score <1. (F) Vaccine secondary structure prediction has been significantly improved using consensus prediction based on multiple alignments.

FIGURE 5

The statistics of the top 10 clusters of TLR5/Vaccine interaction. (A) Diagrams related to electrostatic energy, HADDOCK score, de‐solvation energy and restraints violation energy individually. (B) Graphs related to average electrostatic energy, Van der Waals energy and restraints violation energy. According to the results, Cluster 2 had the best molecular docking result between the vaccine structure and the TLR5 receptor.

FIGURE 6

(A) The vaccine structure, TLR5 receptor and its alignment with the TLR5 receptor are all characterised by their three‐dimensional structure. (B) Molecular docking of vaccine structure and TLR5 receptor. The top 3 clusters were selected as the final constructs. All interacting residues from vaccine are shown in green colour.

FIGURE 7

(A) The process of optimising codons in vaccine constructions targeting Lactococcus lactis. (B) Vaccine cloning using computational methods. The Green region inside the pNZ8123 expression vector represents the multi‐epitope vaccination insert. (C) The corresponding vaccine design map. (D) The mRNA entropy of the vaccine structure indicates the mRNA stability. (E, F) Different mRNA structures predicted from the vaccine structure.