Computational Design of a Chimeric Vaccine against Plesiomonas shigelloides Using Pan-Genome and Reverse Vaccinology

Abstract

The swift emergence of antibiotic resistance (AR) in bacterial pathogens to make themselves adaptable to changing environments has become an alarming health issue. To prevent AR infection, many ways can be accomplished such as by decreasing the misuse of antibiotics in human and animal medicine. Among these AR bacterial species, Plesiomonas shigelloides is one of the etiological agents of intestinal infection in humans. It is a gram-negative rod-shaped bacterium that is highly resistant to several classes of antibiotics, and no licensed vaccine against the aforementioned pathogen is available. Hence, substantial efforts are required to screen protective antigens from the pathogen whole genome that can be subjected easily to experimental evaluations. Here, we employed a reverse vaccinology (RV) approach to design a multi-antigenic epitopes based vaccine against P. shigelloides. The complete genomes of P. shigelloides were retrieved from the National Center for Biotechnological Information (NCBI) that on average consist of 5226 proteins. The complete proteomes were subjected to different subtractive proteomics filters, and in the results of that analysis, out of total proteins, 2399 were revealed as non-redundant and 2827 as redundant proteins. The non-redundant proteins were further checked for subcellular localization analysis, in which three were localized in the extracellular matrix, eight were outer membrane, and 13 were found in the periplasmic membrane. All surface localized proteins were found to be virulent. Out of a total of 24 virulent proteins, three proteins (flagellar hook protein (FlgE), hypothetical protein, and TonB-dependent hemoglobin/transferrin/lactoferrin family receptor protein) were considered as potential vaccine targets and subjected to epitopes prediction. The predicted epitopes were further examined for antigenicity, toxicity, and solubility. A total of 10 epitopes were selected (GFKESRAEF, VQVPTEAGQ, KINENGVVV, ENKALSQET, QGYASANDE, RLNPTDSRW, TLDYRLNPT, RVTKKQSDK, GEREGKNRP, RDKKTNQPL). The selected epitopes were linked with each other via specific GPGPG linkers in order to design a multi-epitopes vaccine construct, and linked with cholera toxin B subunit adjuvant to make the designed vaccine construct more efficient in terms of antigenicity. The 3D structure of the vaccine construct was modeled ab initio as no appropriate template was available. Furthermore, molecular docking was carried out to check the interaction affinity of the designed vaccine with major histocompatibility complex (MHC-)I (PDB ID: 1L1Y), MHC-II (1KG0), and toll-like receptor 4 ((TLR-4) (PDB: 4G8A). Molecular dynamic simulation was applied to evaluate the dynamic behavior of vaccine-receptor complexes. Lastly, the binding free energies of the vaccine with receptors were estimated by using MMPB/GBSA methods. All of the aforementioned analyses concluded that the designed vaccine molecule as a good candidate to be used in experimental studies to disclose its immune protective efficacy in animal models.

Keywords: Plesiomonas shigelloides; immunoinformatics; molecular docking; multi-epitopes.

Figure 1

Methodology flow for designing a novel multi-antigenic epitopes vaccine against P. shigelloides.

Figure 2

The number of surface localized proteins obtained is represented as Venn. Number of virulent proteins (VP), periplasmic proteins (PP), outer membrane proteins (OMP), extracellular proteins (EP), and transmembrane helices (TMH).

Figure 3

Final 10 shortlisted epitopes used for construction of a multi-epitopes-based vaccine.

Figure 4

Schematic diagram of 264 amino acid long vaccine construct sequence. The selected epitopes are represented in green color boxes, GPGPG linkers for linking epitopes with each other while EAAAK linker to join the designed vaccine construct with cholera toxin B subunit adjuvant as shown in the purple color box.

Figure 5

3D structure of chimeric multi-epitopes vaccine construct.

Figure 6

Left side structure represents original structure while right side structure represents the mutated structure of final vaccine construct.

Figure 7

Cloning of vaccine construct into E. coli k12 strains pET28a vector.

Figure 8

(A) 3D structure of the docked complex of chimeric vaccine (green mesh) and receptor MHC-I (yellow mesh). (B). 3D structure of the docked complex of chimeric vaccine (green mesh) and receptor MHC-I (dodger blue mesh). (C). 3D structure of the docked complex of chimeric vaccine (green mesh) and receptor MHC-I (magenta mesh).

Figure 9

Statistical analysis of simulation trajectories; three output values are depicted here: RMSD (A), RMSF (B), and RoG (C).

Figure 10

(A). Immunoglobulin titer (different color peak) in response to multi-epitopes vaccine injection (black color peak). (B). Elicitation of interleukins level after injection of multi-epitopes vaccine construct as represented by C- immune simulation analysis.