Proteomics-based vaccine targets annotation and design of multi-epitope vaccine against antibiotic-resistant Streptococcus gallolyticus

Abstract

Streptococcus gallolyticus is a non-motile, gram-positive bacterium that causes infective endocarditis. S. gallolyticus has developed resistance to existing antibiotics, and no vaccine is currently available. Therefore, it is essential to develop an effective S. gallolyticus vaccine. Core proteomics was used in this study together with subtractive proteomics and reverse vaccinology approach to find antigenic proteins that could be utilized for the design of the S. gallolyticus multi-epitope vaccine. The pipeline identified two antigenic proteins as potential vaccine targets: penicillin-binding protein and the ATP synthase subunit. T and B cell epitopes from the specific proteins were forecasted employing several immunoinformatics and bioinformatics resources. A vaccine (360 amino acids) was created using a combination of seven cytotoxic T cell lymphocyte (CTL), three helper T cell lymphocyte (HTL), and five linear B cell lymphocyte (LBL) epitopes. To increase immune responses, the vaccine was paired with a cholera enterotoxin subunit B (CTB) adjuvant. The developed vaccine was highly antigenic, non-allergenic, and stable for human use. The vaccine's binding affinity and molecular interactions with the human immunological receptor TLR4 were studied using molecular mechanics/generalized Born surface area (MMGBSA), molecular docking, and molecular dynamic (MD) simulation analyses. Escherichia coli (strain K12) plasmid vector pET-28a ( +) was used to examine the ability of the vaccine to be expressed. According to the outcomes of these computer experiments, the vaccine is quite promising in terms of developing a protective immunity against diseases. However, in vitro and animal research are required to validate our findings.

Keywords: Immunoinformatics; Multi-epitope vaccine; Pan-genome; Reverse vaccinology.

Figure 1

Graphical abstract.

Figure 2

S. gallolyticus vaccine population coverage map, 96.15% of the global population is covered.

Figure 3

The S. gallolyticus vaccine construct is shown schematically.

Figure 4

Vaccine Construct Prediction and Validation. (A) The adjuvant sequence is shown in maroon. The EAAAK linker in red, the epitope sequence is black, the AAY linker in golden, the GPGPG connectors in blue, and the KK linkers in green (B) 3D structure of S. gallolyticus vaccine (C) Ramachandran plot analysis of the structure which shows that 9.0% and 86.9% of all amino acids were positioned in the permitted and most favorable regions, respectively, whereas 2.2% were found in the disallowed region.

Figure 5

Disulphide engineering of vaccine structure to enhance stability. The red color represents one mutated pair selected based upon energy and X₃ value.

Figure 6

TLR-4 receptor and vaccine docked together. TLR4 is displayed in cyan, while the vaccine is displayed in pink. Red lines between the residues represents salt bridges, yellow line represents disulphide bonds, blue line represents hydrogen bonds, and orange line represents non-bonded contacts.

Figure 7

Molecular dynamics simulation-based statistical analysis to evaluate the inter-molecular stability and dynamics of the complexes: (A) Root Mean Square Deviation (RMSD) of the vaccine-receptor complex. (B) Root Mean Square Fluctuation (RMSF) of the vaccine-receptor complex.

Figure 8

In silico immune simulation results obtained after administration of three injections of the vaccine. (A) Concentrations of immunoglobulins in proportion to antigen concentrations. (B) The production of various cytokines in response to the administration of c-ImmSim vaccine.

Figure 9

S. gallolyticus vaccine in silico restriction cloning into the pET28a (+) expression vector between between the XhoI and HindIII restriction sites. Vaccine construct is depicted as MCS in blue colour.