Reverse vaccinology-based identification and in silico characterization of immunogenic membrane proteins of Salmonella Typhimurium as novel vaccine targets against multidrug-resistant infections

Abstract

Background: Salmonella enterica serovar Typhimurium (S. Typhimurium) is a leading cause of salmonellosis, gastroenteritis, sepsis, and reactive arthritis. Transmission primarily occurs through contaminated water, eggs, meat, and dairy products. The disease disproportionately affects developing nations, where young children, the elderly, and immunocompromised individuals face high risks of severe morbidity and mortality. Its ability to evade host immune defenses and acquire multidrug resistance (MDR) exacerbates global public health challenges. Currently, no licensed human vaccine is available, underscoring the urgent need for targeted vaccine development.

Methods: This study utilized a reverse vaccinology approach and in silico strategies to identify highly immunogenic membrane proteins as potential vaccine candidates. The complete proteome of S. Typhimurium was screened for membrane-associated candidates using the SOSUI server. Antigenicity was evaluated using VaxiJen v2.0 (threshold ≥ 0.9), and allergenicity was assessed using AllerTOP v1.1. To ensure vaccine safety, homologous proteins were excluded based on PSI-BLAST analysis against the human proteome, and toxicity was predicted using ToxinPred. The immunogenic potential was further evaluated through C-ImmSim immune simulation software. B-cell and T-cell epitopes were predicted using ABCpred and the Immune Epitope Database (IEDB). Physicochemical characteristics were analyzed with ProtParam and TMHMM 2.0. Finally, BLASTp analysis was used to confirm the conservation of the selected proteins across MDR clinical isolates.

Results: Nine membrane proteins were prioritized based on strong antigenicity, non-allergenicity, non-toxicity, favorable epitope profiles, and physicochemical stability. All proteins were highly conserved in MDR isolates, supporting their utility for broad-spectrum vaccine development.

Conclusion: These targets show promising potential for developing a broadly protective multi-epitope vaccine against S. Typhimurium. However, in vitro and in vivo experimental validation is essential to confirm their immunogenicity and protective efficacy.

Keywords: In silico analysis; Salmonella Typhimurium; B-cell epitopes; Immunogenicity; Multidrug-resistance; Reverse vaccinology; T-cell epitopes; Vaccine.

Fig. 1

Computational pipeline for the identification and prioritization of potential vaccine candidates against Salmonella enterica serovar Typhimurium strain LT2. The process begins with sequence retrieval from NCBI, followed by subcellular localization analysis using SOSUI. Antigenicity and allergenicity are evaluated using VaxiJen and AllerTOP, respectively. Homology to human proteins is assessed via BLASTp, and toxicity predictions are made using ToxinPred. Immunogenicity is further validated through C-immsim. B-cell and T-cell epitopes are predicted using ABCpred and IEDB tools, with subsequent antigenicity and allergenicity evaluation. Physiochemical properties are analyzed via ProtParam, TMHMM, UniProt, and SWISS-MODEL. Finally, candidate conservation is confirmed in MDR S. Typhimurium strains

Fig. 2

Antigenicity scores and allergenicity profiles of 15 selected S. Typhimurium proteins. Each bar represents the antigenicity score of an individual protein (identified by UniProt ID). Proteins classified as allergens are marked in red, while non-allergens are shown in green. This visualization highlights potential vaccine candidates based on high antigenicity and non-allergenic properties

Fig. 3

Tracking the immune response over time of a potential candidate (Q7CQG8) via C-immsim tool. (Left panel) The antigen (Ag) level spikes early and quickly drops as the immune system kicks in. IgM appears first, followed by IgG1 and IgG2, with IgG1 + IgG2 sticking around longer, suggesting lasting immunity. (Right panel) Cytokines peak around day 10, with IFN-γ leading the charge. IL-2 surges briefly, as shown in the inset, while overall cytokine levels drop after day 15, signaling immune resolution. The x-axis shows days post-exposure, while the y-axis represent antigen count, antibody levels, and cytokine concentrations

Fig. 4

Antibody titers against selected proteins, measured as IgG + IgM (blue), IgM (orange), IgG1 + IgG2 (gray), IgG1 (yellow), and IgG2 (light blue). Proteins P0A1E5 and Q7CQG8 exhibit the highest immunogenicity, followed by Q7CQW9 and Q8ZMZ1, indicating their potential as vaccine candidates. Low responses were observed for Q7CPZ9, Q8ZN08, and Q7CPM3

Fig. 5

Cytokine concentrations (ng/ml) induced by different proteins, measuring IFN-γ (blue), IL-10 (orange), IL-12 (gray) and IL-2 (yellow). Proteins P0A1E5, Q7CQG8, Q7CQW9, and Q7CPR0 elicit strong IFN-γ and IL-12 responses, indicating potent Th1 (T helper) immunity. Low responses were observed for Q7CPZ9, Q8ZN08

Fig. 6

Predicted B-cell epitopes using IEDB analysis for selected proteins. The yellow regions indicate the predicted linear B-cell epitopes, while the green regions represent non-epitope regions

Fig. 7

Visualization of structural diversity and spatial organization of Selected Proteins obtained from Swiss Model, UniProt IDs of proteins are written below their respective model