Experimental Multi-Epitope Vaccine Shields Mice and Chickens Against Salmonella Enteritidis

A newly designed multi-epitope subunit vaccine has protected mice and chickens against lethal Salmonella Enteritidis challenge in laboratory experiments, according to a study published in the journal Animal Diseases. The vaccine, named FLPD, was built from carefully selected immune-triggering fragments of two Salmonella proteins and conferred 65 percent protection in mice and 70 percent protection in chickens after three immunizations, approaching the 80 percent protection achieved by a commercial live attenuated vaccine in the same chicken trial.

Salmonella Enteritidis is one of the most consequential foodborne pathogens worldwide. It causes acute systemic infections in chickens and can reach humans through contaminated poultry meat and eggs. The researchers note that S. Enteritidis and S. Typhimurium are the serovars most frequently associated with human infections, and a survey conducted in China found them to be the most prevalent serovars in chicken samples, at 43.52 percent and 23.15 percent respectively. With multidrug-resistant Salmonella strains increasingly limiting antibiotic treatment options, the team argues that new preventive tools such as vaccines are urgently needed for both animal and human health.

Existing Salmonella vaccines come with trade-offs. Live attenuated vaccines are effective but raise safety concerns because attenuated strains can theoretically revert to virulence and potentially contaminate food products. Killed vaccines are safer but often fail to elicit sufficiently strong immunity. Subunit vaccines, which contain only defined antigens, offer a middle path, though their limited antigenic repertoire can restrict the breadth of the response they provoke. The new study aimed to overcome this limitation by combining epitopes from two virulence-associated proteins: FliC, the flagellin component involved in bacterial adhesion and invasion, and SipD, the needle-tip protein of the type III secretion system that helps the bacterium penetrate host cell membranes.

Using immunoinformatics tools, the researchers screened the two proteins for linear B-cell epitopes and cytotoxic T-lymphocyte epitopes, excluding segments located in signal peptide regions, hydrophobic stretches, and transmembrane domains. The final construct contains ten B-cell epitopes and three CTL epitopes joined by short peptide linkers, with an N-terminal histidine tag added for purification. The resulting recombinant protein is 273 amino acids long. Computational predictions indicated that FLPD is nonallergenic, highly antigenic with a VaxiJen score of 1.4548, and stable, with an instability index of 27.50 and a strongly hydrophilic character.

Structural modeling supported these predictions. Of five three-dimensional models generated with I-TASSER, the highest-scoring model passed validation checks, with an ERRAT quality score of 82.264 and most residues occupying favored or allowed regions of the Ramachandran plot. Molecular docking using ClusPro 2.0 predicted strong binding interactions between FLPD and the immune receptors MHC-I, MHC-II, and TLR-4, with the lowest binding energies of −948.7, −996.0, and −1000.7 kcal/mol respectively. An in silico immune simulation over roughly 35 days forecast rising immunoglobulin titers, elevated secretion of cytokines including IFN-γ, TGF-β, and IL-2, and expanding populations of memory and activated B and T cells. The authors caution, however, that this simulation platform is calibrated with human parameters, limiting quantitative interpretation for mice and chickens.

The team then moved from computation to the laboratory. The codon-optimized FLPD gene was expressed in E. coli, and the His-tagged protein was purified by nickel-affinity chromatography to 95.6 percent purity. In mice immunized subcutaneously on days 1, 14, and 21 with 20, 50, or 90 micrograms of FLPD in Freund’s adjuvant, serum anti-FLPD IgG titers climbed with each booster dose. The 50-microgram group produced the strongest response, reaching a titer of 1:1,200,000 after three immunizations, while control serum showed no reactivity. Levels of both Th1-associated cytokines, IFN-γ and IL-17A, and Th2-associated cytokines, IL-4 and IL-6, were significantly elevated in vaccinated animals, indicating a mixed humoral and cellular response.

Protective efficacy was tested with lethal intraperitoneal challenges at ten times the lethal dose fifty. All twenty nonimmunized control mice died within eight days of challenge. Among FLPD-vaccinated mice, mortality was 35 percent after Salmonella Enteritidis challenge and 55 percent after Salmonella Typhimurium challenge over fourteen days, corresponding to protection rates of 65 percent and 45 percent. A two-dose regimen performed worse, protecting 50 percent of mice against Enteritidis and 40 percent against Typhimurium, suggesting that the third booster meaningfully improves protection. Vaccinated mice also carried significantly lower bacterial loads in the liver and spleen three days after challenge, and histological examination showed largely preserved tissue architecture with only mild focal inflammation, in contrast to the severe splenic lymphoid loss, hepatic steatosis, and widespread inflammatory infiltration seen in unimmunized animals. The protection against colon injury was significant in Enteritidis-challenged mice but not in Typhimurium-challenged mice, where only splenic lesion scores were significantly reduced.

In chickens, the vaccine’s performance was assessed in specific-pathogen-free White Leghorn chicks given 100 micrograms of FLPD subcutaneously on days 2, 16, and 23. Serum IgG levels rose markedly from fourteen days after the first immunization and continued increasing after each booster, remaining distinctly higher than the plateaued levels seen in chicks receiving a commercial live attenuated vaccine throughout the observation period. Anti-FLPD serum also showed in vitro cross-reactivity against eight clinically prevalent Salmonella serotypes—Enteritidis, Typhimurium, Pullorum, London, Weltevreden, Saintpaul, Kentucky, and Indiana—with the strongest binding to S. Weltevreden and S. Pullorum. When the chicks were challenged intramuscularly with a lethal dose of S. Enteritidis, mortality was 100 percent in unimmunized controls, 30 percent in the FLPD group, and 20 percent in the commercial vaccine group, yielding protection rates of 70 percent and 80 percent respectively.

The authors interpret these results cautiously. They point out that antibody titers do not always correlate directly with protective efficacy, and that the commercial live vaccine’s lower IgG levels may partly reflect a mechanism relying more on cell-mediated and mucosal immunity than on systemic antibodies. Cellular immune responses were not measured in the chickens, so any contribution of T-cell immunity to the observed 70 percent survival remains an inference requiring direct verification. The cross-reactivity against S. Pullorum and other serotypes was demonstrated only in vitro, and challenge experiments against those serotypes have not yet been performed.

Several further limitations frame the findings. The study used specific-pathogen-free animals under controlled laboratory conditions, which do not capture field variables such as concurrent infections, environmental stressors, or varying biosecurity levels. Freund’s adjuvant, used here to reveal FLPD’s intrinsic immunogenicity, causes injection-site reactions and is unsuitable for commercial use, so safer field-applicable adjuvants such as oil emulsions, aluminum hydroxide gel, or CpG oligonucleotides would need to be evaluated. The three-dose schedule may also be impractical for large-scale poultry production, where one- or two-dose regimens are preferred. Because Salmonella initiates infection at the intestinal mucosa, the authors suggest that oral or nasal delivery strategies could strengthen mucosal immunity at the primary site of entry. Large-scale production feasibility, including yield, purification efficiency, and cost, has not yet been assessed. Even so, the dual-species validation distinguishes FLPD from the many Salmonella multi-epitope vaccine designs that have remained purely computational, and the authors position it as a promising, safer candidate for future development against Salmonella Enteritidis.

Subject of Research: Design and experimental testing of a multi-epitope subunit vaccine against Salmonella Enteritidis in mice and chickens

Article Title: A multi-epitope subunit vaccine against Salmonella Enteritidis: design, immunogenicity, and protective efficacy in mice and chickens

Article References: Li, Y., Sui, Y., Yao, J., Li, L., Farhan, M. H. R., Xie, C., Pan, Y., Huang, L., & Cheng, G. (2026). A multi-epitope subunit vaccine against Salmonella Enteritidis: design, immunogenicity, and protective efficacy in mice and chickens. Animal Diseases, 6(1), Article 46. https://doi.org/10.1186/s44149-026-00260-8

Image Credits: AI Generated

DOI: 10.1186/s44149-026-00260-8

Keywords: Salmonella Enteritidis, multi-epitope vaccine, subunit vaccine, FliC, SipD, immunoinformatics, mice, chickens, cross-protection, Salmonella Typhimurium, vaccine design, foodborne pathogen