Introduction: Staphylococcus aureus (S. aureus) is a gram‐positive opportunistic pathogen, there are currently no high effective vaccine against S. aureus in humans and animals, the development of an efficient vaccine remains an important challenge to prevent S. aureus infection. Here, we prepared Als3‐Th‐cell‐epitope‐Target of RNAIII Activating Protein (TRAP) (ATT) proteins plus the novel combined adjuvants to develop a promising vaccine candidate against S. aureus.Methods: The recombinant pET‐28a (+)‐att plasmids were constructed, and the ATT proteins were expressed and obtained, then, ATT plus Freund's adjuvant or the novel combined adjuvants of cytosine‐phosphate‐guanosine oligodeoxynucleotides (CpG), muramyl dipeptides (MDP), and FIA were immunized in mice. After booster immunization, the levels of interferon‐γ (IFN‐γ), interleukin‐4 (IL‐4), IL‐10 and IL‐17A cytokine were evaluated, the humoral immune responses against TRAP were detected in mice, and the survival rate of mice was confirmed by challenge assay.Results: The mice immunized with ATT plus Freund's adjuvant exhibited significantly higher level of IFN‐γ, IL‐4, IL‐10, and IL‐17A, and displayed the stronger humoral immune response against TRAP than control groups, importantly, the survival rate of these mice was significantly higher than control groups. In addition, compared with the control groups, ATT + CpG + MDP + FIA group was elicited significantly higher level of IFN‐γ, IL‐4, IL‐10, and IL‐17A and was triggered the stronger humoral immune responses against TRAP, moreover, generated the higher survival rate of mice.Conclusion: Als3 epitopes significantly enhanced TRAP immunogenicity. ATT plus the novel combined adjuvants of CpG, MDP, and FIA induced the strong immune response and protection against S. aureus, revealing the combination of CpG, MDP, and FIA adjuvant acts the synergistic effect.

Introduction: Staphylococcus aureus seriously threatens human and animal health. IsdB_137‐361 of the iron surface determinant B protein (IsdB) from S. aureus exhibits the strong immunogenicity, but its immunoprotective effect is still to be further promoted. Because PEI‐PLGA nanoparticles are generated by PEI conjugate with PLGA to develop great potential as a novel immune adjuvant, the immunogenicity of IsdB_137‐361 is likely be strengthened by PEI‐PLGA.Methods: Here, PEI‐PLGA nanoparticles containing IsdB_137‐361 proteins were prepared by optimizing the entrapment efficiency. Mice were immunized with IsdB_137‐361‐PEI‐PLGA nanoparticles to assess their anti‐S. aureus effects. The level of IFN‐γ, IL‐4, IL‐17, and IL‐10 cytokines from spleen lymphocytes in mice and generation of the antibodies against IsdB_137‐361 in serum was assessed by ELISA, the protective immune response was appraised by S. aureus challenge.Results: IsdB_137‐361 proteins loaded by PEI‐PLGA were able to stimulate effectively the proliferation of spleen lymphocytes and increase the secretion of IFN‐γ, IL‐4, IL‐17, and IL‐10 cytokine from spleen lymphocytes, and significantly enhance generation of the antibodies against IsdB_137‐361 in serum, reduce the level of bacterial load in liver, spleen and kidney, and greatly improve the survival rate of mice after challenge.Conclusion: These data showed that PEI‐PLGA nanoparticles can significantly enhance the immunogenicity of IsdB_137‐361 proteins, and provide an important reference for the development of novel immune adjuvant.

Introduction. The development of combinatorial adjuvants is a promising strategy to boost vaccination efficiency. Accumulating evidence indicates that manganese exerts strong immunocompetence and will become an enormous potential adjuvant. Here, we described a novel combination of Mn²⁺ plus aluminum hydroxide (AH) adjuvant that significantly exhibited the synergistic immune effect. Methodology. Initially, IsdB3 proteins as the immune-dominant fragment of IsdB proteins derived from Staphylococcus aureus (S. aureus) were prepared. IsdB3 proteins were identified by western blotting. Furthermore, we immunized C57/B6 mice with IsdB3 proteins plus Mn²⁺ and AH adjuvant. After the second immunization, the proliferation of lymphocytes was measured by the cell counting kit-8 (CCK-8) and the level of IFN-γ, IL-4, IL-10, and IL-17 cytokine from spleen lymphocytes in mice and generation of the antibodies against IsdB3 in serum was detected with ELISA, and the protective immune response was assessed through S. aureus challenge. Results. IsdB3 proteins plus Mn²⁺ and AH obviously stimulated the proliferation of spleen lymphocytes and increased the secretion of IFN-γ, IL-4, IL-10, and IL-17 cytokine in mice, markedly enhanced the generation of the antibodies against IsdB3 in serum, observably decreased bacterial load in organs, and greatly improved the survival rate of mice. Conclusion. These data showed that the combination of Mn²⁺ and AH significantly acted a synergistic effect, reinforced the immunogenicity of IsdB3, and offered a new strategy to increase vaccine efficiency.

Here, we prepared the novel combined adjuvants, CTB as intra-molecular adjuvant, CpG and aluminum hydroxide (Alum) to strengthen the immunogenicity of clumping factor A221-550 of Staphylococcus aureus (S. aureus). The protein-immunoactive results showed CTB-ClfA221-550 elicited the strong immune responses to serum from mice immunized with CTB and ClfA221-550, respectively. The mice immunized with CTB-ClfA221-550 plus CpG and Alum adjuvant exhibited significantly stronger CD4+ T cell responses for IFN-γ, IL-2, IL-4, and IL-17 and displayed the higher proliferation response of splenic lymphocytes than the control groups, in addition, these mice generated the strongest humoral immune response against ClfA221-550 among all groups. Our results also showed CTB-ClfA221-550 plus CpG and Alum adjuvant obviously increased the survival percentage of the mice challenged by S. aureus. These data suggested that the novel combined adjuvants, CTB, CpG, and Alum, significantly enhance the immune responses triggered with ClfA221-550, and could provide a new approach against infection of S. aureus.Abbreviations: CTB: Cholera Toxin B; CpG: Cytosine preceding Guanosine; ODN: Oligodeoxynucleotides; Alum: Aluminum hydroxide; TRAP: Target of RNAIII-activating Protein; TLR9: Toll-like Receptor 9; TMB: 3, 3ʹ, 5, 5ʹ-tetramethylbenzidine; mAbs: Monoclonal Antibodies; OD: Optical Densities; S. aureus: Staphylococcus aureus; ClfA: Clumping factor A; FnBPA: Fibronection-binding protein A; IsdB: Iron-regulated surface determinant B; SasA: Staphylococcus aureus Surface Protein A; GapC: Glycer-aldehyde-3-phosphate dehydrogenase-C