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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. [ABSTRACT FROM AUTHOR]

Introduction: Staphylococcus aureus seriously threatens human and animal health. IsdB137‐361 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 IsdB137‐361 is likely be strengthened by PEI‐PLGA. Methods: Here, PEI‐PLGA nanoparticles containing IsdB137‐361 proteins were prepared by optimizing the entrapment efficiency. Mice were immunized with IsdB137‐361‐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 IsdB137‐361 in serum was assessed by ELISA, the protective immune response was appraised by S. aureus challenge. Results: IsdB137‐361 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 IsdB137‐361 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 IsdB137‐361 proteins, and provide an important reference for the development of novel immune adjuvant. [ABSTRACT FROM AUTHOR]

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. 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 The research outline of the combined adjuvants boosted the immunogenicity of clumping factor A221-550. [ABSTRACT FROM AUTHOR]

ATP-binding cassette transporters are ubiquitous in almost all organisms. The Escherichia coli genome is predicted to encode 69 ABC transporters. Eleven of the ABC transporters are presumed to be exporters, of which seven are possible drug export transporters. There has been minimal research on the function of YbhFSR, which is one of the putative drug resistance exporters. In this study, the ybhF gene of this transporter was characterized. Overexpression and knockout strains of ybhF were constructed. The ATPase activity of YbhF was studied using the malachite green assay, and the efflux abilities of knockout strains were demonstrated by using ethidium bromide (EB) as a substrate. The substrates of YbhFSR efflux, examined with the minimum inhibitory concentration (MIC), were determined to be tetracycline, oxytetracycline, chlortetracycline, doxycycline, EB, and Hoechst33342. Furthermore, tetracycline and EB efflux and accumulation experiments confirmed that the substrates of YbhFSR were tetracyclines and EB. The MIC assay and the fluorescence test results showed that tetracyclines are likely to be the major antibiotic substrate of YbhFSR. The existence of the signature NatA motif suggested that YbhFSR may also function as a Na+/H+ transporter. Overexpression of YbhF in E. coli KNabc lacking crucial Na+/H+ transporters conferred tolerance to NaCl, LiCl, and an alkaline pH. Together, the results showed that YbhFSR exhibited dual functions as a drug efflux pump and a Na+ (Li+)/H+ antiporter. [ABSTRACT FROM AUTHOR]

Interferon-γ (IFN-γ), a cytokine produced by activated natural killer cells and T lymphocytes, is an important regulator of innate and adaptive immunity. Interleukin (IL)-18, also known as IFN-γ-inducing factor, is a cytokine that induces T and natural killer cells to produce IFN-γ. In this study, the chicken IL-18 (ChIL-18) and chicken IFN-γ (ChIFN-γ) genes were inserted into the pET28a prokaryotic expression vector, resulting in pET28a-IL-18 and pET28a-IFN-γ, respectively. These plasmids were transformed into Escherichia coli strain BL21, and the ChIL-18 and ChIFN-γ proteins were expressed and purified. To determine their antiviral activities, 200 ng/mL of ChIL-18 and/or ChIFN-γ were inoculated into chicken embryonic fibroblast cells. After 24 h, one 50% tissue culture infective dose (TCID50) of infectious bursal disease virus (IBDV) was inoculated into the chicken embryonic fibroblast cells. The results showed that the antiviral effect of ChIL-18 and ChIFN-γ in combination was better than that of ChIL-18 or ChIFN-γ alone. Next, 14-day-old chicken were injected with 200 µg of ChIL-18 and/or ChIFN-γ and then were challenged with 10³ TCID50 of IBDV via intraperitoneal injection. The results showed that the proliferation of IBDV was inhibited by the injection of the recombinant proteins, especially the combination of ChIL-18 and ChIFN-γ, as evidenced by cytokine detection, quantitative PCR, and pathology analyses. These results indicate that ChIL-18 and ChIFN-γ could inhibit IBDV infection and the combination of ChIL-18 and ChIFN-γ has a better inhibitory effect than either cytokine alone. [ABSTRACT FROM AUTHOR]

The aim of this article was to develop an indirect enzyme-linked immunosorbent assay (ELISA) for efficient detection of the infection ofE. coliin cattle. OmpT, a highly conserved protease in allE. colistrains, was successfully expressed inE. coliXL-1-Blue strain with PET32a vector. Molecular weight of recombinant protein was identified by analyzing SDS-PAGE and the immunogenicity of OmpT was confirmed by Western Blotting. The recombinant OmpT was then employed as capture antigen in the ELISA. The antigen concentration and serum dilution were determined using a checkerboard titration. Results showed that the optimal concentration of coated antigen was 1 μg/ml at a serum dilution of 1:640 and the cut-off value of the assay was 0.335. In addition, the cross-reactivity assay showed that the OmpT wasE. colispecific and the reproducibility experiments displayed good repeatability of the assay. Three hundred and forty cattle serum samples were tested by rOmpT-ELISA and sera coagulation tests. The ELISA has showed relative sensitivity of 100% and specificity of 96.47%. Results of these experiments indicated that the rOmpT-ELISA is a simple, rapid, and convenient method for detection the infection ofE. coliwith different serotype strains. [ABSTRACT FROM PUBLISHER]

ABSTRACT The pathogen Staphylococcus aureus causes a wide range of serious infections, necessitating urgent development of a vaccine against this organism. However, currently developed vaccines are relatively ineffective because of the limited antigenic component that is contained in the vaccine formulations. To develop an effective S. aureus candidate vaccine, overlapping PCR was used to add the truncated immunodominant antigen iron-regulated surface determinant B (IsdB)(N126-P361) (tIsdB) to the N-terminal of intact antigen target of RNAIII activating protein (TRAP) and thus construct a tIsdB-TRAP chimera. The humoral and cellular immune responses against tIsdB-TRAP were compared with those against single or combined formulations. tIsdB-TRAP elicited significantly stronger humoral responses in mice ( P < 0.05). As to cellular immune responses in mice, the tIsdB-TRAP group resulted in a greater IL-4 response than did other groups ( P < 0.05). Greater amounts of IL-2 and IFN-γ were found in the tIsdB-TRAP group. Mouse challenge also showed that tIsdB-TRAP provided better protection against S. aureus than did the control groups. These results suggest that this chimeric protein may be a promising pathogen target for further vaccine development. [ABSTRACT FROM AUTHOR]

• Lipid rafts are required for activation of PI3K/Akt signaling both at early and late stages of CA16 infection. • Lipid raft-associated PI3K/Akt plays critical roles in mediating SREBP1 maturation during late stage of CA16 infection. • This finding of lipid raft-associated PI3K/Akt/SREBP1 will deepen our understanding mechanism of CA16 infection. Coxsackievirus A16 (CA16) is one of predominant Enterovirus that possesses high pathogenicity. Lipid rafts, as cholesterol - and sphingolipid - enriched membrane nanodomains, are involved into many aspects of the virus life cycle. Our previous study found that lipid rafts integrity was essential for CA16 replication, but how lipid rafts regulate CA16 replication through activating downstream signaling remains largely unknown. Thus, in this study, we revealed that lipid rafts were required for activation of PI3K/Akt signaling at early stage of CA16 infection. Treatment with wortmannin significantly reduced the expression of virus protein, indicating PI3K/Akt signaling was beneficial for early stage of virus infection. In addition, lipid rafts integrity was also indispensable for PI3K/Akt activation during the late stage of CA16 infection, which played critical functions in mediating sterol regulatory element-binding proteins 1 (SREBP1) maturation. Whereas, over-expression of SREBP1 exhibited inhibition on virus replication, suggesting that PI3K/Akt signaling and SREBP1 might positively and negatively influence virus replication in two different stages of infection, respectively. Taken together, our study demonstrates an important role of the lipid raft-associated PI3K/Akt/SREBP1 signaling in modulating CA16 replication, which will deepen our understanding mechanism of CA16 infection. [ABSTRACT FROM AUTHOR]

The reoccurrence of successive waves of SARS-CoV-2 variants suggests the exploration of more vaccine alternatives is imperative. Modified vaccinia virus Ankara (MVA) is a virus vector exhibiting excellent safety as well as efficacy for vaccine development. Here, a series of recombinant MVAs (rMVAs) expressing monomerized or trimerized S proteins from different SARS-CoV-2 variants are engineered. Trimerized S expressed from rMVAs is found predominantly as trimers on the surface of infected cells. Remarkably, immunization of mice with rMVAs demonstrates that S expressed in trimer elicits higher levels of binding IgG and IgA, as well as neutralizing antibodies for matched and mismatched S proteins than S in the monomer. In addition, trimerized S expressed by rMVA induces enhanced cytotoxic T-cell responses than S in the monomer. Importantly, the rMVA vaccines expressing trimerized S exhibit superior protection against a lethal SARS-CoV-2 challenge as the immunized animals all survive without displaying any pathological conditions. This study suggests that opting for trimerized S may represent a more effective approach and highlights that the MVA platform serves as an ideal foundation to continuously advance SARS-CoV-2 vaccine development., Importance: MVA is a promising vaccine vector and has been approved as a vaccine for smallpox and mpox. Our analyses suggested that recombinant MVA expressing S in trimer (rMVA-ST) elicited robust cellular and humoral immunity and was more effective than MVA-S-monomer. Importantly, the rMVA-ST vaccine was able to stimulate decent cross-reactive neutralization against pseudoviruses packaged using S from different sublineages, including Wuhan, Delta, and Omicron. Remarkably, mice immunized with rMVA-ST were completely protected from a lethal challenge of SARS-CoV-2 without displaying any pathological conditions. Our results demonstrated that an MVA vectored vaccine expressing trimerized S is a promising vaccine candidate for SARS-CoV-2 and the strategy might be adapted for future vaccine development for coronaviruses., Competing Interests: J.Z., Y.L., Z.Z., W.W., and C.P. are listed as co-inventors on a patent filed by China Agricultural University that contains findings from this study.

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 Mn 2+ 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 Mn 2+ 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 Mn 2+ 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 Mn 2+ and AH significantly acted a synergistic effect, reinforced the immunogenicity of IsdB3, and offered a new strategy to increase vaccine efficiency., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2024 Yuwei Cen et al.)