Identification of CD4+ T cell epitopes on glyceraldehyde-3-phosphate dehydrogenase-C of Staphylococcus aureus in Babl/c mice.

Glyceraldehyde-3-phosphate dehydrogenase-C (GapC) is a highly conserved surface protein of Staphylococcus aureus, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, which represents an excellent vaccine candidate antigen. It can induce protective immune responses to S. aureus infections. However, CD4+ T cell epitopes of GapC that induce CD4+ T cell immune responses are currently unclear. In this study, we used bioinformatics prediction algorithms to predict CD4+ T cell epitopes of GapC. Ten peptides were synthesized to investigate the candidate epitopes. Our results showed that the peptides, G4 (GapC 104-123) and G10 (GapC 314-333) were able to induce proliferation of CD4+ T cells and secrete high levels of interferon (IFN)-γ, respectively. In addition, they significantly reduced bacterial loads in tissue and induced immunoprotective effects. It is suggested that G4 and G10 are Th1-type epitopes of S. aureus GapC. This study provides the potential development of the design of epitope-based vaccine against S. aureus.

The novel combinations of CTB, CpG, and aluminum hydroxide significantly enhanced the immunogenicity of clumping factor A 221-550 of Staphylococcus aureus.

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, 3', 5, 5'-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.

Evaluation of the immunogenicity of an omp A and staphylococcal target of RNAIII activating fusion protein displayed on the surface of Escherichia coli.

The purpose of this investigation was to construct a recombinant Escherichia coli strain displaying the Staphylococcus aureus target of RNAIII activating protein (TRAP) on its surface, and to investigate the strain for its immunogenicity. The lpp'ompA and lpp'ompA-TRAP genes were fused by the overlap polymerase chain reaction and then ligated into expression plasmid pQE30 producing pLO and pLO-TRAP. These two recombinant plasmids were transformed into E. coli XL1-Blue, resulting in XL1-Blue/pLO and XL1-Blue/pLO-TRAP, which were induced to express protein. The expressed TRAP protein was displayed on the surface of XL1-Blue as judged by whole cell ELISA, flow cytometric analysis, and laser scanning confocal microscopy using the lpp'ompA surface display system. ICR mice were intramuscularly immunized with recombinant strains XL1-Blue/pLO and XL1-Blue/pLO-TRAP as well as recombinant protein TRAP. Immunized mice were assessed for anti-TRAP antibody and lymphocytes for secreted IL-4 and IFN-γ by ELISPOT and secreted IL-17A by indirect ELISA. Immunized mice were challenged with S. aureus Newman and HLJ23-1 strains. The results showed both XL1-Blue/pLO-TRAP and TRAP protein immunized mice to produce better cellular and humoral immunity than XL1-Blue/pLO and PBS injected mice.

Identification of a conserved linear B-cell epitope in the Staphylococcus aureus GapC protein.

The GapC protein of Staphylococcus aureus (S. aureus) is a surface protein that is highly conserved among Staphylococcus strains, and it can induce protective humoral immune responses. However, B-cell epitopes in S. aureus GapC have not been reported. In this study, we generated a monoclonal antibody (mAb2A9) targeting S. aureus GapC. Through a passive immunity test, mAb2A9 was shown to partially protect mice against S. aureus infection. We screened the motif 236PVATGSLTE243 that is recognized by mAb2A9 using a phage-display system. The motif sequence exactly matched amino acids 236-243 of the S. aureus GapC protein. Then, we identified the key amino acids in the motif using site-directed mutagenesis. Site-directed mutagenesis revealed that residues P236, G240, L242, and T243 formed the core of the 236PVATGSLT243 motif. In addition, this epitope was proven to be located on the surface of S. aureus, and it induced a protective humoral immune response against S. aureus infection in immunized mice. Overall, our results characterized a conserved B-cell epitope, which will be an attractive target for designing effective epitope-based vaccines against S. aureus infection.

Prokaryotic Expression and Anti-IBDV Activity of Chicken Interleukin-18 and Interferon-γ.

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.

Immunogenicity of amino acids 1-150 of Streptococcus GapC displayed on the surface of Escherichia coli.

Streptococcus is one of the main pathogens that cause bovine mastitis. They includes into S.agalactiae, S.dysgalactiae, and S.uberis. The GapC protein is a virulence factor that is expressed on the surface of Streptococcus species. GapC is highly antigenic and immunization with GapC confers cross-protection against all three species. Our previous data showed that amino acids 1-150 of GapC (GapC1-150) of S. dysgalactiae conferred similar immunoprotection compared to full-length GapC. Thus, the present study aimed to construct a recombinant Escherichia coli XL1-Blue strain that displayed GapC1-150 on its surface, and to investigate the immunogenicity of the surface-localized GapC1-150. To do so, the ompA gene of the E. coli XL1-Blue strain was replaced with the lpp'-ompA-gapC11-150 or lpp'-ompA genes by λ Red recombination, the former of which fused GapC1-150 to an Lpp lipoprotein signal peptide and amino acids 1-159 of OmpA; the recombinant strains were named XL1-Blue/LOG76 and XL1-Blue/LO11, respectively. GapC1-150 was confirmed to localize to the surface of the XL1-Blue/LOG76 strain by an indirect enzyme-linked immunosorbent assay (ELISA), a fluorescence-activated cell sorter analysis, and laser-scanning confocal microscopy. Then, ICR mice were immunized intramuscularly with the XL1-Blue/LOG76 or XL1-Blue/LO11 strains, or recombinant GapC1-150. The sera of the immunized mice were collected and the anti-GapC1-150 antibody levels were detected by ELISA. Lymphocytes secreting interleukin (IL)-4 and interferon-γ were detected by an enzyme-linked ImmunoSpot assay, as was the level of IL-17A level in the supernatant of cultured splenic lymphocytes. The mice immunized with the XL1-Blue/LOG76 strain or GapC1-150 exhibited better cellular and humoral immunity. Lastly, the immunized mice were challenged with S. uberis, S. dysgalactiae, and S. agalactiae strains, and mice that were immunized with the XL1-Blue/LOG76 strain were better protected than those that were immunized with the XL1-Blue/LO11 strain. These results indicate that it is feasible to display GapC1-150 on the E. coli surface as a vaccine against Streptococcus species.

Identification and characterization of CD4+ T cell epitopes on manganese transport protein C of Staphylococcus aureus.

Manganese transport protein C (MntC) of Staphylococcus aureus represents an excellent vaccine-candidate antigen. The important role of CD4+ T cells in effective immunity against S. aureus infection was shown; however, CD4+ T cell-specific epitopes on S. aureus MntC have not been well identified. Here, we used bioinformatics prediction algorithms to evaluate and identify nine candidate epitopes within MntC. Our results showed that peptide M8 emulsified in Freund's adjuvant induced a much higher cell-proliferation rate as compared with controls. Additionally, CD4+ T cells stimulated with peptide M8 secreted significantly higher levels of interferon-γ and interleukin-17A. These results suggested that peptide M8 represented an H-2d (I-E)-restricted Th17-specific epitope.

Identification and characterization of CD4⁺ T-cell epitopes on GapC protein of Streptococcus dysgalactiae.

The GapC protein is highly conserved surface dehydrogenase among Streptococcus dysgalactiae (S. dysgalactiae) and is shown to be involved in bacterial virulence. Immunization of GapC protein can induce specific CD4(+) T-cell immune responses and protect against S. dysgalactiae infection. However, there are no studies to identify immunodominant CD4(+) T-cell epitopes on GapC protein. In this study, in silico MHC affinity measurement method was firstly used to predict potential CD4(+) T-cell epitopes on GapC protein. Six predictive 15-mer peptides were synthesized and two novel GapC CD4(+) T-cell epitopes, GapC63-77 and GapC96-110, were for the first time identified using CD4(+) T-cells obtained from GapC-immunized BALB/c (H-2(d)) and C57BL/6 (H-2(b)) mice spleen based on cell proliferation and cytokines response. The results showed that peptides containing 63-77 and 96-110 induced significant antigen-specific CD4(+) T-cells proliferation response in vivo. At the same time, high levels of IFN-γ and IL-17A, as well as moderate levels of IL-10 and IL-4 were detected in CD4(+) T-cells isolated from both GapC and peptide-immunized mice in vivo, suggesting that GapC63-77 and GapC96-110 preferentially elicited polarized Th1/Th17-type responses. The characterization of GapC CD4(+) T-cell epitopes not only helps us understand its protective immunity, but also contributes to design effective T-cell epitope-based vaccine against S. dysgalactiae infection.

Identification of CD4+ T-cell epitopes on iron-regulated surface determinant B of Staphylococcus aureus.

Iron-regulated surface determinant B (IsdB) of Staphylococcus aureus (S. aureus) is a highly conserved surface protein that can induce protective CD4(+) T-cell immune response. A pivotal role of CD4(+) T-cells in effective immunity against S. aureus infection has been proved, but CD4(+) T-cell epitopes on the S. aureus IsdB have not been well identified. In this study, MHC binding assay was firstly used to predict CD4(+) T-cell epitopes on S. aureus IsdB protein, and six peptides were synthesized to validate the probable epitopes. Two novel IsdB CD4(+) T-cell epitopes, P1 (residues 159-178) and P4 (residues 287-306), were for the first time identified using CD4(+) T-cells obtained from IsdB-immunized C57BL/6 (H-2(b)) and BALB/c (H-2(d)) mice spleen based on cell proliferation and cytokines response. The results showed that P1 and P4 emulsified in Freund's adjuvant (FA) induced much higher cell proliferation compared with PBS emulsified in FA. CD4(+) T-cells stimulated with peptides P1 and P4 secreted significantly higher levels of IFN-γ and IL-17A. However, the level of the cytokine IL-4 almost remained unchanged, suggesting that P1 and P4 preferentially elicited polarized Th1-type responses. In addition, BALB/c mice just respond to P4 not P1, while C57BL/6 mice respond to P1 not P4, implying that epitope P1 and P4 were determined as H-2(b) and H-2(d) restricted epitope, respectively. Taken together, our data may provide an explanation of the IsdB-induced protection against S. aureus and highlight the possibility of developing the epitope-based vaccine against the S. aureus.

Identification of a conserved B-cell epitope on the GapC protein of Streptococcus dysgalactiae.

Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines.

Single-incision versus conventional three-port laparoscopic appendectomy: A meta-analysis of randomized controlled trials.

PURPOSE: The aim of this article was to compare the advantages and disadvantages of single-incision laparoscopic appendectomy (SILA) and conventional three-port laparoscopic appendectomy (CTLA). MATERIAL AND METHODS: A meta-analysis was performed by analyzing all randomized controlled trials (RCTs) published in English that compared SILA and CTLA for appendicitis in adults and children. These studies compared these two methods from different angles including outcomes of interest, patient characteristics, operative time, pain visual analogue scales scores (VAS scores), length of hospital stay, time to return to full activity, resumption of diet, postoperative complications and cosmetic results The risk ratios (RR) and mean difference (MD) with 95% confidence intervals (CIs) were employed to assess the outcome. RESULTS: Seven recent RCTs encompassing 1170 patients (586 SILA and 584 CTLA cases) were included in this meta-analysis. The pooled results demonstrated that conversion rate, drain inserted, reoperation, length of hospital stay, resumption of normal diet and postoperative complications were statistically comparable between the two groups. The postoperative abdominal pain within 24 h was -0.57 in favor of the SILA technique (p = 0.05). Compared with CTLA, SILA showed a better cosmetic satisfaction score (SMD, 0.58; 95% CI, 0.32-0.83; p < 0.0001) and shorter time to recover normal activity (WMD, -0.69; 95% CI, -1.11-0.26; p = 0.001). However, SILA has a longer operative time (WMD, 5.38; 95% CI, 2.94-7.83; p < 0.0001). CONCLUSIONS: In selected patients, SILA was confirmed to be as safe and effective as CTLA. Despite the longer operative time, SILA has higher cosmetic satisfaction and shorter recovery time to normal activity. Due to the limitations of the available data, further research is needed.

Pancreaticogastrostomy is associated with significantly less pancreatic fistula than pancreaticojejunostomy reconstruction after pancreaticoduodenectomy: a meta-analysis of seven randomized controlled trials.

OBJECTIVES: This study aimed to compare pancreaticojejunostomy (PJ) with pancreaticogastrostomy (PG) after pancreaticoduodenectomy (PD). METHODS: A literature search of PubMed and the Cochrane Central Register of Controlled Trials for studies comparing PJ with PG after PD was conducted. The primary outcome for meta-analysis was pancreatic fistula. Secondary outcomes were morbidity, mortality, biliary fistula, intra-abdominal fluid collection, hospital length of stay (LoS), postoperative haemorrhage and reoperation. Outcome measures were odds ratios (ORs) and mean differences with 95% confidence intervals (CIs). RESULTS: Seven recent RCTs encompassing 1121 patients (559 PJ and 562 PG cases) were involved in this meta-analysis. Incidences of pancreatic fistula (10.6% versus 18.5%; OR 0.52, 95% CI 0.37-0.74; P = 0.0002), biliary fistula (2.3% versus 5.7%; OR 0.42, 95% CI 0.03-3.15; P = 0.03) and intra-abdominal fluid collection (8.0% versus 14.7%; OR 0.50, 95% CI 0.34-0.74; P = 0.0005) were significantly lower in the PG than the PJ group, as was hospital LoS (weighted mean difference: -1.85, 95% CI -3.23 to -0.47; P = 0.008). Subgroup analysis indicated that severe pancreatic fistula (grades B or C) occurred less frequently in the PG than the PJ group (8.3% versus 20.5%; OR 0.37, 95% CI 0.23-0.59; P < 0.00001). However, there was no significant difference in morbidity (48.9% versus 51.0%; OR 0.90, 95% CI 0.70-1.16; P = 0.41), mortality (3.2% versus 3.5%; OR 0.82, 95% CI 0.43-1.58; P = 0.56), delayed gastric emptying (16.6% versus 14.7%; relative risk: 1.02, 95% CI 0.62-1.68; P = 0.94), postoperative haemorrhage (9.6% versus 11.1%; OR 0.82, 95% CI 0.54-1.24; P = 0.35) or reoperation (9.9% versus 9.8%; OR 0.93, 95% CI 0.60-1.43; P = 0.73). CONCLUSIONS: Pancreaticogastrostomy provides benefits over PJ after PD, including in the incidences of pancreatic fistula, biliary fistula and intra-abdominal fluid collection and in hospital LoS. Therefore, PG is recommended as a safer and more reasonable alternative to PJ reconstruction after PD.

Development of an indirect ELISA for detection of E. Coli antibodies in cow serum using a recombinant OmpT as antigen.

The aim of this article was to develop an indirect enzyme-linked immunosorbent assay (ELISA) for efficient detection of the infection of E. coli in cattle. OmpT, a highly conserved protease in all E. coli strains, was successfully expressed in E. coli XL-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 was E. coli specific 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 of E. coli with different serotype strains.

Improved protective efficacy of a chimeric Staphylococcus aureus vaccine candidate iron-regulated surface determinant B ( N 126- P 361) -target of RNAIII activating protein in mice.

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.

Construction and Analysis of ceRNA Networks Reveal the Key Genes Associated with Bovine Herpesvirus Type 1 Infection.

Background: Virus infection can cause the changes of lncRNA expression levels to regulate the interaction between virus and host, but the relationship between BHV-1 infection and lncRNA has not been reported. Methods: In this study, in order to reveal the molecular mechanism of RNA in BoHV-1 infection, the Madin-Darby bovine kidney (MDBK) cells were infected with BoHV-1, transcriptome sequencing were performed by next-generation sequencing at 18 h or 24 h or 33 h of viral infection and then based on the competitive endogenous RNA (ceRNA) theory, lncRNA-miRNA-mRNA networks were constructed using these high-throughput sequencing data. The network analysis, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for functional annotation and exploration of ncRNA ceRNAs in BoHV-1 infection. Results: The results showed that 48 lncRNAs, 123 mRNAs and 20 miRNAs as differentially expressed genes, and the mitogen activated protein kinase (MAPK) pathway and calcium signaling pathway were significantly enriched in the ceRNA network. Some differentially expressed lncRNA genes were randomly selected for verification by RT-qPCR, and the results showed that their expression trend was consistent with the results of transcriptome sequencing data. Conclusion: This study revealed that BoHV-1 infection can affect the expression of RNAs in MDBK cells and the regulation of ceRNA network to carry out corresponding biological functions in the host, but further experimental studies are still necessary to prove the hub genes function in ceRNA network and the molecular mechanism in BoHV-1 infection.

Immunogenicity of the Non-toxic Form of Staphylococcus Aureus α-Hemolysin Using a Chimeric Fusion in Mice.

Background: Staphylococcus aureus is an opportunistic pathogen responsible for various infections with diverse clinical presentation and severity. The α-hemolysin is a major virulence factor in the pathogenesis of S. aureus infections. Objective: To produce a chimeric fusion protein for hemolytic detection of the S. aureus isolates and as a component of a multi-antigen vaccine. Methods: The fused strategy employed a flexible linker to incorporate the possible B cell and T cell determinants into one chimera (HlaD). The humoral and cellular response to the HlaD in mice was assessed to reveal a non-significant difference compared with the full-length α-hemolysin mutant (Hla H35L). Results: The results of the protective effect, the mimetic lung cell injury, and bacterial clearness demonstrated that the mice vaccinated with the HlaD alleviated the severity of the infection of the S. aureus, and the HlaD could similarly function with Hla H35L. Conclusion: The chimeric fusion (HlaD) provided a diagnostic antigen for hemolysis of the S. aureus strains and a potential vaccine component.

PEI-PLGA nanoparticles significantly enhanced the immunogenicity of IsdB137-361 proteins from Staphylococcus aureus.

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.

Als3-Th-cell-epitopes plus the novel combined adjuvants of CpG, MDP, and FIA synergistically enhanced the immune response of recombinant TRAP derived from Staphylococcus aureus in mice.

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.

Immunogenicity of multi-epitope vaccines composed of epitopes from Streptococcus dysgalactiae GapC.

Glyceraldehyde-3-phosphate dehydrogenase C (GapC) of Streptococcus dysgalactiae (S. dysgalactiae) is a highly conserved surface protein that can induce a protective immune response against S. dysgalactiae infection. To investigate the immune response and protective efficacy induced by epitope-vaccines against S. dysgalactiae infection, we constructed epitope-vaccines GTB1, GB1B2, and GTB1B2 using a T cell epitope (GapC63-77, abbreviated as GT) and two B cell epitopes (GapC30-36, abbreviated as GB1, and GapC97-103, abbreviated as GB2), which were identified in GapC1-150 of S. dysgalactiae in tandem by a GSGSGS linker. BALB/c mice were immunized via an intramuscular injection with the epitope vaccines. The levels of the cytokines, IFN-γ, IL-4, and IL-17, secreted by splenic lymphocytes and the antibody levels in the sera of the immunized mice were detected by ELISA. The immunized mice were subsequently challenged with S. dysgalactiae, and the bacterial colonization in the immunized-mouse organs was examined using the plate counting method. The results showed that the level of the cytokines induced by GTB1B2 was lower than that induced by GapC1-150, but higher than that induced by other epitope vaccines. The level of IgG induced by GTB1B2 was lower than that induced by GapC1-150, but higher than the levels induced by other epitope vaccines. The bacterial colonization numbers in the organs of the mice immunized with GTB1B2 were higher those of the mice immunized with GapC1-150, but significantly lower than those from the mice immunized with other epitope-vaccines. Our results demonstrated that the T cell and B cell epitopes in the epitope-vaccines worked synergistically against bacterial challenge. The multi-epitope vaccine, GTB1B2, could induce stronger cellular and humoral immune responses, and provide a better protective effect against S. dysgalactiae infection.

Candida albicans CaPTC6 is a functional homologue for Saccharomyces cerevisiae ScPTC6 and encodes a type 2C protein phosphatase.

Type 2C protein phosphatases (PP2C) are monomeric enzymes and their activities require the presence of magnesium or manganese ions. There are seven PP2C genes, ScPTC1, ScPTC2, ScPTC3, ScPTC4, ScPTC5, ScPTC6 and ScPTC7, in Saccharomyces cerevisiae. PTC6 is highly conserved in pathogenic and nonpathogenic yeasts. In the current study we have demonstrated that the Candida albicans CaPTC6 gene could complement the functions of ScPTC6 in the rapamycin and caffeine sensitivities of S. cerevisiae cells, indicating that they are functional homologues. We have also demonstrated that the CaPTC6-encoded protein is a typical PP2C enzyme and that CaPtc6p is localized in the mitochondrion of yeast-form and hyphal cells. However, deletion of CaPTC6 neither affects cell and hyphal growth nor renders Candida cells sensitive to rapamycin and caffeine. Therefore, possibly with a functional redundancy to other mitochondrial phosphatases, CaPtc6p is likely to be involved in the regulation of a mitochondrial physiology.