Development of Different Methods for Preparing Acinetobacter baumannii Outer Membrane Vesicles Vaccine: Impact of Preparation Method on Protective Efficacy.

Acinetobacter baumannii (A. baumannii) is becoming a common global concern due to the emergence of multi-drug or pan-drug resistant strains. Confronting the issue of antimicrobial resistance by developing vaccines against the resistant pathogen is becoming a common strategy. In this study, different methods for preparing A. baumannii outer membrane vesicles (AbOMVs) vaccines were developed. sOMV (spontaneously released AbOMV) was extracted from the culture supernatant, while SuOMV (sucrose-extracted AbOMV) and nOMV (native AbOMV) were prepared from the bacterial cells. Three AbOMVs exhibited significant differences in yield, particle size, protein composition, and LPS/DNA content. To compare the protective efficacy of the three AbOMVs, groups of mice were immunized either intramuscularly or intranasally with each AbOMV. Vaccination via both routes conferred significant protection against lethal and sub-lethal A. baumannii challenge. Moreover, intranasal vaccination provided more robust protection, which may be attributed to the induction of significant sIgA response in mucosal sites. Among the three AbOMVs, SuOMV elicited the highest level of protective immunity against A. baumannii infection, whether intramuscular or intranasal immunization, which was characterized by the expression of the most profound specific serum IgG or mucosal sIgA. Taken together, the preparation method had a significant effect on the yield, morphology, and composition of AbOMVs, that further influenced the protective effect against A. baumannii infection.

Efficacy, safety, and immunogenicity of an oral recombinant Helicobacter pylori vaccine in children in China: a randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Helicobacter pylori is one of the most common gastric pathogens, affecting at least half the world's population, and is strongly associated with gastritis, peptic ulcer, gastric adenocarcinoma, and lymphoma. We aimed to assess the efficacy, safety, and immunogenicity of a three-dose oral recombinant H pylori vaccine in children in China. METHODS: We did this randomised, double-blind, placebo-controlled, phase 3 trial at one centre in Ganyu County, Jiangsu Province, China. Healthy children aged 6-15 years without past or present H pylori infection were randomly assigned (1:1), via computer-generated randomisation codes in blocks of ten, to receive the H pylori vaccine or placebo. Participants, their guardians, and study investigators were masked to treatment allocation. The primary efficacy endpoint was the occurrence of H pylori infection within 1 year after vaccination. We did analysis in the per-protocol population. This trial is registered with ClinicalTrials.gov, number NCT02302170. FINDINGS: Between Dec 2, 2004, and March 19, 2005, we randomly assigned 4464 participants to either the vaccine group (n=2232) or the placebo group (n=2232), of whom 4403 (99%) participants completed the three-dose vaccination schedule and were included in the per-protocol efficacy analysis. We extended follow-up to 3 years. We recorded 64 events of H pylori infection within the first year (14 events in 2074·3 person-years at risk in the vaccine group vs 50 events in 2089·6 person-years at risk in the placebo group), resulting in a vaccine efficacy of 71·8% (95% CI 48·2-85·6). 157 (7%) participants in the vaccine group and 161 (7%) participants in the placebo group reported at least one adverse reaction. Serious adverse events were reported in five (<1%) participants in the vaccine group and seven (<1%) participants in the placebo group, but none was considered to be vaccination related. INTERPRETATION: The oral recombinant H pylori vaccine was effective, safe, and immunogenic in H pylori-naive children. This vaccine could substantially reduce the incidence of H pylori infection; however, follow up over a longer period is needed to confirm the protection of the vaccine against H pylori-associated diseases. FUNDING: Chongqing Kangwei Biological Technology.

Burkholderia pseudomallei survival in lung epithelial cells benefits from miRNA-mediated suppression of ATG10.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease with high mortality, which is prevalent in tropical regions of the world. A recent study shows that B. pseudomallei can survive inside mammalian cells because of its ability to actively evade cell autophagy. However, the underlying mechanisms remain unclear. In the present study, based on microarray screening, we found that ATG10 was downregulated following B. pseudomallei infection in A549 human lung epithelial cells. Forced expression of ATG10 accelerated the elimination of intracellular B. pseudomallei by enhancing the process of autophagy. Moreover, MIR4458, MIR4667-5p, and MIR4668-5p were found, by microarray screening, to be upregulated in response to B. pseudomallei infection. These 3 novel miRNAs, MIR4458, MIR4667-5p, and MIR4668-5p, targeted to the 3'-untranslated region of ATG10 in different time-course and spatial manners. Upregulation of these miRNAs reduced the level of ATG10 and inhibited autophagy, leading to increasing survival rate of intracellular B. pseudomallei. Furthermore, the increase of these miRNAs was correlated with the reduced promoter methylation status in A549 cells in response to B. pseudomallei infection. Our results reveal that 3 novel miRNAs regulate autophagy-mediated elimination of B. pseudomallei by targeting ATG10, and provide potential targets for clinical treatment.

Evaluation of the protective immunity of a novel subunit fusion vaccine in a murine model of systemic MRSA infection.

Staphylococcus aureus is a common commensal organism in humans and a major cause of bacteremia and hospital acquired infection. Because of the spread of strains resistant to antibiotics, these infections are becoming more difficult to treat. Therefore, exploration of anti-staphylococcal vaccines is currently a high priority. Iron surface determinant B (IsdB) is an iron-regulated cell wall-anchored surface protein of S. aureus. Alpha-toxin (Hla) is a secreted cytolytic pore-forming toxin. Previous studies reported that immunization with IsdB or Hla protected animals against S. aureus infection. To develop a broadly protective vaccine, we constructed chimeric vaccines based on IsdB and Hla. Immunization with the chimeric bivalent vaccine induced strong antibody and T cell responses. When the protective efficacy of the chimeric bivalent vaccine was compared to that of individual proteins in a murine model of systemic S. aureus infection, the bivalent vaccine showed a stronger protective immune response than the individual proteins (IsdB or Hla). Based on the results presented here, the chimeric bivalent vaccine affords higher levels of protection against S. aureus and has potential as a more effective candidate vaccine.

A dominant CD4(+) T-cell response to Helicobacter pylori reduces risk for gastric disease in humans.

BACKGROUND & AIMS: Immunodominance is an important feature of antiviral, antitumor, and antibacterial cellular immune responses, but it is not well demonstrated in the immune responses against Helicobacter pylori. Antigen-specific CD4(+) T cells protect mice against infection with H pylori. We investigated the immunodominant CD4(+) T-cell response to neuraminyllactose-binding hemagglutinin (HpaA), which is a conserved, H pylori-specific colonization factor that is being investigated as an antigen for vaccination strategies. METHODS: HpaA-specific CD4(+) T cells were expanded with autologous peripheral blood mononuclear cells that had been incubated with recombinant HpaA and characterized using overlapping synthetic peptides. We compared the percentage of CD4(+) T cells with specificity for HpaA(88-100), restricted to HLA-DRB1*1501, among 59 H pylori-infected subjects with different gastric diseases. RESULTS: We identified and characterized several immunodominant CD4(+) T-cell epitopes derived from HpaA. The immunodominant CD4(+) T-cell responses specific to HpaA(88-100) were observed in most H pylori-infected individuals who expressed HLA-DRB1*1501 and were significantly more abundant in patients with less severe diseases (P < .05). CONCLUSIONS: The HLA-DRB1*1501-restricted immunodominant CD4(+) T-cell response to HpaA(88-100) is associated with reduced risk of severe gastric diseases. Further study of these and other immunodominant CD4(+) T-cell responses to H pylori will provide insight into mechanisms of protective immunity and aid in vaccine design.

CD8(+) T cells that produce interleukin-17 regulate myeloid-derived suppressor cells and are associated with survival time of patients with gastric cancer.

BACKGROUND & AIMS: CD8(+) T cells that produce interleukin (IL)-17 (Tc17 cells) promote inflammation and have been identified in tumors. We investigated their role in the pathogenesis of gastric cancer. METHODS: We used flow cytometry analyses to determine levels and phenotype of Tc17 cells in blood and tumor samples from 103 patients with gastric cancer. We performed multivariate analysis to identify factors associated with overall survival using the Cox proportional hazards model. CD8(+) T cells and monocytes were isolated and cocultured in an assay for induction of Tc17 cells. Tumor cells and myeloid-derived suppressor cells (MDSCs) were isolated and used in assays of Tc17 cell function. RESULTS: Tc17 cells with distinct cytokine and functional profiles were found in gastric tumor samples from patients. The percentage of Tc17 cells increased with tumor progression and was associated with overall survival time. Tumor-activated monocytes secreted IL-6, IL-1ß, and IL-23, which promoted development of Tc17 cell populations. Supernatants from cultured Tc17 cells induced production of the chemokine CXCL12 by tumor cells; this promoted CXCR4-dependent migration of MDSCs and impaired functions of anti-tumor CD8(+) cytotoxic T cells via a cell contact-dependent mechanism. CONCLUSIONS: Percentages of Tc17 cells in gastric tumors are associated with survival times of patients. These cells promote chemotaxis of MDSCs, which might promote tumor progression.

Increased tumor-infiltrating CD8(+)Foxp3(+) T lymphocytes are associated with tumor progression in human gastric cancer.

BACKGROUND: CD8(+)Foxp3(+) T lymphocytes have been detected in tumors. However, the distribution, phenotypic features, and regulation of these cells in gastric cancer remain unknown. METHODS: The levels of CD8(+)Foxp3(+) T lymphocytes in the peripheral blood, tumor-draining lymph nodes, non-tumor tissues, and tumor tissues of patients with gastric cancer were detected by flow cytometry. Foxp3 induction in CD8(+)Foxp3(-) T cells was investigated in vitro. The suppressive function of CD8(+)Foxp3(+) T lymphocytes was analyzed by their effect on CD4(+) T-cell proliferation and IFN-γ production. The percentages of CD8(+)Foxp3(+) T lymphocytes were evaluated for the association with tumor stage. RESULTS: The frequency of CD8(+)Foxp3(+) T lymphocytes in tumor tissues was significantly higher than that in non-tumor tissues, and similar results were also observed in tumor-draining lymph nodes compared with peripheral blood. Most intratumoral CD8(+)Foxp3(+) T lymphocytes were activated effector cells (CD45RA(-)CD27(-)). TGF-ß1 levels were positively correlated with the frequency of CD8(+)Foxp3(+) T lymphocytes in tumor tissues, and in vitro TGF-ß1 could induce the generation of CD8(+)Foxp3(+) T lymphocytes in a dose-dependent manner. Furthermore, intratumoral CD8(+)Foxp3(+) T lymphocytes suppressed the proliferation and IFN-γ production of CD4(+) T cells. Finally, intratumoral CD8(+)Foxp3(+) T lymphocytes were significantly increased with tumor progression in terms of tumor-node-metastasis (TNM) stage. CONCLUSIONS: Our data have shown that increased intratumoral CD8(+)Foxp3(+) T lymphocytes are associated with tumor stage and potentially influence CD4(+) T-cell functions, which may provide insights for developing novel immunotherapy protocols against gastric cancer.

Increased miR-222 in H. pylori-associated gastric cancer correlated with tumor progression by promoting cancer cell proliferation and targeting RECK.

Little is known about the potential role of microRNAs (miRNAs) in the carcinogenesis of gastric cancer induced by Helicobacter pylori (H. pylori). Here, we showed that microRNA-222 (miR-222) was up-regulated in H. pylori-infected gastric mucosa and gastric cancer. Ectopic expression of miR-222 promoted cell proliferation and colony formation in vitro. Mechanistically, we identified RECK as a novel target of miR-222, and also confirmed their relationship by the inverse correlation of mRNA expression ex vivo. Furthermore, we found that RNA interference silencing of RECK can mimic the oncogenic effects of miR-222. Collectively, H. pylori may function as an initiator in the process of carcinogenesis by up-regulating miR-222, which further participates in the progression of cancer by promoting proliferation and inhibiting RECK.

Increased miR-146a in gastric cancer directly targets SMAD4 and is involved in modulating cell proliferation and apoptosis.

MicroRNAs (miRNAs) have emerged as important gene regulators and are recognized as oncogenes or tumor suppressor genes in carcinogenesis. Gastric cancer is one of the most common malignant diseases worldwide. Our previous studies have revealed that miR-146a is upregulated in gastric epithelial cells infected with Helicobacter pylori (H. pylori) and in mucosal tissues from H. pylori-positive patients. However, the role of miR-146a in gastric cancer is largely unknown. In the current study, we showed that miR-146a was upregulated in 20 gastric cancer tissues compared with matched non-tumor adjacent tissues by quantitative RT-PCR. Furthermore, ectopic expression of miR-146a could improve cell proliferation in vitro by using Cell Counting kit 8 (CCK-8). We also found that miR-146a inhibited apoptosis of gastric cancer cells by flow cytometry (FCM) and Caspase-Glo® 3/7 assay. Using target prediction algorithms, luciferase reporter assay and Western blot assay, SMAD family member 4 (SMAD4) was identified as a target gene of miR-146a in gastric cancer. Moreover, an inverse correlation was observed between the expression of SMAD4 mRNA and miR-146a in gastric cancer tissues (R=-0.731, P=0.039, Pearson's correlation). Taken together, our results provide important evidence that miR-146a can directly target SMAD4, and suggest that miR-146a may play a role in the development of gastric cancer by modulating cell proliferation and apoptosis. miR-146a could serve as a potential biomarker and therapeutic target against gastric cancer.

Protection against Helicobacter pylori infection by a trivalent fusion vaccine based on a fragment of urease B-UreB414.

A multivalent fusion vaccine is a promising option for protection against Helicobacter pylori infection. In this study, UreB414 was identified as an antigenic fragment of urease B subunit (UreB) and it induced an antibody inhibiting urease activity. Immunization with UreB414 partially protected mice from H. pylori infection. Furthermore, a trivalent fusion vaccine was constructed by genetically linking heat shock protein A (HspA), H. pylori adhesin A (HpaA), and UreB414, resulting in recombinant HspA-HpaA-UreB414 (rHHU). Its protective effect against H. pylori infection was tested in BALB/c mice. Oral administration of rHHU significantly protected mice from H. pylori infection, which was associated with H. pylori-specific antibody production and Th1/Th2-type immune responses. The results show that a trivalent fusion vaccine efficiently combats H. pylori infection, and that an antigenic fragment of the protein can be used instead of the whole protein to construct a multivalent vaccine.

[Recombinant Helicobacter pylori urease B subunit and its biological properties].

OBJECTIVE: To perform genetic recombination of the urease B subunit (UreB) of Helicobacter pylori (Hp) and examine the biological properties of the recombinant protein. METHODS: The gene fragment encoding Hp UreB was isolated clinically from Chinese subjects by means of PCR, and cloned subsequently into an expression vector pET-11C-UreB for the non-fusion protein expression in E.coli BL21 (DE3) strain. RESULTS: The expression of recombinant UreB was achieved in E.coli BL21 with a relative molecular weight of approximately 62,000 at the expression ratio of 26%, and the first 15 amino acids of recombinant UreB were MKKISREYVSMYGP. The results of peptide mapping and amino acid compositional analysis were consistent with previous theoretical prediction, and enzyme-linked immunosorbent assay together with Western blotting indicated strong immunogenicity and reactivity of the recombinant protein in BalB/c mice, which were specifically recognized by polyclonal BalB/c mice anti-Hp sera or human sera infected with Hp. CONCLUSION: The results of this study has laid an solid immunological foundation for incorporating recombinant UreB as a subunit vaccine component against Hp.