Research progress of bacterial flagellin as a vaccine adjuvant / 中国生物制品学杂志

@#Vaccines play an important role in the prevention and control of infectious diseases. Bacterial flagellin can activate Toll like receptor 5(TLR5) and NOD like receptor C4(NLRC4) in host cells,and has immune adjuvant effect. As a new immune adjuvant,flagellin can not only share with antigen protein,but also fuse with antigen protein,which can significantly improve the immune effect. At the same time,it has achieved good application effect in mucosal immunity and antitumor immunity,becoming a hot spot in the research and development of vaccine adjuvants. This review mainly discussed the research progress on application of bacterial flagellin as vaccine adjuvant,so as to provide new ideas for the development of flagellin adjuvant vaccine.

Comparison of immunogenicity between recombinant flagellins C and B of Salmonella abortus equi / 生物工程学报

To evaluate and compare of the immunogenicity differences of flagellins FliC and FljB of Salmonella abortus equi, and lay the experimental foundation for the further utilization of the two recombinant proteins, FliC and FljB recombinant proteins were induced, expressed and purified. The purified FliC and FljB were used to immunize mice separately. The antibody level, titer and subtype of mice serum were detected after immunization. Immune-related receptors and histopathological changes were observed in immunized mice after challenged. The recombinant proteins FliC and FljB were successfully induced and expressed. Proteins of about 52 kDa and 42 kDa were purified. High levels of specific IgG antibodies were induced in mice immunized with these two proteins, the antibody level of FljB-immunized group was higher than that of FliC-immunized group, and IgG1 was the dominant subtype of antibody. The challenge protection rate of the FljB-immunized group was 87.5%, higher than that of FliC immunized group. Bacterial loads and observation pathological of FljB-immunized group were better than that of FliC immunized group, the levels of TCR2, TCR4, MHC-I and TCR induced by FljB-immunized group were higher than those of FliC-immunized group. The of immune response induced by FljB group was better than that of FliC group.

A Fusion Protein of Derp2 Allergen and Flagellin Suppresses Experimental Allergic Asthma

PURPOSE: The house dust mite (HDM) is one of the most important sources of indoor allergens and a significant cause of allergic rhinitis and allergic asthma. Our previous studies demonstrated that Vibrio vulnificus flagellin B (FlaB) plus allergen as a co-treatment mixture improved lung function and inhibited eosinophilic airway inflammation through the Toll-like receptor 5 signaling pathway in an ovalbumin (OVA)- or HDM-induced mouse asthma model. In the present study, we fused the major mite allergen Derp2 to FlaB and compared the therapeutic effects of the Derp2-FlaB fusion protein with those of a mixture of Derp2 and FlaB in a Derp2-induced mouse asthma model. METHODS: BALB/c mice sensitized with Derp2 + HDM were treated with Derp2, a Derp2 plus FlaB (Derp2 + FlaB) mixture, or the Derp2-FlaB fusion protein 3 times at 1-week intervals. Seven days after the final treatment, the mice were challenged intranasally with Derp2, and airway responses and Derp2-specific immune responses were evaluated. RESULTS: The Derp2-FlaB fusion protein was significantly more efficacious in reducing airway hyperresponsiveness, lung eosinophil infiltration, and Derp2-specific IgE than the Derp2 + FlaB mixture. CONCLUSIONS: The Derp2-FlaB fusion protein showed a strong anti-asthma immunomodulatory capacity, leading to the prevention of airway inflammatory responses in a murine disease model through the inhibition of Th2 responses. These findings suggest that the Derp2-FlaB fusion protein would be a promising vaccine candidate for HDM-mediated allergic asthma therapy.

Enhancement of antigen-specific humoral immune responses and protein solubility through conjugation of bacterial flagellin, Vibrio vulnificus FlaB, to the N-terminus of porcine epidemic diarrhea virus surface protein antigen S0

Porcine epidemic diarrhea (PED) is a highly contagious enteric swine disease. The large economic impact of PED on the swine industry worldwide has made the development of an effective PED vaccine a necessity. S0, a truncated region of the porcine epidemic diarrhea virus (PEDV) spike protein, has been suggested as a candidate antigen for PED subunit vaccines; however, poor solubility problems when the protein is expressed in Escherichia coli, and the inherent problems of subunit vaccines, such as low immunogenicity, remain. Flagellin has been widely used as a fusion partner to enhance the immunogenicity and solubility of many difficult-to-express proteins; however, the conjugation effect of flagellin varies depending on the target antigen or the position of the fusion placement. Here, we conjugated flagellin, Vibrio vulnificus FlaB, to the N- and C-termini of S0 and evaluated the ability of the fusion to enhance the solubility and immunogenicity of S0. Flagellin conjugation in the presence of the trigger factor chaperone tig greatly improved the solubility of the fusion protein (up to 99%) regardless of its conjugation position. Of importance, flagellin conjugated to the N-terminus of S0 significantly enhanced S0-specific humoral immune responses compared to other recombinant antigens in Balb/c mice. The mechanism of this phenomenon was investigated through in vitro and in vivo studies. These findings provide important information for the development of a novel PED vaccine and flagellin-based immunotherapeutics.

Application potential of bacterial flagellin in treatment of autoimmune rheumatic diseases with Treg epitope peptides / 中华微生物学和免疫学杂志

Immunization with regulatory T cell (Treg) epitope peptides to activate and induce Tregs, by which to suppress pathological autoimmune responses and reconstitute a new homeostasis, is a promising therapeutic regimen for autoimmune rheumatic diseases. However, it is usually hard to induce potent peptide-specific immune responses in vivo with small molecular peptides. Bacterial flagellin is one of the agonists triggering innate immune responses. When used as carrier, it shows strong adjuvant activity to its conjugated antigens. In some particular situations, bacterial flagellin can also activate and induce Tregs. Thus if Treg epitope peptides are covalently conjugated to a bacterial flagellin, the conjugates should be able to effectively enhance the Treg-based immune responses via flagellin itself and the adjuvanticity of flagellin to Treg epitope peptides, and thereby enhance the immunotherapeutic effects on autoimmune rheumatic diseases.

Application potential of bacterial flagellin in treatment of autoimmune rheumatic diseases with Treg epitope peptides / 中华微生物学和免疫学杂志

Immunization with regulatory T cell ( Treg ) epitope peptides to activate and induce Tregs, by which to suppress pathological autoimmune responses and reconstitute a new homeostasis, is a promising therapeutic regimen for autoimmune rheumatic diseases. However, it is usually hard to induce po-tent peptide-specific immune responses in vivo with small molecular peptides. Bacterial flagellin is one of the agonists triggering innate immune responses. When used as carrier, it shows strong adjuvant activity to its conjugated antigens. In some particular situations, bacterial flagellin can also activate and induce Tregs. Thus if Treg epitope peptides are covalently conjugated to a bacterial flagellin, the conjugates should be able to effectively enhance the Treg-based immune responses via flagellin itself and the adjuvanticity of flagellin to Treg epitope peptides, and thereby enhance the immunotherapeutic effects on autoimmune rheumatic diseases.

Molecular characterization of virulence genes cctA, nanA, and fliC in Clostridium chauvoei from Rio Grande do Sul and São Paulo State, Brazil / Caracterização molecular dos genes de virulência cctA, nanA e fliC em Clostridium chauvoei isolados no Rio Grande do Sul e São Paulo, Brasil

ABSTRACT: Clostridium chauvoei toxin A (CctA), neuraminidase (NanA), and flagellin (FliC) proteins contribute to the pathogenicity of Clostridium chauvoei, the causative agent of blackleg in cattle. The aim of this study was to analyze the genetic variability of cctA, nanA, and fliC genes in C. chauvoei isolates from the Rio Grande do Sul and São Paulo state- Brazil, during different sampling periods. The presence of these genes was verified through PCR amplification and partial gene sequencing of 17 strains. Alignment of PCR amplicons combined with bioinformatics analysis was used in an attempt to study the variability across C. chauvoei solates. The similarity among the partial sequences of cctA and nanA genes was 100%. The sequencing of fliC revealed three different paralog alleles of flagellin, and two strains were seen to be polymorphic, with amino acid alterations in the predicted protein. Overall, this study indicates that strains of C. chauvoei isolated in Brazil are highly conserved with respect to the virulence factors evaluated.

RESUMO: Toxina A de Clostridium chauvoei (CctA), neuraminidase (NanA) e flagelina (FliC) são proteínas que contribuem para a patogenicidade de Clostridium chauvoei, o agente causador do carbúnculo sintomático em bovinos. O objetivo deste estudo foi analisar a variabilidade genética dos genes cctA, nanA, e fliC em C. chauvoei isolados em diferentes períodos no Rio Grande do Sul e São Paulo. A presença destes genes foi verificada pela amplificação dos produtos da PCR e sequenciamento parcial dos genes de 17 cepas. Os alinhamentos da amplificação dos produtos da PCR combinados com a análise de bioinformática foram utilizados na tentativa de avaliar a variabilidade dos genes entre os isolados de C. chauvoei. A similaridade do sequenciamento parcial dos genes cctA e nanA foi 100%. O sequenciamento do fliC revelou três alelos paralogos diferentes de flagelina e duas cepas mostraram polimorfismos, causando alterações na sequência de aminoácidos. As cepas de C. chauvoei isoladas no Brasil mostraram-se altamente conservadas em relação aos fatores de virulência avaliados neste estudo.

Pathogenic effect of bacterial flagellin in TNBS-induced colitis in mice / 中国病理生理杂志

AIM:To detect the expression of CBir1 in the serum and colon tissue and mast cell degranulation in the tissue of 2,4,6-trinito-benzene-sulfonic acid ( TNBS)-induced colitis in mice with different interventions. ME-THODS:SPF male BALB/c mice were randomized into 6 groups (12 mice in each group):normal control group, normal saline group, 50% alcohol group, 50% alcohol+TNBS group, 50% alcohol+TNBS+lipopolysaccharide (LPS) +ovalbu-min (OVA) group and 50% alcohol+TNBS+ketotifen group. Corresponding treatment was given to each group, and the disease activity index (DAI) of the mice was evaluated. The mice were sacrificed on day 22 after treatment. The colon tis-sues were evaluated by histological index (HI) scoring. Serum concentrations of anti-CBir1, mast cell tryptase (MCT) and histamine were measured by ELISA. The expression of CBir1, toll-like receptor 5 (TLR5) and MCT in the colon tissues was detected by immunohistochemistry. RESULTS:Compared with the normal control group, the DAI score, HI score and CBir1, anti-CBir1, MCT, TLR5, histamine concentrations in colon tissues and serum were all significantly higher in 50% alcohol+TNBS group, 50% alcohol+TNBS+ketotifen group and 50% alcohol+TNBS+LPS+OVA group (P<0.05). The DAI score, HI score and anti-CBir1, CBir1, MCT, histamine levels in 50% alcohol+TNBS group were lower than those in 50% alcohol+TNBS+LPS+OVA group (P<0.05). The DAI score, HI score and anti-CBir1, TLR5, hista-mine, CBir1 Levels in 50% alcohol+TNBS group were higher than those in 50% alcohol+TNBS+ketotifen group ( P<0.05). Normal saline group and 50% alcohol group had no statistically significant difference in comparison with normal control group. In TNBS model group, serum concentration of anti-CBir1 was positively correlated with MCT concentration (r=0.648, P<0.01) and histamine concentration (r=0.751, P<0.01). CONCLUSION:The heavier degree of in-flammation in TNBS-induced colitis, the higher levels of the CBir1 and the degranulation of mast cells. There is a positive correlation between the expression of CBir1 and the degranulation of mast cells in TNBS-induced colitic mice.

Protective effect of MUC2 in mice with colitis and the expression of anti-CBir1 antibody in serum / 中国病理生理杂志

AIM:To investigate the protective effect of mucin 2(MUC2)on intestinal mucosa of colitis model mice,and to explore the correlation between the expression of anti-CBir1 flagellin antibody and MUC2.METHODS:The mice were randomly divided into normal control group,2,4,6-trinitrobenzenesulfonic acid(TNBS)group,lipopolysaccha-ride(LPS)+ovalalbumin(OVA)+TNBS group and ketotifen+TNBS group.The expression of MUC2 in colon tissue was determined by PAS staining and immunohistochemistry, and the anti-CBir1 antibody level in the serum of mice in each group was measured by ELISA.RESULTS:The scores of disease activity index and histological index in TNBS group were higher than those in normal control group(P<0.05).The scores in LPS +OVA+TNBS group were much higher than those in TNBS group(P<0.05).However, the values in ketotifen +TNBS group were lower than those in TNBS group (P<0.05).PAS staining showed a decrease in goblet cells in TNBS group.Compared with TNBS group,the colonic mu-cosa integrity in LPS+OVA+TNBS group was destroyed, and the number of goblet cells in ketotifen +TNBS group in-creased significantly.Immunohistochemical staining showed that the expression of MUC 2 in the intestinal tract of each mo-del group was basically consistent with the results of PAS staining.The serum anti-CBir1 antibody level in TNBS group was higher than that in normal control group(P<0.05), and that in LPS+OVA+TNBS group was significantly higher than that in TNBS group(P<0.05),whereas that in ketotifen +TNBS group was decreased slightly(P<0.05).CONCLU-SION:MUC2 plays a protective role in the pathogenesis of colitis in mice,and there is a negative correlation between the expression of MUC2 and the bacterial flagellin in the intestinal mucosa of mice with colitis.

Production of recombinant flagellin to develop ELISA-based detection of Salmonella Enteritidis

ABSTRACT Food-borne diseases, caused by the pathogenic bacteria, are highly prevalent in the world. Salmonella is one of the most important bacterial genera responsible for this. Salmonella Enteritidis (SE) is one of the non-typhoid Salmonellae that can be transmitted to human from poultry products, water, and contaminated food. In recent years, new and rapid detection methods such as enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) have been developed. In this study, recombinant FliC (rFliC) was produced to be used as an antigen. The immunization was conducted in mice with the purified recombinant FliC (rFliC). The mice were subcutaneously immunized with rFliC and elicited significant rFliC specific serum IgG antibodies. An indirect ELISA system was established for the detection of Salmonella Enteritidis. Our results confirmed that the recombinant flagellin can be one of the excellent indicators for the detection of Salmonella Enteritidis.

Impact of TLR5 and NLRC4 activation on proliferation of different breast cancer cell lines / 中国免疫学杂志

Objective:To explore the impact of TLR5 and NLRC4 activation on the proliferation of different breast cancer cell lines,MCF-7 and MDA-MB-23 i.Methods:Induction,expression,purification and identification of recombiant flagellin,including FliC (activating both TLR5 and NLRC4),FliC△90-97 (unable to activate TLR5),FliC-L3A (unable to activate NLRC4),FliC△90-97:L3A (unable to activate both TLR5 and NLRC4).Using different concentration of recombinant flagellin to stimulate MCF-7 and MDA-MB-231 cell lines,72 h later,the proliferation of tumor cells were detected with CCK8.We also used soft AGAR forming experiments to detect the inhibition ratio of recombinant flagellin on breast cancer cell lines.Briefly,1 000 cells were plated in the 6-well plate,then stimulated with 1 μg/ml recombinant flagellin,14 days later,the number of cloning were counted after crystal violet staining.Results:After stimulation with four recombinant flagellins at the concentration of 0.1 μ,g/ml,the inhibition ratio on MCF-7 reached 30％,and FliC△90-97 were dose-dependent on the inhibition of MCF-7 proliferation.At the concentration of 1 μg/ml,FliC-L3A which only activated TLR5 showed stronger inhibition ratio than FliC.FliC△90-97:L3A which did not activate both TLR5 and NLRC4 also inhibited the proliferation of MCF-7.After adding transfection reagent,four recombinant flagellins showed inhibition effect on MDA-MB-231.Conclusion:Flagellin can inhibit the proliferation of MCF-7 and MDA-MB-231,and the mechanism of inhibition on the proliferation were not TLR5 and NLRC4 pathway dependent.There might exist new mechanisms to explain this phenomenon.

Expression and assembly of chimeric flagellins in Escherichia coli BL21(DE3) and Salmonella / 生物工程学报

Flagellin can be expressed in monomeric or polymeric form based on assembly. The difference of these two forms of flagellin is less studied. In this experiment, recombinant plasmid pET-fliC/M2e2 was transferred into Escherichia coli BL21(DE3) and Salmonella SL5928 to express chimeric flagellin, mfliC/M and pfliC/M, respectively, and then their assembly characteristics were analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results indicated that the two recombinant bacteria could successfully express chimeric flagellin. The transmission electronic microscope observation showed that no flagella were found on the surface of recombinant E. coli, whereas it was found for recombinant Salmonella. After purification, distinct circular dichroism spectra between them were found and pfliC/M showed the similar structure as wild-type flagellin, but not for mfliC/M. The dynamic light scattering assay also indicated that the polymerization of mfliC/M was much lower than that for pfliC/M. Three hours after transfection into mouse peritoneal macrophages, both could induce interleukin 1β secretion, but mfliC/M is stronger than pfliC/M. These data will be helpful for the selection of expression form of flagellin.

Expression, purification and detection of activities of AprA and its inhibitor AprI from Pseudomonas aeruginosa / 军事医学

Objective To clone and express the alkaline protease AprA , one important virulence factor secreted by Pseudomonas aeruginosa(PAE)in Escherichia coli, to clone and express the inhibitor of AprA (AprI) and its substrate flagellin , and to detect the function of AprA and the inhibitory function of AprI .Methods The genes encoding AprA ,AprI and flagellin gene were amplified respectively by PCR using PAE PAO 1 genome DNA as the template .The expression vec-tors (pET-28a-AprA, pET-28a-AprI and pET-28a-Flagellin) were constructed and transformed into E.coli BL21(DE3) respectively.The recombinant AprA protein was expressed by IPTG induction and purified via denaturing and renaturation. The recombinant AprI and flagellin were expressed and purified by Ni 2+affinity chromatography .The cleavage activities of AprA on flagellin were detected by SDS-PAGE.Results Recombinant AprA , AprI and flagellin protein were expressed and purified .It was demonstrated that AprA cleaved flagellin , which was blocked by AprI .Conclusion Recombinant AprA could cleave its substrates as an alkaline protease , and its inhibitor AprI inhibits the activities of AprA .This study will contribute to further investigations on the role of AprA in the pathogenesis of PAE .

Flagellin Modulates the Function of Invariant NKT Cells From Patients With Asthma via Dendritic Cells

PURPOSE: Invariant natural killer T (iNKT) cells play a critical role in the pathogenesis of asthma. We previously reported the association between circulating Th2-like iNKT cells and lung function in asthma patients and the suppressive effect of Toll-like receptor 5 ligand flagellin B (FlaB) on asthmatic in a mouse model. Thus, we investigated whether FlaB modulates the function of circulating iNKT cells in asthmatic patients. METHODS: Peripheral blood mononuclear cells (PBMCs) were treated with FlaB, and the secreted and intracellular cytokines of iNKT cells were evaluated by using ELISA and flow cytometry, respectively, following stimulation with alpha-galactosylceramide. Foxp3+ iNKT cells were also measured. To determine the effect of FlaB-treated dendritic cells (DCs) on iNKT cells, we co-cultured CD14+ monocyte-derived DCs and T cells from patients with house dust mite-sensitive asthma and analyzed intracellular cytokines in iNKT cells. RESULTS: A reduction of IL-4 and IL-17 production by iNKT cells in PBMCs after FlaB treatment was alleviated following blocking of IL-10 signaling. A decrease in the frequencies of IL-4+ and IL-17+ iNKT cells by FlaB-treated DCs was reversed after blocking of IL-10 signaling. Simultaneously, an increase in Foxp3+ iNKT cells induced by FlaB treatment disappeared after blocking of IL-10. CONCLUSIONS: FlaB may inhibit Th2- and Th17-like iNKT cells and induce Foxp3+ iNKT cells by DCs via an IL-10-dependent mechanism in asthmatic patients. In patients with a specific asthma phenotype associated with iNKT cells, FlaB may be an effective immunomodulator for iNKT cell-targeted immunotherapy.

Impact of TLR5 and NLRC4 activation on innate immune cells in mice / 中国免疫学杂志

Objective:To investigate the impact of recombinant flagellin targeting TLR5 and NLRC4 simultaneously or respectively on innate immune cells in mice. Methods: Induction,expression,purification and identification of recombiant FliC,which were FliC(activating both TLR5 and NLRC4);FliCΔ90-97(unable to activate TLR5),FliC-L3A(unable to activate NLRC4),FliCΔ90-97:L3A(unable to activate both TLR5 and NLRC4). The mice were divided into five groups,namely group FliC,FliC-L3A,FliCΔ90-97,FliCΔ90-97:L3A and PBS,which were injected with 100μl PBS or 10μg recombinant flagellin intraperitoneally,three mice in each group. 12 h later,the mice were executed using dislocation of cervical vertebra and the splenic and peritoneal cells were isolated. The spleen was grinded into single-cell suspension. The proportion of neutrophils,NK cells,DCs and the expression level of CD80 and CD86 on DCs were evaluated with flow cytometry. Results:Group FliC,group FliC-L3A and group FliCΔ90-97 shared the similar proportion of neutrophils in peritoneal cavity ( P>0. 05 ) , and all of which were significantly higher than group PBS and group FliCΔ90-97 ( P<0. 01),and NK cells also showed the similar trend. Compared with group FliCΔ90-97 and FliCΔ90-97:L3A,the mean fluorescence intensities(MFIs) of CD80 and CD86 in group FliC and FliC-L3A increased significantly(P<0. 01). The proportion of Treg in spleen was highest among all groups. Conclusion:Activation of TLR5 and NLRC4 had similar chemotaxis of neutrophils and NK cells. The ex-pression of CD80 and CD86 on DCs were upregulated after stimulation by flagellin and TLR5-dependent. Activation of TLR5,but not NLRC4,increased the proportion of Treg in spleen.

Comparative study of the effects of rhKGF, CBLB502 and WR2721 on radiation-induced oral mucositis / 解放军医学杂志

Objective To compare the protective effect of rhKGF, CBLB502 and WR2721 on radiation-induced oral mucositis (ROM). Methods Fifty male 6-8-week-old C57BL/6J mice were randomly divided into normal group, irradiation control group, rhKGF group, CBLB502 group, and WR2721 group (n=10 each). The 30-day survival rate and change in body weight of mice that had received 17Gy irradiation of head and neck area were recorded. In another group of 20 mice, 1% toluidine blue staining and HE staining were used to observe oral ulcers and pathological changes in the tongue tissue. The proliferation of keratinocyte cells was assessed by Ki-67 immunohistochemistry. Results Compared with the irradiation control group, administration of rhKGF and WR2721 could significantly improve the 30-day survival rate, accelerate the recovery of body weight, and promote the proliferation of keratinized epithelial cells of mice after irradiation, without inducing obvious oral mucositis. However, There was no significant difference between CBLB502 group and irradiation control group in survival rate, body weight and pathological changes in tongue tissues of mice. Conclusion rhKGF and WR2721 could alleviate ROM and improve the survival of mice, while CBLB502 has no such effect.

Radioprotective effects of CBLB502 on γ-radiated rhesus monkey / 解放军医学杂志

Objective A Rhesus monkey model was employed to study the radioprotective effects of a Toll-like receptor 5 agonist, CBLB502, against 7.0Gy whole-body irradiation of 60Co gamma-rays. Methods Thirty animals were assigned to a placebo treatment group, a WR-2721 positive control group, and three CBLB502 treatment groups (n=6 animals/group). Each animal was irradiated with 7.0Gy 60Co γ and given CBLB502 at 2.5, 10 and 40μg/kg, respectively in treatment groups, or WR-2721 at 30mg/kg, or physiological saline 0.3ml/kg for the placebo treatment group. The treatment was given once by intramuscular injection 30 min before irradiation. All irradiated animals received symptomatic treatment based on same guidelines. General observation, peripheral blood tests, hemopoietic progenitor cell colony-counting, and histopathological examination were performed. Results We found that 10 or 40μg/kg CBLB502 treatment resulted in 100% survival, while the survival rate was 33% in placebo treatment group. Hematopoietic recovery in the WR-2721 treatment group was marginally superior to the irradiation control group. Nadirs of peripheral white cell and platelet counts of animals treated with 40μg/kg of CBLB502 were significantly higher than those of the placebo treatment group (P60Co γ-rays would suffer from severe acute radiation sickness of hematopoietic system. CBLB502 at 40μg/kg is radioprotective in this model and a single intramuscular injection of CBLB502 in a dose of 40μg/kg 30min before irradiation gives better radioprotective effects than WR-2721.

Tetanus toxin fragment C fused to flagellin makes a potent mucosal vaccine

PURPOSE: Recombinant subunit vaccines provide safe and targeted protection against microbial infections. However, the protective efficacy of recombinant subunit vaccines tends to be less potent than the whole cell vaccines, especially when they are administered through mucosal routes. We have reported that a bacterial flagellin has strong mucosal adjuvant activity to induce protective immune responses. In this study, we tested whether FlaB could be used as a fusion partner of subunit vaccine for tetanus. MATERIALS AND METHODS: We constructed fusion proteins consisted with tetanus toxin fragment C (TTFC), the nontoxic C-terminal portion of tetanus toxin, and a Toll-like receptor 5 agonist from Vibrio vulnificus (FlaB). Mice were intranasally administered with fusion protein and protective immune responses of the vaccinated mice were analyzed. RESULTS: FlaB-TTFC recombinant protein induced strong tetanus-specific antibody responses in both systemic and mucosal compartments and prolonged the survival of mice after challenge with a supra-lethal dose of tetanus toxin. CONCLUSION: This study establishes FlaB as a successful fusion partner for recombinant subunit tetanus vaccine applicable through mucosal route, and it further endorses our previous observations that FlaB could be a stable adjuvant partner for mucosal vaccines.

Salmonella Enteritidis Flagella may Enhance Attachment and Invasion of Hen Ovarian Granulosa Cells and Induce Protective Immune Response in Egg-laying Hens.

Aims: To study the effect of flagellin on bacterial attachment and invasion of avian ovary cells in vitro by comparing the attachment and invasion of wild-type S. Enteritidis with nonmotile mutants. To assess the immunogenic properties of extracted flagellin against Salmonella Enteritidis experimental infection in laying hens. Methodology: Non-flagellated mutants for wild-type S. Enteritidis (phage type 8, 13A and 28) were produced by using a strain of S. Enteritidis, SA4502, which carried an fliC::Tn 10 to transfer fliC::Tn 10 insertion into the wild type strains using phage 22 (P22)-mediated transduction with selection for antibiotic resistance encoded within the mutant alleles. Granulosa cells were harvested from Single Comb White Leghorn hens between 18-45 weeks of age. Flagellin was purified from the studied bacterial cultures of Salmonella Enteritidis following reported methods. Laying hens were immunized with the flagellin with adjuvant Results: Non-motile mutants of S. Enteritidis phage wild types were analyzed to confirm the elimination of H1 flagellin synthesis. Wild-type and fliC mutant strains were assessed for their ability to adhere to hen's ovarian granulosa cells. The adherence of the mutant strain was reduced nearly ten-fold compared with that of the wild-type phage 8. Similarly, light microscopic observation of fixed cover slips from wild-type phage types and its mutant strain revealed fewer numbers of the bacterial mutants adhered to the cultured granulosa cell monolayer. Light microscopy revealed similar findings for mutant phage types 28 and 13 A when compared to the wild-type control. There was five folds rise in the egg yolk antibody during the 2-3 weeks post-immunization. No rise was detected in the egg yolk samples from the control hens injected with the placebo mixture without flagellin. Conclusion: It was concluded that Flagellin has an important role in the attachment and invasion of Salmonella Enteritidis to avian ovary cells and that it can be used as immunogenic components to induce a protective immune response in vaccinated hens against challenge infection with the wild type strains.

Seleção, caracterização e clonagem dos genes fljB e groEL agonistas dos receptores de reconhecimento de padrão do sistema imune inato das aves / Selection, characterization and cloning of the fljB and groEL genes agonists of the innate immune system pattern recognition receptors of birds

The recombinant production of innate immune system pattern recognition receptor agonists has provided a new tool for the production of immunostimulants for animals. The molecular pattern associated with the pathogen (PAMP), flagellin, coded by the fljB gene from Salmonella Typhimirium, and the molecular pattern associated to the damage (DAMP), HSP60, coded by the groEL gene from S. Typhimurium and S. Enteritidis, are recognized by pattern recognition receptors (PRRs) of the innate immune system of birds. In the present study, we performed the cloning of genetic fragments of the genes fljB, from S. Typhimurium, and groEL from S. Typhimurium and S. Enteritidis inserted in expression vector pET100/D-TOPO and transformed in E. coli TO10 cells. The clones were evaluated by colony PCR, plasmidial DNA PCR and genome sequencing in order to confirm the presence of these genes. In the colony PCR, we identified the presence of genes groEL (S. Enteritidis), groEL (S. Typhimurium) and fljB (S. Typhimurium) in 80%, 60% and 80% of the transformed colonies, respectively. The cloning system adopted allowed the production of HSP60 genetic fragment clones and flagellin of Salmonella strains, allowing the posterior use of these clones in gene expression trials, with the future potential of being used as non-specific immunostimulants for birds.

A produção recombinante de agonistas dos receptores do reconhecimento de padrão do sistema imune inato tem fornecido uma nova ferramenta para a produção de imunoestimulantes para animais. O padrão molecular associado ao patógeno (PAMP), flagelina, codificado pelo gene fljB de Salmonella Typhimurium e o padrão molecular associado ao dano (DAMP) HSP60, codificado pelo gene groEL da S. Typhimurium e S. Enteritidis, são reconhecidos por receptores de reconhecimento de padrões (RRPs) do sistema imune inato das aves. No presente estudo, foi feita a clonagem de fragmentos genéticos dos genes fljB de S. Typhimurium e groEL de S. Typhimurium e S. Enteritidis inseridos no vetor de expressão pET100/D-TOPO e transformados em células de E. coli TOP10. Os clones foram avaliados pela PCR de colônia, PCR de DNA plasmidial e sequenciamento genômico para a confirmação da presença desses genes. Na PCR de colônia, foram identificadas em 80%, 60% e 80% das colônias transformadas, a presença dos genes groEL (S. Enteritidis), groEL (S. Typhimurium) e fljB (S. Typhimurium) respectivamente. O sistema de clonagem adotado possibilitou a produção de clones dos fragmentos genéticos da HSP60 e flagelina das cepas de Salmonella, permitindo a utilização posterior desses clones em ensaios de expressão gênica, com potencial futuro de serem utilizados como imunoestimulante inespecífico das aves.