Search

Objective: Antimicrobial resistance is an emerging problem and multi-drug resistant (MDR) Klebsiella pneumoniae ( K. pneumoniae ) represents an enormous risk of failing therapy in hospital-acquired pneumonia. The current study aimed to determine the immunomodulatory effect of topical flagellin in addition to antibiotic treatment during respiratory infection evoked by hypervirulent antibiotic-susceptible and antibiotic-resistant K. pneumoniae in mice., Methods: C57BL6 mice were inoculated intranasally with hypervirulent K. pneumoniae (K2:O1) which was either antibiotic-susceptible or multi-drug resistant. Six hours after infection, mice were treated with antibiotics intraperitoneally and flagellin or vehicle intranasally. Mice were sacrificed 24 hours after infection. Samples were analyzed for bacterial loads and for inflammatory and coagulation markers., Results: Flagellin therapy induced neutrophil influx in the lung during antibiotic-treated pneumonia evoked by either antibiotic-susceptible or -resistant K. pneumoniae . The pulmonary neutrophil response was matched by elevated levels of neutrophil-attracting chemokines, neutrophil degranulation products, and local coagulation activation. The combined therapy of effective antibiotics and flagellin did not impact K. pneumoniae outgrowth in the lung, but decreased bacterial counts in distant organs. Neutrophil depletion abrogated the flagellin-mediated effect on bacterial dissemination and local coagulation responses., Conclusion: Topical flagellin administration as an adjunctive to antibiotic treatment augments neutrophil responses during pneumonia evoked by MDR- K. pneumoniae , thereby reducing bacterial dissemination to distant organs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 van Linge, Kullberg, Chouchane, Roelofs, Goessens, van ‘t Veer, Sirard, de Vos and van der Poll.)

Research on innovative mucosal adjuvants is essential to develop new vaccines for safe mucosal application. In this work, we propose the development of a Lactococcus lactis that expresses a variant of flagellin on its surface (FliC131*), to increase the adjuvanticity of the living cell and cell wall-derived particles (CWDP). We optimized the expression of FliC131*, and confirmed its identity and localization by Western blot and flow cytometry. We also generated CWDP containing FliC131* (CDWP-FliC131*) and evaluated their storage stability. Lastly, we measured the human TLR5 stimulating activity in vitro and assessed the adjuvanticity in vivo using ovalbumin (OVA) as a model antigen. As a result, we generated L. lactis/pCWA-FliC131*, that expresses and displays FliC131* on its surface, obtained the corresponding CWDP-FliC131*, and showed that both activated hTLR5 in vitro in a dose-dependent manner. Furthermore, CWDP-FliC131* retained this biological activity after being lyophilized and stored for a year. Finally, intranasal immunization of mice with OVA plus live L. lactis/pCWA-FliC131* or CWDP-FliC131* induced OVA-specific IgG and IgA in serum, intestinal lavages, and bronchoalveolar lavages. Our work demonstrates the potential of this recombinant L. lactis with an enhanced adjuvant effect, prompting its further evaluation for the design of novel mucosal vaccines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Developing a mucosal vaccine against SARS-CoV-2 is critical for combatting the epidemic. Here, we investigated long-term immune responses and protection against SARS-CoV-2 for the intranasal vaccination of a triple receptor-binding domain (RBD) scaffold protein (3R-NC) adjuvanted with a flagellin protein (KFD) (3R-NC + KFDi.n). In mice, the vaccination elicited RBD-specific broad-neutralizing antibody responses in both serum and mucosal sites sustained at high level over a year. This long-lasting humoral immunity was correlated with the presence of long-lived RBD-specific IgG- and IgA-producing plasma cells, alongside the Th17 and Tfh17-biased T-cell responses driven by the KFD adjuvant. Based upon these preclinical findings, an open labeled clinical trial was conducted in individuals who had been primed with the inactivated SARS-CoV-2 (IAV) vaccine. With a favorable safety profile, the 3R-NC + KFDi.n boost elicited enduring broad-neutralizing IgG in plasma and IgA in salivary secretions. To meet the challenge of frequently emerged variants, we further designed an updated triple-RBD scaffold protein with mutated RBD combinations, which can induce adaptable antibody responses to neutralize the newly emerging variants, including JN.1. Our findings highlight the potential of the KFD-adjuvanted triple-RBD scaffold protein is a promising prototype for the development of a mucosal vaccine against SARS-CoV-2 infection., (© 2024. The Author(s).)

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn's disease. CBir1 TCR transgenic naϊve CD4 + T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1 -/- mice. The disease severity is assessed by histopathology. The CD4 + T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

We generated self-adjuvanted protein nanoparticles of conserved influenza antigens and immunized mice via skin vaccination with dissolvable microneedle patches (MNPs) to increase the strength and breadth of immune responses. We produced M2e nanoparticles via ethanol desolvation, and double-layered NA1/M2e (shell/core), NA1-FliC/M2e, NA2/M2e, and NA2-FliC/M2e protein nanoparticles by chemically crosslinking influenza NA and flagellin (FliC) onto the surfaces of the M2e nanoparticles. The resulting nanoparticles retained FliC TLR5 innate signaling activity and significantly increased antigen-uptake and dendritic cell maturation in vitro . We incorporated the nanoparticles into MNPs for skin vaccination in mice. The nanoparticle MNPs significantly increased M2e and NA-specific antibody levels, the numbers of germinal center B cells, and IL-4 positive splenocytes. Double-layered nanoparticle MNP skin vaccination protected mice against homologous and heterosubtypic influenza viruses. Our results demonstrated that MNP skin vaccination of NA-FliC/M2e nanoparticles could be developed into a standalone or synergistic component of a universal influenza vaccine strategy., Competing Interests: Declarations of interest: none

Acetaminophen (APAP) poisoning is the most common cause of drug-induced acute liver injury (ALI). Our results showed that toll-like receptor 5 (TLR5) was abundantly expressed in hepatocytes and dramatically downregulated in the toxic mouse livers. Hence, we herein investigated the role of TLR5 signaling after APAP overdose. Mice were intraperitoneally (i.p.) injected with APAP to induce ALI, and then injected with flagellin at one hour after APAP administration. Flagellin attenuated APAP-induced ALI based on decreased histopathologic lesions, serum biochemical, oxidative stress, and inflammation. Furthermore, the protective effects of flagellin were abolished by TH1020 (a TLR5 antagonist) treatment. These results suggest that flagellin exerted protective effects on ALI via TLR5 activation. Mechanistically, flagellin injection promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus in hepatocytes. Consistent with the in vivo results, flagellin increased the activation of Nrf2 in hepatocytes, resulting in decreased APAP toxicity. ML385, a selective inhibitor of Nrf2, abolished the flagellin-mediated hepatoprotective effects in damaged livers and hepatocytes. Additionally, the flagellin-induced Nrf2 translocation was dependent upon the activation of TLR5-JNK/p38 pathways. These findings suggest that TLR5 signaling-induced Nrf2 activation, at least partially, contributed to the protection against APAP-induced ALI by flagellin treatment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Vibrio anguillarum is a globally distributed aquatic pathogen, and its flagellin B (FlaB) protein can evoke innate immune responses in hosts. In order to explore the role of FlaB in V. anguillarum infection, we constructed a FlaB-deficient mutant using overlapping PCR and two-step homologous recombination, and gene sequencing confirmed successful knockout of the FlaB gene. Scanning electron microscopy showed no significant differences in the morphological structure of the flagellum between wild-type and FlaB-deficient strains. The mutant was subsequently injected into the freshwater prawn (Macrobrachium rosenbergii) to explore its pathogenicity in the host, and expression of myeloid differentiation factor 88, prophenoloxidase, catalase, superoxide dismutase and glutathione peroxidase was investigated by real-time PCR. The results showed that deletion of FlaB had little effect on V. anguillarum-induced expression of these immune-related genes (p > 0.05). In general, the FlaB mutant displayed similar flagella morphology and immune characteristics to the wild-type strain, hence we speculated that knockout of FlaB might promote the expression and function of other flagellin proteins. Furthermore, this study provides a rapid and simple method for obtaining stable mutants of V. anguillarum free from foreign plasmid DNA., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The combination of photothermal therapy (PTT) and toll-like receptor (TLR)-mediated immunotherapy can elicit antitumor immunity and modulate the immunosuppressive tumor microenvironment (TME). Unlike other TLRs, TLR-5 is a promising target for immune activation, as its expression is well-maintained even during immunosenescence. Here, we developed a unique tumor microenvironment-regulating immunosenescence-independent nanostimulant consisting of TLR-5 adjuvant Vibrio vulnificus flagellin B (FlaB) conjugated onto the surface to an IR 780-loaded hyaluronic acid-stearylamine (HIF) micelles. These HIF micelles induced immune-mediated cell death via PTT when irradiated with a near-infrared laser. In comparison with PTT alone, the combination of in situ -generated tumor-associated antigens produced during PTT and the immune adjuvant FlaB demonstrated enhanced vaccine-like properties and modulated the TME by suppressing immune-suppressive regulatory cells (Tregs) and increasing the fraction of CD103 + migratory dendritic cells, which are responsible for trafficking tumor antigens to draining lymph nodes (DLNs). This combinatorial strategy ( i.e. , applying a TLR-5 adjuvant targeted to immunosenescence-independent TLR-5 and the in situ photothermal generation of tumor-associated antigens) is a robust system for next-generation immunotherapy and could even be applied in elderly patients, thus broadening the clinical scope of immunotherapy strategies.

Bacteria-released components can modulate host innate immune response in the absence of direct host cell-bacteria interaction. In particular, bacteria-derived outer membrane vesicles (OMVs) were recently shown to activate host caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide. However, further precise understanding of innate immune-modulation by bacterial OMVs remains elusive. Here, we present evidence that flagellated bacteria-released OMVs can trigger NLRC4 canonical inflammasome activation via flagellin delivery to the cytoplasm of host cells. Salmonella typhimurium -derived OMVs caused a robust NLRC4-mediated caspase-1 activation and interleukin-1β secretion in macrophages in an endocytosis-dependent, but guanylate-binding protein-independent manner. Notably, OMV-associated flagellin is crucial for Salmonella OMV-induced inflammasome response. Flagellated Pseudomonas aeruginosa -released OMVs consistently promoted robust NLRC4 inflammasome activation, while non-flagellated Escherichia coli -released OMVs induced NLRC4-independent non-canonical inflammasome activation leading to NLRP3-mediated interleukin-1β secretion. Flagellin-deficient Salmonella OMVs caused a weak interleukin-1β production in a NLRP3-dependent manner. These findings indicate that Salmonella OMV triggers NLRC4 inflammasome activation via OMV-associated flagellin in addition to a mild induction of non-canonical inflammasome signaling via OMV-bound lipopolysaccharide. Intriguingly, flagellated Salmonella -derived OMVs induced more rapid inflammasome response than flagellin-deficient Salmonella OMV and non-flagellated Escherichia coli -derived OMVs. Supporting these in vitro results, Nlrc4 -deficient mice showed significantly reduced interleukin-1β production after intraperitoneal challenge with Salmonella -released OMVs. Taken together, our results here propose that NLRC4 inflammasome machinery is a rapid sensor of bacterial OMV-bound flagellin as a host defense mechanism against bacterial pathogen infection., (Copyright © 2020 Yang, Hwang, Lee, Shin, Lee, Rhee and Yu.)

The TLR5 agonist flagellin is a potent adjuvant and is currently being developed for use in vaccines. The mechanisms that drive flagellin's activity are influenced by its administration route. Previous studies showed that lung structural cells (especially epithelial cells lining the conducting airways) are pivotal for the efficacy of intranasally administered flagellin-containing vaccines. In this study, we looked at how the airway epithelial cells (AECs) regulate the flagellin-dependent stimulation of Ag-specific CD4 + T cells and the Ab response in mice. Our results demonstrate that after sensing flagellin, AECs trigger the release of GM-CSF in a TLR5-dependent fashion and the doubling of the number of activated type 2 conventional dendritic cells (cDC2s) in draining lymph nodes. Furthermore, the neutralization of GM-CSF reduced cDC2s activation. This resulted in lower of Ag-specific CD4 + T cell count and Ab titers in mice. Our data indicate that during pulmonary immunization, the GM-CSF released by AECs orchestrates the cross-talk between cDC2s and CD4 + T cells and thus drives flagellin's adjuvant effect., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Acute lung injury (ALI) is a complex condition frequently encountered in the clinical setting. The aim of the present study was to investigate the effect of conditioned media (CM) from human adipose‑derived mesenchymal stromal cells (MSCs) activated by flagellin (F‑CM), a Toll‑like receptor 5 ligand, on inflammation‑induced lung injury. In the in vitro study, RAW264.7 macrophages treated with F‑CM had a higher proportion of cells with the M2 phenotype, lower expression of pro‑inflammatory factors and stronger expression of anti‑inflammatory genes compared with the CM from normal adipose‑derived MSCs. Furthermore, in vivo experiments were performed in mice with ALI induced by intraperitoneal injection of lipopolysaccharide. F‑CM significantly alleviated the lung exudation, inhibited inflammatory cell recruitment in lung tissues and decreased the concentration of inflammatory factors in the bronchoalveolar lavage fluid. These findings indicated that F‑CM has superior anti‑inflammation ability compared with CM, and that it may represent a promising therapeutic approach to the treatment of inflammation‑induced ALI.

Since late 2010, outbreaks of porcine epidemic diarrhea (PED) have been reported in the swine industry in China. A variant PEDV strain that differs from strain CV777 causes prevalent PEDV infections which commercial vaccines based on CV777 cannot provide complete protection. In this study, we designed a new vaccine based on the epidemic PEDV strain AH2012/12, adjuvanted with flagellin, a mucosal adjuvant that induces mucosal and systemic production of IgA. Three groups of pregnant sows were immunized twice, with a 14-day interval, with PEDV adjuvanted with flagellin, PEDV alone, or PBS before farrowing, and newborn piglets from each group were selected and challenged with PEDV. Immunization with this vaccine elicited high levels of IgG, IgA, and neutralizing antibodies in the serum and colostrum of sows, and newborn piglets were protected against PEDV while suckling. This study should guide the prevention and control strategies for PEDV infection, thereby reducing the losses associated with this virus.

Toll-like receptor (TLR) ligands have emerged as the attractive adjuvant for subunit vaccines. However, selection of TLR ligands needs to be rationally chosen on the basis of antigen and adjuvant properties. In the present study, we expressed the Ag473 lipoprotein from Neisseria meningitides, flagellin FlaB from Vibrio vulnificus and heat shock protein 70 from Mycobacterium tuberculosis (mHsp70) in Escherichia coli as single proteins and fusion proteins with VP2 protein of infectious bursal disease virus (IBDV). Both cellular and humoral adjuvanticities of the three TLR ligands were compared by immunization of mice in two different ways. Among the three co-administered TLR ligands, recombinant Ag473 lipoprotein exhibited the highest cellular and humoral adjuvanticities, including promotion of IL-4, IL-12, IFN-γ and IBDV VP2-specific antibody production. Among the three genetically fused TLR ligands, fusion with Ag473 D1 domain exhibited the highest cellular and humoral adjuvanticities. Overall, the adjuvanticities of genetically fused TRL ligands were significantly higher than that of co-administered TLR ligands. Fusion with Ag473 D1 domain exhibited superior adjuvanticity among the three TLR ligands delivered in two different ways., (© 2020 The Author(s).)

Bacterial flagellin, recognized by cell surface of Toll-like receptor (TLR) 5, is a potent activator of many types of cells, leading to the activation of innate or adaptive immunity, which are pivotal in regulating fibrotic process. However, the exact role of TLR5 signaling in hepatic fibrogenesis remains unclear, and this study aims to elucidate its underlying mechanisms. Flagellin was injected to hepatotoxin- and cholestasis-induced liver fibrosis murine models. Flagellin-induced TLR5 activation significantly decreased the severity of liver fibrosis. Interestingly, the expression levels of IL-1 receptor antagonist (IL1RN) and interferon (IFN)β markedly increased in fibrotic livers on flagellin treatment. Consistently, in vivo activation of TLR5 signaling markedly increased IFNβ and IL1RN expression in the livers. Notably, flagellin injection significantly exacerbated the severity of liver fibrosis in IFN-α/β receptor 1 (IFNAR1) knockout mice. Furthermore, hepatic expression of IL1RN in the fibrotic livers of IFNAR1 knockout mice was significantly lower than those of wild-type mice. In support of these findings, flagellin-mediated IL1RN production is not sufficient to alleviate the severity of hepatic fibroinflammatory responses in IFNAR1-deficient milieu. Finally, hepatic stellate cells treated with IL1RN had significantly decreased cellular activation and its associated fibrogenic responses. Collectively, manipulation of TLR5 signaling may be a promising therapeutic strategy for the treatment of liver fibrosis., (Copyright © 2020 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Intranasal mucosal vaccines are an attractive approach to induce protective mucosal immune responses. Activation of lung antigen presenting cells (APCs), a phenotypically and functionally heterogeneous cell population located at distinct mucosal sites, may be key to the immunogenicity of such vaccines. Understanding responsiveness of newborn lung APCs to adjuvants may the inform design of efficacious intranasal vaccines for early life, when most infections occur. Here, we characterized and phenotyped APCs from neonatal (7 days of life) and adult (6-8 weeks of age) mice. Neonatal mice demonstrated a relatively high abundance of alveolar macrophages (AMs), with lower percentages of plasmacytoid dendritic cells (pDCs), CD103 + (cDC1), and CD11b + (cDC2) DCs. Furthermore, neonatal CD103 + and CD11b + DC subsets demonstrated a significantly lower expression of maturation markers (CD40, CD80, and CD86) as compared to adult mice. Upon stimulation of lung APC subsets with a panel of pattern recognition receptor (PRR) agonists, including those engaging TLRs or STING, CD11c + enriched cells from neonatal and adult mice lungs demonstrated distinct maturation profiles. Of the agonists tested, the TLR5 ligand, flagellin, was most effective at activating neonatal lung APCs, inducing significantly higher expression of maturation markers on CD103 + (cDC1) and CD11b + (cDC2) subsets. Intranasal administration of flagellin induced a distinct migration of CD103 + and CD11b + DC subsets to the mediastinal lymph nodes (mLNs) of neonatal mice. Overall, these findings highlight age-specific differences in the maturation and responsiveness of lung APC subsets to different PRR agonists. The unique efficacy of flagellin in enhancing lung APC activity suggests that it may serve as an effective adjuvant for early life mucosal vaccines., (Copyright © 2020 Sharma, Levy and Dowling.)

The immunization of poultry where H5 and H7 influenza viruses (IVs) are endemic is one of the strategies to prevent unexpected zoonoses. Our group has been focused on conserved HA-epitopes as potential vaccine candidates to obtain multivalent immune responses against distinct IV subtypes. In this study, two conserved epitopes (NG-34 and CS-17) fused to flagellin were produced in a Baculovirus platform based on Trichoplusia ni larvae as living biofactories. Soluble extracts obtained from larvae expressing "flagellin-NG34/CS17 antigen" were used to immunize chickens and the efficacy of the vaccine was evaluated against a heterologous H7N1 HPAIV challenge in chickens. The flagellin-NG34/CS17 vaccine protected the vaccinated chickens and blocked viral shedding orally and cloacally. Furthermore, no apparent clinical signs were monitored in 10/12 vaccinated individuals. The mechanism of protection conferred is under investigation., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Alterations in gut microbiota composition are associated with metabolic syndrome and chronic inflammatory diseases such as inflammatory bowel disease. One feature of inflammation-associated gut microbiotas is enrichment of motile bacteria, which can facilitate microbiota encroachment into the mucosa and activate pro-inflammatory gene expression. Here, we set out to investigate whether elicitation of mucosal anti-flagellin antibodies by direct administration of purified flagellin might serve as a general vaccine against subsequent development of chronic gut inflammation. We show, in mice, that repeated injection of flagellin elicits increases in fecal anti-flagellin IgA and alterations in microbiota composition, reduces fecal flagellin concentration, prevents microbiota encroachment, protects against IL-10 deficiency-induced colitis, and ameliorates diet-induced obesity. Flagellin's impact on the microbiota is B-lymphocyte dependent and, in humans, obese subjects exhibit increased levels of fecal flagellin and reduced levels of fecal flagellin-specific IgA, relative to normal weight subjects. Thus, administration of flagellin, and perhaps other pathobiont antigens, may confer some protection against chronic inflammatory diseases.

Influenza infections remain a burden on health care systems despite vaccination programs and marketed antiviral drugs. Immunomodulation through activation of innate sensors could represent innovative approaches to fight the flu. This study evaluated the ability of flagellin, agonist of Toll-like receptor 5 (TLR5), to control the replication of influenza A virus (IAV) in mice. First, we showed that systemic or intranasal administration of flagellin activated transcription of anti-viral genes in lung tissue. Prophylactic and therapeutic flagellin administration resulted in decreased levels of viral RNA and infectious virus in the lungs of H3N2 IAV-infected mice. The effect of the flagellin on viral replication was also observed in Ifnar -/- and Il22 -/- IAV-infected mice, suggesting a mechanism independent of type I interferon and interleukin 22 signaling. In addition, a combination therapy associating the neuraminidase inhibitor oseltamivir and flagellin was more effective than standalone treatments in reducing pulmonary viral replication. Thus, this study highlights the therapeutic potential of the flagellin to control the replication of the influenza virus., (Copyright © 2019. Published by Elsevier B.V.)

We hypothesized that NADPH oxidase 4 (Nox4) is involved in the formation of neointimal atherosclerotic plaques through the migration of smooth muscle cells (SMCs) in response to flagellin. Here, we demonstrate that TLR5-mediated Nox4 activation regulates the migration of SMCs, leading to neointimal plaque formation in atherosclerosis. To investigate the molecular mechanism by which the TLR5-Nox4 cascade mediates SMC migration, we analyzed the signaling cascade in primary vascular SMCs (VSMCs) from wild-type (WT) or Nox4 KO mice. Stimulation of VSMCs from Nox4 KO mice with flagellin failed to induce H 2 O 2 production and Rac activation compared with stimulation of VSMCs from WT mice. Moreover, the migration of Nox4-deficient VSMCs was attenuated in response to flagellin in transwell migration and wound healing assays. Finally, we performed partial carotid artery ligation in ApoE KO and Nox4ApoE DKO mice fed a high-fat diet (HFD) with or without recombinant FliC (rFliC) injection. Injection of rFliC into ApoE KO mice fed a HFD resulted in significantly increased SMC migration into the intimal layer, whereas SMC accumulation was not detected in Nox4ApoE DKO mice. We conclude that activation of the TLR5-Nox4 cascade plays an important role in the formation of neointimal atherosclerotic plaques.

The viral E proteins of dengue virus (DENV) and Zika virus (ZIKV) are the major viral proteins involved in receptor binding and fusion, and for the induction of protective antibodies against viral infections. DIII of the E proteins is an independent domain and stretches out on the virion surface that can elicit type-specific neutralizing antibodies. For recombinant DIII vaccine development, prime-boost immunizations can provide an advantage of eliciting more type-specific neutralizing antibodies by recalling DIII antigens after DIII booster to improve protection. Methods : The DIII of the E genes of DENV and ZIKV were fused with bacterial fliC gene for the expression of flagellin-DIII (FliC-DIII) fusion proteins. Prime-boost immunization strategies by the second-dose booster of four DENV serotype or ZIKV FliC-DIII fusion proteins were used to investigate the induction of neutralizing antibodies and protection against viral infections. Cross-reactive non-neutralizing antibodies in each group of antisera were also examined using in vitro antibody-dependent enhancement (ADE) assay. A series of glycan-masking E antigens were finally constructed for prime-boost immunizations to abolish the elicitation of cross-reactive non-neutralizing antibodies for ADE activity. Results : We showed that inclusion of a bivalent live-attenuated vaccine with a FliC-DIII booster is superior in eliciting neutralization titers and protection in vivo against all four-serotype DENVs. We also demonstrated that recombinant adenovirus vectors encoding four-serotype DENV prMEs with a FliC-DIII prime-boost scheme is capable of eliciting good antibody responses. In contract, recombinant adenovirus vector of ZIKV prME gene priming, followed by ZIKV FliC-DIII booster did not improve vaccine efficacy. The glycan-masking mutation on the ZIKV E protein ij loop (E-248NHT), but not on DENV2 E protein ij loop (E-242NHT), resulted in abolishing the elicitation of cross-reactive antibodies for DENV and ZIKV infection enhancements. Conclusions : Our findings can provide useful information for designing novel immunogens and vaccination strategies in an attempt to develop a safe and efficacious DENV or ZIKV vaccine., Competing Interests: Competing Interests: H-HL, S-PY, M-JT, G-CL, S-CW are inventors on patents that cover prime-boost immunization and glycan masking E antigens for dengue and Zika vaccine development.

Aeromonas veronii is an important type of gram-negative pathogen of human-livestock-aquatic animal and causes great economic losses in the aquaculture industry. Vaccination is an effective method of defence against A. veronii. There are many factors that restrict the use of vaccination, and the development of new oral vaccines is urgently needed. The selection of suitable antigens is of great significance for the development of aquaculture vaccines. Bacterial flagellin can specifically bind to TLR5 and induce the release of cytokines from the organism, which could be used in the development of vaccines. In this study, we constructed two recombinant Lactobacillus casei (L. casei) (surface-displayed or secretory) expressing the flaB of A. veronii and evaluated the effect of immune responses in common carp. The flaB gene (900 bp) of A. veronii was subcloned into the L. casei expression plasmids pPG-1 (surface-displayed) and pPG-2 (secretory). Western blot and immunofluorescence assays confirmed the expression of the recombinant flaB protein. Common carp immunized with Lc-pPG-1-flaB and Lc-pPG-2-flaB via oral administration route exhibited induction of antibody expression and innate immune responses. The results indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB can induce high levels of IgM, ACP, AKP, LZM and SOD activity in organisms, and Lc-pPG-1-flaB can induce even higher levels. The recombinant L. casei may effectively induce humoral immunity and increase the serum immunological index. Furthermore, leukocytes phagocytosis percentage and index of the recombinant L. casei were enhanced. The results of qRT-PCR showed that recombinant L. casei can significantly increase the expression of IL-10, IL-β, IFN-γ and TNF-α in the tissues of immunized common carp, compared with control groups. Viable recombinant L. casei strains, which were delivered directly survived throughout the intestinal tract. Common carp that received Lc-pPG-1-flaB (66.7%) and Lc-pPG-2-flaB (53.3%) exhibited higher survival rates than the controls after challenge with the pathogen A. veronii. Our work indicated that Lc-pPG-1-flaB and Lc-pPG-2-flaB had beneficial effects on immune response and enhanced the disease resistance of common carp against A. veronii infection. The combination of flaB delivery and the Lactic acid bacteria (LAB) approach may be a promising method for the development of oral vaccines for treating A. veronii. In future research, we will focus on the colonization ability of LAB in the intestines and on the impact of these bacteria on intestinal flora., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Problems regarding purification efficacy in recombinant technologies is due to the protein structure. Experimental manipulation of genes and the subsequent proteins may overcome this issue. In order to improve production efficacy and maintain immunestimulatory effect of flagellin, the Toll-like Receptor 5 (TLR5) ligand and a potent adjuvant, we performed a bioinformatic study to find the best model for FliC manipulation. Truncated modified FliC (tmFliC) and full length FliC (flFliC) genes were cloned and expressed in pET-21a vector and protein purification was carried out using an improved His-Tag method. Polyclonal antibodies were generated against flFliC and tmFliC in New Zealand white rabbits. IgG response to the recombinant proteins was determined by ELISA. Cross-reactivity assay was performed by ELISA for all proteins and bacteria. Immunogenicity of tmFliC and flFliC was evaluated in chicken cells, and expression level of tumor necrotic factor-α (TNF-α) and interleukin-6 (IL-6) were relatively analyzed by Real-Time-PCR. Results showed high purification efficacy for tmFliC. Antibody titer of tmFliC was significantly higher than that of flFliC. In addition, the cross-reactivity assay for both proteins and Salmonella was positive which indicates similar epitopic regions. Stimulation of both FliCs significantly increased TNF-α and IL-6 expression in peripheral blood mononuclear cells (PBMCs) and splenocytes, with higher effect observed with flFliC. IL-8 protein level increased after 6 and 24 h stimulation with different concentrations of tmFliC and flFliC. These results suggest that the aimed gene modification in fliC gene produces a bioactive immunostimulant type of flagellin which upregulates TLR5 downstream genes as well as in flFliC., (Copyright © 2018. Published by Elsevier Ltd.)

The adjuvant effects of flagellin on regulation of immune response have been proved; whether flagellin could assist tumor cell lysate (TCL) to enhance anti-glioma immunity remains to be investigated. This study tests a hypothesis that therapeuticly intracranial administration with flagellin plus TCL enhances the effects of specific immunotherapy on glioma in mice. In this study, GL261 cells were transferred into C57BL/6 mice and the GL261-bearing mice were subcutaneously or intracranially inoculated with flagellin plus TCL, flagellin, TCL or saline. Our results showed that prophylacticly subcutaneous administration with TCL and flagellin could induce potent cytotoxic T lymphocyte (CTL) and prolong the survival of GL261-bearing mice significantly, but therapeuticly subcutaneous administration failed to. However, therapeuticly intracranial administration of TCL plus flagellin could prolong the survival. Moreover, intracranial administration of flagellin could recruit CD4 + T cells and CD8 + T cells to brain tissues, induce proliferation of natural killer (NK) cells, CD4 + T cells and CD8 + T cells in peripheral blood mononuclear cells and induce to splenomegaly. The results suggested that flagellin could be acted as an efficient adjuvant for TCL based vaccine., (Copyright © 2018. Published by Elsevier Ltd.)

Considering the increased antibiotic resistance of Pseudomonas aeruginosa, the evaluation of immune response against the antigens of this bacterium seems necessary. In this study, the protective efficacy and immunological properties of P. aeruginosa recombinant PilQ (r-PilQ) and type b-flagellin (FLB) proteins was evaluated in the burn mouse model of infection. The inbred BALB/c mice were immunized with r-PilQ and FLB antigens. To investigate the type of induced immune response, sera were analyzed by ELISA for total IgG, IgG1, and IgG2a isotypes. After the final immunization, the IL-4, IFN-γ, and IL-17 cytokines level were examined in the spleen of non-challenged mice. Fifty days after lethal challenge, the survival rate and bacterial burden in the skin and other internal organs of experimental mice were assessed. The in vivo administration of r-PilQ, FLB and combined antigen resulted in a significant increase in the survival of mice (66%, 75%, and 83%, respectively) infected by the PAO1 strain of P. aeruginosa in the burn model of infection. Immunization of mice with r-PilQ and FLB mixture induced high titers of IL-4 and IL-17 cytokines compared to control groups (P < 0.05). The high titer of antisera raised against combined antigen was able to inhibit the systemic spread of the PAO1 strain from the site of infection to the internal organs. We concluded that the parallel role of IL-4 and IL-17 is necessary for elimination of the bacteria and promotion of survival in the immunized burn mice., (Copyright © 2018. Published by Elsevier Ltd.)

Neonates have an increased susceptibility to infections, particularly those caused by intracellular pathogens, leading to high morbidity and mortality rates. This is partly because of a poor response of neonatal CD4 + T cells, leading to deficient antibody production and a low production of IFN-γ, resulting in deficient elimination of intracellular pathogens. The poor memory response of human neonates has underpinned the need for improving vaccine formulations. Molecular adjuvants that improve the response of neonatal lymphocytes, such as the ligands of toll-like receptors (TLRs), are attractive candidates. Among them, flagellin, the TLR5 ligand, is effective at very low doses; prior immunity to flagellin does not impair its adjuvant activity. Human CD4 + and CD8 + T cells express TLR5. We found that flagellin induces the expression of IFN-γ, IL-1β and IL-12 in mononuclear cells from human neonate and adult donors. When human naïve CD4 + T cells were activated in the presence of flagellin, there was high level of expression of IFN-γ in both neonates and adults. Furthermore, flagellin induced IFN-γ production in Th1 cells obtained from adult donors; in the Th2 population, it inhibited IL-4 cytokine production. Flagellin also promoted expression of the IFN-γ receptor in naive CD4 + T cells from neonates and adults. To test the adjuvant capacity of flagellin in vivo, we used a murine neonate vaccination model for infection with rotavirus, a pathogen responsible for severe diarrhea in young infants. Using the conserved VP6 antigen, we observed an 80% protection against rotavirus infection in the presence of flagellin, but only in those mice previously primed in the neonatal period. Our data suggest that flagellin could be an attractive adjuvant for achieving a Th1 response., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

PEDV variants, causing severe diarrhea in neonatal suckling piglets with a mortality rate up to 100%, have being epidemic since late 2010 in china. To meet the pressing need of safe and cost-efficient PED maternal vaccines against PEDV variant, we vaccinated growing piglets with a flagellin-adjuvanted PED vaccine rSF-COE-3D by injection at Houhai acupoint. The vaccination not only enhanced the antibody responses of serum IgG/IgA, mucosal IgA and serum neutralizing antibody, but also improved the production of IFN-γ and IL-4. Moreover, rSF-COE-3D could provide a better protection against the challenge of a high pathogenic PEDV variant, with less diarrhea pigs, less pigs with detectable PEDV shed, lower rank values of feces and less apparent lesions and inflammation in duodenum of the PEDV infected pigs. The improved protective efficiency of rSF-COE-3D compared with COE was mostly benefited from the enhanced production of serum IgA, mucosal IgA in feces and serum neutralizing antibody titers. Taken together, our data suggest that rSF-COE-3D would be a novel efficient PED vaccine., (Copyright © 2018. Published by Elsevier Ltd.)

Introduction: Bacterial flagellin, as a pathogen-associated molecular pattern (PAMP), can activate both innate and adaptive immunity. Its unique structural characteristics endow an effective and flexible adjuvant activity, which allow the design of different types of vaccine strategies to prevent various diseases. This review will discuss recent progress in the mechanism of action of flagellin and its prospects for use as a vaccine adjuvant., Areas Covered: Herein we summarize various types of information related to flagellin adjuvants from PubMed, including structures, signaling pathways, natural immunity, and extensive applications in vaccines, and it discusses the immunogenicity, safety, and efficacy of flagellin-adjuvanted vaccines in clinical trials., Expert Commentary: It is widely accepted that as an adjuvant, flagellin can induce an enhanced antigen-specific immune response. Flagellin adjuvants will allow more effective flagellin-based vaccines to enter clinical trials. Furthermore, vaccine formulations containing PAMPs are crucial to exert the maximum potential of vaccine antigens. Therefore, combinations of flagellin-adjuvanted vaccines with other adjuvants that act in a synergistic manner, particularly TLR ligands, represent a promising method for tailoring targeted vaccines to meet specific requirements.

Background: Previous studies have demonstrated that flagellin, a component of bacterial flagella, engages Toll-Like receptor 5 (TLR-5) causing the activation of the Myeloid Differentiation Factor-88 (MYD-88) pathway that leads to the production of pro-inflammatory cytokines including Tumor Necrosis Factor-alpha (TNF-α) and Interleukin-12 (IL-12). In physiological levels, cytokines can aid in protection against infectious agents. However, excessive production of cytokines can lead to septic shock during sepsis., Objective: In this study, we aimed at investigating the effect of denatured flagellin on hindering the effects induced by intact flagellin or flagellated Pseudomonas aeruginosa on the Toll-Like Receptor-5 (TLR-5) in mice., Methods: Mouse mononuclear cells (MNCs) were cultured with intact flagellin, heat-denatured flagellin, TLR-5 antagonist, Pseudomonas aeruginosa, and TLR-4 antagonist each alone or in combinations. Supernatants were collected at 4 hours post incubation to assess the levels of IL-12 and TNF-α by Enzyme-Linked ImmunoAssay (ELISA). Furthermore, groups of BALB/c mice were injected intraperitoneally (IP) with Pseudomonas aeruginosa, LPS-RS, intact flagellin, and denatured flagellin, each alone or in different combinations. Serum levels of IL-12 and TNF-α were measured at 2, 4, and 6 hours post injections of Pseudomonas aeruginosa or intact flagellin., Results: Pretreatment with denatured flagellin significantly reduced the amount of TNF-α and IL-12 produced both in vitro and in vivo by intact flagellin or Pseudomonas aeruginosa., Conclusion: Denatured flagellin suppressed the production of the pro-inflammatory cytokines induced by intact flagellin or Pseudomonas aeruginosa both in vitro and in vivo, probably by blocking TLR5. Denatured flagellin might be considered as an anti-septic shock agent., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Introduction: Intentional aerosolization of Yersinia pestis may result in pneumonic plague which is highly fatal if not treated early., Methods: We conducted a phase 1 randomized, double blind (within each group), placebo controlled, dose escalation trial to evaluate a plague vaccine, Flagellin/F1/V, in healthy adults aged 8 through 45years. Vaccine was administered intramuscularly on Days 0 and 28 at a dose of 1, 3, 6 or 10mcg. Subjects were observed for 4h after vaccination for cytokine release syndrome. Reactogenicity and adverse events (AE) were collected for 14 and 28days, respectively, after each vaccination. Serious AE were collected for the entire study. ELISA antibody and cytokines were measured at multiple time points. Subject's participation lasted 13months., Results: Sixty healthy subjects were enrolled; 52% males, 100% non-Hispanic, 91.7% white and mean age 30.8years. No severe reactogenicity events occurred; most AE were mild. No serious AE related to vaccine occurred. A dose response effect was observed to F1, V and flagellin. The peak ELISA IgG antibody titers (95% CI) after two 10mcg doses of vaccine were 260.0 (102.6-659.0) and 983.6 (317.3-3048.8), respectively, against F1 and V antigens. The 6mcg dose group provided similar titers. Titers were low for the placebo, 1mcg and 3mcg recipients. A positive antibody dose response was observed to F1, V and flagellin. Vaccine antigen specific serum IgE was not detected. There were no significant rises in serum or cellular cytokine responses and no significant IgG increase to flagellin after the second dose., Conclusion: The Flagellin/F1/V vaccine exhibited a dose dependent increase in immunogenicity and was well tolerated at all doses. Antibody specific responses to F1, V and flagellin increased as dose increased. Given the results from this trial, testing higher doses of the vaccine may be merited., (Copyright © 2017. Published by Elsevier Ltd.)

Chronic periodontitis is caused by interactions between the oral polymicrobial community and host factors. Periodontal diseases are associated with dysbiotic shift in oral microbiota. Vaccination against periodontopathic bacteria could be a fundamental therapeutic to modulate polymicrobial biofilms. Because oral cavity is the site of periodontopathic bacterial colonization, mucosal vaccines should provide better protection than vaccines administered systemically. We previously reported that bacterial flagellin is an excellent mucosal adjuvant. In this study, we investigated whether mucosal immunization with a flagellin-adjuvanted polypeptide vaccine induces protective immune responses using a Porphyromonas gingivalis infection model. We used the Hgp44 domain polypeptide of Arg-gingipain A (RgpA) as a mucosal antigen. Intranasal (IN) immunization induced a significantly higher Hgp44-specific IgG titer in the serum of mice than sublingual (SL) administration. The co-administration of flagellin potentiated serum IgG responses for both the IN and SL vaccinations. On the other hand, the anti-Hgp44-specific IgA titer in the saliva was comparable between IN and SL vaccinations, suggesting SL administration as more compliant vaccination route for periodontal vaccines. The co-administration of flagellin significantly potentiated the secretory IgA response in saliva also. Furthermore, mice administered a mixture of Hgp44 and flagellin via the IN and SL routes exhibited significant reductions in alveolar bone loss induced by live P. gingivalis infections. An intranasally administered Hgp44-flagellin fusion protein induced a comparable level of Hgp44-specific antibody responses to the mixture of Hgp44 and flagellin. Overall, a flagellin-adjuvanted Hgp44 antigen would serve an important component for a multivalent mucosal vaccine against polymicrobial periodontitis.

Melioidosis is a severe infectious disease with a high mortality that is endemic in South-East Asia and Northern Australia. The causative pathogen, Burkholderia pseudomallei, is listed as potential bioterror weapon due to its high virulence and potential for easy dissemination. Currently, there is no licensed vaccine for prevention of melioidosis. Here, we explore the use of rapid plasmid DNA vaccination against B. pseudomallei flagellin for protection against respiratory challenge. We tested three flagellin DNA vaccines with different subcellular targeting designs. C57BL/6 mice were vaccinated via skin tattoo on day 0, 3 and 6 before intranasal challenge with B. pseudomallei on day 21. Next, the most effective construct was used as single vaccination on day 0 by tattoo or intranasal formulation. Mice were sacrificed 72 hours post-challenge to assess bacterial loads, cytokine responses, inflammation and microscopic lesions. A construct encoding a cellular secretion signal resulted in the most effective protection against melioidosis via tattooing, with a 10-fold reduction in bacterial loads in lungs and distant organs compared to the empty vector. Strikingly, a single intranasal administration of the same vaccine resulted in >1000-fold lower bacterial loads and increased survival. Pro-inflammatory cytokine responses were significantly diminished and strong reductions in markers for distant organ damage were observed. A rapid vaccination scheme using flagellin DNA tattoo provides significant protection against intranasal challenge with B. pseudomallei, markedly improved by a single administration via airway mucosa. Hence intranasal vaccination with flagellin-encoding DNA may be applicable when acute mass vaccination is indicated and warrants further testing.

The aim of the present work was to investigate the synergistic effect between toll-like receptor (TLR) 3 ligand polyinosinic:polycytidylic acid (pI:C) and TLR5 ligand flagellin (FLN) on immune responses induced by nasally delivered hepatitis B virus surface antigen (HBsAg). Mannan and chitosan oligosaccharide-modified, pH-responsive poly(lactic- co -glycolic acid) (MC-PLGA) microparticles (MPs) containing HBsAg, FLN, pI:C or both ligands were prepared with a double-emulsion method. In vitro uptake experiments show that cellular uptake of MC-PLGA MPs by macrophages was through energy-dependent, receptor-mediated endocytosis mechanism. After uptake of MPs by macrophages, MC-PLGA MPs existed both in the endo-some and in the cytoplasm. FLN and pI:C in solution or MP formulation could synergize to activate macrophages and induce higher pro-inflammatory cytokines interleukin (IL)-6, IL-12, interferon-γ and anti-inflammatory cytokines IL-10 compared to single TLR ligand ( P <0.05). In vivo immunogenicity studies indicated that co-delivery of FLN and pI:C within MC-PLGA MPs synergistically induced higher serum anti-HBsAg IgG levels and Th1 cytokine levels compared with MC-PLGA MPs encapsulated single TLR ligand plus MPs encapsulated HBsAg ( P <0.05). These results suggest that synergic TLR3 and TLR5 stimulation might be a promising novel tool for nasally delivered HBsAg., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Vaccination is the most cost-effective means of infectious disease control. Although current influenza vaccines are effective in battling closely matched strains, such vaccines have major limitations such as the requirement to produce new vaccines every season, an egg-dependent production system, long production periods, uncertainty in matching the vaccine to circulating strains, and the inability to react to new influenza pandemics resulting from genetic drift or shift. To overcome the intrinsic limitations of the conventional influenza vaccine, we have designed dual-linker gold nanoparticles (AuNPs) conjugated with both recombinant trimetric A/Aichi/2/68 (H3N2), hemagglutinin (HA) and TLR5 agonist flagellin (FliC) as a novel vaccine approach. Click chemistry and metal-chelating reactions were used to couple the two proteins. The conjugated proteins were found to possess high coupling specificity, high stability in harsh environments, high conjugation efficiency, and the ability to keep the appropriate protein conformations for immunogenicity and immunostimulation. Both AuNPs-HA/FliC and AuNPs-HA formulations induced higher levels of antibody responses than a mixture of soluble HA and FliC proteins when administered via a single intranasal immunization in mice. To further investigate the adjuvancy of these nanoparticles, in vitro experiments were conducted in both the JAWS II dendritic cell (DC) line and bone marrow-derived DC (BMDC) models. The results showed that dual-conjugated AuNPs were rapidly targeted and taken up by DCs. Consequently, DCs were induced toward maturation, as demonstrated by high levels of cytokine secretions and membrane costimulatory molecule expression. T cell proliferation was observed when splenic T cells were cocultured with AuNPs-HA/FliC-primed BMDCs. These results suggest that dual-conjugated AuNPs are effective at simultaneously displaying antigens and adjuvants in an oriented, multivalent format and can promote a strong immune response by activating DCs and T cells., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Ligands NLR2 (muramyldipeptide) and TLR (bacterial LPS, flagellin, CpG-dinucleotide, and Poly I:C) and S. typhimurium antigenic complex by 1.5-3-fold increase the efficiency of cloning and content of multipotent stromal cells (MSC) in the bone marrow of CBA mice as soon as 1 h postinjection. The counts of large colonies (150-500 cells) increased by 2.5-3.3 times in comparison with intact bone marrow cultures at the expense of a decrease in the number of smaller colonies, which attests to enhanced proliferation of stromal cells in the colonies. The efficiency of cloning and hence, MSC content in the femoral bone decreased by 1.2-1.9 times after 3 h and increased again after 24 h to the level 1.3-1.5 times higher than the level 1 h postinjection (LPS, Poly I:C, and S. typhimurium antigenic complex). The dynamics of bone marrow MSC cloning efficiency after 1-3 h corresponded to the dynamics of serum cytokine concentrations during the same period. However, the levels of serum cytokines after 24 h in general were similar to those in intact mice or were lower. The concentrations of osteogenic multipotent stromal cells in the bone marrow decreased 2-3-fold after 3 h and thus persisted by 24 h postinjection. Twofold (at 24-h interval) and a single injection of S. typhimurium antigenic complex to mice led to a significant increase of cloning efficiency, observed as early as just 1 h postinjection (1.9 and 1.5 times, respectively). The same picture was observed for serum cytokines. On the whole, injections of TLR and NLR ligands and of S. typhimurium antigenic complex led to stromal tissue activation within 1 h postinjection, this activation consisting in a significant increase of the efficiency of cloning and of MSC count in the bone marrow, and also in an increase in their proliferative activity and a decrease (after 3 h) of osteogenic MSC concentration.

The aim of this study was to elucidate the expression and functions of IL-24 in C57BL/6 mouse corneas in response to Pseudomonas aeruginosa infection. Among IL-20R cytokines, only IL-24 was induced at both mRNA and protein levels by infection at early time points. The upregulation of IL-24 was dampened by flagellin pretreatment, which protects the corneas from microbial infection. Time course studies revealed bimodal early and later peaks of IL-24 expression, a pattern shared with suppressor of cytokine signaling (SOCS)3 but not IL-1β or IL-6. Silencing of IL-24 enhanced S100A8/A9 expression and suppressed SOCS3, IL-1β, IL-1RN, and matrix metalloproteinase 13 expression at 6 h postinfection. Downregulation of the IL-24 signaling pathway significantly reduced the severity of keratitis, whereas rIL-24 exacerbated P. aeruginosa -mediated tissue destruction. In vitro, rIL-1β induced the expression of SOCS3, IL-24, IL-1β, and IL-6 in primary cultured human corneal epithelial cells. rIL-24, alternatively, stimulated the expression of SOCS3, but not the others. In conclusion, IL-24 promotes P. aeruginosa keratitis through the suppression of early protective mucosal immunity, culminating in increased severity of P. aeruginosa keratitis., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Signaling through Toll-like receptors (TLRs), the main receptors in innate immunity, is essential for the defense of mucosal surfaces. It was previously shown that systemic TLR5 stimulation by bacterial flagellin induces an immediate, transient interleukin-22 (IL-22)-dependent antimicrobial response to bacterial or viral infections of the mucosa. This process was dependent on the activation of type 3 innate lymphoid cells (ILCs). The objective of the present study was to analyze the effects of flagellin treatment in a murine model of oral infection with Yersinia pseudotuberculosis (an invasive, Gram-negative, enteropathogenic bacterium that targets the small intestine). We found that systemic administration of flagellin significantly increased the survival rate after intestinal infection (but not systemic infection) by Y. pseudotuberculosis This protection was associated with a low bacterial count in the gut and the spleen. In contrast, no protection was afforded by administration of the TLR4 agonist lipopolysaccharide, suggesting the presence of a flagellin-specific effect. Lastly, we found that TLR5- and MyD88-mediated signaling was required for the protective effects of flagellin, whereas neither lymphoid cells nor IL-22 was involved., (Copyright © 2017 American Society for Microbiology.)

Burn injury is associated with a loss of gut barrier function, resulting in systemic dissemination of gut-derived bacteria and their products. The bacterial protein and TLR5 agonist, flagellin, induces non-specific innate immune responses. Because we detected flagellin in the serum of burn patients, we investigated whether gut-derived flagellin was a primary or secondary contributor to intestinal dysfunction and systemic inflammation following burn injury. The apical surface of polarized human intestinal epithelial cells (IECs), Caco-2BBe, were exposed to 50 or 500 ng of purified flagellin and 1 x 105 of an intestinal E. coli (EC) isolate as follows: 1) flagellin added 30 min prior to EC, 2) flagellin and EC added simultaneously, or 3) EC added 30 min prior to flagellin. Our results showed that luminal flagellin and EC modulated each other's biological actions, which influenced their ability to induce basolateral secretion of inflammatory cytokines and subsequent translocation of bacteria and their products. A low dose of flagellin accompanied by an enteric EC in the lumen, tempered inflammation in a dose- and time-dependent manner. However, higher doses of flagellin acted synergistically with EC to induce both intestinal and systemic inflammation that compromised barrier integrity, increasing systemic inflammation following burn injury, a process we have termed flagellemia. In a murine model of burn injury we found that oral gavage of flagellin (1 μg/mouse) significantly affected the gut microbiome after burn injury. In these mice, flagellin disseminated out of the intestine into the serum and to distal organs (mesenteric lymph nodes and lungs) where it induced secretion of monocyte chemoattractant protein (MCP-1) and CXCL1/KC (mouse equivalent of human IL-8) at 24 and 48h post-burn. Our results illustrated that gut-derived flagellin alone or accompanied by a non-pathogenic enteric EC strain can function as an initiator of luminal and systemic inflammation following burn injury., Competing Interests: The authors declare they have no conflicting interests regarding the data in this current manuscript.

Young infants are at significantly increased risk of developing severe disease following infection with influenza virus. At present there is no approved vaccine for individuals below the age of six months given previous studies showing a failure of these individuals to efficiently seroconvert. Given the major impact of influenza on infant health, it is critical that we develop vaccines that will be safe and effective in this population. Using a nonhuman primate (NHP) model, we have evaluated the ability of an inactivated influenza virus vaccine adjuvanted with flagellin to result in long term immune responses in neonates. To evaluate this critical attribute, neonate NHP were vaccinated and boosted with inactivated influenza virus in combination with either flagellin or a mutant inactive flagellin control. Our studies show that inclusion of flagellin resulted in a significant increase (5-fold, p=0.04) in influenza virus-specific IgG antibody at 6months post-vaccination. In addition, the antibody present at this late time was of higher affinity (2.4-fold, p=0.02). Finally a greater percentage of infants had detectable neutralizing antibody. These results support the use of flagellin in neonates as an adjuvant that promotes long-lived, high affinity antibody responses., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aim: To evaluate efficacy of sublingual flagellin to treat acute pneumonia., Materials & Methods: Mice were treated sublingually with flagellin and challenged intranasally with a lethal dose of pneumococcus. Flagellins lacking TLR5 or NLRC4 activation domains were used to assess their contribution to protection., Results: Sublingual flagellin protected mice in a TLR5-dependent, NLRC4-independent fashion. Neutrophils were required for protection. Flagellin-stimulated lung epithelial cells recapitulated the lung's transcriptional profile suggesting they could be targeted by flagellin in vivo., Conclusion: Ligation of TLR5, a pathogen recognition receptor not naturally engaged by pneumococcus, protects mice from invasive pneumonia when administered via sublingual route. This can be a highly cost-effective alternative therapy against pneumonia.

Influenza virus infection of neonates poses a major health concern, often resulting in severe disease and hospitalization. At present, vaccines for this at-risk population are lacking. Thus, development of an effective vaccine is an urgent need. In this study, we have used an innovative nonhuman primate neonate challenge model to test the efficacy of a novel TLR 7/8 agonist R848-conjugated influenza virus vaccine. The use of the intact virus represents a step forward in conjugate vaccine design because it provides multiple antigenic targets allowing for elicitation of a broad immune response. Our results show that this vaccine induces high-level virus-specific Ab- and cell-mediated responses in neonates that result in increased virus clearance and reduced lung pathology postchallenge compared with the nonadjuvanted virus vaccine. Surprisingly, the addition of a second TLR agonist (flagellin) did not enhance vaccine protection, suggesting that combinations of TLR that provide increased efficacy must be determined empirically. These data support further exploration of this new conjugate influenza vaccine approach as a platform for use in the at-risk neonate population., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Background: Bacterial flagellin, a Toll-like receptor 5 agonist, is used as an adjuvant for immunomodulation. In this study, we aimed to evaluate the effect and its mechanism following intralymphatic administration of OVA-flagellin (FlaB) mixture in the mouse model of allergic rhinitis., Materials and Methods: BALB/c mice were sensitized with OVA and treated with an OVA-FlaB mixture via intranasal, sublingual, and intralymphatic routes to evaluate the effect of each treatment. Several parameters for allergic inflammation and its underlying mechanisms were then evaluated., Results: Intralymphatic injection of the OVA-FlaB mixture reduced symptom scores, eosinophil infiltration in the nasal mucosa, and total and OVA-specific IgE levels more significantly than intranasal and sublingual administration. Systemic cytokine (IL-4, IL-5, IL-6, IL-17, and IFN-γ) production and local cytokine (IL-4 and IL-5) production were also reduced significantly after intralymphatic injection with OVA-FlaB. Double intralymphatic injection of the mixture was more effective than single injection. Moreover, the expression of innate cytokines such as IL-25 and IL-33 in nasal epithelial cells was reduced, and the expression of chemokines such as CCL24 (eotaxin-2), CXCL1, and CXCL2 was decreased in the nasal mucosa, suggesting the underlying mechanism for intralymphatic administration of the OVA-FlaB mixture., Conclusion: Intralymphatic administration of an OVA-FlaB mixture was more effective in alleviating allergic inflammation than intranasal and sublingual administration in a mouse model of allergic rhinitis. This effect may be attributed to the reduced expression of innate cytokines and chemokines. This treatment modality can be considered as a new therapeutic method and agent., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Vaccination is the most effective method for preventing rabies virus (RABV) infection in both humans and animals; however, no satisfactory vaccine has been developed for use worldwide. In the present study, we investigated the immunoadjuvant properties of Salmonella Typhimurium flagellin (FljB, FliC, and FljB'-FliC) to improve immune responses against the rabies vaccine (RV) and the protective efficacy of the whole-killed rabies vaccine (WKRV) with or without flagellins in BALB/c mice. We also compared the differences among the three flagellins in terms of immunoadjuvant properties to RV. FljB can cause the WKRV to induce stronger humoral and cellular immune responses than WKRV alone or WKRV with FliC or FljB'-FliC can. Mice immunized with WKRV and FljB produced higher levels of virus-neutralizing antibody (VNA) against RABV than those in the other groups did. Although mice in all treatment groups survived RABV challenge, the body weight loss in the group immunized with WKRV and FljB was lower than in the other groups. These results indicate that FljB is a promising adjuvant for use in the development of effective rabies vaccines.

Immunotherapy employs selected prokaryotic elements which are specially targeted because of their designated important role in the pathogenicity of the microbes. Among these is the flagellin of P. aeruginosa, which plays a major role in establishment of urinary tract infections (UTIs). In this study we envisage divalent flagellin (a combination of flagellin subtypes, 'a' and 'b') as an immunotherapeutic candidate against UTIs caused by Pseudomonas aeruginosa. Flagellin proteins were isolated from P. aeruginosa strains and characterized by MALDI-TOF. Their efficacy was checked in an ascending model of UTI. Divalent flagellin ('a' and 'b') when given together (intraperitoneally, i.p.) to female LACA mice at a concentration of 5 μg each, protected mice against pyelonephritis due to P. aeruginosa strains with no bacterial load at peak day of infection. Tissue destruction was minimum, as assessed by MDA levels and renal histopathology. Divalent flagellin immunization also drastically reduced pro-inflammatory cytokine levels (TNF α and IL-1β) in renal homogenates as determined by ELISA. It also prevented UTI caused by heterologous strain Escherichia coli. Antibodies against both flagellin proteins were assessed by ELISA. Passive immunization protected mice against UTI induced by either of the strains, P. aeruginosa and E. coli. These results confirmed homologous and heterologous protection provided by divalent flagellin., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

There are few safe and effective drugs available that protect healthy tissue against radiation-induced damage, highlighting the need to develop such radioprotective agents. We investigated the mechanism underlying the protective effects of the novel, recombinant, flagellin-like protein FlaA N/C against radiation-induced tissue damage in female BALB/c mice. FlaA N/C treatment increased the levels of several pro-proliferative cytokines while inhibiting apoptosis and reducing inflammation. These effects were accompanied by activation of the nuclear factor κB signaling pathway via direct interaction of FlaA N/C with Toll-like receptor 5, as well as enhanced survival of mice after total-body irradiation compared to that observed with treatment with amifostine, a radioprotective agent that is currently being used in clinical practice. These results indicate that FlaA N/C could be further explored as a possible protector of damage to healthy tissue during radiotherapy.

Researchers are currently involved in a strong effort to find a safe and effective vaccine against highly pathogenic avian influenza H5N1 viruses. Toward that goal, we obtained soluble recombinant flagellin (FliC) of Salmonella typhimurium to be used as a mucosal adjuvant for H5HA subunit vaccine development. Intranasal immunization of H5HA antigen with recombinant FliC protein in an oil-in-water emulsion increased H5HA-specific IgG and IgA titers in sera, bronchoalveolar lavage fluids (BALFs), and nasal washes. Use of FliC adjuvant for intranasal immunization further augmented B-cell responses in mucosal environments via increased IgA titers in BALFs and nasal washes. Increases in IgA and IgG titers through the use of FliC adjuvant in intranasal immunization correlated with higher neutralizing antibody titers in sera and BALFs and higher numbers of IgG- and IgA-secreting B cells in spleen and cervical lymph nodes. High levels of IL-17A cytokine production were also found in stimulated T cells of spleen and cervical lymph node cells, only by intranasal immunization particularly with the use of FliC adjuvant in oil-in-water emulsions. These findings may provide useful information toward the development of H5HA mucosal influenza vaccines., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigA(C)) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigA(C), either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigA(C) or LigA(C) coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Influenza virus can cause life-threatening infections in neonates and young infants. Although vaccination is a major countermeasure against influenza, current vaccines are not approved for use in infants less than 6 months of age, in part due to the weak immune response following vaccination. Thus, there is a strong need to develop new vaccines with improved efficacy for this vulnerable population. To address this issue, we established a neonatal African green monkey (AGM) nonhuman primate model that could be used to identify effective influenza vaccine approaches for use in young infants. We assessed the ability of flagellin, a Toll-like receptor 5 (TLR5) agonist, to serve as an effective adjuvant in this at-risk population. Four- to 6-day-old AGMs were primed and boosted with inactivated PR8 influenza virus (IPR8) adjuvanted with either wild-type flagellin or inactive flagellin with a mutation at position 229 (m229), the latter of which is incapable of signaling through TLR5. Increased IgG responses were observed following a boost, as well as at early times after challenge, in infants vaccinated with flagellin-adjuvanted IPR8. Inclusion of flagellin during vaccination also resulted in a significantly increased number of influenza virus-specific T cells following challenge compared to the number in infants vaccinated with the m229 adjuvant. Finally, following challenge infants vaccinated with IPR8 plus flagellin exhibited a reduced pathology in the lungs compared to that in infants that received IPR8 plus m229. This study provides the first evidence of flagellin-mediated enhancement of vaccine responses in nonhuman primate neonates., Importance: Young infants are particularly susceptible to severe disease as a result of influenza virus infection. Compounding this is the lack of effective vaccines for use in this vulnerable population. Here we describe a vaccine approach that results in improved immune responses and protection in young infants. Incorporation of flagellin during vaccination resulted in increased antibody and T cell responses together with reduced disease following virus infection. These results suggest that flagellin may serve as an effective adjuvant for vaccines targeted to this vulnerable population., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The envelope (E) protein of flaviviruses includes three domains, EI, EII, and EIII, and is the major protective antigen. Because EIII is rich in type-specific and subcomplex-specific neutralizing epitopes and is easy to express, it is particularly attractive as a recombinant vaccine antigen. VaxInnate has developed a vaccine platform that genetically links vaccine antigens to bacterial flagellin, a Toll-like receptor 5 ligand. Here we report that tetravalent dengue vaccines (TDVs) consisting of four constructs, each containing two copies of EIII fused to flagellin (R3.2x format), elicited robust and long-lived neutralizing antibodies (geometric mean titers of 200 to 3,000), as measured with a 50% focus reduction neutralization test (FRNT50). In an immunogenicity study, rhesus macaques (n = 2) immunized subcutaneously with 10 μg or 90 μg of TDV three or four times, at 4- to 6-week intervals, developed neutralizing antibodies to four dengue virus (DENV) serotypes (mean post-dose 3 FRNT50 titers of 102 to 601). In an efficacy study, rhesus macaques (n = 4) were immunized intramuscularly with 16 μg or 48 μg of TDV or a placebo control three times, at 1-month intervals. The animals that received 48-μg doses of TDV developed neutralizing antibodies against the four serotypes (geometric mean titers of 49 to 258) and exhibited reduced viremia after DENV-2 challenge, with a group mean viremia duration of 1.25 days and 2 of 4 animals being completely protected, compared to the placebo-treated animals, which all developed viremia, with a mean duration of 4 days. In conclusion, flagellin-EIII fusion vaccines are immunogenic and partially protective in a nonhuman primate model., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)