The GM-CSF Released by Airway Epithelial Cells Orchestrates the Mucosal Adjuvant Activity of Flagellin.

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.

Improving the analytical toolbox to investigate copurifying host cell proteins presence: N-(4)-(ß-acetylglucosaminyl)- l-asparaginase case study.

Levels of host cell proteins (HCPs) in purification intermediates and drug substances (DS) of monoclonal antibodies (mAbs) must be carefully monitored for the production of safe and efficacious biotherapeutics. During the development of mAb1, an immunoglobulin G1 product, unexpected results generated with HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit triggered an investigation which led to the identification of a copurifying HCP called N-(4)-(ß-acetylglucosaminyl)-l-asparaginase (AGA, EC3.5.1.26) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The risk assessment performed indicated a low immunogenicity risk for the copurifying HCP and an ad hoc stability study demonstrated no mAb glycan cleavage and thus no impact on product quality. Fractionation studies performed on polishing steps revealed that AGA was coeluted with the mAb. Very interestingly, the native digestion protocol implemented to go deeper in the MS-HCP profiling was found to be incompatible with correct AGA detection in last purification intermediate and DS, further suggesting a hitchhiking behavior of AGA. In silico surface characterization of AGA also supports this hypothesis. Finally, the combined support of HCP ELISA results and MS allowed process optimization and removal of this copurifying HCP.

Toll-like receptor 4 signaling in hematopoietic-lineage cells contributes to the enhanced activity of the human vaccine adjuvant AS01.

The 3-O-desacyl-4'-monophosphoryl lipid A (MPL) activates immunity through Toll-like receptor 4 (TLR4) signaling. The Adjuvant System AS01 contains MPL and is used in the candidate malaria vaccine and the licensed zoster vaccine. Recent studies reported that AS01 adjuvant activity depends on a transient inflammation at the site of vaccination, but the role of stromal or structural cells in the adjuvant effect is unknown. We investigated this question in mouse models by assessing the role of TLR4 on hematopoietic versus resident structural cells during immunization with AS01-adjuvanted vaccines. We first established that TLR4-deficient animals had a reduced immune response to an AS01-adjuvanted vaccine. Using bone marrow chimera, we consistently found that Tlr4 expression in radio-sensitive cells, i.e., hematopoietic cells, was required for an optimal adjuvant effect on antibody and T-cell responses. At day 1 after injection, the pro-inflammatory reaction at the site of injection was strongly dependent on TLR4 signaling in hematopoietic cells. Similarly, activation of dendritic cells in muscle-draining lymph nodes was strictly associated with the radio-sensitive cells expressing Tlr4. Altogether, these data suggest that MPL-mediated TLR4-signaling in hematopoietic cells is critical in the mode of action of AS01.

Sublingual flagellin protects against acute pneumococcal pneumonia in a TLR5-dependent and NLRC4-independent fashion.

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.

A Toll-Like Receptor 5 Agonist Improves the Efficacy of Antibiotics in Treatment of Primary and Influenza Virus-Associated Pneumococcal Mouse Infections.

Prophylactic intranasal administration of the Toll-like receptor 5 (TLR5) agonist flagellin protects mice against respiratory pathogenic bacteria. We hypothesized that TLR5-mediated stimulation of lung immunity might improve the therapeutic index of antibiotics for the treatment of Streptococcus pneumoniae respiratory infections in mice. Intranasal administration of flagellin was combined with either oral administration of amoxicillin or intraperitoneal injection of trimethoprim-sulfamethoxazole to treat S. pneumoniae-infected animals. Compared with standalone treatments, the combination of antibiotic and flagellin resulted in a lower bacterial load in the lungs and greater protection against S. pneumoniae dissemination and was associated with an early increase in neutrophil infiltration in the airways. The antibiotic-flagellin combination treatment was, however, not associated with any exacerbation of inflammation. Moreover, combination treatment was more efficacious than standalone antibiotic treatments in the context of post-influenza virus pneumococcal infection. Lastly, TLR5 signaling was shown to be mandatory for the efficacy of the combined antibacterial therapy. This report is the first to show that combining antibiotic treatment with the stimulation of mucosal innate immunity is a potent antibacterial strategy against pneumonia.

Indirect Toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract.

The Toll-like receptor 5 (TLR5) agonist flagellin is an effective adjuvant for vaccination. Recently, we demonstrated that the adaptive responses stimulated by intranasal administration of flagellin and antigen were linked to TLR5 signaling in the lung epithelium. The present study sought to identify the antigen presenting cells involved in this adjuvant activity. We first found that the lung dendritic cells captured antigen very efficiently in a process independent of TLR5. However, TLR5-mediated signaling specifically enhanced the maturation of lung dendritic cells. Afterward, the number of antigen-bound and activated conventional dendritic cells (both CD11b(+) and CD103(+)) increased in the mediastinal lymph nodes in contrast to monocyte-derived dendritic cells. These data suggested that flagellin-activated lung conventional dendritic cells migrate to the draining lymph nodes. The lymph node dendritic cells, in particular CD11b(+) cells, were essential for induction of CD4 T-cell response. Lastly, neutrophils and monocytes were recruited into the lungs by flagellin administration but did not contribute to the adjuvant activity. The functional activation of conventional dendritic cells was independent of direct TLR5 signaling, thereby supporting the contribution of maturation signals produced by flagellin-stimulated airway epithelium. In conclusion, our results demonstrated that indirect TLR5-dependent stimulation of airway conventional dendritic cells is essential to flagellin's mucosal adjuvant activity.