NOMV are actively being considered as vaccines against meningococcal disease (van der Ley and van den Dobbelsteen, 2011), we therefore wanted to understand how a further modification may impact on human DC and T-cell responses. Th2 cell differentiation; however, NOMV should be considered further as a vaccine vector, particularly considering the importance of in antigen uptake and further human studies on antigen-specific responses should be considered. Introduction (Nm) is usually a major cause of meningitis and septicaemia worldwide. Effective capsular polysaccharide vaccines have been developed against serogroups A, C, W135 and Y TG-02 (SB1317) (Jodar NOMVs are in fact currently being investigated as potential meningococcal vaccines (Keiser lipid TG-02 (SB1317) A variants are found naturally and appear to be associated with a less severe meningococcal disease (Fransen gene revealing a structural motif that is specifically recognized by DC-SIGN (Steeghs LOS oligosaccharide modification are more readily phagocytosed by human DC than their wild-type strain predominately via DC-SIGN (Steeghs (Querec induce distinct expression patterns of maturation receptors on the surface of DC Human DC were stimulated with NOMV from the LOS altered strains and surface expression of HLA-Class I, HLA-DR, CD40 and CD83 were determined by Flow Cytometry. An increase in the expression of all molecules examined was observed upon stimulation with NOMV when compared to unstimulated DC (Fig. ?(Fig.1).1). These were comparable levels to those expected from Rabbit Polyclonal to Caspase 3 (Cleaved-Ser29) either purified LOS or killed Nm whole bacteria. Similar expression levels were also observed with NOMV from unencapsulated wild-type bacteria (data not shown). The NOMV with a pentacylated lipid A induced modest expression of CD40 compared to unstimulated but induced little to no expression of HLA-Class I and HLA-DR and CD83. This obtaining is consistent with reduced inflammatory potential of lipid A compared to its hexacylated wild-type lipid A counterpart. However, the combination of a pentacylated lipid A and DC-SIGN recognition motif (= 0.05), HLA-DR (= 0.009) and HLA-Class I (= 0.004), but not CD83, when compared to those observed for the TG-02 (SB1317) NOMV derived from the single mutation. This obtaining demonstrates enhanced expression of certain surface markers through the engagement of DC-SIGN in the presence of a mutation. Open in a separate window Physique 1 Dendritic cell surface phenotype following stimulation with LOS altered NOMV. Human monocyte-derived DC were stimulated with 1 TG-02 (SB1317) g ml?1 NOMV for 18C24 h and then analysed by Flow Cytometry for the TG-02 (SB1317) expression of surface proteins CD40, HLA-DR, HLA-Class I and CD83. Data from one representative donor are shown (A) together with a summary of data from eight individual human donors (B). Data are expressed as the mean and standard error of the mean and significance was determined by a paired altered Nm are more readily phagocytosed than Nm expressing wild-type LOS (Steeghs altered NOMV is to increase uptake of meningococcal antigens to DC. Human DC were co-cultured with FITC-labelled LOS-modified NOMV and uptake measured by flow cytometry. As shown in Fig. 2A and B, the NOMV were most readily internalized by DC. In contrast, no obvious uptake was observed for the NOMV even when NOMV concentrations were increased beyond 10 g ml?1. Interestingly, the NOMV were taken up by DC but not to the same extent as those observed for NOMV. More surprisingly, NOMV from unencapsulated Nm with wild-type (WT) LOS (H44/76 NOMV. These data strongly suggest that majority of phagocytosis is usually via conversation with DC-SIGN. In order to confirm further that NOMV were internalized we used a differential antibody staining and confocal microscopy method to distinguish NOMV which were either surface bound and internalized by DC. As Fig. ?Fig.2C2C shows, both and NOMV are internalized, while and WT NOMV were only found on the surface, thus confirming flow cytometric observations. Open in a separate window Physique 2 Internalization of LOS altered NOMV by dendritic cells. Human monocyte-derived DC were co-cultured with 10 g ml?1 FITC labelled NOMV for 4 h. DC were separated into 3 aliquots to distinguish internalized and surface adhered NOMV. One aliquot was left untreated and the second was treated with 0.4% trypan blue to quench FITC signal emitted from extracellular NOMVs. DC were then analysed by flow cytometry. The third aliquot was stained with To-Pro3 (blue) to stain the nucleus and anti-meningococcal serosubtype P1.7 antibody followed by Alex Fluor 568 goat anti-mouse to stain the surface adhered NOMV. Cells were then analysed by Confocal Microscopy. Data are shown from one representative donor (A and C) together with the summary of data from eight donors (B). Data are expressed.