LOS oligosaccharide modification enhances dendritic cell responses to meningococcal native outer membrane vesicles expressing a non-toxic lipid A

Abstract

Outer membrane vesicles (OMV) are released by many bacteria, and contain immunogenic antigens in addition to harmful inflammatory factors, like lipopolysaccharides. Chemically detoxified OMV have been used in vaccines against Neisseria meningitidis (Nm); however, little is known about their interaction with antigen presenting cells. In this study, we investigated the interaction of Nm OMV with human dendritic cells (DC) to gain further understanding of their biological activity. We engineered a novel serogroup B Nm that is unencapsulated (siaD), expresses pentacylated lipid A (lpxL1), hence conferring reduced toxicity, and expresses an lgtB oligosaccharide structure designed to target OMV to DC via DC-SIGN. We show that the lgtB moiety is critical for internalization of NOMV by DC. Furthermore, the lgtB moiety significantly enhances DC maturation, IL-10 and IL-23 production in the presence of a pentacylated lipid A. While different DC phenotypes were observed for each NOMV, this had little effect on Th1 and Th2 cell differentiation; however, lgtBsignificantly increased Th17 cell expansion in the presence of pentacylated lipid A. We believe that lpxL1/lgtB NOMV should be considered further as a vaccine vector, particularly considering the importance of lgtB in antigen uptake and further human studies on antigen-specific responses should be considered.

Figure 1

Dendritic cell surface phenotype following stimulation with LOS modified NOMV. Human monocyte-derived DC were stimulated with 1 μg ml⁻¹ NOMV for 18–24 h and then analysed by Flow Cytometry for the 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 t-test.

Figure 2

Internalization of LOS modified NOMV by dendritic cells. Human monocyte-derived DC were co-cultured with 10 μg 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 as the mean and standard error of the mean and significance was determined by a paired t-test.

Figure 3

Effect of DC-SIGN blocking antibody and GlcNAc on internalization of LOS modified NOMV. Human monocyte-derived DC were stimulated with 10 μg ml⁻¹ FITC-labelled NOMV for 4 h in the presence of either 20 μg ml⁻¹ DC-SIGN blocking antibody or 50 mM N-acetyl glucosamine. DCs were then treated with 0.4% trypan blue to quench any FITC signal from surface bound NOMV. Data are representative of three experiments from three different donors yielding similar results.

Figure 4

The production of inflammatory cytokines by DC in response to LOS modified NOMV. Human monocyte-derived DC were stimulated with 1 μg ml⁻¹ LOS modified NOMV for 18–24 h. Supernatants were collected and then analysed for the presence of inflammatory cytokines TNF-α, IL-1β, IL-6, by ELISA. A summary of data is shown for 8 human donors. Data are expressed as the mean and standard error of the mean. Statistical significance was tested using a paired t-test.

Figure 5

The production of T-cell polarizing cytokines by DC upon stimulation with LOS modified NOMV. Human monocyte-derived DC were stimulated with 1 μg ml⁻¹ LOS modified NOMV for 18–24 h. Supernatants were collected and then analysed for the presence T-cell polarizing cytokines IL-10 (A), IL-23 (B), IL-12p70 (C) and PGE₂ (D) by ELISA. A summary of data is shown for eight human donors. Data are expressed as the mean and standard error of the mean. Statistical significance was tested using a paired t-test.

Figure 6

The effect of blocking phagocytosis of NOMV on DC cytokine production. Human monocyte-derived DC were co-cultured with 1 μg ml⁻¹ NOMV in the presence of 10 μg ml⁻¹ cytochalasin D (cytD) for 18–24 h. Supernatant was collected and analysed for IL-10, IL-12p70 and IL-23 production. Data from one representative donor out of three are shown.

Figure 7

The effect of NOMV stimulated DC on Th1, Th2 and Th17 responses. To assess the generation of Th1 and Th2 responses naïve CD4+ T-cells were co-cultured with NOMV-stimulated DCs (1 μg ml⁻¹ for 18–24 h) for 12 days in the presence of IL-2 and 100 pg ml⁻¹ SEB. On the 12th day T-cells were restimulated with 10 ng ml⁻¹ PMA and 1 μg ml⁻¹ ionomycin for 5 h, cells were then analysed by flow cytometry for the production of IFN-γ and IL-4 (Th1 and Th2 skewing cytokines respectively). To assess Th17 cell responses NOMV-stimulated DC (1 μg ml⁻¹ for 18–24 h) were co-cultured with memory CD4+ cells for 5 days in the presence of IL-2 and SEB. T-cells were then stimulated with PMA and ionomycin and then analysed by flow cytometry for IL-17 production. Each point on the graph represents each separate donor and the mean of each group is also shown. Statistical significance was determined by a Friedman test. Significant differences are indicated on the figure.

Figure 8

The immunogenicity of NOMV in a murine in vivo model. BALB/c mice (15 per group) were immunized with 5 μg of lgtB, lpxL1/lgtB or lpxL1. Serum was collected at day 42 and analysed for specific IgG antibody by ELISA (A). Wild-type H44/76 and PorA-deficient H44/76 were used as target antigens in the ELISA. Specific IgG levels are expressed as log₁₀ titres for individual mice and the geometric mean is shown for each group. Antibody function was assessed by SBA (B). Briefly, mouse serum was incubated with rabbit complement together with either wild-type H44/76 or PorA-deficient H44/76. Highest dilution that resulted in 90% killing of bacteria is shown for individual mice and the geometric mean for each group. Statistical significance was determined by a paired T-test unless stated otherwise. For SBA against PorA-deficient H44/76 the number of responders are indicated on the graph. A responder was defined as a mouse that who serum killed 90% of bacteria at at least 1:5 dilution. *Statistical significance was determined by Fishers Exact Test.