Single lysophosphatidylcholine components exhibit adjuvant activities in vitro and in vivo

Abstract

Improving vaccine immunogenicity by developing new adjuvant formulations has long been a goal of vaccinologists. It has previously been shown that a natural mix of lysophosphatidylcholine (LPC) from chicken eggs promotes mature dendritic cell (DC) generation in vitro and primes antigen-specific immune responses in mice. In the present study, we dissected the adjuvant potentials of five individual LPC components found in the chicken egg mixture. In vitro analyses of the impact of the individual components on the maturation of human DCs were performed by means of phenotypic analysis, chemokine secretion analysis, and analysis of the ability of mature DC to stimulate T lymphocytes. Two components, C16:0-LPC and C18:0-LPC, were identified to be capable of the upregulation of expression of CD86, HLA-DR, and CD40 on in vitro-cultured monocyte-derived DCs from healthy donors. Both induced the release of chemokines to high concentrations (macrophage inflammatory protein 1, monocyte chemoattractant protein 1) or moderate concentrations (interleukin-8 [IL-8], gamma interferon-inducible protein 10). In addition, C16:0-LPC engaged naïve T cells to produce gamma interferon. This suggests that C16:0-LPC and C18:0-LPC have the capacity to promote, at least in vitro, a Th1-oriented response. The intravenous injection of C16:0-LPC or C18:0-LPC into mice resulted in the detectable secretion of IL-6 and IL-5 in sera. Both LPC components were tested for their capacities to act as adjuvants for two selected immunogens: the hepatitis B virus surface antigen and the hepatitis C virus NS3 helicase. The secretion of specific IgG1 was observed with either or both C16:0-LPC and C18:0-LPC, depending on the immunogen tested, and was observed at an efficiency comparable to that of alum. These data identify C16:0-LPC and C18:0-LPC as the active components of the LPC natural mixture. Although discrepancies between the results of the in vitro and in vivo analyses existed, studies with animals suggest that these components can trigger significant and specific humoral-mediated immunity.

FIG. 1.

Effect of fatty acid composition of LPC on DC maturation. The phenotypes of day 6 MoDCs stimulated for 24 h with PBS (control, thick line), egg LPC (thin line), or soybean LPC (dashed line) were analyzed by flow cytometry. The results are representative of those of one of three experiments.

FIG. 2.

Mature DC generation induced by LPC single components. The phenotypes of day 6 MoDCs stimulated for 24 h with LPC components (A and B) or LPS (C) were analyzed by flow cytometry. The expression of surface markers on treated cells (thin lines) was compared to that on control cells receiving only PBS (thick lines). The results are representative of those of 1 of 10 experiments.

FIG. 3.

Chemokines secreted by MoDCs upon stimulation with various LPCs. The supernatants of MoDCs treated with LPC components or PBS (A to D) or with LPS (E) at day 5 for 24 h were collected, and the chemokine concentrations were measured by using the CBA technology. Raw data were normalized to the results for the control (PBS treated), which was arbitrarily set equal to 1. The data for each donor are plotted individually; data for a total of seven donors were assayed independently, and the mean for the donors is indicated by the thick horizontal lines. (A) MIP-1β; (B) MCP-1; (C) IL-8; (D) IP-10. The mean values obtained with LPS-treated cells are indicated separately (E).

FIG. 4.

Allogeneic T-cell stimulation by LPC-treated cells. MoDCs, treated or not treated with an LPC component for 24 h, were cocultured with allogeneic T cells at a ratio of 1:10 for 5 days. The cell supernatants were assessed for the presence of IFN-γ, IL-13, and IL-5 by CBA assays. Cytokine detection was performed in triplicate, and the means plus standard deviations are represented. The results are representative of those of three separate experiments with sera from different donors.

FIG. 5.

Inflammatory profile induced by C16:0-LPC and C18:0-LPC. Sera from anesthetized C57BL/6 mice receiving intravenous injections of either C16:0-LPC (500 nmol), C18:0-LPC (500 nmol), CpG-ODN1826 (10 μg and 25 μg), LPS (10 μg and 25 μg), or poly(I:C) (10 μg and 25 μg) were assayed for the presence of cytokines by using a Milliplex map kit. The data for two mice are included per time point; and sera were collected at 2 h (mouse 1 [M1], M2), 4 h (M3, M4), or 12 h (M5, M6). (A) IL-6. The positive thresholds, defined as two times the mean level of secretion for PBS-injected mice, were 45 pg/ml at 2 h, 45 pg/ml at 4 h, and 0 pg/ml at 12 h. (B) IL-5. The positive thresholds were 61 pg/ml at 2 h, 78 pg/ml at 4 h, and 39 pg/ml at 12 h.

FIG. 6.

Anti-HBsAg humoral response induced in BALB/c mice following immunization with HBsAg combined with C18:0-LPC. Groups of six mice were immunized two times at a 2-week interval with 0.5 μg of HBsAg combined with C18:0-LPC at decreasing doses (from 500 to 75 nmol), and the humoral responses were analyzed by ELISA 6 weeks after the last immunization. The median optical density (O.D.) values (490 nm) for each group at various serum dilutions are represented. The positive threshold was defined as three times the mean for preimmune sera diluted 1/200 for each mouse. One star, P < 0.05 compared with the results for the control group (treatment with HBsAg alone); two stars, P < 0.05 compared with the results for any other group.

FIG. 7.

Anti-NS3 humoral response induced in BALB/c mice following immunization with NS3 helicase combined with C16:0-LPC or C18:0-LPC. Groups of six mice were immunized three times at 3-week intervals with 1 μg of NS3 helicase combined with either C16:0-LPC (A and B) or C18:0-LPC (C and D) at decreasing doses from 500 to 75 nmol (A and C) or 75 to 12.5 nmol (B and D). The humoral responses were analyzed by ELISA 3 weeks after the last immunization. The median optical density (O.D.) values (490 nm) for each group at various serum dilutions are represented. The positive threshold was defined as three times the mean for preimmune sera diluted 1/200 for each mouse. One star, P < 0.05 compared with the results for the control group (treatment with NS3 helicase alone); two stars, P < 0.05 compared with the results for any other group.