Coincorporation of LpxL1 and PagL mutant lipopolysaccharides into liposomes with Neisseria meningitidis opacity protein: influence on endotoxic and adjuvant activity

Abstract

Wild-type lipopolysaccharide (LPS) of Neisseria meningitidis normally contains six acyl chains. Penta-acylated LPS forms were generated through inactivation of the lpxL1 gene or through the expression of the Bordetella bronchiseptica pagL gene in N. meningitidis. The resulting LPS species, designated LpxL1 LPS and PagL LPS, respectively, display reduced endotoxic activity compared to wild-type LPS. Here, we determined the adjuvant potential of PagL LPS by comparison with the broadly used LpxL1 LPS. We also investigated the potential benefit for adjuvanticity of coincorporating these LPS species, together with the meningococcal opacity-associated protein OpaJ as a model antigen, in a liposomal delivery system. PagL LPS showed a higher endotoxic activity than LpxL1 LPS, and their incorporation into liposomes significantly reduced their endotoxic activity as determined by measuring the induction of interleukin-6 (IL-6) production in a murine macrophage cell line. To determine the adjuvant effect, BALB/c mice were immunized with OpaJ-containing liposomes and either free LPS or LPS coincorporated into the proteoliposomes. OpaJ-containing liposomes adjuvanted with AlPO(4) or not adjuvanted at all were included as control groups. In the appropriate dose, PagL LPS showed a superior adjuvant effect compared with LpxL1 LPS, and for both LPS types, free LPS showed a higher adjuvant effect than when coincorporated into the liposomes, as evidenced by higher titers of IgG2a and IgG2b antibodies against OpaJ(+) meningococci and higher bactericidal titers. In conclusion, PagL LPS is a better adjuvant than LpxL1 LPS, but coincorporation of either LPS into proteoliposomes did not improve their adjuvant activity.

FIG. 1.

Incorporation of OpaJ protein and LPS derivatives into liposomes analyzed by flow cytometry and electrophoretic techniques. (A) Reactivity of anti-OpaJ MAb 15-1-P5.5 against OpaJ-containing liposomes measured by FCM analysis. (B) Reactivity of the anti-OpaJ MAb with liposomes containing LpxL1 LPS and OpaJ (continuous line) and with liposomes containing PagL LPS and OpaJ (dotted line). The inset shows the heat modifiability of OpaJ coincorporated with PagL or LpxL1 LPS into liposomes determined in seminative SDS-PAGE as a measure for its correct folding. Before electrophoresis, the samples were treated for 10 min in sample buffer with 0.1% SDS at room temperature (RT) or with 2% SDS at 100°C. The positions of molecular size markers are indicated at the left side of the membrane. (C) Reactivity of anti-L8 LPS MAb 43F8.10 with liposomes containing LpxL1 LPS and OpaJ (continuous line) and with liposomes containing PagL LPS and OpaJ (dotted line). The inset demonstrates the presence of LPS in the liposomes in an LPS-specific silver-stained Tricine-glycine-SDS-polyacrylamide gel. (D) Reactivity of the anti-L8 LPS MAb against liposomes containing PagL LPS and OpaJ in the presence (dotted line) or absence (continuous line) of the anti-OpaJ MAb. In all graphics, the reaction of antibodies with empty liposomes was included as negative control (gray-filled profile).

FIG. 2.

Binding of LpxL1 and PagL LPS to OpaJ protein. (A) Western blot patterns of liposomes containing OpaJ and PagL LPS or only OpaJ incubated with the anti-OpaJ MAb 15-1-P5.5 (left panel) or the anti-L8 LPS MAb 43F8.10 (right panel). Prior to SDS-PAGE, the samples were treated in sample buffer with 0.1% SDS at room temperature (RT) or with 2% SDS at 100°C. A complex of LPS and OpaJ is indicated in the right panel with an arrow. The positions of molecular size markers are indicated at the left side of the membranes. (B) Far-dot blots containing 15 μg of membrane-immobilized OpaJ that was denatured by heating for 30 min (100°C) or not (RT) prior to application on the membrane. The membranes were subsequently incubated with 500 μg of purified PagL LPS (a) or LpxL1 LPS (b) and then with the anti-L8 LPS-specific MAb 43F8.10. Bovine serum albumin (BSA) was used as a control for background binding.

FIG. 3.

IL-6 induction in mouse macrophage cell line J774A.1 by PagL LPS (A) and LpxL1 LPS (B). Tested were free LPS, liposomes with incorporated LPS, and liposomes with coincorporated LPS and OpaJ at LPS/protein ratios of 1:5 and 1:50 (μmol/μg). The IL-6 concentration in the supernatant of cell cultures was quantified by ELISA. The data represent the averages and standard errors from three independent experiments. Statistically significant differences between liposome preparations are marked with one asterisk (P < 0.05).

FIG. 4.

Specific IgG responses in sera obtained after immunization of mice with OpaJ-containing liposomes using different adjuvants. (A) Western blots containing whole-cell lysates of OpaJ⁺ cells of N. meningitidis strain H44/76 with immunotype L3 probed with pooled sera of mice immunized with nonadjuvanted liposomes (lane 1) or liposomes adjuvanted with LpxL1 LPS coincorporated into the liposomes (lane 2), free LpxL1 LPS (lane 3), 1 μg of PagL LPS coincorporated into the liposomes (lane 4), 1 μg of free PagL LPS (lane 5), or nonimmune serum (lane 6). Lane 7 shows the reactivity of the sera of the mice immunized with proteoliposomes and 1 μg of free PagL LPS with a whole-cell lysate of Opa⁻ cells. Prior to SDS-PAGE, the bacterial suspensions were heated in sample buffer with 2% SDS at 100°C. The positions of molecular size markers are indicated at the left. (B) Quantitative total IgG titers against purified L8 LPS measured in ELISAs. Sera from mice immunized with LpxL1 LPS-adjuvanted liposomes and from the control groups were tested against purified LpxL1 L8 LPS, and those from mice immunized with PagL LPS-adjuvanted liposomes were tested against purified PagL L8 LPS. The data show the geometric mean (horizontal line), individual serum titers, and number of responder mice/group. Statistically significant differences between serum groups are marked with one asterisk (P < 0.05). Similar results were obtained when the sera tested here against purified LpxL1 L8 LPS were tested against PagL LPS and vice versa (data not shown). No reactivity was detected with L3 LPS. Note that only LPS of immunotype L8 was used for the immunizations.

FIG. 5.

Functional activity of sera obtained after immunization of mice with several OpaJ-containing liposome formulations tested in SBA assays using Opa⁺ cells of N. meningitidis strain H44/76 of immunotype L3. The data represent the geometric mean (horizontal line), the SBA titers of individual mice, and the number of responder mice/group. Results are given for the sera of mice immunized with OpaJ-containing liposomes alone (control), with OpaJ-containing liposomes adjuvanted with AlPO₄ at 0.5 mg/ml (control + AlPO₄), or with the different LPS species (with dose/mouse indicated in parentheses) incorporated or not into liposomes. Statistically significant differences between sera groups are marked with one asterisk (P < 0.05). SBA titers were considered positive at serum dilutions higher than 10.