Characterization of native outer membrane vesicles from lpxL mutant strains of Neisseria meningitidis for use in parenteral vaccination

Abstract

Native outer membrane vesicles (NOMV) of Neisseria meningitidis consist of intact outer membrane and contain outer membrane proteins (OMP) and lipooligosaccharides (LOS) in their natural conformation and membrane environment. NOMV have been safely used intranasally in P1 studies with encouraging results, but they are too toxic for parenteral vaccination. We now report the preparation and characterization of lpxL mutants that express LOS with reduced toxicity, and the evaluation of the potential of NOMV from these strains for use as a parenteral vaccine. A series of deletion mutants were prepared with knockouts of one or more of the lpxL1, lpxL2, or synX genes. The deltalpxL2 mutants had a reduced growth rate, reduced level of LOS expression, and increased sensitivity to surfactants. In addition, deltasynX deltalpxL2 double mutants had reduced viability in stationary phase. The deltalpxL1 deltalpxL2 double mutant behaved essentially the same as the deltalpxL2 single mutant. LOS from both lpxL mutant strains exhibited altered migration on polyacrylamide gels. The LOS of deltalpxL2 mutants of L3,7 strains were fully sialylated. NOMV prepared from lpxL2 mutants was about 200-fold less active than wild-type NOMV in rabbit pyrogen tests and in tumor necrosis factor alpha release assays. Bactericidal titers induced in animals by deltalpxL2 mutant NOMV were lower than those induced by deltalpxL1 or wild-type NOMV. However, immunogenicity could be largely restored by use of an adjuvant. These results provide evidence that NOMV from deltalpxL2 mutant strains will be safe and immunogenic in humans when given parenterally.

FIG. 1.

Growth curves of wild-type and mutant N. meningitidis strains in liquid culture. (A) Strains with lpxL mutations: wild-type 44/76 (•); ΔlpxL1 44/76 (○); ΔlpxL2 44/76 (▴); ΔlpxL1 ΔlpxL2 44/76 (▵). (B) Strains with ΔsynX and ΔlpxL mutations: wild-type 44/76 (•); 44/76 ΔsynX (○); ΔsynX ΔlpxL1 44/76 (▵); ΔsynX ΔlpxL2 44/76 (▴). OD readings are direct readings without dilution.

FIG. 2.

Silver stained polyacrylamide gels of LOS expressed by ΔlpxL and ΔsynX mutants of N. meningitidis. (A) An equal amount of NOMV protein was loaded in each lane: reference LOS from N. gonorrhoeae ML5, lane 1; ΔsynX 44/76 (L7), lane 2; ΔlpxL1 44/76 (L8), lane 3; wild-type 44/76 (L8), lane 4; ΔlpxL2 44/76 (L8), lane 5. (B) An equal amount of LOS was loaded in each lane: reference ML5 LOS (lanes 1 and 6); ΔsynX L7 44/76 (lane 2); ΔsynX ΔlpxL1 44/76 L7 (lane3); ΔlpxL2 44/76 L3 (lane 4); ΔsynX ΔlpxL2 44/76 L7 (lane 5).

FIG. 3.

Coomassie blue-stained polyacrylamide gel of NOMV from 44/76 mutants with ΔsynX and ΔlpxL mutations: lane 1,wild type; lane 2, ΔlpxL1 mutant; lane 3, ΔsynX ΔlpxL1 mutant; lane 4, molecular weight standards (beginning at the top) 97.4, 66.2, 42.7, 31, 21.5, and 14.4 kDa; lane 5, ΔlpxL2 mutant; lane 6, ΔsynX ΔlpxL2 mutant. Arrowheads on the left, beginning at the top, point to PorA, PorB, RmpM, and Opa proteins.

FIG. 4.

Release of cytokines TNF-α, IL-6, and IL-1β from human monocytes by different concentrations of NOMV from ΔsynX and ΔlpxL mutants of N. meningitidis 44/76: ΔsynX NOMV (•), ΔsynX ΔlpxL1 (○), and ΔsynX ΔlpxL2 (▴). All data were from test 1 (Table 4), which used monocytes from donor 1. LOS concentrations given refer to the amount of LOS in the NOMV that were added to the monocytes cultures.

FIG. 5.

Antibody responses of mice to vaccination with NOMV vaccine prepared from strain 44/76 ΔlpxL2 given with (▨) and without (▪) adsorption to aluminum hydroxide. (A) Bactericidal antibody response against wild-type 44/76. The data are mean log₂ reciprocal titers for groups of eight mice. Error bars indicate means ± the standard error of the mean. (B) IgG antibody responses to wild-type 44/76 NOMV by ELISA. Data are expressed as mean the log₂ μg of IgG antibody/ml for groups of eight mice.