Characterization of Native Outer Membrane Vesicles fromlpxLMutant Strains ofNeisseria meningitidisfor 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.