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Infection and Immunity, July 2005, p. 4070-4080, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4070-4080.2005

Characterization of Native Outer Membrane Vesicles from lpxL Mutant Strains of Neisseria meningitidis for Use in Parenteral Vaccination

Makda Fisseha,{dagger} Ping Chen,{ddagger} Brenda Brandt, Todd Kijek,§ Elizabeth Moran, and Wendell Zollinger*

The Walter Reed Army Institute of Research, Silver Spring, Maryland

Received 20 December 2004/ Returned for modification 25 January 2005/ Accepted 20 February 2005

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 {Delta}lpxL2 mutants had a reduced growth rate, reduced level of LOS expression, and increased sensitivity to surfactants. In addition, {Delta}synX {Delta}lpxL2 double mutants had reduced viability in stationary phase. The {Delta}lpxL1 {Delta}lpxL2 double mutant behaved essentially the same as the {Delta}lpxL2 single mutant. LOS from both lpxL mutant strains exhibited altered migration on polyacrylamide gels. The LOS of {Delta}lpxL2 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 {Delta}lpxL2 mutant NOMV were lower than those induced by {Delta}lpxL1 or wild-type NOMV. However, immunogenicity could be largely restored by use of an adjuvant. These results provide evidence that NOMV from {Delta}lpxL2 mutant strains will be safe and immunogenic in humans when given parenterally.

* Corresponding author. Mailing address: Department of Bacterial Diseases, WRAIR, 503 Robert Grant Ave., Silver Spring, MD, 20910-7500. Phone: (301) 319-9901. Fax: (301) 319-9123. E-mail: wendell.zollinger{at}na.amedd.army.mil.

Editor: D. L. Burns

{dagger} Present address: Nestlé Research Center, Lausanne, Switzerland.

{ddagger} Present address: Sequella, Inc., Rockville, Maryland.

§ Present address: U.S. Army 121 General Hospital, Seoul, South Korea.

Infection and Immunity, July 2005, p. 4070-4080, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4070-4080.2005




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