Elizabeth E. Moran

Safety and immunogenicity testing of an intranasal group B meningococcal native outer membrane vesicle vaccine in healthy volunteers

An intranasal vaccine composed of native outer membrane vesicles (NOMV) not exposed to detergent or denaturing agents was prepared from the group B meningococcal strain 9162 SynX(-)(-:15:P1.3:P5.10,11:L3,7,9) and tested in 32 healthy adult volunteers. Four groups of 8 volunteers were vaccinated intranasally with three doses of vaccine. The vaccine was very well tolerated in all dosing groups, despite the presence of lipo-oligosaccharide in the vaccine at a level of 25% relative to protein. The antibody response as measured by ELISA in serum, saliva and nasal wash fluids was relatively low in all 4 groups, but the induced serum antibodies had strong bactericidal activity. Persistent bactericidal antibodies (> or =4-fold increase) were produced in 75% of the recipients. Some of the bactericidal antibodies were cross reactive against divergent group B strains. Most of the bactericidal antibodies appeared to be specific for PorA and L3,7,9 LOS. The vaccine also produced a local antibody response which was detected in the nasal wash fluids of volunteers. These data suggest that nasal immunization with NOMV is a safe and effective approach to induce systemic and local immunity against the group B meningococcus and deserves further study.

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 ΔlpxL2 mutants had a reduced growth rate, reduced level of LOS expression, and increased sensitivity to surfactants. In addition, ΔsynX ΔlpxL2 double mutants had reduced viability in stationary phase. The ΔlpxL1 ΔlpxL2 double mutant behaved essentially the same as the ΔlpxL2 single mutant. LOS from both lpxL mutant strains exhibited altered migration on polyacrylamide gels. The LOS of ΔlpxL2 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 ΔlpxL2 mutant NOMV were lower than those induced by ΔlpxL1 or wild-type NOMV. However, immunogenicity could be largely restored by use of an adjuvant. These results provide evidence that NOMV from ΔlpxL2 mutant strains will be safe and immunogenic in humans when given parenterally.

An experimental outer membrane vesicle vaccine from N. meningitidis serogroup B strains that induces serum bactericidal activity to multiple serogroups

A trivalent native outer membrane vesicle vaccine that has potential to provide broad based protection against Neisseria meningitidis serogroup B strains has been developed. Preliminary immunogenicity studies in mice showed that the vaccine was capable of inducing an effective broad based bactericidal antibody response against N. meningitidis serogroup B strains. These findings in mice have been repeated with a cGMP trivalent NOMV vaccine and extended to show that the bactericidal antibody response induced by the vaccine in mice is effective against strains belonging to serogroups C, Y, W135, X, and NadA-expressing serogroup A strains. Taken together these results suggest that this experimental vaccine may provide protection against both serogroup B and non-serogroup B N. meningitidis strains.

Phase I study of a Neisseria meningitidis liposomal vaccine containing purified outer membrane proteins and detoxified lipooligosaccharide.

Purified outer membrane proteins and purified deacylated lipooligosaccharide (dLOS) were formulated for use as a vaccine in three formulations for clinical use. The three vaccine formulations included (1) purified outer membrane proteins (OMPs) and L8-5 dLOS adsorbed to aluminum hydroxide; (2) purified OMPs and L8-5 dLOS incorporated into liposomes; and (3) purified OMPs and L7 dLOS incorporated into proteoliposomes. The vaccines were compared for immunogenicity and safety in a phase 1clinical study. Ten adult volunteers were vaccinated with each of the three vaccine formulations. Two 50 μg doses were given six weeks apart, and serum samples were obtained at 0, 2, 6, 8 and 14 weeks. Volunteers were evaluated for reactogenicity 30 min after vaccination and at days 1, 2, and 14 after each vaccination, and laboratory safety tests were done at 0, 2 and 6 weeks. Overall, the vaccines were well tolerated. Bactericidal assays against a homologous strain showed a four-fold or greater increase in titer in 6 of 7 volunteers in group one, 9 of 10 volunteers in group two, and 5 of 10 volunteers in group three. A quantitative enzyme linked immunosorbant assay showed increases in antibody against both OMPs and LOS antigens. The liposome formulation appeared to be particularly effective in presenting the dLOS as an antigen.

Analysis of the Bactericidal Response to an Experimental Neisseria meningitidis Vesicle Vaccine

ABSTRACT Rabbit immunogenicity studies on an experimental trivalent native outer membrane vesicle vaccine derived from three serogroup B strains were conducted to evaluate the effectiveness of this vaccine at inducing an antibody response with serum bactericidal activity against meningococcal strains of other serogroups in addition to serogroup B strains. The results showed that the vaccine was capable of inducing an effective broad-based bactericidal antibody response in rabbits against a small sample of Neisseria meningitidis strains of serogroups C, W135, and X and, to a lesser extent, serogroups A and Y. Analysis of antibody specificity using a bactericidal depletion assay revealed that antibodies to lipooligosaccharide (LOS), PorA, and NadA induced in rabbits by the experimental trivalent outer membrane vesicle vaccine were responsible for most of the bactericidal activity against strains of the other N. meningitidis serogroups. In the case of serogroup A N. meningitidis strains, the outer membrane antigen NadA was primarily responsible for protection. The outer membrane antigens fHbp and OpcA were also effective in removing some bactericidal activity from the sera.

Characterization of an antibody depletion assay for analysis of bactericidal antibody specificity.

ABSTRACT Serum bactericidal antibodies are important for protection against systemic Neisseria meningitidis infections. Consequently, identifying the specific targets of bactericidal antibodies is important for understanding protective immunity to meningococcal disease and for vaccine development and evaluation. We have developed a new assay that can be used to investigate the specificity of serum bactericidal antibodies. Prior to testing for bactericidal activity, antibodies specific for a given antigen or group of antigens are depleted from a serum sample by incubation with the antigen(s) bound to the wells of a 96-well microplate. A dilution series of the antigen is bound to the plate to assess the effectiveness of the antigen in removing the bactericidal antibodies. Removal of antibodies with solid-phase antigen prior to bactericidal testing avoids depletion of complement by soluble immune complexes that can form when soluble antigen is present in the bactericidal test mixture (direct inhibition). The parameters associated with this assay are investigated and compared with those associated with a direct-inhibition assay. The bactericidal depletion assay can be an effective tool for studying the specificity of serum bactericidal antibodies.

Importance of Antibodies to Lipopolysaccharide in Natural and Vaccine-Induced Serum Bactericidal Activity against Neisseria meningitidis Group B

ABSTRACT Analysis of the specificity of bactericidal antibodies in normal, convalescent, and postvaccination human sera is important in understanding human immunity to meningococcal infections and can aid in the design of an effective group B vaccine. A collection of human sera, including group C and group B convalescent-phase sera, normal sera with naturally occurring cross-reactive bactericidal activity, and some postvaccination sera, was analyzed to determine the specificity of cross-reactive bactericidal antibodies. Analysis of human sera using a bactericidal antibody depletion assay demonstrated that a significant portion of the bactericidal activity could be removed by purified lipopolysaccharide (LPS). LPS homologous to that expressed on the bactericidal test strain was most effective, but partial depletion by heterologous LPS suggested the presence of antibodies with various degrees of cross-reactivity. Binding of anti-L3,7 LPS bactericidal antibodies was affected by modification of the core structure, suggesting that these functional antibodies recognized epitopes consisting of both core structures and lacto-N-neotetraose (LNnT). When the target strain was grown with 5′-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA) to increase LPS sialylation, convalescent-phase serum bactericidal titers were decreased by only 2- to 4-fold, and most remaining bactericidal activity was still depleted by LPS. Highly sialylated LPS was ineffective in depleting bactericidal antibodies. We conclude that natural infections caused by strains expressing L3,7 LPS induce persistent, protective bactericidal antibodies and appear to be directed against nonsialylated bacterial epitopes. Additionally, subsets of these bactericidal antibodies are cross-reactive, binding to several different LPS immunotypes, which is a useful characteristic for an effective group B meningococcal vaccine antigen.

The development of an experimental multiple serogroups vaccine for Neisseria meningitidis.

A native outer membrane vesicles (NOMV) vaccine was developed from three antigenically diverse strains of Neisseria meningitidis that express the L1,8, L2, and L3,7 lipooligosaccharide (LOS) immunotypes, and whose synX, and lpxL1 genes were deleted.. Immunogenicity studies in mice showed that the vaccine induced bactericidal antibody against serogroups B, C, W, Y and X N. meningitidis strains. However, this experimental NOMV vaccine was not effective against serogroup A N. meningitidis strains. N. meningitidis capsular polysaccharide (PS) from serogroups A, C, W and Y were effective at inducing bactericidal antibody when conjugated to either tetanus toxoid or the fHbp1-fHbp2 fusion protein fHbp(1+2). The combination of the NOMV vaccine and the N. meningitidis serogroup A capsular polysaccharide (MAPS) protein conjugate was capable of inducing bactericidal antibodies against a limited number of N. meningitidis strains from serogroups A, B, C, W, Y and X tested in this study.