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.

Importance of antibodies to lipopolysaccharide in natural and vaccine-induced serum bactericidal activity against Neisseria meningitidis group B.

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.

Design and evaluation in mice of a broadly protective meningococcal group B native outer membrane vesicle vaccine.

A vaccine based on native outer membrane vesicles (NOMV) that has potential to provide safe, broad based protection against group B strains of Neisseria meningitidis has been developed. Three antigenically diverse group B strains of N. meningitidis were chosen and genetically modified to improve safety and expression of desirable antigens. Safety was enhanced by disabling three genes: synX, lpxL1, and lgtA. The vaccine strains were genetically configured to have three sets of antigens each with potential to induce protective antibodies against a wide range of group B strains. Preliminary immunogenicity studies with combined NOMV from the three strains confirmed the capacity of the vaccine to induce a broad based bactericidal antibody response. Analysis of the bactericidal activity indicated that antibodies to the LOS were responsible for a major portion of the bactericidal activity and that these antibodies may enhance the bactericidal activity of anti-protein antibodies.

Intranasal delivery of group B meningococcal native outer membrane vesicle vaccine induces local mucosal and serum bactericidal antibody responses in rabbits.

We have previously shown that intranasal immunization of mice with meningococcal native outer membrane vesicles (NOMV) induces both a good local mucosal antibody response and a good systemic bactericidal antibody response. However, in the intranasal mouse model, some of the NOMV entered the lung and caused an acute granulocytic response. We therefore developed an alternate animal model using the rabbit. This model reduces the probability of lung involvement and more closely mimics intranasal immunization of humans. Rabbits immunized intranasally with doses of 100 mug of NOMV in 0.5 ml of saline developed serum bactericidal antibody levels comparable to those of rabbits immunized intramuscularly with 25-mug doses, particularly when the primary intranasal immunization was given daily for 3 days. Intranasal immunization also induced a local mucosal response as evidenced by immunoglobulin A antibody in saliva, nasal washes, and lung lavage fluids. NOMV from a capsule-deficient mutant induced higher serum bactericidal antibody responses than NOMV from the encapsulated parent. Meningococcal NOMV could be administered intranasally at 400 mug with no pyrogenic activity, but as little as 0.03 mug/kg of body weight administered intravenously or 25 mug administered intramuscularly induced a pyrogenic response. These data indicate that the rabbit is a useful model for preclinical testing of intranasal meningococcal NOMV vaccines, and this immunization regimen produces a safe and substantial systemic and local mucosal antibody response.

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

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.

Immunogenicity and safety testing of a group B intranasal meningococcal native outer membrane vesicle vaccine.

The presently licensed meningococcal vaccine is a tetravalent capsular polysaccharide vaccine that induces immunity to serogroups A, C, Y, and W-135 but not to group B, which causes nearly half of the meningitis cases in the United States. The purpose of this study was to evaluate the safety and immunogenicity of an intranasal native outer membrane vesicle (NOMV) vaccine prepared from a capsule negative strain of group B of Neisseria meningitidis. In this study all volunteers received the same dose of vaccine, but we evaluated two different immunization schedules and the oropharyngeal and intranasal routes of vaccine delivery, assessed nasal cytology for cellular infiltration, and measured antibody-secreting cells (enzyme-linked immunospot assay [ELISPOT]) as an early marker for systemic immune response. Additionally, both intranasal and serum vaccine-specific antibodies were measured as well as serum bactericidal activity. Four groups with a total of 42 subjects were immunized on days 0, 28, and 56. Group 3 received an additional dose on day 7. Group 2 subjects were immunized both intranasally and oropharyngeally. Group 4 received a different lot of vaccine. All groups received approximately 1,200 microg of vaccine per subject. Patients were evaluated for side effects. The vaccine was well tolerated without evidence of inflammation on nasal cytology. The group receiving the extra vaccine dose showed the maximum increase in bactericidal activity. Thirty of 42 subjects demonstrated an increase in meningococcus-specific intranasal immunoglobulin A (IgA) titers, while 23 of 42 demonstrated an increase in specific IgG titers. The group receiving vaccine intranasally and oropharyngeally showed the highest rise in intranasal titers for both IgA and IgG. Groups 1, 3, and 4 showed a significant increase in antibody-secreting cells on ELISPOT. Eighteen of 42 volunteers demonstrated a fourfold or greater rise in bactericidal titers, with 81% showing an increase over baseline. We have demonstrated the immunogenicity and safety of a group B lipopolysaccharide-containing, intranasal, NOMV vaccine.