Wendell D. Zollinger

Efficacy, safety, and immunogenicity of a meningococcal group B (15:P1.3) outer membrane protein vaccine in Iquique, Chile

A meningococcal group B (15:P1.3) outer membrane protein vaccine was tested for efficacy in a randomized, double-blind controlled study in Iquique, Chile. A total of 40 811 volunteers, ages 1-21 years, enrolled in the study. Volunteers received two doses of vaccine six weeks apart by jet injector. Both the experimental vaccine and the control vaccine (Menomune, A, C, Y and W135 meningococcal polysaccharide vaccine) were well tolerated with minor side-effects. Active surveillance for suspected cases of meningococcal disease was conducted for 20 months in Iquique. Eighteen cases of group B meningococcal disease were confirmed during the 20 months. Efficacy was estimated to be 51% (p = 0.11) for all ages combined. In children aged 1-4 no protection was evident, but in volunteers aged 5-21 vaccine efficacy was 70% (p = 0.045). The IgG antibody response by ELISA was characterized by a large booster effect after the second dose, followed by a substantial drop in antibody levels by 6 months. The youngest children had the highest responses. The bactericidal antibody response, on the other hand, was characterized by the lack of a significant booster response, higher responses in the older children, and an increase in the geometric mean titer in the later months of the study in the older children.

Description and Nomenclature of Neisseria meningitidis Capsule Locus

Pathogenic Neisseria meningitidis isolates contain a polysaccharide capsule that is the main virulence determinant for this bacterium. Thirteen capsular polysaccharides have been described, and nuclear magnetic resonance spectroscopy has enabled determination of the structure of capsular polysaccharides responsible for serogroup specificity. Molecular mechanisms involved in N. meningitidis capsule biosynthesis have also been identified, and genes involved in this process and in cell surface translocation are clustered at a single chromosomal locus termed cps. The use of multiple names for some of the genes involved in capsule synthesis, combined with the need for rapid diagnosis of serogroups commonly associated with invasive meningococcal disease, prompted a requirement for a consistent approach to the nomenclature of capsule genes. In this report, a comprehensive description of all N. meningitidis serogroups is provided, along with a proposed nomenclature, which was presented at the 2012 XVIIIth International Pathogenic Neisseria Conference.

A phase 1 study of a meningococcal native outer membrane vesicle vaccine made from a group B strain with deleted lpxL1 and synX, over-expressed factor H binding protein, two PorAs and stabilized OpcA expression.

This phase I clinical trial assessed the safety and immunogenicity of a native outer membrane vesicle (NOMV) vaccine prepared from an lpxL1(-) synX(-) mutant of strain 8570(B:4:P1.19,15:L8-5) of Neisseria meningitidis. Additional mutations enhance the expression of factor H binding protein variant 1 (fHbp v.1), stabilize expression of OpcA and introduce a second PorA (P1.22,14). Thirty-six volunteers were assigned to one of four dose groups (10, 25, 50 and 75 mcg, based on protein content) to receive three intramuscular injections at six week intervals with aluminum hydroxide adjuvant. Specific local and systemic adverse events were solicited by diary and at visits on days 2, 7, and 14 after each vaccination. Blood chemistries, complete blood count, and coagulation studies were measured on each vaccination day and again 2 and 14 days later. Blood for ELISA and serum bactericidal assays was drawn two and six weeks after each vaccination. The proportion of volunteers who developed a fourfold or greater increase in bactericidal activity to the wild type parent of the vaccine strain at two weeks after the third dose was 27 out of 34 (0.79, 95% C.I. 0.65-0.93). Against four other group B strains the response rate ranged from 41% to 82% indicating a good cross reactive antibody response. Depletion assays show contributions to bactericidal activity from antibodies to lipooligosaccharide (LOS), fHbp v.1 and OpcA.

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.

Use of a zwitterionic detergent for the restoration of the antibody-binding capacity of electroblotted meningococcal outer membrane proteins

A method is described for the partial restoration of the antibody-binding capacity of meningococcal class 2 outer membrane proteins (40-42 K molecular weight) following denaturation (dissociation) in boiling sodium dodecyl sulfate (SDS). The method relies on the presence of 0.1-0.4% zwitterionic detergent in the electrode buffer during the electroblot transfer of proteins from SDS-polyacrylamide gels to nitrocellulose paper. Meningococcal class 2 proteins which had lost their earlier capacity to bind mouse monoclonal antibodies in the normal blot procedure after SDS-polyacrylamide gel electrophoresis, bound monoclonal antibodies following the addition of the zwitterionic detergent to the blot buffer. Human post-vaccination anti-class 2 protein antibodies reacted with serotype 2a class 2 protein in a similar manner. This simple modification to the electroblot procedure proved helpful in identifying mouse monoclonal antibodies and human antibodies specific for native meningococcal class 2 proteins.

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.

Immunogenicity and Safety Testing of a Group B Intranasal Meningococcal Native Outer Membrane Vesicle Vaccine

ABSTRACT 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 μg 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.

GD3/proteosome vaccines induce consistent IgM antibodies against the ganglioside GD3

The gangliosides of melanoma and other tumours of neuroectodermal origin are suitable targets for immune intervention with tumour vaccines. The optimal vaccines in current use contain ganglioside plus bacillus Calmette-Guérin and induce considerable morbidity. We have screened a variety of new adjuvants in the mouse, and describe one antigen-delivery system, proteosomes, which is especially effective. Highly hydrophobic Neisserial outer membrane proteins (OMP) form multimolecular liposome-like vesicular structures termed proteosomes which can readily incorporate amphiphilic molecules such as GD3 ganglioside. The optimal GD3/proteosome vaccine formulation for induction of GD3 antibodies in the mouse is determined. Interestingly, the use of potent immunological adjuvants in addition to proteosomes augments the IgM and IgG antibody titres against OMP in these vaccines but GD3 antibody titres are unaffected. The application of proteosomes to enhance the immune response to GD3 extends the concept of the proteosome immunopotentiating system from lipopeptides to amphipathic carbohydrate epitopes such as cell-surface gangliosides. The demonstrated safety of meningococcal OMP in humans and the data in mice presented here suggest that proteosome vaccines have potential for augmenting the immunogenicity of amphipathic tumour antigens in humans.

A rapid and sensitive PCR strategy employed for amplification and sequencing of porA from a single colony-forming unit of Neisseria meningitidis

The predicted amino acid sequence was determined for the class-1 outer membrane protein, PorA, from a B:15:P1.7,3 strain of Neisseria meningitidis that is currently causing an epidemic of meningitis in Northern Chile. The P1.7,3 PorA showed a unique sequence in the exposed loop 4 of the putative porin structure that is different from all the reported PorA sequences. Based on the nucleotide (nt) sequence of the P1.7,3 porA, we designed two sets of PCR (polymerase chain reaction) primers that specifically amplified porA from any N. meningitidis strain, and a third set of primers that amplified porA only from the P1.7,3 strain. Using these primers, we developed a sensitive double hot-start nested PCR (HNPCR) strategy that could amplify porA and generate nt sequence from as low as a single colony-forming unit. This strategy consisted of three phases of PCR. The first two phases were designed to generate amplified target DNA that could be directly visualized by ethidium bromide staining starting from one to two molecules of Neisseria genome. The third phase was designed to generate a sequence of several hundred nt directly from the amplified DNA. A number of culture-negative cerebrospinal fluid samples from individuals suspected of meningitis during a vaccine trial were analyzed by this strategy to obtain more accurate information on the actual number of cases that occurred in the study and the non-study populations. The basic HNPCR strategy described here could be applied to amplify and sequence target DNAs from any low-copy-number biological sample.

Invasive Meningococcal Disease in Québec, Canada, Due to an Emerging Clone of ST-269 Serogroup B Meningococci with Serotype Antigen 17 and Serosubtype Antigen P1.19 (B:17:P1.19)

ABSTRACT During periods of endemic meningococcal disease, serogroup B Neisseria meningitidis is responsible for a significant percentage of invasive diseases, and no particular clone or strain predominates (F. E. Ashton and D. A. Caugant, Can. J. Microbiol. 47: 293-289, 2001), However, in the winter of 2004 to 2005, a cluster of serogroup B meningococcal disease occurred in one region in the province of Québec, Canada. The N. meningitidis strain responsible for this cluster of cases was identified as sequence type ST-269 with the antigenic formula B:17:P1.19. Retrospective analysis of isolates from 2000 onwards showed that this clone first emerged in the province of Québec in 2003. The emergence of this clone of serogroup B meningococci occurred after a mass vaccination against serogroup C N. meningitidis, suggesting possible capsule replacement.