Betsy Kuipers

Production, characterization and control of a Neisseria meningitidis hexavalent class 1 outer membrane protein containing vesicle vaccine.

An experimental serogroup B meningococcal vaccine was prepared from two genetically engineered strains; each expressing three different class 1 outer membrane proteins (OMPs) (PorA). The two strains expressed the subtypes P1.7,16;P1.5,2;P1.19,15 and P1.5c,10;P1.12,13;P1.7h,4, respectively. Outer membrane vesicles (OMV) were prepared from these strains by deoxycholate extraction, mixed with aluminiumphosphate as adjuvant and formulated to final vaccines. The class 1 OMPs represent ca 90% of the protein in the vaccine. The vaccine was found safe for human use and induced a bactericidal immune response in mice against five of the six wild type strains, which served as donors for the various por A genes.

Immunogenicity and safety of a hexavalent meningococcal outer-membrane-vesicle vaccine in children of 2-3 and 7-8 years of age.

To study the reactogenicity and immunogenicity of a hexavalent meningococcal outer-membrane-vesicle vaccine (OMV), two different dosages of this vaccine (7.5 and 15 microg of individual PorA proteins) consisting of vesicles expressing class 1 outer-membrane proteins (OMPs) of subtypes P1.7,16; P1.5,2; P1.19,15 and P1.5(c), 10; P1.12,13; P1.7(h),4 were administered to a group of 7-8 year (n=165) and a group of 2-3 year old children (n=172). Control groups of children with similar ages were vaccinated against hepatitis B. All participants received three injections. Pre- and postimmunisation sera were tested for bactericidal antibodies against six isogenic meningococcal vaccine strains expressing different PorA proteins. Antibody titres against OMP of the two different vesicles (PL16215 and PL10124) were measured by ELISA. The meningococcal hexavalent OMV vaccine was well tolerated. No statistically significant differences were seen between the high and low dose of hexavalent meningococcal OMV vaccine. The percentage of children showing a fourfold increase of bactericidal antibody titres against the specific serosubtype varied in toddlers from 28 to 98% and in older children from 16 to 100%. Both ELISA antibody titres and bactericidal activity showed the highest level in the youngest age-group.

The genetic basis of the phase variation repertoire of lipopolysaccharide immunotypes in Neisseria meningitidis

Neisseria meningitidis strains express a diverse range of lipopolysaccharide (LPS) structures that have been classified into 12 immunotypes. A feature of meningococcal LPS is the reversible, high-frequency switching of expression (phase variation) of terminal LPS structures. A number of studies are strongly suggestive of a key role for these terminal structures, and their phase-variable expression, in pathogenesis. In a previous study, a locus of three LPS biosynthetic genes, IgtABE, involved in the biosynthesis of one of these terminal structures, lacto-N-neotetraose, was described. The molecular mechanism of phase-variable expression of this structure is by high-frequency mutation in a homopolymeric tract of G residues in the IgtA gene. To investigate the genetic basis of the structural differences between the immunotypes, and the potential for strains to express alternative immunotypes, this locus was examined in all of the immunotype strains. Initially, the Igt locus of strain 126E, an L1 immunotype strain, was cloned and sequenced, revealing two active genes, IgtC and IgtE. The remnants of the IgtA and IgtB genes and an inactive IgtD gene were also present, indicating that the locus may have once contained five active genes, similar to a locus previously reported in Neisseria gonorrhoeae strain F62. Probes based on each of the Igt genes (ABCDE), and the recently reported IgtG gene, were used to determine the presence or absence of Igt genes within individual strains, allowing the prediction of the phase variation repertoire of these strains. Sequencing to determine the nature of homopolymeric tract regions within the Igt genes was carried out to establish the potential for LPS switching. In general, the set of strains examined could be sorted into two distinct groups: one group which phase-vary the alpha-chain extension via IgtA or IgtC but cannot make beta-chain; the second group phase-vary the beta-chain extension via IgtG but do not vary alpha-chain (lacto-N-neotetraose).

Expression of the Lipopolysaccharide-Modifying Enzymes PagP and PagL Modulates the Endotoxic Activity of Bordetella pertussis

ABSTRACT Lipopolysaccharide (LPS) is one of the major constituents of the gram-negative bacterial cell envelope. Its endotoxic activity causes the relatively high reactogenicity of whole-cell vaccines. Several bacteria harbor LPS-modifying enzymes that modulate the endotoxic activity of the LPS. Here we evaluated whether two such enzymes, i.e., PagP and PagL, could be useful tools for the development of an improved and less reactogenic whole-cell pertussis vaccine. We showed that expression of PagP and PagL in Bordetella pertussis leads to increased and decreased endotoxic activity of the LPS, respectively. As expected, PagP activity also resulted in increased endotoxic activity of whole bacterial cells. However, more unexpectedly, this was also the case for PagL. This paradoxical result may be explained, in part, by an increased release of LPS, which we observed in the PagL-expressing cells.

Serum bactericidal activity and isotype distribution of antibodies in toddlers and schoolchildren after vaccination with RIVM hexavalent PorA vesicle vaccine

A clinical phase II trial with the RIVM hexavalent OMV vaccine containing six different PorAs was carried out in toddlers (2-3 years) and schoolchildren (7-8 years) in The Netherlands. Children were vaccinated three times (0, 2, 8 months). Sera after two and three vaccinations were analysed for serum bactericidal activity (SBA) and isotype distribution in whole cell enzyme linked immunosorbent assay (ELISA). The SBA after vaccination against the six PorAs was significantly different. We investigated whether the age specific and PorA specific differences in SBA titers correlated with differences in PorA specific IgG isotype distribution. The SBA titers were higher in toddlers compared with schoolchildren. After vaccination, IgG1 antibodies dominated the response followed by IgG3 antibodies. IgG2 levels were low, whereas IgG4 was not detected. Irrespective of PorA, IgG total and isotype specific titers after two and three vaccinations were significantly higher in toddlers than in schoolchildren. A weak correlation was found between IgG total or IgG1 and SBA. Although the immunogenicity of the six PorAs is very different, the isotype distribution was similar for all six tested PorAs. We conclude that the RIVM hexavalent PorA vesicle vaccine induces bactericidal antibodies mainly of the IgG1 and IgG3 isotypes that are considered to be most important for protection against disease. The isotype distribution of the response is not age-dependent.

Relative Immunogenicity of PorA Subtypes in a Multivalent Neisseria meningitidis Vaccine Is Not Dependent on Presentation Form

ABSTRACT The hexavalent meningococcal vaccine HexaMen, containing six PorAs on two vesicles, was tested in clinical studies. Although fourfold increases in serum bactericidal activity (SBA) titers against all of the PorAs were observed, there were significant differences between PorA-specific SBA titers. SBA titers were mainly directed against one PorA from each vesicle, P1.5-2,10 and P1.5-1,2-2, and were lower against the other PorAs, especially P1.7-2,4 and P1.19,15-1. We investigated whether these differences were due to immunological interference that resulted in competition between the three PorAs on the same vesicle or whether they were caused by a difference in the immunogenicities of the separate PorAs. Therefore, mice were immunized either with HexaMen, with six monovalent outer membrane vesicles (OMVs) representing the same six PorAs simultaneously (HexaMix), or with only one of the monovalent OMVs. The immunoglobulin G and SBA titers after HexaMen immunization in mice resembled the results obtained in clinical studies. Although immunization with HexaMix gave higher titers than immunization with HexaMen for some PorAs, the pattern of high and low titers was the same. Similar differences in immunogenicity between subtypes were seen after monovalent immunization when interference was eliminated as a cause of the differences. Monovalent immunization resulted in higher titers for P1.5-1,2-2 and P1.7,16 than immunization with HexaMen. However, no significant differences were found for the weakly immunogenic PorAs, P1.7-2,4 and P1.19,15-1. Since immunization with the six PorAs in the trivalent presentation form (HexaMen) and in the mixture of monovalent vesicles (HexaMix) resulted in the same pattern of high and low titers, we concluded that the differences between the PorA-specific responses are due to differences in the immunogenicities of the various PorAs and not due to interference that results in competition between different PorAs.

Description of a hybridoma bank towards Bordetella pertussis toxin and surface antigens

This paper describes the development of a murine bank of monoclonal antibodies against Bordetella pertussis toxin, filamentous hemagglutinin (FHA), pili, lipopolysaccharide (LPS), or outer membrane proteins (OMPs). Subunits S1, S2, S3 of pertussis toxin (PT) bound immunoglobulins and glycoproteins such as fetuin and haptoglobin in an unspecific manner. The specificity of monoclonal antibodies towards subunits S1, S2, S3 or S4 of PT could be demonstrated by using purified immunoglobulins or their Fab2 fragments. A set of FHA-specific monoclonal antibodies could be differentiated on the basis of their binding to the various breakdown products present in FHA preparations. Pili-specific monoclonal antibodies reacted with either native pili or denatured pilin, and both demonstrated serotype specificity. Monoclonal antibodies to Bordetella pertussis OMPs were directed to either the virulent phase-regulated trypsin-sensitive, detergent-extractable OMPs 92 kDa, 32 kDa, and 30 kDa or the non-virulent phase-expressed, not-trypsin sensitive OMPs 38 kDa, 33kDa, and 18 kDa.

Antibody specificities and effect of meningococcal carriage in icelandic teenagers receiving the Norwegian serogroup B outer membrane vesicle vaccine.

ABSTRACT Antibody specificities of pre- and postvaccination serum samples from 40 (53%) teenagers who received three doses of the Norwegian Neisseria meningitidis serogroup B vaccine (B:15:P1.7,16) during a previous trial in Iceland (Perkins et al., J. Infect. Dis. 177:683-691, 1998) were analyzed with serum bactericidal activity (SBA) and immunoblotting assays with reference and isogenic meningococcal H44/76 vaccine strains. The H44/76 variants demonstrated significant vaccine-induced SBA to P1.7,16 PorA and Opc but not to PorB, Opa5.5, and a heterologous PorA protein. On blots, immunoglobulin G levels to all these proteins increased significantly after vaccination. Measurement of SBA to the two main variable regions (P1.7 and P1.16) on the P1.7,16 PorA with PorA deletion mutants revealed significantly higher activity to the P1.7,− and P1.−,16 mutants compared to the P1.7,16 strain, indicating exposure of new accessible epitopes. Only 12 (30%) serum samples showed distinct decreases with these or the P1.−,− mutant, with most samples containing SBA to the P1.7 and P1.16 combination. In contrast, P1.16-specific antibodies were mainly found on blots. Thirteen of the vaccinees (32.5%) were carriers of meningococci at the time of the third dose, of whom four (30.8%) harbored strains of the ET-5 complex. Carriage of P1.15 strains was generally reflected in ≥4-fold increases in SBA and distinct immunoglobulin G binding to the P1.19,15 PorA on blots. Although vaccination did not elicit bactericidal activity to the serotype 15 PorB, most carriers of serotype 15 strains showed ≥4-fold increases in SBA to this antigen.

Mass Tag-Assisted Identification of Naturally Processed HLA Class II-Presented Meningococcal Peptides Recognized by CD4+ T Lymphocytes

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of 14N- and 15N-labeled isoforms of the neisserial porin A serosubtype P1.5–2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities (∼30–4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4+ T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4+ T cell epitopes relevant for vaccine development.

Differential Effect of TLR2 and TLR4 on the Immune Response after Immunization with a Vaccine against Neisseria meningitidis or Bordetella pertussis

Neisseria meningitidis and Bordetella pertussis are Gram-negative bacterial pathogens that can cause serious diseases in humans. N. meningitidis outer membrane vesicle (OMV) vaccines and whole cell pertussis vaccines have been successfully used in humans to control infections with these pathogens. The mechanisms behind their effectiveness are poorly defined. Here we investigated the role of Toll-like receptor (TLR) 2 and TLR4 in the induction of immune responses in mice after immunization with these vaccines. Innate and adaptive immune responses were compared between wild type mice and mice deficient in TLR2, TLR4, or TRIF. TRIF-deficient and TLR4-deficient mice showed impaired immunity after immunization. In contrast, immune responses were not lower in TLR2−/− mice but tended even to be higher after immunization. Together our data demonstrate that TLR4 activation contributes to the immunogenicity of the N. meningitidis OMV vaccine and the whole cell pertussis vaccine, but that TLR2 activation is not required.