Biomedical subjects

Betsy Kuipers

Publications and source records attributed to Betsy Kuipers.

8 recordsLinked to original sources

PubMed2006

Expression of the lipopolysaccharide-modifying enzymes PagP and PagL modulates the endotoxic activity of Bordetella pertussis.

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.

Acylation↗

PubMed2005

Multi-antigen immunization using IgG binding domain ZZ as carrier.

This article describes a method in which multiple vaccine candidates can be tested in parallel for their immunogenicity. Antigens derived from the genome sequence of Neisseria meningitidis group B strain MC58 were cloned and expressed as recombinant proteins fused to the IgG-binding domain ZZ or to a His-tag. Immunization of mice with a mixture of 22 ZZ-fusion antigens applied with the adjuvant QuilA, induced an enhanced immune response as compared to the same antigen mixture without QuilA or a mixture containing the corresponding His-tagged antigens with QuilA. The enhanced immune response of the ZZ-fusion antigens/QuilA preparation was apparent from 1) the higher number of antigens in the mixture that elicited an antibody response and 2) the much lower antigen dose needed to get this response. Our approach using ZZ-fusion antigens/QuilA mixtures may serve as a high throughput discovery tool for new vaccine candidates.

Adjuvants, Immunologic↗

PubMed2005

Crystal structure of an Anti-meningococcal subtype P1.4 PorA antibody provides basis for peptide-vaccine design.

In various western countries, subtype P1.4 of Neisseria meningitidis serogroup B causes the greatest incidence of meningococcal disease. To investigate the molecular recognition of this subtype, we crystallised a peptide (P1HVVVNNKVATH(P11)), corresponding to the subtype P1.4 epitope sequence of outer membrane protein PorA, in complex with a Fab fragment of the bactericidal antibody MN20B9.34 directed against this epitope. Structure determination at 1.95 A resolution revealed a unique complex of one P1.4 antigen peptide bound to two identical Fab fragments. One Fab recognises the putative epitope residues in a 2:2 type I beta-turn at residues P5NNKV(P8), whereas the other Fab binds the C-terminal residues of the peptide that we consider a crystallisation artefact. Interestingly, recognition of the P1.4 epitope peptide is mediated almost exclusively through the complementarity-determining regions of the heavy chain. We exploited the observed turn conformation for designing conformationally restricted cyclic peptides for use as a peptide vaccine. The conformational stability of the two peptide designs was assessed by molecular dynamics simulations. Unlike the linear peptide, both cyclic peptides, conjugated to tetanus toxoid as a carrier protein, elicited antibody responses in mice that recognised meningococci of subtype P1.7-2,4. Serum bactericidal assays showed that some, but not all, of the sera induced with the cyclic peptide conjugates could activate the complement system with titres that were very high compared to the titres induced by complete PorA protein in its native conformation administered in outer membrane vesicles.

Animals↗

PubMed2005

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 (14)N- and (15)N-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 ( approximately 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.

Amino Acid Sequence↗

PubMed2003

Immunogenicity of peptide-vaccine candidates predicted by molecular dynamics simulations.

We present an in silico, structure-based approach for design and evaluation of conformationally restricted peptide-vaccines. In particular, we designed four cyclic peptides of ten or 11 residues mimicking the crystallographically observed beta-turn conformation of a predicted immunodominant loop of PorA from Neisseria meningitidis. Conformational correctness and stability of the peptide designs, as evaluated by molecular dynamics simulations, correctly predicted the immunogenicity of the peptides. We observed a peptide-induced functional antibody response that, remarkably, exceeded the response induced by the native protein in outer membrane vesicles, without losing specificity for related strains. The presented approach offers tools for a priori design and selection of peptide-vaccine candidates with full biological activity. This approach could be widely applicable: to outer membrane proteins of Gram-negative bacteria, and to other epitopes in a large range of pathogens.

Amino Acid Sequence↗

PubMed2003

Relative immunogenicity of PorA subtypes in a multivalent Neisseria meningitidis vaccine is not dependent on presentation form.

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.

Animals↗

PubMed2003

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

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 > or =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 > or =4-fold increases in SBA to this antigen.

Adolescent↗

PubMed1999

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).

Bacterial Proteins↗