Biomedical subjects

Peter van der Ley

Publications and source records attributed to Peter van der Ley.

At least 19 recordsLinked to original sources

PubMed2006

Llama single-chain antibody that blocks lipopolysaccharide binding and signaling: prospects for therapeutic applications.

Sepsis is a considerable health problem and a burden on the health care system. Endotoxin, or lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria, is responsible for more than 50% of the sepsis cases and is, therefore, a legitimate target for therapeutic approaches against sepsis. In this study, we selected and characterized a llama single-chain antibody fragment (VHH) directed to Neisseria meningitidis LPS. The VHH, designated VHH 5G, showed affinity to purified LPS as well as to LPS on the surfaces of the bacteria. Epitope mapping using a panel of N. meningitidis mutants revealed that VHH 5G recognizes an epitope in the inner core of LPS, and as expected, the VHH proved to have broad specificity for LPS from different bacteria. Furthermore, this VHH blocked binding of LPS to target cells of the immune system, resulting in the inhibition of LPS signaling in whole blood. Moreover, it was found to remove LPS efficiently from aqueous solutions, including serum. The selected anti-LPS VHH is a leading candidate for therapies against LPS-mediated sepsis.

Animals↗

PubMed2006

Shielding of immunogenic domains in Neisseria meningitidis FrpB (FetA) by the major variable region.

The meningococcal iron-limitation-inducible outer membrane protein FrpB (FetA) has been shown to induce bactericidal antibodies, and is, therefore, considered a vaccine candidate. However, these antibodies are strain specific and, consistently, epitope mapping showed that they are directed against a region, located in a surface-exposed loop, L5, that displays considerable sequence variability between strains. Here, we attempted to redirect the immune response to more conserved domains of the protein by deleting L5. Immunization with an FrpB protein lacking L5 resulted in a bactericidal antibody response, and epitope mapping showed that these antibodies were directed against loop L3, which also displays considerable sequence variability. To re-direct the immune response further, immunizations were performed with an FrpB protein lacking both L5 and L3. The antibodies obtained were not bactericidal. Furthermore, the bactericidal antibodies against L3 were only bactericidal in the absence of L5, and immunofluorescence microscopy experiments showed that L5 efficiently shields other immunogenic cell surface-exposed epitopes outside of this region on living cells. Whereas the ability of micro-organisms to vary surface-exposed domains that are targets for protective immunity has long been established, the current work shows that such domains can be remarkably efficient in shielding other, more conserved epitopes.

Amino Acid Sequence↗

PubMed2006

A novel phase-variable autotransporter serine protease, AusI, of Neisseria meningitidis.

The sequenced genomes of pathogenic Neisseria meningitidis strains contain up to eight genes putatively encoding autotransporters, which are secreted proteins implicated in virulence. Here, we have characterized one of these genes, designated ausI, which encodes an autotransporter of the serine protease family. It was found to be specific for N. meningitidis and present in 14 out of 20 isolates, although only six of them expressed the gene. We show that expression of the gene is subject to phase variation as a result of a variable number of cytosines in a poly-C tract in the coding region. The open reading frame went out-of-phase at the poly-C tract in seven strains that did not express AusI. In the eighth strain, the open reading frame remained in frame at the poly-C tract, but it was disrupted by a premature stop codon further downstream. In accordance with its assignment as an autotransporter, a secreted AusI passenger domain was released into the extracellular milieu. This release was influenced by another autotransporter, NalP, as different forms of AusI were produced in the presence or absence of NalP. In silico sequence analysis suggested several putative functions for AusI, which, however, could not be confirmed experimentally.

Blotting, Western↗

PubMed2006

Crystal structure and catalytic mechanism of the LPS 3-O-deacylase PagL from Pseudomonas aeruginosa.

Pathogenic gram-negative bacteria can modify the lipid A portion of their lipopolysaccharide in response to environmental stimuli. 3-O-deacylation of lipid A by the outer membrane enzyme PagL modulates signaling through Toll-like receptor 4, leading to a reduced host immune response. We found that PagL is widely disseminated among gram-negative bacteria. Only four residues are conserved: a Ser, His, Phe, and Asn residue. Here, we describe the crystal structure of PagL from Pseudomonas aeruginosa to 2.0-A resolution. It consists of an eight-stranded beta-barrel with the axis tilted by approximately 30 degrees with respect to the lipid bilayer. The structure reveals that PagL contains an active site with a Ser-His-Glu catalytic triad and an oxyanion hole that comprises the conserved Asn. The importance of active site residues was confirmed in mutagenesis studies. Although PagL is most likely active as a monomer, its active site architecture shows high resemblance to that of the dimeric 12-stranded outer membrane phospholipase A. Modeling of the substrate lipid X onto the active site reveals that the 3-O-acyl chain is accommodated in a hydrophobic groove perpendicular to the membrane plane. In addition, an aspartate makes a hydrogen bond with the hydroxyl group of the 3-O-acyl chain, probably providing specificity of PagL toward lipid A.

Bacterial Outer Membrane Proteins↗

PubMed2006

Neisseria meningitidis expressing lgtB lipopolysaccharide targets DC-SIGN and modulates dendritic cell function.

Neisseria meningitidis lipopolysaccharide (LPS) has been identified as a major determinant of dendritic cell (DC) function. Here we report that one of a series of meningococcal mutants with defined truncations in the lacto-N-neotetraose outer core of the LPS exhibited unique strong adhesion and internalization properties towards DC. These properties were mediated by interaction of the GlcNAc(beta1-3)-Gal(beta1-4)-Glc-R oligosaccharide outer core of lgtB LPS with the dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) lectin receptor. Activation of DC-SIGN with this novel oligosaccharide ligand skewed T-cell responses driven by DC towards T helper type 1 activity. Thus, the use of lgtB LPS may provide a powerful instrument to selectively induce the desired arm of the immune response and potentially increase vaccine efficacy.

Antigen Presentation↗

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

Well-defined and potent liposomal meningococcal B vaccines adjuvated with LPS derivatives.

Potent liposomal PorA formulations containing various lipopolysaccharide (LPS) derivatives were developed. The following adjuvants were compared: the commonly used aluminum phosphate (AlPO(4)), and three LPS like adjuvants: monophosphoryl lipid A (MPL), lipopolysaccharide (galE LPS) and the less toxic LPS mutant lpxL1. The immunogenicity in mice was evaluated and compared with that against an outer membrane vesicle (OMV) vaccine. The IgG isotype distribution and bactericidal activity were determined. Furthermore, PorA specific proliferation of lymph node cells after immunization and restimulation in vitro was studied with selected formulations. Both AlPO(4) and MPL were unable to improve the functional immunogenicity (i.e. bactericidal response) of liposomal PorA. Besides, when these adjuvants were used, the percentage of responders in the groups did not reach 100%. This was also observed with non adjuvated PorA-liposomes or OMV. Of the adjuvants studied, only galE LPS and lpxL1 LPS were capable of increasing the immunogenicity and avoid non responsiveness against PorA-liposomes. Importantly, the adjuvant activity of lpxL1 LPS was accompanied by an improved PorA specific proliferation of lymph node cells and a concomitant increase in IL-2 production. In conclusion and considering its lower toxicity, lpxL1 LPS adjuvated liposomes are superior to other formulations tested.

Adjuvants, Immunologic↗

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

Working mechanism of immunoglobulin A1 (IgA1) protease: cleavage of IgA1 antibody to Neisseria meningitidis PorA requires de novo synthesis of IgA1 Protease.

Neisseria meningitidis secretes a protease that specifically cleaves the hinge region of immunoglobulin A1 (IgA1), releasing the effector (Fc) domain of IgA1 from the antigen binding (Fab) determinants. Theoretically, the remaining Fab fragments can block pathogen receptors or toxins and still provide protection. Here, we describe binding of V-gene-matched human IgA1 and IgA2 to PorA of strain H44/76. On live meningococci, efficient cleavage of IgA1, but not cleavage of IgA2, was observed, and up to approximately 80% of the IgA1 Fc tails were lost from the meningococcal surface within 30 min. No cleavage of IgA1 was found on an isogenic H44/76 strain lacking IgA1 protease. Furthermore, our data indicate that PorA-bound IgA1 is masked by the serogroup B polysaccharide capsule, rendering the IgA1 less accessible to degradation by secreted IgA1 protease present in the bacterial surroundings. Experiments with protein synthesis inhibitors showed that de novo production of IgA1 protease was responsible for cleavage of PorA-bound IgA1 on encapsulated bacteria. Finally, our data suggest that cleavage of IgA1 by IgA1 protease releases a significant proportion of Fab fragments from the bacterium, probably as a result of their reduced avidity compared to that of whole antibodies.

Bacterial Capsules↗

PubMed2004

Dissemination of lipid A deacylases (pagL) among gram-negative bacteria: identification of active-site histidine and serine residues.

Lipopolysaccharide (LPS) is one of the main constituents of the Gram-negative bacterial outer membrane. It usually consists of a highly variable O-antigen, a less variable core oligosaccharide, and a highly conserved lipid moiety, designated lipid A. Several bacteria are capable of modifying their lipid A architecture in response to external stimuli. The outer membrane-localized lipid A 3-O-deacylase, encoded by the pagL gene of Salmonella enterica serovar Typhimurium, removes the fatty acyl chain from the 3 position of lipid A. Although a similar activity was reported in some other Gram-negative bacteria, the corresponding genes could not be identified. Here, we describe the presence of pagL homologs in a variety of Gram-negative bacteria. Although the overall sequence similarity is rather low, a conserved domain could be distinguished in the C-terminal region. The activity of the Pseudomonas aeruginosa and Bordetella bronchiseptica pagL homologs was confirmed upon expression in Escherichia coli, which resulted in the removal of an R-3-hydroxymyristoyl group from lipid A. Upon deacylation by PagL, E. coli lipid A underwent another modification, which was the result of the activity of the endogenous palmitoyl transferase PagP. Furthermore, we identified a conserved histidine-serine couple as active site residues, suggesting a catalytic mechanism similar to serine hydrolases. The biological function of PagL remains unclear. However, because PagL homologs were found in both pathogenic and nonpathogenic species, PagL-mediated deacylation of lipid A probably does not have a dedicated role in pathogenicity.

Amino Acid Sequence↗

PubMed2004

Mannose binding lectin enhances IL-1beta and IL-10 induction by non-lipopolysaccharide (LPS) components of Neisseria meningitidis.

Mannose binding lectin (MBL) is a key molecule in the lectin pathway of complement activation, and likely of importance in our innate defence against meningococcal infection. We evaluated the role of MBL in cytokine induction by LPS or non-LPS components of Neisseria meningitidis, using a meningococcal mutant deficient for LPS. Binding experiments showed that MBL exhibited low, but significant binding to encapsulated LPS+ meningococci (H44/76) and LPS-deficient (LPS-) meningococci (H44/76lpxA). Experiments with human mononuclear cells (PBMCs) showed that MBL significantly augmented IL-1beta production after stimulation with LPS+ and LPS- meningococci, in a dose-dependent fashion. In addition, IL-10 production was enhanced after stimulation with LPS- meningococci. In contrast, TNFalpha, IL-6 and IFNgamma productions were unaffected. No effect of MBL was observed on cytokine induction by meningococcal LPS. MBL enhanced cytokine production at concentrations >10(7) meningococci. It is concluded that MBL interacts with non-LPS components of N. meningitidis and in this way modulates the cytokine response.

Cells, Cultured↗

PubMed2004

Intranasal immunisation of mice with liposomes containing recombinant meningococcal OpaB and OpaJ proteins.

The opacity (Opa) proteins of Neisseria meningitidis are outer membrane proteins involved in adhesion and invasion of host epithelial cells and are therefore expected to play an important role in colonisation of the nasopharynx. The majority of meningococcal Opa proteins bind to members of the CEACAM receptor family, such as CEA. Blocking of the Opa-CEACAM interaction by mucosal anti-Opa antibodies could thus constitute an important protective mechanism for novel meningococcal vaccines. In this study we analysed the specific anti-Opa antibody responses after intranasal immunisation of mice with liposomes containing purified and native OpaB (recognising the CEA receptor) and OpaJ (no affinity for CEA) proteins. These antigens were combined with or without one of three different adjuvants, i.e. purified meningococcal LPS, monophosphoryl lipid A (MPL) or the B-subunit of Escherichia coli heat-labile enterotoxin (EtxB). After intranasal immunisation with any of these formulations, anti-Opa IgA antibodies were found in nasal lavages and in some cases anti-Opa IgA and IgG antibodies were also found in lung lavages. With OpaJ but not OpaB, significant bactericidal serum titres were obtained. Of the different adjuvants used, meningococcal LPS gave the strongest overall immune response. Non-adjuvated liposomal Opa formulations were poorly immunogenic. No differences were found between the immune response in transgenic mice expressing the CEA-receptor and non-transgenic mice, showing that the CEA-Opa interaction does not influence the antibody response.

Adjuvants, Immunologic↗

PubMed2004

Human lipoproteins have divergent neutralizing effects on E. coli LPS, N. meningitidis LPS, and complete Gram-negative bacteria.

The use of lipoproteins has been suggested as a treatment for Gram-negative sepsis because they inhibit lipopolysaccharide (LPS)-mediated cytokine production. However, little is known about the neutralizing effects of lipoproteins on cytokine production by meningococcal LPS or whole Gram-negative bacteria. We assessed the neutralizing effect of LDLs, HDLs, and VLDLs on LPS- or whole bacteria-induced cytokines in human mononuclear cells. A strong inhibition of Escherichia coli LPS-induced interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, and IL-10 by LDL and HDL was seen, whereas VLDL had a less pronounced effect. In contrast, Neisseria meningitidis LPS, in similar concentrations, was neutralized much less effectively than E. coli LPS. Effective neutralization of meningococcal LPS required a longer interaction time, a lower concentration of LPS, or higher concentrations of lipoproteins. The difference in neutralization was independent of the saccharide tail, suggesting that the lipid A moiety accounted for the difference. Minimal neutralizing effects of the lipoproteins were observed on whole E. coli or N. meningitidis bacteria under all conditions tested. These results indicate that efficient neutralization of LPS depends on the type of LPS, but a sufficiently long interaction time, a low LPS concentration, or high lipoprotein concentration also inhibited cytokines by the less efficiently neutralized N. meningitidis LPS. Irrespective of these differences, whole bacteria showed no neutralization by lipoproteins.

Cytokines↗

PubMed2004

Human dendritic cell activation by Neisseria meningitidis: phagocytosis depends on expression of lipooligosaccharide (LOS) by the bacteria and is required for optimal cytokine production.

Group B Neisseria meningitidis is a human pathogen, for which a universally effective vaccine is still not available. Immune responses to bacteria are initiated by dendritic cells (DC), which internalize and process bacterial antigens for presentation to T cells. We show here that optimal IL-12 and TNF-alpha production by human monocyte derived DC in response to killed serogroup B N. meningitidis depends on physical contact and internalization of the bacteria by DC. The majority of DC producing cytokines had internalized N. meningitidis while inhibition of bacterial internalization markedly impaired IL-12 and TNF-alpha, but not IL-6 production. Internalization of N. meningitidis was shown to depend on lipooligosaccharide (LOS) expressed by the bacteria with poor internalization of LOS deficient bacteria compared to wild-type bacteria. Restoration of LOS biosynthesis in a LOS regulatory strain also restored both internalization and cytokine production and was enhanced in the presence of LPS binding protein (LBP). These results suggest that DC phagocytosis depends on expression of LOS within the bacteria and that optimal cytokine production, particularly IL-12, requires internalization of the bacteria. These findings have important implications for designing vaccines that will induce protective immune responses to group B N. meningitidis.

Cells, Cultured↗

PubMed2004

Teasing apart structural determinants of 'toxicity' and 'adjuvanticity': implications for meningococcal vaccine development.

The use of lipopolysaccharide (LPS) as an adjuvant is limited by its high endotoxic activity. In particular, the fatty-acyl pattern of the lipid A part of LPS has been demonstrated to determine its biological activity. By genetic modification of the lipid A biosynthesis pathway in Neisseria meningitidis, a panel of recombinant strains with specific alterations in the lipid A acylation pattern, as well as a strain completely lacking LPS were isolated. Whereas all variations in the fatty-acyl pattern resulted in reduced endotoxic activity, as measured by TNF-alpha induction in the human macrophage cell line MM6, the adjuvant activity of the modified LPS was, in most cases, barely affected. The in vivo adjuvant properties of N. meningitidis wild-type and mutant LPS was found to correlate with induction of co-stimulatory molecules, in particular CD80 and CD40, and with IL-12 production by LPS-stimulated bone marrow-derived BALB/c dendritic cells in vitro. Our results suggest that the ability of LPS to stimulate pro-inflammatory cytokine induction is not necessarily linked to its adjuvant activity. The availability of this novel set of lipid A variants with improved pharmacological properties will be of great importance for the improvement of future outer membrane vesicle vaccines against N. meningitidis.

Animals↗

PubMed2003

The Omp85 protein of Neisseria meningitidis is required for lipid export to the outer membrane.

In Gram-negative bacteria, lipopolysaccharide and phospholipid biosynthesis takes place at the inner membrane. How the completed lipid molecules are subsequently transported to the outer membrane remains unknown. Omp85 of Neisseria meningitidis is representative for a family of outer membrane proteins conserved among Gram-negative bacteria. We first demonstrated that the omp85 gene is co-transcribed with genes involved in lipid biosynthesis, suggesting an involvement in lipid assembly. A meningococcal strain was constructed in which Omp85 expression could be switched on or off through a tac promoter-controlled omp85 gene. We demonstrated that the presence of Omp85 is essential for viability. Depletion of Omp85 leads to accumulation of electron-dense amorphous material and vesicular structures in the periplasm. We demonstrated, by fractionation of inner and outer membranes, that lipopolysaccharide and phospholipids mostly disappeared from the outer membrane and instead accumulated in the inner membrane, upon depletion of Omp85. Omp85 depletion did not affect localization of integral outer membrane proteins PorA and Opa. These results provide compelling evidence for a role for Omp85 in lipid transport to the outer membrane.

Amino Acid Sequence↗

PubMed2003

Involvement of lipooligosaccharides of Haemophilus influenzae and Neisseria meningitidis in defensin-enhanced bacterial adherence to epithelial cells.

Stimulated neutrophils release a variety of antimicrobial peptides, including neutrophil defensins (HNP1-4). We have previously reported that neutrophil defensins enhanced the adherence of Haemophilus influenzae and Neisseria meningitidis to cultured respiratory epithelial cells. In this study, the effect of defensins on the adherence of H. influenzae and N. meningitidis lipooligosaccharide (LOS) mutants to epithelial cells was tested. Neutrophil defensins enhanced the adherence of the oligosaccharide mutants of H. influenzae and N. meningitidis, whilst the adherence of the lipid A mutants B29 of H. influenzae and lpxL1 and lpxL2 of N. meningitidis was not or only moderately stimulated by neutrophil defensins. The adherence of the N. meningitidis LOS negative mutant lpxA was not enhanced by defensins. These findings suggested that the secondary fatty acids of lipid A were involved in the defensin-enhanced adherence. LOS from strain H44/76 or HNP-LOS complexes did not affect or stimulate the adherence of N. meningitidis, although the defensin-enhanced adherence is specific for certain bacterial species having LOS in their outer membrane. These results indicated that LOS is involved in the defensin-enhanced adherence. However, the mechanism by which defensins and LOS interact with epithelial cells to promote bacterial adherence remains to be resolved.

Bacterial Adhesion↗

PubMed2003

Mapping the binding domains on meningococcal Opa proteins for CEACAM1 and CEA receptors.

The opacity (Opa) proteins of pathogenic Neisseria spp. are adhesins, which play an important role in adhesion and invasion of host cells. Most members of this highly variable family of outer membrane proteins can bind to the human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). Several studies have identified the Opa-binding region on the CEACAM receptors; however, not much is known about the binding sites on the Opa proteins for the corresponding CEACAM-receptors. The high degree of sequence variation in the surface-exposed loops of Opa proteins raises the question how the binding sites for the CEACAM receptors are conserved. Neisseria meningitidis strain H44/76 possesses four different Opa proteins, of which OpaA and OpaJ bind to CEACAM1, while OpaB and OpaD bind to CEACAM1 and CEA. A sequence motif involved in binding to CEACAM1 was identified by alanine scanning mutagenesis of those amino acid residues conserved within the hypervariable (HV) regions of all four Opa proteins. Hybrid Opa variants with different combinations of HV-1 and HV-2 derived from OpaB and OpaJ showed a reduced binding to CEACAM1 and CEA, indicating that particular combinations of HV-1 and HV-2 are required for the Opa binding capacity. Homologue scanning mutagenesis was used to generate more refined hybrids containing novel combinations of OpaB and OpaJ sequences within HV-1 and HV-2. They could be used to identify residues determining the specificity for CEA binding. The combined results obtained with mutants and hybrids strongly suggest the existence of a conserved binding site for CEACAM receptors by the interaction of HV-1 and HV-2 regions.

Alanine↗