Hendrik Jan Hamstra

Modification of Lipid A Biosynthesis in Neisseria meningitidis lpxL Mutants: Influence on Lipopolysaccharide Structure, Toxicity, and Adjuvant Activity

ABSTRACT Two genes homologous to lpxL andlpxM from Escherichia coli and other gram-negative bacteria, which are involved in lipid A acyloxyacylation, were identified in Neisseria meningitidis strain H44/76 and insertionally inactivated. Analysis by tandem mass spectrometry showed that one of the resulting mutants, termed lpxL1, makes lipopolysaccharide (LPS) with penta- instead of hexa-acylated lipid A, in which the secondary lauroyl chain is specifically missing from the nonreducing end of the GlcN disaccharide. Insertional inactivation of the other (lpxL2) gene was not possible in wild-type strain H44/76 expressing full-length immunotype L3 lipopolysaccharide (LPS) but could be readily achieved in agalE mutant expressing a truncated oligosaccharide chain. Structural analysis of lpxL2 mutant lipid A showed a major tetra-acylated species lacking both secondary lauroyl chains and a minor penta-acylated species. The lpxL1mutant LPS has retained adjuvant activity similar to wild-type meningococcal LPS when used for immunization of mice in combination with LPS-deficient outer membrane complexes from N. meningitidis but has reduced toxicity as measured in a tumor necrosis factor alpha induction assay with whole bacteria. In contrast, both adjuvant activity and toxicity of the lpxL2 mutant LPS are strongly reduced. As the combination of reduced toxicity and retained adjuvant activity has not been reported before for eitherlpxL or lpxM mutants from other bacterial species, our results demonstrate that modification of meningococcal lipid A biosynthesis can lead to novel LPS species more suitable for inclusion in human vaccines.

Naturally Occurring Lipid A Mutants in Neisseria meningitidis from Patients with Invasive Meningococcal Disease Are Associated with Reduced Coagulopathy

Neisseria meningitidis is a major cause of bacterial meningitis and sepsis worldwide. Lipopolysaccharide (LPS), a major component of the Gram-negative bacterial outer membrane, is sensed by mammalian cells through Toll-like receptor 4 (TLR4), resulting in activation of proinflammatory cytokine pathways. TLR4 recognizes the lipid A moiety of the LPS molecule, and the chemical composition of the lipid A determines how well it is recognized by TLR4. N. meningitidis has been reported to produce lipid A with six acyl chains, the optimal number for TLR4 recognition. Indeed, meningococcal sepsis is generally seen as the prototypical endotoxin-mediated disease. In the present study, we screened meningococcal disease isolates from 464 patients for their ability to induce cytokine production in vitro. We found that around 9% of them were dramatically less potent than wild-type strains. Analysis of the lipid A of several of the low-activity strains by mass spectrometry revealed they were penta-acylated, suggesting a mutation in the lpxL1 or lpxL2 genes required for addition of secondary acyl chains. Sequencing of these genes showed that all the low activity strains had mutations that inactivated the lpxL1 gene. In order to see whether lpxL1 mutants might give a different clinical picture, we investigated the clinical correlate of these mutations in a prospective nationwide observational cohort study of adults with meningococcal meningitis. Patients infected with an lpxL1 mutant presented significantly less frequently with rash and had higher thrombocyte counts, consistent with reduced cytokine induction and less activation of tissue-factor mediated coagulopathy. In conclusion, here we report for the first time that a surprisingly large fraction of meningococcal clinical isolates have LPS with underacylated lipid A due to mutations in the lpxL1 gene. The resulting low-activity LPS may have an important role in virulence by aiding the bacteria to evade the innate immune system. Our results provide the first example of a specific mutation in N. meningitidis that can be correlated with the clinical course of meningococcal disease.

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.

Coincorporation of LpxL1 and PagL Mutant Lipopolysaccharides into Liposomes with Neisseria meningitidis Opacity Protein: Influence on Endotoxic and Adjuvant Activity

ABSTRACT Wild-type lipopolysaccharide (LPS) of Neisseria meningitidis normally contains six acyl chains. Penta-acylated LPS forms were generated through inactivation of the lpxL1 gene or through the expression of the Bordetella bronchiseptica pagL gene in N. meningitidis. The resulting LPS species, designated LpxL1 LPS and PagL LPS, respectively, display reduced endotoxic activity compared to wild-type LPS. Here, we determined the adjuvant potential of PagL LPS by comparison with the broadly used LpxL1 LPS. We also investigated the potential benefit for adjuvanticity of coincorporating these LPS species, together with the meningococcal opacity-associated protein OpaJ as a model antigen, in a liposomal delivery system. PagL LPS showed a higher endotoxic activity than LpxL1 LPS, and their incorporation into liposomes significantly reduced their endotoxic activity as determined by measuring the induction of interleukin-6 (IL-6) production in a murine macrophage cell line. To determine the adjuvant effect, BALB/c mice were immunized with OpaJ-containing liposomes and either free LPS or LPS coincorporated into the proteoliposomes. OpaJ-containing liposomes adjuvanted with AlPO4 or not adjuvanted at all were included as control groups. In the appropriate dose, PagL LPS showed a superior adjuvant effect compared with LpxL1 LPS, and for both LPS types, free LPS showed a higher adjuvant effect than when coincorporated into the liposomes, as evidenced by higher titers of IgG2a and IgG2b antibodies against OpaJ+ meningococci and higher bactericidal titers. In conclusion, PagL LPS is a better adjuvant than LpxL1 LPS, but coincorporation of either LPS into proteoliposomes did not improve their adjuvant activity.

The Structure of Neisseria meningitidis Lipid A Determines Outcome in Experimental Meningococcal Disease

ABSTRACT Lipopolysaccharide (LPS), a major component of the meningococcal outer membrane, is sensed by the host through activation of Toll-like receptor 4 (TLR4). Recently, we demonstrated that a surprisingly large fraction of Neisseria meningitidis disease isolates are lipid A mutants, due to inactivating mutations in the lpxL1 gene. The lpxL1 mutants activate human TLR4 much less efficiently than wild-type bacteria, which may be advantageous by allowing them to escape from the innate immune system. Here we investigated the influence of lipid A structure on virulence in a mouse model of meningococcal sepsis. One limitation, however, is that murine TLR4 recognizes lpxL1 mutant bacteria much better than human TLR4. We show that an lpxL2 mutant, another lipid A mutant lacking an acyl chain at a different position, activates murine TLR4 less efficiently than the lpxL1 mutant. Therefore, the lpxL2 mutant in mice might be a better model for infections with lpxL1 mutants in humans. Interestingly, we found that the lpxL2 mutant is more virulent in mice than the wild-type strain, whereas the lpxL1 mutant is actually much less virulent than the wild-type strain. These results demonstrate the crucial role of N. meningitidis lipid A structure in virulence.

LOS oligosaccharide modification enhances dendritic cell responses to meningococcal native outer membrane vesicles expressing a non‐toxic lipid A

Outer membrane vesicles (OMV) are released by many bacteria, and contain immunogenic antigens in addition to harmful inflammatory factors, like lipopolysaccharides. Chemically detoxified OMV have been used in vaccines against Neisseria meningitidis (Nm); however, little is known about their interaction with antigen presenting cells. In this study, we investigated the interaction of Nm OMV with human dendritic cells (DC) to gain further understanding of their biological activity. We engineered a novel serogroup B Nm that is unencapsulated (siaD), expresses pentacylated lipid A (lpxL1), hence conferring reduced toxicity, and expresses an lgtB oligosaccharide structure designed to target OMV to DC via DC‐SIGN. We show that the lgtB moiety is critical for internalization of NOMV by DC. Furthermore, the lgtB moiety significantly enhances DC maturation, IL‐10 and IL‐23 production in the presence of a pentacylated lipid A. While different DC phenotypes were observed for each NOMV, this had little effect on Th1 and Th2 cell differentiation; however, lgtBsignificantly increased Th17 cell expansion in the presence of pentacylated lipid A. We believe that lpxL1/lgtB NOMV should be considered further as a vaccine vector, particularly considering the importance of lgtB in antigen uptake and further human studies on antigen‐specific responses should be considered.

Meningococcal Outer Membrane Vesicle Composition-Dependent Activation of the Innate Immune Response

ABSTRACT Meningococcal outer membrane vesicles (OMVs) have been extensively investigated and successfully implemented as vaccines. They contain pathogen-associated molecular patterns, including lipopolysaccharide (LPS), capable of triggering innate immunity. However, Neisseria meningitidis contains an extremely potent hexa-acylated LPS, leading to adverse effects when its OMVs are applied as vaccines. To create safe OMV vaccines, detergent treatment is generally used to reduce the LPS content. While effective, this method also leads to loss of protective antigens such as lipoproteins. Alternatively, genetic modification of LPS can reduce its toxicity. In the present study, we have compared the effects of standard OMV isolation methods using detergent or EDTA with those of genetic modifications of LPS to yield a penta-acylated lipid A (lpxL1 and pagL) on the in vitro induction of innate immune responses. The use of detergent decreased both Toll-like receptor 4 (TLR4) and TLR2 activation by OMVs, while the LPS modifications reduced only TLR4 activation. Mutational removal of PorB or lipoprotein factor H binding protein (fHbp), two proteins known to trigger TLR2 signaling, had no effect, indicating that multiple TLR2 ligands are removed by detergent treatment. Detergent-treated OMVs and lpxL1 OMVs showed similar reductions of cytokine profiles in the human monocytic cell line MM6 and human dendritic cells (DCs). OMVs with the alternative penta-acylated LPS structure obtained after PagL-mediated deacylation showed reduced induction of proinflammatory cytokines interleukin-6 (IL-6) and IL-1β but not of IP-10, a typical TRIF-dependent chemokine. Taken together, these data show that lipid A modification can be used to obtain OMVs with reduced activation of innate immunity, similar to what is found after detergent treatment.

Prevalence and clinical course in invasive infections with meningococcal endotoxin variants.

Background Meningococci produce a penta-acylated instead of hexa-acylated lipid A when their lpxL1 gene is inactivated. Meningococcal strains with such lipid A endotoxin variants have been found previously in adult meningitis patients, where they caused less blood coagulopathy because of decreased TLR4 activation. Methods A cohort of 448 isolates from patients with invasive meningococcal disease in the Netherlands were screened for the ability to induce IL-6 in monocytic cell Mono Mac 6 cells. The lpxL1 gene was sequenced of isolates, which show poor capacity to induce IL-6.. Clinical characteristics of patients were retrieved from hospital records. Results Of 448 patients, 29 (6.5%) were infected with meningococci expressing a lipid A variant strain. Lipid A variation was not associated with a specific serogroup or genotype. Infections with lipid A variants were associated with older age (19.3 vs. 5.9 (median) years, p = 0.007) and higher prevalence of underlying comorbidities (39% vs. 17%; p = 0.004) compared to wild-type strains. Patients infected with lipid A variant strains had less severe infections like meningitis or shock (OR 0.23; 95%CI 0.09–0.58) and were less often admitted to intensive care (OR 0.21; 95%CI 0.07–0.60) compared to wild-type strains, independent of age, underlying comorbidities or strain characteristics. Conclusions In adults with meningococcal disease lipid A variation is rather common. Infection with penta-acylated lipid A variant meningococci is associated with a less severe disease course.

Bordetella pertussis Naturally Occurring Isolates with Altered Lipooligosaccharide Structure Fail To Fully Mature Human Dendritic Cells

ABSTRACT Bordetella pertussis is a Gram-negative bacterium and the causative agent of whooping cough. Despite high vaccination coverage, outbreaks are being increasingly reported worldwide. Possible explanations include adaptation of this pathogen, which may interfere with recognition by the innate immune system. Here, we describe innate immune recognition and responses to different B. pertussis clinical isolates. By using HEK-Blue cells transfected with different pattern recognition receptors, we found that 3 out of 19 clinical isolates failed to activate Toll-like receptor 4 (TLR4). These findings were confirmed by using the monocytic MM6 cell line. Although incubation with high concentrations of these 3 strains resulted in significant activation of the MM6 cells, it was found to occur mainly through interaction with TLR2 and not through TLR4. When using live bacteria, these 3 strains also failed to activate TLR4 on HEK-Blue cells, and activation of MM6 cells or human monocyte-derived dendritic cells was significantly lower than activation induced by the other 16 strains. Mass spectrum analysis of the lipid A moieties from these 3 strains indicated an altered structure of this molecule. Gene sequence analysis revealed mutations in genes involved in lipid A synthesis. Findings from this study indicate that B. pertussis isolates that do not activate TLR4 occur naturally and that this phenotype may give this bacterium an advantage in tempering the innate immune response and establishing infection. Knowledge on the strategies used by this pathogen in evading the host immune response is essential for the improvement of current vaccines or for the development of new ones.

Emerging Bordetella pertussis Strains Induce Enhanced Signaling of Human Pattern Recognition Receptors TLR2, NOD2 and Secretion of IL-10 by Dendritic Cells

Vaccines against pertussis have been available for more than 60 years. Nonetheless, this highly contagious disease is reemerging even in countries with high vaccination coverage. Genetic changes of Bordetella pertussis over time have been suggested to contribute to the resurgence of pertussis, as these changes may favor escape from vaccine-induced immunity. Nonetheless, studies on the effects of these bacterial changes on the immune response are limited. Here, we characterize innate immune recognition and activation by a collection of genetically diverse B. pertussis strains isolated from Dutch pertussis patients before and after the introduction of the pertussis vaccines. For this purpose, we used HEK-Blue cells transfected with human pattern recognition receptors TLR2, TLR4, NOD2 and NOD1 as a high throughput system for screening innate immune recognition of more than 90 bacterial strains. Physiologically relevant human monocyte derived dendritic cells (moDC), purified from peripheral blood of healthy donors were also used. Findings indicate that, in addition to inducing TLR2 and TLR4 signaling, all B. pertussis strains activate the NOD-like receptor NOD2 but not NOD1. Furthermore, we observed a significant increase in TLR2 and NOD2, but not TLR4, activation by strains circulating after the introduction of pertussis vaccines. When using moDC, we observed that the recently circulating strains induced increased activation of these cells with a dominant IL-10 production. In addition, we observed an increased expression of surface markers including the regulatory molecule PD-L1. Expression of PD-L1 was decreased upon blocking TLR2. These in vitro findings suggest that emerging B. pertussis strains have evolved to dampen the vaccine-induced inflammatory response, which would benefit survival and transmission of this pathogen. Understanding how this disease has resurged in a highly vaccinated population is crucial for the design of improved vaccines against pertussis.