Outer membrane vesicles (OMVs) are released spontaneously during growth by many Gram‐negative bacteria. They present a range of surface antigens in a native conformation and have natural properties like immunogenicity, self‐adjuvation and uptake by immune cells which make them attractive for application as vaccines against pathogenic bacteria. In particular with Neisseria meningitidis, they have been investigated extensively and an OMV‐containing meningococcal vaccine has recently been approved by regulatory agencies. Genetic engineering of the OMV‐producing bacteria can be used to improve and expand their usefulness as vaccines. Recent work on meningitis B vaccines shows that OMVs can be modified, such as for lipopolysaccharide reactogenicity, to yield an OMV product that is safe and effective. The overexpression of crucial antigens or simultaneous expression of multiple antigenic variants as well as the expression of heterologous antigens enable expansion of their range of applications. In addition, modifications may increase the yield of OMV production and can be combined with specific production processes to obtain high amounts of well‐defined, stable and uniform OMV particle vaccine products. Further improvement can facilitate the development of OMVs as platform vaccine product for multiple applications.
Outer membrane vesicles (OMVs) are naturally non-replicating, highly immunogenic spherical nanoparticles derived from Gram-negative bacteria. OMVs from pathogenic bacteria have been successfully used as vaccines against bacterial meningitis and sepsis among others and the composition of the vesicles can easily be engineered. OMVs can be used as a vaccine platform by engineering heterologous antigens to the vesicles. The major advantages of adding heterologous proteins to the OMV are that the antigens retain their native conformation, the ability of targeting specific immune responses, and a single production process suffices for many vaccines. Several promising vaccine platform concepts have been engineered based on decorating OMVs with heterologous antigens. This review discusses these vaccine concepts and reviews design considerations as the antigen location, the adjuvant function, physiochemical properties, and the immune response.
Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract.
The virulence plasmid pJM1 enables the fish pathogen Vibrio anguillarum, a gram-negative polarly flagellated comma-shaped rod bacterium, to cause a highly fatal hemorrhagic septicemic disease in salmonids and other fishes, leading to epizootics throughout the world. The pJM1 plasmid 65,009-nucleotide sequence, with an overall G؉C content of 42.6%, revealed genes and open reading frames (ORFs) encoding iron transporters, nonribosomal peptide enzymes, and other proteins essential for the biosynthesis of the siderophore anguibactin. Of the 59 ORFs, approximately 32% were related to iron metabolic functions. The plasmid pJM1 confers on V. anguillarum the ability to take up ferric iron as a complex with anguibactin from a medium in which iron is chelated by transferrin, ethylenediamine-di(o-hydroxyphenyl-acetic acid), or other iron-chelating compounds. The fatDCBA-angRT operon as well as other downstream biosynthetic genes is bracketed by the homologous ISV-A1 and ISV-A2 insertion sequences. Other clusters on the plasmid also show an insertion element-flanked organization, including ORFs homologous to genes involved in the biosynthesis of 2,3-dihydroxybenzoic acid. Homologues of replication and partition genes are also identified on pJM1 adjacent to this region. ORFs with no known function represent approximately 30% of the pJM1 sequence. The insertion sequence elements in the composite transposon-like structures, corroborated by the G؉C content of the pJM1 sequence, suggest a modular composition of plasmid pJM1, biased towards acquisition of modules containing genes related to iron metabolic functions. We also show that there is considerable microheterogeneity in pJM1-like plasmids from virulent strains of V. anguillarum isolated from different geographical sources.The fish pathogen Vibrio anguillarum strain 775 is the causative agent of vibriosis, a highly fatal hemorrhagic septicemic disease (3). This bacterium disseminates in the vertebrate host by using the otherwise unavailable iron bound by high-affinity iron binding proteins, such as transferrin and lactoferrin. Furthermore, V. anguillarum 775 has the ability to grow in vitro in media in which iron is chelated by transferrin, ethylenediamine-di(o-hydroxyphenyl-acetic acid), and other iron chelators (15, 40). The metabolic pathway supporting the ability of this bacterium to grow under iron-limiting conditions is linked to the presence in the bacterial cells of the virulence plasmid pJM1 (15).Iron metabolic plasmids are rare; in addition to the pJM1-like plasmids only the pColV-K30 family of plasmids, identified in human clinical strains of Escherichia coli and other enteric bacteria, have been associated with iron metabolism. However, the pJM1 and pColV-K30 plasmid-mediated iron uptake systems are unrelated (17, 49). pJM1-like-plasmids, usually around 65 to 67 kbp, have been reported by us and others in different virulent V. anguillarum strains isolated from many epizootics throughout the world (30,32,46). In this family of plasmids the best characterized is t...
The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as “nutritional immunity.” The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn2+ and Mn2+ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.
We have identified two functional tonB systems in the marine fish pathogen Vibrio anguillarum, tonB1 and tonB2. Each of the tonB genes is transcribed in an operon with the cognate exbB and exbD genes in response to iron limitation. Only tonB2 is essential for transport of ferric anguibactin and virulence.Outer membrane receptors for ferric siderophores require energy to internalize the complexed iron. This energy is transduced from the proton motive force in the inner membrane to the outer membrane receptor by the TonB protein in complex with the ExbB and ExbD proteins (4, 10). The presence of tonB has been associated with bacterial virulence in bacteria such as Vibrio cholerae (16), Shigella dysenteriae (15), and Bordetella pertussis (13).To be able to cause an infection in a vertebrate fish host, the marine fish pathogen Vibrio anguillarum requires an active iron uptake mechanism (2,6,8,17,21) mediated by the siderophore anguibactin (1); however, it can also acquire iron via transport of heme and siderophores secreted by other microorganisms, like ferrichrome and enterobactin (5). Anguibactin is synthesized via a nonribosomal peptide synthetase mechanism and secreted to the extracellular environment (9). Once bound to iron, the ferric siderophore is transported back into the cell cytosol through the specific outer membrane receptor FatA and a transport complex consisting of the periplasmic binding lipoprotein FatB and the integral membrane proteins FatC and FatD (3,7,12).Ferric-anguibactin transport via the FatA receptor requires energy, suggesting the existence of a tonB complex in V. anguillarum (7).The tonB, exbB, and exbD cluster in V. anguillarum 775 also includes the heme transport genes (Fig. 1). This tonB system shows homology to the V. cholerae tonB1 cluster: TonB1, 48% (GenBank accession number NP_233295.1); ExbB1, 74% (NP_233296.1); ExbD1, 72% (NP_233297.1). When the tonB1 gene was replaced with a chloramphenicol resistance cassette (MS533), no phenotypic difference in iron uptake could be detected by bioassays with different iron sources (Table 1). This indicates that at least one other tonB system must be present in V. anguillarum. By Tn10 mutagenesis (11) of MS533, the tonB1 knockout, we identified a mutant, MS570, that was unable to transport any of the iron sources tested (Table 1). The Tn10 insertion was cloned and sequenced, showing that it occurred at bp 935 of an open reading frame homologous (66% identity) to open reading frame 1547 (tolR) of the V. cholerae genome (accession no. AF047974.1). This V. anguillarum tolR homologue is located upstream of a second tonB cluster (Fig. 1), which shares homology with the tonB2 system in V. cholerae: TonB2, 68% (NP_231186.1); ExbB2, 87% (NP_231185.1); ExbD2, 62% (NP_231184.1). We then generated a tonB2 knockout strain by inserting the kanamycin resistance (Km r ) cassette (18) into the chromosomal locus of tonB2, with the suicide vector pTW-MEV (19) in the wild-type V. anguillarum strain, generating MS801. We repeated this mutagenesis with the tonB1 ...
The iron transport-biosynthesis (ITB) operon in Vibrio anguillarum includes four genes for ferric siderophore transport, fatD, -C, -B, and -A, and two genes for siderophore biosynthesis, angR and angT. This cluster plays an important role in the virulence mechanisms of this bacterium. Despite being part of the same polycistronic mRNA, the relative levels of transcription for the fat portion and for the whole ITB message differ profoundly, the levels of the fat transcript being about 17-fold higher. Using S1 nuclease mapping, lacZ transcriptional fusions, and in vitro studies, we were able to show that the differential gene expression within the ITB operon is due to termination of transcription between the fatA and angR genes, although a few transcripts proceeded beyond the termination site to the end of this operon. This termination process requires a 427-nucleotide antisense RNA that spans the intergenic region and acts as a novel transcriptional terminator.Small RNAs, some of them antisense, have been found to regulate a wide variety of genes, either in a positive or negative fashion, although some RNAs can act both positively and negatively at different loci. Most work has been carried out in Escherichia coli, although with the explosion in the knowledge on bacterial genomes, small RNAs appear to be ubiquitous in bacteria (3,17,28,30,32). Functions that have been ascribed to some of these RNAs fall into one or more of the following processes: control of plasmid DNA replication (27), blocking of the ribosome binding site (2), freeing of the ribosome binding site (20), or targeted degradation of mRNA (21). In certain plasmids, such as pT181, harbored by some gram-positive bacteria, small RNAs have been associated with transcription attenuation by binding and folding the target RNA so that a Rho-independent terminator structure forms (7).We have previously identified two small RNAs in the fish pathogen Vibrio anguillarum: RNA␣, which acts on the iron transport gene expression, and RNA (11,22,23). These two RNAs are transcribed as countertranscripts at different loci within the iron uptake-biosynthesis (ITB [iron transport-biosynthesis]) operon of the virulence plasmid pJM1 (15). This operon encodes the proteins FatD, -C, -B, and -A, which are involved in the transport of the ferric siderophore across the membranes, and AngR and -T, which are both active in the synthesis of the siderophore anguibactin and thus responsible for the ability of this bacterium to cause septicemia in salmonid fishes (26).Although transcribed as one operon, the level of fatDCBA mRNA is higher than the level of the full-length mRNA (fatDCBA angRT), thus causing differential expression of the genes encoding the ferric siderophore transport proteins as compared to the genes encoding the proteins involved in the siderophore biosynthesis. In this work, we report that the differential expression within this operon is mediated by RNA and discuss its biological significance. MATERIALS AND METHODSBacterial strains and growth conditions. Vibrio...
Conjugation chemistry is among the most important parameters governing the efficacy of glycoconjugate vaccines. High robustness is required to ensure high yields and batch to batch reproducibility. Herein, we have established a robust bioconjugation protocol based on the thiol-maleimide addition. Major variables were determined and acceptable margins were investigated for a synthetic pentadecasaccharide-tetanus toxoid conjugate, which is a promising vaccine candidate against Shigella flexneri serotype 2a infection. The optimized process is applicable to any thiol-equipped hapten and provides an efficient control of the hapten:carrier ratio. Moreover, comparison of four S. flexneri 2a glycoconjugates only differing by their pentadecasaccharide:tetanus toxoid ratio confirmed preliminary findings indicating that hapten loading is critical for immunogenicity with an optimal ratio here in the range of 17 ± 5. In addition, the powerful influence of alum on the immunogenicity of a Shigella synthetic carbohydrate-based conjugate vaccine candidate is demonstrated for the first time, with a strong anti-S. flexneri 2a antibody response sustained for more than one year.
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