Continuous production of Neisseria meningitidis outer membrane vesicles

Outer membrane vesicles (OMVs) are nanoparticles secreted by Gram-negative bacteria that can be used for diverse biotechnological applications. Interesting applications have been developed, where OMVs are the basis of drug delivery, enzyme carriers, adjuvants, and vaccines. Historically, OMV research has mainly focused on vaccines. Therefore, current OMV production processes have been based on batch processes. The production of OMVs in batch mode is characterized by relatively low yields and high costs. Transition of OMV production processes from batch to continuous processes could increase the volumetric productivity, reduce the production and capital costs, and result in a higher quality product. Here, we study the continuous production of Neisseria meningitidis OMVs to improve volumetric productivity. Continuous cultivation of N. meningitidis resulted in a steady state with similar high OMV concentrations as are reached in current batch processes. The steady state was reproducible and could be maintained for at least 600 h. The volumetric productivity of a continuous culture reached 4.0 × 1014 OMVs per liter culture per day, based on a dilution rate of 1/day. The tested characteristics of the OMVs did not change during the experiments showing feasibility of a continuous production process for the production of OMVs for any application.publishedVersionPaid Open Acces

Continuous production of Neisseria meningitidis outer membrane vesicles

Outer membrane vesicles (OMVs) are nanoparticles secreted by Gram-negative bacteria that can be used for diverse biotechnological applications. Interesting applications have been developed, where OMVs are the basis of drug delivery, enzyme carriers, adjuvants, and vaccines. Historically, OMV research has mainly focused on vaccines. Therefore, current OMV production processes have been based on batch processes. The production of OMVs in batch mode is characterized by relatively low yields and high costs. Transition of OMV production processes from batch to continuous processes could increase the volumetric productivity, reduce the production and capital costs, and result in a higher quality product. Here, we study the continuous production of Neisseria meningitidis OMVs to improve volumetric productivity. Continuous cultivation of N. meningitidis resulted in a steady state with similar high OMV concentrations as are reached in current batch processes. The steady state was reproducible and could be maintained for at least 600 h. The volumetric productivity of a continuous culture reached 4.0 × 1014 OMVs per liter culture per day, based on a dilution rate of 1/day. The tested characteristics of the OMVs did not change during the experiments showing feasibility of a continuous production process for the production of OMVs for any application

Continuous production of Neisseria meningitidis outer membrane vesicles

Outer membrane vesicles (OMVs) are nanoparticles secreted by Gram-negative bacteria that can be used for diverse biotechnological applications. Interesting applications have been developed, where OMVs are the basis of drug delivery, enzyme carriers, adjuvants, and vaccines. Historically, OMV research has mainly focused on vaccines. Therefore, current OMV production processes have been based on batch processes. The production of OMVs in batch mode is characterized by relatively low yields and high costs. Transition of OMV production processes from batch to continuous processes could increase the volumetric productivity, reduce the production and capital costs, and result in a higher quality product. Here, we study the continuous production of Neisseria meningitidis OMVs to improve volumetric productivity. Continuous cultivation of N. meningitidis resulted in a steady state with similar high OMV concentrations as are reached in current batch processes. The steady state was reproducible and could be maintained for at least 600 h. The volumetric productivity of a continuous culture reached 4.0 × 1014 OMVs per liter culture per day, based on a dilution rate of 1/day. The tested characteristics of the OMVs did not change during the experiments showing feasibility of a continuous production process for the production of OMVs for any application.</p

Meningococcal outer membrane vesicle composition-dependent activation of the innate immune response

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 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 standard OMV isolation methods using detergent or EDTA with 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 TLR4 and TLR2 activation by OMVs, while the LPS modifications only reduced TLR4 activation. Mutational removal of PorB or fHbp, two proteins known to trigger TLR2 signaling, had no effect indicating that multiple TLR2 ligands are removed by detergent treatment. Detergent treated OMV and lpxL1 OMV showed similar reduction of cytokine profiles in the human monocytic cell line MM6 and human DCs. OMVs with the alternative penta-acylated LPS structure obtained after PagL-mediated deacylation showed reduced induction of pro-inflammatory cytokines 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

Differentially expressed genes in <i>ptxP3</i> vs. <i>ptxP1 Bordetella pertussis</i> strains.

<p>Differentially expressed genes in <i>ptxP3</i> vs. <i>ptxP1 Bordetella pertussis</i> strains.</p

Role of the <i>ptxP3</i> and <i>ptxP1</i> mutations and the genetic background of <i>ptxP1</i> and <i>ptxP3</i> strains in colonizing the trachea (A) and lungs (B) in mice.

<p>Mice were intranasally infected with the wildtype <i>ptxP1</i> strain, the <i>ptxP3</i> strain, isogenic strains carrying the <i>ptxP3</i> allele in the <i>ptxP1</i> genetic background (P1 gb:<i>ptxP3</i>) or the <i>ptxP1</i> allele in the <i>ptxP3</i> genetic background (P3 gb:<i>ptxP1</i>). CFUs were determined in the trachea and lungs four days post-infection. The mean is indicated by a thin line. P-values (uncorrected for multiple tests) were shown if greater than 0.05. The experiment was performed two times representative result is shown.</p

Genome-Wide Gene Expression Analysis of <i>Bordetella pertussis</i> Isolates Associated with a Resurgence in Pertussis: Elucidation of Factors Involved in the Increased Fitness of Epidemic Strains

<div><p><i>Bordetella pertussis (B. pertussis)</i> is the causative agent of whooping cough, which is a highly contagious disease in the human respiratory tract. Despite vaccination since the 1950s, pertussis remains the most prevalent vaccine-preventable disease in developed countries. A recent resurgence pertussis is associated with the expansion of <i>B. pertussis</i> strains with a novel allele for the pertussis toxin (ptx) promoter <i>ptxP3</i> in place of resident <i>ptxP1</i> strains. The recent expansion of <i>ptxP3</i> strains suggests that these strains carry mutations that have increased their fitness. Compared to the <i>ptxP1</i> strains, <i>ptxP3</i> strains produce more Ptx, which results in increased virulence and immune suppression. In this study, we investigated the contribution of gene expression changes of various genes on the increased fitness of the <i>ptxP3</i> strains. Using genome-wide gene expression profiling, we show that several virulence genes had higher expression levels in the <i>ptxP3</i> strains compared to the <i>ptxP1</i> strains. We provide the first evidence that wildtype <i>ptxP3</i> strains are better colonizers in an intranasal mouse infection model. This study shows that the <i>ptxP3</i> mutation and the genetic background of <i>ptxP3</i> strains affect fitness by contributing to the ability to colonize in a mouse infection model. These results show that the genetic background of <i>ptxP3</i> strains with a higher expression of virulence genes contribute to increased fitness.</p></div

Functional categories of differentially expressed genes between the <i>ptxP1</i> and <i>ptxP3</i> strains.

<p>The gene count (absolute number of genes) of differentially expressed genes per category (blue). And the gene count of differentially expressed genes relative to the total number of genes (red) present in the genomes of the analyzed strains.</p

Genes from the vir-core regulon were significantly upregulated in the <i>ptxP3</i> strains compared to the <i>ptxP1</i> strains.

<p>Genes from the vir-core regulon were significantly upregulated in the <i>ptxP3</i> strains compared to the <i>ptxP1</i> strains.</p

Relative differences in gene expression between <i>ptxP3</i> and <i>ptxP1</i> strains, as detected by microarrays and Q-PCR.

<p>Relative differences in gene expression between <i>ptxP3</i> and <i>ptxP1</i> strains, as detected by microarrays and Q-PCR.</p