High dissolved oxygen tension triggers outer membrane vesicle formation by Neisseria meningitidis.

BACKGROUND: Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria and can be used as vaccines. Often, detergents are used to promote release of OMVs and to remove the toxic lipopolysaccharides. Lipopolysaccharides can be detoxified by genetic modification such that vesicles spontaneously produced by bacteria can be directly used as vaccines. The use of spontaneous OMVs has the advantage that no separate extraction step is required in the purification process. However, the productivity of spontaneous OMVs by bacteria at optimal growth conditions is low. One of many methods for increasing OMV formation is to reduce the linkage of the outer membrane to the peptidoglycan layer by knocking out the rmpM gene. A previous study showed that for Neisseria meningitidis this resulted in release of more OMVs. Furthermore, cysteine depletion was found to trigger OMV release and at the same time cause reduced growth and oxidative stress responses. Here we study the effect of growth rate and oxidative stress on OMV release. RESULTS: First, we identified using chemostat and accelerostat cultures of N. meningitidis that increasing the growth rate from 0.03 to 0.18 h-1 has a limited effect on OMV productivity. Thus, we hypothesized that oxidative stress is the trigger for OMV release and that oxidative stress can be introduced directly by increasing the dissolved oxygen tension of bacterial cultures. Slowly increasing oxygen concentrations in a N. meningitidis changestat showed that an increase from 30 to 150% air saturation improved OMV productivity four-fold. Batch cultures controlled at 100% air saturation increased OMV productivity three-fold over batch cultures controlled at 30% air saturation. CONCLUSION: Increased dissolved oxygen tension induces the release of outer membrane vesicles in N. meningitidis cultures. Since oxygen concentration is a well-controlled process parameter of bacterial cultures, this trigger can be applied as a convenient process parameter to induce OMV release in bacterial cultures. Improved productivity of OMVs not only improves the production costs of OMVs as vaccines, it also facilitates the use of OMVs as adjuvants, enzyme carriers, or cell-specific drug delivery vehicles.

Cysteine depletion causes oxidative stress and triggers outer membrane vesicle release by Neisseria meningitidis; implications for vaccine development.

Outer membrane vesicles (OMV) contain immunogenic proteins and contribute to in vivo survival and virulence of bacterial pathogens. The first OMV vaccines successfully stopped Neisseria meningitidis serogroup B outbreaks but required detergent-extraction for endotoxin removal. Current vaccines use attenuated endotoxin, to preserve immunological properties and allow a detergent-free process. The preferred process is based on spontaneously released OMV (sOMV), which are most similar to in vivo vesicles and easier to purify. The release mechanism however is poorly understood resulting in low yield. This study with N. meningitidis demonstrates that an external stimulus, cysteine depletion, can trigger growth arrest and sOMV release in sufficient quantities for vaccine production (±1500 human doses per liter cultivation). Transcriptome analysis suggests that cysteine depletion impairs iron-sulfur protein assembly and causes oxidative stress. Involvement of oxidative stress is confirmed by showing that addition of reactive oxygen species during cysteine-rich growth also triggers vesiculation. The sOMV in this study are similar to vesicles from natural infection, therefore cysteine-dependent vesiculation is likely to be relevant for the in vivo pathogenesis of N. meningitidis.

Identification and optimization of critical process parameters for the production of NOMV vaccine against Neisseria meningitidis.

Outer membrane vesicles (OMV) are used as a vaccine against Neisseria meningitidis serogroup B and are traditionally produced with detergent-extraction to remove toxic lipopolysaccharide. Engineered strains with attenuated lipopolysaccharide allowed the use of native vesicles (NOMV) with improved stability and immunogenicity. In the NOMV production process detergents are omitted and vesicle release is stimulated with EDTA extraction (a chelating agent) to enable a higher yield. Many process parameters may change the EDTA extraction efficiency, but it is unknown what the optimal ranges for these parameters are in terms of quality. The present study systematically optimized EDTA extraction and was representative for production at large-scale. Two critical process parameters were identified, harvest point of the cultivation (harvest) and pH of the extraction buffer (pH), which significantly affected yield (7-fold) and bacterial lysis (35-fold). The other quality attributes remained unchanged. Optimization of harvest and pH revealed that the desired low bacterial lysis coincided with intermediate but sufficient yield. High functional immunogenicity and low toxicity of the optimized vaccine were also confirmed. The EDTA extraction is therefore a robust process step which produces high quality OMV if harvest and pH are controlled accurately.