Production of bioengineered outer membrane vesicles as a vaccine platform

Development of new vaccines based on vaccine platforms forms an interesting opportunity to significantly reduce the development time. New vaccines are required to keep up with newly emerging diseases that spread quickly in the interconnected global world. The traditional development of new vaccines is a lengthy process, as a new production process must be developed for each vaccine. A vaccine platform allows the use of the existing production process for new vaccine targets. Bacterial outer membrane vesicles (OMVs) produced by Neisseria meningitidis are highly suitable candidates to form a vaccine platform. N. meningitidis OMVs have been safely used as meningococcal vaccines. OMVs are non-replicative nanoparticles derived from the bacterial membrane, that can display heterologous antigens. N. meningitidis OMVs have been produced by extraction of vesicle like structures from bacterial cells. However, OMVs spontaneously released from bacteria have advantages over extracted OMVs as they can be directly purified from the supernatant of the bacterial culture, have enhanced quality, and trigger broader immune responses. On the downside, the yields of spontaneously released OMVs are low. The aim of this thesis was to obtain a better understanding of outer membrane vesicle formation by Neisseria meningitidis and OMV quality, and use this to develop improved OMV production processes that can become a cost-effective basis for an OMV-based vaccine platform. A vaccine platform should be versatile and adaptable for the addition of heterologous antigens onto the OMV. In Chapter 2 we explored existing methods for antigen decoration of OMVs through a comprehensive literature review. We distinguished two approaches of OMV platforms, based on either separate production of antigen and OMV followed by coupling or production of the antigen directly by the OMV producing bacterium. Separate antigen production seems more suitable for viral and therapeutic targets as it allows coupling of complex glycosylated targets to the OMV. Production of antigens directly by the OMV producing bacterium is probably more suited for microbial targets and allows for the most straightforward production process. To optimize OMV production processes, a new method for OMV quantification was needed as current quantification methods of OMVs were indirect and elaborate. In Chapter 3 we successfully used nanoparticle tracking analysis to quantify OMVs directly from sterile filtered culture samples, in a high-throughput manner. Now that we had a more reliable method available to quantify OMV release, our next step was to improve the OMV yields by studying the release of OMVs from the bacterium. A previous study had shown that cysteine depletion causes a stationary growth phase in which OMVs are released. In Chapter 4 we show that sulfur depletion in general resulted in OMV release of N. meningitidis cultures and found that sulfate depltion results in an even higher level of OMV release. Mechanistically, OMVs were enriched in phospholipids following sulfate depletion, suggesting that enrichment of phospholipids is an important factor in the OMV release process. A second parameter is oxidative stress that had been previously observed in cysteine depleted cultures, as well as in the sulfate depleted cultures described in Chapter 4. We found that high dissolved oxygen tension could mimick this situation and trigger increased OMV release. Because dissolved oxygen tension is a well-controlled process parameter, high dissolved oxygen concentrations could be conveniently used to stimulate OMV release. This was demonstrated in Chapter 5 where we showed that sulfur depletion and high dissolved oxygen tension stimulate the OMV release per bacterium and can be applied in batch production processes. Chapter 6 presents a proof of concept of the OMV-based vaccine platform in which the findings from the previous chapters were combined. We expressed outer surface protein A and outer surface protein C of Borrelia burgdorferi, the cause of Lyme disease, on N. meningitidis OMVs. These OMVs with heterologous model antigens were produced in a batch production process. In this process, sulfur depletion and high-dissolved oxygen concentrations were combined to establish high OMV yields. Purification based on scalable unit-operations resulted in a recovery of 90 mg OMV associated protein per liter culture. This production proces could be used as a basis for the development of novel Lyme disease vaccines. Lastly, in chapter 7 we suggest that OMV production can be further improved by adopting continuous production processes. Continuous production results in increased volumetric productivities, enhanced process control, and reduced variability. However, before continuous OMV production can be used for OMV vaccines, a method to assure OMV quality in the lengthy cultivations needs to be developped. This thesis shows that high yields of spontaneously released N. meningitidis OMVs can be obtained by stimulating release of OMVs from the bacterium by process parameters. These OMVs show potential as modular production platform and could boost future vaccine development. OMV based vaccine platforms will reduce the time required to develop new vaccines, which is urgently needed to meet the demand for new vaccines.</p

Bioengineering bacterial outer membrane vesicles as vaccine platform

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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

Sulfate depletion triggers overproduction of phospholipids and the release of outer membrane vesicles by Neisseria meningitidis

Outer membrane vesicles (OMVs) produced by bacteria are interesting vaccine candidates. OMVs are nanoparticles that contain many immunogenic components, are self-adjuvating, and non-replicative. Despite recent insights in the biogenesis of OMVs, there is no consensus on a conserved mechanism of OMV release and the OMV yield from bacterial cultures remains low. For Neisseria meningitidis, a Gram-negative human pathogen causing meningitis and sepsis, a feasible OMV production method based on triggering OMV release by cysteine depletion has been described. In this study, we investigated the mechanism behind this external trigger for OMV release to improve the production process. Since enhanced OMV release upon cysteine depletion was associated with oxidative stress and redox responses, we investigate the influence of more oxidized sulfur sources on OMV release. We show that N. meningitidis grows similarly on sulfate, the most oxidized sulfur source, and OMV release is triggered by sulfur depletion in general. Sulfate depletion induced increased release of OMVs over cysteine depletion. Proteomics showed that sulfur depletion resulted in oxidative stress responses and upregulated phospholipid and LPS biosynthesis. Furthermore, OMVs produced by sulfur depletion were enriched in phospholipids. Mechanistically, we hypothesize that sulfur depletion results in overproduction of phospholipids causing increased bulging of the outer membrane and subsequent OMV release

Spontaneously released Neisseria meningitidis outer membrane vesicles as vaccine platform: production and purification

Outer membrane vesicles (OMVs) are nanoparticles produced by Gram-negative bacteria that can be used as vaccines. The application of OMVs as vaccine component can be expanded by expressing heterologous antigens on OMVs, creating an OMV-based antigen presenting platform. This study aims to develop a production process for such OMV-based vaccines and studies a production method based on meningococcal OMVs that express heterologous antigens on their surface. As a proof of concept, the Borrelia burgdorferi antigens OspA and OspC were expressed on Neisseria meningitidis OMVs to create a concept anti-Lyme disease vaccine. Production of OMVs released in the culture supernatant was induced by high dissolved oxygen concentrations and purification was based on scalable unit operations. A crude recovery of 90 mg OMV protein could be obtained per liter culture. Expressing heterologous antigens on the OMVs did result in minor reduction of bacterial growth, while OMV production remained constant. The antigen expression did not alter the OMV characteristics. This study shows that production of well characterized OMVs containing heterologous antigens is possible with high yields by combining high oxygen concentrations with an optimized purification process. It is concluded that heterologous OMVs show potential as a vaccine platform.</p

Sulfate depletion triggers overproduction of phospholipids and the release of outer membrane vesicles by Neisseria meningitidis

Outer membrane vesicles (OMVs) produced by bacteria are interesting vaccine candidates. OMVs are nanoparticles that contain many immunogenic components, are self-adjuvating, and non-replicative. Despite recent insights in the biogenesis of OMVs, there is no consensus on a conserved mechanism of OMV release and the OMV yield from bacterial cultures remains low. For Neisseria meningitidis, a Gram-negative human pathogen causing meningitis and sepsis, a feasible OMV production method based on triggering OMV release by cysteine depletion has been described. In this study, we investigated the mechanism behind this external trigger for OMV release to improve the production process. Since enhanced OMV release upon cysteine depletion was associated with oxidative stress and redox responses, we investigate the influence of more oxidized sulfur sources on OMV release. We show that N. meningitidis grows similarly on sulfate, the most oxidized sulfur source, and OMV release is triggered by sulfur depletion in general. Sulfate depletion induced increased release of OMVs over cysteine depletion. Proteomics showed that sulfur depletion resulted in oxidative stress responses and upregulated phospholipid and LPS biosynthesis. Furthermore, OMVs produced by sulfur depletion were enriched in phospholipids. Mechanistically, we hypothesize that sulfur depletion results in overproduction of phospholipids causing increased bulging of the outer membrane and subsequent OMV release.</p

Spontaneously released Neisseria meningitidis outer membrane vesicles as vaccine platform : production and purification

Outer membrane vesicles (OMVs) are nanoparticles produced by Gram-negative bacteria that can be used as vaccines. The application of OMVs as vaccine component can be expanded by expressing heterologous antigens on OMVs, creating an OMV-based antigen presenting platform. This study aims to develop a production process for such OMV-based vaccines and studies a production method based on meningococcal OMVs that express heterologous antigens on their surface. As a proof of concept, the Borrelia burgdorferi antigens OspA and OspC were expressed on Neisseria meningitidis OMVs to create a concept anti-Lyme disease vaccine. Production of OMVs released in the culture supernatant was induced by high dissolved oxygen concentrations and purification was based on scalable unit operations. A crude recovery of 90 mg OMV protein could be obtained per liter culture. Expressing heterologous antigens on the OMVs did result in minor reduction of bacterial growth, while OMV production remained constant. The antigen expression did not alter the OMV characteristics. This study shows that production of well characterized OMVs containing heterologous antigens is possible with high yields by combining high oxygen concentrations with an optimized purification process. It is concluded that heterologous OMVs show potential as a vaccine platform

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

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.</p