Process for producing outer membrane vesicles

Abstract

The present invention relates to the fields of medical microbiology and vaccines. In particular the invention relates to a process wherein the spontaneous release of bacterial outer membrane vesicles (OMV) of Gram-negative bacteria is stimulated by application of a dissolved oxygen tension (DOT) that is higher than a physiological DOT. The thus produced OMVs are for use in vaccines. The invention further relates to OMV obtainable by said process, and to a pharmaceutical composition comprising such OMV. The present invention further relates to the use of OMV of the present invention as a medicament in particular for use in a method for eliciting an immune response.

Claims

1. A process for producing spontaneously released bacterial outer membrane vesicles (OMV), wherein the process comprises the steps of: a) cultivating a population of a Gram-negative bacterium, which cultivation comprises stimulation of the release of OMV by application of a dissolved oxygen tension (DOT) that is higher than a physiological DOT of 40% air saturation measured at 35° C.; and, b) recovering the OMV released in a), wherein the recovery at least comprises removal of the bacteria from the OMV.

2. The process according to claim 1, wherein the DOT applied to stimulate the release of OMV is at least 50, 55, 60, 70, 80, 90, 100, 125, 150 or 200% air saturation measured at 35° C.

3. The process according to claim 1, wherein said cultivating comprises a mode that employs adding a feeding medium, wherein said mode is selected from fed-batch mode, semi-continuous mode, and continuous mode.

4. The process according to claim 1, wherein the process comprises: a) a first phase wherein biomass of the Gram-negative bacterium is accumulated at a first DOT; and, b) a second phase wherein release of OMV from the biomass accumulated in a) is stimulated by the application of a second DOT that is higher than the first DOT.

5. The process according to claim 1, wherein the Gram-negative bacterium has at least one of: a) a genetic modification which causes the bacterium to produce an LPS with reduced toxicity but which LPS retains at least part of its adjuvant activity; b) a genetic modification which causes the bacterium to overproduce OMV as compared to a corresponding wild-type bacterium without the genetic modification, wherein the genetic modification is a modification that attenuates the peptidoglycan-binding activity of one or more proteins comprising a peptidoglycan-associated site; and c) a genetic modification that decreases or knocks-out expression of a gene product, and wherein the gene product is selected from the group consisting of cps, a lipid A biosynthesis gene product, PorA, PorB and opA.

6. The process according to claim 1, wherein the Gram-negative bacterium belongs to a genus selected from the group consisting of the genera Neisseria, Bordetella, Helicobacter, Salmonella, Vibrio, Shigella, Haemophilus, Pseudomonas, Escherichia, Moraxella, Klebsiella and Acinetobacter.

7. The process according to claim 1, wherein the Gram-negative bacterium expresses an antigen foreign to said Gram-negative bacterium.

8. The process according to claim 1, wherein the Gram-negative bacterium expresses multiple antigens.

9. The process according to claim 1, wherein the OMV are sterilized.

10. The process according to claim 1, further comprising the step of combining the OMV with a pharmaceutically accepted excipient and optionally an adjuvant.

11. The process according to claim 1, wherein the wherein the process comprises incorporating the OMV into a vaccine composition.

12. The process according to claim 1, wherein the DOT applied to stimulate the release of OMV is less than 350, 325, 300, 275, 250, 225, 205 or 185% air saturation measured at 35° C.

13. The process according to claim 4, wherein the first DOT is a physiological DOT.

14. The process according to claim 13, wherein the physiological DOT is a DOT of less than 50, 40, 35 or 32% air saturation measured at 35° C.

15. The process according to claim 5, wherein the Gram-negative bacterium has at least one of: a) the genetic modification which causes the bacterium to produce an LPS with reduced toxicity but which LPS retains at least part of its adjuvant activity, wherein the modification is a modification that decreases or knocks-out expression of one or more genes selected from the lpxL1 and lpxL2 genes or homologues thereof and the lpxK gene or a homologue thereof and/or is a modification that effects the expression of one or more lpxE and/or pagL genes; and b) the genetic modification which causes the bacterium to overproduce OMV as compared to a corresponding wild-type bacterium without the genetic modification, wherein the modification is a modification that decreases or knocks-out expression of one or more genes selected from the group consisting of the tolQ, tolR, tolA, tolB, tolRA, rmpM and ompA genes.

16. The process according to claim 6, wherein the Gram-negative bacterium is of a species selected from the group consisting of Neisseria meningitidis, Neisseria lactamica, Neisseria gonorrhoeae, Helicobacter pylori, Salmonella typhi, Salmonella typhimurium, Vibrio cholerae, Shigella spp., Haemophilus influenzae, Bordetella pertussis, Pseudomonas aeruginosa, Escherischia coli, Moraxella catarrhalis, Klebsiella pneumoniae and Acinetobacter baumannii.

17. The process according to claim 1, wherein the Gram-negative bacterium population comprises more than one strain of the Gram-negative bacterium, and wherein each strain expresses different antigens.

18. The process according to claim 1, wherein the OMV are sterilized by filter sterilization.

19. The process according to claim 2 wherein the OMV are sterilized by filter sterilization using a filter with pores of less than about 0.3 micrometer.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1. Accelerostat cultivation of N. meningitidis. Graph A shows the increase of the dilution rate (black line, a.sub.D of 0.0055 h.sup.−2), the actual measured dilution rate (diamonds), and the carbon dioxide evolution rate in time (grey line). Graph B shows the resulting specific sOMV productivity at different dilution rates for the accelerostat (black) and chemostats (grey).

(2) FIG. 2. The influence of increased dissolved oxygen tension on the growth of N. meningitidis. Graph A shows the control of the dissolved oxygen in the DOT-changestat, where the DOT setpoint was increased by 1% per hour. The effect of the elevated DOT on the growth is shown in Graph B, whereas both bacteria are capable of handling high levels of DOT. The release of vesicles was found to increase with higher DOT (Graph C).

(3) FIG. 3. High dissolved oxygen tension induces OMV release in N. meningitidis batch cultures. Growth curves of N. meningitidis batch cultures controlled at 30% and 100% air saturation show similar growth (Graph A). The increased oxygen concentration showed to induce a higher level of vesicle release (Graph B). Graphs are the overlay of two replicate cultures to practically allow for sufficient data points covering 24 h. The first replicate consists of data points at 0 h to 12 h cultivation and at 24 h cultivation, and the second replicate at 0 h and 15 h to 22 h.

(4) FIG. 4. DOT triggers OMV release in Escherichia coli and Bordetella pertussis. DOT changestat of E. coli (A) shows growth at DOT levels up to 200% in a DOT changestat with α.sub.DOT=1.5%/h. OMV release is directly related to the increased DOT. B. pertussis (B) shows growth up to DOT of 180% in a DOT changestat experiment with α.sub.DOT=1.0%/h, after which carbon dioxide concentrations in the off-gas showed to decrease.

EXAMPLES

Example 1

(5) Materials and Methods

(6) Bacterial Strains

(7) A recombinant derivate of the N. meningitidis serogroup B isolate H44/76 (Holten 1979) was used in this study. The selected strain was a PorA lacking derivate of the H44/76 isolate. This strain has a non-encapsulated phenotype due to the siaD knockout, lpxL1 deletion to attenuate LPS-toxicity, rmpM deletion to improve vesicle formation (unless indicated otherwise) and IgtB mutation to promote interactions with dendritic cells (Steeghs et al. 2006; van de Waterbeemd et al. 2010). This strain was stored in glycerol as working seedlots. All cultivations were performed in chemically defined growth medium (Baart et al. 2007b). Growth on sulfate was performed after adaptation since cysteine is the preferred sulfur source (Port et al. 1984). Adaptation was performed by sub-culturing the strain in shaker flasks with lower cysteine concentration and subsequent growth in medium without cysteine.

(8) Bioreactor Cultivations

(9) Batch cultivations were performed in 5-liter dished bottom Applikon bioreactors with an H/D ratio of 1.6 based on total volume. Cultivations were operated with 3 liter working volume on a Pierre Guerin Tryton.sup.i controller. Temperature was controlled at 35±0.5° C. and pH was controlled at pH 7.2±0.05 using 1M HCl and 1M NaOH. Dissolved oxygen tension (DOT) was controlled at 30% unless indicated otherwise. The membrane covered amperometric oxygen sensor (InPro 6850i, Mettler Toledo) was calibrated at 100% in air-saturated sterile culture medium of 35° C. In the first phase of the cultivation, DOT is controlled by increasing the agitation rate (300-1000 RPM) and next the fraction of oxygen is increased in the headspace aeration (1 NL/min) by the addition of pure oxygen. The agitation rate of the 100% DOT cultures was set at 1000 RPM directly after inoculation after which the DOT was controlled by the addition of pure oxygen in the headspace. Samples were taken for optical density measurements and used for nutrient and sOMV measurements after sterile filtration (0.22 μm) and storage at 4° C. Off-gas composition was analyzed by a Thermo Prima δb process mass spectrometer.

(10) Continuous Cultivations

(11) Continuous cultivations were performed in a similar setup as the batch cultivation setup. The working volume of the 5-liter bioreactor was decreased from 3.0 liter to 2.0 liter to reduce the feed medium required for the experiments. The vessel was equipped with a medium inlet and two outlet pipes, one submerged in the cultivation broth at the height of the stirrer and one directly at the liquid-gas interphase. The latter allowed the control of the working volume to be exactly 2.0 liter at a fixed maximum stirrer speed, independent of foaming. The weight of the bioreactor, the feed medium and the pH titrant solutions was measured by balances and used for verification of the dilution rate. Samples were taken for optical density measurements and off-gas analysis was similar to the batch cultivation. The bioreactor was controlled with the same control loops as used in the batch cultivations. After 8 hours of growth the feed and the bleed pumps were started to initiate a continuous culture. Steady state of the culture was assumed based on stable bacterial density values and stable carbon dioxide emission for at least 3 dilutions of the bioreactor volume.

(12) Accelerostat and DOT-Changestat Cultivation

(13) An accelerostat was started from a chemostat fermentation in steady state at D=0.03 h.sup.−1, operated as described in the previous section, by increasing the dilution rate linearly with a.sub.D=0.0055 h.sup.−2. The dilution rate was changed by increasing the medium inflow rate and equally increasing the broth outflow rate. From the culture broth, 50 mL samples were drawn to purify sOMVs. The sample was centrifuged at 4000×g for 30 min at 4° C. and the sterile filtered supernatant (Nalgene RapidFlow 0.2 μm PES filter unit) was concentrated on 100 kDa spin filters. The concentrated sOMVs were washed with 3% sucrose buffered by TrisHCl (pH 7.4) to wash out contaminating proteins. Next, the diafiltrated sOMVs were centrifuged at 125.000×g for 2 h. The sOMV containing pellet was dissolved in 1 mL TrisHCl pH 7.4 with 3% sucrose.

(14) The DOT-changestat was started from a chemostat culture. For this, a continuous culture in steady state with μ=0.04 h.sup.−1 was obtained as described previously. During this steady state, the DOT was controlled at 30%, the starting point for the DOT-changestat. From the start of the DOT changestat, the DOT was increased linearly with a DOT=1.0%/h.

(15) Quantification of sOMVs and Metabolites

(16) Culture samples were sterile filtered (0.22 μm) before the sOMVs were measured. sOMVs were measured with a phospholipid specific probe FM 4-64 (SynaptoRed C2, Biotium) by mixing 50 μL of diluted samples or OMV with a known concentration with 50 μL of dye solution (0.05 mM FM 4-64). Fluorescence was measured directly after mixing this solution using a plate fluorometer (Synergy MX, Biotek ex480, em650). The concentration of sOMVs in the culture supernatants was calculated from a calibration curve which was based on the responses of the standards (sOMVs corresponding with 0-2.5 mg/L total protein and eOMVs corresponding with 0-10 mg/L total protein). In the DOT-changestat experiments nanoparticle tracking analysis (Malloy and Carr 2006) was used for sOMV quantification. Static measurements (10 captures of 30-seconds) were made on a NanoSight NS500 with 488 nm laser module and sCMOS camera, that was calibrated with the concentration upgrade (Malvernlnstruments 2015). Temperature was controlled at 25° C. and captures were analyzed with the NTA 3.2 software build 3.2.16. Automated flow measurements were made as described previously (Gerritzen et al. 2017).

(17) OMV size was assessed by dynamic light scattering in a Zetasizer Nano-ZS with Zetasizer 7.11 software (Malvern Instruments). Measurements were performed using a SOP that takes three measurements in backscatter mode, with auto measurement duration and “seek for optimal position” as positioning setting. The sample was assumed to be protein with a refractive index of 1.450 and 0.001 absorption, in water as dispersant with a viscosity of 0.8872 cP and refractive index of 1.330. Data was processed with the normal analysis model.

(18) Results

(19) sOMV Release as a Function of Growth Rate

(20) The increased productivity of OMVs during the stationary phase of a batch cultivation (van de Waterbeemd et al. 2013b) raised the question what the direct influence of the growth rate on the OMV release was. Here we assess the influence of growth rate on OMV release in an accelerostat, by slowly increasing the dilution rate of a chemostat culture of N. meningitidis. The slow change in dilution rate (a.sub.D) should keep the culture in steady state in this accelerostat approach (Paalme et al. 1995). In this accelerostat an a.sub.D of 0.0055 h.sup.−2 was used (FIG. 1). The carbon dioxide evolution rate (CER) increased simultaneously with the dilution rate up to a dilution rate of 0.18 h.sup.−1, indicating accordingly increased growth rate. OMVs were produced throughout the accelerostat and these were similar in size and protein composition (data not shown). Altered growth rate from 0.03 h.sup.−1 to 0.18 h.sup.−1 showed not to influence the specific sOMV productivity (FIG. 1). Chemostat cultures of N. meningitidis at three different growth rates confirmed these results. From these results, we conclude that reducing the growth rate (e.g. from 0.18 h.sup.−1 to 0.03 h.sup.−1) is not a trigger for sOMV release.

(21) Influence of Oxidative Stress Assessed by a DOT-Changestat

(22) Since reduced growth rate alone was not applicable as trigger of sOMV release, we hypothesized that oxidative stress might be used to directly induce sOMV release. We therefore assessed the effect of oxidative stress on the sOMV release. We have previously shown the release of vesicles under hydrogen peroxide addition (van de Waterbeemd et al. 2013b). This method of hydrogen peroxide addition, however, is not feasible for scalable production processes of OMVs since local hydrogen peroxide addition to a bacterial culture will result in significant cell death and lysis of bacteria. We next tested whether extracellular oxidative stress could be induced by high concentrations of dissolved oxygen, which is one of the controlled parameters in bioreactor cultivations. The DOT is typically kept low, to minimize the stress from hyperoxia and to prevent oxygen inhibition (Haugaard 1968). Especially for a facultative anaerobic pathogen it is standard practice to design the cultivation with low DOT. For example, our N. meningitidis cultivation for both the vaccine concepts Hexamen and Nonamen has been designed with DOT levels of 30% air saturation (Baart et al. 2007a; Claassen et al. 1996). Here we assessed the impact of increased DOT on the bacterial growth and the OMV release with a changestat approach. For this DOT-changestat, the DOT of a chemostat culture is linearly increased to maintain a steady state culture (FIG. 2A). N. meningitidis shows to be capable of growth up to 150% DOT without significant impact on online measurements (FIG. 2B). Higher levels of DOT result in a rapid reduction of carbon dioxide production and a lower biomass concentration due to wash-out and lysis. Carbon dioxide production is observed up to a DOT of 220%. The release of sOMVs is linearly linked to the concentration of oxygen in the culture broth (FIG. 2C). OMV production can be increased by a factor of 4 by high DOT, while preserving the growth of bacteria. DOT-changestat experiments of E. coli and B. pertussis in Example 2 showed a similar correlation of increased OMV release at increased DOT levels (FIG. 4). Inducing OMV production at high DOT is thus applicable to Gram-negative bacteria in general.

(23) Improved Productivity of Batch Cultures at Increased Oxygen Concentrations

(24) The high oxygen concentration was then applied to batch cultivation to assess the feasibility of increased sOMV yield in a batch culture. A dissolved oxygen tension of 100% air saturation was used since this value showed increased OMV release while maintaining similar growth characteristics as at 30% air saturation in the changestat (FIG. 2). Bacteria were grown in chemically defined medium that triggers sOMV release from the onset of the stationary phase. The bacterial growth profile was similar for the batch cultures at 30% and 100% air saturation, showing the capability of N. meningitidis to deal with higher oxygen concentrations (FIG. 3A). The higher oxygen concentration triggered an increased release of vesicles resulting in a three-times higher productivity at the end of the culture compared to the standard level of 30% (FIG. 3B). The size of OMVs remain constant throughout the culture and is similar between the two concentrations (data not shown). High dissolved oxygen levels also showed to be a potent inducer of sOMV release in batch cultures.

Example 2

(25) Materials and Methods

(26) Escherichia coli

(27) E. coli JC8031 (TolRA) was used for the DOT-changestat of E. coli (Espesset et al. 1994). A shaker flask culture was started by adding 10 μL of frozen glycerol stock (−80° C.) to 100 mL LB medium (Large Capsules: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, MP Biomedicals) and incubating the shaker flask at 37° C. for 16 hours. Bioreactor cultivations were performed on LB medium without antifoam with a maximum stirrer speed of 600 RPM at 37° C.

(28) Bordetella pertussis

(29) The B. pertussis vaccine strain BP509 was used in this study (van Hemert 1967). A chemically defined medium was used without magnesium sulfate (Metz et al. 2017; Thalen et al. 1999). The DOT-changestat was started similarly to the N. meningitidis cultivation described above, with a dilution rate of the DOT-changestat of 0.05 h.sup.−1. A 7 L Applikon bioreactor with 5.4 L working volume was used with H/D ratio of 2.2 based on total volume.

(30) Results

(31) The release of OMVs by increased DOT levels is not limited to N. meningitidis but also extend to other Gram-negative bacteria. We tested the influence of oxidative stress on B. pertussis and E. coli in DOT-changestat experiments (FIG. 4). Both bacteria showed enhanced vesicle formation under increased DOT. E. coli was capable of handling DOT of over 200% after which the experiment was stopped. B. pertussis showed a reduction in carbon dioxide evolution rate at DOT of over 180%, after which the cultivation was terminated.

(32) Furthermore, the biomass concentrations of both cultures showed a clear decrease upon higher DOT. This relation was also observed for N. meningitidis. We hypothesize that relatively more energy is required for maintenance under oxidative stress. Since the experimental setup was a chemostat with a fixed nutrient supply and dilution rate, the increased energy requirement for maintenance is show as decrease in biomass concentration since less energy is available for bacterial growth.

(33) During the B. pertussis DOT changestat, the pH control was affected by blockage of the pH titrants during the experiment at the DOT setpoint of around 80% DOT. This has resulted in a linear increase in pH from 7.2 to 7.4 within 3 hours, followed by a rapid drop in pH to 5.5 due to over addition of acid. After this rapid reduction, the setpoint of pH 7.2 was quickly maintained again by the controller (data not shown). Remarkably, at this time-point a high sOMV concentration was observed, but the exact effects of this deviation are unknown. This deviation did not impact the conclusion on the relation between increased DOT on the vesicle release of B. pertussis, since the effect was already observed between 30% and 80% DOT.

Discussion Regarding Examples 1 and 2

(34) In this study we investigated external signals to induce OMV formation. Results show that growth rates from 0.03 h.sup.−1 to 0.18 h.sup.−1 do not influence the biogenesis of OMVs. Oxidative stress did trigger OMV release and could be applied directly to bioreactor cultures by increasing the dissolved oxygen tension of these cultures. Oxidative stress could be applied directly to bioreactor cultures by increasing the DOT of these cultures. DOT-changestat cultures show the Gram-negative bacteria to be capable of handling dissolved oxygen concentrations of up to 200% air saturation. Elevated DOT directly increased OMV release. OMV productivity was increased four-fold in a DOT-changestat culture at 120% DOT and three-fold in a batch culture controlled at 100% DOT. Applying increased DOT on E. coli and B. pertussis results in similar increases in OMV release indicating that this method is broadly applicable to Gram-negative bacteria.

(35) The production of OMVs by oxidative stress could be triggered in the bioreactor by controlling the oxygen concentration in the culture broth. The oxidative stress trigger was observed to trigger OMV release on top of the known mutations on increased OMV formation (Bernadac et al. 1998; Deatherage et al. 2009; van de Waterbeemd et al. 2010). These mutations reduce the linkage between the outer membrane and the peptidoglycan layer. For the N. meningitidis DOT-changestat we used a rmpM knockout strain to assess the effect of oxygen on vesicle release, although we also tested the effect of high DOT on a related strain that contained RmpM (data not shown). High DOT levels in a DOT-changestat did trigger increased vesicle release in N. meningitidis that contained RmpM too, although the OMV yield per liter culture remained lower than in the rmpM knockout cultures.

(36) Oxidative stress may be a general mechanism to induce OMV release. Here we showed this relation for N. meningitidis, E. coli and B. pertussis. Sabra et al. showed by electron micrographs that Pseudomonas aeruginosa forms more vesicles under extreme oxidative stress (pO.sub.2˜350% of air saturation) conditions compared to anoxic (pO.sub.2˜0) conditions (Sabra et al. 2003). Biologically the response of forming OMVs by the bacterium could be explained as a response to avoid phagocytosis by macrophages. During infection, sOMV release probably contribute to disease progression and the severity of fulminant meningococcal sepsis (Brandtzaeg et al. 1989; van Deuren et al. 1995). N. meningitidis encounters oxidative stress upon oxidative bursts of phagocytes (Moslen 1994; Ng et al. 2004). Lappann et al. showed that sOMVs of N. meningitidis induced the formation of neutrophil extracellular traps (NETs) and binding of OMVs to NETs served as a decoy for the bacteria to circumvent binding to the NETs (Lappann et al. 2013a). The role of OMVs in the interaction with phagocytes should gain more interest.

(37) The growth rate showed to influence the oxidative stress responses. Production of oxidative stress is a characteristic of aerobic bacterial growth as components of the respiratory chain are oxidized (Storz and Imlay 1999). Neisseria spp. are oxidase positive pathogens containing a mitochondrial like respiratory chain (Bøvre 1984). Neisseria species typically show high levels of respiration (Archibald and Duong 1986). The N. meningitidis genome encodes multiple small c-type cytochromes and a single terminal cytochrome oxidase of the cbb3 type (Aspholm et al. 2010; Deeudom et al. 2008; Li et al. 2010; Seib et al. 2006). Li et al. hypothesized that the high respiratory capacity of Neisseria spp. and the excess capacity for oxygen reduction acts as defence against endogenous reactive oxygen species (ROS) (Li et al. 2010). SodA and MntC are the major effectors involved in the Neisseria spp. oxidative stress response (Seib et al. 2004; Tseng et al. 2001). In the accelerostat experiments in this study, the growth rate increases linearly with the CER up to a growth rate of 0.18 h.sup.−1. At higher dilution rates, a reduction in CER was observed and the experiment was stopped. The maximum specific growth rate of N. meningitidis on this medium is 0.5 h.sup.−1 (van de Waterbeemd et al. 2010) and wash-out is thus not expected at this dilution rate. The chosen acceleration rate was moderately high (a.sub.D=0.0055 h.sup.−2) and possibly more time for adaptation was required to adapt to the increased growth rates. The chosen acceleration rate possibly resulted in an underestimation of the effect of the higher dilution rates. These chemostats showed depletion of the carbon sources glucose and glutamate and the cultures were likely carbon limited. At lowered growth rates, a lower bacterial density was observed that can be explained by the increased energy requirement for maintenance.

(38) Our initial results show that OMV-size was not affected although oxidative stress can cause damage to bacteria. In general, elevated oxygen concentrations could affect bacterial growth and the production of biological compounds (Baez and Shiloach 2014). Neisseria spp. are adapted to ROS production, caused by the respiratory system since reactive oxygen species accumulate as byproducts of the aerobic respiration (Imlay 2008; Korshunov and Imlay 2006), and contain several methods to handle ROS (Seib et al. 2004; Seib et al. 2006). The DOT changestat experiments showed that increased DOT can be controlled such that growth remains possible. Applications, such as the additions of enzymes on OMVs (Alves et al. 2015; Su et al. 2017), could also benefit from this production method.

(39) This disclosure expands the knowledge on sOMV productivity and enhances the process control. We used the dissolved oxygen tension of bacterial cultivations to induce oxidative stress to test the influence of oxidative stress on the vesicle release. Though, it is not obvious to design a fermentation process with a high DOT for a facultative aerobic micro-organism (Hewitt et al. 2000), but it showed to be a convenient process parameter to induce outer membrane vesicle formation. Besides the induced oxidative stress by altering the metabolism, increased DOT may be a more simplistic and better controllable approach. With this approach, it becomes possible to feasibly produce sOMV from Gram-negative cultures for many applications.

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