JP2018131416A - Immunopotentiators and methods of making the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel adjuvant to solve the following problems: LPS, a known vaccine adjuvant, has strong toxicity so that modification is necessary to reduce toxicity; lipid A being an active center of LPS, e.g., monophosphoryl lipid A (MPL), is effective as an adjuvant but requires combination use with other basal materials; and many of the outer membrane vesicles (OMVs), which are now studied as an adjuvant, are derived from pathogens such as Escherichia coli so that safety problem should be solved.SOLUTION: The invention provides an immunopotentiator comprising an outer membrane vesicle (OMV) derived from acetic acid bacteria. The immunopotentiator of the invention can be used alone or as a vaccine adjuvant.SELECTED DRAWING: None

Description

  The present invention relates to a novel immune enhancer and a method for producing the same. More specifically, the present invention relates to an immunopotentiator comprising an outer membrane vesicle derived from acetic acid bacteria that can be used for vaccine adjuvants, immunogenic compositions, foods and drinks that can be expected to have an immune enhancing effect, and supplements. And a preferred manufacturing method thereof.

  Production of vaccines to enhance immune responses in animal hosts, including humans, for the purpose of preventing infections caused by bacteria, viruses, etc., and in recent years for the treatment and prevention of parasitic infections, cancer and immune diseases And development is underway. A vaccine generally contains an antigen and an adjuvant for enhancing its antigenicity.

  As an adjuvant, for example, an aluminum compound, saponin, emulsion, lipid A and the like have been put into practical use. As one form of adjuvant, lipopolysaccharides (LPS) derived from bacteria containing lipid A have attracted attention. However, LPS has a strong toxicity, and when it enters the circulatory system, it is known that it is difficult to use as an adjuvant as it is because it causes excessive side reactions such as excessive activation of the immune system and development of sepsis. ing. For this reason, conventionally, the chemical structure of lipid A, which is the active center of LPS, has been improved and attenuated by chemical synthesis or genetic modification (Patent Document 1). In recent years, the presence of a Toll-like receptor (TLR) having an important function in the immune system has been reported in relation to the action mechanism of an adjuvant (Non-patent Document 1).

  In recent years, outer membrane vesicles (OMV), which are vesicles with a diameter of about 20-300 nm, released by many gram-negative bacteria, have been incorporated into cells by endocytosis (Non-patent Document 2). ), And having a component derived from the parent strain such as LPS and protein in one structure suggests the possibility of use as a vaccine (Non-patent Document 3), for example, a vaccine against Neisseria meningitidis, etc. (Patent Documents 2 and 3, Non-Patent Document 4). However, OMVs need to be attenuated in some way in order to be similarly toxic due to the LPS involved. For example, removal of LPS by a surfactant, improvement of lipid A structure by genetic modification, and the like have been implemented.

  On the other hand, black vinegar, a brewed vinegar produced in Kagoshima Prefecture, is known to be a functional food useful for maintaining health, such as improving lifestyle-related diseases such as the reduction of cholesterol and neutral fat, and allergic diseases. ing.

  Black vinegar is fermented by various microorganisms such as saccharification fermentation, alcohol fermentation, lactic acid fermentation, and acetic acid bacteria that are responsible for acetic acid fermentation. From previous researches by the group of the present inventors, black vinegar has been found to contain microorganism-derived hydrophobic components, which activate the innate immune system (Non-patent Document 5). In addition, the LPS fraction of acetic acid bacteria, one of the fermenting microorganisms of brewed vinegar, activates TLR4 weakly, and lipid A, the active center, has a strong structure against acid (non- Patent Document 6). Since the acetic acid bacteria LPS is similar to the hydrophobic fraction derived from black vinegar, the constituent sugars, and the molecular weight, it is highly possible that the acetic acid bacteria LPS is related to the functionality of black vinegar. However, it is not clear what form LPS-like components exist in black vinegar.

Special table flat 8-508722 Special table 2005-514388 Special table 2014-520801

Toussi DN and Massari P, Vaccines 2014, 2, 323-353 O'Donoghue EJ et al., Cell Microbiol. 2016, 18 (11), 1508-1517 Baker JL et al., Curr Opin Biotechnol. 2014, 29, 76-84 Asevedo R et al., Front Immunol. 2014, 5: 121 Hashimoto M et al., J. Biosci. Bioeng. 2013, 116 (6), 688-696 Hashimoto M et al., J. Biol. Chem. 2016, 291 (40), 21184-21194

  As described above, LPS, which is known to be used as an adjuvant, has strong toxicity, and thus requires modification such as reduction of toxicity. Lipid A, which is the active center of LPS, such as monophosphoryl lipid A (MPL), is effective as an adjuvant, but needs to be combined with other bases. Furthermore, many OMVs currently being investigated as adjuvants are derived from pathogenic bacteria such as Escherichia coli, and their safety remains a problem due to their toxicity.

  In examining the functions of fermenting microorganisms used in black vinegar brewing, the present inventors can ultimately leave these microorganism-derived components in black vinegar, and in particular, immunomodulation derived from acetic acid bacteria. Focusing on the possibility of the inclusion of sex components, we examined whether OMV was produced in the culture of acetic acid bacteria.

  As a result, it was found that acetic acid bacteria produce OMV containing LPS, and that the obtained outer membrane vesicles are weakly toxic. Furthermore, it was also found that the obtained OMV has acid resistance, and it has been found that it can be effective as an adjuvant and as a vaccine, particularly as an oral vaccine.

That is, the present invention provides the following.
1. An immunopotentiator comprising outer membrane vesicles (OMV) derived from acetic acid bacteria.
2. 2. The acetic acid bacterium belongs to the genus Acetobacter, Gluconobacter, Gluconacetobacter, Asaia, and / or Komagateibacter Immune enhancer.
3. Acetic acid bacteria are Acetobacter pasteurianus, Acetobacter aceti, Acetobacter orleanensis, Acetobacter orientalis, Acetobacter porum (rum) Gluconobacter oxydans, Gluconobacter frateurii, Gluconobacter japonicus; Gluconacetobacter liquefaciens, Gluconacetobacter liquefaciens, Gluconacetobacter liquefaciens (Gluconacetobacter diazotrophicus); Asaia bogorensis, Asaia siamensis; Komagataeibacter europaeus, Ko Komagataeibacter hansenii, Komagataeibacter maltaceti, Komagataeibacter medellinensis, Komagataeibacter medellinensis (Komagataeibacter oboediens, Komagataeibacter oboediens, Komagataeibacter oboediens) 3. The immunopotentiator according to 2 above, which is one or more selected from Komagataeibacter xylinus).
4). 4. The immunopotentiator according to any one of 1 to 3 above, wherein the OMV has a particle size of 50 to 200 nm.
5. 5. The immunopotentiator according to any one of 1 to 4 above, wherein OMV is stable in an aqueous solution having a pH of 1 to 8.
6). 6. The immunopotentiator according to any one of 1 to 5 above, which is a vaccine adjuvant.
7). An adjuvant composition comprising the immunopotentiator according to any one of 1 to 6 above.
8). 8. The adjuvant composition according to 7 above, which is administered in combination with an antigen.
9. An immunogenic composition comprising the immunopotentiator according to any one of 1 to 6 above and administered as part of a vaccine composition.
10. 10. The immunopotentiator according to any one of 1 to 6, the adjuvant composition according to 7 or 8, or the immunogenic composition according to 9 described above, which is orally administered or ingested.
11. 11. The immunopotentiating agent, adjuvant composition, or immunogenic composition according to the above 10, which is provided as a liquid, semi-solid or solid pharmaceutical product, food or drink, or supplement.
12 Less than:
culturing the acetic acid bacteria to a late logarithmic growth phase at a temperature ranging from 25 ° C. to 37 ° C. in a pH 1-8 buffer;
A method for producing acetic acid-derived OMV, comprising the steps of separating cells from a culture solution, and obtaining OMV released from the cells.
13. A method for producing an immunopotentiator comprising the step of formulating OMV derived from acetic acid bacteria obtained by the method described in 12 above.

  According to the present invention, OMV can be effectively recovered from acetic acid bacteria. Since OMV derived from acetic acid bacteria is stable even under strongly acidic conditions, the immunopotentiator of the present invention passes through the stomach to reach the intestinal tract and is effective not only for infectious symptoms in the digestive tract, Absorption from the intestine can also be expected.

In 804 medium (High Polypeptone (Wako Pure Chemical Industries, Ltd.) 5 g, yeast extract 5 g, glucose 5 g, MgSO ₄ · 7H ₂ O 1 g, ± agar 15 g, distilled water 1 L, pH 6.6-7.0) The growth curve of Acetobacter pasteurianus NBRC 3283 strain cultured at 27 ° C. in FIG. The hexose content in OMV derived from Acetobacter pasteurianus NBRC 3283 strain is shown in relation to the number of culture days. The purification of OMV by density gradient centrifugation using Optiprep is shown. A: It shows that the sugar contained in the OMV derived from acetic acid bacteria is almost the same as the sugar in LPS. B: Indicates that the protein contained in OMV is an outer membrane protein derived from acetic acid bacteria. MP: Marker protein, Ap LPS: LPS derived from Acetobacter pasturiana, Ap OMV: OMV derived from Acetobacter pasturiana, OmpA: Membrane protein derived from Acetobacter pasturiana. It shows that fraction 3 containing a large amount of OMV derived from acetic acid bacteria has TLR2 activation ability (A) and weak TLR4 activation ability (B). The horizontal axis represents each fraction fractionated with Optiprep, and the vertical axis represents relative luciferase activity compared to the case without fraction. The black bar shows the result when a 1/10 dilution of the fraction is used, and the white bar shows the result when the 1/100 dilution of the fraction is used. It shows that OMV derived from acetic acid bacteria has TLR2 (A) and TLR4 activation ability (B) in a dose-dependent manner. The horizontal axis represents the OMV concentration in 1 ml of the sample, and the vertical axis represents the relative luciferase activity compared to the case where OMV is not included. It shows that OMV derived from acetic acid bacteria is stable under acidic conditions. A: Stability in simulated gastric juice (pH 1.6), B: Stability in HEPES buffer.

  The present invention provides an immunopotentiator comprising outer membrane vesicles (OMV) derived from acetic acid bacteria. OMV contains LPS derived from acetic acid bacteria in its membrane, but has been proven to be safe because it has been used safely in the food industry in the past, but has an immunopotentiating effect by LPS and the vesicle itself It was found.

  Acetic acid bacteria are gram-negative aerobic bacteria that produce acetic acid by oxidative fermentation of ethanol. Examples of the acetic acid bacteria that can be preferably used in the present invention include, for example, Acetobacter, Gluconobacter, Gluconacetobacter, Asaia, and Komagateibacter. The thing which belongs to.

  Although not particularly limited, specific examples of the bacterium belonging to the genus Acetobacter that can be suitably used in the present invention include Acetobacter pasteurianus, Acetobacter aceti, Acetobacter oletienen. Examples thereof include cis (Acetobacter orleanensis), Acetobacter orientalis, and Acetobacter pomorum.

  Although not particularly limited, examples of bacteria belonging to the genus Gluconobacter that can be preferably used in the present invention include Gluconobacter oxydans, Gluconobacter frateurii, and Gluconobacter frateurii. (Gluconobacter japonicus).

  Further, although not particularly limited, specific examples of the bacterium belonging to the genus Gluconacetobacter that can be suitably used in the present invention include Gluconacetobacter liquefaciens, Gluconacetobacter liquefaciens (Gluconacetobacter liquefaciens) Gluconacetobacter diazotrophicus) and the like.

  Further, although not particularly limited, specific examples of the bacteria belonging to the genus Asia that can be suitably used in the present invention include Asaia bogorensis and Asaia siamensis.

  Although not particularly limited, the bacteria belonging to the genus Komagataeibacter that can be preferably used in the present invention include Komagataeibacter europaeus, Komagataeibacter hansenii, Komagataeibacter hansenii, and Komagataeibacter hansenii. Komagataeibacter maltaceti, Komagataeibacter medellinensis, Komagataeibacter oboediens, Komagataeibacter xylin, and others.

  Acetic acid bacteria can be obtained, for example, from the NITE Biological Resource Center (NBRC) of the National Institute of Technology and Evaluation, for example, Acetobacter Pasteuriana is NBRC3283 strain, Acetobacter aceti is NBRC14818 strain, Acetobacter The Orientalis can use NBRC16606 strain etc. suitably.

  Similarly, NBRC14819 strain and the like for Gluconobacter oxydans, NBRC3285 strain and the like for Gluconobacter frateuri, and NBRC3272 strain and the like for Gluconobacter japonicus can be preferably used.

  Moreover, NBRC12388 strain etc. can be used suitably for Gluconacetobacter liquefaciens, and NBRC110704 strain etc. can be used suitably for Gluconacetobacter diazotrophicus.

  NBRC16594 strain and the like can be suitably used for Asaia Bogorensis, and NBRC16457 strain and the like can be suitably used for Asaia Xiamensis.

  NBRC3261 strain or the like can be suitably used for Komagata Teibutor Europaeus, and NBRC14820 or the like can be suitably used for Komagata Teibutor Hansenii.

  OMV derived from acetic acid bacteria can be obtained by culturing the above acetic acid bacteria. As a medium for culturing acetic acid bacteria, a conventionally used medium containing a carbon source, a nitrogen source, an inorganic salt, and optionally vitamins, amino acids, nucleic acids and the like necessary for the growth of acetic acid bacteria may be appropriately used. There is no particular limitation.

  Examples of the carbon source that can be contained in the medium include sugars such as glucose, sucrose, fructose, trehalose, mannose, mannitol, and maltose; alcohols such as ethanol, propanol, butanol, pentanol, hexanol, and isobutanol. .

  Examples of the nitrogen source include potassium nitrate, ammonium nitrate, ammonium chloride, ammonium sulfate, ammonium phosphate, ammonia, urea, and the like.

  Inorganic salts include magnesium sulfate, potassium phosphate, sodium phosphate, magnesium chloride, iron sulfate, iron chloride, manganese sulfate, manganese chloride, zinc sulfate, zinc chloride, copper sulfate, calcium chloride, calcium carbonate, sodium carbonate, etc. Can be mentioned.

  Extraction / enzymatic degradation / purification / drying from raw materials derived from microorganisms such as yeast, animal and plant such as casein / soybean / brown rice / wheat, etc., including carbon sources, nitrogen sources, inorganic salts, and amino acids and nucleic acids It is also possible to use what has been processed. For example, yeast extract, casein degradation product, soybean degradation product, cereal degradation product and the like can be mentioned.

  The medium for culturing the acetic acid bacteria can be either a liquid medium or a solid medium such as an agar medium. In the present invention, since the OMV released from the acetic acid bacteria is recovered and purified, the liquid medium Is preferred. In the practice of the present invention, for example, 804 medium, 350 medium, and the like available from NBRC can be suitably used.

  The culture temperature is not particularly limited, but for example, acetic acid bacteria can suitably grow if the temperature is in the range of 25 ° C to 37 ° C. The pH during the culture may be in the range of about 1 to about 8, and considering the pH change during the culture, it is preferable to use a medium having a pH of about 5.5 to about 7.0. As is usually done in the art, a buffer is preferably used for adjusting the pH.

  It was confirmed that OMV derived from acetic acid bacteria was released into the medium when the acetic acid bacteria were cultured until the late logarithmic growth phase under the medium composition and culture conditions as described above. On the other hand, it was also confirmed that when the culture was continued after the stationary phase, the amount of OMV gradually decreased (FIG. 2). Therefore, in order to increase the recovery amount of OMV derived from acetic acid bacteria, it is preferable that the acetic acid bacteria grow from the late logarithmic growth phase to the stationary phase.

  Therefore, in order to suitably recover OMV derived from acetic acid bacteria, the acetic acid bacteria are cultured at least in the logarithmic growth phase at a temperature ranging from 25 ° C. to 37 ° C. in a buffer solution having a pH of about 1 to about 8. Is preferred.

  In the art, the terms “logarithmic growth phase” and “stationary phase” in bacterial culture are generally understood and used. These terms in this specification also have ordinary meanings in the art, and specifically, the “logarithmic growth phase” means that cells divide and proliferate at regular intervals, It is understood that the logarithm of the number becomes a straight line that rises to the right, and the “stationary phase” is understood to mean a phase where the growth apparently stops after the logarithmic growth phase. The stage before the logarithmic growth phase is called the “induction phase” where the growth rate is low, and after passing the stationary phase, the phase shifts to a stage called “death phase” in which the number of bacteria killed exceeds the growth. Whether the growth of the bacteria is in the induction phase, logarithmic growth phase, stationary phase, or death phase, a growth curve is created by counting the number of cells in culture or measuring the optical density (OD600) at 600 nm. Can be determined.

  In addition, the logarithmic growth phase includes the first phase in which the cell growth rate starts to increase from the induction phase, the middle phase in which cells grow exponentially and exhibits the linear growth described above, and the growth rate starts to decrease. It can be divided into the latter period before reaching the stationary period. In the present specification, “late logarithmic growth phase” means that the cell growth rate starts to decrease, and strictly speaking, the actual growth is lower than the above-mentioned linear growth in the growth curve and shifts to the stationary phase. It shall refer to the previous stage. Further, the late logarithmic growth phase can correspond to a stage where an OD value of about 3/4 or more of the maximum OD value achieved by the bacterium can be obtained when OD600 is measured.

  The logarithmic growth phase and stationary phase in the culture vary depending on the bacteria to be cultured, and may vary depending on the culture conditions.

  For example, when cultivating Acetobacter pasteuriana as an acetic acid bacterium, OMV is released into the culture by culturing for 3 days or more after inoculation in a buffer solution having a pH of about 1 to about 8. This corresponds to the late growth of the acetic acid bacteria in the culture in the late logarithmic growth phase. When the inoculum is cultured for 2 days or less, almost no OMV is released during the culture. On the other hand, the amount of OMV does not increase after 7 days of culture, so there is no limitation. However, when culturing Acetobacter pasturianas, it is beneficial to shorten the culture to 7 days or less. .

In this case, it is preferable to obtain OMV from a culture solution having an OD600 of about 0.6 to about 0.8. This may correspond to a cell concentration of acetic acid bacteria in the culture medium of about 3-4 × 10 ⁸ cells / ml.

  As the OMV that can be used in the immunopotentiator of the present invention, the OMV released in culture can be used as it is, and it is not particularly limited, but those having a particle size in the range of 50 to 200 nm are preferably used. can do.

  The immune enhancing agent of the present invention can be used alone. Alternatively, the immunopotentiator of the present invention can be used as a vaccine adjuvant. Accordingly, the present invention also provides an adjuvant composition comprising the immunopotentiator of the present invention.

  OMVs derived from acetic acid bacteria are stable in aqueous solutions having a pH of about 1 to about 8. Therefore, the immunopotentiator of the present invention can be advantageous particularly in the case of oral administration among the conventionally used administration routes of adjuvants.

  The function of an adjuvant can generally be divided into two. One provides a suitable delivery system that, when administered with an antigen, provides a sustained release by storing the antigen at the site of administration, and the other is directly related to immune cell activation and function. It enhances the immune response by acting on (Toussi DN and Massari P, Vaccines 2014, 2, 323-353).

  Without intending to be bound by theory, the immunopotentiator of the present invention contains LPS derived from acetic acid bacteria in OMV, which acts as a TLR agonist and leads to activation of NF-κB. Can be induced. Furthermore, OMV has a lipophilic membrane portion similar to liposomes and emulsions that have been used as conventional adjuvants, which can contribute to the sustained release of antigens.

  It is known that immunity includes natural immunity that occurs immediately after contact with an antigen and specific acquired immunity that subsequently occurs due to activation of T cells and B cells. Innate immunity is one of the systems that recognize and eliminate microorganisms that enter the living body, and plays an important role in the forefront of the biological defense system. A host cell responsible for innate immunity recognizes a molecular structure unique to a microorganism that does not exist in the host, and activates this system to initiate biological defense. In recent years, it has been revealed that TLR, a receptor protein on the surface of animal cells, is a receptor involved in this molecular recognition. Furthermore, in vertebrates, it is said that innate immunity via TLR or the like is necessary for acquired immunity to work. Currently, 10 types of TLRs have been identified in humans, but many TLRs are known to act as homodimers or heterodimers. A component derived from a microorganism binds to TLR as a ligand, and the activated TLR activates the transcription factor NF-κB through a cell signaling pathway to induce cytokines and the like, thereby inducing immunity or inflammation.

  Among the above TLRs, it has been reported that TLR2 recognizes lipoprotein or the like as a ligand, and TLR4 recognizes Gram-negative bacterium LPS as a ligand.

  The immunopotentiator of the present invention contains OMV containing LPS, and is not bound by theory, but it is believed that it can act as a ligand for both TLR2 and TLR4. According to the knowledge of the present inventors, activation of both was confirmed by contact with OMV derived from acetic acid bacteria.

  The ability to activate TLR2 / TLR4-dependent cells can be confirmed, for example, by a luciferase assay using cells that co-express TLR2 or TLR4 and luciferase. When a sample is added to such cells and cultured, when TLR2 or TLR4 is stimulated by the sample, the transcription factor NF-κB is activated by signal transduction and expresses the luciferase gene. Chemoluminescence is generated by the translated luciferase. TLR2 / TLR4-dependent cell activity can be measured by quantifying this chemiluminescence amount using a luminometer.

  The present inventors have previously found that LPS or lipid A derived from acetic acid bacteria has TLR4 activation ability (J. Biol. Chem. 2016, 291 (40), 21184-21194). When examined in a similar assay system, these immunopotentiators have not only the ability to activate TLR4, but also the ability to activate TLR2, which was hardly detected with LPS or lipid A, but rather the ability to activate TLR2. It has been confirmed that No. has a feature of being significantly higher. That is, the immunopotentiator of the present invention can not only activate both TLR2 and TLR4, but also has an advantageous effect of having acid resistance.

  The adjuvant composition of the present invention can contain one or more other adjuvants in addition to the above-described immunopotentiator of the present invention. Examples of other adjuvants include aluminum compounds such as aluminum phosphate, aluminum hydroxide, and alum, LPS derived from bacteria other than acetic acid bacteria, lipid A, and the like, but saponins, liposomes, and the like that can change the OMV structure It is preferable not to use these together. In this case, it is preferable to select LPS derived from other bacteria that has been modified to reduce toxicity in some cases.

  The adjuvant composition of the present invention can be administered alone or in combination with an antigen. When administered in combination with an antigen, the adjuvant composition may be administered at the same time as the antigen, or before or after the antigen. The adjuvant composition may be administered during primary immunization or during booster immunization.

  The immunopotentiator of the present invention can also be prepared as an immunogenic composition together with an antigen. Accordingly, the present invention also provides an immunogenic composition comprising the above-described immunopotentiator of the present invention and administered as part of a vaccine composition. In this case, an antigen having lipophilicity can be incorporated into OMV. Alternatively, an OMV containing the intended antigen together with LPS can be produced by culturing an acetic acid bacterium modified so as to express a specific antigen by genetic engineering. Such OMV can be produced based on the description in this specification and common general technical knowledge in the field.

  The adjuvant composition and immunogenic composition of the present invention may optionally contain pharmaceutically acceptable carriers, excipients, diluents and the like in addition to OMV derived from acetic acid bacteria and optionally an antigen.

  The immunopotentiating agent, adjuvant composition and immunogenic composition of the present invention can be administered by various administration routes. Either the oral or parenteral route of administration can be used, and either systemic administration or local administration may be used. For example, it can be administered using routes such as oral, intravenous, intramuscular, intraperitoneal, intradermal, transdermal, nasal, inhalation, and intralesional routes. However, since the composition of the present invention has been demonstrated to be safe by ingestion and stable in the digestive tract, it can exhibit particularly advantageous effects when orally administered.

  It has been found that OMVs derived from acetic acid bacteria are stable over time in acidic to neutral conditions, specifically in aqueous solutions having a pH of about 1 to about 8. Therefore, the immunopotentiator, adjuvant composition, and immunogenic composition of the present invention can be administered orally orally to effectively act against infection in the digestive tract, and can also be administered from the intestine. Absorption can also be expected.

  Therefore, the immunopotentiator, adjuvant composition, or immunogenic composition of the present invention is provided, for example, as a liquid, semi-solid, or solid pharmaceutical product, and as a food or drink or supplement in the hope of its immune enhancing effect. can do. In this case, it can be of any form that can be used orally. The immunopotentiator of the present invention utilizes OMV derived from acetic acid bacteria, which has been proven to be safe, and can be suitably added to any food or drink.

  The immunopotentiating agent, adjuvant composition, or immunogenic composition of the present invention is not particularly limited. In a range of about 1 μg to about 1 mg per dose or intake. However, since the immunopotentiator of the present invention is very safe, it can be administered or ingested in a larger amount beyond the above range.

The present invention also provides a method for producing the above novel immune enhancing agent.
Specifically, the present invention provides the following:
culturing the acetic acid bacteria to a late logarithmic growth phase at a temperature ranging from 25 ° C. to 37 ° C. in a pH 1-8 buffer;
There is provided a method for producing an OMV derived from acetic acid bacteria, comprising a step of separating cells from a culture solution and a step of obtaining OMV released from the cells.

  The buffer solution may be a liquid medium containing a carbon source, a nitrogen source, an inorganic salt, and the like necessary for culturing acetic acid bacteria. A person skilled in the art can select a buffer solution that can be suitably used. For example, the above-described 804 medium, 350 medium (available from NBRC), and the like can be used.

The above-mentioned culturing step includes culturing until the late stage of the logarithmic growth phase of acetic acid bacteria, preferably OD600 is about 3/4 of the maximum OD value. For example, when cultivating Acetobacter pasturianas, it is preferable to culture for 3 days or more after inoculation, in other words, OD600 is about 0.6, and in this case, the cell concentration of acetic acid bacteria in the culture solution is about 3 × 10 Can correspond to ⁸ / ml. Thus, in this embodiment, the methods of the invention can include culturing until the cell concentration in the buffer exceeds about 3 × 10 ⁸ cells / ml.

In addition, the culture is preferably terminated during the stationary phase, and it may be suitable, for example, within 14 days from inoculation, for example, within 10 days, for example, within 7 days. The cell concentration at this point can be about 4 × 10 ⁸ cells / ml when the OD600 is about 0.8.

Separation of the bacterial cells from the culture solution can be performed by a usual method such as centrifugation.
In addition, OMV released from the cells can be obtained by a method such as ultracentrifugation.

  The OMV-derived OMV obtained by the above-described method of the present invention can be formulated as an immunopotentiator by, for example, purification, concentration, sterilization, freezing and the like. The purification and / or concentration step includes, for example, operations such as centrifugation and dialysis. The sterilization step includes operations such as sterilization filtration. A person skilled in the art can select an appropriate means for formulation based on the description of the present specification and the common general technical knowledge in the field.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited to these.

[Examination of OMV production conditions of Example 1 acetic acid bacteria]
First, the presence or absence of OMV production by acetic acid bacteria was examined.
Acetobacter pasteurianus NBRC 3283, a kind of acetic acid bacteria, 804 medium (high polypeptone (Wako Pure Chemical Industries, Ltd.) 5 g, yeast extract 5 g, glucose 5 g, MgSO ₄ · 7H ₂ O 1 g, ± agar 15 g, distilled water 1 L, pH 6.6-7.0). First, frozen stock bacteria were inoculated on an agar medium and cultured at 27 ° C. for 4 days to obtain a single colony. Subsequently, colonies were inoculated with 1 μl of Dispoloop (As One Co., Ltd.) in 5 ml of liquid medium, and precultured for 4 days while shaking at 27 ° C. The obtained preculture was inoculated into 25 ml of liquid medium and cultured at 27 ° C for 1 day. When OD600 reached about 0.8, it was further inoculated into 800 ml of liquid medium, and at 27 ° C, 0-14 Cultured with shaking for 1 day.

  When a growth curve was prepared by measuring OD600 using a spectrophotometer, logarithmic growth was observed on days 1-3, and a stationary phase was reached after day 5 (FIG. 1). Therefore, each culture broth was centrifuged at 4 ° C, 9000 rpm for 20 minutes using a C0650 rotor (manufactured by Beckman Coulter), separated into supernatant and cells, and filtered through a 0.22 µm bottle top filter. The cells were removed. The culture supernatant was ultracentrifuged at 4 ° C. and 40000 rpm for 2 hours using a Type 50Ti rotor (manufactured by Beckman Coulter) to separate the supernatant and the pellet. The pellet was washed with 50 mM HEPES buffer (pH 7.1-7.5) and obtained as crude OMV.

  The presence or absence of OMV in the culture was observed using a transmission electron microscope (TEM) and dynamic light scattering (DLS). TEM was measured using HT7700 (manufactured by Hitachi High-Technologies Corporation) at an acceleration voltage of 80 kV. The measurement sample was negatively stained with 1% sodium phosphomolybdate on a carbon-coated collodion membrane grid. DLS was measured using Zeta sizer Nano-ZS90 (manufactured by Malvern Instruments), and the particle size and particle distribution of particles in the solution were calculated from the average of three measurements.

  As a result, it was clarified that the presence of OMV particles can be confirmed from the third day after inoculation into an 800 ml liquid medium and the beginning of the culture, and OMV can be recovered from the third day (Table 1).

  Furthermore, the amount of OMV derived from acetic acid bacteria in the culture solution was quantified. Since OMV is expected to contain LPS, the amount of OMV was estimated from the amount of hexose. Hexose was quantified by a colorimetric method using the anthrone sulfuric acid method, and converted to an amount of glucose. As a result, as shown in FIG. 2, it was found that the largest amount of OMV was present in the culture solution on the seventh day of culture. From these results, it was found that acetic acid bacteria are optimally cultured for 7 days to recover OMV.

[Example 2 Purification of OMV derived from acetic acid bacteria]
Since the crude OMV contains impurities, the crude OMV obtained in Example 1 was purified by density gradient centrifugation using Optiprep (trademark, Cosmo Bio).

  Mix 250 μl sample containing crude OMV derived from acetic acid bacteria cultured in HEPES buffer for 7 days with 750 μl Optiprep (trademark, Cosmo Bio Inc.) containing 60% iodixanol (1: 3 (v / v)) and centrifuge Placed in the lower layer of the tube. Next, the 15-45% OptiPrep density gradient solution shown in FIG. 3 (35% (750 μl), 30% (1000 μl), 25% (500 μl), 20% (500 μl), 15% (500 μl), 10% (250 μl) Of iodixanol) in order to form a density gradient. This was ultracentrifuged at 4 ° C. and 50000 rpm for 3 hours (10,000 × g) using an MLS50 rotor (manufactured by Beckman Coulter), and then 500 μl was collected from the upper layer and separated into fractions 1-9. Each fraction was diluted with 3 times the amount of HEPES buffer and then further ultracentrifuged at 50,000 rpm for 3 hours using a TLA100.3 rotor (manufactured by Beckman Coulter). The crude OMV and each fraction pellet were suspended in HEPES buffer and used for further analysis.

  The crude OMV and each fraction were subjected to SDS-PAGE using 15% polyacrylamide gel, and then sugars were separated and visualized by periodate-silver staining, and fractions 1-9 were compared (FIG. 3). As a result, the bands of fractions 3 and 4 were deeply seen. In addition, when measured by TEM, spherical vesicles were observed in fractions 3 and 4, indicating the presence of OMV. That is, it was found that purified OMV can be recovered at an Optiprep concentration of around 25%.

  Fractions 3 and 4 were combined and ultracentrifuged, and the recovered pellet was used as a purified OMV in subsequent experiments.

[Example 3 Analysis of purified OMV]
The purified OMV obtained in Example 2 was separated again by SDS-PAGE together with LPS derived from the same acetic acid bacteria obtained separately. After electrophoresis, sugars were visualized by periodate-silver staining, and proteins were visualized by CBB staining.

  As a result, in the purified OMV, almost the same band as that of LPS derived from acetic acid bacteria was obtained (FIG. 4A). As a result of monosaccharide analysis using GC-MS, the constituent sugars were glucose, galactose, and rhamnose as well as LPS derived from acetic acid bacteria (data not shown). Further, the band obtained as a result of staining the protein was subjected to MALDI-TOF-MS analysis (autoflex speed; Bruker Daltonics) after digestion with trypsin. As a result, an outer membrane protein OmpA derived from acetic acid bacteria was detected (FIG. 4B).

  From these results, it is shown that the obtained OMV contains LPS and membrane proteins derived from acetic acid bacteria.

[Example 4: Evaluation of TLR activation by luciferase assay]
The ability of OMV derived from acetic acid bacteria to activate innate immunity was evaluated by NF-κB-dependent luciferase assay. As cells used in the assay, NF-κB / DNA binding activity-dependent luciferase reporter construct p55IgκLuc, mouse TLR2 or mouse TLR4 / MD-2 and stably expressed mouse Ba / F3 cells (Ba / mTLR2 and Ba / mTLR4, Cultivated in RPMI-1640 medium containing 10% FBS and 1% penicillin / streptomycin solution.

First, 10 μl of each fraction diluted to 1/10 or 1/100 as a sample was added to each well of a 384 well plate, and then 2.5 × 10 ⁵ cells / 10 μl of cells suspended in a medium were seeded. . After culturing at 37 ° C. for 4 hours in 5% CO ₂ and saturated steam atmosphere, 20 μl of Bright-Glo luciferase reagent (Promega) was added to lyse the cells. The amount of chemiluminescence was quantified with a luminometer Mithras LB940 (Berthold Japan). The result was calculated as a relative luciferase activity by calculating the ratio of each cell to the amount of unstimulated luminescence.

  As a result, when the diluted fraction 1-9 obtained in Example 2 was used as a sample, the ability to activate Toll-like receptor 2 (TLR2) was confirmed in fractions 3 and 4, particularly fraction 3. (FIG. 5A). Fraction 3 also slightly activated Toll-like receptor 4 (TLR4) (FIG. 5B).

  Moreover, as a result of examining the fractions 3 and 4 together, it was found that OMV derived from acetic acid bacteria activates TLR2 in a dose-dependent manner (FIG. 6A). TLR4 activation was relatively weak (FIG. 6B).

[Example 5: Stability evaluation of OMV derived from acetic acid bacteria in simulated gastric juice]
To estimate the dynamics of OMV in vivo, add OMV suspension to simulated gastric juice (34.2 mM NaCl-HCl pH 1.6, 1 mg / ml pepsin) to determine the stability of OMV under acidic conditions. investigated. When the change in the particle size of OMV was observed using DLS, the particle size of OMV in the simulated gastric juice hardly changed for 8 hours, which was the same as that of OMV in HEPES buffer (FIG. 7). This indicates that orally ingested OMV derived from acetic acid bacteria is stable even under acidic conditions and may pass through the stomach while maintaining the particle form.

  Currently, various vaccines are manufactured and inoculated, but the prevention against infectious diseases is still insufficient, and there is a demand for reducing the side effects of existing vaccines. Acetic acid bacteria have a long track record as a food product and have many advantages such as being safe and widely used in the brewing vinegar industry and being easy to handle and culture. By using OMV derived from acetic acid bacteria, safe and reliable adjuvants and vaccines can be provided, and completely new products can be industrialized.

Claims (13)

  An immunopotentiator comprising outer membrane vesicles (OMV) derived from acetic acid bacteria.   2. The acetic acid bacterium belongs to the genus Acetobacter, Gluconobacter, Gluconacetobacter, Asaia, and / or Komagateibacter. Immune enhancer.   Acetic acid bacteria are Acetobacter pasteurianus, Acetobacter aceti, Acetobacter orleanensis, Acetobacter orientalis, Acetobacter porum (rum) Gluconobacter oxydans, Gluconobacter frateurii, Gluconobacter japonicus; Gluconacetobacter liquefaciens, Gluconacetobacter liquefaciens, Gluconacetobacter liquefaciens (Gluconacetobacter diazotrophicus); Asaia bogorensis, Asaia siamensis; Komagataeibacter europaeus, Ko Komagataeibacter hansenii, Komagataeibacter maltaceti, Komagataeibacter medellinensis, Komagataeibacter medellinensis (Komagataeibacter oboediens, Komagataeibacter oboediens, Komagataeibacter oboediens) The immunopotentiator according to claim 2, which is one or more selected from Komagataeibacter xylinus).   The immunopotentiator according to any one of claims 1 to 3, wherein the OMV has a particle size of 50 to 200 nm.   The immunopotentiator according to any one of claims 1 to 4, wherein the OMV is stable in an aqueous solution having a pH of 1 to 8.   The immunopotentiator according to any one of claims 1 to 5, which is a vaccine adjuvant.   An adjuvant composition comprising the immunopotentiator according to any one of claims 1 to 6.   The adjuvant composition according to claim 7, which is administered in combination with an antigen.   An immunogenic composition comprising the immunopotentiator according to any one of claims 1 to 6 and administered as part of a vaccine composition.   The immunopotentiator according to any one of claims 1 to 6, the adjuvant composition according to claim 7 or 8, or the immunogenic composition according to claim 9, which is orally administered or ingested.   The immunopotentiating agent, adjuvant composition or immunogenic composition according to claim 10, which is provided as a liquid, semi-solid or solid pharmaceutical product, food or drink, or supplement. Less than:
culturing the acetic acid bacteria to a late logarithmic growth phase at a temperature ranging from 25 ° C. to 37 ° C. in a pH 1-8 buffer;
A method for producing acetic acid-derived OMV, comprising the steps of separating cells from a culture solution, and obtaining OMV released from the cells.   The manufacturing method of an immunopotentiator including the step which formulates the acetic acid bacteria origin OMV obtained by the method of Claim 12.