Carrier based on membrane vesicles of bacillus bacteria and product using the same

Abstract

To provide vaccines, therapeutic agents, or the like, free of mutagenic substances such as colibactin and endotoxins, by using membrane vesicles derived from Bacillus bacteria as a carrier.SOLUTION: The foregoing problem is solved by a carrier using membrane vesicles produced by Bacillus subtilis or Bacillus natto.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a vaccine / drug delivery vehicle consisting of highly safe membrane vesicles derived from Bacillus bacteria. [Background technology]

[0002] With the spread of viral infectious diseases, the development of safer and more effective vaccine and drug delivery vehicles is required. One candidate is membrane vesicles, which have long been known to be produced by gram-negative and gram-positive bacteria, and their use as vaccine and drug carriers has been investigated.

[0003] To date, the use of outer membrane vesicles derived from the probiotic Escherichia coli Nissle 1917 strain as vaccine adjuvants and delivery carriers has been investigated, and the outer membrane vesicles of this bacterium have been found to have strong mucosal adjuvant activity (see non-patent document 1).

[0004] Furthermore, the use of membrane vesicles isolated from a double mutant strain of the probiotic Escherichia coli Nissle 1917, which has two genetic mutations: a deletion mutation (ΔflhD) in a gene encoding flagella that is often contaminating membrane vesicle fractions recovered from Escherichia coli culture medium, and a mutation (ΔwbbL) in a gene involved in the production of O-side chain polysaccharides (O-antigens) in the sugar chain portion of lipopolysaccharide, is being considered for use in vaccines (see Patent Document 1).

[0005] However, it has been revealed that these probiotic E. coli strains contain the mutagenic substance colibactin. Furthermore, membrane vesicles derived from Gram-negative bacteria generally contain endotoxins (lipopolysaccharides). Therefore, some researchers have pointed out the need for safety considerations when using outer membrane vesicles derived from Gram-negative bacteria, including probiotic E. coli (see Non-Patent Document 2). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2020-089292 [Non-patent literature]

[0007] [Non-Patent Document 1] Hirayama S and Nakao R.: Microb Biotechnol.,13(4),1162-1178 (2020). [Non-patent document 2] Nougayrede JP, Chagneau CV, Motta JP, Bossuet-Greif N, Belloy M, Taieb F, Gratadoux JJ, Thomas M, Langella P, Oswald E.: mSphere 6(4).,e0062421 (2021). Summary of the Invention [Problem to be solved by the invention]

[0008] In light of this background, an objective of the present invention is to provide vaccines, therapeutic drugs, etc. that do not contain mutagens such as colibactin and endotoxins, using membrane vesicles obtained from Bacillus bacteria as carriers. [Means for solving the problem]

[0009] The present inventors conducted studies using various Bacillus bacteria to solve the above-mentioned problems, and as a result, found that the above-mentioned problems can be solved by producing membrane vesicles from Bacillus subtilis and Bacillus natto (hereinafter referred to as Bacillus subtilis, etc.) among these Bacillus bacteria, and using these as carriers to form complexes with antigens or drugs, thereby completing the present invention.

[0010] The present invention has the following configuration. [1] A carrier consisting of membrane vesicles produced by Bacillus subtilis or Bacillus subtilis natto. [2] A complex comprising the carrier of [1] above and at least one member selected from the group consisting of an antigen and a drug. [3] A vaccine comprising the carrier of [1] above and an antigen. [4] A therapeutic agent comprising the carrier of [1] above and a drug. [Effects of the Invention]

[0011] The membrane vesicles produced by Bacillus subtilis or Bacillus subtilis natto used in the present invention do not contain genotoxic substances (such as colibactin, a risk factor for colon cancer) or endotoxins (lipopolysaccharides), and are produced by bacteria that are familiar to us in our diet. Therefore, when used as a carrier for antigens or drugs, they are extremely safe, and their high adjuvant activity can improve the performance of the resulting vaccines and drugs. Furthermore, the complexes of the present invention can be used in the development and production of new drugs and vaccines. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1 shows membrane vesicles of Bacillus subtilis (top and bottom left) and Bacillus subtilis natto (bottom right and bottom right) photographed with a scanning electron microscope. [Figure 2] Figure 2 is a graph showing the production of ovalbumin-specific IgG and IgA, as detected by ELISA, when BALB / c mice were nasally immunized with bacterial membrane vesicles together with ovalbumin as a test antigen. DETAILED DESCRIPTION OF THE INVENTION

[0013] The following describes in detail embodiments of the present invention. However, these embodiments are intended to facilitate understanding of the principles of the present invention. The scope of the present invention is not limited to the following embodiments, and other embodiments in which a person skilled in the art appropriately replaces the configuration of the following embodiments are also included in the scope of the present invention.

[0014] Bacillus subtilis, a member of the Bacillus genus, is a gram-positive rod-shaped bacterium that forms endospores (blastocysts). Bacillus natto is a type of Bacillus subtilis that produces fermented soybean foods such as natto. Bacillus natto and Bacillus subtilis form endospores when exposed to harsh conditions such as high temperatures, making them very stable, acid-resistant, heat-resistant, and non-toxic.

[0015] The membrane vesicles used in the present invention are roughly spherical bodies made of the cell membrane of Bacillus subtilis or the like, with a diameter of about 20 to 400 nm, which are produced from Bacillus subtilis or the like and released outside the cells of Bacillus subtilis or the like.

[0016] In the present invention, membrane vesicles can be used as carriers to form complexes composed of the membrane vesicles and at least one selected from the group consisting of antigens and drugs. By incorporating a drug into the complex, it can be used as a drug delivery system. Furthermore, by incorporating an antigen into the complex, it can be used as a vaccine.

[0017] The drugs used in the present invention include compounds such as antibiotics and anticancer drugs. The drugs can be held in membrane vesicles as carriers. Methods for holding the drugs in the carriers include binding by bioconjugation and filling the drug into the lumen of the membrane vesicles. Among these, bioconjugation is preferred for its simplicity.

[0018] The composition ratio of the drug to the membrane vesicles can be 1:1000 to 100:1, more preferably 1:100 to 50:1, and even more preferably 1:10 to 10:1. In the present invention, when used as a nasal medication, it can be administered to humans or animals as a mixture of membrane vesicles bound to the drug.

[0019] Antigens that can be used in the present invention include sugar chains and proteins. When the complex of the present invention is used as a vaccine, the antigen can be mixed with membrane vesicles and used. Alternatively, the antigen can be bound to membrane vesicles and used. Methods for the binding include bioconjugation, protein ligation, and Bacillus subtilis strains expressing antigen-fusion membrane proteins. Among these, bioconjugation is preferred for its versatility.

[0020] The composition ratio of the antigen to the vaccine carrier can be 1:10,000 to 10:1, more preferably 1:100 to 1:1, and even more preferably 1:10 to 5:1. When used as a nasal vaccine in the present invention, the antigen and vaccine carrier can be mixed as a liquid and administered to humans or animals.

[0021] Method for culturing Bacillus bacteria Bacillus bacteria are streaked onto a plate containing LB agar medium and cultured overnight at 37°C in an incubator. A single colony of Bacillus subtilis or other bacteria is then picked from the streaked plate and inoculated into a centrifuge tube containing liquid LB medium. The tube is then incubated overnight at 37°C and 130 rpm in a water bath. The culture solution after incubation is inoculated into liquid BHI medium. This is then incubated for 24 hours at 37°C and 130 rpm in a water bath. However, Bacillus pumilus is cultured at 30°C.

[0022] Method for recovering Bacillus bacteria The supernatant of a culture solution of Bacillus subtilis or other bacteria incubated for 24 hours is filtered using a PVDF membrane with a pore size of approximately 0.45 μm, and the filtrate is ultracentrifuged. The resulting sediment is then further subjected to iodixanol density gradient ultracentrifugation to remove flagella, allowing the cells to be recovered as membrane vesicles.

[0023] How to check for the presence of flagella SEM images of membrane vesicles derived from the culture medium of Bacillus subtilis are taken, and the presence or absence of flagella is visually confirmed. The magnification of the SEM image is preferably 100,000 times or higher. [Example]

[0024] The present invention will be described in detail with reference to examples.

[0025] 1. Bacteria ·168 strains of Bacillus subtilis ·BEST 195 strains of natto bacteria Bacillus subtilis natto strain Miyagino Bacillus pumilus (hereinafter abbreviated as B. pumilus) Bacillus licheniformis (hereinafter referred to as B. licheniformis)

[0026] 2. Culture Medium The composition of LB liquid medium is 1.0% Tryptone, 0.5% yeast extract, and 1.0% NaCl. The composition of LB agar medium is 1.0% Tryptone, 0.5% yeast extract, 1.0% NaCl, and 1.5% agar. The composition of BHI liquid medium is 7.5% Calf Brain, 9.8% Beef Heart, 10% Proteose Peptone, 2% Dextrose, 5% NaCl, and 2.5% Disodium Phosphate.

[0027] 3.Measurement method ·Antibody production amount Measurement was performed by ELISA using alkaline phosphatase-labeled anti-mouse IgG and IgA antibodies.

[0028] 4.Membrane vesicle collection method The membrane vesicles were collected as a precipitate by filtering the supernatant of the culture medium of Bacillus bacteria using a 0.45 μm pore size PVDF membrane and then ultracentrifuging the filtrate (4°C, 150,000 × g, 1 hour). Note that Bacillus bacteria can grow in the temperature range of 20 to 45°C, with the optimum growth temperature being 37°C for most species.

[0029] We cultured Bacillus subtilis strain 168, Bacillus subtilis (BEST195 strain, Miyagino strain), B. pumilus and B. licheniformis, and purified membrane vesicles from these bacteria. The five types of bacteria preserved in 20% glycerol were scraped with a sterile loop, streaked onto LB agar medium, and cultured overnight at 37°C in an incubator, except for B. pumilus, which was cultured at 30°C. Next, 2.0 mL of liquid LB medium was aseptically dispensed into a 15 mL centrifuge tube. The tip of a sterilized platinum loop was contacted with a single bacterial colony on the streaked plate. The tip of the platinum loop that had contacted the bacterial colony was lightly immersed in the liquid LB medium dispensed into the 15 mL centrifuge tube. This was incubated overnight in a water bath at 37°C (30°C for B. pumilus) with shaking at 130 rpm. 1 mL of the incubated preculture was added to 100 mL of liquid BHI medium. This was incubated for 24 hours in a water bath at 37°C (30°C for B. pumilus) with shaking at 130 rpm. After 24 hours of incubation, the supernatant was filtered using a 0.45 μm pore size PVDF membrane, and the filtrate was further ultracentrifuged (4°C, 150,000 × g, 1 hour). The resulting sediment was separated and purified by iodixanol density gradient ultracentrifugation (4°C, 100,000 × g, 3 hours) to obtain purified membrane vesicles.

[0030] Examples 1 to 3, Comparative Examples 1 to 4 Mucosal adjuvant effects of protein antigens in mice Using 6-week-old female BALB / c mice, membrane vesicles of Bacillus subtilis (Example 1), Bacillus subtilis BEST195 strain (Example 2), Bacillus subtilis Miyagino strain (Example 3), B. pumilus (Comparative Example 1), B. licheniformis (Comparative Example 2), Escherichia coli Nissle (Comparative Example 3), which is known to have high adjuvant activity, and P. gingivalis (Comparative Example 4), which has low adjuvant activity, were mixed with a test protein antigen, and the adjuvant activity was examined. Mice were immunized three times, three weeks apart, intranasally with 5 μg of ovalbumin as a test protein antigen. 3 μg of membrane vesicles of the five bacterial species were also administered along with the ovalbumin. Two weeks after the third immunization, serum, bronchoalveolar lavage fluid, saliva, and nasal washes were collected. The serum, bronchoalveolar lavage fluid, saliva, and nasal washes collected from the mice were subjected to ELISA to examine the production of ovalbumin-specific antibodies. Figure 2 shows the production of ovalbumin-specific IgG and IgA, as detected by ELISA, when bacterial membrane vesicles were immunized intranasally in BALB / c mice together with the test antigen ovalbumin. Figure 2 shows ELISA graphs showing the amounts of IgG derived from mouse serum (top left), IgG derived from mouse saliva (bottom left), IgG derived from lung lavage fluid (top right), and IgA derived from nasal lavage fluid (bottom right). When membrane vesicles derived from any of the five bacillus species were administered intranasally to mice together with ovalbumin, ovalbumin-specific IgG and IgA were produced. In particular, membrane vesicles derived from B. licheniformis and two Bacillus species (natto) tended to produce higher levels of IgG in serum and bronchoalveolar lavage fluid. On the other hand, membrane vesicles derived from B. subtilis and Bacillus subtilis Miyagino strain (natto) produced higher levels of IgA in saliva and nasal lavage fluid. These results demonstrate that membrane vesicles derived from these five bacillus species, including B. subtilis and Bacillus subtilis (natto), possess exceptional adjuvant activity. [Industrial Applicability]

[0031] The membrane vesicles of the present invention can be used for the production of vaccines, pharmaceuticals used in cancer treatment and immunotherapy, quasi-drugs, cosmetics or foods. [Explanation of symbols]

[0032] EcN: Escherichia coli Pg: P. gingivalis Bsu: Bacillus subtilis Bpu: B. pumilus Bli: B. licheniformis BEST: Bacteria natto BEST195 Miya-1: Bacillus subtilis natto strain Miyagino

Claims

1. A carrier consisting of membrane vesicles produced by Bacillus subtilis or Bacillus subtilis natto.

2. A complex comprising the carrier according to claim 1 and at least one member selected from the group consisting of an antigen and a drug.

3. A vaccine comprising the carrier according to claim 1 and an antigen.

4. A therapeutic agent comprising the carrier according to claim 1 and a drug.

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