JP6709488B2 - Adjuvant and method for producing adjuvant - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel adjuvant extracted from black spores (Kurobokin: Ustilago esculenta) and Pantoea (Pantoea) isolated from Macomo.

It has been experimentally known that when a tumor antigen is expressed in cancer tissue, an immune response is generated against this tumor antigen. A cancer vaccine is a technique that utilizes this property to specifically destroy cancer cells. Specifically, cancer-specific antigens are administered from the outside of the body to activate immune-presenting cells (especially dendritic cells (DC cells)), and cancer-cell-specific CTLs are activated by the activated dendritic cells. (Cytotoxic T cells) are induced. CTL is an immunotherapy with few side effects that attacks cancer cells and specifically kills them.
In 2011, Provenge of Dendron, Inc., the first immunotherapy agent in the US, was approved as a drug for patients with prostate cancer. Provenge is a protein preparation containing a fusion of prostatic acid phosphotase (PAP) antigen and granulocyte macrophage colony stimulating factor (GM-CSF). An immunotherapy is employed in which the preparation is cultured with dendritic cells collected from the peripheral blood of the patient, and the activated dendritic cells are infused into the patient. In a multicenter, randomized controlled trial, overall survival (OS) was extended by 4.1 months compared to the control group, and similar effects were observed in the Phase 2 trial. Was approved with. In April 2014, Dendron reported a preliminary result of a long-term analysis of a Phase II STAND study on prostate cancer patients using Provenge, which was antigen-specific in patients with metastatic high-risk recurrent prostate cancer treated with Provenge. T cell responses were shown to be enhanced and maintained.

The peptide vaccine uses a full-length PAP protein as an antigen, but a normal peptide vaccine is a short chain of 8-10 amino acid length (mer) that is CD-8-restricted and can induce cancer cell-specific CTL. Many combinations of peptides and adjuvants have been adopted. Short-chain peptides have the advantage that they can be chemically synthesized more easily than long-chain peptides.
The administered antigenic protein/peptide is taken into professional antigen-presenting cells such as dendritic cells by an action such as endocytosis and cleaved into peptides of various lengths by protease and the like. In particular, dendritic cells decompose foreign antigens into peptides of 8-10 amino acids in length by the action of specific cross-presentation, and the major histocompatibility complex MHC (HLA in humans) class I molecule on the cell surface. Present above. Dendritic cells that have taken up the antigen migrate to the T cell region of lymph nodes, and during migration, they become mature dendritic cells that are capable of presenting antigens to naive T cells. For maturation, dendritic cells need to express costimulatory substances such as CD86 molecule and adhesion molecules such as CD40 molecule.

Immature dendritic cells in which co-stimulators such as CD86 and CD40 molecules are not induced only give stimulation (first signal) by MHC antigen signal during naive T cell stimulation, and co-stimulatory signals (such as CD86 molecule) ( The second signal) does not enter, and finally the T cell falls into a state (anagy) incapable of responding to the antigen. Therefore, in order for cancer vaccines that use tumor antigens to avoid the anergy state, that is, the immune-tolerance state and to function CTL, inducing the second signal by inducing expression of CD86 molecule, CD40 molecule, etc., and dendritic cell maturation Is important.
As a result, CTL activated by mature dendritic cells carrying the MHC class I/tumor antigen peptide complex detected the MHC class I/antigen peptide complex present on the tumor cells, and the CTLs such as granzyme and perforin were detected. Tumor cells are destroyed using effector molecules.

However, after Provenge's approval, the results of late-stage clinical trials of multiple peptide vaccines have been reported, but none of them have been favorable. Specifically, the oncotherapy science pancreatic cancer vaccine "OTS-102" did not meet the main evaluation criteria, and as a result, Merck/Serono's MUC-1 peptide vaccine for non-small cell lung cancer "L-BLP25" (25 amino acids, liposomal formulation) was unable to achieve the extension of OS, the main evaluation item. As a result, Phase III development of Oncotherapy Science's pancreatic cancer vaccine "C01" was discontinued and GlaxoSmithKline's MAGE-A3 vaccine for non-small cell lung cancer is disease-free survival (DFS) (first primary endpoint), and DFS of this drug for MAGE-A3-positive patients who did not receive chemotherapy (second It was reported that no significant prolongation was shown in any of the primary endpoints) compared with the placebo group that was administered a drug containing no active ingredient. In particular, MAGE-A3 is a clinical trial failure in post-administration of cancer tissue, which was expected to have the most effective vaccine function, using the full-length protein as an antigen, not a short-chain peptide, and it is the most effective cancer vaccine. Was the expected test.
On the other hand, a cancer vaccine targeting the receptor DEC-205 that is specifically expressed on dendritic cells was developed, and cell-mediated immunity was efficiently induced against the antigen NY-ESO-1 introduced by the cancer vaccine. Was reported (Non-patent document: Sci Transl Med 6, 231ra51 (2014)). That is, it has been suggested that a cancer vaccine can effectively function by efficiently introducing an antigen into dendritic cells and inducing a costimulatory substance or the like. In addition, the document reported that the combined use of an anti-CTLA-4 antibody, which is an immune checkpoint inhibitor, with ipilimumab was effective in 4 out of 6 melanoma patients, that is, the response rate was 67%. Compared with the response rate of 11% when ipilimumab was administered alone, it was confirmed that the response rate was significantly increased by the combined use of the immune checkpoint inhibitor and the cancer vaccine. The combination of immune checkpoint inhibitors and cancer vaccines is expected to become a trend in cancer immunotherapy in the future, and the development of effective cancer vaccines is important.

In the practice of cancer vaccine, usually, an adjuvant that activates immunity of an antigen is used in combination, instead of the administration of the vaccine alone. In addition, in the case of infectious disease vaccines, the number of cases in which an adjuvant is added is increasing in order to increase the vaccine titer.
Until recently, emulsion type and aluminum salt type adjuvants, which have the effect of retaining the antigen at the administration site, were generally used, but as the understanding of the immune mechanism deepened, the development of adjuvants with diverse immune functions was vigorous. is there.
For example, in the case of an influenza vaccine, particularly a split vaccine in which a virus component is purified, it is often used in combination with an adjuvant having a property of enhancing the tolerability and immunogenicity of the vaccine, and an adjuvant having such a property is actively developed. .. Since an excellent adjuvant can significantly reduce the amount of antigen required for the induction of neutralizing antibodies, it has a great influence on the construction of a large-scale manufacturing plant for preparing a large amount of antigen for several millions or more. Recently, squalene has come to be used as an adjuvant material, and it has been approved as a new anjuvant such as an oil adjuvant (GSK: AS03, Novartis: MF59) emulsified with the surfactant polysorbate 80, and its effect is drawing attention. There is.

Toll-like receptor (TLR) stimulators such as CpG and Poly I:C are highly safe clinically available adjuvant candidates with high interest in dendritic cell costimulator (second signal) activation adjuvant, Furthermore, cytokines such as GM-CSF and IFN-α/γ, and heat shock proteins are being studied.
The present inventor has developed an antigen delivery vector capable of efficiently introducing a vaccine antigen into a target and activating a second signal in dendritic cells in order to address various problems of cancer vaccines using peptide antigens. It was The vector is a highly safe virosome type vector in which a virus vector in which an antigenic polypeptide gene is incorporated into the virus structural gene of Paramyxoviridae human parainfluenza type 2 vector is inactivated, and the virus is inactivated ( Patent Document 1). The vector can significantly increase the expression of costimulatory substances such as CD86 molecule and adhesion molecules such as CD40 molecule in dendritic cells, and can efficiently introduce the antigen into antigen presenting cells and the like. The effectiveness of the model mouse against infectious diseases and cancer has already been confirmed using a vector into which the M2e antigen of pandemic influenza, the cancer antigen peptide gp100, and the wt1 peptide antigen have been introduced.

Makomo is a perennial plant that belongs to the genus Ricosidae and belongs to the genus Raccoonia that forms communities on waterside wetlands. The auxin secreted by the coexisting black spores (Kurobokin: Ustilago esculenta) causes the larvae to grow, and the enlarged part of them. Is served as edible mushrooms. Regarding the mushroom extract, the extract product is used as a bath agent by heat extraction or hydrophilic solvent such as alcohol than dried mushroom powder, it has an excellent warming effect on the body, improves blood circulation, and removes anti-waste from the body. It is said that it has an effect and also has a cosmetic effect (Patent Document 2). In addition, an immunostimulatory composition that can be ingested by ingestion of the product extracted from dried powder of Momotake mushrooms with hot water or chloroform or ethanol, and does not impair the flavor of the food or drink even when added to food, and food and drink containing it The provision of goods is shown (patent document 3).
Kurobuchin (Ustilago esculenta), which parasitizes Makomo, is a basidiomycete-type yeast belonging to the Kurokin web (Ustilaginomycetes). The cell wall of basidiomycetes is composed of chitin, which is a polymer of N-acetylglucosamine, as a skeleton, and glucan or mannan is bound to it, and the major cell-constituting sugar of clovokin is glucose, which is a 6-monosaccharide. It has been reported that β-glucan composed of glucose is contained and that 5-monosaccharide xylose is not contained (Non-Patent Document 1).

It has been reported that β-glucan, which is a kind of polysaccharide contained in mushrooms, yeasts, fungi, grasses, etc., has various immunostimulatory effects. In β-glucan, glucose has β-(1,3) bonds in its main chain and β-(1,6) bonds as side chains, and many substances have a molecular weight of several hundred thousand or more. The ratio of β-(1,3)-bonded side chains to the number of β-(1,3)-bonded main chains and the molecular weight of β-glucan may vary depending on the mushroom, yeast, fungus, gramineous plant, etc. from which they are derived. Are known. In order for β-glucan to have bioactive properties, it is important that three β-glucan molecules associate and have a triple helix structure. Lentinan (a physiologically active substance derived from Lentinus edodes) and schizophyllan (a physiologically active substance derived from Suehirodake) are highly purified β-glucans, and lentinan is 2 for every 5 β-(1,3)-linked glucose residues. Has β-(1,6) bonds, Schizophyllan has one β-(1,6) bond for approximately three β-(1,3)-linked glucose residues, and the molecular weight is several. It is an extremely large physiologically active substance of one million. In addition, sophibeta-glucan extracted from black yeast having 90% or more of β-(1,6) binding side chains has been reported, and its versatile physiological activity has been noted.
Pantoea is a gram-negative bacterium that is widely symbiotic with plants such as wheat, fruits, potatoes, rice, and shiitake mushrooms. It is known that lipopolysaccharide (LPS) contained in Pantoea is safer than conventional LPS.

International publication 2011/052771 JP-A-2-52827 JP, 2007-126382, A

Microbiol. Cult. Col. Dec. 2000, P41-50

As one of the factors that the conventional vaccine does not work effectively against the infectious diseases that are difficult to treat, and the cancer vaccine with the combination of the antigen peptide/protein and the oil-based adjuvant does not function effectively. It has been pointed out that these immune cells cannot be fully activated.
The present invention has been made in view of these circumstances, and an object thereof is to provide a novel adjuvant capable of significantly activating dendritic cells during vaccine administration and enhancing vaccine effect. That is.

The present inventor has added an extract from black rot fungus (Kloboquine: Ustilago esculenta) and Pantoea fungi (Pantoea) isolated from the gramineous plant Makomo to a vaccine as an adjuvant to express co-stimulants and the like in dendritic cells. It was found that the antibody-producing ability and the cytotoxic cell (CTL)-inducing ability are significantly increased by inducing the increase and the efficient maturation of dendritic cells associated therewith, compared with the administration of the vaccine alone, and thus the present invention was completed Came to do.
Thus, the adjuvant according to the invention for achieving the above object, (i) after crushing clovokin, centrifuged, and extracted from the supernatant thereof, (ii) after crushing Pantoea bacteria, centrifuged The extract is extracted from the supernatant thereof, or (iii) is extracted from a dried powder of mackerel plant using a solution (preferably water), and the activity is maintained by heat treatment. When extraction is performed using a solution (particularly water), the extraction treatment is performed at a temperature of 20°C to 150°C and a time period of 5 minutes to 24 hours. When the temperature is low, the extraction takes a long time, but the equipment and energy are small. On the other hand, if the temperature is raised, the extraction time will be short, but the equipment will be large.
An adjuvant is also called an antigenicity-enhancing agent and means a substance used to enhance the antigenicity of an antigen when administered together with the antigen. Adjuvants include sedimentary ones (eg sodium hydroxide, aluminum hydroxide, calcium phosphate, aluminum phosphate, alum, carboxyvinyl polymer, etc.) and oily ones (eg liquid paraffin, lanolin, complete Freund's, incomplete Freund's). Etc.) are known.

Although the mechanism of action of the adjuvant is not always clear, (1) by insolubilizing the antigen, it stays in the tissue for a long time, gradually releasing the antigen over a long period of time, and (2) by causing inflammation in the administration site, It is said that macrophages gather, antigens are easily phagocytosed, antigen presentation is effectively performed, and (3) activation of T cells and B cells in the administration local area and the lymph nodes to which they are administered is strengthened. In order to exert such a mechanism of action, it is advantageous that the molecular weight of the adjuvant is relatively large. Considering the adjuvant of the present invention, it is extracted with water and is not inactivated even at 120° C. for 20 minutes. From this, it is considered that the extract from cloboquine (Ustilago esculenta) that is symbiotic to Macomo, which is the adjuvant of the present invention, is a polysaccharide (for example, β-glucan (β1,3 glucan)). β-Glucan is a substance in which glucose has β-(1,3) bonds in its main chain, β-(1,6) bonds as side chains, and a molecular weight of several hundred thousand or more. However, the structure varies depending on the raw material. In addition, the extract from Pantoea bacteria is considered to be highly safe lipopolysaccharide (LPS) (hereinafter referred to as "Pantoair LPS" in order to distinguish it from the LPS for immunity-inducing positive control), because it is normally dietary. Be done. It was not known that Pantoea was symbiotic with Makomo.

Makomo (Zizania latifolia, also known as hanagatsumi) means a perennial plant of the genus Makomo of the Poaceae family. The sprouting new shoots that coexist with Ustilago esculenta, which is a kind of black spore fungus (Kurobokin), are called mackerel mushrooms, and are used as food materials. In Komono-cho, Mie-gun, Mie Prefecture, we are promoting the spread of locally-produced macaque mushrooms under the name of "the Kono no Makoto".
The adjuvant of the present invention will be extracted from dried powder of mackerel plant, Krobokin or Pantoea, or three or any two.
Makomo is a large, perennial swamp plant that forms communities and grows on waterside wetlands. Makomo's roots have rhizomes that grow thick and short in the mud, and branch roots that extend laterally from them. Also, many leaves and stems are emitted from the root. The leaves are 2-3 cm wide, long with rough edges and a round sheath at the bottom. Kurobuchin (Ustilago esculenta), which parasitizes Makomo, is a basidiomycete-type yeast belonging to the Kurokin web (Ustilaginomycetes), and β-glucan is contained in the main components of the yeast cell wall and the like. The Pantoea bacteria that co-exist in Makomo that we found this time are gram-negative bacteria and include Pantoea LPS. This Pantoair LPS can be contained and used as an adjuvant. Regarding the immunostimulation of the present invention, it is preferable to extract from dry powder of mackerel plant body from a part where Kloboquine or Pantoea bacteria coexist. In addition, among these, it is preferable to use the black ear fungus (Kurobokin: Ustilago esculenta) or Pantoea which coexist in Maccomo in an isolated state free from other microorganisms. With respect to the suppression of influenza virus growth, extraction from dried leaves or leaf powder components of Maccomo is preferable. Since this extract has influenza growth inhibitory activity, it can be provided as an anti-influenza composition. The composition can be used, for example, as a medicine, health food, and the like.

In order to prepare an adjuvant sample from the dried powder of macaque plant according to the present invention, for example, the following procedure can be performed.
For extraction from mackerel, (A) dry the leaf part or macaw mushroom part of macomo, and then make powder. (B) add water to this powder, extract at a suitable temperature, and precipitate by solid-liquid separation. (B') If necessary, water is added to the precipitate again, extraction is performed at a suitable temperature, and solid-liquid separation is performed to separate the precipitate from the supernatant liquid. A crude extract is obtained by separating the supernatant (C), the supernatant (C) as it is or after filtering with a membrane filter, and then concentrating the filtrate. This crude extract is used as it is, or after water is removed, it becomes a residue, and an appropriate amount of water is added again to this residue to dissolve it, and this is used as an adjuvant sample.
In the above (A), it is not always necessary to dry the part of the leaf of Makomo or the part of Bombyx mori. However, it is preferable to dry the raw materials from the viewpoint of stable supply of the raw materials and storage. Moreover, it is not always necessary that the leaf portion or the mushroom leaf portion of Macomo is powder. However, from the viewpoint of easy handling and extraction rate, powder is preferable.

In the above (B), the temperature of water is not particularly limited, but is 5°C to 120°C, preferably 10°C to 90°C, and more preferably 20°C to 40°C. Since the adjuvant sample of the present invention is not inactivated even by the pressurization/heating treatment at 120° C. for 20 minutes, it is preferable to extract at a high temperature in order to prevent contamination (contamination) of other substances. However, low-temperature water is preferable because a large amount of inexpensive treatment can be performed. In that case, it is preferable to perform sterilization treatment after extraction.
In the above (B), the solid-liquid separation method is not particularly limited, but examples thereof include filter filtration, centrifugation, vacuum dehydration, pressure dehydration and the like.

For example, the following procedure can be used to prepare an adjuvant sample from the smut (Kurobokin: Ustilago esculenta) or Pantoea bacterium of the present invention.
(C) Isolation of Kurobikin (Ustilago esculenta) or Pantoea bacterium is carried out by (C) isolation of Kurobikin (Ustilago esculenta) or Pantoea bacterium from the black spots of the enlarged raw mushrooms (D). (Kurobokin: Ustilago esculenta) or Pantoea bacterium is cultivated, and (F) black ear fungus (Kurobokin: Ustilago esculenta) or Pantoea bacterium is extracted.
Isolation of Kurobuchin (Ustilago esculenta) or Pantoea isolates the black spots of the enlarged flesh mushroom and isolates it on an agar medium suitable for the growth of Kurobuchin (Ustilago esculenta) or Pantoea. To do. The separation operation is repeated until the morphology of the microorganism becomes single. Furthermore, increasing the number of fungi and yeast strains by culturing the isolated black spores (Kurobokin: Ustilago esculenta) or Pantoea bacteria on the same agar medium is an effective means for increasing the purity and increasing the production amount. It is also possible to co-cultivate both the black spore (Kurobokin: Ustilago esculenta) and the Pantoea bacterium, or to use them as an adjuvant without separating them. It is also effective to mix isolated and cultivated Kroboquine or Pantoea in a ratio of 1:1 to 1:10.
In addition, black spore fungus (Kurobokin: Ustilago esculenta) or Pantoea bacterium may be found in plants other than Makomo. In that case, in the plant, each fungus is isolated from a site containing (A) black ear fungus (Kurobokin: Ustilago esculenta) or Pantoea fungus, (B) the bacterial cells are grown by culture, and (C) the supernatant An adjuvant can be manufactured by obtaining a crude extract from a liquid or a microbial cell.

According to the present invention, it is possible to provide a novel adjuvant that can significantly activate dendritic cells during vaccine administration and enhance the vaccine effect.

4 is a graph showing the results of induction of CD86 molecules in Example 1. 3 is a graph showing the induction results of I-A/I-E molecules in Example 1. 7 is a graph showing the induction results of CD86 molecules (A) and I-A/I-E molecules (B) of mackerel extract extracted by pressurization/heating treatment in Example 2. 7 is a graph showing the induction results of CD86 molecule (A), HLA-A molecule (B) and HLA-DR molecule (C) in Example 3. 9 is a graph showing the results of induction of CD86 molecules and I-A/I-E molecules when separated with a 100K filter in Example 4. 8 is a graph showing the results of confirming the adjuvant activity when the macaque extract was administered intranasally and intramuscularly together with the BC-PIV vaccine in Example 5. 5 is a photographic diagram and a graph showing the results of confirming the adjuvant activity when the macaque extract was intratumorally administered with the BC-PIV vaccine in Example 6. FIG. The photograph shows tumor growth 13 days after administration. 5 is a photographic diagram and a graph showing the results of confirming the adjuvant activity when the macaque extract is administered to a remote site (intradermal) together with the BC-PIV vaccine in Example 6. The photograph shows tumor growth 13 days after administration. FIG. 9 is a photograph showing the hemagglutination activity of the virus showing the result that the dried mackerel leaf powder extract suppresses the growth of influenza virus in Vero cells in Example 7. FIG. 3 is a micrograph showing the morphology of three types of microorganisms separated from raw mushrooms. 11 is a graph showing the expression of CD86 molecules in mouse dendritic cells of the microbial extract shown in FIG.

Next, embodiments of the present invention will be described with reference to the drawings, but the technical scope of the present invention is not limited to these embodiments, and various forms can be applied without changing the gist of the invention. Can be implemented in.
<Example 1> An extract from dried powder of macaque plant increases the expression of CD86 molecule and IA/IE molecule.
The CD86 molecule required for signal transduction in dendritic cells, which are antigen-presenting cells, in order for a substance to function as an adjuvant that contributes to the acquisition of antigen-specific immunity to T cells and B cells of vaccines It is necessary to increase the expression of co-stimulators.
Therefore, we examined the expression of co-stimulant CD86 molecule and class II molecule IA/IE molecule in mouse dendritic cells in an extract from macaque.
The Makomo extract sample was prepared by the following method. 20 mL of water was added to 2 g of the powder prepared by drying and cutting Makomo, and the mixture was stirred at 25° C. for 24 hours. Then, the suspension was centrifuged (5000 rpm, 10 minutes) to obtain a supernatant. Ion-exchanged water (5 mL) was added to the precipitate, and the mixture was stirred and centrifuged (5000 rpm, 10 minutes) to obtain a supernatant. This operation was repeated 3 times, and the supernatant was filtered with a membrane filter having a diameter of 0.45 μm. The filtrate was concentrated under reduced pressure to obtain 0.49 g of a pale yellow residue. A solution obtained by dissolving the residue in 10 mL of ion-exchanged water was used as a crude extract. 0.5 mL of the crude extract was subjected to centrifugal separation (2500×g, 30 minutes) using 3K, 10K, and 50K Amicon Ultra ultrafiltration membranes, and fractionated. Fractions that passed or did not pass through each filter were collected, and ion-exchanged water was added to prepare 1 mL of sample liquid.

Mouse dendritic cells were prepared as follows based on the ethical rules. Bone marrow was collected from the femur of B57BL/6/J mouse, and the residue was removed and cultured. As a culture medium, RPM-1640 medium containing 10% FBS was used. The medium was exchanged every 2 days of culture, and GM-CSF and IL-4 were added at 20 ng/mL each. To a cell culture sample that had been cultivated for 8 days, the Makomo extract was added in an amount of 1/10 of the medium, and after culturing for 2 days, the expression of CD86 molecule and IA/IE molecule was analyzed by FACS.
As a positive control, lipopolysaccharide (LPS), which is a constituent of the cell wall outer membrane of Gram-negative bacteria at 1 μg/mL, was used.
The results are shown in FIGS. 1 and 2. The expression of CD86 molecule and IA/IE molecule was increased by the addition of mackerel extract. Details are given below.

1. CD86 molecule Fig. 1 shows the induction result of the CD86 molecule. Increased expression of the CD86 molecule was observed in all the non-passage fractions (concentrated fractions) of the ultrafiltration membranes 3K, 10K and 50K. On the other hand, since no increase in expression was observed in the ultrafiltration membrane passage fraction, the passage fraction did not contain a component that increases the expression of the CD86 molecule. As described above, the ultrafiltration-enriched fraction of Macomo contained a component that promotes the increased expression of costimulatory substances of dendritic cells. Although not shown in the figure, when the same measurement test was performed for the CD40 molecule, an increase in expression was observed.

2. IA/IE molecule Fig. 2 shows the induction result of the IA/IE molecule. In all non-passage fractions (concentrated fractions) of the ultrafiltration membranes 3K, 10K, and 50K, an increase in the expression of IA/IE molecules, which are MHC class II components, was observed. Makomo extract is a non-passage fraction of all ultrafiltration membranes and has a high IA/IE molecule expression-inducing ability equivalent to or higher than LPS, and can efficiently activate helper T cells. Was expected. In addition, no increase in expression was observed in all ultrafiltration membrane passage fractions.
From these results, it was found that the extract component of Maccomo that does not pass through the 50K ultrafiltration membrane of Amicon increases the expression of IA/IE molecules, which are MHC class II molecules of dendritic cells.
Although not shown in the figure, when the same measurement test was performed for CD86 molecule and CD40 molecule, the result of increased expression was obtained.
As described above, the soluble fraction obtained by extracting water from mackerel dried matter contained a co-stimulator of dendritic cells and a component that increased the expression of MHC class II molecules.

Example 2 Extracts from Maccomo are heat stable.
The Makomo extract sample was prepared as follows. That is, 18 mL of distilled water was added to 2 g of the powder prepared after drying and cutting Makomo, and the mixture was stirred and heated/pressurized at 120° C. for 20 minutes (so-called autoclave treatment). After pressurizing and heating, centrifuge at 2500 × g, collect approximately 7 mL of supernatant so that precipitates are not included, remove the residue with a 0.45 μm filter, and add distilled water. To give 20 mL of crude extract. 1 mL of the crude extract was centrifuged (2500 xg, 30 minutes) using a 3K Amicon Ultra ultrafiltration membrane centrifuge, and each fractionated, and the fractions that passed or did not pass through the filter were collected. In addition, 1 mL of sample solution was prepared.
With respect to this sample, a test for confirming the CD86 molecule and IA/IE molecule expression inducing ability of mouse dendritic cells was carried out in the same manner as in Example 1.
The results are shown in Fig. 3. The sample solution that had been subjected to pressure/heat treatment at 120°C for 20 minutes contained components capable of inducing expression of CD86 molecules and IA/IE molecules. A certain effect can be expected in the range of 100 to 130°C and 10 to 30 minutes in the range of 120°C and 20 minutes.
Thus, the adjuvant component was extremely stable to heat.

<Example 3> The macaque extract also efficiently activates human dendritic cells.
Next, the macaque extract was examined for its ability to induce the expression of costimulatory molecules on human dendritic cells.
Peripheral blood was collected from a healthy person who obtained informed consent, and peripheral blood mononuclear cells (PBMC) were collected using Ficoll-Paque PLUS (GE Healthcare). CD14-positive monocytes were collected from PBMC using CD14 macro beads. CD14-positive monocytes were cultured at 37°C for 7 days using RPMI1640 medium containing 10% FBS, 50 μM 2-mercaptoethanol, 50 ng/mL GM-CSF and 50 ng/mL IL-4, and monocyte-derived dendritic cells were obtained. Recovered.
To the cells obtained, the Makomo extract collected at 37°C was added to the medium at a volume of 1/10 and 1/100, and after culturing for 2 days, the CD86 molecule, HLA-A molecule and HLA-DR molecule were added. Was analyzed using FACS.
As a positive control, 1 μg/mL LPS was used.

The results are shown in Fig. 4. When Makomo extract was added to 1/10 volume of the medium, it was higher than the LPS of the positive control, and the expression of CD86 molecule was induced. The CD86 molecule was also induced when 1/100 was added. HLA class I HLA-A had less induction compared to LPS. On the other hand, HLA class II HLA-DR showed higher expression inducibility than LPS.
As described above, the Makomo extract was able to efficiently activate not only mouse but also human dendritic cells. It was found that HLA class II molecules can be activated higher than HLA class I molecules as compared with LPS. That is, it was shown that helper T cells also have a function as an adjuvant that can be efficiently activated.

<Example 4> Most of the CD86 molecule expression inducing component is present in the 100K filter non-passage fraction (each concentrated fraction).
The supernatant fraction of the Amicon centrifugal filter 3K prepared by the method of Example 2 was fractionated using a 100K filter having a larger fractionated molecular weight to examine higher molecular weight fraction components.
The fractionation method was the same as in Example 2, and the sample solution was extracted by centrifuging at 2500×g for 30 minutes using an Amicon 100K filter. The Makomo extract fraction fractionated with a 100K filter was added to a mouse-derived dendritic cell preparation solution in an amount 1/10 of the medium, and the expression of CD86 molecule and IA/IE molecule was analyzed by FACS.
As a positive control, 1 μg/mL LPS was used.

The results are shown in Fig. 5. Most of the CD86 molecule expression inducing component was present in the 100K filter non-passage fraction (each concentrated fraction). The fraction passed through the 100K filter was higher than that in the negative control PBS-added group, but the expression of the CD86 molecule could not be effectively induced compared with the non-passed fraction (each concentrated fraction). It was On the other hand, the expression of MHC class II (IA/IE molecule) was also abundantly contained in the filter passage fraction. In the fractionation with a 50K filter, it was confirmed that the I-A/I-E molecule expression inducing component was present in the Amicon Ultra ultrafiltration membrane (50K) impermeable fraction (Fig. 1). In this example, it was found that the 50K to 100K fractions of the Amicon Ultra ultrafiltration membrane also contained a large amount of components capable of inducing the expression of MHC class II I-A/I-E molecules.

<Example 5> Maccomo extract exhibits an adjuvant effect against BC-PIV vaccine (influenza vaccine).
The present inventor has developed a novel inactivated virus vaccine vector (BC-PIV) and confirmed its efficacy in model mice against infectious diseases and cancer (Patent Document 1). Specifically, in infectious diseases, BC-PIV/M2e introduced with M2e (24 amino acid residues), which is an antigen in the extramembrane region of influenza virus M2 protein known as an antigen capable of responding to the pandemic influenza virus. Was constructed and the vaccine effect against influenza virus (PR-8 strain) was examined using Balb/C mice. It has been confirmed that BC-PIV/M2e has a high virus infection/growth inhibitory effect against influenza virus. To date, many pharmaceutical companies have been working on the development of a universal influenza vaccine using the M2e antigen, but it has been reported that extrapolation to large animals resulting from small animals is not easy. Specifically, it has been reported that it is difficult to raise the antibody titer in monkeys and the like even when the antibody titer against M2e and the virus growth inhibitory effect are confirmed in the infection test of mice, ferrets and the like. Thus, the universal influenza antigen M2e is not easy to raise the antibody titer in higher animals such as monkeys, which indicates the difficulty of developing the universal influenza in humans.

On the other hand, the host of human paraffinphena type 2 virus, which is the mother of the inactivated BC-PIV vector, is human, and the virus is permissive in humans. However, it is known that this virus has a very poor proliferative property in mice (poor permissive). Therefore, it is considered that the mouse results are highly extrapolable to humans, as inferred from the infection characteristics.
Therefore, we added Makomo extract to BC-PIV/M2e vaccine containing M2e antigen and evaluated the antibody production by intranasal or intramuscular administration. The test method is as follows: After anaesthetizing a 5-week-old Balb/C mouse (female), nasal (vaccine amount 20 μL, 6×10 ⁷ particles) or muscle (quadriceps femoris 50 μL, 1.5×10 ⁷ particles) The increase in antibody titer was measured under the presence or absence of BC-PIV/M2e and MacMomo extract (Nasal: Macomo extract 4 μL, Muscle: Macomo extract 10 μL). Nasal: BC-PIV group, BC-PIV/M2e group, BC-PIV/M2e group + Makomo sample solution group, and BC-PIV group, BC-PIV/M2e group, BC as intramuscular administration group -PIV/M2e group + Maccomo sample solution group. The control was the non-administration group.

Spleen cells were collected from the sample-administered mice. Cut the spleen into 5 mm width in RPMI1640 medium (10% FBS, 1% penicillin-streptomycin solution and 50 μM 2-mercaptoethanol), spleen cells by rubbing spleen fragments with a glass slide surface, and collecting spleen cells, After centrifugation, the supernatant was discarded. After adding 1 mL of erythrocyte removal buffer (150 mM ammonium chloride, 10 mM potassium hydrogen carbonate and 100 μM EDTA), 9 mL of RPMI1640 medium was added and centrifuged to collect cells and suspend the cells in RPMI1640 medium. 5 μg/mL of Me2 antigen or BC-PIV protein was added to the collected spleen cells and cultured for 3 days. The culture supernatant was collected and the induced IgG1, IgG2, and IgA were measured by ELISA.

The test results are shown in FIG. Nasal administration of the vaccine induced IgA mainly against the universal influenza antigen M2e. IgA was induced about 1.8 times in the BC-PIV/M2e group and about 2.3 times in the BC-PIV/M2e group + Makomo sample solution group, compared with the non-sensitized control. Already, in a test using mice, the protective effect against influenza virus by BC-PIV/M2e administration has been confirmed, and since the anti-M2e/IgA antibody titer was increased by adding the Makomo extract fraction this time, It was confirmed that nasal administration has an adjuvant effect on BC-PIV vaccine.

On the other hand, for administration to the quadriceps femoris, the vector and the Makomo extract were administered at 2.5 times the nasal dose. Upon administration to quadriceps femoris, the anti-IgG2a antibody titer was mainly increased. Compared to the non-sensitized control, about 2 times as much IgG2a antibody was induced in the BC-PIV/M2e group and about 2.5 times as much in the BC-PIV/M2e group+Makomo sample solution group. Similar to IgA, in the case of IgG2a, the anti-M2e IgG2a antibody titer was increased by adding the Makomo extract fraction. It was confirmed that nasal administration has an adjuvant effect on BC-PIV. In addition, an adjuvant effect against the BC-PIV vaccine was confirmed even in the case of intramuscular administration.
On the other hand, the antibody titer to the hPIV2 vector was increased both intranasally and intramuscularly. Since the vector as a whole contains various antigens, an increase in antibody titer is naturally expected, but the Makomo extract fraction did not raise the antibody titer against the vector itself. In other words, it was suggested that the Makomo extract has an action of increasing the immunogenicity against the antigen having low antigenicity, without increasing the immunogenicity against the antigen having high antigenicity.

<Example 6> Maccomo extract exhibits an adjuvant effect against BC-PIV vaccine (mouse melanoma cancer vaccine).
Next, it was confirmed whether or not the extract component of Makomo could enhance the antitumor effect as a cancer vaccine. Vaccine and Makomo extract were administered directly into the tumor or distant from the tumor site, and their efficacy was examined. As test animals, B6 mice (female) aged 4-5 weeks were used. B16 cells, which are mouse melanocytes transformed into cancer, were used as the cancer cells. In addition to BC-PIV/gp100 which holds the gp100 peptide, BC-PIV/wt1 which holds the WT1 peptide (SEQ ID NO: 1: N-RMFPNAPYL-C) was prepared and added.

1. Intratumoral administration test The back of the mouse was shaved, and 2×10 ⁶ B16 cells suspended in 100 μL of physiological saline were intradermally transplanted. Four days after the transplantation, B16 cell-engrafted mice were selected and subjected to intratumoral vaccination test. As the vaccine, BC-PIV/gp100 (1×10 ⁷ particles) and BC-PIV/wt1 (8×10 ⁶ particles) were administered, respectively. The sample prepared in Example 2 was used as the Maccomo extract sample. The administration groups consisted of (i) PBS (70 μL) control group, (ii) BC-PIV group, (iii) BC-PIV/wt1 (35 μL) + BC-PIV/gp100 (35 μL) group, (iv) BC- There were 4 groups of PIV/wt1 (35 μL)+BC-PIV/gp100 (35 μL)+macomo extract (20 μL). The vaccine was administered intratumorally four times after cell transplantation, 4, 8, 11, and 14 days. The tumor volume (mm ³ ) was calculated by measuring the minor axis (mm) and major axis (mm) of the tumor and calculating major axis x minor axis x minor axis x 0.52.
The results are shown in Fig. 7. Tumor volume increased in the PBS-administered group, but tumor growth was suppressed in all the other three groups compared to the PBS-administered group. Regarding intratumoral administration, BC-PIV was the only common component in the tumor suppression group, and BC-PIV alone was considered to have a tumor suppression effect.

2. Remote site (intradermal) administration test Next, the effect of administering the vaccine to the remote site from the tumor transplant site was examined. The back of the mouse was shaved, and 2×10 ⁶ B16 cells suspended in 100 μL of physiological saline were transplanted into the right back skin. The day after transplantation, the sample was administered intradermally on the dorsal side of the skin opposite to the site of transplantation. The composition of the administration group was (i) PBS (70 μL) control group, (ii) Maccomo extract sample (100 μL) group, (iii) BC-PIV group, (iv) BC-PIV/wt1 (35 μL; 1×10 ⁷ (Particles) + BC-PIV/gp100 (35 μL; 8×10 ⁶ particles) group, (v) BC-PIV/wt1 (35 μL) + BC-PIV/gp100 (35 μL) + Maccomo extract (100 μL) group .. There were multiple administration sites. The administration was performed 4 times, 1 day, 4 days, 8 days and 11 days after cell transplantation. The tumor diameter was measured in the same manner as the above intratumoral administration.

The results are shown in FIG. 8 (ulcers were generated as tumors increased, which affected the measurement of tumor growth, and in some animals with large tumor growth, an accurate tumor growth volume could not always be measured). (v) The tumor growth was strongly suppressed only in the group administered with BC-PIV/gp100+BC-PIV/wt1+macomo extract combined. No tumor suppressive effect was observed in the (ii) macaque extract administration group and (iv) BC-PIV/wt1+BC-PIV/gp100 vaccine administration group. From the above, it was confirmed that the tumor growth was suppressed by the synergistic effect of both the macaque extract and BC-PIV/wt1+BC-PIV/gp100. In the vaccine-only group (BC-PIV/gp100 (1 × 10 ⁷ particles) and BC-PIV/wt1 (8 × 10 ⁶ particles)), it was confirmed that increasing the dose had a tumor suppressive effect. , The effect of suppression as high as that of the vaccine/macomo extract combination group was not observed. Therefore, it was considered that the Makomo extract enhances the effect of BC-PIV vaccine to suppress the tumor growth by the adjuvant action.

<Example 7> Among mackerel extracts, particularly, an extract from dried mackerel leaf powder suppresses the growth of influenza virus infecting Vero cells.
Dried macaque leaf extract does not act directly on the influenza virus and cannot inhibit it. Therefore, it was investigated whether or not the virus growth in influenza virus infected cells could be suppressed.
Water was added in an amount 10 times the weight of the dried powder of Makomo leaf and heated at 37°C, 60°C, and 80°C for 6 to 16 hours. The supernatant after the heat treatment was centrifuged was used as the sample solution. Alternatively, the mixture was autoclaved at 121° C. for 20 minutes, and the supernatant after centrifugation was used as a sample solution in the following tests.
The cells used were Vero cells derived from African green monkey kidney epithelial cells, which are known to grow well in influenza virus.
Influenza virus (PR-8 strain) was infected with 1/100 (MOI=0.01) virus to the cell number for 1 hour after removing the medium. After infection, the medium and the sample solution were added to 1/10 or 1/100 volume of the medium and cultured for 3 days. After infection, add 10% or 1/100 volume of the culture medium of macomo extract treated with 10% volume at 37°C (overnight) or autoclave and incubate for 3 days. It was evaluated by measuring the activity (HA activity).

FIG. 9 shows the results of suppressing the influenza virus growth of the dried powdery extract of mackerel leaf in Vero cells infected with influenza virus. When 1/10 volume of Makomo leaf extract was added, the HA value was 2 or less in any of the extraction temperature samples, and the HA value of the negative control containing no virus was 2 or less. It was strongly suppressed (HA value of virus positive control 64 (positive control in the figure). The growth of virus was suppressed to a certain extent even when 1/100 volume of Makomo extract was added. It was found that the growth was suppressed.
Similarly, in a test using 293 cells derived from human fetal kidney, the Makomo extract suppressed the growth of infuenza virus.

<Example 8> Adjuvant effect of Kurobikin (Ustilago esculenta) or Pantoea fungus isolated from raw Momotake mushrooms: From the results obtained so far, the extract from Makomo showed activation of dendritic cells and action of BC-PIV vaccine. It was found to have an adjuvant effect of activating. However, in the process of performing the extraction operation a plurality of times, it was not always possible to obtain a result showing the dendritic cell activation effect from the extract. Therefore, it was examined whether the phenomenon was caused by a component derived from mackerel or a microorganism such as Kurobuchin (Ustilago esculenta) coexisting in macomo.
A modified medium of Czapeck-Dox medium described in Natural Medicine 48(4), 272-281 (1994) was used for the isolation of the symbiotic fungus. Specific media contained (g), as a 1% Agar maintenance medium (pH 6.0 ), (NH 4) 2 SO 4 3.0; K 2 HPO 4 1.0; MgSO 4 · 7H 2 0 0.5; KCI 0.5; FeSO 4・7H ₂ O 0.01; Sucrose 20.0; Agar powder 10.0; Macaw mushroom extract 50 (ml); make up to 1 liter with purified water, sterilize by autoclave, put about 12 mL of agar medium on 10 cm plate, and use as a solid plate for separation. did.
Regarding bacteria expected to be bacteria among the bacteria obtained from the above culture solution, DSMZ medium (malt extract 30.0 g, soybean peptone 3 g, agar 15.0 g (in the case of plate), distilled water (1000 mL)) was added to pH 5.6. And sterilized at 121° C. for 10 minutes, and subjected to a culture test. The culture temperature was mainly 23°C.

Separation The black spots, which are thought to contain the spores of Kurobikin (Ustilago esculenta), were taken out from the swelled raw mushrooms, and were sterilized, placed on a solid plate for separation, and incubated at 25-27°C. Microorganisms considered to be molds were excluded, three types of grown microorganisms were isolated, and subjected to subsequent tests.

Separation of Bacteria The morphology of the three types of separated bacteria is shown in Figures 10-1, 10-2, and 10-3. Regarding the isolates, PCR primers were set to the nucleotide sequence reported as 28S rRNA of Klovokine and its neighboring ITS region, and PCR was performed using the recovered genome. For the ITS2 and 28S rRNA regions, PCR was performed using the sequences 5'-gaa ttg ttt cga acg aca gc-3' (SEQ ID NO: 2) and 5'-taa agg tgc ccg aag gcc cg-3' (SEQ ID NO: 3). A 919 bp long PCR product was expected (accession number: AB211904.1 and AF453937 sequences used). For the intergenic spacer region, a PCR product of 575 bp in length was used with 5'-tga gtg agt tgg ctg gtt gg-3' (SEQ ID NO: 4) and 5'-gac aag tgt gag gtt ccc gg-3' (SEQ ID NO: 5). Was expected (accession number: using the sequence of AB211931). PCR using the genome of the bacteria shown in FIGS. 10-1 and 10-2 confirmed a PCR product of a desired length. The fungus shown in Figures 10-1 and 10-2 grows from the area where spores of Kurobuchin (Ustilago esculenta) are accumulated, but they are the same fungus due to the hyphal extension form. It was difficult to think that, and it was assumed that the bacteria had similar sequences. The bacterium shown in 10-1 is a bacterium that germinated from spores, and the nucleotide sequence of the PCR product is in agreement with the reported sequence of Kurobikin (Ustilago esculenta). (Kurobokin: Ustilago esculenta).
The bacteria shown in 10-3 prepared in DSMZ medium were identified. A primer for 16S ribosomal RNA for bacteria was set, PCR was carried out using the genome recovered from the isolated bacterial cells by the usual method, and the nucleotide sequence of the sequence was examined. As the primer sequences, 5'-agagtttgatcmtggctcag-3' (SEQ ID NO: 6) and 5'-ggttaccttgttacgactt -3' (SEQ ID NO: 7) were used (m is a or c). By comparing the nucleotide sequences with homology, a match was found at 599 bases out of 604 bases compared with Pantoea agglomerans strain WSA 16S ribosomal RNA (accession number:KT075208).
The cultured cells on the three types of flat plates were collected and examined for the expression of CD86 molecule in mouse dendritic cells. As for the method, the bacterial cells of 10-1 and 10-3 samples were suspended in 2 ml of sterile platinum water and sonicated for 30-40 seconds. The ultrasonic treatment did not completely solubilize the 10-1 sample. The 10-3 sample was significantly solubilized by sonication. In the 10-2 sample, since the entire colony was hard and it was difficult to collect the bacterial cells with a platinum loop, the bacterial cells were picked up with a toothpick and suspended in sterile water. The cells were hardly decomposed even after the ultrasonic treatment. The mixture was treated at 60°C for 2 hours, and the residue was removed by centrifugation. The supernatant was taken, passed through a 0.45 μm filter, and the residue was further removed, and subjected to the following test. The main component of the extracted component of Kroboquine 10-1 was β-glucan as measured by a glucan measurement kit. The main component of the Pantoea extract of 10-3 was not β-glucan.

Confirmation of CD86 expression Recovery of dendritic cells from mice was performed by the same method as described above. The expression of 10-1, 10-2 and 10-3 CD86 molecules was examined. From FIG. 11-1, the extracts from each of the bacteria 10-1, 10-2, and 10-3 showed the expression of the CD86 molecule as shown in the figure as compared with the control LPS. It was found that 10-1 (a sample derived from Kurobuchin (Ustilago esculenta)) and 10-3 pantoea sample liquids can significantly increase CD86 molecules.
According to the present embodiment, it is possible to provide a novel adjuvant that can significantly activate dendritic cells during vaccine administration and enhance the vaccine effect.
It is also possible to co-cultivate both Kurobikin (Ustilago esculenta) and Pantoea, or use them as an adjuvant without separating them. Further, it is also effective to mix the isolated and cultivated black spores (Kurobokin: Ustilago esculenta) or Pantoea at a ratio of 1:1 to 1:10. Further, it can be expected to be applied not only as an adjuvant but also as a food additive, a functional food or a supplement, to a food or the like that enhances the immune function.
Further, as described above, with respect to the inhibition of influenza virus growth, extraction from dried leaves or leaf powder components of mackerel is preferable. Since this extract has influenza virus growth inhibitory activity, it can be provided as an anti-influenza composition. The composition can be used, for example, as a medicine, health food, and the like.
Examples of the Gramineae plants that can be used above include rice, wheat, millet, and corn.

As described above, a component extracted from a microorganism symbiotic with Pleurotus cornucopiae or gramineous plants is extremely effective as an adjuvant for enhancing the vaccine action against cancer, influenza and the like. As described above, the adjuvant of the present invention is an adjuvant that enhances the costimulate function of increasing the expression of CD86 molecule and IA/IE molecule, and enhances the function of the vaccine antigen to enhance the vaccine body. The healing effect of can be enhanced.

Claims (4)

It was extracted by treating Makomo with a solution at 20°C to 150°C for 5 minutes to 24 hours, which did not pass through a 50K ultrafiltration membrane, and was pressed and heated at 121°C for 20 minutes. features and to luer adjuvant that contains lipopolysaccharide obtained from Pantoea bacterium from wild rice to maintain the activity at process (LPS). The method for producing an adjuvant according to claim 1 , wherein (A) dried Enlarged Pleurotus cornucopiae containing Pantoea bacterium derived from Macomo is made into a powder, and (B) water is added to this powder to perform an extraction operation. A method for producing an adjuvant, in which a solid extract is separated into a precipitate and a supernatant liquid to obtain a crude extract from the supernatant liquid. It is a manufacturing method of the adjuvant of Claim 1, Comprising: (C) Pantoea bacterium is isolated from the black spot part of the enlarged raw mushrooms, (D) Pantoea bacterium is cultured, and (F) Pantoea bacterium is extracted. A method for producing an adjuvant to be performed. A health food containing the adjuvant according to claim 1 for enhancing immune function.