JP6681449B2 - Vγ1Vδ1 T cell activator and therapeutic agent for viral infection - Google Patents

Description

  The present invention relates to a Vγ1Vδ1-type T cell activator and a method for activating Vγ1Vδ1-type T cells in a living body using the same. The present invention also relates to a therapeutic agent for a viral infection containing an active ingredient having a Vγ1Vδ1-type T cell activating effect, and to the use of the therapeutic agent for a viral infection for treating HIV infection.

HIV (human immunodeficiency virus) is a virus that infects and destroys human immune cells. AIDS (AIDS: Acquired Immunodeficiency Syndrome) is a disease that presents with a severe immunodeficiency state or severe immunocompromised symptoms at the onset stage, which is the final stage of HIV infection.
HIV is a type of retrovirus that targets CD4-positive immune system-related cells and infects, for example, CD4-positive helper T cells, CD4-positive macrophages, CD4-positive dendritic cells, and damages the immune system by destroying them. It is known to give. In particular, it directly or indirectly destroys CD4-positive helper T cells that belong to the acquired immune system, causing serious damage to the immune system.
The stage of HIV infection is divided into an acute infection stage, a symptom-free period of 5 to 10 years after the acute infection period, and an onset stage after the asymptomatic period. During the acute infection stage, the viral load increases and presents symptoms that are indistinguishable from other infectious diseases. However, when the production of antibodies is activated, the viral load drops sharply, and the condition shifts to the asymptomatic stage. During the asymptomatic period, an equilibrium between HIV growth and inhibition of virus growth by the immune system is maintained, so that the apparent virus concentration in the blood is kept low and asymptomatic, but the exhaustion of the immune system continues to progress and CD4 Positive helper T cells gradually decrease. When the number of CD4-positive helper T cells in the peripheral blood decreases to some extent, the state of equilibrium between HIV proliferation and the immune system is disrupted, and the disease enters the onset stage. In the onset stage, immunodeficiency occurs, and AIDS develops when the disease becomes severe.
The progress of the infection is determined by measuring the amount of the virus by the HIV-1 PCR method and examining the number of CD4-positive cells by the flow cytometry method. The number of CD4 positive cells is a numerical value reflecting the current disease state. It is 800-1200 cells / μl under normal conditions, but gradually decreases when infected with HIV, and when it is lower than about 200 cells / μl, AIDS develops and symptoms such as opportunistic infections and opportunistic tumors appear.

The human immune system exists mainly on the body surface, which is the site of entry of foreign substances such as mucous membranes and skin, and the immune system mainly exists in the blood and in immune organs such as the spleen and lymph nodes. Acquisition immune system ".
In the former group of cells responsible for innate immunity, information on invading foreign substances is recognized and grasped via Toll-like receptors (TLRs) expressed on the surface, the information is transmitted to the entire immune system, and the surrounding network for foreign substance control Dendritic cell group that directs formation, NKT cells (Natural Killer T-cells) responsible for retaining and processing foreign substances, and γδ T cells that control foreign substance invasion and repair damage caused by invasion Groups etc. are included. The γδ T cell group is mainly classified into Vγ1Vδ1 and Vγ2Vδ2, and is thought to be responsible for controlling invading pathogens and eliminating locally generated tumors.
Until now, cells that have HIV sensitivity in the immune system have been considered to be CD4 molecule-positive T cells that belong to the adaptive immune system, such as helper T cells, but recently, CD4 positive cells that belong to the innate immune system. It was also found that the NKT cell population of E. coli is also sensitive to HIV (Non-patent Document 1).

At present, HAART (Highly Active Anti-retroviral Therapy) is known as an effective treatment for HIV infection, and is widely applied to actual treatment. HAART is a treatment that suppresses viral growth and prevents the onset of AIDS by administering a plurality of anti-HIV-1 drugs in combination according to the symptoms and constitution of individual HIV-infected individuals.
However, even if no HIV particles are found in the patient's peripheral blood by performing HAART on an HIV-infected person, the HIV particles may appear again in the peripheral blood immediately after the treatment is interrupted. (Non-Patent Document 2).
Currently known anti-HIV drugs can reduce HIV particles in the peripheral blood of patients to below the detection limit, but it is almost impossible to completely eliminate HIV particles from the patient's body. Even if is successful, it is difficult to completely cure the HIV infection because HIV remains latently in somewhere in the individual patient and remains proliferative.

In a previous study, the present inventors performed anti-retroviral therapy (ART), and used a large intestine fiberscope to produce a product derived from intestinal mucosa in patients whose virus could not be detected in peripheral blood. Examination of the test tissue confirmed the presence of HIV-releasing p24 antigen-positive cells in the ileal mucosal tissue, and confirmed that the cells expressed a Vα24-positive T cell receptor unique to NKT cells (non Patent Document 3). The results of this study indicate that NKT cells of the innate immune system present at the mucosal site may be a source of HIV-1 release when HIV-infected persons are successfully treated with antiretroviral therapy and then interrupted. Suggests high.
Focusing on the results of this study, the present inventors further attempted to identify cells that regulate virus replication in HIV-infected CD4-positive NKT cells. As a result, the CD8αα-positive T cell group, particularly the Vγ1Vδ1 type γδT cell group, was identified as NKT. It was found that it exhibits an intracellular HIV growth inhibitory action (Non-Patent Document 4).

J. Exp. Med., 195: 869-879, 2002 Nature Med., 6: 757-879, 2002 Biomed. Res., 35: 1-8, 2014 Immunology, 141: 596, 2014

  The present invention has been accomplished based on the above findings, and Vγ1Vδ1 can inhibit virus replication in virus-infected cells by activating Vγ1Vδ1 T cells in the body of a virus-infected person. Infection therapy that can prevent the virus from remaining at the latent site based on a different principle of action from conventional antiviral drugs by using the T cell activator of the type and the Vγ1Vδ1 type T cell activator It is intended to provide an agent.

The therapeutic agent for viral infection provided as at least a part of the present invention comprises, as an active ingredient, a compound represented by the following formula (1) or (2) and a pharmacologically acceptable salt or ester thereof. A therapeutic agent for HIV infection , which comprises at least one flavonoid glycoside selected from the group consisting of: (a) a compound used to suppress the proliferation of HIV-infected patients in an asymptomatic period by activating Vγ1Vδ1-type T cells.

[In the formulas (1) and (2), R ¹ represents an alkyl group having 1 to 3 carbon atoms, and R ² represents a hydrogen atom or a hydroxyl group. R ³ is a substituent which may be bonded on the ring A, and represents a substituent selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, a hydroxyl group and a halogen, and R ⁴ is bonded on the ring B. Represents a substituent selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, a hydroxyl group and a halogen, and R ⁵ is a substituent which may be bonded to the C ring. Represents a substituent selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, a hydroxyl group, and a halogen. n1 is the number of R ³ and represents an integer of 0 to 2, n2 is the number of R ⁴ and represents an integer of 0 to 3 in the case of the formula (1), and 0 or 3 in the case of the formula (2). represents the integer 1, n3 represents an integer of from 0 to a number of R ⁵ 3. When there are a plurality of substituents represented by the same symbol, each substituent may be the same or different. ]

The active ingredient of the therapeutic agent for virus infection according to the present invention includes, for example, hesperidin represented by the following formula (3), linaline represented by the following formula (4), and a pharmacologically acceptable salt thereof. It may be at least one selected from the group consisting of esters.

  The therapeutic agent for viral infection according to the present invention can be used for treating HIV infection in combination with an antiviral agent selected from the group consisting of a reverse transcriptase inhibitor and a viral protein enzyme inhibitor.

The Vγ1Vδ1-type T cell activator and the therapeutic agent for viral infection of the present invention activate Vγ1Vδ1-type T cells in the body of a patient with a viral infection or any immune disease to enhance or regulate immune function, thereby reducing these diseases. Can be treated.
In particular, since the Vγ1Vδ1 T cell activator and the agent for treating a viral infection of the present invention inhibit the proliferation of HIV that continues to hide in NKT cells of HIV-infected persons by the action of activated Vγ1Vδ1 T cells, It is also effective in treating HIV infection.

FIG. 1 shows the results of examining the TCR-dependent stimulus of the T cell clone (1C116) into which Vγ1 and Vδ1 were transferred and the T cell clone (2C21) into which Vγ2 and Vδ2 were transferred. In FIG. 1, FIG. 1A shows the result of the TCR blocking analysis. FIG. 1B is the result of a TCR-dependent stimulation analysis, showing that both the 1C116 and 2C21 clones secreted IL-2 upon stimulation with the anti-CD3 antibody. Figures 1C, 1D and 1E are the results of a response specificity analysis for a TCR-dependent stimulus, showing that clone 1C116 has a different response specificity than clone 2C21. FIG. 2 shows the results of screening flavonoid glycosides for stimulating activity on clones 1C116 and 2C21. Figure 2A shows that hesperidin or linaline exhibited strong stimulatory activity on IL-2 secretion of clone 1C116. Figure 2B shows that the stimulatory activity of hesperidin and linaline on clone 1C116 was completely lost by the anti-γδ TCR-specific antibody. FIG. 3 shows the results of analyzing the stimulating activity of flavonoid glycosides on human-derived natural γ1δ1 ⁺ T cells. The upper graph in Figure 3A and the graph on the left side of Figure 3B show that hesperidin and linalin increased the number of γ1δ1 ⁺ T cells derived from human PBMC. Further, the lower graph in FIG. 3A and the graph on the right side of FIG. 3B show that hesperidin and linaline significantly enhanced CD25 expression of γ1δ1 ⁺ T cells derived from human PBMC. FIG. 4 shows cytokine and chemokine secretion profiles from native human γ1δ1 ⁺ T cells stimulated with hesperidin and linaline. FIG. 4A is a graph showing the process and results of selecting Vγ1Vδ1-TCR-expressing T cells from human PBMCs. FIG. 4B shows that the major cytokines observed in the supernatant of Vγ1Vδ1 T cells stimulated with hesperidin or linaline were unexpectedly IL-5 and IL-13. Figure 4C shows that when the amount of hesperidin and linaline is increased, the secretion of the chemokines MIP-1α, MIP-1β, and RANTES increases, and the secretion of the cytokines IL-5 and IL-13 also increases. Indicates that it has grown. 5, R5 to type CD4 ⁺ NKT and CD4 ^{+ T} cells infected with HIV-1, when added hesperidin or Rinarin in the presence of a human-derived natural γ1δ1 ^{+ T} cells, of the infected CD4 ⁺ within NKT It is the result of examination on whether to suppress HIV-1 virus replication. The graph on the right side of FIG. 5A shows that NKT cell growth stopped when the amount of hesperidin and linaline was too high. The black bars in Figure 5B indicate that stimulation of Vδ1 ⁺ T cells with hesperidin or linaline clearly reduced the amount of p24 antigen detected, indicating the presence of the R5-HIV-1-NL (AD8) virus. The white bar in Figure 5B indicates that the flavonoid glycosides themselves have some degree of viral replication inhibitory activity. FIG. 5C shows that Vδ1 ⁺ T cells activated by hesperidin or linaline also significantly suppressed R5-HIV-1-NL (AD8) virus replication in CD4 ⁺ T cells.

1. The flavonoid glycoside of the present invention HAART therapy has an excellent therapeutic effect by combining a plurality of anti-HIV drugs and administering them to an HIV-infected patient. However, it is almost impossible to completely eliminate HIV particles from the patient, and it is difficult to completely cure HIV infection.
The present inventors have found that Vγ1Vδ1 type T cells belonging to the innate immune system suppress the proliferation of HIV latent in NKT cells in the body of an HIV-infected person (Non-patent Document 4). By activating Vγ1Vδ1 type T cells present in the living body, it becomes possible to treat viral infection by a different principle of action from conventional antiviral drugs. In view of the fact that it is possible to prevent the proliferation of HIV that continues to remain in the body of humans and improve the therapeutic effect of HAART therapy, search for a substance that activates Vγ1Vδ1 type T cells, and activate Vγ1Vδ1 type T cells of the present invention. And the active ingredient of a therapeutic agent for viral infections have been discovered.

  The Vγ1Vδ1-type T cell activator and the therapeutic agent for virus infection of the present invention comprise, as active ingredients, a compound represented by the following general formula (A) or (B) and a pharmacologically acceptable salt or ester thereof. At least one flavonoid glycoside selected from the group consisting of: In the following, the flavonoid glycoside as the active ingredient may be referred to as “flavonoid glycoside of the present invention”.

  The flavonoid glycoside is a compound having a structure in which a saccharide is glycoside-bonded on a flavone skeleton that becomes an aglycone. The flavone skeleton is represented by the following general formula, but one or more of the double bonds contained in the flavone skeleton may be changed to a single bond. In addition, in the following general formula, the numbers described around the flavone skeleton are numbers indicating the substitution positions.

The flavonoid glycoside of the present invention comprises an aglycone having a flavone skeleton and a saccharide moiety composed of a specific disaccharide bonded to the 7-position of the flavone skeleton.
The general formula (A) does not have a single bond between the 2-position and the 3-position of the flavone skeleton, whereas the general formula (B) differs only in having a double bond at the same position. Are the same in structure.

In the general formulas (A) and (B), R ¹ represents an alkyl group having 1 to 3 carbon atoms, and R ² represents a hydrogen atom or a hydroxyl group.
R ³ is a substituent which may be bonded to the A ring, but may not be present on the A ring. R ³ represents a substituent selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, a hydroxyl group, and a halogen.
R ⁴ is a substituent that may be bonded on the B ring, but may not be present on the B ring. R ⁴ represents a substituent selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, a hydroxyl group, and a halogen.
R ⁵ is a substituent that may be bonded on the C ring, but may not be present on the C ring. R ⁵ represents a substituent selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, a hydroxyl group, and a halogen.
n1 is the number of ^{R 3} substituting on the A ring. When R ³ is present on the A ring, one bond can be attached to each of the 6-position and 8-position, that is, up to two bonds. Therefore, n1 represents an integer of 0 to 2.
n2 is the number of ^{R 4} substituting on the B ring. When R ⁴ is present in the general formula (A), one bond can be bonded to the 2-position on the B ring, and two bonds can be bonded to the 3-position, that is, up to three bonds. On the other hand, when R ⁴ is present in the general formula (B), only one R ⁴ can be bonded to the 3-position on the B ring. Therefore, n2 represents an integer of 0 to 3 in the case of the general formula (A), and represents an integer of 0 or 1 in the case of the general formula (B).
n3 is the number of ^{R 5} substituting on C ring. When R ⁵ is present on the C ring, it can be attached to the C ring by one at the 2 ′ position, one at the 3 ′ position and one at the 6 ′ position, that is, a maximum of three. Therefore, n3 represents an integer of 0 to 3.
R ³ , R ⁴ or R ⁵ may be a substituent selected from the group consisting of a methyl group (that is, an alkyl group having 1 carbon atom), a hydroxyl group and a halogen. Further, n1, n2 and n3 representing the number of these substituents may be an integer of 0 or 1.
Each of R ³ , R ^{4 and} R ⁵ may be present in plural on the flavone skeleton. Each substituent represented by the same symbol may be the same or different.

In the general formulas (A) and (B), the sugar moiety linked to the 7-position of the flavone skeleton, which is an aglycone, has an aldohexose on the base side (that is, the side that binds to the aglycone) and a deoxyhexose on the terminal side. It has a disaccharide structure that is linked as follows, the aldohexose on the base side is β- (1 → 7) C glycosidic bond to the flavone skeleton, and the deoxyhexose on the terminal side is the aldohexose. Α (1 → 6) O glycosidic bond. That is, on the base side of the sugar moiety, the 1-position of aldohexose is linked to the 7-position of the flavone skeleton by a β-C glycoside bond. On the other hand, on the terminal side of the sugar moiety, the 1-position of deoxyhexose is O-glycoside linked to the 6-position of aldohexose.
Examples of the base aldohexose include, for example, D-glucose, D-mannose, D-galactose, D-allose, D-altrose, D-gulose, D-idose, D-talose and the like. -Glucose is a typical example.
Examples of the terminal deoxyhexose include, for example, L-rhamnose (ie, 6-deoxy-L-mannose), L-fucose (ie, 6-deoxy-L-galactose), L-talomethylose (ie, 6-deoxy-L-). Talose), D-deoxyribose (i.e., 2-deoxy-D-ribose), D-allomethylose (i.e., 6-deoxy-D-allose), D-quinobose (i.e., 6-deoxy-D-glucose), D-antiallose (I.e., 6-deoxy-D-gulose), D-talomylose (i.e., 6-deoxy-D-talose), D-digitalolose (i.e., 6-deoxy-3-O-methyl-D-galactose), D-digitoxose (i.e., 2,6-dideoxy-D-ribo-hexose), D-simalow (Ie, 2,6-dideoxy-3-O-methyl-D-ribo-hexose), tiberose (ie, 3,6-dideoxy-D-mannose), avecose (ie, 3,6-dideoxy-D-galactose), Examples include paratose (i.e., 3,6-dideoxy-D-glucose), coritolose (i.e., 3,6-dideoxy-L-galactose), and ascalylose (i.e., 3,6-dideoxy-L-mannose). -Rhamnose is a typical example.

  Examples of the pharmacologically acceptable salt or ester of the compound represented by the general formula (A) or (B) include those in which the hydroxyl group on the flavone skeleton or the hydroxyl group of the sugar moiety has a salt or ester structure. Can be As the salt, for example, hydrochloride, sulfate, acetate, succinate, oxalate, maleate, fumarate, sodium salt, potassium salt, cesium salt, calcium salt, magnesium salt, ammonium salt, or glycine , Alanine, lysine or amino acid salts such as glutamic acid. Examples of the ester include an ester to which a carboxylic acid, phosphoric acid, or the like is bonded.

It is known that Vγ1Vδ1 T cells belong to the innate immune system of a living body, play a role in controlling pathogens and eliminating locally generated tumors, and mediate innate immunity and acquired immunity. In addition, a study by the present inventors has revealed that a Vγ1Vδ1-type γδT cell group exhibits an HIV proliferation inhibitory effect in NKT cells, and has already been reported (Non-Patent Document 4).
In the present invention, it has been discovered that flavonoid glycosides having a specific structure have an effect of stimulating Vγ1Vδ1 type T cells. From the experimental results described below, it was discovered that the flavonoid glycoside of the present invention activates Vγ1Vδ1 type T cells and releases chemokines. Furthermore, according to the experimental results described later, the flavonoid glycosides of the present invention were found to show that CD4 ⁺ NKT cells (natural killer T cells) infected with R5-tropic HIV-1 and CD4 ⁺ T cells infected with R5-tropic HIV-1 Inhibits HIV-1 replication. Since CD4 ⁺ NKT cells belong to the innate immune system and CD4 ⁺ T cells belong to the adaptive immune system, the flavonoid glycosides of the present invention have an immunomodulatory action and viral growth on both the innate immune system and the adaptive immune system. Has a suppressive effect.
Therefore, the flavonoid glycosides of the present invention can suppress the viral growth or restore the immune dysfunction by activating Vγ1Vδ1 type T cells in the body of patients with viral infections or some immune disorders. The disease can be treated.

In order for the flavonoid glycoside of the present invention to exert the effect of activating Vγ1Vδ1 T cells, the following two conditions are particularly important.
(1) At the 7-position of the flavone skeleton, aldohexose and deoxyhexose are connected in this order from the base side (that is, the flavone skeleton side) to the terminal side, and the aldohexose on the base side is β with respect to the flavone skeleton. A (1 → 7) C glycoside bond, and the terminal deoxyhexose has a disaccharide structure sugar portion having an α (1 → 6) O glycoside bond to the aldohexose.
(2) The lower alkoxyl group having 1 to 3 carbon atoms is substituted at the 4'position of the phenyl-based substituent bonded to the 2-position of the flavone skeleton.

Among the compounds represented by the above general formula (A) or (B) and esters or salts thereof, compounds represented by the following formula (1) or (2) and pharmacologically acceptable salts or At least one flavonoid glycoside selected from the group consisting of esters is preferred.
The formula (1) corresponds to the general formula (A), and has no single bond between the 2-position and the 3-position of the flavone skeleton, whereas the formula (2) corresponds to the general formula (B). And has a double bond at the same position.

[In the formulas (1) and (2), R ¹ represents an alkyl group having 1 to 3 carbon atoms, and R ² represents a hydrogen atom or a hydroxyl group. R ³ is a substituent which may be bonded on the ring A, and represents a substituent selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, a hydroxyl group and a halogen, and R ⁴ is bonded on the ring B. Represents a substituent selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, a hydroxyl group and a halogen, and R ⁵ is a substituent which may be bonded to the C ring. Represents a substituent selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, a hydroxyl group, and a halogen. n1 is the number of R ³ and represents an integer of 0 to 2, n2 is the number of R ⁴ and represents an integer of 0 to 3 in the case of the formula (1), and 0 or 3 in the case of the formula (2). represents the integer 1, n3 represents an integer of from 0 to a number of R ⁵ 3. When there are a plurality of substituents represented by the same symbol, each substituent may be the same or different. ]

  The compounds of the above formulas (1) and (2) have a structure in which β-rutinose as a sugar moiety is bonded to the 7-position of the flavone skeleton in the general formulas (A) and (B). β-rutinose is 6-O-α-L-rhamnosyl-D-β-glucose. That is, in the compounds of formulas (1) and (2), the 1-position of glucose is β-bonded to the 7-position of the flavone skeleton, and the 1-position of rhamnose is α-bonded to the 6-position of glucose (α-1 when viewed from the rhamnose side). , 6 bonds).

  Examples of the compound represented by the above formula (1) or (2) include hesperidin represented by the following formula (3) included in the formula (1) and the following formula included in the formula (2). Linalin represented by (4) may be mentioned. Hesperidin (CAS registry number 520-26-3) and linaline (CAS registry number 480-36-4) are known compounds.

  The compounds represented by the general formulas (1) and (2) can be synthesized by synthesizing a flavone skeleton by a known method and modifying the flavone skeleton with a substituent such as a sugar moiety. As a method for synthesizing a flavone skeleton, for example, Allan-Robinson Flavone Synthesis according to the following reaction scheme is known. In this method, a flavone derivative is obtained from an o-hydroxyaryl ketone and an acid anhydride. be able to.

Modification of the sugar moiety at the 7-position of the flavone skeleton can be achieved by introducing a hydroxyl group at the 7-position of the flavone skeleton and dehydrating and condensing the sugar to be modified. The introduction of a hydroxyl group at the 5-position of the flavone skeleton, the introduction of a substituent at the 2-position of the flavone skeleton, or the modification of the substituent can also be appropriately performed by a known method.
Of the compounds represented by the general formula (1), hesperidin represented by the above formula (1) and linalin represented by the above formula (2) can be purchased from a manufacturer. For example, hesperidin can be purchased from Tokyo Chemical Industry Co., Ltd., and linalin can be purchased from Extrasynthese (France).

2. Use of flavonoid glycosides of the present invention as Vγ1Vδ1 type T cell activator and therapeutic agent for viral infections and therapeutic method Vγ1Vδ1 type T cells belong to the innate immune system of living organisms, control pathogens and locally generated tumors It is responsible for the elimination of immunity and bridging innate immunity and acquired immunity. Since the flavonoid glycoside of the present invention activates Vγ1Vδ1 type T cells, the flavonoid glycoside is administered to a patient with a viral infection or any immune disease, or is contacted with the flavonoid glycoside in vitro. By administering the Vγ1Vδ1 type T cells to a patient with a viral infection or any immune disease, these diseases can be treated.
The Vγ1Vδ1-type T cell activator and the therapeutic agent for virus infection of the present invention may be a simple substance containing only the above-mentioned flavonoid glycoside as an active ingredient, but may be selected according to the administration route, administration method, or use method. It is generally prepared into a suitable therapeutic composition using known additives, excipients, antioxidants, solvents and the like. As an example of an injection, a flavonoid glycoside as an active ingredient is dissolved in an aqueous solvent, and if necessary, additives such as a buffer, a pH adjuster, and a stabilizer are added to prepare an injection. be able to.

The therapeutic agent for viral infection of the present invention, when administered to a person infected with a virus, activates Vγ1Vδ1 type T cells present in the body of the person infected with the virus, and the activated Vγ1Vδ1 type T cells become infected with the virus. Since it inhibits virus growth in cells, it has a therapeutic effect on viral infection. The route of administration of the therapeutic agent for viral infection of the present invention is not particularly limited, such as oral administration and injection.
The therapeutic agent for viral infections of the present invention is effective for the treatment of HIV infection because it inhibits the proliferation of HIV that continues to hide in NKT cells of HIV-infected persons even with current HAART therapy.
In order for HIV-1 to invade target CD4-positive cells, one of CXCR4 (X4) or CCR5 (R5) must be expressed on the surface of CD4-positive cells. is there. X4-tropic HIV-1 (T cell-tropic HIV-1 strain) invades cells using CXCR4, and R5-tropic HIV-1 (macrophage-tropic HIV-1 strain) invades cells using CCR5. .
R5-tropic HIV-1 is usually detected early after infection, and X4-tropic HIV-1 is often detected when AIDS has progressed. In order to prevent the progression of HIV infection and improve the patient's prognosis, it is important to prevent the proliferation of R5-tropic HIV-1.
NKT cells are CD4 positive cells belonging to the innate immune system. NKT cells express the CCR5 receptor, and HIV that has invaded and remained in NKT cells is R5-tropic HIV-1. Therefore, by administering the therapeutic agent for a viral infection of the present invention in combination with HAART therapy to an HIV-infected person, it is possible to suppress the replication of R5-tropic HIV-1 remaining in NKT cells. It is possible to improve the therapeutic effect on HIV infection by the therapy. Furthermore, complete elimination of HIV particles from the patient's body, which was impossible with conventional HAART therapy, can also be expected.
The therapeutic agent for viral infection of the present invention can also suppress the replication of R5-tropic HIV-1 even when CD4 ⁺ T cells belonging to the acquired immune system are infected with R5-tropic HIV-1. Therefore, the therapeutic agent for viral infections of the present invention not only eliminates viruses that remain latent and replicates in NKT cells, but also inhibits R5-tropic cells against CD4-positive cells belonging to both the innate immune system and the acquired immune system. Since it has a virus suppressing action at the time of HIV-1 infection, it can be applied to all treatments for HIV infection.

The Vγ1Vδ1-type T cell activator of the present invention is administered to a virus-infected person as a therapeutic agent for viral infections, and also modulates immune functions having Vγ1Vδ1-type T-cell activation for immune disease patients other than viral infection. It may be administered as an agent.
Further, the Vγ1Vδ1-type T cell activator of the present invention can be used in a method not directly administering to a patient. For example, after stimulating the Vγ1Vδ1 T cell activator of the present invention by adding the Vγ1Vδ1 T cell activator to the Vγ1Vδ1 T cell in vitro, after confirming that the Vγ1Vδ1 T cell has been activated by the stimulation, or without confirming, A therapeutic method in which Vγ1Vδ1 T cells are administered to a patient suffering from viral infection or some immune disease by injection or the like, or a Vγ1Vδ1 T cell activator of the present invention is added to Vγ1Vδ1 T cells in vitro to obtain the same. The mixture can be used in a therapeutic method of administering to a patient with a viral infection or some immune disease by injection or the like.

3. Embodiments Included in the Present Invention The present invention particularly includes the following embodiments.
(1) as an active ingredient, at least one flavonoid glycoside selected from the group consisting of a compound represented by the following formula (1) or (2) and a pharmacologically acceptable salt or ester thereof: Vγ1Vδ1-type T cell activator.

[The meaning of each code included in the formulas (1) and (2) is the same as that described above. ]

  (2) The flavonoid glycoside is selected from the group consisting of hesperidin represented by the following formula (3), linalin represented by the following formula (4), and a pharmacologically acceptable salt or ester thereof. The Vγ1Vδ1 type T cell activator according to (1) above, which is at least one of the following:

(3) as an active ingredient, at least one flavonoid glycoside selected from the group consisting of the compound represented by the formula (1) or (2) and a pharmacologically acceptable salt or ester thereof; A therapeutic agent for viral infections.
(4) The flavonoid glycoside is selected from the group consisting of hesperidin represented by the formula (5), linaline represented by the formula (6), and a pharmacologically acceptable salt or ester thereof. The therapeutic agent for a viral infection according to the above (3), which is at least one selected from the group.
(5) The therapeutic agent for viral infection according to the above (3) or (4), which is used for treatment of HIV infection.
(6) The viral infection according to (3) or (4), which is used for treating HIV infection in combination with another antiviral selected from the group consisting of a reverse transcriptase inhibitor and a viral protein enzyme inhibitor. Therapeutic agent.
(7) At least one flavonoid glycoside selected from the group consisting of the compound represented by the above formula (1) or (2) and a pharmacologically acceptable salt or ester thereof for treating an immune disease. Body use.
(8) At least one flavonoid compound selected from the group consisting of the compound represented by the above formula (1) or (2) and a pharmacologically acceptable salt or ester thereof for treating a viral infection. Use of glycosides.
(9) A method for treating an immune disease, comprising at least one flavonoid moiety selected from the group consisting of the compound represented by the formula (1) or (2) and a pharmacologically acceptable salt or ester thereof. A therapeutic method comprising: contacting a saccharide with a Vγ1Vδ1 T cell in vitro and then administering the Vγ1Vδ1 T cell to a patient with an immune disease.
(10) A method for treating a viral infectious disease, comprising at least one flavonoid selected from the group consisting of the compound represented by the formula (1) or (2) and a pharmacologically acceptable salt or ester thereof. A therapeutic method comprising: contacting a glycoside with a Vγ1Vδ1 T cell in vitro; and then administering the Vγ1Vδ1 T cell to a patient infected with a virus.

1. Establishment of T cell clones (1C116) into which Vγ1 and Vδ1 TCR genes have been transferred and T cell clones (2C21) having Vγ2 and Vδ2 TCR genes transferred into TCR-deficient T cells (induction of immortalized T cell clones)
Peripheral blood collected from healthy subjects was centrifuged to obtain PBMCs (peripheral blood mononuclear cells). After labeling PBMCs with an anti-panγδTCR antibody (product name B6.1; manufacturer name BD Bioscience; location San Diego, CA, USA), γδT cell groups were selected using FACS (flow cytometry).
The obtained γδ T cell group was cultured in a complete medium containing the following components in the following base medium at 1 μg / mL phytohemagglutinin (PHP-P) (manufacturer name: Sigma-Aldrich; location St. Louis, MO). ) For 3 days.
Base medium: RPMI-1640 (product name; manufacturer name: Thermo Fisher Scientific; location: Waltham, Mass., USA)
Ingredients:
10% heat-inactivated fetal calf serum (FCS) (manufacturer name: Hyclone, Logan, UT)
5 mM HEPES buffer (Manufacturer name: Thermo Fisher Scientific)
100 U / ml penicillin (manufacturer name: Thermo Fisher Scientific)
100 μg / ml streptomycin (Manufacturer name Thermo Fisher Scientific)
2 mM L-glutamine (Manufacturer name: Thermo Fisher Scientific)
2 mM sodium pyruvate (Manufacturer name: Thermo Fisher Scientific)
2 mM non-essential amino acids (manufacturer name: Thermo Fisher Scientific)
2 mM modified vitamins (manufacturer name: Thermo Fisher Scientific)
0.5 μM 2-mercaptoethanol (2-ME) (Manufacturer name: Thermo Fisher Scientific)
After the stimulated γδ T cell group was infected with 488 strains of Herpes saimiri (available under the product name VR-1396 from the manufacturer name ATCC (American Type Culture Collection)), 10 U / ml recombination was performed. The cells were cultured in the presence of interleukin 2 (rIL-2) (manufacturer name: Yoshishio Shiono; location: Osaka, Japan) to induce immortalized γδT cells having stable proliferation ability.
The obtained immortalized γδ T cell group was labeled with a Vδ1 antibody, sorted using FACS (flow cytometry), and an immortalized Vγ1Vδ1 ⁺ (Vδ1 ⁺ ) T cell clone was isolated. This immortalized T cell clone showed a stable proliferation ability for several months without mitogen stimulation.

(Amplification of TCR-γ1 cDNA and TCR-δ1 cDNA using plasmid)
The full sequence cDNA encoding both TCR-γ1 and TCR-δ1 was isolated from immortalized Vγ1Vδ1 ⁺ T cell clone.
The TCR-γ1 cDNA was amplified using the following sense primer and the following antisense primer, cloned into the following plasmid, and cloned.
Sense primer (SEQ ID NO: 1):
5'-GGCGGCGGCCGCGAAGGCATGCGGTGGGCCCT-3 '
Antisense primer (SEQ ID NO: 2):
5'-GGGCTCGAGCTGTTATGATTTCTCTCCATT-3
Plasmid: pEF1 / Myc-His A (manufacturer name: Thermo Fisher Scientific; location: Waltham, MA)
The TCR-δ1 cDNA was amplified using the following sense primer and the following antisense primer, cloned into the following plasmid.
Sense primer (SEQ ID NO: 3):
5'-GGCGGCGGCCGCCTTCAGGCAGCACAACTC-3 '
Antisense primer (SEQ ID NO: 4):
5'-GGGCTCGAGGGAGTGTAGCTTCCTCAT-3 '
Plasmid: pREP4 (manufacturer name: Thermo Fisher Scientific).

(Transfer of Vγ1 and Vδ1 TCR genes into TCR-deficient T cells)
Both the TCR-γ1 cDNA-incorporated primer and the TCR-δ1 cDNA-incorporated primer were transferred into the TCR-defective Jurkat T cell line JRT3-T3.5 by electroporation at 240-270V. Select by culturing the transfected cells for 2 to 3 days in a selective medium containing 1 mg / mL G418 sulfate (Thermo Fisher Scientific) and 0.5 mg / mL hygromycin B (Wako Pure Chemical Industries, Osaka, Japan). did. The sorted cells were seeded on a 96-well microplate by limiting dilution to give 0.5 to 1 cell / well, separated, and then analyzed by flow cytometry for γδTCR expression, and Vγ1 and Vδ1 were transferred. A T cell clone (1C116) was established.

(Separation of Vγ2Vδ2 ⁺ T cells from PBMCs)
Vγ2Vδ2 ⁺ T cells are known to be stimulated by the drug risedronate used for treating osteoporosis. Therefore, Vγ2Vδ2 ⁺ T cells in PBMCs were stimulated and proliferated using risedronate. Specifically, the following procedure was performed.
Peripheral blood collected from healthy subjects was centrifuged to obtain PBMCs (peripheral blood mononuclear cells). The PBMCs were frequently stimulated with 2.5 μg / mL risedronate (manufacturer name: Ajinomoto Pharmaceutical. Co .; location: Tokyo, Japan) in the presence of 10 U / mL rIL-2, and then labeled with Vδ2 antibody and FACS ( (Flow cytometry) to isolate a Vγ2Vδ2 ⁺ (Vδ2 ⁺ ) T cell line. The desired full-length cDNAs were obtained from the separated Vγ2Vδ2 ⁺ (Vδ2 ⁺ ) T cell line.

(Amplification of TCR-γ2 cDNA and TCR-δ2 cDNA using plasmid)
The full sequence cDNA encoding both TCR-γ2 and TCR-δ2 was isolated from Vγ2Vδ2 ⁺ T cells.
TCR-γ2 cDNA was amplified using the following sense primer and the following antisense primer, cloned into the following plasmid, and cloned.
Sense primer (SEQ ID NO: 5):
5'-GGGCTCGAGGACACCGCTTTACAACGA-3 '
Antisense primer (SEQ ID NO: 6):
5'-GGGTCTAGAGTGAGGTTCTCTGTGT-3 '
Plasmid: pEF1 / Myc-His A.
The TCR-δ2 cDNA was amplified using the following sense primer and the following antisense primer, cloned into the following plasmid, and cloned.
Sense primer (SEQ ID NO: 7):
5'-GGGCTCGAGAACACTTGTGTGTTGGTTCA-3 '
Antisense primer (SEQ ID NO: 8):
5'-GGGGGATCCAGTGTATCACTTGTAGGAG-3 '
Plasmid: pREP4

(Transfer of Vγ2 and Vδ2 TCR genes into TCR-deficient T cells)
In the same procedure as the procedure for transferring the Vγ1 and Vδ1 TCR genes, both the primer incorporating the TCR-γ2 cDNA and the primer incorporating the TCR-δ2 cDNA were placed in JRT3-T3.5, a TCR-deficient Jurkat T cell line. The cells were transferred, selected and cultured, and analyzed to establish a T cell clone (2C21) into which Vγ2 and Vδ2 were transferred.

2. TCR blocking analysis, TCR-dependent stimulation analysis, and reaction specificity of TCR-dependent stimulation (TCR blocking analysis) of 1C116 and 2C21 clones
A T cell clone (1C116) into which Vγ1 and Vδ1 were transferred and a T cell clone (2C21) into which Vγ2 and Vδ2 were transferred were subjected to an anti-pan-γδ-PE antibody (manufacturer name, BioLegend; San Diego, CA) and Vδ1-APC. After incubating with an antibody (manufacturer name Miltenyi Biotec GmbH, Bergisch Gladbach; location Germany), an anti-Vδ2-PE antibody (manufacturer name Biolegend), or an anti-CD3 antibody at 4 ° C. for 30 minutes, the treated cells are washed and 3% The cells were resuspended in phosphite buffer artharine (PBS) containing FCS and 0.01 M NaN ₃ (FACS buffer). The cells were analyzed by flow cytometry.
Figure 1A shows the results. The open histogram (white frequency distribution graph) means staining with each antibody, and the shade histogram (shaded frequency distribution graph) means staining with the isotype control.
Clone 1C116 was stained by pan-γδ and Vδ1, but not Vδ2. On the other hand, clone 2C21 was stained by pan-γδ and Vδ2, but not by Vδ1. Therefore, both clone 1C116 and clone 2C21 were confirmed to be γδ T cell clones.

(TCR-dependent stimulation analysis)
The 1C116 clone and 2C21 clone were stimulated with anti-CD3 antibody 500 ng / mL in the presence of PMA 100 ng / mL at 37 ° C. for 24 hours to test the secretion of interleukin 2 (IL-2).
Both clones expressed CD3 (Figure 1A) and secreted IL-2 when stimulated with anti-CD3 antibody in the presence of PMA (Figure 1B). Therefore, the amount of IL-2 secreted from these γδ T cell clones was considered to be an excellent indicator of TCR-dependent stimulation.

(Reaction specificity of TCR-dependent stimulation)
Various types of alkylamines, such as isopentenyl pyrophosphate (IPP), ethylamine, propylamine, or butylamine, and amino-bisphosphonates, such as risedronate, used in the treatment of osteoporosis, are Vγ2Vδ2. It is already known to stimulate ⁺ T cells. Thus, 1C116 and 2C21 clones were stimulated with IPP, ethylamine and risedronate to test for differences in reactivity.
200 μL of complete medium (CCM) was added to each well of a 96-well U-bottom tissue culture plate. In each well filled with CCM, 1 × 10 ⁵ 1C116 clones or 2C21 clones were seeded, and either of the following test compounds or dimethylsulfoxide (DMSO) that was a substrate control (vehicle control), in the presence of PMA 100 ng / mL. The cells were incubated at 37 ° C for 24 hours. After the incubation, the supernatant of the medium was collected, and the concentration of IL-2 was analyzed using an ELISA kit (manufacturer name: BD Biosciense; location: San Diego, CA).
<Test compound>
0 to 1000 μM isopentenyl pyrophosphate (IPP) (manufacturer name: Sigma-Aldrich)
0 to 2000 μM risedronate (Manufacturer name Ajinomoto Co. Ltd; location Tokyo, Japan)
0 to 33.5 μM ethylamine (Manufacturer name Sigma-Aldrich)
As shown in Figure 1C, clone 2C21 secreted IL-2 stimulated by IPP, whereas clone 1C116 did not secrete IL-2. Similarly, clone 2C21 secreted IL-2 stimulated by ethylamine, whereas clone 1C116 did not secrete IL-2 (Figure 1D). Also, clone 2C21 secreted IL-2 stimulated by risedronate, whereas clone 1C116 did not secrete IL-2 (Figure 1E).
These results indicate that clone 2C21 is specifically stimulated by the γ2δ2 stimulating antigen molecule via the Vγ2Vδ2 TCR, but clone 1C116 has the Vγ1Vδ1 TCR and does not have the Vγ2Vδ2 TCR, and thus is different from clone 2C21. This shows that they have different reaction specificities.

3. Screening of flavonoid glycosides for stimulatory activity on clones 1C116 and 2C21 Several plant metabolites are known to have bioactivity. Recently, such natural compounds have been termed "phytochemicals". Therefore, the present inventors screened flavonoids, which are plant-derived aromatic compounds having a C6-C3-C6 structure, for their stimulating activity on clones 1C116 and 2C21.
This test was carried out in the same manner as in the above-mentioned test “Response specificity of TCR-dependent stimulation”. That is, 200 μL of complete medium (CCM) was added to each well of a 96-well U-bottom tissue culture plate. In each well filled with CCM, 1 × 10 ⁵ 1C116 clones or 2C21 clones were seeded, and either of the following test compounds or dimethylsulfoxide (DMSO) that was a substrate control (vehicle control), in the presence of PMA 100 ng / mL. The cells were incubated at 37 ° C for 24 hours. After the incubation, the supernatant of the medium was collected, and the concentration of IL-2 was analyzed using an ELISA kit (manufacturer name: BD Biosciense, San Diego, CA).
<Test compound>
Each flavonoid at 0 to 100 μg / mL The test flavonoids are as follows: quercetin quercetin, isoquercetin isoquercitrin, hyperoside hyperoside, galangin, camphenol, morin morin, myricetin myricetin, hesperetin hesperetin, hesperidin, acetasetin Linarin and isorhoifolin (all of these suppliers Extrasynthese, Lyon, France)
The chemical structure of each test compound is shown below.

(Test results)
Table 1 shows the results of the screening test.

As shown in Table 1, some flavonoids stimulated clone 1C116 and secreted large amounts of IL-2. In contrast, none of the flavonoids stimulated clone 2C21.
Particularly, a disaccharide having a 6-O-α-L-rhamnosyl-D-glucose structure called rutinose is substituted on the A ring, and a methoxy group (-OCH ₃ ) is substituted on the B ring. For example, hesperidin and linaline showed strong stimulatory activities on the IL-2 secretion of clone 1C116 (Figure 2A). The fact that such stimulatory activity was completely lost by treatment with an anti-γδ TCR-specific antibody strongly indicates that recognition of flavonoid glycosides was initiated via the Vγ1Vδ1 TCR (Figure 2B).
In addition, hesperetin having a structure in which rutinose was removed from hesperidin, or acacetin having a structure in which rutinose was removed from linalin, showed no or only weak secretion of IL-2 from clone 1C116 (FIG. 2A). ).
In addition, when the disaccharide rutinose is substituted on the A ring of the flavonoid, but the methoxy group (-OCH3) is not substituted on the B ring on the B ring like isorhoifolin. , IL-2 secretion from clone 1C116 was not observed (Fig. 2A).
Taken together, these findings indicate that clone 1C116, into which the Vγ1 and Vδ1 TCR genes have been transferred, has Vγ1Vδ1 due to a flavonoid glycoside having both rutinose on the A ring and a methoxy group (—OCH ₃ ) on the B ring. It clearly shows that it is specifically and strongly stimulated through the TCR. In addition, hesperidin and linaline showed very remarkable stimulating activity against clone 1C116.
Therefore, the present inventors have focused on hesperidin and linalin, and these flavonoid glycosides have R5-type HIV-1 in CD4 ⁺ NKT cells via the activation effect of Vγ1δ1-TCR-expressing γ1δ1 T cells. We proceeded with the study on whether or not to suppress the replication of.

4. Analysis of stimulating activity of flavonoid glycosides on human-derived natural γ1δ1 ⁺ T cells It is found that a part of compounds belonging to flavonoid glycosides has stimulating activity on T cell clone 1C116 into which Vγ1 and Vδ1 TCR genes have been transferred. Indicated by screening tests. Therefore, whether the flavonoid glycosides that showed a specific stimulatory activity for Vγ1Vδ1 TCR in the screening test have the same stimulatory activity for human-derived natural γ1δ1 T cells as in the case of the Vγ1Vδ1 TCR gene transferer clone. investigated.

Peripheral blood collected from healthy subjects was centrifuged to obtain PBMCs (peripheral blood mononuclear cells). PBMCs were labeled using Cell Trace CFSE Cell Proliferation Kit (carboxy-fluorescein diacetate succinimidyl ester (CFDA-SE, also known as CFSE)) (manufacturer name: Thermo Fisher Scientific).
Next, 0.5 mL of complete medium (CCM) was added to each well of the 48-well plate. For CCM, RPMI-1640 (product name; manufacturer name Thermo Fisher Scientific) was supplemented with 5% AB human serum (manufacturer name Biowest Nuaille; location France) and 100 U / mL recombinant IL-2 (manufacturer name Shionogi). Having a composition. Labeled PBMCs are seeded in each well filled with CCM, together with 100 μg / mL hesperetin, hesperidin, heceperidin, acacetin or linarin, or 0.01% dimethyl sulfoxide (DMSO: vehicle). control) and cultured for 14 days.
After the culture, the PBMCs were incubated with an anti-Vδ1-APC antibody (manufacturer name Miltenyi Biotec GmbH; location Bergisch Gladbach, Germany) or an anti-CD25-PE / Cy7 antibody (manufacturer name Biolegend) for 30 minutes at 4 ° C. The incubated PBMCs were washed, suspended in PBS containing 3% FCS and 0.01 M NaN ₃ (FACS buffer), and analyzed by flow cytometry.

The upper graph in Figure 3A shows the results of analysis by flow cytometry after reacting anti-Vδ1-APC antibody with cultured PBMCs, and shows the proliferative activity of each test compound on γ1δ1 ⁺ T cells present in PBMCs. ing. The graph on the left side of FIG. 3B shows the results for each compound in the upper part of FIG. 3A as one graph. Compared to unstimulated control or DMSO (vehicle control), hesperetin and acacetin did not significantly increase γ1δ1 ⁺ T cells present in PBMCs, whereas hesperidin and linalin were present in PBMCs γ1δ1 ^+. The number of T cells could be increased.
In addition, the lower part of Figure 3A shows the results obtained by reacting the anti-Vδ1-APC antibody and the anti-CD25-PE / Cy7 antibody with the cultured PBMCs and analyzing them by flow cytometry. The results are shown for the γ1δ1 ⁺ T cells present in the PBMCs The CD25 expression level of each test compound is shown. The graph on the right side of FIG. 3B shows the results for each compound in the lower part of FIG. 3A as one graph. As compared to unstimulated control or DMSO (vehicle control), hesperetin and acacetin did not favorably enhance the CD25 expression of γ1δ1 ⁺ T cells present in PBMCs, whereas hesperidin and linaline were present in PBMCs. CD25 expression of existing γ1δ1 ⁺ T cells was significantly enhanced.

5. Cytokine and chemokine secretion profiles from human-derived natural γ1δ1 ⁺ T cells stimulated with hesperidin and linaline The above experiments showed that hesperidin and linaline have stimulatory activity on human-derived natural γ1δ1 ⁺ T cells.
In general, the immune system cells perform immunoregulation between host cells by cytokine secretion and immunomodulation against foreign microorganisms by chemokine secretion. We believe that chemokines, such as CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES) secreted by Vγ1Vδ1 T ⁺ cells, are capable of producing R5-HIV- in CD4 ⁺ NKT cells. We have previously reported that 1-NL (AD8) may suppress viral replication. Furthermore, we have previously reported that Vγ1Vδ1 T cells are activated via MICA / MICB, which is highly expressed on NL (AD8) -infected CD4 ⁺ NKT cells.
Therefore, the present inventors examined whether Vγ1Vδ1 T cells stimulated with hesperidin or linaline secrete cytokines and chemokines.

Peripheral blood collected from healthy subjects was centrifuged to obtain PBMCs (peripheral blood mononuclear cells). VB1-APC antibody (manufacturer name Miltenyi Biotec GmbH, Bergisch Gladbach; address Germany) was reacted with PBMCs, and γ1δ1 ⁺ T cells were selected by a flow cytometry method.
The sorted Vδ1 ⁺ T cells (cell group represented by Vγ1Vδ1 T cells) were incubated with 2 μg / mL PHA (manufacturer name: Sigma-Aldrich) containing 1 × 10 ⁶ cells / mL of irradiated PBMCs. Then, 5% AB human serum (manufacturer name Biowest Nuaille; location France) and 100 U / mL recombinant IL-2 (manufacturer name Shionogi; location Osaka, Japan) were added to each well of the 24-well culture plate. Complete medium (CCM) was added, and each well was seeded with pre-incubated Vδ1 ⁺ T cells, and cultured for 14 days while changing the medium by half every 3 to 4 days.
After the above initial culture for 14 days, Vδ1 ⁺ T cells were restimulated with 2 μg / mL PHA (manufactured by Sigma-Aldrich) containing 1 × 10 ⁶ cells / mL of irradiated PBMCs. Then, the cells were cultured in the same manner as described above. Restimulation of Vδ1 ⁺ T cells began at least 7 days before use in the experiment.
Vδ1 ⁺ T cells that had completed restimulation were stimulated with hesperidin or linalin according to the following procedure, and their cytokine and chemokine profiles were examined.

200 μl of complete medium (CCM) was placed in each well of a round-bottom type 96-well plate. CCM has a composition supplemented with 5% AB human serum (manufacturer name Biowest Nuaille; location France) and 100 U / mL recombinant IL-2 (manufacturer name Shionogi). In each well filled with CCM, 5 × 10 ⁴ cells / mL of Vγ1 ⁺ T cells are seeded, and 1 μg / mL of ionomycin or 10, 30, or 100 μg / mL together with 25 ng / mL of PMA. Of hesperidin, the same amount of linalin as described above, or 0.01, 0.03, or 0.1% of dimethyl sulfoxide (DMSO: vehicle control). Forty-eight hours later, 150 μl of media supernatant was collected from each well,
Stored at -80 C until measurement of cytokines and chemokines.
In order to quantify various kinds of cytokines (IFN-γ, IL-4, IL-5, IL-10, IL-13 and IL-17A), BD CBA kit (“BD” is a registered trademark) Manufacturer name BD Bioscience) and FACSCanto II (product name; manufacturer name Becton Dickinson) Further, among cytokines, the amount of IL-5 can be determined by BD CBA kit and IL-5 ELISA kit (manufacturer name: R & D Systems; Location: Minneapolis , MN), and the amount of IL-13 was measured using a BD CBA kit and an IL-13 ELISA kit (manufacturer name: ThermoFisher). In addition, chemokines such as MIP-1α, MIP-1β, and RANTES were quantified using respective ELISA kits (manufacturer name R & D Systems).

FIG. 4A is a graph showing the process and results of selecting Vγ1Vδ1-TCR-expressing T cells from PBMCs. As shown on the left side of Figure 4A, 0.47% of Vδ1 ⁺ T cells was contained in PBMCs, from which Vδ1 ⁺ T cells were sorted as shown on the right side of Figure 4A.
The major cytokines observed in the supernatant of Vγ1Vδ1 T cells stimulated with hesperidin or linalin are surprisingly IL-5 and IL-13, which affect unstimulated normal lymphocytes 2 (ILC-2). The behavior was very similar. IL-5 and IL-13 are known to be secreted during allergic reaction and infection. However, they found that IL-17 and IFN-γ, which are known to be the major cytokines secreted by the majority of γδ T cells, were not observed (Figure 4B).
Furthermore, the amounts of hesperidin and linaline were changed in three stages (10 μg / mL, 30 μg / mL, 100 μg / mL) to examine the difference in effect, and the secretion amounts of cytokines and chemokines were measured. Compared with DMSO (vehicle control), the secretion of chemokines MIP-1α, MIP-1β and RANTES increased, and the secretion of cytokines IL-5 and IL-13 also increased (Figure 4C). ).
From these results, human-derived natural γ1δ1 ⁺ T cells present in PBMCs can secrete cytokines and / or chemokines and perform immunomodulation when stimulated by flavonoid glycosides such as hesperidin and linaline. confirmed.
In addition, chemokines secreted from γ1δ1 ⁺ T cells stimulated by hesperidin and linalin are thought to suppress R5-HIV-1-NL (AD8) virus replication in CD4 ⁺ NKT cells, and It was suggested that human-derived natural γ1δ1 ⁺ T cells stimulated by such flavonoid glycosides suppress R5-HIV-1-NL (AD8) virus replication in CD4 ⁺ NKT cells.

6. Inhibition of R5-type of HIV-1 replication in CD4 ⁺ natural killer T (CD4 ⁺ NKT) cells via human-derived natural γ1δ1 ⁺ T cells activated by hesperidin and linalin. It was suggested that human-derived natural γ1δ1 ⁺ T cells stimulated with various flavonoid glycosides suppress R5-HIV-1-NL (AD8) virus replication in CD4 ⁺ NKT cells.
Based on the above suggestions, the present inventors found that when hesperidin or linalin was added to R4 type HIV-1 infected CD4 ⁺ NKT in the presence of human-derived natural γ1δ ⁺ T cells, infected CD4 ⁺ NKT It was examined whether HIV-1 virus replication in HIV was suppressed.
To induce NKT cells, 20 ng / ml α-galactosylceramide (α-GalCer) (product name KRN7000: manufacturer name Funakoshi) in 1 mL complete medium (CCM) containing 2 × 10 ⁶ PBMCs Co., Ltd., location Tokyo, Japan). Seven days later, half of the medium was replaced with new CCM containing 20 U / mL IL-2 (manufacturer Shionogi) and no α-GalCer. This culture scheme induced CD4 ⁺ Vα24 ⁺ NKT cells from human PBMCs (Figure 5A, left graph).
Induced CD4 ⁺ Vα24 ⁺ NKT cells were stimulated for 3 days with 2 μg / mL PHA in complete medium (CCM) containing 20 U / mL IL-2. Subsequently, the PHA-stimulated CD4 ⁺ Vα24 ⁺ type-INKT cells described above were washed to remove free PHA. After washing, 1-2 × 10 ⁵ cells were plated in the presence of 30 μg / mL DEAE-Dextran (manufacturer name Sigma-Aldrich) at a multiplicity of infection of 0.1 (0.1 MOI) and at 37 ° C. for 2 hours. Under the condition of time, NL (AD8) HIV-1 (R5-type) was infected. Here, the multiplicity of infection (MOI) is the ratio of infectious virus to cells to be infected, and 0.1 MOI means that one virus particle is cultured for 10 cells.

A base medium containing RPMI-1640 (product name; manufacturer name: Thermo Fisher Scientific; location: Waltham, Mass., USA) containing 2% FCS was prepared. Using this base medium, NL (AD8) -infected NKT cells were washed three times repeatedly. The washed cells were incubated in a medium containing 20 U / ml IL-2 using a round-bottom type 96-well plate.
Then, 2 x 10 ⁴ NL (AD8) -infected NKT cells were used in a round-bottom 96-well plate in 200 μl CCM containing 20 U / mL IL-2. The cells were cultured in the presence or absence of 1 × 10 ⁴ Vδ14 ⁺ cells. Add 3,10 or 30 μg / mL hesperidin, or 3,10 or 30 μg / mL linaline, or 0.003, 0.01 or 0.03% DMSO (vehicle control) to each well of the plate during culture. did.
Four days after the start of the culture, 100 μl of the medium supernatant was collected from each well, stored at −80 ° C. until the measurement of p24 antigen, and measured using an ELISA kit (manufacturer name: Sino Biological; location: Beijing, China). .

Growth of NKT cells was arrested when hesperidin or linalin was added in excess of 100 μg / mL in the medium, indicating that hesperidin and linalin were toxic when the amount exceeded 100 μg / mL. It was an unexpected result (graph toward the right of Figure 5A).
For the evaluation of cell viability, CD4 ⁺ Vα24 ⁺ NKT cells (1 × 10 ⁴ ) derived from human PBMCs were converted to 3, 10, 30 or 100 μg / mL hesperidin or 3,3 The cells were cultured for 3 days with 10, 30 or 100 μg / mL of linalin or 0.003, 0.01 or 0.03% DMSO (vehicle control). After culturing, the cell viability was calculated by the trypan blue exclusion test.
When Vδ1 ⁺ T cells were stimulated with hesperidin or linaline, it was observed that the amount of detection of the p24 antigen, which indicates the presence of the R5-HIV-1-NL (AD8) virus, was clearly reduced, and these flavonoid glycosides were R5-HIV-1-NL (AD8) virus replication in CD4 ⁺ NKT cells was significantly suppressed in a dose-dependent manner (black bars in Figure 5B).
When a medium containing no Vδ1 ⁺ T cells was used, a measurable level of viral replication inhibition was observed in the flavonoid glycoside-added group, indicating that the flavonoid glycoside itself has some viral replication inhibitory activity. It was also shown (white bar in Figure 5B).

7. Inhibition of R5-type of HIV-1 replication in CD4 ⁺ T cells via human-derived natural γ1δ1 ⁺ T cells activated by hesperidin and linalin Stimulated by flavonoid glycosides such as hesperidin and linalin It was suggested that the human-derived natural γ1δ1 ⁺ T cells suppressed the R5-HIV-1-NL (AD8) virus replication in CD4 ⁺ NKT (natural killer T) cells.
Thus, the present inventors, when stimulating human-derived natural γ1δ1 ⁺ T cells with hesperidin or linalin, examined the replication of R5-HIV-1-NL (AD8) in CD4 ⁺ T cells as in CD4 ⁺ NKT. Was also examined.
CD4 ⁺ T cells derived from PBMCs were infected with NL (AD8) HIV-1 (R5-type) by the same procedure as described above for CD4 ⁺ NKT. The obtained NL (AD8) -infected CD4 ⁺ T cells were cultured with hesperidin or linalin by the same procedure as above. Then, on the fourth day after the start of the culture, the p24 antigen present in the culture supernatant was measured.
We observed that Vδ1 ⁺ T cells activated by hesperidin or linaline also significantly suppressed R5-HIV-1-NL (AD8) virus replication in CD4 ⁺ T cells (Figure 5C).
Taken together, these findings show that Vδ1 ⁺ T cells only suppress R5 tropic HIV-1 replication in CD4 ⁺ NKT cells when activated by flavonoid glycosides such as hesperidin and linaline. No, suggesting that it also suppresses R5 tropic HIV-1 replication in CD4 ⁺ T cells.

Claims (3)

As an active ingredient, at least one flavonoid glycoside selected from the group consisting of a compound represented by the following formula (1) or (2) and a pharmacologically acceptable salt or ester thereof, and Vγ1Vδ1 type T A therapeutic agent for HIV infection , which is used to suppress HIV proliferation in a subclinical stage of an HIV-infected patient by activating cells.
[In the formulas (1) and (2), R ¹ represents an alkyl group having 1 to 3 carbon atoms, and R ² represents a hydrogen atom or a hydroxyl group. R ³ is a substituent which may be bonded on the ring A, and represents a substituent selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, a hydroxyl group and a halogen, and R ⁴ is bonded on the ring B. Represents a substituent selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, a hydroxyl group and a halogen, and R ⁵ is a substituent which may be bonded to the C ring. Represents a substituent selected from the group consisting of an alkyl group having 1 to 3 carbon atoms, a hydroxyl group, and a halogen. n1 is the number of R ³ and represents an integer of 0 to 2, n2 is the number of R ⁴ and represents an integer of 0 to 3 in the case of the formula (1), and 0 or 3 in the case of the formula (2). represents the integer 1, n3 represents an integer of from 0 to a number of R ⁵ 3. When there are a plurality of substituents represented by the same symbol, each substituent may be the same or different. ] The flavonoid glycoside is at least one selected from the group consisting of hesperidin represented by the following formula (3), linalin represented by the following formula (4), and a pharmacologically acceptable salt or ester thereof. The HIV infection therapeutic agent according to claim 1, which is
3. The therapeutic agent for HIV infection according to claim 1, which is used for treating HIV infection in combination with an antiviral agent selected from the group consisting of a reverse transcriptase inhibitor and a viral protein enzyme inhibitor.