JPH03188872A - Method for deactivating aids virus - Google Patents

Abstract

PURPOSE:To deactivate AIDS viruses and prevent its infection to other persons via the blood or blood-made agent by processing the circulating blood at ultra high pressure outside the body under vacuum while water or another liquid component is used as a medium. CONSTITUTION:During the process that blood is extracted from an artery 10 to the outside of a body with a catheter and circulated to a vain 9, a pressurizing tank in a pressurization type high pressure device 11 is operated under a low temperature, and the blood is processed at ultra high pressure in days in sequence while water or another liquid is used as a medium. The envelope protein of AIDS viruses 1 in the blood undergoes protein denaturation and is broken, and the AIDS viruses 1 are deactivated. When the gp120 (tentacles) 4 on the surface of AIDS virus cells are processed at ultra high pressure under a low temperature, they undergo protein denaturation and can be broken, thus the bonding with helper T4 cells, and the infection to the helper T4 cells can be prevented. For the infected cells in the blood, reverse transcriptase is singularly deactivated at ultra high pressure, and the proliferation of the AIDS viruses is prevented.

Description

[Detailed Description of the Invention] (a) Purpose of the Invention (Field of Industrial Application) The present invention relates to the medical industry, and is particularly intended to be used for inactivating the AIDS virus that has invaded blood products and human blood. be.

<Conventional technology> As a result of research, it has become clear that AIDS is an intractable disease, and treatment methods for this disease have also been researched.
Three methods are currently being considered.

■Use of antiviral agents Several types of antiviral agents are currently in use, but none of them have sufficient efficacy or cannot be used due to side effects.

■Immunity-enhancing means A method of normalizing the immune function weakened by the AIDS virus, in other words, an immune-enhancing means.There are drugs already used for malignant tumors and autoimmune diseases, and there are also other methods and means. Plasmaphnilysis, leukocyte transfusion, bone marrow transplantation, etc. are performed. However, immunodeficiency occurs as a result of AIDS virus infection, and adjusting it is only one symptomatic treatment. Herein lies the limitation of immunomodulatory therapy.

■Treatment for opportunistic infections and malignant tumors Treatment for opportunistic infections that occur as a result of AIDS infection is still insufficient. Additionally, many opportunistic infections are difficult to treat.

■Vaccines Vaccines are considered the most useful method for preventing virus patients, and in the past, virus patients have been eliminated or significantly reduced by vaccines. However, the AIDS virus mutates frequently, so even if a vaccine is injected, the antibodies generated by the vaccine cannot be expected to be effective. Developing a vaccine for the AIDS virus is considered extremely difficult.

<Problems to be solved by the invention> The difficulty in eradicating (killing) the AIDS virus is that the AIDS virus invades the lower four helper cells (3) and destroys the cells, lowering the immune system and damaging the human body. It makes you more susceptible to cancer and infections. However, when trying to kill the AIDS virus (1) that has invaded the 4 helper cells (3), which are immune cells, conventional therapy cannot kill only the AIDS virus (1), and the immune cells There is a problem that the AIDS virus (1) cannot be killed unless all four helper cells are killed and the living body is destroyed at the same time.
There is a very difficult problem whether it is possible to distinguish between T helper cells and kill them.

Also, the tentacles of the AIDS virus (1) g p 120 (2)
The shape of Gp120 (2) changes easily, and even if the shape of Gp 120 (2) changes, causing resistance to drugs and rendering the drug ineffective, helper F cells (T, lymphocytes) (3)
Since it easily combines with the receptor (4), there is a problem that it can be infected with the AIDS virus (1). Therefore, there is a major problem in that AIDS treatment requires the adoption of other methods that do not rely on drugs.

Therefore, the present invention aims to inactivate the AIDS virus itself that has entered the body (in the blood) or only the AIDS virus in AIDS-infected cells (helper cells infected with the AIDS virus) before entering the body. In addition to preventing infection with the AIDS virus, it can also be used to inactivate the infected virus and prevent the onset of AIDS, or to prevent the progression of AIDS, as well as to treat the disease without side effects. This is what we aim to contribute to.

(b) About the structure of the invention In the process of temporarily extracting blood from the body to the outside and circulating it back into the body, an ultra-high pressure generator is used in the circulation path to generate water or other liquid components under vacuum conditions. Ultra-high pressure treatment is carried out using the blood as a pressure medium, and the treated blood is circulated into the body. Note that the blood to be subjected to ultra-high pressure treatment may be vacuum-packed blood for transfusion, or may be a blood product. In this case, it goes without saying that the above-mentioned blood circulation path is not particularly required, and it is sufficient to treat the blood for transfusion or blood products under high pressure under vacuum conditions.

In such a case, the AIDS virus present in the blood circulating within the body or the AIDS virus contained in vacuum-packed transfusion blood or blood products is completely inactivated.

The results are particularly noticeable at low temperatures (below 0°C).

(c) Considering the infection mechanism of AIDS regarding the effect of the invention, AIDS R/L
/ When (orV) (1) enters the body, l + IV (
The part (tentacle) called g p 120 (2) that protrudes from the surface of 1) binds to the receptor (4) of the lymphocyte (helper T cell) (3), and helper T invades the cell (3). (infection). After that, it remains latent, but eventually becomes active and destroys helper T9 cells (3) one after another, resulting in the loss of the human body's immune function and the onset of AIDS.

CD is the receptor (4) for helper Ty cells.

However, a group led by Professor Nobuhiko Katsunuma and Assistant Professor Hiroshi Kido of the Tokushima University Enzyme Science Research Center has discovered that in addition to the helper T and CD, which is the receptor (4) of cells (3), there is another receptor. body (5) was discovered, and this receptor (5) was found to exist.
) has also been found to specifically bind to a site called the beta epitope (6) of g p 120 (2). (Figure 1 (A) (B)).

If we consider the mechanism of AIDS infection in detail, the AIDS virus (1) is an infectious obligate parasite that uses RNA as a nucleic acid, and forms an envelope protein on the cell surface membrane, and from this envelope protein (7) it spreads outward It has a structure with tentacles g p 120 (2) directed toward the .

Therefore, when the AIDS virus (1) invades the human body, the receptors (4) (5) of helper Tl cells (3) in T lymphocytes
), helper T binds to its central nucleus (
8), and then by the action of the chromosome of the virus and reverse transcriptase, the R of the AIDS virus (1) is
NA (ribonucleic acid = virus genes) is DNA (deoxyliponucleic acid = mostly present in the cell nucleus and forms the main body of genes. The genetic code is incorporated into its base sequence, and it is used for inheritance (copying) and protein synthesis (transcription). ), which undergoes a reverse change to ) and is incorporated into the genes of the cell's nucleus. In other words, it is inserted into the chromosome of the nucleus of the helper Tl cell (3) (referred to as transcription).
be done.

In this way, the AIDS virus (1) that invades the helper Tl cell (3), which is a host cell, synthesizes and modifies viral proteins within the cell (3) to create a central nucleus (8).
The AIDS virus is multiplied, buds off from helper Tl cells (3), separates, and exits. Therefore, the AIDS virus (1
) helper T cells (3), which are host cells invaded by
The patient's function deteriorates and eventually dies. Therefore, the immune system of the human body decreases, the disease develops, and eventually death occurs.

As mentioned above, the basic composition of the AIDS virus (1) consists of nucleic acids and surrounding proteins (envelope proteins), but when a protein solution is heated, the molecular movement becomes intense, the weak non-covalent bonds are torn, and the proteins are separated. It is easy to understand that the three-dimensional structure collapses and denatures.

On the other hand, a collection of water molecules has a property that the volume is smaller when they are coordinated around the amino acid side chains of a protein than when they exist as free water, and the volume reduction effect based on this is large. Therefore, when high pressure is applied to a protein solution, the volume decreases more than the volume reduction due to the filling effect, and the protein tries to settle down to expose the amino acid side chains from the inside into the water. The result is protein denaturation.

Envelope proteins are easily destroyed by strong solvents such as ether and chloroform, and weak surfactants, and are also destroyed by changes in osmotic pressure, freezing and thawing, heat, and drying. However, these methods are expected to affect other blood components.

However, in ultra-high pressure treatment, no heat is generated at all, so other blood components are not affected.

Therefore, in the present invention, the envelope protein (7) formed on the cell surface membrane of the AIDS virus (1)
to inactivate the AIDS virus (1) by destroying it;
Second, the first step in AIDS virus infection is adsorption to gp120(2), which consists of protein, and infection is prevented by destroying this gp120(2).

Third, it inactivates the reverse transcriptase of the AIDS virus (1) to promote healing.

We focused on the above three points.

FIG. 3 is an explanatory diagram of the AIDS virus pressure inactivation processing device (extracorporeal circulation continuous processing system), in which blood is taken out of the body from a vein (9) using a catheter, in accordance with conventional hemodialysis. In the process of circulating the blood to the artery (10), the blood is superimposed over time using water or other liquid as a medium by passing it through a pressurized tank in a pressurized high-pressure device (continuous treatment device) (11) at low temperature. Treat with high pressure.

In such a case, the envelope protein of the AIDS virus (1) in the blood undergoes protein denaturation and is destroyed, thereby inactivating the AIDS virus (1).

Furthermore, if g p 120 (tentacles) (4) on the surface of AIDS virus cells are treated with ultra-high pressure at low temperatures, the protein can be denatured and destroyed in the same way as described above. ) and can prevent infection of helper Tl cells.

Furthermore, in infected cells in the blood, reverse transcriptase is specifically inactivated under ultra-high pressure. Therefore, the proliferation of the AIDS virus is inhibited. In the figure, (12) is an air bubble detector that checks the inflow of air bubbles into the blood, and (14)
is a pump that pressurizes with a hydraulic pump.

(15) is an infusion pump, which is a pump that injects anticoagulant and the like into the blood. (16) is a blood pump that circulates blood, and (17) is a depressurization detector.

Next, FIG. 4 is an explanatory diagram of a batch processing apparatus for blood for transfusion and blood products, in which blood (18) is collected, for example, in a blood set (blood collection bag, blood collection tube, blood collection needle) made of vinyl chloride resin. After blood collection, a pretreatment process (treatment with anticoagulant, etc.) is performed, and the blood is filled and sealed in a pressure-resistant plastic pouch under vacuum.Then, this is cooled at 5°C or less in a cooling tank (21) (24). After that, it is placed in a pressurized container (22) and placed in a pressurized treatment device (25).
) and ultra-high pressure treatment (26) using water as the pressure medium (27).

After treatment, the plastic pouch is taken out from the pressurized container, air-dried (27), and then backed (28) in a barrel.

At such times, the envelope protein or gp120 of the AIDS virus (1) in the blood is destroyed and inactivated, and in helper T cells and cells infected with the AIDS virus, reverse transcriptase, etc. are inactivated, and the proliferation of the AIDS virus is inhibited. is prevented.

Next, the experimental results regarding the effect of ultra-high pressure treatment on the AIDS virus are shown in the following table.

1-12- As can be seen in the table above, it has been revealed that at room temperature and high pressure, it is inactivated in 8 minutes, and at low temperature, it is inactivated in 5 minutes even at 2000 atm.

In addition, the AIDS virus invades helper T cells, and then the RNA is converted into D by the action of the virus's reverse transcriptase.
When converting to NA, 3H-thymidine is incorporated, but when the treatment of the present invention is performed, the amount of 3H-thymidine incorporated is as shown in Figure 5 (A), when the pressure treatment time exceeds 3 minutes. The rest remains almost constant, indicating that DNA is not synthesized.

Table 3 of the grammar shows the effect of ultra-high pressure treatment on the activity of reverse transcriptase (RT) of HIV released outside the cells.

(Table 3) Culture of MOLI/LAV cells, which are persistently infected cells of HIV.The two supernatants were subjected to ultracentrifugation, and the precipitate was mixed with 10% of the original virus solution.
One minute amount of virus solubilization buffer was added to prepare a solution with reverse transcriptase activity. Several steps (0 to 40
After treatment at ultra-high pressure (00 atm) and reacting at room temperature for 30 minutes, 90 μρ of R7 reaction buffer was added. 95 of them
μQ to 3H thymidine triphosphate (3H-dTTP) 10μ
Ci and temperate primer [Po1. y(rA)・
It was added to a tube containing 2.5 μg of P(dT)7a-zP (15 μm), covered with a lid, and reacted at 37° C. for 22 hours. 1
After stopping the reaction by adding 0% trichloroacetic acid aqueous solution,
The precipitated DNA was collected on a glass filter, and the 3H- incorporated into the DNA was measured using a liquid scintillation counter.
The amount of dTTP was measured.

As a result, by high pressure treatment, the RT of old V outside the cells was
The activity was suppressed in a high pressure-dependent manner and was also correlated with treatment time. 2000 atm, 5 minutes to 30% level, 4000 atm, 5 minutes to 4% level, 4000 atm, 10 minutes,
The RT binding property was reduced to 0%.

(d) Effects of the invention As described above, when the method of the present invention is applied to the AIDS virus or helper T cells infected with the AIDS virus, the helper T cells do not bind to the helper T cells before they bind to the cells. Helper T, after binding to cells, inhibits threshing, reverse transcription, halts translation, inhibits protein modification, and finally assembly. In other words, the AIDS virus in the body can be inactivated by applying ultra-high pressure, no matter what stage the AIDS virus is in from the time it invades helper T and 7 cells until budding. Furthermore, the therapeutic effect of AIDS can be enhanced by the combined use of immune enhancers, immunostimulants, anti-AIDS virus drugs, antibiotics, anti-malignant tumor drugs, vaccines, etc.

Furthermore, by treating vacuum-packed blood for transfusion and blood products with the method of the present invention, the AIDS virus can be inactivated, thereby preventing infection to others via blood or blood products. I can do it.

However, since this method does not cause fever and has no effect on blood components other than the AIDS virus, there are no side effects at all.

[Brief explanation of drawings]

Figure 1 explains the infection mechanism of the AIDS virus. (A) shows the state before the AIDS virus and helper T and 7 cells (lymphocytes) combine, and (B) shows the state after they have combined and become infected. This shows that. FIG. 2 is an explanatory diagram showing a state in which the AIDS virus and helper T cells combine, the AIDS virus multiplies, and exits from the helper T cells. FIG. 3 is an explanatory diagram of an AIDS virus pressure inactivation processing device (extracorporeal circulation continuous processing system), and FIG. 4 is an explanatory diagram of a treatment device for transfusion blood and blood products. . FIG. 5 is a graph showing the relationship between the amount of 3H-thymidine taken in, which is required when the AIDS virus invades HelperF cells and changes from RNA to DNA, and the pressure treatment time. (1)・・・・・・AIDS virus (HIV) (2)
・・・・・・・・・g p 120 (Tentacles of the AIDS virus) (3)・・・・・・・・・Helper T cells (lymphocytes, helper T cells) (4)・・・・・・...Receptor (lymphocyte receptor) (
CD4) (5)... Newly discovered receptor for lymphocytes (6)... Newly discovered beta shrimp 1-p of gp120 (7)...・Envelope protein (8)・・・Central nucleus of the AIDS virus (9)・・・Vein (10)・・・Artery (11)・・・...Pressure type high pressure device (12)...
...... Air bubble detector (13) ...... Venous pressure measuring device (14) ...... Hydraulic pump (1
5) Infusion pump (16)
)・・・・・・Blood pump (17)・・・・・・
...Removal of pressure detector (18) ...Blood (19) ...Pretreatment step (20) ...
...Filling and sealing (21) ...Cooling tank (22) ...Cooling container (of pressure treatment equipment) (24) ...・・・・・・Cooling (25)・・・・・・Pressure treatment device (26)・・・・
・・・・・・Pressure treatment (27)・・・・・・Drying

Claims (3)

[Claims] (1) In the process of temporarily taking blood out of the body and circulating it back into the body, the circulating blood is subjected to ultra-high pressure treatment outside the body under vacuum conditions using water or other liquid components as a medium. Characteristic method of inactivating the AIDS virus. (2) A method for inactivating the AIDS virus, which comprises subjecting vacuum-packed blood or blood products for transfusion to ultra-high pressure treatment using water or other liquid components as a pressure medium. (3) Blood for transfusion and blood products characterized by inactivating the AIDS virus by ultra-high pressure treatment using water or other liquid components as a pressure medium.