JPH02233619A - Virus infection defense agent - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed Description of the Invention] Skin coming to the top ■■Ko! The present invention relates to a virus infection prevention agent containing an iron-binding protein as an active ingredient. Viral diseases are currently one of the greatest challenges remaining in the medical field. Research has been conducted on many antiviral drugs to date, but since the virus relies on the proliferation function of cells to proliferate, it has been difficult to treat the virus with drugs. So far, drugs that have been recognized as having therapeutic effects on viral diseases include amantadine for influenza type At, encephalitis caused by herpes simplex virus,
There are only acyclovir and vidarabine for zoster rash. Of these, amantadine is not approved for use as an antiviral agent in Japan. Also, recently, infectious diseases caused by HIV, so-called AIDS
This has become a problem, and a large number of compounds have been screened, and as a result, it has been confirmed that azidothymidine (AZT) has a life-prolonging effect on people infected with AIDS, and is now being used to treat people infected with HIV. However, these drugs are expensive, have strong side effects, and have a limited therapeutic spectrum, so they still have problems as antiviral agents. On the other hand, interferon is an antiviral agent that has recently attracted attention. Interferon is 195
This substance was discovered in 1971, and research has been carried out as a substance that protects cells from being infected by viruses. Interferon can be obtained by culturing leukocytes and fibroblasts, but recently it has also become possible to mass produce it through genetic recombination. Regarding the use of interferon as an antiviral agent, there have been reports of its application to the treatment of respiratory infections, such as influenza, through nasal administration, but its metabolism and dynamics in the body are unknown, and it is difficult to use it clinically. The effectiveness has not yet been confirmed. Furthermore, even though mass production through genetic recombination has become possible, the production cost of interferon is high, and it is still too expensive to be used for the treatment of general viral diseases such as influenza or for infection prevention.

At present, the most popular measure against viral diseases is the administration of vaccines to prevent infection. These include live vaccines in which each virus is attenuated in some way, inactivated vaccines created by treating the virus with formalin, and component vaccines in which only the antigenic part of the virus is purified. These vaccines can prevent most diseases. However, in the case of influenza, which is the most common viral disease, it is difficult to prevent infection through vaccination. Influenza viruses have antigens on a part called the envelope on the surface of the virus, and this antigen is used as a vaccine, but this antigen part often mutates, and older vaccines cannot be used against mutated viruses. , it is clear that it has no effect. Also, H
It is difficult to prevent infection with vaccines against viruses such as IV, for which the antigen is unknown, and against cytomegalovirus infections, which often occur when the immune system is weakened by the administration of immunosuppressive drugs after organ transplantation. ．． In recent years, research in virology has progressed, and it has become clear that during virus infection, the virus binds to virus receptors present on the cell surface, and the virus invades into the cell from this part. For example, HIV binds to the CD4 receptor present on the surface of T4 lymphocytes. Therefore, by administering large amounts of CD4 into the blood, AIDS
It is said that it is possible to prevent the onset of the disease. Research on viruses and virus receptors thus shows the possibility of developing new virus therapeutics. The influenza virus has an enzyme protein called hemagglutinin on its surface that aggregates red blood cells. This hemagglutinin is an important factor in determining the viral antigen. This hemagglutinin is generally H-rich, Hs
Three types are known, but these types often mutate. This reduces the effectiveness of the vaccine.
When influenza infects cells, hemagglutinin recognizes and binds to sialic acid-linked sugar chains present on the surface of the cell membrane and begins to invade the cell, and this binding to cells is an indicator of the agglutination reaction of red blood cells. It can be evaluated as Therefore, if it has a sugar chain structure that inhibits hemagglutination, it can inhibit the binding of influenza virus to cells, prevent infection, and further prevent the spread to other cells after infection. As disclosed in JP-A-63-284-133, the present inventors have already obtained a substance that protects against influenza virus infection and filed a patent application. In addition, Yamakawa et al. isolated a substance that inhibits the hemagglutination of influenza viruses from human blood cells, and this substance is called I!, which has sialic acid as a constituent sugar. It has been revealed that it is the white matter (Yamakawa et al.
"Biochemistry" Vol. 31, pp. 416-421, 1959). However, the regularity of inhibition of viral receptor binding and glycoproteins is not yet clear. As mentioned above, important factors in protecting against viral infection include receptors on the cell surface to which viruses bind and substances that antagonize these receptors. Various receptors have been confirmed. "Proteins, Nucleic Acids, Enzymes vol. 32.
117-135, 1988'' discloses hemagglutinin and its receptor. According to these studies, it is not necessarily possible to find a certain rule regarding the sugar chain structures of glycoproteins and glycolipids and the roles of receptors. As a result of intensive research into virus infection prevention, the present inventors discovered that sialic acid and mannose were used in I! It was discovered that the iron-binding protein contained in the chain structure competes with the receptors of various viruses and prevents viral infection. Therefore, the object of the present invention is to provide a virus infection protective agent containing an iron-binding protein as an active ingredient. The feature of the present invention is that sialic acid and mannose are used as virus infection protective agents. The purpose is to use iron-binding proteins contained in the chain structure. Examples of iron-binding proteins having sialic acid and mannose in their W chain structure include lactoferrin and ovotransferrin. It is also possible to use partially enzymatically hydrolyzed proteins. Lactoferrin is generally an iron-binding protein isolated from the milk of chewing mammals, but in the practice of the present invention, it may be of any species or origin. or,
If necessary, glycoproteins produced by genetic recombination can also be used. Currently, the cheapest and most easily available product is one separated from milk. When separating from milk, a method using an anti-lactoferrin antibody disclosed in JP-A No. 61-145200 can be adopted. Ovotransferrin is a glycoprotein contained in chicken egg white with a molecular weight of approximately 77,000 to 87,000, and is an iron-binding protein similar to lactoferrin. In order to obtain ovotransferrin, separation and purification such as known chromatography is possible. For example, the carboxymethyl cellulose method (Garian et al., Journal
of Food Science” vol. 45, p. 460, 1980
), method using metal-immobilized affinity chromatography (Arumashiki et al., Agricultural Biological Chemistry J51, pp. 2881-2887, 198
7 years) may be adopted.

Iron-binding proteins such as lactoferrin and ovotransferrin obtained as described above can also be partially hydrolyzed with protease and used in the present invention. When enzymatically hydrolyzing lactoferrin and ovotransferrin, the degradation rate must be reduced to 60% in order to maintain the ability to protect against virus infection and to achieve the effects of enzymatic degradation.
It is preferable to keep it below %. The above-mentioned iron-binding protein or its partial enzymatic hydrolyzate can be used alone or in combination as a virus infection protective agent.The iron binding protein or its partial enzymatic hydrolyzate can be used alone or in combination as a virus infection protective agent. When the present invention is used for the purpose of preventing or treating cytomegalovirus (CVM) infection, It is necessary for the effect that the iron-binding protein, which is a component, exists in an amount of 0.1 μg or more per 1 g of the administered composition.
Filtration using a Menpuran filter below can be performed. Lactofurin and ovotransferrin, which are iron-binding proteins according to the present invention, are contained in milk and egg white, respectively, and their safety has been approved. Next, the effectiveness of the present invention in preventing viral infection will be explained using a test example using reaction (II) to inhibit red blood cell agglutination caused by influenza virus. Test Example 1 The following viruses were used as influenza viruses. Inactivated influenza virus obtained from Denka Seiken, A/Yamagata/120/86 (H,N,), A/Niigata/1
02/81 (H3N Tomi), A/Sichuan/α/ 87/ (
H s N *), A/Fukuoka/C29/85 (H s N
Mushroom), B/Nagasaki/1/87, B/Singapore/2
22/79, and uninactivated influenza virus A/PR/8/34 transferred from Shizuoka Pharmaceutical University.
(HIN+), and A/Aichi/2/6B (HsNg). The agglutination inhibition (HI) activity of these viruses against chick-stabilized red blood cells (manufactured by Takeda Pharmaceutical Co., Ltd.) was measured.
l activity was determined by the method of Yamakawa et al. ("Biochemistry" 31@,
416-421). The sample used for measurement was bovine lactoferrin (b L
f), human lactoferrin (hLf), goat lactoferrin (gLf), ovotransferrin (OTf)
and 20%, 40%, 60% of these with tribucin.
% decomposition rate. The results are shown in Table 1. As shown in Table 1, all samples showed strong H-alpha activity. Also, A/Yamagata/120/86 did not agglutinate chick red blood cells. Note that (A) in Table 1 is for chick stabilized red blood cells, and (B6) is for human type 0 red blood cells. Test Example 2 Each sample used in Test Example 1 was dissolved in physiological saline,
The concentration was adjusted to 0.5% (w/v). To this solution, add the human type 0 red blood cells prepared in Test Example 1 or the chick stabilized red blood cells to reach a red blood cell concentration of 1% (V/V) or 10%.
This was suspended at room temperature for 1 hour with occasional stirring, then centrifuged at 1500 rpm for 10 minutes, and the supernatant was purified by the same procedure as Test Example 1.
Activity was measured. As shown in Table 2, no change was observed in the Hl activity of each sample even after such treatment. From the results of Test Examples 1 and 2, iron binding, binding proteins, and their enzymatic decomposition products inhibit red blood cell aggregation caused by viruses, and this effect is not caused by nonspecific adsorption to red blood cells. It was determined that this was caused by the specific affinity of each active ingredient with the hemagglutinin of the virus. Furthermore, it was confirmed that the affinity between the iron-binding protein and the virus is not affected by mutations in the viral antigen. Note that (A) in the table is for chick stabilized red blood cells, and (B) is for human type 0 red blood cells. Examples are shown below to further specifically explain the present invention. Example 1: Bovine lactoferrin (b L f)
of Daily Science” Volume 20, 752-759
It was prepared from milk using the anti-bovine lactoferrin monoclonal antibody affinity column disclosed in P. (1987). Skimmed milk was loaded onto an anti-bovine lactoferrin monoclonal antibody affinity column, and bovine lactoferrin (b
L f) was adsorbed and then thoroughly washed with phosphate buffered saline (PBS) at pH 7.3.

Thereafter, washing was performed with a phosphate buffer of pH 7.3 containing 0.5M sodium chloride, followed by washing with a 0.2M sodium acetate buffer (
The bLf adsorbed on the column was eluted with pu 3.7 (containing 0.15M sodium chloride). After elution, the pi was adjusted to around neutrality, dialyzed against deionized water for 3 days, and then freeze-dried.
bLf was obtained. The purity of the obtained bLf was confirmed by electrophoresis, but it showed a single band. Example 2 Preparation of hLf: Human lactoferrin (hLf) was prepared from large milk using a heparin-Sepharose CL-6B column. Skimmed human milk was loaded onto a Hebarin-Sepharose CL-6B column, hLf in the skimmed human milk was adsorbed into the column, and then p
H7.3 0. Washed with OIM phosphate buffer. Then, 0.0.0% at pH 7.3 containing 1.0M NaCl. The hLr adsorbed in the column was eluted with OIM phosphate buffer. Furthermore, the eluate was dialyzed against deionized water for 3 days and then lyophilized to obtain hlJ. The purity of the obtained hLf was confirmed by electrophoresis and showed a purity of 98% or more.

Example 3 Preparation of 07f: Proteins were precipitated by adding ammonium sulfate to egg white at a concentration of 2.5M. The precipitate was collected by centrifugation and adjusted to pH 6.0. O
It was redissolved in IM phosphate buffer and loaded onto a diethylaminoethyl cellulose column. After adsorbing OTf in egg white onto the column, 0. Wash with OIM phosphate buffer, then pn 6 with 0.1 M saline.
．． OTf was eluted with 0 phosphate buffer. The eluate was collected and dialyzed against deionized water for 3 days, followed by lyophilization to obtain OTf. The purity of the obtained OTf was confirmed by electrophoresis, which showed a purity of 95% or more. Example 4 Preventing the spread of influenza virus? III: Uninactivated influenza virus A used in Test Example 1
/P R /8/34 and A/Aichi/2/6B were diluted in stages and inoculated into the allantoic cavity of five 10-day-old chicken eggs at a dose of 0.1. Urine fluid 50μE
This was mixed with 0.5% Hyco-stabilized red blood cells, stirred, and the infection rate was determined by agglutination of the red blood cells. 100
0.1 of the virus dilution solution with a dilution ratio that showed an infection rate of %.
- blJ, hLf, OTf obtained in Examples 12 and 3,
After dissolving 1.01 g and 0.5 sg of 2.0 mg% and incubating at room temperature for 30 minutes, 0.1 g and 0.5 sg of each were inoculated into the allantoic cavity of 10-day-old chicken eggs. After 3 days, 50 μl of allantoic fluid from each egg was taken and the infection rate was determined by hemagglutination. Five eggs per group were used to obtain the infection rate for each group. In addition, the anti-infection effects of enzymatically degraded products of each protein were measured in the same manner. The results are shown in Table 3, and each sample showed a significantly strong protective effect against influenza virus. Table 3 Example 5 Effect of bLf on inhibiting cytomegalovirus infection: The effect of bLf obtained in Example 1 on inhibiting cytomegalovirus (CMV) infection was confirmed. btr in MEM supplemented with 2% serum
It was dissolved in a medium at a concentration of 5 mg/l, and sterilized by filtration using a 0.45 μm filter to obtain a stock solution. This stock solution was diluted with MEM medium supplemented with 2% serum as necessary. Human CMV (Tonne strain) was incubated with culture medium containing blJ at various concentrations for 1 hour, and then infected into human fetal fibroblasts (HEL cells). After culturing for 24 hours, the cells were fluorescently stained with human CMV-positive serum to measure the adsorption ability of human CMV to the cells. As a result, Table 4
As shown in Figure 2, CMV was completely depleted by ink evacuation with lmg/1 bLf for 1 hour.
It was confirmed that the adsorption and penetration ability was lost. Table 4 Example 6 CMV growth inhibition test by hLf: After incubating human CMV with hLf dissolved and diluted in the same manner as in Example 5 for 1 hour, HEL cells were infected with CMV. Next, the above-mentioned infected HEL cells were cultured in hLf-containing soft agar medium (0.8% soft agar, manufactured by Difco) adjusted to the same concentration as the hLf diluted solution, and the plaques that appeared on the 9th day were counted and compared. The soft agar mound was layered every three days. On the other hand, a similar test was conducted under conditions where hLf was not included in the soft agar medium, and the number of plaques formed was counted. Table 5: CMV proliferation was almost completely suppressed. Table 6 Effect of 0Tf on human CMV growth inhibition As shown in Table 5, when CMV was treated with hLf-containing medium at a concentration of lsg/d and then cultured in hlJ soft agar medium at the same concentration, strong inhibition of CMV growth was observed. Ta. Example 7 Effect of inhibiting CMV proliferation by 0Tf: The OTf obtained in Example 3 was treated in the same manner as described in Example 6, and the bullaks produced in the OTf-containing soft agar medium were counted. As shown in Table 6, HEL cells were cultured in a 96-sized plastic plate (manufactured by Falcon), and h}CMV from 10-1 to 10-'
A series of O-fold dilutions of the virus was added, the cells were infected, and the titer was measured. As in Example 5, the virus solution of each dilution series was incubated with the bLf culture solution of each concentration series for 1 hour,
HEL was then grown on 96 large plastic plates.
Virus solution was added to the cells to infect them, and the number of wells in which infection was established was counted. As a result, CMV with a titer of 5X10'
is 0.1 fog god(7)bLf and titer 7.5X10'
D, 0.5mg/ad (DbLf titer is 2.5X10
It was revealed that the titer significantly decreased to 50 at 1.0 mg/1 bLf (Table 7). Example 9 Treatment effect for preventing CMV infection in animals: In this example, The following is an example in which the prevention of CMV infection and the therapeutic effect on infected animals were confirmed. blJ obtained in Example 1 was dissolved to a concentration of 1000 μg per 1 portion of physiological saline solution, and after dissolution, it was filtered through a 0.22 μ filter. On the other hand, thymectomized mice (C57B16J
) The 20 animals were divided into two groups; one group received the above bLf solution intravenously for 7 days, and the other group received physiological saline in the same manner. Thereafter, mouse CMV was extracted using the method of Grundy et al. (Transplantation, Vol. 37, 4).
84-490, 1984). That is, mice were infected by intraperitoneal administration of CMV Smith salivary gland virus. After raising the mice for an additional month, CMVI in the urine of the mice was measured using an enzyme-linked immunoassay method. All mice administered only physiological saline solution were positive for mouse CMV in their urine, whereas 2 out of 10 mice in the bLf administration group were positive. In addition, 10 mice to which only this physiological saline solution was administered were divided into two groups, and the bLf solution was administered intravenously every day for 15 days, and one group was similarly administered only the physiological saline solution. After 15 days, both groups were anesthetized and dissected, and the state of the organs was observed. Table 8 As shown in Table 8, this was only observed due to mouse CMV infection, and the other three mice had clearly recovered. 11IB Results As stated above, the iron-binding protein of the present invention clearly protects against viral infection and also exhibits a therapeutic effect, so the present invention can be effectively used as a protective agent against viral infection. Furthermore, it is clear that the iron-binding protein of the present invention is not affected by antigenic changes in influenza viruses, and a wide range of preventive effects can be expected. Furthermore, the above-mentioned substance according to the present invention is a component contained in foods, and can be supplied safely and at low cost. Therefore, by carrying out the present invention, a virus infection protective agent that is safe and has a protective effect against a wide range of viruses can be stably and inexpensively supplied. Procedural Amendment June 5, 1999 l. Display of case: 1999 Patent Application No. 53679 2. Title of the invention Virus infection protective agent 3. Relationship with the case of the person making the amendment Patent applicant name (669) Snow Brand Milk Products Co., Ltd. (and 1 other person) 4. Agent address: 7th floor, Crossside Kojimachi Building, 5-4 Kojimachi, Chiyoda-ku, Tokyo

Claims (4)

[Claims] (1) A virus infection protective agent containing an iron-binding protein as an active ingredient. (2) The agent for protecting against viral infection according to claim (1), wherein the iron-binding protein is lactoferrin or ovotransferrin. (3) Claim (1) wherein the iron-binding protein is a partial enzymatic hydrolyzate of lactoferrin or ovotransferrin.
).The virus infection protective agent described in ). (4) Claims (1), (2), and (3) in which the virus is influenza virus or cytomegalovirus.
The virus infection protective agent described in any of