JPH01265034A - Preventive for viral diarrhea - Google Patents

Abstract

PURPOSE:To obtain the title preventive by adding egg antibody which is prepared from egg yolk which is hyperimmunized with the virus of viral diarrhea. CONSTITUTION:One or more of pathogenic viruses of viral diarrhea such as rotavirus or enteric adenovirus are mixed, when needed, emulsified with Freund's complete adjuvant. Chickens are hyperimmunized by injecting the emulsion 3-5 times once every week or 2 weeks and the subject preventive containing egg antibody is obtained from the egg yolk. The egg antibody bonds to the pathogenic virus specifically to prevent the pathogenic virus from sticking to the epithelial tissues of the intestinal tracts whereby the development of diarrhea can be stopped. Since the egg antibody loses its activity, when it is digested by pepsin under acidic conditions, the antibody is preferably prepared in an enteric form in order to avoid the acidic digestion in stomach.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention was made for the purpose of preventing viral diarrhea. Regarding preventive agents.

[Conventional technology]

Viral diarrhea is a common disease encountered on a daily basis, but only recently has the causative virus been isolated and identified.

At present, rotavirus, positive adenovirus, norouk virus, calicivirus, astrovirus, coronavirus, predavirus, and the like are known to be pathogenic viruses for viral diarrhea that frequently occurs in humans and livestock. These viruses are particularly highly infectious to infants and young livestock whose immune systems have not yet fully developed. or,
Symptoms of this infection are characterized by diarrhea accompanied by vomiting, and affected individuals often become extremely dehydrated, severely affecting their development.

Currently, there are approximately 500
More than 10,000 infants and children die from diarrhea. Many of these cases have been found to be caused by the above-mentioned viruses, especially rotavirus, and their eradication is considered an urgent issue by the World Health Organization (W.
It is being promoted as a global project including HO).

Conventionally, vaccines have been commonly used and have been effective in preventing viral infections. Naturally, vaccines can also be used to prevent viral diarrhea. In recent years, a test administration of a live vaccine to infants in developing countries has been conducted for rotavirus, a diarrheal virus that has become a problem worldwide. However, it has been reported that no effect was observed (Demol, P, e
tal,: Lancet, I [: 108, 1986
). In this case, the reason for the lack of efficacy was thought to be that the age of rotavirus infection was less than one year old, which is immunologically immature, suggesting the necessity of passive immunization.

Additionally, the effectiveness of passive immunization was tested for the prevention of rotavirus diarrhea in infants, and researchers succeeded in preventing rotavirus diarrhea by giving pregnant colostrum hyperimmunized with rotavirus orally to infants. It is reported that (Ebina,
T. et al.

Med, Microbiol, Immunol.

:174:177-185.1985).

[Problem that the invention seeks to solve]

There have been many reports that oral administration of specific antibodies against the causative virus is effective in preventing viral diarrhea. In these reports, bovine colostrum antibodies were used as specific antibodies against diarrheal viruses.

Serum antibodies and monoclonal antibodies have been used.

However, these specific antibodies have not been put into practical use because their preparation requires a great deal of cost and mass production is difficult.

[Means to solve the problem]

The present inventors hyperimmunized egg-laying hens with a diarrhea virus and succeeded in preparing a large amount and high titer of egg antibodies that specifically bind to the virus from egg yolks produced by the hens. Furthermore, the present invention was completed based on the discovery that oral administration of the chicken egg antibody to mouse pups significantly prevents infection by the corresponding diarrheal virus.

That is, the present invention relates to a preventive agent for viral diarrhea containing an egg antibody prepared from the egg yolk of a chicken hyperimmunized with a virus that causes viral diarrhea.

[Effect]

In the present invention, the viruses causing viral diarrhea include rotavirus and positive adenovirus.

Infects viruses that cause viral diarrhea that frequently occurs in humans and livestock, such as Norou virus, calicivirus, astrovirus, coronavirus, and predavirus. One or more of these viruses are mixed and, if necessary, emulsified with Friend Completed Ashband to hyperimmunize the laying hens. Generally, it is desirable to inject immunity into chickens into their leg muscles for ease of operation. Repeating this immunization injection three to five times, once every week or two, is usually sufficient to hyperimmunize the chicken. The amount of virus during immunization may be sufficient to sensitize chickens to the virus. Generally, a viral amount of 1 μg to 1 m (1 μg to 1 m) is often used.

Egg yolks from chickens hyperimmunized with the virus that causes viral diarrhea contain egg antibodies that specifically bind to the virus. Therefore, the egg antibodies used in the present invention are anti-viral egg antibodies produced by chickens hyperimmunized with the virus that causes viral diarrhea, and are obtained from whole chicken eggs, egg yolks, or whole chicken eggs containing the egg antibodies. It can be used in any form, such as chicken egg antibodies purified from chicken egg yolk, and is not particularly limited.

The egg antibody can be prepared from egg yolk using a simple and industrial method previously developed by the present inventors (Patent Application No. 62-4).
5900.62-194083), but the manufacturing method is not particularly limited.

Infection with viral diarrhea begins when the diarrhea virus attaches to the intestinal epithelial tissue.

It is known that this attachment takes place via receptors. A specific antibody against the diarrhea virus given orally binds to the corresponding diarrhea virus particles in the intestinal tract and prevents the infection by inhibiting the attachment of the diarrhea virus to the intestinal epithelial tissue. It is estimated that

Therefore, the dosage form of the present invention in actual use must be a dosage form that can orally provide specific chicken egg antibodies against diarrheal viruses and effectively transport them into the intestinal tract. The egg antibodies used in the present invention do not lose their antigen-binding ability even when digested with trypsin and chymotrypsin, but lose their antigen-binding ability when digested with pepsin under acidic conditions. , preferably in enteric-coated form.

It is known that the stomachs of domestic animals in their infant and young stages are not yet as developed as those of adults, and their pH is around 4.0. In addition, the digestive power of pepsin is also very incomplete. Therefore, when the viral diarrhea preventive agent of the present invention is used for these infants and young livestock animals, it is not necessary to consider pepsin digestion in the stomach as much as possible.
There are no particular limitations on the dosage form.

The content of the chicken egg antibody in the present invention should normally be determined in a timely manner based on the infection neutralizing power (titer) of the prepared chicken egg antibody measured against the corresponding diarrhea virus. From experiments using mice, we have found that when using chicken egg antibodies obtained from egg yolks of chickens hyperimmunized against diarrheal viruses in the usual way as powdered milk, the amount of chicken egg antibodies in 1 mj2 of drinking milk is 1μ… We believe that by including the above, we can prevent viral diarrhea that infants and young children encounter on a daily basis.

The specific antibody against diarrhea virus used in the present invention is a chicken egg antibody obtained from chicken egg yolk. 25 chickens per year
It is known that they lay 0 to 300 eggs, and each egg contains about 100 mg of chicken egg antibodies. In experiments in which we used rabbits and chickens and simultaneously overimmunized them with human rotavirus, the neutralizing antibody titers against human rotavirus were almost the same for rabbit serum antibodies and chicken egg antibodies. And at this time rabbit 1
The serum antibody obtained from the wings was 800 mg.

These correspond to the world of chicken egg antibodies contained in eight chicken eggs. As described above, in the present invention, since a chicken egg antibody is used as a specific antibody for a diarrhea virus, the agent for preventing viral diarrhea of the present invention can be prepared in large quantities at a very low cost.

The effects obtained in animal tests of the present invention will be shown below using test examples conducted using human rotavirus, which is a worldwide problem as a causative virus of infant diarrhea, as an antigen.

Test Example 1゜One egg-laying hen was infected with human rotavirus Wa strain (serotype 1).
), Kun strain (serotype 2), and Mo strain (serotype 3) at 1.0×10′ TCID, respectively.

Human rotavirus mixture containing (tissue culture infectious units) 1
ml and 1 ml of Friend Combry Adjuvant were emulsified and injected into the leg muscles of chickens. The same immunization was performed once every two weeks, a total of 4 times. Every two weeks after the first immunization, egg yolks from four immunized chickens and four control chickens were collected, diluted 10 times with a lambda carrageenan aqueous solution (1 mg/mj2), and then diluted with 3.0 O
Or, p, m.

The egg yolk lipoprotein was removed as a precipitate by centrifugation for 10 minutes. A dilution series of the centrifuged supernatant (crude egg antibody fraction) was prepared, and 50μ of each dilution series was prepared. 100 FCFU each of human rotavirus Wa strain, Kun strain, and Mo strain (
50μ of virus solution containing fluorescent antibody (intestinal focus unit)
After mixing with i and incubating at 37°C for 1 hour, monkey kidney cells (MA104 cells) tx1o' cells/well were infected. After culturing at 37°C for 20 hours, the FCFU of the wells was measured, and the maximum dilution rate that inhibited 100 FCFU of each human rotavirus strain by 50% was defined as the neutralizing antibody titer. The results are shown in Table 1.

Table 1. Changes in neutralizing antibody titer (titer) against each strain of human rotavirus in immunized chicken eggs and control chicken egg yolk Test Example 2゜To 5 kg of chicken egg yolk produced by the immunized part of Test Example 1, a lambda raginan aqueous solution (1.2 mg /mp) 25Kg and stirred, centrifuged for 1 minute (s, o.

The egg yolk lipoproteins were removed as a precipitate using 10 min). 23.25 kg of the obtained supernatant (crude egg antibody fraction) was concentrated 10 times using an ultrafiltration membrane with a molecular weight cutoff of 1o and 00, and lyophilized to obtain 127.0 g of crude egg antibody powder. In the obtained powder, the purity of egg antibody (amount of egg antibody/amount of protein x 100) was 2.
It was 0%. 1 mg of this crude chicken egg antibody powder is MA10
Human rotavirus Wa strain 2.5X10'F for 4 cells
CFU, Kun strain 2. lXl0' FCFU, MO strain 3
．． Completely inhibited the infectivity of 6X10' FCFU.

Test Example 3゜The crude egg antibody powder against human rotavirus obtained in Test Example 2 was dissolved in physiological saline, and 100μ of the powder was dissolved in physiological saline. was given orally to BALB/C mice on the fifth day after birth. After 5 to 10 minutes, the same mice were treated with human rotavirus Wa strain,
5 each for Kun strain and Mo strain, OXI O' FC
A challenge test was performed by orally infecting the cells with 100 μρ of a virus solution containing FU.

Thereafter, the presence or absence of diarrhea was observed every day for 5 days, and the results were expressed as the number of mice with diarrhea divided by the number of test mice.

As a control, a similar challenge test was performed on mice given 100 μρ of physiological saline.

Test Example 4 The crude chicken egg antibody powder obtained in Test Example 2 was dissolved in physiological saline, and ddy mice (1 group of 10 Orally administered to each group of animals),
LD by Litchfield-Wilcochran method,
I asked for The results are 5.5g/Kg or more for females and 6.2 for males.
g/Kg or more.

Example 1゜1g of specific crude chicken egg antibody powder (purity 20%) against human rotavirus obtained in Test Example 2 is mixed with 10Kg of whole milk powder to prevent viral diarrhea, which can be used as formula milk powder for infants. obtained the drug.

Example 2゜1 g (purity 20%) of the specific crude chicken egg antibody powder against human rotavirus obtained in Test Example 2 was mixed with 10 kg of commercially available infant formula powder to obtain a virus-resistant powder that can be used as infant formula powder. A diarrheal disease preventive agent was obtained.

Example 3 1 g of the specific crude chicken egg antibody powder (purity 20%) against human rotavirus obtained in Test Example 2 was added to lactose.

After mixing with IKg of a filler consisting of dextrin and the like, the mixture was filled into intestinal fluid capsules to obtain a viral diarrhea preventive agent.

Example 4 100 mg of crude chicken egg antibody powder (purity 20%) that specifically binds to diarrhea virus and neutralizes its infectivity was mixed with 0.0 mg of fructose.
7% by weight, powdered citric acid 0.15% by weight, vitamin C0
, 002% by weight and powdered natural flavor 0.12% by weight to obtain a viral diarrhea preventive agent IP to be used as an infant drink.

Example 5 A viral diarrhea preventive agent used as feed for young livestock was prepared by mixing IKg of whole egg powder produced by chickens hyperimmunized with diarrhea virus with 100Kg of whole milk powder. Obtained.

〔Effect of the invention〕

The viral diarrhea preventive agent of the present invention contains a chicken egg antibody that specifically binds to the diarrhea virus and neutralizes its infectivity, which is prepared from the egg yolk of a chicken hyperimmunized with the diarrhea virus. Therefore, by orally giving the viral diarrhea preventive agent of the present invention to infants, the elderly with reduced autoimmunity, sick people, young livestock, etc., it is possible to prevent infection by diarrhea viruses. . In the present invention, chicken egg antibodies, which can be obtained in large quantities and with high titer through simple operations, are used as antibodies that specifically bind to diarrheal viruses. Therefore, the agent for preventing viral diarrhea of the present invention is inexpensive to produce and is very practical. Furthermore, since it utilizes a specific antibody obtained from the yolk of eggs that have been eaten since ancient times, the agent for preventing viral diarrhea of the present invention is extremely safe. With the viral diarrhea preventive agent of the present invention, passive immunity to prevent diarrheal virus infections has become practical for the first time, contributing to the eradication of viral diarrhea, which is currently a major problem worldwide. It is something.

Claims (1)

[Claims] (1) A prophylactic agent for viral diarrhea containing an egg antibody prepared from the egg yolk of a chicken hyperimmunized with a virus that causes viral diarrhea.