WO1989002278A1

WO1989002278A1 - Bivalent live vaccine for marek's disease and process for its preparation

Info

Publication number: WO1989002278A1
Application number: PCT/JP1987/000677
Authority: WO
Inventors: Yukio Sekiya
Original assignee: Ghen Corporation
Priority date: 1987-09-14
Filing date: 1987-09-14
Publication date: 1989-03-23
Also published as: JPS62212326A

Abstract

Bivalent live vaccine for prophylaxis of fowl Marek's disease and a process for its preparation are disclosed. The vaccine is stored in an ampule in a lyophilized state wherein both turkey herpes virus (HVT) FC-126 strain effective as a vaccine for fowl Marek's disease and Marek's disease virus (MDV) SB-1 strain are present. This vaccine can be easily used by merely diluting the contents of the ampule. This bivalent vaccine can be prepared by culturing respective viruses separately in two steps using fowl embryo cells while controlling initiation of the cultures so that the two cultures are completed at the same time, mixing the obtained two virus culture liquors with each other, pouring it into ampules, sealing and lyophilizing it.

Description

Akira Sho Marek's disease 2 fl & raw kuchin and how to make it

Technical field

The present invention relates to a live vaccine for preventing Maret's disease of nitriles and a method for producing the same. More specifically, two types of effective pectin viruses are contained in a single amble and can be easily used. One possible bivalent nitrile Marek's disease live vaccine and its production method are described below.

Technology

Marek's disease (MD) is a virally transmitted species of malignant limb disease that occurs in poultry, such as chicks, and is associated with nerve palsy, mainly in the lower limbs of young chicks, and convulsions. It is a troublesome illness for Yi Wei because of its symptoms and high mortality.

The virus that causes Marek's disease is a type of carcinogenic virus called Marek's disease virus (hereinafter abbreviated as NDV). It exists in the public and is persistently infected.

Various pectins have been proposed as preventive measures against Marek's disease.

The earliest commercial application was a vaccine prepared from a non-toxic turkey herpes virus called HVT. HVT is MDV MDV is a closely related, but not the only, antigen, and is effective in preventing Marek's disease when inoculated, so it was adopted as a vaccine instead of MDV, which is highly carcinogenic. In particular, a virus strain called HVT PC-126 has been used as a cell-associated (cell-associated) antagonist vaccine or a cell-free (cell-free) freeze-dried pectin. This HVT vaccine was very effective, and the financial loss due to Marek's disease was drastically reduced. However, since 1978, the emergence of highly virulent MDV, which demonstrates the immunity of the HVT vaccine, has become a problem as a Marek's disease vaccine break.

Various vaccines using attenuated MDV have been proposed so far. * For example, it has been proposed to use virus strains called C! J-2 and CV i988 as vaccines. Although they have been put to practical use, they have not been confirmed to have no carcinogenesis [low virulent MDV, serum type 1], and safety issues have not been resolved.

U.S. Pat.No. 4,160,024 describes a non-carcinogenic MDV called SB strain and its clone, HDy SB-1 strain, a method for producing a Marek's disease vaccine using these vaccines, and Marek's administration of this vaccine. A method for preventing disease is described.> A live male vaccine using the MDV SB and SB-1 strains as seed viruses is non-toxic and non-carcinogenic MDV. The conventional weakened Marek This vaccine is different from a live vaccine (for example, a Marek's disease live vaccine using CVI 988 strain), and can be used advantageously because it has no safety problems.

Under the above circumstances, at present, in the United States, as measures against the Marek's disease vaccine break, HVT vaccine using FC-126 strain as seed virus and MDV protein using SB-1 strain as seed virus have been developed. A bivalent vaccine that is used by dissolving it in one lysis solution immediately before use is used.

The details of the above MDV SB and SB-1 strains are described in the above-mentioned U.S. patent, and this U.S. patent also describes in detail various conventional MDVs such as HV TFC-126. Please refer to it,

Currently, in Japan, Marek's disease vaccine, which uses the above-mentioned HVT FC-126 strain as a seed virus, is mainly used as a vaccine for the prevention of Marek's disease. There is a demand for the development of bivalent peptides to which the vaccine of the present invention is further added. As mentioned above, a bivalent Marek's disease vaccine has already been developed and actually used in the United States, but it must be dissolved and mixed in one lysis solution immediately before use, as described above. It is a mixed bivalent lectin.

Mixing at the time of use in this way makes the operation complicated by that much, increases the chance of contamination by various bacteria, and forgets to mix. By inoculating only one virus strain or mixing the contents of two ambles of the same virus, inoculating only one virus strain may cause a difference in the scale, which is not preferable.

Therefore, it is highly desirable to produce a bivalent vaccine in which one of the above two virus strains coexists in one amble because vaccination against Marek's disease is simple and reliable.

However, in the past, such bivalent vaccines were not known at all because the problems in vaccine production for the rainy people may be involved. When a bivalent vaccine is produced, both species of virus are cultured, and the virus is collected and mixed at the time when the virus titer of each species is highest, but flDV is a cell-binding vaccine. Therefore, it is essential to collect both viruses at the same time and freeze them. One of the previously adapted viruses is ligated and stored, and the other virus is converted and mixed with this virus at a special feature where it is adapted, and the mixture is frozen and stored. The virus would freeze twice, causing a significant drop in the Dilka value, which is practically impossible. Therefore, as described above, simultaneous collection of both viruses is necessary.However, the culture time should be adjusted so that the virus can be collected at the same time when the respective virus has the highest value. It has been difficult or impossible to produce a bivalent Marek's disease vaccine that covers the two viruses described above in a single ampoule, or has not been successful it is conceivable that. For example, one of the reasons is that the cultivation period of both viruses was quite long, and the cultivation period was so different that it was troublesome to be able to collect cultured viruses at the same time.

Another reason, discloses that the objective method of measuring the content of each of the two Wirusu such as HVT and SB contained in the final product has not been established. _Ft invention is举down

The present inventor produced a seed virus by culturing a prototypic virus by using 筠 embryo cells (CBF), and also inoculating and cultivating this seed virus into ¾ embryo cells. As a result of various meter readings on the relationship between the amount of inoculated virus and the cultivation time, a production method that can collect the undiluted vaccine solution for HVT and SB-1 in a shorter period of time than before has been studied. Established. In addition, by using this method, it was found that the HVT FC-126 strain was almost completely cultivated in the half of the MDV SB-1 strain during the culture period. It was found that it was extremely easy to adjust the pH and that the above-mentioned divalent Marek's disease protein could be easily produced. As a result, the two types of virus are stored together in one sample. The first successful development of a Marek's disease bivalent vaccine.

Furthermore, in order to commercialize the above vaccine, it is necessary to measure the content of each virus in this bivalent protein and display its titer. Conventionally, HVT FC-126 and MVD SB -Virus content was determined based on the morphological differences of the 1 black. Although the content of HVT virus has little problem with this method due to the specificity of its black form, SB-1 is very difficult to discriminate when mixed with HVT, and Reliable and accurate determination of the content of each virus was not possible with the difference method,

The present inventors have developed a ΡΑΡ (peroxidase-anti-peroxidase) staining method using a type-specific monoclonal monoclonal antibody that specifically reacts with only one of HVT FC-126 virus and MDV SB-1 virus. Was used for the determination of the viral content of the bivalent pectin of the present invention, thereby finding a means for individually and surely measuring the amount of each virus in the bivalent vaccine. The reliability of this bivalent vaccine was secured, and commercialization was actually possible.

Here, on one side, the present invention comprises cryopreserving the HVT FC-126 strain and the MDV SB-1 strain, which are effective as a vaccine for Maret's disease of tutri, in a single ampoule. To provide live bivalent vaccines for Nitrimatic disease

According to another aspect, the present invention provides:

(a) HVT PC-126, effective as Maret's disease vaccine in Nitris A first-stage culturing step of separately culturing the virus of each strain of MDV SB-1 strain and each strain of MDV SB-1 using Wei germ cells to collect the seed virus of each virus strain,

(b) a second-stage culturing step in which each of the obtained seeds is separately cultured with embryonic cells to prepare an infected culture containing each culture virus as a stock solution for producing each vaccine; And

(c) mixing each stock solution containing the cultured virus of each of the HVT PC-126 strain and MDV SB-1 strain thus prepared, dispensing into 7 ampoules, sealing, and storing by freeze-drying;

And wherein the steps (a) and (b) are performed so that the second-stage culture of the HVT FC-126 strain and the MDV SB-1 strain is completed substantially simultaneously.する It also provides a method for producing a live bivalent vaccine for preventing Marek's disease in Tris.

The above culturing time, that is, the culturing time until the virus efficiency becomes the highest, is that the first stage culturing is performed for 40 to 50 hours for the HVT FC-126 strain, particularly 43 to 48 hours, for example, 45 hours, MDV For the SB-1 strain, it is preferably 85-100 hours, especially 90-94 hours, for example, 93 hours, and the second stage culture is 38-48 hours, especially for the HVT FC-126 strain. Preferably, the time is 40 to 45 hours, for example, 41 hours, and for the MDV SB-1 strain, 85 to 100 hours, particularly 90 to 94 hours, for example, 92 hours. The culturing time varies depending on the culturing conditions such as the inoculum volume, the concentration of the culture solution, and the type of medium. By adopting the conditions, such a culture time can be obtained. The culture conditions can be changed as appropriate to achieve an appropriate culture time.

A simple statement of the drawing

In the attached drawings,

FIG. 1 is a process diagram showing one example of a method for producing a divalent vaccine for Marek's disease of nitrile according to the present invention, and

The first is a photomicrograph showing the plaques of HVT and HDV SB-1 virus stained using a type-specific monoclonal antibody, and FIG. 2 (a) shows the specifically stained MDV SB-1 virus. Fig. 2 (b) shows the virus black, and Fig. 2 (b) shows the specifically stained HDV SB-1 virus when HVT and MDV SB-1 overlap. (c) shows the specific HVT virus plug stained. '

The form of prayer for applying the invention

Hereinafter, the present invention will be described in detail with reference to the embodiment shown in FIG. However, the steps in the attached drawings are not limited to one example of the method of the present invention, and it is needless to say that one kind of change is possible within the scope of the present invention. It is needless to say that it can be changed as appropriate because it is only an example.

In the method of the present invention, both the HVT "FC 126 strain and the MDV SB-1 strain 两 virus are cultured using ¾ embryo cells. Prepare chicken embryo cells from the above. Use SPP fertilized eggs as seed eggs and culture them at 37Ϊ for 10 days to grow embryos. In the present invention, Wei embryos collected from the eggs cultured for 10 days are used as primary cells for culture.筠 Embryo cells are diluted with MB (minimum essential medium) containing about 5% fetal bovine serum, and inoculated as a culture containing about 3 to 4 x 108 ⁸ embryo cells per 100 strokes. use.

The culture of the MDV SB-1 strain is carried out by inoculating the above-mentioned primary embryo cell culture solution with the virus of the MDV SB-1 strain progenitor. This inoculation is carried out, for example, by adding 1 to 1 of the original virus (about 4 to 8 × 10 ⁵ PFU of virus) to 100 ml of the above culture solution.

¾ Embryo cells inoculated with the virus of the MDV SB-1 strain were cultivated at 37 days for 85 to 100 hours, preferably 90 to 94 hours. The first stage culture was performed. Used as seed virus for MDV SB-1 strain. This virus is inoculated into another chicken embryo primary cell that has been adapted as described above. This inoculation can be performed under the same conditions as the above-mentioned seed virus, that is, for example, lml of seed virus (about 4 to 8 × 10 ⁵ PFU of virus) is cultured in the above-mentioned embryo cell culture. It is carried out by adding the mixture to 100 l> l of the solution (including about 3 to 4 cells). Seed virus is also cultured at 37'C for 85 to 100 hours, preferably 90 to 94 hours in the same manner as described above (second stage culture), and the obtained infected cell culture solution is used as a stock solution for vaccine production. . Therefore, the seed virus of the MDV SB-1 strain is obtained through a two-stage culturing process in which the seed virus is collected from the original virus by the first-stage cultivation according to the method of the present invention, and cultivated in the second stage to obtain a stock solution. The incubation period from inoculation to the collection of the undiluted solution is about 4 days for each step, a total of about 8 days in total. Conventionally, culture of the HDV SB-1 strain has already been performed in two stages before the seed virus collection in the first stage. Culture was required, and the entire culture period was about 13 days.However, the present inventor learned that under the above conditions, only two stages of culture from collection of the original virus to collection of the undiluted solution for vaccine production were required. The culture period was shortened.

As with the MDV SB-1 strain described above, the HVT FC-126 strain is also cultivated in a two-step cultivation process via a seed virus, and the culturing conditions may be the same. Progeny of HVT PC-126 strain The first stage culture from inoculation of the virus to collection of the seed virus is 40 to 50 hours, preferably 43 to 48 hours. The length of the two-stage column is 38-48 hours, preferably 40-45 hours. Therefore, the cultivation period 1 of the HVT FC-126 strain is about 2 days in each step, and about 4 days in total. The culture period of the HVT FC-126 strain has conventionally required about 7 days.

As a result, according to the method described above, the first stage culture of the MDV SB-1 strain is completed, and the first stage culture of the HVT FC-126 strain is started before and after inoculation and inoculation of the seed virus. The two-stage tower の of this rainy person was finished at the same time, and the cell culture solution of the rainy people was used for vaccine production. It can be collected as a stock solution at the same time, making it very easy to control the culture period.

MDV SB-1 and ffVT FC-126 stained cultured cell stock solutions are mixed at an appropriate ratio, for example, in a ratio of 1 stock solution of SB-1 to 1 stock solution of HVT, and a cryoprotectant (eg, dime Add tyl sulfoxide, DMS0) to make the final pulp. This is dispensed into ampoules according to a conventional method, sealed, sealed, and stored in a liquid nitrogen tank.

After the digestion, the bivalent vaccine of the present invention is prepared using an appropriate diluent (eg, a solution prepared by dissolving TPB (tributose 'phosphate' broth) in water and sterilizing the solution, with appropriate instructions). It is diluted and administered to cranes, for example, in a volume of 0.2 ml. In practice, each ampoule and lysing solution for dilution as described above will be provided in a set. Since it is desirable to have at least the SB-1 1000 PFU and the HVT FC-126 1000 PFU at the above-mentioned dose of 0.1 al, the above mixing operation is carried out so that each virus is possessed at such a ratio. Adjust the mixing ratio of the stock solution as necessary.

The amount of each virus in the 2 俩 vaccine produced by the above method can be measured as follows.

First, dissolve one vaccine ampule, dissolve in 200 ml of the above-mentioned dissolution solution, and dilute 50-fold with the same dissolution solution.Use this as an inoculum, add 0.2 ml / ml to CEF cultured in a petri dish. Inoculate in a Petri dish and incubate for 13 days at 37 ° C for 5-7 days. The target blacks were counted under a microscope and HVT virus virus was found.

Next, the amount of SB-1 virus was measured by the peroxidase anti-peroxidase (hereinafter referred to as PAP) method using an SB-1-specific monoclonal antibody as follows.

Forestry used

Culture solution: Eagle i! EM with 5% fetal calf serum

Antibodies: Type 2 and 3 monoclonal antibodies, and anti-mouse IgG antibodies

Staining reagents: ethanol, mouse PAP, DAB solution, cold PBS,

Measurement method

ω fixation: HVT virus content measurement is morphological as described above :: Petri dishes that have been prepared by the method are washed with cold PBS. • After drying, dry for 4 minutes with 4 to 5 ml of ethanol 2% formalin for 30 minutes. Fix,

(2) -Next antibody reaction: Type 2 monoclonal α-nal antibody (mouse ascites antibody) was diluted 300-fold with PBS, and 1,5,1 were dispensed into the above-mentioned jar, and 37 minutes for 60 minutes ( Or 4x overnight> After washing, wash with cold PBS 3-4 times,

(3) Secondary antibody reaction: Commercially available hidge anti-mouse IgG serum was diluted 30-fold with 1% BSA-added PBS, and 1.5 1.5I was dispensed into the above-mentioned petri dish, followed by simple reaction at 37 ° C for 60 minutes. ♦ Wash as above

(4) ΡΑΡ Reaction: Commercially available mouse により was treated with PBS containing 1% BSA. Dilute 300-fold, dispense 1.5 ml of the mixture into the Petri dish, react at 37 * C for 60 minutes, and wash as above.

(5) DAB reaction: 5 ml of DAB solution (0.05 M Tris buffer containing 0.1% 3,3'-diaminopentidine ♦ 4-basic salt and 0.01% hydrogen peroxide, pH 7 * 6) Is dispensed into the above-mentioned petri dish, reacted at 37'C for 10 minutes, and washed twice with cold PBS.

(6) Inspection: Inspect the above petri dish with an inverted microscope on a cold PBS. Since only the MDV SB-1 virus is specifically stained by the above-mentioned staining method, counting the number of stained blacks allows the amount of MM SB-1 virus to be determined in consideration of the dilution factor. Can be issued. The virus content is preferably determined by averaging the results obtained using a plurality of petri dishes. FIG. 2 (a) is a microscopic photograph of the black SB-1 stained in this manner.

In the above primary antibody reaction, the HVT FC-126 virus in the bivalent vaccine is specifically stained by performing the above method using a type 3 monoclonal antibody, if necessary, and its content. Can also be measured. Fig. 2 (c) is a rifle photograph of the HVT FC-126 black stained in this way. Even if both virus plaques overlap, the above method was used. As a result, only one virus was specifically stained, indicating that the measurement was highly reliable. In Fig. 2 (b), the black of SB-1 and FC-126 overlapped, but the type 2 monoclonal resistance This is a photomicrograph of a black sample of SB-1 in which only SB-1 was specifically stained due to the use of the body. It was confirmed by reference to the results of the known virus measurement method,

Industrial applicability

The bivalent vaccine of the present invention thus produced is an unprecedented kind provided for the first time by the present invention, in which one sample contains both the HVT FG-126 strain and the flDV SB-1 strain. Is a Marek's disease bivalent live vaccine. This divalent pectin can be used for vaccination against poultry marek's disease simply by dissolving it as is and dissolving it in a lysis solution. Traditionally, a crane farmer would prepare ampoules of each of these vaccines and inoculate the contents of a single digestion solution (200 ml) to inoculate both the MMSB-1 strain and the HVT FG-126 strain.・ 1) The mixture was mixed and dissolved, and 0.2 ml was inoculated per bird, but the bivalent vaccine of the present invention was dissolved in the attached lysis solution (200 ml) without any mixing operation. Similarly, since it can be used with 0.2 dl inoculation per bird, it is easy to use by farmers, and there is no mistake in use, and the effect in actual use is great.

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Claims

Scope of IS CC request

(1) Coexistence of the seven-headed crow herpesvirus (flVT) FC-126 strain and the Marek's disease virus (MDV) SB-1 strain, which are effective as a vaccine for Marek's disease in tutus, in i ampoules A bivalent actin for preventing Maret's disease in a tree, which is preserved after storage.

(2) (a) The HVT FC-126 strain and MDV SB-1 prototypic virus, which are effective as a vaccine against Marek's disease in Nitris, were separately cultured in Wei germ cells, and each of the virus strains was cultured. The first stage of the culture process,

(b) a second stage culturing step of separately cultivating each of the obtained seed viruses with crane germ cells and netting a stained cell culture containing each culture virus as a stock solution for each vaccine production; And

(c) mixing the stock solutions each containing the HVT PC-126 strain and MDV SB-1 strain-cultured virus prepared in this way, dispensing the sample into a sample,

And wherein the step (a) and the step (b) are performed so that the second-stage culture of the HVT FC-126 strain and the MDV SB-1 strain is substantially simultaneously completed. A method for producing a live bivalent vaccine for preventing Marek's disease in cotton.

(3) The culture time of the first stage culture is 40 to 50 hours for the HVT FC-126 strain, and 85 to 100 hours for the MDV SB-1 strain. The culture time of the second stage culture is 38 to 48 hours for the HVT PC-126 strain and 85 to 100 hours for the MDV SB-1 strain. Method,

(4) The culture time of the first stage culture is 43 to 48 hours for the HVT PC-126 strain, 90 to 94 hours for the MDV SB-1 strain, and the culture time of the second stage culture is The method according to claim 2, wherein the HVT FC-126 strain is 40-45 hours, and the MDV SB-1 strain is 90-94 hours.

(5) Disassemble the live bivalent vaccine described in claim 1 and dilute it with an appropriate lysis solution, and then add 1,000 PFU of MDV SB-1 and HVT FC-126 virus per bird. A method for preventing Marek's disease in chickens, which comprises administering the drug to a tutri at a prescribed dose.

(6) The method according to claim 5, wherein the administration is performed for ¾ at the age of one day after birth.

(7) Clarify the bivalent live vaccine described in claim 2 of the claim and appropriate: After dilution with a suitable lysis solution, the virus of MDV SB-1 and HVT PC-126 with 1000 PFU or more per bird each. A method for preventing Marek's disease consisting of administering chickens at doses that provide

(8) The method according to claim 7, wherein the administration is performed to a 1-day-old mouse.