JPS62212326A - Bivalent live vaccine for marek's disease and preparation thereof - Google Patents

Abstract

PURPOSE:To obtain the titled easily usable vaccine having high safety, by freezing Marek's disease vaccine SB-1 strain together with turkey herpes virus FC-126 strain effective as a chicken Marek's disease vaccine. CONSTITUTION:Stock strains of Marek's disease vaccine (MDV) SB-1 and turkey herpes virus (HVT) FC-126 (effective as chicken Marek's disease vaccine) are separately cultured in chicken embryo cells to obtain seed virus of each virus strain. The seed viruses are separately cultured in chicken embryo cells and the infected culture cell liquid containing each virus is prepared as a stock liquid for the preparation of vaccine. The stock liquid is pipetted and sealed in an ampule and stored in frozen state to obtain the objective bivalent live vaccine of chicken Marek's disease. It can be used simply by thawing in the ample and dissolving in a dissolution solvent in vaccination. Consequently, the vaccine is convenient to use preventing mis-handling.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to a live vaccine for preventing Marek's disease in chickens and a method for producing the same. It contains
The present invention relates to a bivalent chicken Marek's disease live vaccine that can be easily used and a method for producing the same.

(Prior Art) Marek's disease (M) is a type of virally transmitted malignant lymphomatosis that occurs in poultry such as chickens.
It is a troubling disease for the poultry industry because it causes symptoms such as nerve paralysis and convulsions, mainly in the lower limbs of young chicks, and has a high mortality rate.

The causative virus of Marek's disease is 141, a carcinogenic herpes virus called Marek's disease virus (hereinafter abbreviated as MDV), which is commonly present in the bodies of chickens and is persistently infected.

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

The earliest product to be put into practical use was cotton prepared from a non-toxic turkey herpesvirus called IIVT.

Although IIVT is not MDV itself, it is a virus that is closely related to MDV as an antigen, and vaccination with this virus is effective in preventing Marek's disease, so it was adopted as a vaccine instead of MDV, which is highly carcinogenic. be.

In particular, a virus strain called IIVT FC-126
It is used as a cell-associated frozen vaccine or a cell-free (CO11-free) freeze-dried vaccine. This +1VT
The vaccine is highly effective and the economic losses from Marek's disease have been drastically reduced. However, since 1978, highly pathogenic MDV that overcomes the immunity of HVT vaccine has been introduced.
has appeared and is causing some problems as a vaccine break for Marek's disease.

Various vaccines using attenuated MDV have been proposed so far, such as C11-2 and C11-2.
The use of a virus strain called VI 98B as a vaccine has been proposed and put into practical use, but it has not been confirmed that they have no carcinogenicity [low toxicity].
low virulent) MDV, serotype 1]
, the safety Φ problem has not been resolved.

U.S. Patent No. 4,160.024 describes a non-oncogenic MDV called strain 38 and its clone MDV.
The VSB-1 strain and a method for producing a Marek's disease vaccine using these are described. The Marek's disease live vaccine, which uses the MDV 58 strain and the SB-1 strain as seed viruses, is non-toxic and non-carcinogenic IIDV, so it is used as a seed virus without being attenuated, and it is different from the conventional attenuated Marek's virus. It is different from a live disease vaccine (for example, Marek's live vaccine using the CVI988 strain) in this respect, and can be advantageously used since there is no safety problem.

Due to the above situation, currently in the United States, the IIVT vaccine, which uses the PC-126 strain as a seed virus, and the MDV vaccine, which uses the SB-1 strain as a seed virus, are being used immediately before use to prevent Marek's disease vaccine break. Bivalent vaccines, which are dissolved in a single dissolution solution, are used.

The details of MDV 58 strain and 511-1 strain are explained in the above-mentioned U.S. patent, and this U.S. patent also describes in detail various conventional MDV strains such as IIVT FC-126 strain. So,
Please refer.

(Problems to be solved by the invention) Currently, in Japan, the Marek's disease vaccine using the above-mentioned HVT PC-126 strain as a seed virus is mainly used as a vaccine for preventing Marek's disease. As a countermeasure against vaccine breakout, it is desired to develop a bivalent vaccine in which a different type of vaccine is further added. As mentioned above, a bivalent Malefuku disease vaccine has already been developed and actually applied in the United States, but as mentioned above, it is necessary to dissolve and mix in one dissolution solution immediately before use. It is a combination type bivalent vaccine.

Mixing at the time of use in this way not only makes the operation more complicated, but also increases the chance of contamination with bacteria, forgetting to mix and inoculating only one virus, or inoculating two ampoules of the same virus. By mixing the contents of the two strains, there is a possibility that a mistake may be made such as inoculating only one virus strain or the other virus strain, which is not preferable.

Therefore, in view of this problem, if a bivalent vaccine in which the two types of virus strains described above coexist in one ampoule was manufactured, vaccination against Marek's disease would be easier and more reliable.
Highly desirable.

However, until now, such bivalent vaccines were completely unknown. The reason for this is thought to be related to problems in the manufacturing process of both vaccines. In other words, when producing such a -bivalent vaccine, both seed viruses are cultured, and the viruses are collected and mixed when each virus titer is at its highest. Since IDV is a cell-binding vaccine, cryopreservation is essential, and for this purpose, it is essential that both viruses be collected at the same time and cryopreserved. However, it is difficult to adjust the culture timing so that the viruses can be collected at the same time when the titer of each virus is at its highest. It is believed that no attempt has been made to produce a Marek's disease vaccine of this value, or that attempts have been unsuccessful. for example,
One of the reasons seems to be that the culture period for both viruses was quite long, and there was a large discrepancy between the culture periods, making it troublesome to collect cultured viruses at the same time.

(Means for Solving the Problems) The present inventor has developed a method of preparing a seed virus by culturing an original virus in chicken embryo cells, and inoculating and culturing this seed virus into chicken embryo cells. We conducted various studies on the relationship between the amount of inoculated virus and the culture time, and established a manufacturing method for each of the single vaccines, HVT and SB-1, that allows the collection of stock solutions for virus production in a shorter period of time than in the past. Moreover, with this method, the IIVT FC-126 strain is almost MDV 5B.
- By discovering that a single strain can proliferate well in half the culture period, it becomes extremely easy to adjust the culture start time so that cultured viruses can be collected at the same time.
I learned that it is easy to produce a vaccine for Marek's disease with a similar amount.

As a result, we succeeded for the first time in developing a bivalent vaccine for Marek's disease, which is made by freezing and preserving the two types of viruses mentioned above together in one ampoule, and completed the present invention.

Here, the present invention provides a bivalent chicken Marek's disease vaccine in which HVT FC-126 strain and MDV 5B-1 strain, which are effective as a chicken Marek's disease vaccine, are coexisting and cryopreserved in one ampoule. It is a live vaccine.

According to another aspect, the present invention provides: (a) +1 effective as a chicken Marek's disease vaccine;
A first stage culture step in which each of the original viruses of the VT FC-126 strain and the MDV SB-1 strain is cultured separately in chicken embryo cells and the seed viruses of each virus strain are collected (
A second culture step in which each of the obtained seed viruses is separately cultured in chicken embryo cells and an infected cultured cell solution containing each cultured virus is prepared as a stock solution for producing each vaccine; IIVT PC-1
26 strain and MDV 5B-1 strain cultured viruses are mixed, dispensed into ampoules, sealed, and cryopreserved;
The second strain of IVTPC-126 and MDV 5B-1
There is also provided a method for producing a live bivalent vaccine for preventing Marek's disease in chickens, which is characterized in that the stage cultures are completed substantially simultaneously.

The culture time shown in Σ, that is, the culture time until the virus titer reaches the highest level, is that the first stage culture is HVTFC-126.
For strains, 40-50 hours, especially 43-48 hours, M
85 to 100 hours for DV SB-1 strain, especially 9
The second stage culture is preferably 0 to 94 hours.
For TFC-126 strain, 38 to 48 hours, especially 4
0-45 hours, 85-1 for MDV 5B-1 strain
00 hours, especially 90 to 94 hours. Although the culture time varies depending on the culture conditions such as the amount of inoculation, the concentration of the culture solution, and the type of medium, such a culture time can be achieved, for example, by adopting the conditions described below. Culture conditions can be changed as appropriate to provide an appropriate culture time.

(Operation) Hereinafter, the present invention will be described in detail based on embodiments shown in the accompanying drawings. However, the process diagrams in the accompanying drawings are merely one specific example of the method of the present invention, and it goes without saying that various changes can be made within the scope of the present invention.

In the method of the present invention, the IIVT FC-126 strain and MDV
Since both viruses of the 5B-1 strain are cultured using chicken embryo cells, chicken embryo cells are first prepared for culture. S
A PF fertilized egg is used as a spawning egg, and it is cultured at 37° C. for 10 days to grow an embryo. In the present invention, a chicken embryo collected from the egg cultured for 10 days is used as a primary cell for culture. Chicken embryo cells are diluted in MEM (minimum essential medium) containing about 5% fetal bovine serum and used for inoculation as a culture containing about 3-4 XIO'' chicken embryo cells per 100 ml.

The M[lV 5B-1 strain is cultured by first inoculating the original virus of the MDV 5B-1 strain into the chicken embryo primary cell culture medium. This inoculation is carried out, for example, by inoculating 100 ml of the above culture solution with [1 original virus (approximately 4 to 8
containing a viral load of PFU).

The chicken embryo cells inoculated with MDV 5B-1 original virus are cultured at 37°C for 85 to 100 hours, preferably 90 to 94 hours (first stage culture), and the resulting infected culture cell suspension is incubated with MDV 5B-1. Use as a seed virus for stocks. This type of virus is inoculated into another chicken embryo primary cell prepared as described above. This inoculation may be carried out under the same conditions as for the original virus described above, i.e., 1 ml of seeds,
virus (containing approximately 4-8 x 10 SPFu virus load)
100w1 of the above chicken embryo cell culture medium (approximately 3 to 4
Similarly to the above, culturing of type 0 virus by adding IO" cells to
The culture is carried out for 0 hours, preferably 90 to 94 hours (second stage culture), and the obtained infected cultured cell fluid is used as a stock solution for vaccine production.

Therefore, by the method of the present invention, the first virus can be extracted from the original virus.
By going through a two-step culture process in which a seed virus is collected by stage culture and then cultured in a second stage to obtain a stock solution, the culture period from inoculation of the stock virus of MDV SB-1 strain to collection of the stock solution is , each stage takes about 4 days, for a total of about 8 days. Conventionally, the cultivation of the MDVSa- strain required two stages of cultivation before the first stage of seed virus collection, and the total cultivation period was approximately 13 days. After learning that only two stages of culturing were required from the original virus to collecting the stock solution for vaccine production, they sought to shorten the culturing period.

+1VT PC-126 strain Nitsui, but the above-mentioned MDV
As with the SB-1 strain, it is cultured in a two-step culture process using a seed virus, and the culture conditions are the same, however,
The culture period was as follows: The first stage of culture from inoculation of the original virus of the IIVT FC-126 strain to collection of the seed virus was 40 minutes.
-50 hours, preferably 43-48 hours, and the second stage culture from seed virus inoculation to collection of vaccine stock solution is 38-48 hours, preferably 40-45 hours.

Therefore, the culture period for the IIVT FC-126 strain is approximately 2 days at each stage, for a total of approximately 4 days. IIVT P
Conventionally, the culture period for C-126 strain has been required to be about 7 days.

As a result, according to the method described above, if the first stage culture of the MDV SB-1 strain is completed and the first stage culture of the IIVT FC-126 strain is started before or after collecting and inoculating the seed virus, the These two stages of culture are completed at the same time, and the infected cultured cell fluids of both can be collected at the same time as stock solutions for vaccine production, making it very easy to control the culture period.

Mix stock solutions of infected cultured cells of MDV SB-1 strain and IIVT FC-126 strain in an appropriate ratio, for example, a volume of 1 SB-1 stock solution to 1 HVT stock solution, and add a cryoprotectant to prepare the final bulk. shall be. This is dispensed into ampoules by a conventional method, melt-sealed, frozen, and stored frozen in a liquid nitrogen tank.

(Effect of the invention) The bivalent vaccine of the present invention produced in this way has one (7
) 77bu/L, IIVT FC-126 strain and MDV
This is an unprecedented type of live bivalent Marek's disease vaccine provided by the present invention for the first time, containing both the 5B-1 strain and the 5B-1 strain.

This bivalent vaccine can be used to vaccinate poultry against Marek's disease by simply thawing it and dissolving it in a dissolution solution. Traditionally, poultry farmers use MDV so- as stocks and HVT F
To inoculate both strains of C-126, prepare ampoules of each of these vaccines, dissolve the contents in one bottle of dissolution solution (200 ml), mix, and add 0.0 ml of each vaccine.
However, the bivalent vaccine of the present invention does not require any mixing operation and can be directly added to the attached dissolution solution (200ml).
Since it can be used in the same way by dissolving it in 1) and inoculating 0.2 ml per batch, it is easy to use by poultry farmers, and there are no errors in use, so it is highly effective in actual use.

[Brief explanation of drawings]

The accompanying drawings are process diagrams showing one example of the method for producing a bivalent chicken Marek's disease vaccine according to the present invention.

Claims (4)

[Claims] (1) One ampoule contains turkey herpesvirus (HVT) F, which is effective as a chicken Marek's disease vaccine.
C-126 strain and Marek's disease virus (MDV) SB-1
A bivalent live vaccine for the prevention of Marek's disease in chickens, made by coexisting with the strain and cryopreserved. (2) (a) The original viruses of HVT FC-126 strain and HDV SB-1 strain, which are effective as chicken Marek's disease vaccines, are cultured separately in chicken embryo cells, and the seed virus of each virus strain is collected. 1-stage culture step, (b) 2nd-stage culture step in which each of the obtained seed viruses is separately cultured in chicken embryo cells to prepare an infected cultured cell solution containing each cultured virus as a stock solution for each vaccine production. , and (c) each HVT FC-12 thus prepared.
6 strains and MDV SB-1 strain cultured viruses are mixed, dispensed into ampoules, sealed, and cryopreserved, and the steps (a) and (b) are
A method for producing a bivalent live vaccine for preventing Marek's disease in chickens, characterized in that the second stage cultivation of VTFC-126 strain and MDV SB-1 strain is completed substantially simultaneously. (3) The culture time of the first stage culture is HVT FC-1
For the HVT FC-126 strain, the culture time was 40 to 50 hours, for the MDV SB-1 strain, it was 85 to 100 hours, and for the HVT FC-126 strain, the culture time was 38 to 100 hours.
48 hours, 85-100 for MDV SB-1 strain
3. The method of claim 2, wherein the time is: (4) The culture time of the first stage culture is HVT FC-1
The culture time for the second stage culture was 43 to 48 hours for the 26 strain, 90 to 94 hours for the MDV SB-1 strain, and 40 to 45 hours for the HVT FC-126 strain.
3. The method of claim 2, wherein the time is 90 to 94 hours for MDV SB-1 strain.