JPH0267227A - Vaccine for infectious swine or bovine gastroenteritis - Google Patents

Abstract

PURPOSE:To provide the subject vaccine produced by treating an inactivated or attenuated virus strain with a surfactant and capable of accomplishing effective milk immunization of newly born pigling or calf by inoculating to a pregnant animal before delivery. CONSTITUTION:Inactivated or attenuated virus of infectious swine or bovine gastroenteritis is treated with a surfactant, preferably a nonionic surfactant (e.g. polyoxyethylene sorbitan fatty acid ester surfactant) and the treated virus is used as the objective vaccine as it is or preferably as a mixture with an adjuvant. The concentration of the surfactant in the liquid in the above treatment is preferably >=0.01%, especially 0.5-5%. A neutral antibody against the virus is induced in the milk of the mother swine after delivery by the immunization of the pregnant swine with the vaccine before delivery. The lowering of the neutral antibody titer in the transfer from the colostrum to the normal milk is slow and the mild antibody value is maintained at a level comparable or superior to the level attained by the inoculation of strongly toxic strain. Effective milk immunization can be established by this process.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an infectious gastroenteritis vaccine for pigs and cattle and a method for producing the same.

[Prior art] Porcine infectious gastroenteritis has been reported by Doyle and Hutchings (
It is an infectious disease first reported by (1946) et al.
It has been proven that this is caused by the coronavirus. Symptoms of this disease include vomiting, watery diarrhea, and dehydration, and it infects all pigs regardless of age. Older pigs suffer the greatest economic damage. Subsequent research has revealed that rotavirus can also cause infectious gastroenteritis in pigs, which exhibits similar symptoms, although the symptoms are milder. Also, regarding cows,
Infectious gastroenteritis caused by coronavirus or rotavirus is also known, and the symptoms are similar to those in pigs.

In order to simplify the explanation, the following explanation will be based on porcine transmissible gastroenteritis (TGE) caused by coronavirus. In addition, in the following explanation, the coronavirus derived from pigs is referred to as TG.
It is abbreviated as E-virus.

The occurrence of TGE is widely distributed in the Northern Hemisphere, excluding Alaska and Ireland, especially in temperate to cold regions north of 30 degrees north latitude. There is a close relationship between the distribution of TGE occurrence and temperature, and outbreaks have also been reported in subtropical Taiwan and tropical Malaysia.

Since TGE is a disease with a high mortality rate in young suckling pigs, it is most important to rescue pigs during this period (generally about one week after birth). For this reason, attempts have been made to protect newborn pigs from TOH infection by immunizing pregnant pigs with TGE vaccine before farrowing. By drinking the milk of an immunized sow, suckling pigs are passively immunized by oral inoculation of neutralizing antibodies to the TGE virus contained in the milk. Therefore, even if a field virus invades the intestines of suckling pigs, it is neutralized by the antibodies that remain in the intestines before it can multiply, thus avoiding death from the disease. This mode of immunity is called milk immunity, and research on TGE vaccines has been based on the idea of milk immunity.

Table 1 shows the results of the TGE vaccine protection test on suckling pigs using the milk immunization method conducted at the Ministry of Agriculture, Forestry and Fisheries Livestock Hygiene Test Station.
The challenge was 100 PID per pig on 3 days old.
se (Pig In4active Doses')
This was done by oral inoculation with a highly virulent virus. The antibody titer at the time of challenge is the antibody titer in the mother pig's serum and milk. The results can be summarized as follows.

(1) When formalin-inactivated virus or live attenuated virus is inoculated intramuscularly into pregnant pigs, sufficient neutralizing antibody titer cannot be induced in the milk;
Nearly 00% of pigs die from the disease.

(2) When a live attenuated virus is inoculated into the udder of a pregnant pig, the neutralizing antibody titer in the milk is slightly higher than in (1), and the overall mortality rate after challenge with the highly virulent virus is also slightly lower.

(3) When pregnant pigs are intramuscularly injected with a live attenuated virus and then intranasally inoculated with the same virus, the overall mortality rate after challenge is slightly lower than in (2). In particular, infectious titer 109.3re
tos. Immunization with a live virus of 1.0% results in the highest milk antibody titer at the time of challenge, resulting in a nearly complete protective effect.

Table 1 also lists the results of a milk immunity test conducted by the present inventor using a commercially available oral live vaccine manufactured in the United States. 100 PIDs as shown in Table 1. When pigs were challenged with a highly virulent virus (small intestine emulsion) at 3 days of age, all pigs developed disease and died, and the milk antibody titer at the time of challenge was 32 times lower.

Based on these test results, the most promising vaccine is based on method (3). However, the major problem with this method is that it requires producing a vaccine containing a highly concentrated live attenuated virus, which has a very high infectious titer of 10"5 TCIIlso/l, and storing it in a freeze-dried state for a long period of time. This is difficult in practice, which is why this type of vaccine has not actually been commercialized.

It is generally accepted that the rapid decrease in milk antibody titer after delivery is strongly dependent on the antibody isotype (IgA, IgG). That is, the antibody titer of IgA decreases much more slowly than that of IgG.

For example, when a sow is infected with a field virus during pregnancy, the neutralizing antibody secreted in the colostrum is IgA, and it is known that the antibody titer lasts for more than two weeks with almost no decline after parturition. . Even if piglets born from such mother pigs are attacked with a highly virulent virus, they will not die from the disease.

In contrast, neutralizing antibodies in colostrum induced by intramuscular injection of the inactivated virus or live attenuated virus in Table 1 (1) above, or intramammary inoculation of the live attenuated virus in Table 1 (2) are IgG. However, since the antibody titer rapidly decreases when transferred to conventional milk, it is thought that it is not possible to prevent all disease and death after challenge. 10 shown at the end of Table 1 (3)
9.'tctos. In the example of intramuscular inoculation and then intranasal inoculation of a highly concentrated live attenuated virus, the antibody isotype is overwhelmingly IgG, but IgA is also present. The change in colostrum was 10.228 times, 3 days later (at the time of challenge) 512 times, 98
Although the decrease in antibody titer was rapid, the antibody titer was 40 times higher than the
This is slower than in cases 1) and (2), and not all piglets died due to the attack with the highly virulent strain.

Thus, it has been shown that when the antibody isotype in milk is IgA, more effective milk immunity can be obtained than with IgG. One of the reasons for this is that IgA secreted into milk has greater resistance to proteolysis by digestive enzymes in the gastrointestinal tract than IgG, and because it adheres to the intestinal mucosa, the decline in antibody titer is extremely slow. (It is generally believed that it acts more effectively in the defense against intestinal infections than IgG.

However, as a result of investigating the resistance of IgM, IgA, and IgG isolated and fractionated from the colostrum of TGE-immunized sows to proteolytic enzymes detected in the gastrointestinal tract, Stone et al. It is reported that no significant difference was observed between isotypes. Furthermore, it was reported that when neonatal pigs were orally administered antibodies of each isotype and then challenged with a highly virulent strain of TGE virus, protection against infection was established in all cases, and differences in isotype were not recognized as differences in protection against infection. ing.

On the other hand, it is also known that suckling piglets have weak proteolytic enzyme activity.

From the above, if the antibody isotype of milk is IgA, the milk antibody titer will last and it will be advantageous, but in order to establish effective milk immunity, regardless of the type of antibody isotype detected in milk, it is necessary to neutralize the antibody. It is considered essential that the antibody titer does not drop rapidly during the lactation period and remains above a certain level.

The following three types of milk immunization methods are adopted for TGE vaccines currently used in Japan and abroad.

(1) A live attenuated vaccine is intramuscularly injected twice into the sow before farrowing (Europe, America, Japan).

(2) Inactivated vaccine is injected intramuscularly once before delivery, followed by intranasal inoculation of live vaccine (Japan).

(3) A live attenuated vaccine is administered orally twice before delivery, followed by one intramuscular injection (US).

However, when immunized with these vaccines, most of the pigs become ill and die when challenged at 3 days of age with a highly virulent virus at 100 PIDso per pig, similar to the above test. Since the amount of infectious virus is small in the field, some degree of vaccine efficacy can be expected, but it is clear that it is not satisfactory as a vaccine.

On the other hand, as shown in the examples below, when pregnant pigs are inoculated with a highly virulent strain of the virus, neutralizing antibodies mainly composed of IgA are induced in the milk, as in the case of natural infection with the above-mentioned field virus. and the antibody titer persists.

[Problems to be Solved by the Invention] As described above, conventional commercially available vaccines for milk immunization are not sufficiently effective because they cannot induce sufficient antibody titers in milk. This is because the neutralizing antibody titer in milk decreases rapidly as it transitions from colostrum to regular milk.
It is clear that the cause is that the intestine does not contain enough antibodies to neutralize the invading virus.

In addition, vaccines that can produce neutralizing antibody titers equivalent to or higher than those in the case of inoculation with the virulent strain described above in milk and that can persist even after one week have not only been commercially available but also have been shown in academic literature. Never been.

The purpose of the present invention is to maintain the neutralizing antibody titer in milk without a sudden drop during the first week of life when newborn pigs or cows are most susceptible to disease and death, and to prevent disease and death even when attacked by a highly virulent virus. The aim is to develop a vaccine for infectious swine or bovine gastroenteritis that is far more effective than conventional commercially available vaccines and can almost completely prevent gastroenteritis. In particular, to provide a swine or bovine infectious gastroenteritis vaccine that exhibits a characteristic that cannot be obtained with conventionally known vaccines, such as being able to maintain milk neutralizing antibody titer to the same extent or higher than when inoculated with a virulent strain. It is.

[Means for Solving the Problems] As a result of repeated studies to achieve the above object, the inventors have found that:
Immunization with an inactivated or attenuated TGE virus treated with a surfactant maintains the antibody titer at least as long as immunization with a highly virulent virus, and the isotype of the induced antibodies is mainly IgG; I learned that it also contains some IgA. When we actually immunized sows with this surfactant-treated virus, we were able to reliably achieve effective milk immunity in suckling pigs during the neonatal period, resulting in a TGE vaccine that is more effective than conventional commercially available vaccines. The present invention was completed based on the discovery that

Here, the gist of the present invention is a method for producing a swine or bovine infectious gastroenteritis vaccine, which is characterized by treating an inactivated or attenuated swine or bovine infectious gastroenteritis virus strain with a surfactant; The vaccine obtained using this method.

[Effect] The present invention will be explained in detail below.

The virus used in the production of the vaccine of the present invention is
It is a strain of swine or bovine infectious gastroenteritis virus. Specifically, it is a swine-derived coronavirus or rotavirus strain that causes porcine infectious gastroenteritis, or a bovine-derived coronavirus or rotavirus strain that causes bovine infectious gastroenteritis. These virus strains may be either inactivated viruses or live, attenuated viruses, but as shown in Examples below, even inactivated viruses exhibit sufficient immunological effects.

The production of the vaccine according to the present invention is carried out in the same manner as conventional vaccine production methods, except for the surfactant treatment, which is a feature of the present invention. - In the Funatsuri production method, the virus strain to be used is first cultured, and then, when inactivation is performed, the live virus is inactivated by a conventional method. Inactivation is usually performed by formalin treatment. For example, the virus is separated from the culture medium by an appropriate means such as centrifugation, and then the concentration is adjusted by dissolving the virus in an appropriate buffer to a predetermined concentration. Inactivation can be performed by preparing an antigen solution and adding formalin to this solution. The preferred virus concentration is from about 10".O to about 1O before inactivation.
It is about the same level as ``TCIDs*.

The antigen solution containing the inactivated or live attenuated virus is then treated with a surfactant. Various types of surfactants are effective, and the type is not particularly limited, but since it is used in the production of vaccines for animals used as meat, it is recommended to use surfactants that are approved as pharmaceutical materials. Among these, nonionic surfactants are particularly preferred. An example of a preferred surfactant is a polyoxyethylene sorbitan fatty acid ester type nonionic surfactant, for example, commercially available from IC1 under the registered trademark 1%4een. There is something that has been done. Tween in it
80, Tween 20, etc. are surfactants approved as pharmaceutical materials. Examples of other nonionic surfactants that can be used include polyoxyethylene alkyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene castor oils, and the like.

Surfactant treatment is performed by adding a surfactant to an antigen solution containing an inactivated or attenuated virus. The amount of surfactant added is not particularly limited, and it is preferable to determine an appropriate amount by experiment depending on the type of virus and surfactant used. When using Tween 80, the amount of surfactant added is an amount such that the surfactant concentration in the liquid is 0.01% or more, preferably 0.05% or more, particularly preferably 0.5% or more. The upper limit of the surfactant concentration is not particularly limited, but is generally 5% or less,
Preferably it is 3% or less.

The virus treated with a surfactant in this way can be used as a vaccine as is, but as is well known in the vaccine technical field, it is preferable to add an appropriate adjuvant (immune enhancer) to enhance the immune effect. Any known adjuvant can be used, but when the vaccine of the present invention is administered by injection, an adjuvant that is negative for tissue reactions at the injection site (that is, does not cause tissue reactions) is used. Examples of such adjuvants preferably include:
Carboball, aluminum gel, etc. The amount of adjuvant added may be the same as conventional ones.

The liquid vaccine produced in this manner can be dispensed into appropriate containers, manufactured into a product, and used.

When the vaccine of the present invention is used to immunize pregnant pigs before farrowing, virus-neutralizing antibodies can be induced in the milk of the sow after farrowing. This new and remarkable effect, which has not been observed with conventionally known vaccines, is achieved, in that the titer decreases slowly and the milk antibody titer lasts at the same level or longer than in cases inoculated with a virulent strain.

The isotype of antibodies induced in milk by the vaccine of the invention is predominantly IgG, with some IgA included. The reason why the antibody titer persists even though the antibody titer is mainly IgG is that, contrary to conventional knowledge, the IgG antibody titer in milk induced by the vaccine of the present invention does not rapidly decrease. It turns out that this is due to the characteristics shown. The reason why such a remarkable difference in the persistence of antibody titer and the properties of IgG occurs due to surfactant treatment is currently not clear, but it is possible that treatment with surfactant causes some changes in the three-dimensional structure of the virus surface. It is speculated that this may be related to the occurrence of

As shown in the Examples below, with the vaccine of the present invention, piglets are effectively passively immunized by suckling antibody-containing milk from mother pigs immunized with this vaccine, and are able to immunize against the virus contained in the vaccine. The onset of infectious gastroenteritis caused by the same virus can be completely prevented. In other words, effective milk immunity is established.

When the vaccine of the present invention is used for milk immunization, the vaccine is administered at an appropriate time before delivery, followed by one or more boosters. One time is enough. Inoculation is preferably performed by intramuscular injection, although other routes may also be used. For pigs, the first immunization is given by intramuscular injection, about 4 weeks before parturition, and one booster is given about 2 weeks before parturition.For cattle, vaccination is generally given earlier than for pigs. For example, the first immunization period is 15 to 5 weeks (eg, 10 weeks) before delivery, and the booster immunization is 4 to 1 week (eg, 2 weeks before delivery) before delivery.

The amount of inoculation varies depending on the type of virus strain used, the weight of the individual, and the route of inoculation, and can be appropriately determined through experiments.

By using the above-mentioned milk immunization method, the vaccine of the present invention can effectively prevent infectious gastroenteritis in pigs or cattle within one week after birth, which has a high mortality rate, and has a remarkable effect. .

The vaccine of the present invention can also be a bivalent vaccine. In other words, by mixing a vaccine manufactured from a swine coronavirus strain and a vaccine manufactured from a swine rotavirus strain, it is possible to prevent infectious gastroenteritis caused by either swine coronavirus or rotavirus. An effective bivalent vaccine can be obtained. A bivalent vaccine for bovine infectious gastroenteritis can be similarly produced.

The following experimental examples and examples are exemplified to demonstrate the effects of the present invention, and the present invention is not limited thereto.

As mentioned above, effective milk immunity is established when the antibody titer decreases slowly or when the antibody isotype includes IgA. Furthermore, the antibody titer and antibody isotype in milk show the same trends as those in serum.

In this example, we investigated the pathogenic coronavirus (T) that causes swine infectious gastroenteritis.
The purpose of this study was to use the GE virus strain as an immunogen and investigate the effects of various treatments on the neutralizing antibody titer in serum and the antibody titer of each antibody isotype. Since it is already known that similar results can be obtained with serum neutralizing antibodies and milk neutralizing antibodies, we first preliminarily tested the effect of surfactant treatment using serum, which is easy to test.

Using Nonidet NP-40 (manufactured by Gyudon Kagaku Co., Ltd.), which has a strong surfactant effect, as a surfactant, the serum neutralizing antibody titer of the virus antigen treated with surfactant or ultrasonication was compared to that of untreated virus antigen. compared with the case of

Test results (1) Test pigs Three to four month old pigs brought in from a TOE virus-negative farm were used.

(2) The immunogenic TGE virus strain (To-163 attenuated strain) was grown using CPK cells, and the culture supernatant was collected at 35. OOOrp
Viral pellets were obtained by centrifugation for 2 hours at m. This was added to TNEII at a concentration that gave a virus titer of 10" TCIDso/ml as measured by the method (3) below.
After dissolving in a buffer solution, it was treated as follows.

Group ■: No treatment group ■: Surfactant treatment (NP-40) group ■: Ultrasonic treatment (200W, 60 seconds) group The surfactant treatment for ■ is Nonident NP-40 (hereinafter abbreviated as NP-40). ) to virus-containing buffer at 0.5%
This was done by adding at a final concentration of .

An equal volume of Freund's complete adjuvant was mixed with each virus-containing solution (antigen solution) to prepare an immunogen for inoculation.

For the initial immunization, 1 ml of each antigen solution was intramuscularly injected, and three weeks later, a booster injection of the same amount of the antigen solution was given.

(3) Virus titer (infectious titer) measurement method A CPK monolayer culture microplate was used for titer measurement. The virus solution was serially diluted 10 times, and 0.
11 cells were inoculated into 5 wells, cultured at 37°C for 5 days, and the infectious titer (TCIDso) was determined using Behrens-Karb.
It was determined by the er method. In addition, the cell growth medium and cell maintenance medium were Eagle ME supplemented with 10% bovine inactivated serum, respectively.
M medium and Eagle's MEM medium supplemented with 5% inactivated bovine serum were used.

(4) Neutralization Test Method All test materials were immobilized by heat treatment at 56° C. for 30 minutes. 2-fold serially diluted test material 0.5 + * 1 in l
00 TCIDs. Add 0.5 ml of neutralizing virus solution adjusted to 10.1 ml, shake well, and add 3.
Sensitization was carried out at 7°C for 60 minutes. Then, the inoculum was added to each of 4 wells of CPK monolayer culture microplate at 0.2 + *1
The cells were inoculated and allowed to adsorb to the cells at 37°C for 90 minutes. After removing the liquid from each well, 0.1 ml of cell maintenance medium was added to each well and cultured at 37°C for 5 days. The reciprocal of the highest dilution of the test material that inhibited the expression of CPE (cytopathic effect) of 2 μl or more after 5 days of culture was defined as the neutralizing antibody titer.

(5) Antibody fractionation method In order to investigate the isotype of antibodies, 5ephacr
Antibodies were fractionated by gel filtration using ylS-300 (manufactured by Famacia Fine Chemicals).

A 2.6×120 cm column was used. After adsorbing the test material 3++1 onto the gel, 3 ml of the 0 fraction eluted with phosphate buffered saline was collected using a fraxilon collector.
Collected one by one. The IgA fraction and IgG fraction were identified by the neutralizing antibody titer of both fractions, which was performed by agar gel precipitation reaction using rabbit anti-pig α and T chain serum, using the neutralization test method described in (4) above. Measured by.

The results of the wiping test are shown in Table 2. From the results in Table 2, the neutralizing antibody titer in the IgA fraction for both serum after 1 week and 3 weeks after sterilization tends to be higher for the NP-40 treated antigen than for the untreated and sonicated antigens. was recognized. Also, Ig
The G fraction was also high for the NP-40 treated antigen. That is, T
It has been found that treatment of GE virus with a detergent has an advantageous effect on the induction of neutralizing antibody titers including IgA.

(Left below) NP-40, which was used as a surfactant in Experiment A, has strong cytotoxicity, so in this example, Tween 80 was selected as a surfactant suitable for pharmaceutical materials, and the same test as Experiment A was repeated. Ta.

Test method (1) Test line Pigs at the fourth postnatal age brought in from a TGE virus-negative farm were used.

(2) As in immunogen experiment A, CPK cells were infected with the attenuated strain of TGE virus and cultured, and the culture supernatant was ultracentrifuged at 35.00 rpm for 2 hours to obtain a pellet. TNE this pellet
Dissolve the virus infectivity in buffer to 10” TCIDs.
It was adjusted to 0/ml. The virus antigen solution was treated using Tween 80 (final concentration 0.05% and 0.5%) manufactured by Junsei Kagaku Co., Ltd. The reason why the virus infectivity value before treatment was set at 10'' TCIDss/ml is because it is a concentration that can be easily recovered in actual manufacturing operations.

An equal amount of Freund's complete adjuvant was mixed with each treated antigen solution to prepare an immunogen. For the initial immunization, 1 ml of each antigen was intramuscularly injected, and three weeks later, a booster injection of the same amount of antigen was given.

(3) Infectious titer measurement method It was conducted according to Experiment A.

(4) Neutralization test method It was conducted according to Experiment A.

(5) Antibody fractionation method The gel filtration method was used in the same manner as in Experiment A.

The results of the pear test are shown in Table 3. A similar tendency was observed when NP-40 in Table 2 was used. That is, Tween 80 is a surfactant that is permitted as a pharmaceutical material.
Even when changing to 1gA fraction, the neutralizing antibody titer of the 1gA fraction could be increased. Therefore, it is conceivable that immunization with surfactant-treated viruses can provide higher immunization effects than conventional ones. The IgA neutralizing antibody titer was higher when the Tween 80 concentration was 0.5% than when it was 0.05%.

Table 3 Serum neutralizing antibody titer and antibody isotype when pigs were immunized with surfactant (Tween 80) treated antigen No treatment 1 , 024 8.192 4 , 096 Tween 80 2 0.05X 3 512 4.096 8 256 4,09
6512 4.096 8 12B 4.
096Tween80 4 512 8
．． 192 16 5120.5$ 5
1.024 16.384 16 >512
8.192 8.192 * Neutralizing antibody titer in gel filtration fraction with 5ephacryl S-300 1 test day In this example, a trial vaccine was prepared using a different adjuvant from a Tween 80-treated virus, and compared with a vaccine using an untreated virus. did. That is, Freund's complete adjuvant (FCA) used in Experiments A and B caused severe tissue reactions at the injection site, so it was not retained for use as an adjuvant in general vaccines. Therefore, a test similar to the above experiment was conducted by changing the adjuvant to Carbopol, which has been shown to produce negative tissue reactions at the injection site in many toxicity tests.

Extraction of lame limbs (1) Test pigs Pigs at the fourth postnatal age brought in from a farm that was negative for TG virus were used.

(2) Similar to immunogen experiment B, the centrifuged pellet of TGE virus culture solution was dissolved in TNE buffer, and the virus infectivity titer was determined to be 100
・'TCIDs. Adjusted to /+wl. This virus-containing antigen solution was treated with Tween 80 so that the final concentration was 0.5%. Thereafter, an equal amount of the following adjuvant was mixed with each antigen solution to prepare an immunogen.

■Untreated antigen: FCAC Freund's complete adjuvant) ■Tween 80 treated antigen: FCA ■Tween 80 treated antigen: Carbopol adjuvant (1% solution) For the initial immunization of pigs, 1ml of each antigen is injected intramuscularly.
Three weeks later, a booster injection of the same amount of antigen was given.

(3) Infectious titer measurement method It was conducted according to Experiment A.

(4) Neutralization test method It was conducted according to Experiment A.

(5) Antibody fractionation method The gel filtration method was used in accordance with Experiment A.

The results of the axillary retention test are shown in Table 4. From Table 4, it can be seen that the neutralizing antibody titer of the IgA fraction tends to be higher for the antigen treated with Tween 80 than for the untreated antigen.

In the case of Tween 80-treated antigen, when the adjuvant was changed from Freund's complete adjuvant to Carbopol adjuvant, both the antibody titer before fractionation and the antibody titer of the IgA fraction decreased slightly; The IgA fraction antibody titer was higher than when mixed.

Table 4 Neutralizing antibody titer and antibody isotype when pigs were immunized with Tween 80 treated vaccine FCA I 4.096 4
2562 4.096 2 25
6Tween80 FCA 3 1
6,384 16 >5124 4.09
6 8 512Tiien 80 Carbobol 5 8,1926 2.048 +k 5ephacryl Neutralizing antibody titer in gel filtration fraction with S-300 ) We actually immunized pregnant pigs by intramuscularly injecting a vaccine consisting of 10-carbopol adjuvant, examined the duration of neutralizing antibody titer secreted in milk and the isotype of the antibody, and tested the newborn piglets 3 days after delivery. 100 PIDs on the day. /We investigated the efficacy of a milk-immunized vaccine by challenging pigs with a virulent virus.

For comparison, immunization with a highly virulent strain to approximate field immunization and immunization with a commercially available oral attenuated live vaccine were also performed.

Wiping method (1) Test experimental pigs Using 6 pregnant pigs brought in from a TGE-negative farm, it was confirmed that the serum of each animal did not contain neutralizing antibodies against the TGE virus before being brought in.

(2) Immunogen sow 1 has approximately lOS·' pros. A highly virulent intestinal emulsion of the virus was orally inoculated 4 weeks before the expected delivery date. After this inoculation, sows experienced transient loss of appetite, depression of energy, and vomiting. This is because a large amount of data has proven that this holds true.

Sows 2.3.4 and 5 were vaccinated with the Tween 80 treated vaccine according to the invention prepared as follows.

That is, a TOE virus culture solution cultured using CPK cells was microcentrifuged by ultracentrifugation, then lysed with TNE buffer to adjust the infectious titer to 10"TCIns./1, and then 0.2% of formalin was added to inactivate the virus. Then, 1% Tween 80 was added to perform detergent treatment. An equal volume of 1% Carbopol may be added as an adjuvant to this virus-containing antigen solution. This vaccine was mixed and used as a test vaccine. Vaccination of this vaccine was performed by intramuscularly injecting 21 doses of each sow 4 weeks before expected farrowing (first immunization) and 1 week before expected farrowing (puster). No particular abnormalities were observed in the clinical symptoms of the sows afterwards.

Sow 6 was vaccinated with a commercially available live attenuated vaccine made in the United States that is commonly used in the field around the world. The vaccination was carried out by orally inoculating the vaccine on the 35th and 21st days before scheduled delivery, and then intramuscularly on the 7th day thereafter, according to the instructions for use. The sows were fasted the day before oral inoculation. When the virus infectivity of this vaccine was measured using a pig testicular cell line, it was 10S1TCIDs. /1.

(3) Treatment of milk Before the neutralization test, the milk to be tested was mixed with an appropriate amount of rennet, left at 37°C for several hours, and then heated at 14,000 rpm.
After centrifugation at +m for 30 minutes, the supernatant was inactivated by heat treatment at 56° C. for 30 minutes, and then subjected to a neutralization test.

(4) Attack method Piglets born from each sow were attacked on the third day after birth.
0 PID,. The test was carried out by orally administering a virulent strain of TGE virus in an amount of 1/pig. Clinical symptoms were observed for 7 days thereafter. In addition, in order to measure the neutralizing antibody titer in milk, milk was collected at the time of delivery, on day 1, 2, 3, 4, 5, 6, and 7 days.

(5) Virus titer measurement method This was carried out according to the method described in Example 1.

(6) Neutralization test method This was carried out according to the method described in Example 1.

(7) Milk antibody fractionation method According to the method described in Example 1, 5ephacryl
Milk was fractionated by gel filtration using S-300, and I
gA and IgG fractions were identified, and the neutralizing antibody titer of each fraction was determined.

Of the test results, the neutralizing antibody titers in milk and the mortality rate of piglets after challenge are shown in Table 5, and the neutralizing antibody titers in IgA and IgG fractions after milk gel filtration fractionation are shown in Table 5. They are summarized in Table 6.

Sow 1 inoculated with virulent virus strain as shown in Table 5
The milk neutralizing antibody titer at the time of delivery was 2048-fold, and the antibody titer in the milk decreased slowly after delivery, and after the second day, the antibody titer did not decrease and remained at 256-fold. The incidence of disease and death of piglets after challenge was 0%.

In four sows 2 to 5 inoculated with the test vaccine treated with a surfactant according to the present invention, the neutralizing antibody titers at parturition were all higher than those in Example (1) inoculated with a virulent strain. 4096
The subsequent decline in antibody titer was relatively slow, with antibody titers of 256 to 2048 times, which is equivalent to or higher than that of case (1) inoculated with a virulent strain, maintained even on the 7th day after delivery. Ta. Therefore, effective antibody production in milk was observed in all of the sows 2 to 5. This 4! A total of 38 piglets were born from sow I, but 0% of the piglets born to either sow died due to disease after challenge.

On the other hand, in sow 6 inoculated with a commercially available live attenuated vaccine made in the United States, the milk antibody titer at parturition was 128 times lower than the above, and on the 7th day the antibody titer was only 8 times as high. dropped sharply to. In addition, due to attack of piglets with a highly virulent strain on the 3rd day of life, the piglets of the chairman were infected with T.
The first piglet exhibited severe watery diarrhea characteristic of GE, and died by day 7 (mortality rate 100%). Sow 6 also exhibited symptoms of transient loss of appetite, depression of energy, and vomiting after the attack. was confirmed. This is because no disease or death was observed in the cases inoculated with the virulent strain and in the group vaccinated with the test vaccine according to the present invention, effective milk immunity was established, and no abnormal clinical findings were observed in the sows after challenge. The results were in complete contrast.

As can be seen from the measurement results of milk antibody isotypes for each inoculation example shown in Table 6, highly virulent virus inoculation example (1)
The isotype of antibodies is mainly IgA, and IgA is the main isotype of antibodies.
Although G was observed at the time of delivery, IgC antibodies were not detected after the third day due to a rapid decline.

On the other hand, in surfactant-treated vaccination examples (2) to (5) of the present invention, the isotype of the milk neutralizing antibody was I
Although gG was the main component, some IgA was also found to be present. Even in this vaccination example, the IgA antibody titer decreased very slowly. Unexpectedly, in these inoculation examples (2) to (5), unlike the highly virulent strain inoculation example (1),
There was no rapid decrease in IgG antibody titer, and IgG was maintained even on the 7th day after delivery.

In the case of attenuated vaccination (6), analysis could not be performed because the antibody titer in the milk was low.

As already explained, in order to induce effective milk immunity, it is important that an antibody titer at least equivalent to that of a virulent strain inoculated is detected in the milk and that it persists without decreasing during the lactation period. In the case of vaccination with the surfactant-treated vaccine according to the present invention, the antibody titer was equivalent to or higher than that in the case of vaccination with the virulent strain and lasted for at least 7 days after delivery. As shown in Table 5, the piglet mortality rate of 0% and the establishment of effective milk immunity may be due to the isotype containing some IgA; This is thought to be largely due to the effect of the vaccine of the present invention in that the antibody titer of the present invention persists.

As shown in Table 6, this persistence of milk antibody titer is different from that of conventional attenuated or inactivated vaccines, in which the IgG antibody titer decreases slowly with the vaccine of the present invention and lasts for at least 7 days after delivery. Based on contradictory and unexpected results. In conventional vaccines, the isotype of antibodies in milk is mainly IgG, similar to the vaccine of the present invention, but since the IgG antibody titer drops rapidly, it is necessary to maintain the antibody titer necessary for effective milk immunity. I couldn't do it.

To date, regarding the TGE vaccine, there have been no reports showing that neutralizing antibodies with an antibody titer equal to or higher than those inoculated with a virulent strain are detected in the milk and that this persists for one week after delivery. In that sense, the vaccine of this example is an excellent vaccine that has never existed before.

(Margin below) Fruit 1111 The vaccine according to the present invention obtained by treating swine-derived rotavirus with a surfactant using Tween 80 was injected intramuscularly into pregnant pigs, and the duration of the neutralizing antibody titer secreted into milk was examined. 10' within 24 hours after farrowing the newborn piglets.
TCIDS. The efficacy of the vaccine was investigated by challenge with the virus.

■Gohiro Soda (1) Experimental Pig SPF Four pregnant pigs were used, and blood was collected before delivery to confirm that the pigs did not have neutralizing antibodies against rotavirus.

(2) The culture solution of rotavirus cultured using immunogenic MA104 cells was reduced by ultracentrifugation to 10" TCItls.7.
After adjusting the virus infectivity titer by diluting with TNEEI solution to give a ring of 1, the virus was inactivated with 0.2-0.3% formalin, and then treated with 1% Tween 80. To this was added an equal amount of 1% carbobol and mixed well to obtain a test vaccine of the present invention. Sows 1, 2 and 3 are scheduled to farrow 4
Two ml of each test vaccine was intramuscularly injected one week before and one week before. Sow 4 served as a non-vaccinated control.

(3) Attack method Piglets born from test sows are 10" within 24 hours of birth.
rctos. / Orally challenged with a virulent strain of bovine rotavirus at a viral load of a pig. The condition of diarrhea was then observed for 7 days. In addition, in order to measure the neutralizing antibody titer, milk was collected from the time of delivery until day 7.

(4) Treatment of milk The milk to be tested was immobilized by the same treatment as in Example 2, and then a neutralization test was conducted.

(5) Virus infectious titer measurement method Inoculate 0.1 ml of the test virus serially diluted 10 times into four MA104 monolayer culture test tubes, and inoculate 0.1 ml of the test virus serially diluted 10 times at 37°C.
Allowed to adsorb for hours. After adsorption, cells were washed twice with Eagle FiEFI medium containing 0.5 g/ml trypsin (Sigma+ tVp@III), and then incubated with 0.5 m
1 of the same medium was added, and rotational culture was performed at 37°C for 7 days. The infectious titer depends on the presence or absence of CPE (C11). It was displayed in

(6) Neutralization test method Add an equal amount of virus solution (200TC) to 2-fold serially diluted milk.
IDs. 10.1 ml) was added and incubated at 37°C for 90 minutes.

Then, add 0.1 ml of each diluted solution to two bottles of H^104.
It was inoculated into a monolayer culture test tube and allowed to adsorb at 37°C for 1 hour.

'After I&i, cells were treated with 0.5 μg/ml trypsin (
Eagle including Sigma+type m)? IBM
After washing twice with the medium, add 0.51 of the same medium and
Rotation culture was performed at 7°C for 7 days. The neutralizing antibody titer was expressed as the reciprocal of the highest dilution of the test material at which the expression of CPH was inhibited in one or more monolayer culture test tubes.

The test results are shown in Table 7. As shown in Table 7, the colostrum neutralizing antibody titer of sows immunized with the vaccine of the present invention was 8192.
The antibody titer was extremely high, over 2,000 times as high, and it hardly decreased even after transitioning to regular milk, and even on the 7th day after delivery, the antibody titer remained as high as over 2,000 times. On the other hand, the colostrum neutralizing antibody titer of the control non-immunized sow was less than twice as high. On the other hand, when piglets born to each sow were challenged within 24 hours of birth, piglets born to the vaccinated sows did not develop the disease and all tolerated the disease, whereas piglets born from non-immune sows survived the disease. After the challenge, the pigs showed depression and vomiting, and watery diarrhea persisted in the first pig until at least 7 days.

(Left below) Tween 80
The vaccine obtained by surfactant treatment was injected intramuscularly into pregnant women, and the persistence of neutralizing antibody titers secreted into milk was investigated.
CIns. The efficacy of the vaccine was investigated by challenge with the virus.

Limb Removal (1) Six pregnant cows of the meat-only breed were used in the experiment, and blood samples were taken before delivery, and it was confirmed that they did not have antibodies against rotavirus.

(2) Bovine rotavirus culture broth cultured using immunogen 1^104 cells is fIA-contracted by ultracentrifugation, and then diluted with TNE buffer to give a bad staining titer of 10"↑Cl0S,/ml. After inactivation with 0.2-0.3% formalin, 1% Tween 80 was added for surfactant treatment.To this, an equal volume of 1% Carbopol may be added. The test vaccines according to the present invention were mixed together to form the test cotton of the present invention. 51 doses of the above test vaccine were injected intramuscularly for 1, 2, and 3, respectively, 8 weeks and 2 weeks before scheduled delivery.
．． Groups 5 and 6 were used as non-vaccinated controls.

(3) Attack method The calves born at the test site were attacked within 24 hours of birth.
' TCITIs/Cows were challenged orally with a virulent strain of Ushiyamaki rotavirus at a viral load of a cow. The condition of diarrhea was then observed for 7 days. In addition, milk was collected from the time of delivery to day 7, and the neutralizing antibody titer was measured.

(4) Treatment of Milk Before performing the neutralization test, the milk to be tested was immobilized by the same treatment as described in Example 2, and then the neutralization test was performed.

(5) Virus infectious titer measurement method Cultivate the test virus according to the method described in Example 3,
The infectious titer was expressed as TCIDS@ depending on the presence or absence of CPE.

(6) Neutralization Test Method Milk was sensitized and cultured according to the method described in Example 3. Neutralizing antibody titer is CP for one or more monolayer culture test tubes.
It is expressed as the reciprocal of the highest dilution factor of the test material that inhibited the expression of E.

Fushin 1 The test results are shown in Table 8. As shown in Table 8, the neutralizing antibody titer in the colostrum of the tested vaccine-immunized infants was extremely high, at more than 8192 times, and hardly decreased even when transferred to regular milk, and was 4096 times higher on the 7th day after delivery. Antibody titers above these levels were maintained. In contrast, the colostrum neutralizing antibody titer in non-immune breast milk was less than twice as high.

On the other hand, when calves born in each company were challenged, calves born in the vaccinated group did not develop any disease, whereas all calves in the control group developed severe watery diarrhea and eventually One calf died.

Sugo 11 [Cow-derived coronavirus is transformed into Tweer+8 by the present invention]
The vaccine obtained by surfactant treatment at 0 was injected intramuscularly into pregnant women, and the persistence of the neutralizing antibody titer secreted into the milk was investigated.
The efficacy of the vaccine was tested by challenge with IDse virus.

(1) Six pregnant cows of the meat-only breed were used in the test, and blood samples were taken before delivery to confirm that they did not have antibodies against the coronavirus.

(2) A bovine coronavirus culture solution cultured using the immunogenic bovine embryonic kidney cell line (REK-1) was concentrated by ultracentrifugation, and then diluted with TNE buffer to reach a viral infectivity of 10
1・'tctos. /ml, then inactivated with 0.3% formalin, and 0.5% NP-
40 was added for surfactant treatment. 1% equal to this
Carboball was added and mixed well to prepare a test vaccine according to the present invention. For health groups 1, 2, and 3, 5 ml of the above test vaccine was intramuscularly injected 10 weeks and 2 weeks before scheduled delivery, respectively. Health 4.5 and 6 were used as non-vaccinated controls.

(3) Attack method A calf born from a sample company was attacked on the 3rd day after birth.
rc+os. / challenged orally with a virulent strain of bovine coronavirus at a viral load of a bovine. Thereafter, the condition of diarrhea was observed for 7 days. In addition, milk was collected at the time of delivery and on the 3rd, 5th, and 7th day after delivery, and the neutralizing antibody titer was measured.

(4) Treatment of Milk Before performing the neutralization test, the milk to be tested was immobilized by the same treatment as described in Example 2, and then the neutralization test was performed.

(5) Virus infectious titer measurement method Four BEK-1 monolayer culture test tubes were each inoculated with 0.1 ml of the test virus serially diluted 10 times, and after adsorption at 37°C for 90 minutes, the virus solution was was removed. Add 5% bovine serum and 10% tryptose phosphinyl to each culture tube.
Eagle's MEM medium supplemented with Tobros was added in 0.5 ml portions, and rotational culture was performed at 37°C for 7 days. Infectious value is CP
TCIns depending on the presence or absence of E. It was displayed in

(6) Neutralization test Add an equal amount of virus solution (200TC
IDso (10.1 ml) was added and sensitized at 37°C for 90 minutes.

Then, add 0.1 ml of each diluted solution to two bottles of BEK-1.
Added to monolayer culture test tubes and allowed to adsorb for 1 hour at 37°C.
Next, 0.5 ml each of Eagle's MEII medium supplemented with 5% bovine serum and 10% tryptose phosphate prosthesis was added, and rotational culture was performed at 37°C for 7 days. The neutralizing antibody titer was expressed as the reciprocal of the highest dilution of the test material at which the expression of CPE was inhibited in one or more monolayer culture test tubes.

Trial folded boar fruit The test results are shown in Table 9.

As shown in Table 9, the neutralizing antibody titer in the colostrum of the tested vaccine immunizer was extremely high, at over 4,096 times, and hardly decreased even when transferred to regular milk, with an antibody titer of over 2,048 times. was maintained. In contrast, the colostrum neutralizing antibody titer in non-immune breast milk was less than twice as high.

On the other hand, when calves born from each welfare group were challenged, calves born from the vaccinated group did not develop any disease, whereas calves from the control group developed yellow-brown, heavily watery stools.

[Effects of the Invention] As explained and exemplified above, by inoculating pregnant animals before parturition using coronavirus or rotavirus derived from pigs or cows treated with a surfactant according to the present invention, Effective milk immunity is established in the newly born piglets or calves, and they can be reliably protected from disease and death due to the attacking virus. As shown in Example 2, the neutralizing antibody titer in the colostrum of the immunized sows exceeded that of the cases inoculated with the virulent strain, and the antibody titer persisted without much of a decline even after parturition, and on the 7th day. However, we were still able to maintain antibody titers higher than those inoculated with the virulent strain. This is because in mother pigs vaccinated with existing vaccines, the antibody titer in the milk drops rapidly after parturition, which is why when piglets born are challenged with a virulent strain, the
This result is in stark contrast to the 0% mortality rate. In other words, with conventional vaccines, milk antibody titers drop rapidly and are not effective at all in protecting against attacks by highly virulent viruses, whereas with the vaccine of the present invention, milk antibody titers persist and are effective against such attacks. The results showed that the vaccine had an excellent effect on disease prevention that was completely unexpected from conventional vaccines.

Therefore, by using the vaccine of the present invention, it has become possible to almost completely protect piglets within the first year of life from infectious porcine gastroenteritis, which is the most damaging.

What is particularly noteworthy about the vaccine of the present invention is that the isotype of antibodies induced in milk by the vaccine of the present invention is Ig.
Although it is not much different from conventional vaccines in that it is mainly composed of G and contains some IgA, the IgG antibody titer does not drop sharply. According to conventional wisdom, Ig
Considering that it was previously thought that the G antibody titer decreases rapidly and does not contribute to the sustainability of the immune effect, surfactant treatment was able to induce an IgG antibody titer that does not easily decrease in this way. is completely unexpected. As shown in Example 2, even when inoculated with a highly virulent strain, Ig
In the present invention, the G antibody titer is rapidly decreasing.
Milk antibodies contain some IgA, and the persistence of this IgG antibody is thought to achieve the above-mentioned sustained immune effect.

The vaccine of the present invention, as illustrated in Examples 3 to 5,
In addition to TGE virus (swine-derived coronavirus), when applied to swine-derived rotavirus and bovine-derived coronavirus and rotavirus, effective milk immunity is established in the same way as above, and the mortality rate is reduced for a while after birth. For the longest period of time, piglets or calves can be almost completely protected from the onset of infectious gastroenteritis and death.

Infectious gastroenteritis in pigs and cattle accounts for a significant portion of deaths in piglets and calves, but no highly effective vaccine has hitherto existed.

Therefore, it is expected that by applying the vaccine of the present invention, which shows a perfect preventive effect, it will be possible to considerably reduce the mortality caused by diarrheal spasm in suckling piglets and calves, and its usefulness will be extremely high. There is something big.

Claims (5)

[Claims] (1) A method for producing a porcine or bovine infectious gastroenteritis vaccine, which comprises treating an inactivated or attenuated porcine or bovine infectious gastroenteritis virus strain with a surfactant. (2) The method according to claim 1, wherein the surfactant is a nonionic surfactant. (3) A swine or bovine infectious gastroenteritis vaccine produced by the method according to claim 1 or 2. (4) The swine or bovine infectious gastroenteritis vaccine according to claim 3, wherein an adjuvant that is negative for tissue reaction at the injection site is added. (5) Mixing a vaccine obtained from a swine coronavirus strain and a vaccine obtained from a swine rotavirus strain, or a vaccine obtained from a bovine coronavirus strain and a vaccine obtained from a bovine rotavirus strain. The bivalent vaccine according to claim 3 or 4, obtained by.