JPS5817169B2 - Shinsei Koshigeri Vaccine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a vaccine against a novel coronavirus-like agent causing diarrhea in newborn calves, and a method for producing this vaccine.

More particularly, the present invention relates to a method for attenuating a coronavirus-like virus by serial passage culture in fetal bovine kidney cells, and a vaccine obtained thereby.

Furthermore, the present invention relates to a method for producing a vaccine in which the coronavirus-like factor is grown on a cell culture medium and then inactivated.

neonatal calf diarrhea
The etiological factors of rhea (abbreviated as NCD) are complex.

Many types of bacteria are involved as the cause of this disease, including bacteria, fungi (mycoplasma), and chlamydia.
mydia) and viruses.

Particularly mentioned viruses include bovine diarrhea virus, infectious bovine rhinotracheitis virus, adenovirus, enterovirus and LADEN virus.

Other viruses that have been reported as etiological factors for NCD include bovine pneumoenteritis virus and a reovirus-like agent called NCD virus.

A method for producing a vaccine effective against this NCD reovirus-like agent is described in U.S. Patent Application No. 197,520;
Filed on November 10, 1971.

In the course of field trials of vaccines produced from reovirus-like agents, inadequate control of the disease in cattle was observed in herds of Western Nebulas (Vet,
Med,/Small Anim, CI in,.

67(2), p. 173 (1,972)).

Typically, in cows administered this vaccine within the first 24 hours of birth, some herds still developed diarrhea even when the cows were 5-20 days old.

The diarrheal feces of these cows were tested for reovirus-like viruses using a fluorescent antibody method, but the results were negative.

Diarrhea in one of the vaccinated groups, in which 90% of the cows were infected when they were 7 to 12 years old, was removed by duodenal injection into calves born through uterine incision (Caesarean) and not given colostrum. Inoculated.

The calf developed diarrhea, and a bacteria-free fecal fluid was prepared from the diarrheal stool, which was then used for gutbiosis (g
(notobiotic) calves were inoculated orally.

These calves developed diarrhea, and the presence of other viral agents was considered as the cause of the diarrhea.

When diarrheal stool from laboratory calves was subjected to electron microscopy, coronavirus-like agents were observed.

The same type of virus particles were observed in the diarrheal stools of both the vaccine and chin-administered groups and the non-vaccine-administered group in the field test.

This new coronavirus-like agent has been purified and identified in great detail.

The inventors have discovered that this coronavirus-like virus can be grown and transformed into an effective vaccine in cell culture media, and that an effective vaccine can be obtained by growing this virus in cell culture media. discovered that it can be manufactured by inactivating it.

Accordingly, the present invention relates to a vaccine effective against this new coronavirus-like agent and methods for producing and administering this vaccine.

Am for purification and characterization of this factor. J,Ve
t, Res, vol. 33, pp. 1147-1156 (19
72).

Purification of coronavirus-like agents: Stool samples were collected from diarrheal calves from 19 herds in Western Nebulas.

Calves in groups 12-19 were pre-orally vaccinated with NCD reovirus-like virus vaccine, while calves in other groups were not vaccinated.

Fecal smears from calves with diarrhea were stained with an immunofluorescent complex for NCD virus testing [Nebraska State University, Ag.
ri, Exper, Sta.

Res, Bul I, p. 233 (1969)) and only samples negative for NCD virus were subjected to testing.

Diarrheal fecal material collected from naturally infected and artificially infected calves on the farm was stored at -20°C to -60°C until virus purification was undertaken.

Various sucrose concentration gradient solutions were prepared and left overnight at 4° C. to directly form gradients before use.

Each gradient consisted of 8 mA containing 400,300,200 and 100, respectively, of sucrose per ml of distilled deionized water.

The canned liquids were placed in a 2.5×8.9 crfL nitrocellulose centrifuge tube in descending order of concentration.

All virus purification procedures were performed at 4°C and used medical centrifugation and ultracentrifugation.

Mix diarrheal stool material with 3 volumes of deionized water and incubate at 500 ml for 30 minutes.
Centrifuged at 0G.

The supernatant was then centrifuged for 3 hours at 25.00 Orpm in a type 30 rotor.

The resulting pellets were dispersed in a 5% (w/■) sucrose solution by sonication for 30 seconds.

This suspension is called a crude virus suspension.

5 ml of the crude virus suspension was layered onto the gradient in the tube and centrifuged at 25,000 rpm for 2 hours in an SW27 rotor.

After centrifugation, each zone was located using a light scattering method.

The material in each zone was collected from the top of the centrifuge tube using a Pasteur pipette equipped with a propipette.

Each sample was dialyzed against a 1% ammonium acetate solution before electron microscopy.

The fraction containing the virus (semi-purified virus from the zone 6C1rL from the liquid surface) is further purified by sucrose gradient centrifugation or cesium chloride (CsC1) gradient centrifugation.

The semi-purified virus preparation was further purified by repeated sucrose gradient centrifugation as described above.

The zone is located again, and the zone at 6.0 CI'rL from the liquid surface is collected and dialyzed against 1% ammonium acetate to obtain purified virus.

The same concentration gradient centrifugation of the semi-purified virus preparation was carried out in a 30% solution (15 g of CsC1' + 35 rrLl of deionized water).
I started and went.

Transfer this solution (4 InJ) to a 21-o-1z rulose centrifuge tube, and then add 1.4 r/l of virus suspension to the tube.
l was added.

Centrifugation for 18 hours at 60.00 Orpm using a 5W65 rotor has reached full equilibrium at this point.

The refractive index of CsC1 in the zone where the virus is present is measured using a TS meter.

A band of 2.9 CrrL is collected from the liquid surface and dialyzed against a 1% ammonium acetate solution to obtain purified virus.

In addition, the crude virus suspension (15rrLl) described above was applied to a 4.2 x 42crIL gel column (Sepharos).
e 2B: Pharmacia Fine Chemistry
Cals, Inc., Piscataway, New Jersey) for fractionation by gel filtration.

The column was equilibrated in advance with 0.9% NaC] and developed with this saline solution.

The flow rate was 6.0 ml/hour. A fraction of 5 rrLl was collected and the absorbance was measured spectrophotometrically at 260 μm.

The material from each peak is combined and then concentrated separately by ultrafiltration.

A simplified method for preparing virus from diarrheal material from test calves is also applied.

The fecal material is directly centrifuged at 3,400 G for 30 minutes.

A portion of the supernatant portion is extracted twice with dichlorodifluoromethane.

The aqueous layer (5d) and untreated supernatant (5d) material are subjected to sucrose gradient centrifugation as described above.

Virus concentrated by concentration gradient centrifugation is used for electron microscopy studies and as an antigen for immunization of rabbits for the preparation of conjugates for immunofluorescence analysis.

Blood was collected from the heart of a white rabbit about 6 months old (14 mJ
)j,, then add a mixture of 0.4vr,l of antigen and 0.6ml of complete Freund's supplement to 0.25m per site.
Intramuscular injections were made at 4 sites at L.

Five weeks after inoculation, 70 ml of blood is collected by cardiac puncture.

Known methods for preparing gamma globulin labeled with fluorescein Cproc, U, S, Livestock San, A
, ssn, October 1968, pp. 139-144) from post-inoculation serum.

The presence of coronavirus-like agents can be detected in the infected intestine by immunofluorescence using the formulation prepared above.

The specificity of this formulation against coronavirus-like agents is clearly demonstrated by the test method presented below.

When a section of the intestine of a calf with diarrhea infected with a coronavirus-like agent was stained with the combination, fluorescence of trophoblastic cells was observed.

Intestines from the same calf showed no fluorescence when stained with the complex for the reovirus-like factor described above.

Intestinal sections from diarrheal calves infected with reovirus-like viruses were positive when stained with a complex for reovirus-like factors and negative when stained with a complex for coronavirus-like factors.

Intestine sections from calves with normal gutbiosis showed no fluorescence when stained with a complex for coronavirus-like agents.

Virus morphology: The virus-containing zone obtained by concentration gradient ultracentrifugation purification method is examined by electron microscopy.

Droplets from the band are applied to a 200 mesh copper grid previously coated with collodion and reinforced with vapor deposited carbon.

These droplets range from 3 to 2 depending on the degree of light scattering of the zone under test.
Leave on grid for 5 minutes.

Excess liquid is blotted with filter paper and then a small drop of vanadium molybdate-phosphotungstic acid solution is added onto the grid.

The dye is allowed to remain on the grid for 1 to 15 minutes before its excess is removed with P paper as a blotter.

The samples are examined using an electron microscope with a magnification of 32,000x or higher.

Dimension measurements are performed by dimensional analysis of the electron micrograph of the sample and by using a diffraction grating replica taken immediately after taking the electron micrograph of the sample without changing the equipment set.

The dimensions of a complete virus particle are 1 in the same micrograph.
They ranged in size from 0.07 to 160 μm and had surface protrusions similar to those seen on coronaviruses.

The average size of the grains, including surface protrusions, was 126 μm, as determined by electron microscopy.

Nucleocapsids were pleomorphic, varying in size and shape from circular to oblong.

The capsid envelope becomes particularly distinct when the fringe is lost.

The width of the outer edge varies up to 23 μm.

The surface protrusions of well-retained grains are petal-shaped, connected to the grains by elongated stalks, and have an average length of 11
It is μm.

The coronavirus-like agents described in this invention are easily distinguished from other viruses that have been proven or proposed to be pathogenic agents of diarrhea in newborn calves on the basis of size and morphological characteristics.

The coronavirus-like agents described here have morphological characteristics different from enteroviruses, bovine pneumonia-enteritis virus, bovine diarrhea virus, neonatal calf diarrhea virus (reoform), and Haden virus, and these It is larger than.

This new agent is smaller than bovine rhinotracheitis virus and lacks its typical morphological features.

Buoyant density of purified coronavirus-like agent
gnt density) is 1.2 if CsC1 is used.
4, while reovirus-like agents have a buoyant density of 135
9 (Can, J.

Comp, Med, p. 35 (1971)).

The physiological effects of coronavirus-like agents are also different from those of reoviruses.

The incubation period after oral inoculation of reovirus-like agents to gutbiosis cattle can be as short as 13.5-14 hours, whereas the minimum incubation period observed for coronavirus-like agents is 18 hours.

Furthermore, gutbiosis cows infected with reovirus exhibit diarrhea symptoms for 5 to 8 hours, then return to normal 24 hours after the onset of diarrhea.

In contrast, gutbiosis cows infected with coronavirus-like agents may have severe diarrhea, continue to have diarrhea for five or more days, or even die two to three days after the onset of diarrhea.

Methods for producing and using vaccines: Effective methods for carrying out the present invention will be explained below by way of examples, but these are not intended to limit the present invention in any way.

The coronavirus-like agent obtained from infected cattle multiplies on tallow kidney cells.

The virus can also be grown on cells from other tissues or cell lines of bovine or other origin.

A monolayer cell culture medium is prepared by known methods and inoculated with the coronavirus-like agent.

Typically, the monolayer medium is washed with Hanks' balanced salt solution, then the virus inoculum is added and allowed to absorb for several hours, usually 2 hours, at about 37°C.

0.5% lactalbumin thawed product (abbreviated as LAH)
, 0.1% yeast extract, and ], rul.

Lipenicillin 100 units and streptomycin 2
Earle's balanced salt solution stock medium containing 0.00 μg is added and then incubated at 30-40° C., preferably about 37° C., for 2-10 days.

Drain the supernatant from the cells, e.g.

Collect.

The virus inoculum can be stool collected from a coronavirus infection or a culture fluid from a coronavirus-like virus culture medium.

Feces can be collected as is or in phosphate buffer (pH 7, 2).
) and centrifuge it at 100OG for 20 minutes.
.

Kokichi is produced by using the supernatant as an inoculum.

Other storage media can be used, such as modified Eagle medium containing lactalbumin hydrolyzate.

The selection of the medium can be done according to the common sense of those skilled in the art.

Effectively attenuated corona virus vaccines are obtained by subculturing the purified virus in tallow vesicle cells a sufficient number of times to ensure that inoculated cattle do not become ill when challenged with the virulent virus. It will be done.

Usually, it is necessary to subculture the virus 5 to 60 times.
This passage is carried out in primary and secondary cells (primary cells have been passaged once) or in higher passage cells or cell lines.

High passage cells that have been subcultured 10 times or more may be considered a cell line.

A preferred method involves the combination of primary or secondary cells and subculturing on higher passage cells or cell lines.

Virus titer is determined by standard methods.

For example, cells inoculated with virus dilutions are cultured for 5 days in tubes containing fetal bovine kidney cells.

The presence of virus is determined, for example, by cytopathic effect, immunofluorescence, or blood absorption.

More specifically, an effectively attenuated vaccine is produced by the method described below.

Prepare bovine fetuses and prepare large amounts of primary kidney cells.

Several bottle and coverslip media were prepared from the primary cells by adding Earle's Balanced Salt Solution containing 0.5% LAH, 1.0% adult bovine serum, and 10% dimethyl sulfoxide to the remaining primary cells. to freeze.

The bottle and cover glass medium are inoculated with a virus-containing solution as described herein and incubated at 37°C.

Once a few fluorescent cells are observed on the coverslip medium, thaw the frozen-substituted cells from the same fetus and form a monolayer medium by standard methods.

Fluid from this infected primary cell culture is subcultured into these secondary cell cultures.

Six to seven days after inoculation, the liquid from the secondary cell medium is used to inoculate another secondary cell medium.

For subculture from generation 1 to generation 15, use 2ml of liquid as described above! is used to inoculate 8 oz. spins.

For passages 15 to 25, add 2 ml of liquid to the storage medium in the bottle.

After 25 passages, add 1 rul of inoculum to the storage medium in each bottle.

Once the coronavirus-like agent has been passaged in tissue culture for passages 5-20, a series of serial passages is begun with cells from the secondary cells.

At each passage, only a small portion of the cells become infected, and other uninfected cells are subcultured to provide cells for subsequent higher passages.

This type of cell culture medium is called a passage cell culture medium.

A suitable cell-virus system is formed by continuing serial subculture passages of the virus in cell culture media.

After subculturing the virus 5 to 20 times in a secondary cell culture medium, if the virus is subcultured 5 to 40 times or more in a subculture cell culture medium, an effectively attenuated coronavirus-like virus can be obtained. Get the vaccine.

The culture period for virus passage decreases as the number of passages increases, and passage on higher passage cells or cell lines can be carried out for 2 to 7 days.

Each passage is carried out until cytopathic effects occur.

The vaccine uses cell lines from fetal bovine kidney or other tissues or cell lines of bovine or other origin, such as bovine lung, bovine turbinate cell lines or porcine kidney cells or cell lines, which are incubated in tissue culture at about 37°C. It can be produced by culturing until a suitable virus titer is obtained and then collecting the virus.

A titer of 102-106.5, preferably 104-106 is obtained.

Vaccines can be used in liquid or lyophilized form.

For subsequent use, it can be reconstituted by diluting with a sterile diluent such as distilled water.

A preferred product is applied to secondary cells 12 times and then itself 9 to 10 times.
It consists of a virus that has been attenuated by being cultured 15 times on cells that have been passaged 27 times.

The total subculture level is thus 27 times.

Vaccines have been produced that have been passaged up to 35 times.

Additionally, effective inactivated vaccines are produced by inactivating coronavirus-like agents.

Usually, this is produced by first culturing the factor on tallow kidney cells until a suitable titer is obtained, as described above.

The titer should be as high as possible within the above range.

Viruses at any level of cell subculture, as well as
Even non-attenuated viruses can be used.

The virus then exposes it to treatments known to those skilled in the art, such as formalin, beta-propiolactone, ultraviolet light, or heat.
Inactivation is achieved by treatment with an inactivating agent or means at 20-40° C. for a time and at a concentration that effectively inactivates the virus.

These details are known to those skilled in the art.

Adjuvants can be added to promote antigenicity.

The adjuvants may be any known to those skilled in the art, such as aluminum hydroxide gel, potassium alum, alginates, or other adjuvants such as oil bases or mineral or organic oils.

A typical method for producing an inactivated vaccine is as follows.

A coronavirus-like agent at the level of 27 passages as described above is used.

Formalin is added to the virus-containing culture medium to a concentration of 0.2%.

These liquids are then left at 37° C. for 1 to 2 days.

Aluminum hydroxide gel adjuvant is added to the inactivated virus to a concentration of 10%.

The pH is adjusted to about 7.2 with aluminum hydroxide.

This attenuated vaccine is 102-106.5 TCID/I
Doses of 1 to 5 ml containing Ll can be administered to calves, preferably newborn calves, to immunize against virulent viruses.

The inactivated vaccine is administered parenterally in a dose of 1 to 5 ml to pregnant cows 30 to 90 days before calving or to newborn calves as soon as possible after calving, and confers a protective effect against coronavirus-like agents.

Additionally, a combination vaccine, preferably consisting of an inactivated coronavirus and any other viral agent, is used to immunize calves or adult cattle.

Preferred composite examples include inactivated coronaviruses and reoviruses, both of which are produced by inactivating non-attenuated or live attenuated viruses with chemicals, radiation, or heat.

Coronaviruses can also be caused by bovine adenovirus, bovine diarrhea virus, or Escherichia coli.
ia coli) or Clostridium species (C
1ostridia Sp,).

Combined vaccines containing coronavirus and reovirus are prepared by mixing equal amounts of both inactivated viral agents.

Next, formalin at a concentration of 0.2% and aluminum hydroxide gel at a concentration of 10% by volume are added thereto.

This vaccine is administered parenterally to pregnant cows for 1 to 5 hours.
Administration of ul.

This combination vaccine can be administered to both calves and pregnant cows.

Combined vaccines confer protection to newborn calves via colostrum and/or milk antibodies.

Furthermore, protective effects against both factors can be achieved by immunizing pregnant cows with an inactivated reovirus-like vaccine and then administering an attenuated coronavirus-like vaccine to the calves.

Protective effects against both factors can also be conferred by immunizing pregnant cows with an inactivated coronavirus-like vaccine and then administering an attenuated reovirus-like vaccine to the calves.

Additionally, a combination vaccine containing an attenuated coronavirus and a reovirus-like agent can be administered to pregnant cows.

Vaccines prepared at various cell culture subculture levels were administered to calves of Guttobiosis at approximately 7 hours of age;
Seven tests were conducted to test the efficacy of an attenuated virus vaccine by challenging calves with a virulent virus.

The results of these tests are shown in Tables 1 and 2.

Calf 1, which was orally inoculated with 10 ml of cell culture fluid obtained by subculturing the virus 14 times on secondary bovine fetal kidney cells, developed diarrhea and then recovered.

9 years old Usuda, this calf has diarrhea (toxic virus')
], Oml added to 20ml of PBS was orally inoculated, but the disease did not develop.

Calf 2 was inoculated with 10 rILl, a 15-passage culture of the virus on secondary fetal bovine kidney cells, but did not develop diarrhea and did not become ill even after being challenged with virulent virus.

Virus 1 cultured 16 times on secondary fetal bovine kidney cells
When calves 3 and 4 were inoculated with 0rnl, a weak onset of diarrhea was observed.

Calf 3 was sacrificed some time after the onset of diarrhea, and sections of the small intestine and colon were stained with a compound for coronavirus-like agents, and immunofluorescence was observed in the intestinal cilia and epithelium of the colon.

Calf 4 was inoculated with a toxic virus found in diarrheal stool at the sixth white mark of its life, but it did not develop any disease.

Virus 5 cultured 19 times on secondary fetal bovine kidney cells
When calf 6 was orally inoculated with rI'll, it developed diarrhea.

Viruses cultured for at least 5 passages on high-passage fetal bovine kidney cells and 13 to 25 passages were added to 12 calves (/16.7-13 and pain 15-19).
When calves were orally inoculated, no diarrhea developed, and 18 calves
With the exception of , no illness occurred even after oral inoculation with feces containing the virulent virus.

A combined vaccine containing inactivated coronavirus and reovirus was tested on adult cattle from two farms.

This vaccine is a 27-passage formalin-inactivated coronavirus and a 201-passage formalin-inactivated coronavirus (on secondary kidney and bovine kidney cell lines).
It was produced from reovirus.

Mix equal amounts of each inactivated virus with each other,
10% aluminum hydroxide gel was added.

Explanation of the test results in Tables 1 and 2: D-
Diarrhea onset, N-No abnormality, b-The number of passages of the virus is the number of passages on primary or secondary cells and on subcultured cells.

Embodiments of the present invention are listed below: 1. When coronavirus adult bovine diarrhea virus is inoculated into a newborn calf in a tissue culture medium that maintains the growth of the virus, the virus will be protected from subsequent attack by the virulent virus. A method for attenuating the coronavirus adult bovine diarrhea virus, which method comprises inoculating the coronavirus adult bovine diarrhea virus 5 to about 60 times until the onset of the disease is suppressed.

2. The method described in j above, in which the subculture is carried out in a tissue culture medium of fetal bovine kidney cells at 30 to 40°C.

3. In 2 above, the total number of passages is 10 to 40, of which 5 to 10 passages are carried out in primary or secondary bovine fetal kidney cell culture medium, and the remaining passages are carried out in higher A method performed using subcultured fetal bovine kidney cell culture medium.

4. A bovine diarrhea vaccine containing attenuated coronavirus adult bovine diarrhea virus produced by the method in 1 above.

5. A bovine diarrhea vaccine produced by the method of 2 above and containing attenuated coronavirus adult bovine diarrhea virus.

6. A bovine diarrhea vaccine produced by the method of 3 above and containing attenuated coronavirus adult bovine diarrhea virus.

7. Attenuated coronavirus production, characterized by growing the coronavirus adult bovine diarrhea virus in a fetal bovine kidney cell culture medium for a period sufficient to proliferate a large amount of the virus, and then collecting the viral material produced. Mass production method for bovine diarrhea virus.

8. A bovine diarrhea vaccine produced by the method described in 7 above and containing attenuated coronavirus adult bovine diarrhea virus.

9. Inactivates coronavirus adult bovine diarrhea virus,
A method for producing an inactivated coronavirus adult bovine diarrhea vaccine, which comprises incorporating this into an adjuvant.

10. The method according to 9 above, wherein the inactivating agent is formalin.

11. An inactivated coronavirus adult bovine diarrhea vaccine produced by the method described in 9 above, containing an inactivated coronavirus adult bovine diarrhea virus and its adjuvant.

12. Combined bovine diarrhea vaccine containing inactivated coronavirus adult bovine diarrhea virus and other immunizing agents effective against cattle pathogens.

13. A combined bovine diarrhea vaccine containing an inactivated coronavirus adult bovine diarrhea virus and an inactivated reovirus adult bovine diarrhea virus.

14. A method for immunizing newborn cows against newborn cow diarrhea caused by coronavirus adult bovine diarrhea virus, which comprises orally administering the vaccine described in 1 above to the newborn cow.

15. A method for immunizing newborn cows against newborn cow diarrhea caused by coronavirus adult bovine diarrhea virus, which comprises parenterally administering the vaccine described in 9 above to adult female cows before birth.

16. To prevent newborn cow diarrhea caused by coronavirus-like or reovirus adult cow diarrhea virus, which is characterized by parenterally administering the combination vaccine described in 13 above to adult female cows before birth. Immunization methods in.

Claims (1)

[Scope of Claims] 1. A neonatal product characterized in that an attenuated coronavirus-like bovine diarrhea virus is obtained by subculturing coronavirus-like bovine diarrhea virus 5 to about 60 times in a tissue culture of fetal bovine kidney cells. Production method for bovine diarrhea vaccine. 2. A coronavirus-like bovine diarrhea virus is subcultured 5 to about 60 times in tissue culture of fetal bovine kidney cells to obtain an attenuated strain of the virus, which is then further subcultured into fetal bovine kidney, bovine lung, and bovine nasal passages. A cell selected from the group consisting of a bone cell line, a pig kidney cell line, and a pig kidney cell line. A method for producing a diarrhea vaccine for newborn calves, which comprises growing a large amount of the attenuated strain with a titer of 102 to 106.5 in Orizen's post, and then collecting the generated virus.