JPS6124370B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a vaccine against a new coronavirus-like agent that causes 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 corona vaccine-like factor is grown on a cell culture surface and then inactivated. Neonatal calf diarrhea (NCD)
(abbreviated as )) are complex. Many types of bacteria are implicated as causes of this disease, including bacteria, fungi, mycoplasma, chlamydia, and viruses.
Particularly mentioned viruses include bovine diarrhea virus, infectious bovine rhinotracheitis virus, Aday virus, enterovirus and HADEN virus. Other viruses that have been reported as etiological factors for NCD include bovine pneumonia enteritis virus and a reovirus-like agent called NCD virus. this
For methods of making vaccines effective against NCD reovirus-like agents, see U.S. Patent Application No. 197520;
Filed on November 10, 1971. During field trials of vaccines produced from reovirus-like agents, inadequate control of disease in cattle was observed in some herds in western Nebraska [Vet.Med./Small Anim.Clin., 67
(2), p. 173 (1972)]. As a typical example, cases of diarrhea were observed in several groups of cows that were administered this vaccine within the first 24 hours of birth, even when the cows were 5 to 20 days old. The diarrheal feces of these cows were tested for reovirus-like viruses using the 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 reached 7 to 12 days of age, was removed by duodenal infusion into calves born by uterine incision (Caesarean) and not fed colostrum. Inoculated. The calf developed diarrhea and bacteria-free faecal fluid was prepared from the diarrheal stool, which was then orally inoculated into the gnotobictie calf. 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 found in the diarrheal stools of both the vaccinated and non-vaccinated groups 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 propagated and transformed into an effective vaccine in a cell culture medium, and that an effective vaccine can be obtained by growing this virus in a cell culture medium. 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.
The purification and characterization of this factor is described in Am.J.Vet.
Res., Vol. 33, pp. 1147-1156 (1972). Purification of coronavirus-like agents: Stool samples were collected from diarrheal calves from 19 ranch herds in western Nebraska. Calves in groups 12-19 were pre-vaccinated orally with NCD reovirus-like virus vaccine, while calves in other groups were not vaccinated. Faecal smears from calves with diarrhea = NCD
Stained with immunofluorescent complex for virus testing [Nebracas State University, Agri.Exper.Sta.Res.
Bull. p. 233 (1969)] and only samples negative for NCD virus were subjected to the test. Diarrheal fecal material collected from naturally infected and artificially infected calves on the farm was kept at −20°C to −20°C until virus purification began.
Stored at 60°C. Various sucrose concentration gradient solutions were prepared and each solution was left at 4° C. to form a linear gradient before use. Each gradient contains 400, 300, 200 and 400 sucrose per ml of distilled deionized water, respectively.
It is made up of 8ml containing 100mg. Each solution was placed in a 2.5 x 8.9 cm 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 for 30 minutes.
Centrifuged at 5000G. The supernatant was then centrifuged for 3 hours at 25000 rpm in a type 30 rotor. 5% of the obtained pellets
(w/v) in sucrose solution by sonication for 30 seconds to disperse. 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 25000 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 6 cm above the liquid surface) is further purified by sucrose gradient centrifugation or cesium chloride (CsCl) gradient centrifugation. The semi-purified virus preparation was further purified by repeated sucrose gradient centrifugation as described above. Locate the zone again, collect the zone 6.0 cm from the liquid surface, and dialyze against 1% ammonium acetate to obtain purified virus. Isogradient centrifugation of semi-purified virus preparations
This was done starting with a 30% solution (15 g CsCl + 35 ml deionized water). This solution (4 ml) was aliquoted into nitrocellulose centrifuge tubes, and then 1.4 ml of the virus suspension was added to each tube. Using SW65 rotor
If centrifuged at 60,000 rpm for 18 hours, complete equilibrium has been reached at this point. The refractive index of CsCl in the zone where the virus exists is measured using a TS meter. A layer of 2.9 cm from the liquid surface was collected,
Purified virus is obtained by dialysis against 1% ammonium acetate solution. In addition, the crude virus suspension (15 ml) described above was added to
4.2 x 42 cm gel column (Sepharose 2B:
The fractions were fractionated by gel filtration on Pharmacia Fine Chemicals, Inc., Piscataway, New Jersey). The column was equilibrated with 0.9% NaCl in advance and developed with this saline solution. flow rate
The flow rate was 6.0ml/hour. 5 ml of fraction was collected and the absorbance was measured spectrophotometrically at 260 μm. The material from each peak is combined and then separately concentrated by ultrafiltration. A simplified method for preparing virus from diarrheal stool material from test calves is also applied.
Centrifuge the stool material directly at 3400G for 30 minutes. A portion of the supernatant liquid was diluted with dichlorodifluoromethane.
Extract times. The aqueous layer (5 ml) and untreated supernatant (5 ml) 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 formulations for immunofluorescence analysis. Blood was collected (14 ml) from the viscera of a 6-month-old white rabbit, and then 0.4 ml of antigen was collected.
and 0.6ml of complete Freund's auxiliary solution.
Intramuscular injections were given at 4 sites at 0.25 ml per site. Five weeks after inoculation, 40 ml of blood is collected by cardiac puncture. Gamma globulin labeled with fluorescein was prepared using a known method (Proc.USLivestoek San.Assn.
, October 1968, pp. 139-144) from post-inoculation serum. The presence of the coronavirus-like agent 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 described below. When sections of the intestines of calves with diarrhea infected with a coronavirus-like agent were 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. Intestinal sections from calves with normal gnotobiosis 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 remain on the grid for 3 to 25 minutes depending on the degree of light scattering of the band under test. Excess liquid is blotted with paper and then a small drop of vanadium molybdate-phosphotungstic acid solution is added onto the grid. The dye is allowed to distill over the grid for 1 to 1.5 minutes before the excess is removed with a blotter. The sample is 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 the complete virus particles ranged from 107 to 160 μm in the same micrograph and had surface protrusions similar to those seen on coronaviruses. As measured by electron microscopy,
The average size of the particles including surface protrusions was 126 μm. The nuclear capsid was plesiomorphic, varying in size and shape from circular to oblong. The outer skin of the capsid is the fringe.
This becomes especially clear when the The width of the outer edge is maximum
It varies up to 23μm. The surface projections of the well-retained grains are petal-shaped, connected to the grains by elongated pods, and have an average length of 11 μm. The coronavirus-like agents described in this invention are easily distinguished from other viruses that have been demonstrated or proposed to be pathogenic in newborn calves on the basis of size and morphological characteristics. . The coronavirus-like agents mentioned here include enterovirus, bovine pneumonia-enteritis virus, bovine diarrhea virus,
It has morphological characteristics different from neonatal calf diarrhea virus (ioform) and Haeden virus, and is larger than these. This new agent is smaller than bovine infectious rhinotracheitis virus and lacks its typical morphological features. Buoyant density of purified coronavirus-like agent
density) is 1.24 using CsCl, while the buoyant density of reovirus-like factor is 1.359 [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 gnotobiotic cattle can be as short as 13.5-14 hours, whereas the minimum incubation period observed for coronavirus-like agents is 18 hours. Furthermore, gnotobiosis cows infected with reovirus exhibit symptoms of diarrhea for 5 to 8 hours, and then return to normal 24 hours after the onset of diarrhea. In contrast, gnotobiosis 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 fetal bovine kidney cells. The virus can also be grown on cells from 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 hydrolyzate (abbreviated as LAH), 0.1% yeast extract, and 100 units of penicillin and 200μ of streptomycin per ml.
Earle's Balanced Salt Solution storage medium containing 100 g is then added to 30-40°C, preferably about
Incubate for 2-10 days at ℃. The virus is collected, for example, by draining the supernatant from the cells. The virus inoculum can be feces collected from coronavirus-infected cows or culture fluid from coronavirus-like virus cultures. Stool can be collected as is or diluted with phosphate buffer (PH7.2).
Centrifuge at 1000G for 20 minutes and the supernatant can be used as inoculum. Other storage media can be used, such as modified Eagle's 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 coronavirus vaccines are obtained by subculturing purified virus in fetal bovine kidney cells a sufficient number of times to ensure that inoculated cattle do not become ill when challenged with the virulent virus. . Typically, the virus needs to be passaged 5 to 60 times, and this passage is carried out in primary or 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 cell lines. Preferred methods include a combination of subculturing on primary or secondary cells and 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 viruses can be detected by e.g. cytopathic effects, immunofluorescence,
Or measured by blood absorption method. 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 and the remaining primary cells were treated with 0.5% LAH, 10% adult bovine serum, and 10% LAH.
Add Earle's balanced salt solution containing % dimethyl sulfoxide and freeze. The bottles and coverslip 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 primary 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 passages 1 through 15, 2 ml of liquid is used to inoculate 8 oz. bottles as described above. For passages 15 to 25, add 2 ml of liquid to the storage medium in the bottle. After 25 passages, add 1 ml 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 are infected, and the remaining uninfected cells are subcultured to serve as cells at a subsequent high-passage culture level. This type of cell culture medium is called a passage cell culture medium. A suitable cell-virus system is formed by continuing to subculture the virus in continuous cell culture medium. Virus in secondary cell culture medium
After ~20 passages, the virus can be passaged 5 to 40 times or more in subculture cell culture to obtain an effectively attenuated coronavirus-like virus vaccine. The culture period for subculturing the virus decreases as the number of passages increases, and subculturing on higher passage cells or cell lines can be performed 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 nasal turbinate cell lines or porcine kidney cells or cell lines, in which the tissue culture contains approximately 37
Culture at °C until an appropriate virus titer is obtained.
It can then be produced by collecting the virus. A titer of 10 ² to 10 ^{6 .5} , preferably 10 ⁴ to 10 ⁶ 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 consists of a virus that has been attenuated by being passaged 12 times on secondary cells and then 15 times on cells that have themselves been passaged between 9 and 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,
It is produced by first culturing this factor on fetal bovine kidney cells as described above until a suitable titer is obtained. The titer should be as high as possible within the above range. Viruses at any level of cell subculture, even non-attenuated viruses, can be used. The virus is then inactivated using inactivating agents or means known to those skilled in the art, such as formalin, β-propiolactone, ultraviolet light, or heat, at 20-40°C to effectively inactivate the virus. It is inactivated by treatment for a time and concentration. 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. Add formalin 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. Add aluminum hydroxide gel adjuvant to this inactivated virus to a 10% concentration. Adjust the pH to approximately 7.2 with aluminum hydroxide. This attenuated vaccine can be administered to calves, preferably newborn calves, in doses of 1 to 5 ml containing 10 ² to ^{10 6.5} TCID/ml to confer immunity 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 production or to newborn calves as soon as possible after birth, 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 composites include inactivated coronaviruses and reoviruses, both of which are produced by inactivating non-attenuated or live attenuated viruses by chemical agents, radiation or heat. Coronaviruses can also be complexed with bovine adenovirus, live diarrhea virus, or other bovine pathogens such as Escherichia coli or Clostridia Sp. Combined vaccines containing coronavirus and reovirus are produced by mixing equal amounts of both inactivated viral agents. Next, formalin at a concentration of 0.2% and aluminum hydroxide gel were added to this.
Add 10% by volume. This vaccine is administered parenterally in 1 to 5 ml to pregnant cows. 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, protection 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 calf.
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 approximately 7-hour-old gnotobiotic calves, and the calves were then exposed to a virulent virus (a virulent coronavirus-like virus collected from the diarrheal feces of infected cows). ) to test the efficacy of the attenuated virus vaccine. These test results are shown in Tables 1 and 2.
Shown in the table. Calf 1 was orally inoculated with 10 ml of cell culture fluid obtained by subculturing the virus 14 times on secondary fetal bovine kidney cells.
The patient developed diarrhea and then recovered. On the 9th day of life, this calf was given 10 ml of diarrheal stool (toxic virus).
The animal was orally inoculated with 20ml of PBS, but no symptoms occurred. 15 Virus on Secondary Bovine Fetal Kidney Cells
When calf 2 was inoculated with 10 ml of the subculture, it did not cause diarrhea and did not become ill even after being challenged with the virulent virus. When calves 3 and 4 were inoculated with 10 ml of the virus that had been cultured 16 times on secondary fetal bovine kidney cells, a weak onset of diarrhea was observed. When 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, immunofluorescence was observed in the intestinal villi and epithelium of the colon. Calf 4 was inoculated with a toxic virus found in diarrheal stool on the 6th day after birth, but did not develop any disease. When calf 6 was orally inoculated with 5 ml of the virus that had been cultured 19 times on secondary fetal bovine kidney cells, diarrhea developed. Viruses cultured at least 5 times on high-passage fetal bovine kidney cells, 13 to 25 times cultured, and 5 ml were added to 12 calves (No. 7 to 13 and No. 15).
-19) did not develop diarrhea after oral inoculation, and, with the exception of calf 18, no disease occurred even after oral inoculation with feces containing the toxic virus. A combined vaccine containing inactivated coronavirus and reovirus was tested on adult cattle from two farms (farms contaminated with virulent coronavirus-like viruses). The vaccine was produced from formalin-inactivated 27-passage cultured coronavirus and formalin-inactivated 201-passage cultured reovirus (on primary bovine kidney and bovine kidney cell lines). Equal amounts of each inactivated virus were mixed with each other, and 10% aluminum hydroxide gel was added. Explanation of the table for the test results in Tables 1 and 2: D = Diarrhea onset, N = No abnormality, b = Number of passages of virus on primary or secondary cells + on higher passage cells Number of passages at.

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[Table] Embodiments of the present invention are listed below. 1. Passage the coronavirus-like bovine diarrhea virus 5 to about 60 times in a tissue culture medium that maintains the growth of the virus until the virus is inoculated into newborn calves and the onset of the disease is suppressed against attack by the virulent virus. A method for attenuating coronavirus-like bovine diarrhea virus, which comprises inoculation. 2. The method according to 1 above, wherein 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 subcultures is 10 to 40 times, of which 5 to 10 subcultures are performed in primary or secondary bovine fetal kidney cell culture medium, and the remaining subcultures are carried out in high-order subcultures. A method performed using subcultured bovine fetal kidney cell culture medium. 4. A bovine diarrhea vaccine containing an attenuated coronavirus-like bovine diarrhea virus produced by the method in 1 above. 5. A bovine diarrhea vaccine produced by the method of 2 above and containing an attenuated coronavirus-like bovine diarrhea virus. 6. A bovine diarrhea vaccine produced by the method of 3 above and containing an attenuated coronavirus-like bovine diarrhea virus. 7. An attenuated coronavirus-like bovine diarrhea virus characterized by growing the coronavirus-like 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 generated viral material. A method for mass production of diarrhea virus. 8. A bovine diarrhea vaccine produced by the method described in 7 above and containing an attenuated coronavirus-like bovine diarrhea virus. 9. A method for producing an inactivated coronavirus-like bovine diarrhea vaccine, which comprises inactivating the coronavirus-like bovine diarrhea virus and incorporating it into an adjuvant. 10 The method according to 9 above, wherein the inactivating agent is formalin. 11. An inactivated coronavirus-like bovine diarrhea vaccine produced by the method described in 9 above, containing an inactivated coronavirus-like bovine diarrhea virus and its adjuvant. 12 A combined bovine diarrhea vaccine containing inactivated coronavirus-like bovine diarrhea virus and other immunizing agents effective against the pathogen in cattle. 13 A combined bovine diarrhea vaccine containing inactivated coronavirus-like bovine diarrhea virus and inactivated reovirus-like bovine diarrhea virus. 14. A method for immunizing newborn cows against newborn cow diarrhea caused by coronavirus-like bovine diarrhea virus, which comprises orally administering the vaccine described in 1 above to newborn cows. 15. A method for immunizing newborn cows against newborn cow diarrhea caused by coronavirus-like bovine diarrhea virus, which comprises parenterally administering the vaccine described in 9 above to adult pregnant cows before the birth of the newborn cow. 16 A method for treating newborn cow diarrhea caused by coronavirus-like or reovirus-like bovine diarrhea virus, which is characterized by parenterally administering the combination vaccine described in 13 above to adult pregnant cows before the birth of the new cow. Immunization methods.

Claims (1)

[Claims] 1. A non-attenuated coronavirus-like bovine diarrhea virus was treated with formalin, β. Propiolactone, inactivated by treatment with UV irradiation or heat, and combined with adjuvants selected from the group consisting of aluminum hydroxide gel, potassium alum, alginates, oil bases, mineral oils and organic oils. A method for producing a newborn calf diarrhea vaccine characterized by the following.