Method of egg-yolk antibody for feline coronavirus and egg-yolk antibody manufactured by the same

Abstract

The present invention relates to a method for producing a yolk antibody against feline coronavirus and a yolk antibody produced thereby. Specifically, the invention provides a method for producing a feline coronavirus yolk antibody and a feline coronavirus yolk antibody produced by the method, comprising the steps of: inoculating a feline coronavirus into a CRFK cell line to obtain a feline coronavirus-infected cell line; culturing the cell and isolating a target antigen through ultracentrifugation; inoculating a laying hen with the isolated target antigen; collecting eggs from the laying hen; and isolating a yolk antibody against feline coronavirus from the eggs.

Description

Technology Field

[0001] The present invention relates to a method for producing a yolk antibody against feline coronavirus and a yolk antibody produced thereby. Specifically, the invention provides a method for producing a feline coronavirus yolk antibody and a feline coronavirus yolk antibody produced by the method, comprising the steps of: inoculating a feline coronavirus into a CRFK cell line to obtain a feline coronavirus-infected cell line; culturing the cell and isolating a target antigen through ultracentrifugation; inoculating a laying hen with the isolated target antigen; collecting eggs from the laying hen; and isolating a yolk antibody against feline coronavirus from the eggs. Background Technology

[0002] Feline coronavirus is known as a major causative virus of gastroenteritis. In particular, it is known to progress to chronic infection and proliferate in the intestines, but when the tissue affinity of the virus changes due to a mutated coronavirus, it migrates to tissues outside the intestines to cause granulomatous inflammation, leading to feline infectious peritonitis, which is very fatal to cats.

[0003] Looking at the incidence rates of these, it is known that most cats (about 80% or more) exposed to feline coronavirus become acutely infected, about 20% of them progress to chronic infection and intermittently excrete the coronavirus in their feces, and about 10% of them—that is, about 1-3% of all cats—progress to feline infectious peritonitis (FIP).

[0004] Feline coronavirus is known to cause chronic inflammation in the body's parenchymal organs due to coronaviruses mutated from enteric coronaviruses, which can progress to highly fatal infectious peritonitis. Therefore, diagnosis and analysis of feline coronaviruses prevalent in Korea (such as type 1 and type 2 coronaviruses and infectious peritonitis virus) are the most important core areas for minimizing the fatal clinical course of companion cats caused by infectious peritonitis.

[0005] In follow-up tests on coronavirus-positive samples, it was confirmed that feline coronavirus had been chronically infected for several months in more than 30% of cases, and coronavirus was detected in various non-intestinal tissue specimens, such as ascites, pleural effusion, or intra-abdominal masses, among suspected FIP cases, confirming that FIP ​​is the most threatening infectious disease among companion cats.

[0006] Feline Infectious Peritonitis (FIP) is a disease akin to a death sentence for cats, and it is a group of diseases with a very poor clinical prognosis where treatment is almost always abandoned once a diagnosis is made.

[0007] According to current knowledge, FIP occurs when a cat becomes infected with enteric coronavirus and the infection progresses chronically. During this process, the enteric coronavirus binds to antibodies and is phagocytosed by leukocytes. The coronavirus phagocytosed by leukocytes undergoes genetic mutation, causing a change in the tissue affinity (Tropism) of the virus that replicates in the intestines to a form that allows it to replicate in other parenchymal organs.

[0008] When the disease progresses to FIP, macrophages and multinucleated neutrophils in various parenchymal organs become involved in inflammation, causing granulomatous inflammation accompanied by suppuration, leading to fluid retention in the abdominal or thoracic cavity (wet form FIP), or it progresses to a dry form of FIP in which masses are formed in parenchymal organs such as the kidneys, lymph nodes, brain, and liver, but fluid retention is not observed. In such cases, treatment is difficult, and it has been reported that the mortality rate reaches nearly 100%.

[0009] Due to the high clinical importance of FIP, it is difficult to provide appropriate treatment once it has progressed to FIP; therefore, preventing enterocoronavirus infection at the source or suppressing the continuous replication of the virus during chronic infection can serve as a good means to block the intermediate process leading to FIP. Prior art literature

[0010] Republic of Korea Published Patent No. 10-2019-0122255 The problem to be solved

[0011] The inventors of the present invention provide a method for producing egg yolk that forms antibodies specifically binding to feline coronavirus by inoculating birds with feline coronavirus antigens, and to provide a composition for the prevention and treatment of feline coronavirus suitable for use as an injectable preparation by purifying and isolating the antibodies from the egg yolk to a high degree of purity. means of solving the problem

[0012] To solve the above technical problem, the present invention comprises the step of inoculating a feline coronavirus into a CRFK cell line to obtain a feline coronavirus-infected cell line;

[0013] A step of culturing the above cells and isolating the target antigen through ultracentrifugation;

[0014] Step of inoculating the above-described separated target antigen into laying hens;

[0015] A step of collecting eggs from the above-mentioned laying hen; and

[0016] A method for manufacturing feline coronavirus yolk antibodies is provided, comprising the step of isolating yolk antibodies against feline coronavirus from the above-mentioned egg.

[0017] The present invention obtains yolk antibodies from eggs obtained from laying hens by inoculating them with feline coronavirus as an antigen. The inoculation is preferably carried out by administering a first dose of feline coronavirus antigen at a dose of 0.1 ml or more and 0.5 ml or less, followed by additional doses at a dose of 2 to 4 times after 2 weeks. More preferably, an immune response can be induced by administering 0.3 ml at a time intramuscularly as a first dose and then administering two additional doses at 2-week intervals from the date of the first inoculation.

[0018] The above feline coronavirus antigen is preferably isolated by inoculating a feline coronavirus into a CRFK cell line and culturing it, followed by ultracentrifugation, but is not limited thereto.

[0019] The above feline coronavirus is characterized by causing infectious peritonitis in cats, but is not limited thereto.

[0020] In order to inoculate laying hens with the above-mentioned feline coronavirus antigen, an inoculum containing the feline coronavirus antigen may be prepared, and preferably, an inoculum containing the feline coronavirus antigen may be prepared in an amount of 20% to 40% by volume and inoculated into laying hens. More preferably, an inoculum containing the feline coronavirus antigen may be prepared in an amount of 30% by volume and inoculated into laying hens. In addition to the antigen, the inoculum may additionally include an adjuvant to ensure the desirable action of the vaccine. The adjuvant that may be included in the inoculum of the present invention may use any adjuvant known in the art without limitation, but as a preferred example, ISA70 may be used as an adjuvant. However, such a composition is not necessarily limited thereto and may vary depending on the condition of the individual and the type and degree of progression of the disease.

[0021] In addition, when administering the above feline coronavirus antigen, an adjuvant may be administered together, and the adjuvant is characterized by being one or more selected from the group consisting of complete / incomplete Freud's adjuvant, aluminum hydroxide gel, aluminum phosphate gel, mineral oil, and polysorbate 80.

[0022] The step of separating the egg yolk antibody may further include the step of centrifuging the egg yolk containing the antibody, followed by purification through ion exchange chromatography and gel permeation chromatography.

[0023] In another embodiment of the present invention, a feline coronavirus yolk antibody prepared by the above-described yolk antibody preparation method is provided.

[0024] In another embodiment of the present invention, a composition for the prevention and treatment of feline infectious peritonitis is provided, comprising the egg yolk antibody as an active ingredient.

[0025] The egg yolk antibody composition can be prepared in the form of an injectable with a purity of 90% or higher by centrifuging the egg yolk containing the antibody and then purifying it through ion exchange chromatography and gel permeation chromatography. This is because if the purity is less than 90%, it is not suitable for use as an injectable.

[0026] The egg yolk antibody composition of the present invention for the prevention and treatment of feline infectious peritonitis caused by feline coronavirus can be used as an injectable preparation when its purity is 90% or higher, and can also be used as a functional food by adding it to feed when its purity is less than 90%.

[0027] The antibody obtained from the above purification process can be used as an injectable preparation by diluting it in phosphate buffer solution (PBS) at an appropriate ratio. Effects of the invention

[0028] The method for producing a feline coronavirus yolk antibody according to the present invention can produce an antibody that specifically binds to the feline coronavirus, and the antibody specifically binds to the feline coronavirus that causes feline infectious peritonitis, thereby maximizing the immune function and therapeutic effect against the said disease. Brief explanation of the drawing

[0029] Figure 1 is a graph showing the difference in virus detection titers for feline coronavirus-positive samples (enteric coronavirus vs. FIP). Figure 2 is a photograph showing the results of virus isolation showing cytopathic effects after inoculating a feline coronavirus-positive sample into a CRFK cell line. Figure 3 is a photograph of a hybridoma cell line that produces monoclonal antibodies showing reactivity to feline coronavirus antigens. Figure 4 is a photograph showing the ELISA results for virus-specific antigens performed after the fourth immunization of laying hens. Specific details for implementing the invention

[0030] The present invention will be explained in more detail below through examples. However, these examples are intended only to aid in understanding the invention and do not limit the scope of the invention in any way.

[0032] Examples

[0033] 1. Securing a coronavirus-positive sample

[0034] Among the various specimens submitted to PopAniLab for testing, the presence and amount of coronavirus were quantified using a real-time RT-qPCR diagnostic kit equipped with a feline coronavirus-specific probe provided by PostBio Co., Ltd., targeting feline gastroenteritis specimens (feces) and suspected FIP specimens.

[0035] From January 1 to November 30, 2018, the presence of feline coronavirus was confirmed in 642 fecal samples submitted for gastroenteritis testing among cat specimens submitted to the pet entrusted testing service (Pop Ani Lab) operated by Post Bio. As a result of the test, feline coronavirus was detected in 316 out of 642 cases, and the average Ct value of the detected coronavirus was confirmed to be 27.71.

[0036] Analysis of 316 confirmed cases of feline coronavirus infection revealed that 81 cases (25.6%) were confirmed as feline coronavirus infections alone, while 235 cases (approximately 74.4%) involved mixed infections with 18 additional infectious disease pathogens, including bacteria, viruses, and protozoa, that can cause gastroenteritis in cats, along with feline coronavirus infection.

[0037] A comparative analysis of the quantity and positive rate of specimens in which enteric coronavirus was detected among cases of gastroenteritis referred between January and November 2018 revealed that, unlike the general pattern where viral diseases are prevalent in winter and bacterial diseases are prevalent in summer, the period with a high positive rate was approximately 56% and the period with a low positive rate was 42.3%. Although there were slight differences depending on the time period, it was confirmed that the incidence rate was very high throughout the year and there was no difference in the infection rate according to the time period.

[0038] From January 1 to November 30, 2018, the presence of feline coronavirus was confirmed in 43 cases of body fluids (ascites or pleural fluid, etc.) submitted for Feline Infectious Peritonitis (FIP) testing among cat specimens submitted to the pet testing service (Pop Ani Lab) operated by Post Bio. As a result of the test, feline coronavirus was detected in 14 of the 43 specimens, and the average Ct value of the detected coronavirus was confirmed to be 32.68.

[0039] When comparing the relative quantitative values ​​of the virus using Realtime RT-PCR on positive samples in which enteric coronavirus was detected and positive samples in infectious peritonitis, it was confirmed that the viral titer in positive cases of enteric coronavirus infection was about 36 times lower than in infectious peritonitis. It was found that in the case of infectious peritonitis, the viral titer is low because inflammation progresses chronically, and in addition, there were about 67.4% of cases where infectious peritonitis was suspected based on clinical symptoms but the coronavirus was not detected (Fig. 1).

[0041] 2. Feline Coronavirus Gene Sequencing Analysis

[0042] RT-PCR was performed on structural and non-structural protein genes of feline coronavirus obtained from various clinical cases, and the amplified products were analyzed using standard DNA sequencing methods to identify genotypes and genetic mutations of feline coronavirus circulating in Korea.

[0043] The primers used for RT-PCR have the sequences listed in [Table 1].

[0044] Target gene Primer name order NSP3 FCoV nsp3 F1 (sequence number 1) 5′-ATCCATATGGTTCTGGCATGG-3 FCoV nsp3 R1 (sequence number 2) 5′-TTTAGCYGTACTATAATCATTGAGCA-3′ NSP12 FCoV nsp12 F1 (sequence number 3) 5′-CCCACAATGACTCAAATGAA-3′ FCoV nsp12 R1 (sequence number 4) 5′-TCTGGTTCYACCCAACACTT-3′ spike FCoV S F1 (Sequence No. 5) 5′-TCTGTKGCCATCAAAATCAC-3′ FCoV S R1 (Sequence No. 6) 5′-CATTAACATCHACCATTACATCTG-3′ Membrane glycoprotein FCoV M F1 (Sequence No. 7) 5′-GCGGTTMTAAACGAAATTGA-3′ FCoV M R1 (Sequence No. 8) 5′-TGAGTAATCACCRGCTTTAGATTT-3′

[0045] Primers for performing real-time PCR on target gene sequences for virus detection were added to the PCR reaction mixture at a final concentration of 500 nM, and fluorescently labeled probes to confirm the amplification reaction results were added at a final concentration of 250 nM. RT-PCR was performed by amplifying the target gene sequences for each virus detection by performing a reverse transcription reaction at 15°C, followed by denaturation at 95°C for 10 minutes, and repeating the conditions of 95°C for 10 seconds and 55°C for 30 seconds for 45 cycles. For the RT-PCR amplification reaction, the templates used were prepared in six stages by serially diluting the RNA extracted from the feline coronavirus obtained above tenfold. In addition, to verify the results of the real-time PCR amplification reaction and to analyze them quantitatively, the probe is double-labeled with a fluorescent substance and a quenching substance. As previously mentioned, in the case of the probe used to detect the coronavirus, the 5' end was labeled with the fluorescent substance FAM (maximum absorption wavelength: 495 nm, maximum emission wavelength: 520 nm), and the 3' end was labeled with the quenching substance ZEN-IBFQ from IDT.

[0046] Serial No. Symptom classification Ct Value RT-PCR results NSP3 NSP12 Spike Membrane 1 FIP 18.59 + + 2 FIP 34.86 + + 3 FIP 33.87 + + 4 FIP 33.50 + + 5 FIP 33.80 6 FIP 32.51 7 FIP 22.51 + + 8 Enteric FCoV 29.22 + + 9 Enteric FCoV 23.75 + + + + 10 Enteric FCoV 26.61 + + + 11 Enteric FCoV 19.05 + + + + 12 Enteric FCoV 27.05 + + + 13 Enteric FCoV 24.59 + + + 14 Enteric FCoV 21.67 + + + + 15 Enteric FCoV 26.00 + + + +

[0047] RT-PCR was performed on four genes of coronaviruses confirmed positive in the two clinical conditions (gastroenteritis and infectious peritonitis) used in the study. As a result, gene amplification was confirmed for the non-structural protein NSP3 and the structural protein Spike protein in both disease groups among the samples used in the study, whereas gene amplification for NSP12 and Membrane proteins could not be confirmed in the infectious peritonitis samples, whereas gene amplification was confirmed in the gastroenteritis samples. In the case of the Spike protein, which was detected in both clinical cases, gene amplification occurred in both clinical cases, but the size of the amplified product differed depending on the clinical sample, so it was expected that there would be significant genetic mutations between the feline coronaviruses detected in infectious peritonitis and acute gastroenteritis.

[0049] 3. Securing feline coronavirus isolates

[0050] Using specimens selected for the production of target antigens based on clinical symptoms, an attempt was made to isolate virus strains using two cell lines (FCWF4, CRFK) susceptible to feline coronavirus.

[0051] The sample selected for virus isolation is filtered through a 0.2 µm filter for cell culture, then inoculated into two cell lines that have been prepared in advance and are forming a monolayer, and the occurrence of cytotoxic effects is checked for one week.

[0052] Even if no cytopathic effects occur, after undergoing freezing and thawing about two times, inoculate the two prepared cell lines again and observe for an additional week.

[0053] Among various clinical symptom specimens, a specimen confirmed to be positive for feline coronavirus antigen was used to inoculate a cell line and attempt virus culture as described above, but virus culture from feline gastroenteritis specimens failed, and one case of feline infectious peritonitis virus culture was successful from the CRFK cell line (Fig. 2).

[0055] 4. Purification of viral antigens for antibody production

[0056] After securing feline coronavirus isolates, the target antigens necessary for the production of feline coronavirus-specific antibodies were purified through ultracentrifugation and specific gravity-based ultracentrifugation processes to obtain large quantities of viral antigens through mass culture, and utilized as immunosources for antibody production.

[0058] 5. Production of Feline Coronavirus-Specific Antibodies

[0059] Virus-specific monoclonal antibodies are produced using various viral antigens obtained through the previous step, employing technologies such as mouse immunity and antibody library technology.

[0060] To produce monoclonal antibodies, mice were immunized three times intraperitoneally with inactivated antigens obtained from a feline coronavirus clinical isolate, followed by final boosting via microvenous injection. Hybridoma cell lines responding to the immunogen were obtained through the fusion of splenic immune cells and mouse myeloma cells and the screening of positive cell lines using a generally known monoclonal antibody production method, and 11 cell lines showing an ELISA OD of 0.6 or higher were obtained (Fig. 3).

[0061] [Table 3] ELISA results for virus-specific antigens performed after mouse antigen immunization

[0062]

[0064] Chickens are inoculated multiple times using viral antigens, and once sufficient immune antibodies are secured, yolk antibodies specific to the coronavirus are obtained from the yolks of eggs obtained from the immunized chickens.

[0065] Coronavirus antigens were immunized to five laying hens four times, and blood samples were collected before, after the second, and after the fourth immunization to evaluate the degree of immunity in the laying hens. As shown in [Figure 4] and [Table 4], the evaluation results confirmed an increase in virus-specific antibodies in the serum.

[0066] [Table 4] ELISA results for virus-specific antigens performed after 4th immunization of laying hens

[0067]

Claims

Claim 1 A method for producing feline coronavirus yolk antibodies, comprising the steps of: inoculating a feline coronavirus into a CRFK cell line to obtain a feline coronavirus-infected cell line; culturing the cell and isolating a target antigen through ultracentrifugation; inoculating a laying hen with the isolated target antigen; collecting eggs from the laying hen; and isolating yolk antibodies against feline coronavirus from the eggs, wherein the inoculation is characterized by a first inoculation of 0.1 ml or more and 0.5 ml or less, followed by additional inoculations of 2 to 4 times at 2-week intervals, and wherein the coronavirus causes feline infectious peritonitis. Claim 2 delete Claim 3 delete Claim 4 Feline coronavirus egg yolk antibody manufactured according to Article 1. Claim 5 delete

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