KR20210056018A - Method of producing porcine reproductive and respiratory syndrome virus for vaccine production - Google Patents

Abstract

The present invention provides a method for producing a porcine reproductive and respiratory syndrome virus for a vaccine, a porcine reproductive and respiratory syndrome virus prepared by the production method, and an immunogenic composition comprising the virus. According to the present invention, it is possible to obtain high titer of a virus and mass-produce a virus for a vaccine.

Description

Method of producing porcine reproductive and respiratory syndrome virus for vaccine production

The present invention relates to a method for producing a swine genital respiratory syndrome virus for vaccine production.

Porcine reproductive and respiratory syndrome (PRRS), along with swine circovirus infection and foot-and-mouth disease, is an infectious disease that causes the most damage to the domestic pig industry. PRRS induces reproductive disorder symptoms such as miscarriage, premature birth, and stillbirth in sows, and causes respiratory symptoms such as sneezing, fever, and so on in piglets and fattening pigs. In general, severe respiratory symptoms are caused by secondary infections such as bacteria after being infected with the virus, but in the case of chronic infection, weight gain decreases and mortality increases without characteristic clinical symptoms.

Pathogens that cause PRRS is the PRRS virus belonging to the genus Arterivirus, the Arteriviridae, Nidovirales neck. The PRRS virus has a positive-sense single stranded RNA genome and is approximately 15.4 kilobases in size. The genome of the PRRS virus has 9 ORFs (Conzelmann et al., 1993; Meulenberg et al., 1993). Among them, ORF1a and ORF1b, which encode non-structural proteins (NSP), occupy about 80% of the viral genome (Bautista et al., 2002; Meulenberg et al., 1993; Snijder and Meulenberg, 1998, 2001 ). The glycosylated structural proteins GP2, GP3, GP4, and GP5, and the non-glycosylated membrane (M) protein and nucleocapsid (N) protein are encoded by ORF, which accounts for the remaining 20%. The auxiliary structural proteins GP2, GP3, GP4 form a heterotrimer and act when the virus invades into the host cell, and the main structural proteins GP5 and M form a heterodimer to increase the infectivity of the virus.

The PRRS virus is highly mutated due to the nature of the RNA virus, so there are many differences between viruses. PRRS virus is largely divided into North American type and European type, and it is known that there is a gene difference of up to 40% between North American type and European type, so that cross-protection is not possible with each other. In addition, cross-protection is not often performed even between mutants belonging to the same type (Meng, X. J. et al., 2000). For this reason, vaccines based on standard mutant strains have been produced for each, but because of poor cross-protection, PRRS is not effectively prevented. In order to overcome this, various attempts have been made to produce a vaccine with effective safety, immunogenicity, and protective ability (application number 10-2011-7004020, title of invention: highly pathogenic porcine genital and respiratory syndrome (HP PRRS) ) Against the vaccine).

Therefore, PRRS vaccine technology is required to produce a vaccine PRRS virus to activate the pig farming business.

One aspect provides a method for the production of porcine genital respiratory syndrome virus.

One aspect provides a porcine genital respiratory syndrome virus produced by a method of producing a virus of porcine genital respiratory syndrome.

One aspect provides a composition comprising a porcine genital respiratory syndrome virus produced by a method for producing a virus of porcine genital respiratory syndrome.

One aspect provides an immunogenic composition comprising a porcine genital respiratory syndrome virus produced by a method for producing a virus of porcine genital respiratory syndrome.

One aspect provides a vaccine comprising a porcine genital respiratory syndrome virus produced by a method of producing a virus of porcine genital respiratory syndrome.

One aspect provides a method for the production of porcine genital respiratory syndrome virus.

The production method comprises the steps of: (a) culturing adherent cells in a bioreactor; (b) inoculating the cells in a bioreactor with the porcine genital respiratory syndrome virus under conditions in which the porcine genital respiratory syndrome virus infects the cells; (c) culturing the infected cells in a bioreactor under conditions in which the infected cells produce porcine genital respiratory syndrome virus; And (d) recovering the porcine genital respiratory syndrome virus produced by the cells.

In the production method, the bioreactor may include a matrix vessel including a carrier and a medium, a pump of the matrix container, and a pump controller.

The matrix container may contain a carrier. The carrier may be a fine carrier, a large carrier, or a combination thereof. The carrier may be porous, non-porous, or a combination thereof. The carrier is about 60% to about 99%, about 70% to about 99%, about 80% to about 99%, about 90% to about 99%, about 60% to about 90%, about 70% to about 90% , Or may have a porosity of about 80% to about 90%. The carrier may be one having a surface area accessible to the cells. The pores may have a surface area for cell expansion. The carrier may be a three-dimensional matrix. The bioreactor may be a three-dimensional bioreactor. The carrier may include cells. The cells can be attached to the carrier. The bioreactor may be formed from a biocompatible material.

The medium may vibrate up and down the matrix container. This may be using tide motion. The medium can move up and down the matrix container. The medium may be vibrated through tidal motion, thereby intermittently exposing cells in the matrix container to air and supplying nutrients. The upward vibration of the medium exposes the cells to nutrients in the medium, and the downward vibration of the medium may expose the cells to air and/or wash products and by-products produced from the cells. The bioreactor using the tidal motion can be used in the art. Culture of cells and/or viruses and inoculation of viruses through vibration of the medium may not create air bubbles in the matrix container containing cells and/or viruses, and the generation of shear stress in the cells. Can be minimized. The air bubbles and/or shear stress may be a detrimental factor to the culture and/or growth of cells and/or viruses.

In the bioreactor, the vibration speed of the medium may be adjusted. The bioreactor may comprise a pump regulator. The pump regulator may be a matrix vessel, or an oscillation regulator of a medium in the matrix vessel. The vibration regulator may adjust the vibration of the pump. The vibration regulator may cause the medium to vibrate in an upward oscillation or downward oscillation of the matrix container. The vertical vibration may be vibration in a direction perpendicular to an axis of the matrix container including the medium.

In the bioreactor, the medium may be a cell culture medium. The medium may contain serum, for example fetal bovine serum. Any type of serum suitable for the growth of cultured cells can be used. Serum may include, for example, fetal bovine serum, fetal calf serum, horse serum, goat serum, rabbit serum, rat serum, mouse serum, or human serum. In one example, the medium may comprise about 10%, or up to about 10% serum, such as fetal bovine serum.

In addition, the medium may be a serum-free medium. The medium may be a protein-free medium. It may be a medium free of additives of serum of human or animal origin.

The cell may be an adherent cell. Adherent cells refer to any cells that are attached or immobilized to a substrate during culture. The cells may be cells capable of proliferating porcine genital respiratory syndrome virus. The cells may be, for example, monkey kidney cells, MARC-145 cells, or porcine alveolar marcrophage (PAM) cells isolated and cultured directly from pigs. The cells may be injected into the matrix container.

The production method may include (a) culturing adherent cells in a bioreactor. In step (a), about 1×10^8 cells to about 5×10^8 cells may be cultured. for step (a), about 1 to about 10 x 10 5 cells/ml; About 2 to about 9 x 10^5 cells/ml; About 3 to about 8 x 10^5 cells/ml; About 4 to about 7 x 10^5 cells/ml; About 5 to about 6 x 10^5 cells/ml; Alternatively, about 5.4 × 10^5 cells/ml may be cultured.

The production method may include (b) inoculating the cells with the porcine genital respiratory syndrome virus in a bioreactor under conditions in which the porcine genital respiratory syndrome virus infects the cells.

In step (b), the cells may be inoculated with the porcine genital respiratory syndrome virus at a multiplicity of infection (MOI) of about 0.25 to about 0.009. The MOI is consistent with the art-accepted meaning, for example a known or predicted ratio of viral agents to viral targets, for example cells, for example porcine genital respiratory syndrome virus. In one embodiment, the MOI is lower than any of the following MOIs: 0.25, 0.22, 0.20, 0.18, 0.15, 0.12, 0.10, 0.08, 0.05, 0.02, or 0.010. In one embodiment, the MOI is greater than any of the following MOIs: 0.009, 0.010, 0.02, 0.05, 0.08, 0.10, 0.12, 0.15, 0.18, 0.20, or 0.22. That is, the MOI has an upper limit of 0.25, 0.22, 0.20, 0.18, 0.15, 0.12, 0.10, 0.08, 0.05, 0.02, or 0.010 and an independently selected 0.009, 0.010, 0.02, 0.05, 0.08, 0.10, 0.12, 0.15, 0.18, It can be any range of MOI with a lower limit of 0.20, or 0.22, where the lower limit is less than the upper limit. In certain embodiments, the cells are inoculated with porcine genital respiratory syndrome virus at an MOI of about 0.01. The bioreactor may be as described above.

The production method may include (c) culturing the infected cells in a bioreactor under conditions in which the infected cells produce swine genital respiratory syndrome virus. In step (c), the bioreactor may be as described above.

The production method may include (d) recovering the porcine genital respiratory syndrome virus produced by the cells. (d) greater than about 2.0 × 10^7 TCID ₅₀ /mL, from about 3.0 × 10^7 to about 1.0 × 10^9 TCID ₅₀ /mL, from about 4.0 × 10^7 to about 1.0 × 10^9 TCID ₅₀ /mL, from about 5.0 × 10^7 to about 1.0 × 10^9 TCID ₅₀ /mL, from about 6.0 × 10^7 to about 1.0 × 10^9 TCID ₅₀ /mL, from about 7.0 × 10^7 to about 1.0 × 10^9 TCID ₅₀ /mL, about 8.0 × 10^7 to about 1.0 × 10^9 TCID ₅₀ /mL, about 9.0 × 10^7 to about 1.0 × 10^9 TCID ₅₀ /mL, about 1.0 × 10^8 To about 1.0×10^9 TCID ₅₀ /mL, or greater than about 1.0×10^8 TCID ₅₀ /mL swine genital respiratory syndrome virus can be recovered.

One aspect provides a porcine genital respiratory syndrome virus produced by the method for producing the porcine genital respiratory syndrome virus described above.

In one embodiment, the porcine genital respiratory syndrome virus may be a chimeric variant of the porcine genital respiratory syndrome virus. The chimeric mutant may include a DNA sequence described in SEQ ID NO: 1 or RNA transcribed from the DNA.

One aspect provides a composition comprising a porcine genital respiratory syndrome virus produced by the method for producing the porcine genital respiratory syndrome virus described above.

One aspect provides an immunogenic composition comprising a porcine genital respiratory syndrome virus produced by the method for producing the porcine genital respiratory syndrome virus described above.

One aspect provides a vaccine comprising a porcine genital respiratory syndrome virus produced by the method for producing the porcine genital respiratory syndrome virus described above.

The vaccine may be an immunogenic substance, that is, a veterinary vaccine containing an antigenic substance. It is specific for porcine genital respiratory syndrome and can be administered for the purpose of inducing active or passive immunity.

The vaccine may be a live venom vaccine or a dead venom vaccine.

The vaccine may contain, in addition to the active ingredient, one or more adjuvants, excipients or carriers suitable for constituting the vaccine. The adjuvant may contain a substance that enhances an individual's immune response. For example, the vaccine may comprise a water-in-oil emulsion adjuvant. It may also further contain one or more emulsifiers. In addition, a protective agent may be additionally included.

In one embodiment, the vaccine may be a vaccine comprising a chimeric variant of the swine genital respiratory syndrome virus. The chimeric mutant strain is as described above. The vaccine containing the chimeric mutant may be a live vaccine.

The production method of the porcine genital respiratory syndrome virus for vaccine production according to the present invention can obtain a virus having a high titer, and can be used for mass production of a vaccine virus.

The production method of porcine genital respiratory syndrome virus by a bioreactor using tidal motion according to the present invention exposes cells in a matrix container to air and supplies nutrients by using the vibration of a medium, while washing products and waste products, It can be suitable for mass vaccine production by suppressing factors harmful to cell growth by minimizing the occurrence of shear stress in cells without creating air bubbles through vibration.

The method for producing vaccine viruses through a bioreactor using tidal movement according to the present invention is suitable for mass production of swine genital respiratory syndrome virus, and chimeric of swine genital respiratory syndrome virus that can be used as a vaccine through previous studies of this research team. It may be suitable for mass production of mutant stocks.

1 shows the growth of MARC-145 cells.
2 shows viral titers grown in a two-dimensional cell culture system and a three-dimensional cell culture system.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Culture and growth of MARC-145 cells using an adherent bioreactor using tidal motion

The adherent bioreactor CelCradle-500AP (Esco Aster, Singapore) is a matrix container containing a carrier and capable of injecting media and cells, pumping the matrix container, e.g., pumping by vibrating up and down, and controlling the speed and time of the pumping. It includes a pump regulator that does. In the bioreactor, a packed bed included in the matrix container is a BioNOC II carrier composed of 100% PET fibers. These carriers not only provide a large area for attachment and growth, but also form a 3D matrix similar to an in vivo environment.

When culturing MARC-145 cells, 10% fetal bovine serum DMEM medium to which antibiotics were added was used. MARC-145 cells spread as a single layer in two T175 flasks were collected through trypsin EDTA, centrifuged at 1,200 rpm for 3 minutes, and then resuspended in the medium. After adding the medium to a total volume of 120 ml, the cells were attached to the vehicle for 3 hours. After 3 hours, when the cells were properly attached to the carrier, the medium was added so that the total volume became 500 ml, and then cultured using the tidal motion shown in Table 1 below. In the bioreactor, the matrix vessel was pumped, i.e., vibrated vertically. Table 1 shows the rising speed and retention time of the medium during the rising vibration of the medium in the bioreactor, the falling speed and the holding time of the medium during the falling vibration of the medium in relation to the conditions for culturing MARC-145 cells. The ascending speed and its holding time, and the descending speed and its holding time were controlled through a pump controller.

Ascent speed Upside hold time Descent speed Down hold time 1.0 mm/s 0 minutes 1.0 mm/s 1 minute

1 shows the growth of MARC-145 cells. According to FIG. 1, on the 5th day of cell culture, the total number of cells reached 2.7×10^8, which was 5.4×10^8 cells/ml.

Example 2: Culture and growth of PRRS virus using an adherent bioreactor using tidal motion

On the 5th day of cell culture, PRRS virus was inoculated at 0.01 MOI for 6 hours using the tidal motion shown in Table 2. Table 2 shows the conditions for viral inoculation, the rising speed and the holding time of the medium in the bioreactor during the rising vibration, the falling speed and the holding time of the medium during the descending vibration of the medium.

Ascent speed Upside hold time Descent speed Down hold time 2.0 mm/s 10 minutes 0.25 mm/s 1 minute

After inoculation, it was cultured using the tidal motion shown in Table 3. The cultivation was done under 0% CO2. Table 3 shows the conditions for viral growth after virus inoculation, as well as the rising speed and holding time of the medium in the bioreactor during the rising vibration, the falling speed and the holding time of the medium during the downward vibration of the medium.

Ascent speed Upside hold time Descent speed Down hold time 1.0 mm/s 1 minute 1.0 mm/s 1 minute

The titer of the PRRS virus was measured using cell titration. As a result of observing CPE by PRRS virus, the amount of virus collected on the 2nd day of virus inoculation ^{reached 1.35×10 8} TCID/ml 500 ml (total 6.74×10 ¹⁰ TCID).

Table 4 and FIG. 2 show viral titers grown in a two-dimensional cell culture system and a three-dimensional cell culture system. The PRRS virus has been shown to exhibit higher titers in the three-dimensional cell culture system than in the two-dimensional cell culture system. While it is possible to collect ^{2.04 × 10 7} TCID/ml 25 ml (total 5.10 × 10 ⁸ TCID) under plate culture conditions, a two-dimensional cell culture system, ^{1.35 × 10 8} TCID/ml 500 ml (total 6.74 × 10 ¹⁰ TCID) was found to be collectable. It can be used to mass-produce PRRS virus for use as a vaccine through the increase in the titer of the virus.

Two-dimensional cell culture system 3D cell culture system Surface area (cm ² ) 175 15,000 Total amount of virus (TCID) 5.10 × 10 ⁸ 6.74 × 10 ¹⁰ Two-dimensional cell culture system comparison efficiency One 132

Claims (9)

As a production method of Porcine Reproductive and Respiratory Syndrome virus (PRRS virus),
(a) culturing adherent cells in a bioreactor;
(b) inoculating the cells in a bioreactor with the porcine genital respiratory syndrome virus under conditions in which the porcine genital respiratory syndrome virus infects the cells;
(c) culturing the infected cells in a bioreactor under conditions in which the infected cells produce porcine genital respiratory syndrome virus; And
(d) recovering the porcine genital respiratory syndrome virus produced by the cells. The method of claim 1,
The bioreactor includes a matrix container including a carrier and a medium and a pump, wherein the pump is capable of vibrating the medium up and down the matrix container. The method of claim 1,
1 × 10^8 cells to 5 × 10^8 cells are cultured in the step (a). The method of claim 1,
The cell is inoculated with porcine genital respiratory syndrome virus at an MOI of 0.25 to 0.009. The method of claim 4,
The cell is inoculated with porcine genital respiratory syndrome virus at an MOI of 0.001. The method of claim 2,
The method wherein the carrier is a matrix. The method of claim 1,
2.0 × 10^7 TCID ₅₀ /mL in step (d) How excess swine genital respiratory syndrome virus is recovered. Porcine genital respiratory syndrome virus produced by the method of any one of claims 1 to 7. An immunogenic composition comprising a porcine genital respiratory syndrome virus produced by the method of any one of claims 1 to 7.

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