WO2014046410A1 - Method for producing virus-like particles of nodavirus, yeast cell line expressing same, and vaccine composition containing same - Google Patents

Abstract

The present invention relates to a method for producing virus-like particles of nodavirus, a yeast cell line expressing the same, and a vaccine composition containing the same. According to the method of the present invention, it is possible to mass produce high-quality virus-like particles of nodavirus by expressing a nodavirus capsid protein through a yeast expression system and purifying the same by chromatography. The virus-like particles of nodavirus prepared by the method of the present invention can be very useful as an antigen of a vaccine against nodavirus infection. In addition, the present invention provides a recombinant yeast strain expressing a nodavirus capsid protein, and a vaccine composition containing the recombinant yeast strain or a cultured product thereof.

Description

Production method of virus like particles of nodavirus, yeast cell line expressing the same and vaccine composition comprising the same

The present invention relates to a method for producing virus-like particles (VLP) of nodavirus, a yeast cell line expressing the same, and an immunogenic composition and vaccine composition comprising the same as an active ingredient.

The nodaviridae family consists of alphanodavirus and betanodavirus [1]. Alpha nodaviruses infect insects and beta nodaviruses are known to infect fish [1]. Betanoviruses can infect more than 40 species of fish and can cause nerve damage (nervous necrosis) resulting in 100% death [2]. Betanovirus is mainly infected with the central nerve and is known to infect fish during the growing season [2]. Betanodavirus species known to date include stripped jack nervous necrosis virus (SJNNV), barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV), and red spotted grouper nervous necrosis virus (RGNNV) [3]. BFNNV is mainly found in fish inhabiting the Korean Wave, while RGNNV is found mainly in fish inhabiting the turbulent ocean [4]. Fish infected with nodaviruses exhibit abnormal swimming and brain lesions that form vacuole in brain tissue [5].

Species susceptible to beta-daviruses include convict surgeonfish (Acanthurus triostegus), lined surgeonfish (A. lineatus), narrowstripe cardinalfish (Apogon exostigma), and three-spotted dome (threespot). dascyllus; Dascyllus trimaculatus), Scopas tang; Zebrasoma scopas, blueband goby; Valenciennea strigata, tiger puffer; Takifugu rubripes, lemon peel angelfish (lemonpeel angelfish; Centropyge flavissimus) Orbicularis batfish (Platax orbicularis) is known [6, 7].

Nodavirus has 3.1 kb of RNA 1 and 1.4 kb of RNA2. RNA 1 encodes an RNA-dependent RNA polymerase [8] and RNA 2 encodes a capsid protein [9]. During RNA replication, an additional 0.4 kb of RNA 3 is synthesized and RNA 3 is known to contain two open reading frames (ORFs). Both ORFs encode a B1 protein of 111 amino acids and a B2 protein of 75 amino acids [10, 11].

To date, prophylactic vaccines against nodaviruses have been tried in various forms, including virus inactivated virus vaccines, synthetic peptide vaccines, DNA vaccines, and recombinant capsid protein vaccines [12]. Among them, the vaccine using capsid protein showed a good preventive effect in fish and also induced high virus neutralizing activity in blood [13, 14]. The amino acid regions 1-32, 91-162, 181-212, 254-256 of betanovirus virus capsid proteins are known to be important in the formation of neutralizing antibodies in host fish [15, 16]. Thus, the capsid protein of nodavirus is considered to be the most useful antigen candidate.

Capsid proteins produced by cDNA cloning of RNA 2 are known to self-assemble to form virus-like particles (VLPs) similar to the structure of nodaviruses [17]. The tertiary structure of the beta-novirus virus like particles, revealed by cryo-electron microscopy, is in the form of T-30 capsid, 25-30 nm in size [18]. To date, viral analogues of nodavirus have been produced in Escherichai coli and insect cells (spodoptera frugiperda) [17, 19]. However, there have not been reported examples of producing virus analog particles of nodavirus using the yeast production system.

Ultracentrifugation using sucrose cushion or cesium chloride cushion is commonly used to purify virus like particles of nodaviruses [17, 19, 20]. However, these purification methods have limitations that can be applied only to the production of antigens on a laboratory scale. Purification using column chromatography is essential for the actual production of viral vaccines [21]. However, no method for the high efficiency purification of virus particles of Nodavirus using column chromatography has been developed.

In order to induce protective immunity against pathogens, a method of injecting purified antigen subcutaneously or intramuscularly has been used. Immunization of antigens by injection can induce high levels of antibody titers and cellular immune responses, but much effort has been put into securing safety for vaccines during the manufacturing process. High purity purified antigens are required for the preparation of injectable vaccines, and intense care is required for the removal of cell line derived impurities and toxins.

Mass vaccination of animals and fish by injection has many limitations in economic terms. Injectable vaccines are expensive to produce, which can put economic burdens on livestock and fish farmers when inoculated in large quantities. In particular, injecting immunity against aquaculture fish requires a lot of time and effort.

Vaccine immunization through mucous membranes, such as oral or nasal, has been considered the most ideal way to immunize fish and animals in large quantities. Mucosal immunity, however, induces a significantly lower immune response than injection-induced immunity and in many cases fails protective defense. Low pH of the digestive system and enzymes for proteolysis are major factors that make oral immunity difficult. Most vaccine antigens break down or lose antigenicity as they pass through the digestive tract. Even if the antigenicity remains, the digestive organs often recognize these antigens as food and cannot cause an immune response. Therefore, oral immunization requires a significantly larger amount of antigen than immunization, and even when a large amount of antigen is used, a lower antibody response is induced than immunization through injection. As a barrier to the development of such oral vaccines, vaccination of farmed fish is still carried out by injection.

Meanwhile, Korean Patent Publication No. 10-2011-0135206 discloses a polypeptide comprising an epitope of a fish nodavirus protein, a recombinant expression vector comprising the polypeptide, a host cell transformed with the vector, and a production by the host cell. Although recombinant proteins have been described, there is no mention of expression in yeast strains as in the present invention.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have tried to develop a method for stably mass production of virus analog particles (VLP) of nodavirus for vaccine production. As a result, by expressing the capsid protein of nodavirus using a yeast cell expression system and purifying the expressed yeast cell lysate by column chromatography method, it is possible to obtain a large amount of virus-like particles of high quality nodavirus. Confirmed that the present invention was completed. In addition, when the yeast cell line expressing the nodavirus capsid protein itself is used as a vaccine composition, it shows a superior protective immune inducing effect than when the purified capsid protein is used as a vaccine composition, which is superior to the yeast strain containing the capsid protein itself. It was confirmed that the immune inducing ability and completed the present invention.

It is therefore an object of the present invention to provide a method for producing virus like particles of nodavirus.

Another object of the present invention is to provide a virus like particle of nodavirus assembled from a nodavirus capsid protein.

It is another object of the present invention to provide an immunogenic composition comprising virus like particles of nodavirus.

Another object of the present invention is to provide a recombinant yeast strain expressing the nodavirus capsid protein.

Still another object of the present invention is to provide a vaccine composition comprising a virus like particle of nodavirus, a recombinant yeast strain expressing a nodavirus capsid protein, or a culture thereof.

The objects and advantages of the invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for producing a nodavirus virus analogous particle comprising the steps of: (a) transformed with an expression vector comprising a DNA sequence encoding a nodavirus capsid protein Culturing yeast cells expressing the nodavirus capsid protein; (b) lysing the cultured yeast cells to obtain cell lysates; And (c) subjecting the cell lysate to chromatography to purify the nodavirus capsid protein.

According to another aspect of the present invention, the present invention provides a virus-like particle (VLP) of nodavirus assembled from a nodavirus capsid protein.

According to another aspect of the present invention, the present invention comprises a virus-like particle (VLP) of nodavirus assembled from a nodavirus capsid protein, and is immunogenic capable of inducing an immune response against nodavirus. It provides a composition (immunogenic composition).

According to another aspect of the present invention, the present invention provides a recombinant yeast strain expressing the nodavirus capsid protein.

According to another aspect of the invention, the present invention (a) expresses an immunogenic composition comprising a virus-like particle (VLP) of nodavirus assembled from the nodavirus capsid protein, nodavirus capsid protein Immunologically effective amount of a recombinant yeast strain or culture thereof; And (b) a pharmaceutically acceptable carrier.

The present invention relates to a method for producing virus-like particles (VLP) of nodavirus, a yeast cell line expressing the same, and an immunogenic composition and vaccine composition comprising the same as an active ingredient. According to the method of the present invention, the capsid protein of nodavirus is expressed through a yeast expression system, and then purified through a chromatographic purification method to mass produce high quality nodavirus virus analogous particles. In addition, the prepared nodavirus virus analogous particles can be very usefully used as an antigen of a vaccine against nodavirus infection.

Meanwhile, according to the results of the present invention, when the yeast cell line expressing the nodavirus capsid protein itself was used as the vaccine composition, the protective capsid protein exhibited a superior protective immune inducing effect than the vaccine composition. Therefore, this means that the yeast strain containing the capsid protein itself has excellent immune inducing ability. When using this type of recombinant yeast strain as an oral vaccine, the process for preparing the vaccine antigen can be omitted. In addition, using it for oral vaccines can save a great deal of time, effort and money for vaccination.

Figure 1 shows the sequence of DNA encoding the capsid protein of RGNNV nodavirus of the present invention.

FIG. 2 shows the amino acid sequence of 338 aa translated from the DNA sequence of FIG. 1.

Figure 3 shows the configuration of the recombinant vector YEGα-MCS-NNVcp inserted with the DNA sequence (NNV capsid protein gene, 1017 bp) encoding the nodavirus capsid protein. The DNA sequence was inserted at the HindIII and SalI restriction enzyme sites of the vector YEGα-MCS.

Figure 4 is the result of analyzing the main section in which nodavirus capsid protein is eluted in heparin chromatography. The concentration of NaCl from which nodavirus capsid protein was eluted was confirmed by SDS-PAGE and Western blotting. Cell lysates containing capsid proteins were bound to heparin resin under buffer conditions containing 0.15 M NaCl. Subsequently, an elution buffer containing 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, and 1.2 M NaCl, respectively, was flowed sequentially. As a result, the capsid protein was clearly reduced in impurities from the NaCl containing elution buffer of 0.5M to 0.6M, it was confirmed that the largest amount is eluted when flowing the buffer containing 1.2M NaCl. It was confirmed that a large amount of impurity protein was eluted with the capsid protein in the elution buffer section containing 0.4-0.5 M NaCl.

5 shows the process of purifying the nodavirus capsid protein. Cell lysates were dialyzed against heparin binding buffer after crushing Saccharomyces cerevisiae expressing nodavirus capsid proteins. Capsid proteins in dialysis completed cell lysate were purified by heparin chromatography. Fractions containing the capsid protein were concentrated using a centrifugal filter device and then dialyzed with a buffer containing 0.5 NaCl.

6 is a result of performing heparin chromatography under optimized binding conditions based on the result of FIG. 4. LS and FT refer to a loading sample (LS) loaded on heparin resin, and a flow-throug (FT) flowed out without binding. Cell lysates were prepared by dialysis with a buffer containing 0.5 M NaCl. The purified cell lysate was passed through heparin resin and purified, and the high purity capsid protein was confirmed in the elution fraction of 1.2 M NaCl. The heparin chromatography fraction containing the capsid protein was concentrated and dialyzed against a buffer containing 0.5 M NaCl.

FIG. 7 shows the results of 6 μg of the purified nodavirus capsid protein developed by SDS-PAGE, followed by silver staining (Panel A) and Western blotting (Panel B). Capsid protein was identified as the major band 37 kDa band, 110 kDa band was also identified. This is consistent with the SDS-PAGE results of previously known nodavirus capsid proteins [19].

8 shows the results of observing the purified capsid protein with a transmission electron microscope (80 kv). It was confirmed that the capsid protein forms a virus analog of 25-30 nm size similar to that of nodavirus.

9 is a result of confirming the purified capsid protein by an energy transmission electron microscope (120 kv). The results of energy transmission electron microscopy also confirmed that the capsid protein forms a virus like particle of 25-30 nm size similar to that of nodavirus.

10 shows the results of dynamic light scattering analysis of purified capsid proteins. Dynamic light scattering analysis shows the size of capsid proteins present in solution. In dynamic light scattering analysis, the capsid protein was found to have an average size of 103 nm.

FIG. 11 shows Western RGNNV capsid protein expression levels of recombinant Saccharomyces cerevisiae (rs) expressing RGNNV capsid protein and recombinant Yarrowia lipolytica (rY) expressing RGNNV capsid protein. This is the result of the blot analysis. Panels (a) and (b) show the results of rS and rY expression analysis. 48 μg of rS was found to contain 200 ng of RGNNV capsid protein. 140 μg of rY was found to contain 60 ng of RGNNV capsid protein. Thus, 1 mg of rS contained 4.1 μg of RGNNV capsid protein and 1 mg of rY contained 0.4 μg of RGNNV capsid protein.

12 shows anti-RGNNV capsid protein IgG titers in blood of mice after the 3rd and 4th immunization. PBS means orally immunized phosphate buffered saline (PBS), rS means orally immunized Saccharomyces cerevisiae expressing the RGNNV capsid protein. rY refers to a group orally immunized Yarrowia lipolytica expressing the RGNNV capsid protein. VLP-S and VLP-Y refer to a group orally administered capsid proteins purified according to the amount of capsid proteins administered to the rS and rY groups.

Figure 13 shows the result of neutralizing activity against RGNNV of mouse serum after the fourth oral immunization. Cytopathic effect (CPE) was observed when serum of PBS, VLP-S and VLP-Y groups were reacted with RGNNV and then infected with E-11 cells. On the other hand, when the serum of the rS and rY groups were reacted with RGNNV and then infected with E-11 cells, no cell lesion was observed. Therefore, it was confirmed that rS and rY effectively induced neutralizing antibodies against RGNNV.

According to one aspect of the present invention, the present invention provides a method for producing a nodavirus virus analogous particle comprising the steps of: (a) transformed with an expression vector comprising a DNA sequence encoding a nodavirus capsid protein Culturing yeast cells expressing the nodavirus capsid protein; (b) lysing the cultured yeast cells to obtain cell lysates; And (c) subjecting the cell lysate to chromatography to purify the nodavirus capsid protein.

Hereinafter, the method of the present invention will be described in detail with each step.

(a) culturing yeast cells transformed with an expression vector comprising a DNA sequence encoding the nodavirus capsid protein to express the nodavirus capsid protein.

As used herein, the term “nodavirus” is a virus belonging to the nodaviridae family and includes both an alphanodavirus that hosts insects and a betanodavirus that infects fish. Nodaviruses do not have an envelope, the virion is 29-35 nm in size, and the capsid consists of 32 capsomers. The genome of nodaviruses is a linear, positive sense, single-stranded RNA of about 4500 nucleotides, consisting of two sites of RNA 1 and RNA 2.

RNA 1, about 3.1 kb in size, encodes a protein with a multi-functional domain, wherein the multi-functional domain comprises a mitochondrial targeting domain, a transmembrane domain, an RNA-dependent RNA polymerase (RdRp) domain, self-interaction Functional domain and RNA capping domain. RNA 1 also encodes a subgenomic RNA3 translated to protein B2, which is an RNA silencing inhibitor.

About 1.4 kb of RNA 2 encodes protein α, a viral capsid protein precursor, which is cleaved into two mature proteins, 38 kDa β protein and 5 kDa γ protein, during viral assembly.

Preferably the "nodavirus" of the present invention is a beta nodavirus (betanodavirus) that infects fish, the beta noda virus is SJNNV (striped jack nervous necrosis virus), BFNNV (barfin flounder nervous necrosis virus), TPNNV (tiger puffer) nervous necrosis virus (RGNNV), red spotted grouper nervous necrosis virus (RGNNV), malabaricus grouper nervous necrosis virus (MGNNV), and dragon grouper nervous necrosis virus (DGNNV).

In the present invention, "a DNA sequence encoding nodavirus capsid protein" can be obtained through a suitable method known in the art. For example, total viral RNA can be used as a template for amplification into cDNA through suitable primers and reverse-transcriptase polymerase chain reaction (RT-PCR). Total viral RNA can be isolated from, for example, the brains of fish infected with nodaviruses. The cloned cDNA is inserted into a suitable expression vector and transformed into host cells for expression of the capsid protein.

In the present invention, the “nodavirus capsid protein” is an amino acid sequence of SEQ ID NO: 2, an amino acid sequence having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99% identity with this sequence It consists of a nodavirus capsid protein.

According to another preferred embodiment of the present invention, the "DNA sequence encoding the nodavirus capsid protein" of the present invention is the base sequence of SEQ ID NO: 1 codon optimized for expression in S. cerevisiae (S. cerevisiae) It consists of.

The specific construction of the vector expressing the nodavirus capsid protein of the present invention is described in the following examples and illustrated in FIG. 3.

In the present invention, the cell used as a host cell is a yeast, and a preferred yeast strain genus includes, for example, Saccharomyces, Candida, Cryptococcus (Cryptococcus). ), Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia, and more preferably. Are Saccharomyces, Candida, Hansenul, Peachia and Ski Research Carromamyses, most preferably Saccharomyces.

On the other hand, preferred yeast strain species (Species) Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces carlsbergensis, Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcus laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Klutoluco morphus Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus var.lactis, Pichia pastoris, Rhodotorula Rhodotorula rubra, Schizosaccharomyces pombe, Yarrowia lipolytica, and the like. Most preferably the host yeast of the present invention is Saccharomyces cerevisiae .

Transformed yeast of the present invention refers to yeast cells transformed with an expression vector that successfully expresses the nodavirus capsid protein. The expression vector may include transcriptional or translational regulatory elements and other marker genes known in the art.

Transformed yeasts expressing the nodavirus capsid proteins of the invention can be readily prepared using methods known in the art, such methods being described in US Pat. Nos. US 7250170, US 6613557, US 5888516, US 5871998, US 5618536, US5437951 and the like, the contents of these patent documents are incorporated herein by reference.

Cultivation of the transformed yeast of the present invention is carried out using a medium known to those skilled in the art in which the yeast strain can be cultured. An effective medium is generally a water soluble medium containing anabolic carbohydrates, nitrogen and phosphate sources, as well as suitable salts, minerals, metals and other nutrients such as vitamins and growth factors. The medium may be a complex nutrient or a minimal minimal medium. Transformed yeast of the present invention may be cultured in a variety of vessels including, but not limited to, bioreactors, flasks, test tubes, microtiter dishes, and petri plates. Cultivation of yeast is carried out at temperatures, pH and oxygen concentrations suitable for yeast cells. Culture conditions for yeast cells are known to those skilled in the art, for example Guthrie et al. (eds.), 1991, Methods in Enzymolgy, vol. 194, Academic Press, San Diego.

(b) lysing the cultured yeast cells to obtain cell lysate.

The method for lysing the yeast cells cultured in the present invention may be any method capable of obtaining a total lysate of the yeast cells, and is not limited to a specific method. Dissolution methods that can be used in the present invention may use, for example, crushing by ultrasonication, crushing by glass beads, crushing by using a surfactant, and combinations of these methods, but It is not limited.

(c) chromatography of the cell lysate to purify the nodavirus capsid protein.

The host yeast cells expressing the nodavirus capsid protein are lysed and the lysate is chromatographed to purify the nodavirus capsid protein. In the previous method, ultracentrifugation using a sucrose cushion or cesium chloride cushion was used to purify the capsid protein of nodavirus, but the method of the present invention is characterized by using a chromatography method for mass purification of the capsid protein.

Chromatographic methods usable in the present invention include cation-exchange chromatography, anion-exchange chromatography, affinity chromatography and size-exclusion chromatography. In particular, the method of the present invention can be used heparin chromatography having both cation exchange chromatography characteristics and affinity chromatography characteristics.

In the present invention, when the nodavirus capsid protein is purified by heparin chromatography, it is preferable to use an elution buffer containing NaCl at a concentration of 0.15 M-1.2 M. More preferably, the use of an elution buffer containing 0.5 M-1.2 M NaCl can effectively purify the capsid protein in terms of purity and amount. In the present invention, the concentration of NaCl contained in the elution buffer is preferably It is at a concentration of 0.6 M-1.2 M, more preferably 0.7 M-1.2 M, even more preferably 0.8 M-1.2 M, most preferably 0.9 M-1.2 M. Elution buffers with concentrations of NaCl in this range can be used to obtain pure nodavirus capsid protein fractions that do not contain contaminants.

In the present invention, when the nodavirus capsid protein is purified by heparin chromatography, the concentration of NaCl included in the binding buffer is not particularly limited. However, 0.5 M is most suitable according to the following specific examples.

According to one specific example below and the results shown in FIGS. 8 to 10, the nodavirus capsid protein obtained by the production method of the present invention successfully infected virus analog particles having a size of 25 to 35 nm, similar in size to that of the nodavirus. Formed. Therefore, according to the method of the present invention, a virus like particle composed of nodavirus capsid can be obtained.

According to another aspect of the present invention, the present invention provides a virus-like particle (VLP) of nodavirus assembled from a nodavirus capsid protein.

According to another aspect of the present invention, the present invention comprises a virus-like particle (VLP) of nodavirus assembled from a nodavirus capsid protein, and is immunogenic capable of inducing an immune response against nodavirus. It provides a composition (immunogenic composition).

According to another aspect of the present invention, the present invention provides a recombinant yeast strain expressing the nodavirus capsid protein.

According to another aspect of the invention, the present invention (a) expresses an immunogenic composition comprising a virus-like particle (VLP) of nodavirus assembled from the nodavirus capsid protein, nodavirus capsid protein Immunologically effective amount of a recombinant yeast strain or culture thereof; And (b) a pharmaceutically acceptable carrier.

According to a preferred embodiment of the present invention, the virus analog particles of the nodavirus are prepared by the method for producing nodavirus analog particles of the present invention described above.

According to a preferred embodiment of the invention, the nodavirus analogue of the present invention is an amino acid sequence of SEQ ID NO: 2, at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or at least 99 It consists of a nodavirus capsid protein consisting of an amino acid sequence having% identity.

According to another preferred embodiment of the present invention, the vaccine composition of the present invention is for preventing or treating nodavirus infection in fish.

The nodavirus includes alpha nodavirus and beta nodavirus, more preferably beta nodavirus. The beta nodal virus is, for example, striped jack nervous necrosis virus (SJNNV), barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV), red spotted grouper nervous necrosis virus (RGNNV), or Malabaricus grouper nervous (MGNNV). necrosis virus), and dragon grouper nervous necrosis virus (DGNNV).

According to another preferred embodiment of the present invention, the vaccine composition is a vaccine composition for fish, and the fish is a fish, flounder, perch, or rockfish, but is not limited thereto.

According to a preferred embodiment of the invention, the immunogenic composition of the invention is adjusted or buffered within the range of pH 7.0 to 8.0, or pH 7.2 to 7.6.

The vaccine composition of the present invention may comprise one or more pharmaceutically acceptable carriers. A pharmaceutically acceptable carrier in the present invention means any component suitable for delivering the virus analog particles of the antigen nodavirus to an in vivo site, for example, water, saline, phosphate buffered saline, Ringer's solution, dextrose. Solutions, serum-containing solutions, Hans' solutions, other water-soluble physiological equilibrium solutions, oils, esters and glycols, and the like.

Carriers of the present invention may include suitable auxiliary ingredients and preservatives to enhance chemical stability and isotonicity, and may include temperature stabilizers or freezes by including stabilizers such as trehalose, glycine, sorbitol, lactose or monosodium glutamate (MSG). The vaccine composition can be protected against drying. The vaccine composition of the present invention may comprise a suspension liquid, such as sterile water or saline (preferably buffered saline).

The immunogenic composition or vaccine composition of the present invention may contain any adjuvant in an amount sufficient to enhance the immune response to the immunogen. Suitable adjuvants are described in Takahashi et al. (1990) Nature 344: 873-875, for example, aluminum salts (aluminum phosphate or aluminum hydroxide), squalene mixtures (SAF-1), muramyl peptides, saponin derivatives, mycobacterial cell wall preparations, monophos Polyl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunits, polyphosphazenes and derivatives, and immunostimulatory complexes (ISCOMs).

As with all other immunogenic compositions or vaccine compositions, an immunologically effective amount of an immunogen should be determined empirically, in which case factors that can be considered include immunogenicity, route of administration and the number of immune doses administered.

Nodavirus virus like particles in the vaccine composition of the present invention may be present in various concentrations in the composition of the present invention, but typically, the virus like particles of the nodavirus are required to induce an appropriate level of antibody formation in vivo. Include. In this sense, an “immunologically effective amount” of the vaccine composition of the invention means an amount capable of inducing a suitable immune response against nodavirus in vivo, wherein the immunogenic composition or vaccine composition is preferably 1-100 μg. , More preferably 5-50 μg, even more preferably 5-25 μg of virus analog particles.

The vaccine composition of the present invention can be used to protect or treat animals sensitive to nodavirus infection by administering the vaccine composition via the systemic or mucosal route. Administration of the vaccine composition can include injection via the intramuscular, intraperitoneal, intradermal or subcutaneous route, oral / meal, respiratory, mucosal administration to the genitourinary tract.

The preparation of a vaccine or vaccine composition is generally described in Vaccine Design ("The subunit and adjuvant approach" (eds Powell MF & Newman MJ) (1995) Plenum Press New York), which is hereby incorporated by reference. Is inserted into.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Production and Purification of Virus-like Particles (VLPs) of Noda Viruses

< Experiment Method>

1. Construction of Nodavirus capsid protein expression vector and host cell transformation

DNA sequence (NNVcp) encoding capsid protein of RGNNV (red spotted grouper nervous necrosis virus) was synthesized by Blueheron biotechnology Inc. (USA). NNVcp was constructed to have codon configuration with optimized expression in Saccharomyces cerevisiae (FIG. 1). The prepared NNVcp was inserted into the HindIII and SalI restriction sites of the YEGα-MCS vector to construct a recombinant vector YEGα-MCS-NNVcp (see FIG. 3). The recombinant vector YEGα-MCS-NNVcp prepared above was transformed using Saccharomyces cerevisiae Y2805 as a host cell according to a known method [25]. The transformant into which the recombinant vector YEGα-MCS-NNVcp was introduced was selected by culturing in a synthetic medium lacking uracil.

2. Cell Culture

S. cerevisiae Y2805 expressing a nodavirus capsid protein was cultured according to a known method [26]. More specifically, the transformant into which the recombinant vector YEGα-MCS-NNVcp was introduced was selected by the above method and inoculated in SD-ura liquid medium and cultured for 2 days. The cultured cells were cultured in YPDG medium containing 1% yeast extract (Duchefa, Netherlands), 2% peptone (Duchefa), 7% glucose (Duchefa) and 1% galactose (Duchefa). After completion of the culture, the cultured cells were centrifuged to remove the culture solution, and washed with PBS (phosphate-buffered saline). The washed cells were collected by centrifugation and stored at -70 ° C until protein purification.

3. Purification of Virus Analog Particles

The cultured cells were mixed 1: 1 with cell disruption buffer (10 mM sodium phosphate dibasic, 0.15 M NaCl, 0.05% Tween 80 pH 7.2). The mixed solution was again mixed with 0.5 mm glass beads (glass bead, Biospec Product, USA) and vortexed to crush the cells. Cell residue was removed by centrifugation for 15 min at 12,000 × g. The supernatant from which cell residues have been removed is mixed with the supernatant in a ratio of 1: 5 to 1:10 (supernatant: binding buffer) or dialyzed against binding buffer to make the composition of heparin chromatography binding buffer. It was. Heparin binding buffer consists of 10 mM Tris, 0.15-0.5 M NaCl, 5% glycerol, 0.05% Tween 80, 0.1% β-mercaptoethanol, 50 mM L-glutamine, 50 mM L-arginine pH 7.2-8.0 It was.

POROS50 HE heparin resin (Applied Biosystems, USA) was equilibrated by flowing heparin binding buffer at least 5 times the resin volume. The cell lysate prepared with the heparin binding buffer composition was passed through the heparin resin, and then the binding buffer was flowed at least 5 times the resin volume to wash the resin. The elution buffer was then flowed to elute the nodavirus capsid protein. Elution buffer was 10 mM Tris, 5% glycerol, 0.05% Tween 80, 0.1% β-mercaptoethanol, 50 mM L-glutamine, 50 mM L-arginine pH 7.2-8.0 in NaCl 0.65 M, 0.75 M, respectively. , 0.85 M, 0.95 M, 1.2 M was prepared to flow sequentially. The capsid protein of the eluate was identified by SDS-PAGE and Western blot by the method described below. The eluate containing the capsid protein was concentrated using Ultracel-100K (Millipore, USA), the concentrate was 10 mM Tris, 0.5 M NaCl, 0.05% Tween 80, 0.1% β-mercaptoethanol, 50 mM L-glutamine, Dialysis against 50 mM L-arginine pH 8.0 buffer. Protein concentrations in purification steps were measured using a protein quantification kit (Bio-Rad, USA).

4. SDS-PAGE and Western Blotting

SDS-PAGE was performed according to Laemmli's method [27], and the developed protein was visualized by silver staining. Proteins were run on SDS-PAGE gels for Western blotting and then transferred to PVDF membranes (Millipore, USA). PVDF membranes were blocked with PBS-T (PBS + 0.05% Tween 20) containing 5% skim milk (Bioworld, USA). To detect nodavirus capsid proteins, rabbit anti-betanodavirus capsid serum as primary antibody and goat HRP-attached anti-rabbit IgG polyclonal antibody (Bethyl, USA) were used as secondary antibody. Antibody-bound betanovirus capsid proteins were visualized on X-ray films using the ECL kit (Santacruz, USA). Anti-betanovirus virus capsid serum was purchased from Gendocs Inc. South Korea.

5. Electron Microscope Analysis

The purified nodavirus capsid protein was adsorbed onto a carbon-coated grid and then stained with phosphotungstic acid or uranyl acetate. Transmission electron micrographs were taken using JEM1010 (JEOL, Japan) [25], and energy transmission electron micrographs were taken using LIBRA 120 (Carl Zeiss, Germany).

6. Dynamic Light Scattering Analysis

 Purified capsid proteins for dynamic light scattering analysis of nodavirus capsid proteins were prepared in buffer of 10 mM Tris, 0.5 M NaCl, 0.05% Tween 80, 50 mM L-glutamine, 50 mM L-arginine pH 8.0. It was then analyzed using an ELSZ-2 system (Otsuka Electronics, Japan).

< Experiment Result>

1. Analysis of synthesized nodavirus capsid coding DNA sequence

DNA sequences encoding nodavirus RGNNV capsid proteins (NNVcp DNA, 1017 bp) and amino acid sequences translated from NNVcp DNA are shown in FIGS. 1 and 2, respectively. The amino acid sequence of FIG. 2 was confirmed to match the NCBI reference sequence YP_611157.1 (capsid protein of RGNNV, 338 aa). The NNVcp DNA encodes the YP_611157.1 amino acid sequence, but the codon is constructed to optimize expression in S. cerevisiae . Recombinant expression vector for the expression of nodavirus capsid protein was prepared by inserting the prepared NNVcp DNA into the vector YEGα-MCS (Fig. 3).

2. Characterization of Heparin Binding of Nodavirus Capsid Protein

To characterize the binding of the nodavirus capsid protein to the heparin resin, the cultured cells were disrupted and then prepared in binding buffer containing 0.15 M NaCl. In addition to the NaCl composition, the remaining composition of the binding buffer and the elution buffer was the same as that presented in the experimental method. After loading the cell lysate in the heparin resin equilibrated with the buffer, the concentration of NaCl was confirmed while eluting the nodavirus capsid (FIG. 4). A buffer containing 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.2 M NaCl was flowed sequentially to the capsid-bound heparin resin. As shown in the results of Figure 4, the purity and amount of the Nodavirus capsid protein eluted in 1.2 M NaCl was the best, the fraction eluted with a buffer of 0.4-0.5 M NaCl was found to contain a lot of impurity protein . Therefore, according to the results of FIG. 4, the ideal concentration of NaCl in the elution buffer for separating the nodavirus capsid protein in heparin chromatography is shown. It was confirmed that the concentration of 0.5 M or more and 1.2 M or less.

3. Purification of Nodavirus Capsid Protein Using Heparin Chromatography

The purification process of the nodavirus capsid protein is shown in FIG. 5. Cell lysates were prepared by dialysis on heparin binding buffer containing 0.5 M NaCl, and then loaded on heparin resin. The concentration of NaCl in heparin binding buffer was determined under 0.5 M NaCl conditions based on the results in FIG. 4. As shown in the results of FIG. 6, high purity nodavirus capsid proteins were identified in the fraction of the elution buffer containing 1.2 M NaCl. Elution buffer fractions containing 1.2 M NaCl were concentrated and dialyzed against 0.5 M NaCl containing buffer.

The finally obtained nodavirus capsid protein was confirmed on SDS-PAGE. As shown in the results of Figure 7, 37 kDa capsid protein was identified as the main band, a band of 110 kDa was further confirmed. A band of 110 kDa is also shown in the SDS-PAGE and Western blotting results of the 1.2 M NaCl fractions of FIGS. 4 and 6. The band patterns of 37 kDa and 110 kDa are consistent with the band patterns of known nodavirus capsid proteins [19]. In conclusion, these results show that the purity of the nodavirus capsid protein purified by the above method is excellent.

Saccharomyces serevisiae ( S. cerevisiae ) expressing the Nodavirus capsid protein was purified from 1 L culture results are shown in Table 1. Table 1 shows that most impurity proteins were removed after heparin chromatography.

Table 1

step	Volume (ml)	Total protein amount (mg)
Cell lysate	90	1074
Heparin Chromatography Sample Loading	200	1072
Heparin Chromatography Elution	10	8.2
Final product	One	1.2

4. Electron Microscopy Analysis of Purified Nodavirus Capsid Protein

The results of electron microscopic analysis of the purified nodavirus capsid proteins are shown in FIG. 8. The horizontal bar in FIG. 8 means a size of 100 nm. The result of analyzing the purified nodavirus capsed protein using an energy transmission electron microscope is shown in FIG. In Figure 9, the horizontal bars represent 50 nm size. Electron microscopic analysis showed that the purified capsid proteins were in the form of virus pseudoparticles of 25-30 nm size. This is similar to the form of known nodaviruses [5].

5. Dynamic Light Scattering Analysis of Purified Nodavirus Capsid Proteins

When the purified nodavirus capsid was in solution, the size was examined by dynamic light scattering analysis. 10 shows the results of the dynamic light scattering analysis. In the dynamic light scattering assay, the average size of purified nodavirus capsid protein was found to be 103 nm. This is similar to the results of dynamic light scattering analysis of known virus mimics [28].

Example 2 Construction of Recombinant Yeast Cell Line Expressing Nodavirus Capsid Protein and Induction of Protective Immune

< Experiment Method>

1. Preparation of RGNNV Capsid Protein Expressing Yeast

Nucleic acid sequences for encoding RGNNV capsid proteins in the present invention are shown in FIG. 1. YEG-αMCS-opt-RGNNV-cp vector was constructed by inserting the YEG-αMCS vector into the nucleic acid sequence of FIG. 1. Saccharomyces cerevisiae Y2805 was transformed with YEG-αMCS-opt-RGNNV-cp and transformants were selected from synthetic media lacking uracil. Selected transformants were cultured in YPDG medium containing 1% yeast extract (Duchefa, Netherlands), 2% peptone (Duchefa), 4% glucose (Duchefa) and 4% galactose (Duchefa). Incubation was for 48 hours at 30 ° C.

The nucleic acid sequence of FIG. 1 was inserted into pINATX2 and pIMR53 vectors to express RGNNV capsid proteins in Yarrowia lipolytica. Yarrowia lipolytica was transformed with the two vectors prepared above, and transformants containing both vectors were selected from synthetic media lacking uracil and leucine. Selected transformants were cultured in a medium containing 1% yeast extract (Duchefa), 2% peptone (Duchefa), 2% glucose (Duchefa). Incubation was for 48 hours at 28 ℃.

2. Mouse Oral Immunity of RGNNV Capsid Protein and RGNNV Capsid Protein Expressing Yeast

Cultured cells were lyophilized after freezing to -70 ℃. After weighing the lyophilized cells, Western blot was used to determine the amount of capsid protein expression per dry weight. 1 mg capsid protein expression Saccharomyces cerevisiae (rs) was found to express 4.1 μg of capsid protein. 1 mg of capsid protein expression Yarrowia lipolytica (rY) was found to express 0.43 μg of capsid protein. The amount of capsid protein expression for each cell line is shown in detail in FIG. 11.

The rS group received 10 mg (dry weight) of rS per immunization. 10 mg rS contains 40 μg of capsid protein. The VLP-S group was set up to administer the same amount of capsid protein as the dose of rS. The VLP-S group was orally administered 40 μg of purified capsid protein (according to Bradford protein quantitative concentration). The rY group received 35 mg (dry weight) of rY orally per immunization. 35 mg rY contains 15 μg of capsid protein. The VLP-Y group was set up to administer the same amount of capsid protein as the dose of rY. The VLP-Y group was orally administered 15 μg of purified capsid protein (according to Bradford protein quantitative concentration).

The amount of antigen administered per immunization is shown in Table 2. Immunization progressed four times every two weeks. Each antigen per immunization was administered in combination with 5 mg of saponin (Saponin from quilaja bark, Sigma). Three consecutive dose regimens were made for one immunization. Therefore, the VLP-S, rS, VLP-Y, and rY groups received 40 μg, 10 mg, 15 μg, and 35 mg of antigen divided into 3 days. After the third and fourth oral immunizations, blood was collected through the tail vein of the mouse. Sera were collected by centrifuging mouse blood at 12000 × g and stored at −70 ° C. until neutralizing antibody activity and anti-RGNNV capsid protein IgG measurements.

TABLE 2

Mouse group	Dose
PBS	300 uL
VLP-S	40 ug (Bradford Protein Quantitation)
rS	10 mg (dry weight)
VLP-Y	15 ug (Bradford Protein Quantitation)
rY	35 mg (dry weight)

3. Western blot for RGNNV capsid protein detection

Purified RGNNV capsid proteins and cell lysates were transferred onto PVDF membranes (Millipore, USA) after being run on a 12% acrylamide gel. Protein-adsorbed PVDF membranes were blocked with a buffer containing 5% skim mik (Bioworld, USA) (tris-buffered saline with 0.1% Tween 20, TBST). PVDF membranes were then reacted with rabbit anti-RGNNV capsid protein polyclonal antibodies. Rabbit anti-RGNNV capsid protein polyclonal antibodies were awarded from Gendocs Inc., South Korea. The membrane was washed three times with TBST for 10 minutes, and then reacted with HRP-labeled goat anti-earth IgG antibody (Bethyl, USA) and washed four times for 10 minutes. RGNNV capsid proteins were detected using the ECL kit (Santacruz, USA).

4. Determination of Anti-RGNNV Capsid Protein IgG Titers

Purified RGNNV capsid proteins were coated with 200 ng per well in 96 well ELISA plates. Coating was carried out at 4 ° C. for 16 hours. Capsid proteins were diluted in PBS for coating. After coating each well was blocked with PBS-T (PBS containing 0.05% Tween 20) containing 3% bovine serum albumin. Blocking was performed for 1 hour at room temperature. The mouse serum was then serially diluted three times with PBS-T containing 0.3% bovine serum albumin and applied to the wells and reacted at 37 ° C. for 1 hour. The plate was washed three times with PBS-T and then reacted with HPR attached goat anti-mouse Ig antibody (Bethyl, USA) for 1 hour at 37 ° C. Color development was carried out using o-phenylenediamine dihydrochloride (Sigma, USA) as substrate and color reaction was measured at 492 nm. End-point titers were determined to be 1.5 times the optical density values of mouse serum immunized with PBS.

5. Measurement of RGNNV Neutralizing Activity in Mouse Serum Using E-11 Cells

Neutralizing antibody titers were determined by modifying known methods. E-11 cells were dispensed into 96 well cell culture plates at 1 × 10 ⁴ cells per well and incubated at 25 ° C. As a medium for cell culture, L-15 medium (Gibco, USA) containing 10% FBS and 1% penicillin-streptomycin was used. When cells formed 80-90% confluency, a mixture of mouse serum and RGNNV was infected with the prepared cells. Mouse serum was diluted 1:50 or 1: 200 in L-15 medium with 5% FBS. 75 μL of mouse serum dilutions were mixed with 75 μL of RGNNV (10 ² TCID ₅₀ ) and allowed to stand at room temperature for 30 minutes. Serum + RGNNV mixed solution was removed from the culture medium of E-11 cells prior to infection with the cells. Thereafter, 75 μL of mouse serum and RGNNV mixed solution were infected with E-11 cells, and cultured at 25 ° C. for 4 days. After 4 days of culture, the cell lesion was observed under a microscope.

< Experiment Result>

1.Determination of Dose of Recombinant Yeast Cell Line and Purified RGNNV Capsid Antigen for Mouse Oral Immunity

Western blot was performed to evaluate the amount of RGNNV capsid proteins expressed by recombinant Saccharomyces cerevisiae (rS) and recombinant Yarrowia lipolytica (rY) (FIG. 11). To confirm the expression level of recombinant yeast, rS was loaded with 48, 24, 12, 6, 3 μg (dry weight) of cells and the capsid amount contained in these cells was 200, 100, 50, 25, 12 ng (Bradford protein Quantitative) of purified RGNNV capsid protein. rY was loaded with 140, 70, 35, 17 μg (dry weight) of cells and the amount of capsid contained in these cells was compared with 60, 30, 15, 7.5 ng (Bradford protein quantification) of purified RGNNV capsid proteins. 48 μg of rS contained 200 ng of capsid protein and 140 μg of rY contained 60 ng of capsid protein. Thus, 1 mg of rS expresses 4.1 μg of capsid protein and 1 mg of rY expresses 0.43 μg of capsid protein.

2. Comparison of immunogenicity between purified RGNNV capsid protein and RGNNV capsid protein in oral immunity

12 shows the result of measuring antibody titers in blood after the third and fourth immunization of purified RGNNV capsid protein and yeast (rS, rY) expressing RGNNV capsid protein by mouse oral. The VLP-S group is a group immunized with purified RGNNV capsid protein in accordance with the amount of capsid protein expressed by rS. The VLP-Y group is an orally immunized RGNNV capsid protein purified to the amount of capsid protein expressed by rY. Antibody titers in each mouse group were calculated as median values. Anti-RGNNV capsid protein IgG antibody titers were measured after tertiary oral immunity. The VLP-S group showed an antibody titer of 450 while the rS group showed 4050 antibody titers. The antibody titer of the VLP-Y group showed 0 while the antibody titer of the rY group showed 4050. The antibody titers after the 4th immunity were found to have an antibody titer of 450 while the VLP-S and VLP-Y groups showed an antibody titer of 12150. Therefore, oral immunization of the yeast cell line expressing the RGNNV capsid protein itself was confirmed to induce anti-RGNNV capsid protein antibody titers 10 to 30 times higher than when orally immunized the purified capsid protein.

3. Evaluation of neutralizing antibody inducing ability of purified RGNNV capsid protein and RGNNV capsid protein expressing yeast

Purified RGNNV capsid protein and RGNNV capsid protein expressing yeast were orally immunized up to 4th, and neutralizing antibody activity of mouse serum was evaluated (FIG. 13). Mouse serum of the PBS, VLP-S, and VLP-Y groups were reacted with RGNNV and then infected with E-11 cells. Cell lesions (CPE) were observed, indicating that the serum of these groups had no neutralizing activity. On the other hand, when the mouse sera of the rS and rY groups were reacted with RGNNV and then infected with E-11 cells, no cell lesions were observed (CPE). Therefore, oral immunization with rS and rY confirmed that antibodies capable of neutralizing RGNNV could be induced.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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According to the method of the present invention, the capsid protein of nodavirus is expressed through a yeast expression system, and then purified through a chromatographic purification method to mass produce high quality nodavirus virus analogous particles. In addition, the prepared nodavirus virus analogous particles can be very usefully used as an antigen of a vaccine against nodavirus infection. Meanwhile, according to the results of the present invention, when the yeast cell line expressing the nodavirus capsid protein itself was used as the vaccine composition, the protective capsid protein exhibited a superior protective immune inducing effect than the vaccine composition. Therefore, this means that the yeast strain containing the capsid protein itself has excellent immune inducing ability. When using this type of recombinant yeast strain as an oral vaccine, the process for preparing the vaccine antigen can be omitted. In addition, using it for oral vaccines can save a great deal of time, effort and money for vaccination.

Claims (17)

1. Method for producing virus-like particles of nodavirus comprising the following steps:

   (a) culturing yeast cells transformed with an expression vector comprising a DNA sequence encoding the nodavirus capsid protein to express the nodavirus capsid protein;

   (b) lysing the cultured yeast cells to obtain cell lysates; And

   (c) chromatography of the cell lysate to purify the nodavirus capsid protein.
2. The method of claim 1, wherein the chromatography in step (c) is cation exchange chromatography, anion exchange chromatography, affinity chromatography, size exclusion chromatography, or a combination thereof.
3. The method of claim 1, wherein the chromatography in step (c) is heparin chromatography.
4. 4. The method of claim 3, wherein the purification of the nodavirus capsid protein by heparin chromatography in step (c) is carried out using an elution buffer containing 0.15-1.2 M NaCl.
5. 4. The method of claim 3, wherein the purification of the nodavirus capsid protein by heparin chromatography in step (c) is performed using a binding buffer containing 0.5 M NaCl.
6. The method of claim 1, wherein the yeast in step (a) is Saccharomyces cerevisiae .
7. The method of claim 1, wherein the DNA sequence encoding the nodavirus capsid protein of step (a) is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1.
8. Virus-like particles (VLPs) of nodavirus assembled from nodavirus capsid proteins.
9. An immunogenic composition comprising viral analogs of nodavirus assembled from nodavirus capsid proteins and capable of eliciting an immune response against nodaviruses.
10. 10. The immunogenic composition of claim 9, wherein the virus like particle of nodavirus is prepared by the method of any one of claims 1-7.
11. A recombinant yeast strain expressing the nodavirus capsid protein represented by SEQ ID NO: 2.
12. 12. The recombinant yeast strain according to claim 11, wherein the strain is transformed with the expression vector YEGα-MCS-NNVcp shown in FIG.
13. 12. The recombinant yeast strain according to claim 11, wherein the yeast strain is Saccharomyces cerevisiae or Yarrowia lipolytica .
14. (a) an immunogenic composition comprising a virus-like particle (VLP) of nodavirus assembled from a nodavirus capsid protein, an immunologically effective amount of the recombinant yeast strain of claim 11 or a culture thereof; And (b) a pharmaceutically acceptable carrier.
15. 15. The vaccine composition according to claim 14, wherein the virus like particle of the nodavirus is prepared by the method of any one of claims 1 to 7.
16. The vaccine composition according to claim 14, wherein the vaccine composition is for preventing or treating nodavirus infection in fish.
17. The vaccine composition according to claim 16, wherein the fish is a fish, flounder, perch, or rockfish.

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