JP6432896B2 - Japanese encephalitis virus vaccine and production method thereof - Google Patents

Description

  The present invention relates to a vaccine against Japanese encephalitis virus classified into the genus Flavivirus, and a method for producing the same.

Japanese encephalitis is a mosquito-borne viral infection caused by the Japanese encephalitis virus classified into the genus Flavivirus, and its patients are widely distributed from East Asia to Southeast Asia and South Asia. In Japan, with the spread of Japanese encephalitis vaccination, the number of Japanese encephalitis patients has decreased, and since 1990, the number of such patients has been less than 10 per 100,000 population per year. However, Japanese encephalitis is still a serious health threat, and there are many confirmed cases of symptoms in recent years, especially in Southeast Asia. Moreover, even with effective vaccines, the Japanese encephalitis virus is actively and quietly circulating in the mosquito swine system, increasing the incidence of Japanese encephalitis in many parts of Asia and other Pacific countries. (See Non-Patent Documents 1 and 2).
Japanese encephalitis virus is classified into five types (GI to GV) of gene types I to V based on the genome sequence. In the production of Japanese encephalitis vaccines that have been sold so far, the Beijing strain virus derived from the mouse brain or the Nakayama strain virus derived from the mouse brain belonging to the Japanese encephalitis virus gene type III (GIII) is used. (See Patent Documents 3 and 4). Conventionally, Japanese encephalitis vaccines have been made from the brain emulsion of virus-infected mice. However, allergic central nervous system disorders and contamination with pathogenic microorganisms due to contaminants derived from the mouse brain are necessary, and because of the large amount of mice required, the cost is high and animal cells and insect cells are used for animal protection. Attempts have been made to produce a Japanese encephalitis vaccine in an in vitro system (see Patent Documents 1 and 2).
Under such circumstances, development of a new vaccine that is even cheaper, safer and more effective is underway.
Here, according to recent research, Japanese encephalitis vaccines derived from viruses classified as Japanese encephalitis virus gene type III (GIII) are classified into types I to IV (GI to GIV) based on cross-reactions, etc. Is effective, but has not been shown to be effective against viruses classified into the same type V (GV) (see Non-Patent Documents 3 and 4). Specifically, as shown in Table 3 of Non-Patent Document 3 (the data in Table 3 is shown as Table 1 below), in the case of Beijing strain vaccine (2-fold dilution), the homo titer is higher than 640. Furthermore, the virus neutralizing antibody titer is 320 for Nakayama strain virus classified as the same gene type 3 (GIII), 160 to 320 for GI virus, and 80 to 320 for GIII virus. As shown, the neutralizing antibody titer of 20 was shown to be very low against the GV type Muar virus.

The same applies to the Nakayama strain vaccine. Therefore, there is a concern that the Beijing vaccine, which is a Japanese encephalitis vaccine that is currently widely used, has very little effect on GV virus.
It has also been shown that GV type viruses behave differently than other types of viruses. In addition, GV virus was first confirmed only in Indonesia-Malaysia, but GV virus was confirmed in China in 2009 and in Korea in 2010 and 2012. Has been.

JP 2004-65118 A JP 2000-83657 A

Neji and others, clinical examination vol 48 no. 4 April 2004 Nerome et al., Journal of General Virology (2007), 88, 2762-2768 Tajima et al., Journal of General Virology (2015), 96, 2661-2669 Lei Cao et al., PLOS Neglected Tropical Diseases May 3, 2016

  There is a need for new vaccines that are highly effective not only for viruses classified as Japanese encephalitis virus gene type III (GIII) but also for viruses classified as type V (GV).

The present invention is a Japanese encephalitis virus-like particle derived from a virus classified into Japanese encephalitis virus gene type III expressed in silkworm silkworms unexpectedly, not only for GIII type virus, but also for GV type virus, It was made based on the knowledge that the unexpectedly high effect was shown.
That is, the present invention relates to (i) a Japanese encephalitis virus-like particle containing M protein and E protein derived from a virus classified as Japanese encephalitis virus gene type III, and (ii) a virus classified as Japanese encephalitis virus gene V type. Provided is a Japanese encephalitis vaccine of a mixture of two types, comprising Japanese encephalitis virus-like particles containing the derived M protein and E protein.
In the present invention, the neutralizing antibody titer against the virus classified into the Japanese encephalitis virus gene type V of the antibody produced by immunizing an animal with the Japanese encephalitis virus-like particle (i) is the Japanese encephalitis virus gene type III of the antibody. It is preferable that it is equal to or more than the neutralizing antibody titer against the virus classified into the above.
The present invention also includes (i) a Japanese encephalitis virus-like particle containing M protein and E protein derived from a virus classified as Japanese encephalitis virus gene type III, and immunizes animals with the Japanese encephalitis virus-like particle (i). The Japanese encephalitis vaccine wherein the neutralizing antibody titer against the virus classified as Japanese encephalitis virus gene type V of the antibody prepared in the above is equal to or higher than the neutralizing antibody titer of the antibody classified as Japanese encephalitis virus gene type III I will provide a.

In the present invention, the Japanese encephalitis virus-like particles (i) and / or (ii) are preferably produced by silkworm-derived cells or silkworm silkworms, and viruses classified as Japanese encephalitis virus gene type III are Nakayama strain viruses. Alternatively, it is preferably a Beijing strain virus and / or a virus classified as Japanese encephalitis virus gene V type is a Muar strain virus.
The present invention also relates to a method for producing the above Japanese encephalitis vaccine, which encodes the pr e M protein in Japanese encephalitis virus classified into Japanese encephalitis virus gene type III or V type using a silkworm protein expression system. In the DNA fragment comprising the M gene encoding the pr e M gene, the M gene encoding the M protein, and the E gene encoding the E protein in this order, the encoded amino acid sequence is the corresponding amino acid sequence in the wild type Japanese encephalitis virus as does not occur mutation compared to the step of the modifying codons of the DNA fragment is inserted to obtain a codon optimized DNA fragment for the expression of silkworm, the resulting codon optimized DNA fragment in base compactors, a base compactors obtained, and baculovirus-derived DNA linearized, be co-transfected into Bombyx mori cells A step of obtaining a baculovirus recombinant containing a codon-optimized DNA fragment from the obtained silkworm cells, a step of infecting a silkworm cocoon with the baculovirus recombinant and breeding a silkworm cocoon, and the silkworm cocoon Provides a method for producing a Japanese encephalitis vaccine comprising the step of isolating Japanese encephalitis virus-like particles.
In the present invention, the sequence of the codon optimized DNA fragment is preferably the base sequence of SEQ ID NO: 4 and / or 7.

Provided is a novel Japanese encephalitis vaccine, in particular, a Japanese encephalitis vaccine that has a high effect on both viruses classified into Japanese encephalitis virus gene type III and viruses classified into the same type V.
Also provided is a method for producing a Japanese encephalitis vaccine that is safe and provides a very high yield.

The codon usage frequency in Bombux Mori (scientific name: Bombyx mori) is shown. It is the table | surface which investigated the usage frequency in 450043 codon in the 1180 coding region (CDS) of Bombux Mori. The frequency of appearance per 1000 codons is shown. The numbers in parentheses indicate the total number of occurrences. The bold text indicates that it is the most frequently used gene codon for each amino acid. FIG. 3 shows an optimized codon correspondence table based on the most frequently used gene codons shown in FIG. Serine (S) is UCA and its complementary strand is TGA, which is a stop codon. Therefore, the complementary strand of serine serves as a stop codon and no large frame appears in the complementary strand. It is a figure which shows the relationship of the antibody-attack virus made by immunizing a rabbit with Nakayama strain virus-like particle | grains (JEV-NVLP) in confirmation of the neutralizing antibody titer. It is a figure which shows the relationship of the antibody-attack virus made by immunizing a rabbit with Muar strain virus-like particle | grains (JEV-VLP) in confirmation of the neutralizing antibody titer. It is a figure which shows the antibody production ability in a mouse | mouth at the time of using JEV-NVLP for the immunization in a mouse | mouth. The correspondence between the Nakayama strain virus codon optimized PreM-ME fusion protein gene DNA sequence (SEQ ID NO: 4) and amino acid sequence (SEQ ID NO: 3) is shown. A surrounding line shows a FLAG tag. Based on the base sequence of the Nakayama strain PreM protein gene, M protein gene, and E protein gene, considering the introduction of the FLAG tag at the C-terminal and the optimization of the codon usage of Bombux Mori cells, the base sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. PreM / M protein: DNA sequence 1 to 501; E protein: 502 to 2001; FLAG tag: 2002 to 2025. It is a continuation of FIG. FIG. 6-2 is a continuation of FIG. FIG. 6-3 is a continuation of FIG.

<Virus-like particles>
The virus-like particle of the present invention contains M protein and E protein derived from Japanese encephalitis virus classified into the genus Flavivirus, but does not contain Japanese encephalitis virus C protein and RNA therein. Means particles that are sexual. The structure of the virus-like particle is preferably 30 nm to 300 nm, more preferably 50 to 150 nm, and even more preferably 60 nm to 120 nm. The particle has a structure in which clear HA spikes are densely arranged on the surface of the particle. And it is very similar in shape to virus particles.
The virus-like particle of the present invention is preferably expressed by silkworm-derived cells or silkworm cocoons, and may have silkworm-derived lipids or sugar chain modifications.
Examples of the lipid include glyceroglycolipid, glycosphingolipid, cholesterol, phospholipid and the like.

Examples of the glyceroglycolipid include sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride and the like.
Examples of the glycosphingolipid include galactosyl cerebroside, lactosyl cerebroside, ganglioside and the like.
Examples of the phospholipid include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, lysophosphatidylcholine, sphingomyelin and the like.
Fatty acids derived from the above lipids by hydrolysis or the like may be included. Examples of the fatty acid include myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linolenic acid, and the like.
The vaccine containing the virus-like particle of the present invention has a structure with densely-arranged spikes of recombinant virus membrane protein having immunogenicity, so that even if an adjuvant is not used, an unprecedented high immunogen is used. It becomes possible to show sex. Since no adjuvant is used, side effects such as allergies and anaphylactic shock can be reduced.

<Neutralizing antibody titer>
The neutralization titer of the present invention is obtained using the plaque neutralization test method. In the plaque neutralization test method, first, a cell plate is prepared as follows. First, use a cell growth medium to adjust the number of cells to 2 to 3 x 10 ⁵ cells / mL, dispense 2 mL / well into a 6-well plate, and place the plate back and forth, left and right to form a uniform cell sheet. After shaking well, static culture is performed in a 35% C, 100% humidity, 5% CO ₂ incubator and used after 1 day of culture. If a cell sheet of about 80 to 90% is formed, it can be used.
Next, a specific procedure of the plaque neutralization test method will be described. Test serum (ie, VLP vaccine immune serum and each immune serum) is inactivated at 56 ° C. for 30 minutes, and then diluted 4-fold in steps (40 ×, 160 ×, 640 ×, 2560 ×) with a diluent. A stock virus (Nakayama strain, Beijing strain, Muar strain) is diluted to 200 PFU / 100 μL and used as an attack virus (in an ice bath). Add 300 μL of challenge virus to 300 μL of 40x-2560x diluted serum (in ice bath). For the control virus, add 1.8 mL of the attack virus to an equal volume of 1.8 mL (in an ice bath). Mix each tube well and perform a neutralization reaction in a constant-temperature water bath at 37 ° C for 90 minutes. Immediately transfer each tube after completion of the neutralization reaction to an ice bath and place it on the cell plate. Qing is completely excluded. Inoculate 100 μL per well of the control virus and serum and virus mixture from the wall. Use 6 to 12 wells for the control virus, and 3 to 4 wells for the serum and virus mixture. Lightly move the plate so that the inoculum spreads over the entire cell surface immediately after inoculation. The virus adsorption time is left to stand in a 35 ° C., 5% CO ₂ incubator for 90 minutes, but the plate is moved every 15 minutes so that the inoculum is moistened on the entire cell surface. After completion of the virus adsorption reaction, without removing the inoculum, 2 mL of the overlay medium is added to each well and cultured at 35 ° C. under 5% CO ₂ for 4 to 6 days. Then, add 1.5 mL of 3.7% neutral buffered formalin solution (diluted formalin solution (37%) 10-fold with PBS (-)) 10 times on the overlay medium of each well, and gently shake for 1 hour or more at room temperature. put. After completion of formalin fixation, wash the medium and formalin solution under running tap water, add 1.5 mL of methylene pull-staining solution to each well, leave it at room temperature for 1 hour, and after staining, wash off the staining solution with tap water to determine the number of plaques. Calculate. Store the stained plate for 1 month from the date of final pass / fail judgment.

Here, the “neutralizing antibody titer” in the present invention is defined as the reciprocal of the maximum dilution factor of serum that results in a 50% plaque number reduction compared to the control virus (non-serum control).
In the “antibody produced by immunizing an animal with virus-like particles” of the present invention, the “animal” has an immune system and can acquire humoral immunity or cellular immunity against the virus by vaccination. Although it will not specifically limit if it is an animal which can be used, it can be animals, such as a human, a pig, a mouse, a rat, a rabbit, a bird, a horse, a dog, a cat. In addition, in “an antibody made by immunizing an animal with virus-like particles”, “an antibody made by immunization” means, for example, that virus-like particles are placed in the abdominal cavity in the case of mice, and subcutaneous or groin in the neck in the case of rabbits. It is injected into the muscle, blood is collected after 14 days to (28 days (1 month), 2 months, 3 months) to 4 months, etc., and can be contained in the serum.

<Method for producing virus-like particles>
In the present invention, it is preferable to produce virus-like particles using a protein expression system using silkworm-derived cells or silkworm cocoons.
In the present invention, codon optimization is performed for the purpose of improving protein expression using silkworms. Bonbux Mori's codon usage (URL: http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=7091) obtained from Codon Usage Database provided by Kazusa DNA Research Institute (Figure) Based on 1), a correspondence table (FIG. 2) between the prepared amino acid sequences and gene sequences was prepared. In this correspondence table, in order to prevent unexpected by-products from being synthesized due to the formation of a long coding frame in the complementary strand, the Ser sequence is UCA (most frequently used in Bombux Mori), and the complementary strand is used. Were designed to generate many UGAs (stop codons).
A virus PreM-M, which is a base for nucleic acid design, by adding a tag sequence such as FLAG tag to the amino acid sequence of preM protein-M protein-E protein in the amino acid sequence registered in Genbank et al. -Obtain the amino acid sequence of the E fusion protein. Code based on the amino acid sequence of this virus PreM-M-E fusion protein, full pr e M genes encoding the complete pr e M proteins of the virus, the complete M gene and a complete E protein encoding M protein The virus-like particle gene sequence is determined, and the virus-like particle gene sequence is modified in consideration of optimization of the codon usage frequency of silkworms. By such a procedure, a codon optimized DNA fragment for silkworm expression is obtained.

Here, in the present invention, in a DNA fragment including the pr e M gene encoding the pr e M protein in the target virus, the M gene encoding the M protein, and the E gene encoding the E protein in this order, The codons of the DNA fragments are modified so that the amino acid sequence to be generated does not mutate as compared with the corresponding amino acid sequence in the wild-type target virus. “The encoded amino acid sequence does not mutate compared to the corresponding amino acid sequence in the wild-type target virus” means that the PreM-ME fusion protein encoded by the codon-optimized DNA fragment of the target virus It means that the amino acid sequence is substantially identical to the corresponding amino acid sequence in the wild type target virus. Note that “substantially the same” means having a homology of 90% or more, more preferably 95% or more, and more preferably 99% or more.

Thereafter, the resulting codon optimized DNA fragment, using any method known, inserted into a known base compactors suitable in baculovirus, a base compactors obtained, linearized baculovirus DNA from And a step of co-transfecting silkworm cells, a step of obtaining a baculovirus recombinant in which a codon-optimized DNA fragment is recombined from the obtained silkworm cells, and a silkworm silkworm infected with the baculovirus recombinant. Virus-like particles can be obtained through a step of breeding silkworm cocoons and a step of isolating virus-like particles from the silkworm cocoons.

The time of introduction (inoculation) of the recombinant baculovirus into Bonpukus Mori is not particularly limited as long as it is in the leap period, but is preferably early in the leap period. (Day 1 of molting) to 4th day (day 5 of molting) are preferred, and the following day (day 2 of molting) to the next day (day 3 of molting) are more preferred.
After inoculating the silkworm silkworm pupae with the above recombinant baculovirus, these pupae are bred for a predetermined period. The temperature conditions at that time are preferably 20 ° C to 30 ° C, more preferably 22 ° C to 28 ° C, more preferably 24 ° C. ˜27 ° C. is more preferred, and 26 ° C. is particularly preferred. The period of raising the upper wing is not particularly limited as long as it is from the inoculation to before the pupae transform into adults (pupae), but preferably 2 to 6 days after the inoculation, more preferably 2 to 4 days. Preferably, 3 days is more preferable.
Recovery of virus-like particles from silkworm cocoons can use any method known to those skilled in the art. For example, silkworm cocoons can be homogenized in an isotonic buffer solution and then recovered using immobilized erythrocytes or a sialic acid column (fetuin column). In addition, a fraction containing virus-like particles can be obtained using a fractionation method such as sucrose density gradient centrifugation.

  In the vaccine of the present invention, since the virus-like particle of the present invention containing the virus-like particle of the present invention has high immunogenicity, it is possible to exhibit high immunogenicity without using an adjuvant. Since no adjuvant is used, side effects such as allergies and anaphylactic shock can be reduced. Of course, the virus-like particles of the present invention are non-pathogenic because no virus-derived nucleic acid is present in the hollow interior of the spherical outer shell. The vaccine of the present invention can effectively prevent virus infection of animals including humans. Moreover, the vaccine of this invention can shorten the period required for manufacture remarkably compared with the manufacturing method etc. which used the conventional egg. As a result, vaccines can be procured in a short period of time during the epidemic of viral infections such as influenza, which can greatly contribute to protecting people from infections and maintaining their health. Furthermore, in the production of vaccines using conventional eggs, it is necessary to produce highly safe facilities such as P3 facilities to handle seed viruses used for vaccines, and to produce large quantities of vaccines because of the low immunogenicity of vaccines. Whereas a huge manufacturing cost is necessary due to the fact that the manufacturing method is present, the manufacturing method of the present invention can remarkably reduce the manufacturing cost.

<Vaccine composition>
In one embodiment, the present invention provides a method for inoculating an animal with a vaccine against viral infection, comprising an effective amount of a vaccine comprising a polypeptide according to the present invention or produced according to a production method according to the present invention. Is administered to said animal.
In one embodiment, the present invention provides a method for inducing an immune response against a virus in an animal, comprising a polypeptide according to the invention or produced by a production method according to the invention. A method comprising administering an amount to said animal. In another aspect, the present invention relates to a viral vaccine composition. In addition to the virus-like particles, the vaccine composition can include additional components and a pharmaceutically acceptable carrier. Examples of the additional component include an adjuvant and a component that enhances an anti-influenza virus action such as an anti-influenza virus agent. Adjuvants can include aluminum hydroxide, aluminum phosphate, saponin, water-in-oil emulsion, oil-in-water emulsion, oil-in-water emulsion, acrylic or methacrylic acid polymers, maleic anhydride, alkenyl derivatives, carbomers, block copolymers. . Examples of the antiviral agent include neuramitase inhibitors such as zanamivir, oseltamivir, peramivir, and laninamivir, amantadine, rimantadine, RNA polymerase inhibitors, and the like. The pharmaceutically acceptable carrier contained in the composition of the present invention includes a solvent, a dispersant, a coating agent, a stabilizer (albumin, ethylenediaminetetraacetic acid alkali salt), a diluent (allowed for use as a pharmaceutical product) Contains water, saline, dextrose, ethanol, glycerol, etc.), preservatives, excipients, antibacterial agents, antifungal agents, isotonic agents (sodium chloride, dextrose, mannitol, sorbitol, lactose, etc.), absorption delaying agents .

For example, the composition of the present invention can be in a dosage form suitable for parenteral administration or oral administration. Examples of compositions for parenteral administration include injections and nasal drops, and injections include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. The dosage form is included. Such an injection can be prepared according to a known method, for example, by dissolving, suspending or emulsifying the antigen protein in a sterile aqueous or oily liquid usually used for injection. The prepared injection solution is usually filled in appropriate ampules, vials, and syringes. In addition, parenteral pharmaceutical compositions are prepared in dosage unit form that is compatible with the dosage of the active ingredient.
An effective amount of vaccine refers to an amount sufficient to achieve a biological effect such as inducing sufficient humoral or cellular immunity against the virus. Administration methods also include inhalation, intranasal, oral, parenteral (eg, intradermal, intramuscular, intravenous, intraperitoneal, and subcutaneous administration). The effective amount and method of administration may depend on the age, sex, condition, weight of the human being administered. For example, in the case of an influenza vaccine, generally, a vaccine containing 15 μg or more of HA protein per strain in 1 ml; Inject 0.5 ml subcutaneously twice, approximately 2-4 weeks apart. For those 13 years and older, 0.5 ml is injected subcutaneously once or twice at approximately 1 to 4 week intervals.
EXAMPLES Next, although an Example demonstrates this invention in detail, it is not limited to this.

[Used cells and viruses]
Vero cells (catalog number JCRB 9013, lot number 11292002) were purchased from the Japanese Collection of Research Bioresources [JCRB] cell bank.
The Japanese encephalitis viruses, Zhongshan strain virus, Beijing strain virus, and Muar strain virus, were obtained from National Institute of Infectious Diseases (Tokyo, Japan).
Silkworm Bm-N cells were obtained from Baculo Technologies, Inc. (Kagawa Prefecture).
The P6E strain of baculovirus Bombyx mori nuclear polyhedrosis virus-BmNPV was obtained from Baculo Technologies, Inc. (Kagawa Prefecture).

[Example 1] Method for producing Japanese encephalitis virus-like particles classified into Japanese encephalitis virus gene type III <Construction of baculovirus recombinants having a gene expressing Japanese encephalitis-Nakayama strain virus-like particles (JEV-NVLP) >
Based on the literature content such as J Biosci Bioeng 2012; 114: 657-62 by Yamaji H et al., Hum Vaccin Immunother 2014 by Yun SI et al .; J Virol 2012 by Luca VC et al .; 86: 2337-46 Thus, a baculovirus recombinant used for expressing virus-like particles containing M protein and E protein derived from Japanese encephalitis virus gene type III was prepared.
First, the FLAG tag sequence (SEQ ID NO: 2) is added to the amino acid sequence (SEQ ID NO: 1) from the preM protein in the amino acid sequence registered in Genbank under Accession No. ABQ52691 to the M protein and E protein. The amino acid sequence of the Nakayama strain virus PreM-ME fusion protein, which is the basis of the design, was obtained (SEQ ID NO: 3). Based on the amino acid sequence of the Nakayama strain virus PreM-M-E fusion protein, full pr e M genes encoding the complete pr e M protein of Nakayama strain viruses, a M gene and a complete full coding for M protein Regarding JEV-NVLP gene sequence encoding E protein, codon optimization of the JEV-NVLP gene sequence is performed so that expression of the Nakayama strain virus-like particle can be enhanced in the silkworm silkworm used in the subsequent step. The modified JEV-NVLP gene sequence was obtained (SEQ ID NO: 4). In addition, Dragon Genomics (Shiga, Japan) was used for the synthesis | combination of the DNA fragment of a codon optimization JEV-NVLP gene sequence.

The synthesized DNA fragment In-Fusion Kit (Clontech, Mountain View, CA, USA) was inserted into the ppm-8 plasmid is base restrictor was used to prepare a pBm-8-JEV-NVLP vector. Subsequently, linearized DNA derived from the pBm-8-JEV-NVLP vector and the baculovirus Bombyx mori nuclear polyhedrosis virus-BmNPV strain P6E was cotransfected into silkworm Bm-N cells in serum-free medium. . After 5 hours of incubation, the serum-free medium was replaced with minimal essential medium containing TC-100 medium and 10% fetal calf serum, and the cells were further incubated for 5-7 days. Thereafter, the medium was collected and centrifuged at 3000 rpm for 5 minutes to separate the precipitated cell debris. The same centrifugation was repeated to obtain a JEV-NVLP baculovirus recombinant concentrate.

  Here, silkworm Bm-N cells infected with JEV-NVLP baculovirus recombinants were confirmed based on microscopic observation of the appearance and morphology of the cells. Furthermore, a fluorescent antibody method using a mouse antiserum or rabbit antiserum against Japanese encephalitis virus (Nakayama strain virus) and a fluorescein isothiocyanate (FITC) -conjugated anti-mouse IgG antibody (West Grove, PA, USA), JEV-NVLP Silkworm Bm-N cells infected with the baculovirus recombinant were reconfirmed. In addition, silkworm Bm-N cells infected with JEV-NVLP baculovirus recombinants are producing Japanese encephalitis virus-like particles containing E protein derived from viruses classified as Japanese encephalitis virus gene type III. Mouse antiserum or rabbit antiserum against Nakayama strain virus, goat polyclonal anti-JEV rabbit serum (obtained from NIID, Tokyo, Japan), FITC-conjugated rabbit anti-goat IgG-AP antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) ) And Western blot analysis using ECL plus reagent (GE Healthcare, Port Washington, NY, USA).

<Generation and purification of Nakayama strain virus-like particles in silkworm cocoons>
The obtained JEV-NVLP baculovirus recombinant concentrate (1 × 10 ¹⁰ pfu / ml) was adjusted to 1 × 10 ⁶ / 蛹 with PBS (diluted) and 20 μl was injected into silkworm cocoons to give JEV-NVLP baculo Infected with virus recombinants. Silkworm cocoons were collected and crushed 1 to 5 days after infection, and 0.01% formalin and phenylthiourea were added to the crushed material. The mixture was then homogenized by 3 cycles of sonication. The resulting homogenate is centrifuged at 16,000 rpm for 30 minutes using a SW28 rotor equipped with a Beckman centrifuge, followed by an additional 10-50% (w / w) linear sucrose gradient using the same rotor. Centrifugation yielded 20 fractions. In order to detect the presence of virus-like particle antigens, all fractions were subjected to protein content quantification, enzyme-linked immunosorbent assay (ELISA) and Western blot analysis by known procedures. The antibodies used in these assays are the same as described above. As a result, it was found that the E protein yield obtained from silkworm cocoons on the third day after infection was the highest. Specifically, it was confirmed that 9,480 μg of E protein could be recovered from the homogenate of 30 silkworm pupae on the third day after infection. Therefore, in calculation, each silkworm silkworm was able to produce more than 300 μg of E protein.
In a separate experiment, 15,840 μg E protein was recovered from 30 pupae, producing 396 μg E protein per cocoon. Further, when the obtained virus-like particle (VLP) antigen was observed with an electron microscope, particles having a diameter of 30 to 200 nm were confirmed.
From the above, it was found that the virus-like particle antigen can be safely obtained with an unexpectedly high yield in the silkworm cocoon expression system of the present invention.

[Example 2] Method for producing Japanese encephalitis virus-like particles classified into Japanese encephalitis virus gene type V <Construction of a baculovirus recombinant having a gene expressing Japanese encephalitis-Muar strain virus-like particles (JEV-VLP) >
The FLAG tag sequence (SEQ ID NO: 3) is added to the amino acid sequence (SEQ ID NO: 5) from the preM protein to the M protein and E protein in the amino acid sequence registered in Genbank with ACCESSION ADX31663, The resulting amino acid sequence of the Muar strain virus PreM-ME fusion protein was obtained (SEQ ID NO: 6). Among the amino acid sequences of the Muar strain PreM / ME fusion protein corresponding to the amino acid sequence of the Nakayama strain PreM / ME fusion protein, the first Val protein is changed to Met in the same manner as the Nakayama strain, so that it becomes a start codon. The TAG amino acid sequence was added, and the JEV-VLP gene sequence was codon-optimized so that the expression of the Muar strain virus-like particles could be enhanced to obtain a codon-optimized JEV-VLP gene sequence (SEQ ID NO: 7).
All other procedures were the same as in Example 2.

[Example 3] Method for producing two-type mixed vaccine The virus-like particles obtained in Example 1 and the virus-like particles obtained in Example 2 are defined by the amount of protein (20 μg / ml), respectively. Were prepared by mixing equal amounts.

[Example 4] Confirmation of neutralizing antibody titer of Japanese encephalitis virus-like particles expressed in silkworm (plaque neutralization test)
Based on the plaque neutralization test method described above, the following experiment was conducted.
Immunogenicity was examined by measuring the neutralizing antibody titer of the antibody produced by immunizing rabbits with Nakayama strain virus-like particles (JEV-NVLP) produced in silkworm silkworms. Specifically, Rabbits were injected (immunized) with 0.5 to 1 ml of Nakayama strain virus-like particles (JEV-NVLP) 100 to 200 μg / ml seven times at 2-week intervals, and the collected blood was removed at 1200 rpm for 10 minutes. The neutralizing antibody titer was confirmed using the antiserum obtained with care.
Test anti-rabbit sera against JEV-NVLP was serially diluted 10-fold and then serially diluted and mixed with the same amount of challenge virus. For plaque inhibition assay, challenge virus was mixed with 10-fold diluted antisera prior to inoculation. After 6 days incubation in 6 well plates, the agarose medium was removed and viable plaques were counted.
The plaques produced by Vero cells infected with Zhongshan strain were very small, but the number of plaques obviously decreased as the serum concentration increased. This trend was also observed for the number of plaques produced by Beijing and Muar strain viruses. This antibody test anti-rabbit antibody-attack virus relationship is shown in FIG. A correlation was observed between antibody dilution and plaque number. Based on the curve in FIG. 3, the neutralization antibody titer, which is the maximum serum dilution factor required to reduce the number of plaques to 50%, was calculated (Table 2). The antiserum against JEV-NVLP has a high inhibitory effect on the Zhongshan strain virus and Beijing strain virus classified as type III, respectively. In particular, the antiserum against JEV-NVLP had a higher titer in the Muar strain classified as type V than in the Nakayama strain.

[Example 5] Confirmation of neutralizing antibody titer of Japanese encephalitis virus-like particles expressed in silkworms Japanese encephalitis of antibodies made by immunizing rabbits with Muar strain virus-like particles (JEV-VLP) produced in silkworm silkworms The immunogenicity was examined by measuring the neutralizing antibody titer against the virus. The procedure was the same as in Example 4. This antibody test anti-rabbit antibody-attack virus relationship is shown in FIG. A correlation was observed between antibody dilution and plaque number. Based on the curve of FIG. 4, the average number of plaques in each well was set to 100%, and the neutralizing antibody titer that was the maximum serum dilution factor required to reduce the number of plaques to 50% was calculated (Table 3). Antisera against JEV-VLP had a high inhibitory effect on Muar strain viruses classified as type V. The antiserum also exerted a high inhibitory effect against the Nakayama strain virus. This was an unexpected effect that was not obtained with conventional vaccines.

[Example 6] Confirmation of neutralizing antibody titer of Japanese encephalitis virus-like particles expressed in silkworms (2 types of mixed vaccine)
Immunogenicity was examined by measuring the neutralizing antibody titer of the antibody produced by immunizing rabbits with the two-mixed vaccine prepared in Example 3 against Japanese encephalitis virus. The procedure for confirming the neutralizing antibody titer was the same as in Example 4. And the neutralizing antibody titer obtained by 2 types of mixed vaccine was computed (Table 4). Table 4 reflects the results of Tables 2 and 3 and the prior art shown in Table 1.

  Currently, the neutralizing antibody titer of JEV GV type Muar strain reacts as low as 20 against the immune sera of Nakayama strain and Beijing strain vaccine, which are widely marketed, and its preventive effect cannot be expected. In contrast, vaccines containing Nakayama strain virus-like particles are highly reactive with Nakayama and Muar strains, but are slightly less reactive with Beijing strains. On the other hand, vaccines containing Muar strain virus-like particles react very high with homo muar strain virus and also with Zhongshan strain, but show extremely low response with Beijing strain, and neutralizing antibody titer is slightly 76. From the above, two types of mixed vaccines containing Nakayama strain virus-like particles and Muar strain virus-like particles have 9,600, 8,000, 8,000 and almost the same antibody titer. Vaccines will be able to control GI, GIII, and GV Japanese encephalitis viruses that are currently prevalent in the world. According to the present invention, two mixed vaccines of Nakayama strain and Muar strain, or Beijing strain and Muar strain virus are expected to appear.

[Example 7] Vaccination with JEV-NVLP vaccine Partially purified JEV-NVLP was used for immunization in mice. First, JEV-NVLP was mixed with complete adjuvant. The resulting adjuvant vaccine was used for the first immunization (first dose) and the incomplete adjuvant vaccine was used for the second immunization (second dose) after 2 weeks. In addition, a third immunization (third dose) without adjuvant was given after 4 weeks and blood samples were collected after 2 weeks. The results are shown in FIG. The basic rules for animal experiments were in accordance with the basic rules for animal experiments (Okinawa Institute for Bioresources) and guidelines for the management and use of laboratory animals (Japan Science Society).
As shown in FIG. 5, higher neutralization titers were observed for the JEV-Muar strain in mice immunized with a series of three doses. Subsequent high plaque neutralization titers were confirmed in the homologous Nakayama strain. In contrast, the powerful antisera described above against JEV-NVLP did not affect the inhibition of plaque formation by Beijing strains as much as the other two.

Claims (9)

Japanese encephalitis vaccine against Nakayama strain virus and Beijing strain virus, viruses classified as Japanese encephalitis virus gene type III, and Muar strain virus, a virus classified as Japanese encephalitis virus gene type V,
(i) Japanese encephalitis virus-like particles containing M protein and E protein derived from Nakayama strain virus, produced by silkworm-derived cells or silkworm cocoons, and (ii) containing M protein and E protein derived from Muar strain virus Including Japanese encephalitis virus-like particles
Two types of Japanese encephalitis vaccine.   The antibody produced by immunizing an animal with the Japanese encephalitis virus-like particle (i) has a neutralizing antibody titer against the Muar strain virus which is a virus classified into the Japanese encephalitis virus gene type V. The mixed Japanese encephalitis vaccine according to claim 1, which is equal to or more than a neutralizing antibody titer against Nakayama strain virus or Beijing strain virus, which is a virus classified as a type. Japanese encephalitis vaccine against Muar strain virus, a virus classified as Japanese encephalitis virus gene V type,
A codon-optimized PreM-ME fusion protein comprising an amino acid sequence expressed in the DNA sequence of Nakayama strain virus codon optimized PreM-ME fusion protein shown in SEQ ID NO: 4 in a silkworm-derived cell or silkworm silkworm , Including Japanese encephalitis virus-like particles (i),
Neutralizing antibody titer against Muar strain virus, which is a virus classified into Japanese encephalitis virus gene V type of antibody made by immunizing animals with Japanese encephalitis virus-like particles (i), Higher than neutralizing antibody titer against Zhongshan strain virus or Beijing strain virus, which is a virus classified as
Japanese encephalitis vaccine. (i) Japanese encephalitis virus-like particles containing M protein and E protein derived from a virus classified as Japanese encephalitis virus gene type III; and (ii) a virus derived M protein and E classified as Japanese encephalitis virus gene V type. A method for producing a mixed Japanese encephalitis vaccine comprising Japanese encephalitis virus-like particles containing protein,
Using silkworm protein expression system,
A DNA fragment comprising, in this order, a preM gene encoding a preM protein, an M gene encoding an M protein, and an E gene encoding an E protein in each of Japanese encephalitis viruses classified into Japanese encephalitis virus genes type III and V A codon-optimized DNA fragment for expression of silkworms by modifying the codon of the DNA fragment so that the encoded amino acid sequence does not mutate compared to the corresponding amino acid sequence in wild-type Japanese encephalitis virus Obtaining a step,
Inserting the obtained codon optimized DNA fragment into a vector;
Co-transfecting the obtained vector and linearized baculovirus-derived DNA into silkworm cells,
Obtaining a baculovirus recombinant containing a codon-optimized DNA fragment from the obtained silkworm cell,
Infecting silkworm cocoons with the baculovirus recombinant, and breeding silkworm cocoons, and isolating Japanese encephalitis virus-like particles from the silkworm cocoons,
A method for producing a Japanese encephalitis vaccine, wherein the sequence of the codon optimized DNA fragment is the base sequence of SEQ ID NOs: 4 and 7 . (i) Japanese encephalitis virus-like particles containing M protein and E protein derived from a virus classified into Japanese encephalitis virus gene type III,
A virus whose neutralizing antibody titer against a virus classified as Japanese encephalitis virus gene type V of an antibody made by immunizing an animal with Japanese encephalitis virus-like particle (i) is classified as Japanese encephalitis virus gene type III of the antibody More than neutralizing antibody titer against
A method for producing a Japanese encephalitis vaccine against Muar strain virus, which is a virus classified as Japanese encephalitis virus gene V type ,
Using silkworm protein expression system,
In a Japanese encephalitis virus gene type III classified into the Japanese encephalitis virus gene III, the preM gene encoding the preM protein, the M gene encoding the M protein, and the E gene encoding the E protein are encoded in this order. Modifying the codons of the DNA fragment so that the amino acid sequence is not mutated compared to the corresponding amino acid sequence in wild-type Japanese encephalitis virus, obtaining a codon-optimized DNA fragment for silkworm expression,
Inserting the obtained codon optimized DNA fragment into a vector;
Co-transfecting the obtained vector and linearized baculovirus-derived DNA into silkworm cells,
Obtaining a baculovirus recombinant containing a codon-optimized DNA fragment from the obtained silkworm cell,
Infecting silkworm cocoons with the baculovirus recombinant, and breeding silkworm cocoons, and isolating Japanese encephalitis virus-like particles from the silkworm cocoons,
A method for producing a Japanese encephalitis vaccine, wherein the sequence of the codon optimized DNA fragment is the base sequence of SEQ ID NO: 4. A method for producing the Japanese encephalitis vaccine according to claim 1 or 2 ,
Using silkworm protein expression system,
A DNA fragment comprising, in this order, a preM gene encoding a preM protein, an M gene encoding an M protein, and an E gene encoding an E protein in each of Japanese encephalitis viruses classified into Japanese encephalitis virus genes type III and V A codon-optimized DNA fragment for expression of silkworms by modifying the codon of the DNA fragment so that the encoded amino acid sequence does not mutate compared to the corresponding amino acid sequence in wild-type Japanese encephalitis virus Obtaining a step,
Inserting the obtained codon optimized DNA fragment into a vector;
Co-transfecting the obtained vector and linearized baculovirus-derived DNA into silkworm cells,
Obtaining a baculovirus recombinant containing a codon-optimized DNA fragment from the obtained silkworm cell,
Infecting silkworm cocoons with the baculovirus recombinant, and breeding silkworm cocoons, and isolating Japanese encephalitis virus-like particles from the silkworm cocoons,
A method for producing a Japanese encephalitis vaccine, comprising: The method for producing a Japanese encephalitis vaccine according to claim 6, wherein the sequence of the codon optimized DNA fragment is the base sequence of SEQ ID NOs: 4 and 7 .   It is a manufacturing method of the Japanese encephalitis vaccine of Claim 3,
  Using silkworm protein expression system,
  In the Japanese encephalitis virus gene type III classified into the Japanese encephalitis virus gene III, the preM gene encoding the preM protein, the M gene encoding the M protein and the E gene encoding the E protein are encoded in this order Modifying the codons of the DNA fragment so that the amino acid sequence is not mutated compared to the corresponding amino acid sequence in wild-type Japanese encephalitis virus, obtaining a codon-optimized DNA fragment for silkworm expression,
  Inserting the obtained codon optimized DNA fragment into a vector;
  Co-transfecting the obtained vector and linearized baculovirus-derived DNA into silkworm cells,
  Obtaining a baculovirus recombinant containing a codon-optimized DNA fragment from the obtained silkworm cell,
  Infecting silkworm cocoons with the baculovirus recombinant, and breeding silkworm cocoons, and
  Isolating Japanese encephalitis virus-like particles from the silkworm cocoon,
A method for producing a Japanese encephalitis vaccine, comprising:   The method for producing a Japanese encephalitis vaccine according to claim 8, wherein the sequence of the codon optimized DNA fragment is the base sequence of SEQ ID NO: 4.

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