Novel recombinant antigen protein for japanese encephalitis, and use thereof

Abstract

The present invention relates to a novel recombinant antigen protein for Japanese encephalitis and, more specifically, to a recombinant antigen protein for Japanese encephalitis, designed through protein structural stability analysis and introduction of point mutations. It has been identified that a composition comprising the recombinant antigen protein for Japanese encephalitis, according to the present invention, exhibits immunogenicity and protective efficacy against challenge inoculation that are superior to those of an approved live-attenuated vaccine. In particular, it has been experimentally identified that the recombinant antigen protein for Japanese encephalitis, of the present invention, exhibits excellent protective efficacy against Japanese encephalitis virus G5 and other genotypes. Therefore, the recombinant antigen protein for Japanese encephalitis, of the present invention, can be variously used in the field of preventing infection by Japanese encephalitis virus G1 to G5.

Description

Novel Japanese encephalitis recombinant antigen protein and its uses

[0001] The present invention relates to a novel recombinant Japanese encephalitis antigen protein, and more specifically, to a recombinant Japanese encephalitis antigen protein designed through protein structural stability analysis and the addition of point mutations.

[0002] Japanese encephalitis is a zoonotic disease transmitted by mosquitoes that also affects humans. The Japanese encephalitis virus belongs to the Flavivirus family and is similar to dengue fever, yellow fever, and West Nile virus. Japanese encephalitis occurs mostly in Asian countries, and while the major genotypes of the virus have different geographical distributions, the symptoms upon onset are similar.

[0003] Japanese encephalitis virus is currently subdivided into five genotypes based primarily on viral gene sequences encoding premembrane (prM) and envelope (E) proteins. In 1952, GV (genotype 5, G5) Muar, a serologically distinct Japanese encephalitis virus, was isolated from the brain of a JE patient in Malaysia. Based on a series of serological studies using monoclonal antibodies, it was confirmed that G5 Muar belongs to a separate antigenic group. Early phylogenetic analyses also identified Muar as a distinct G5, and evolutionary studies revealed that G5 is the ancestor of other genotypes (i.e., G1–G5). It was not reported anywhere else in the world until it was rediscovered in 2009 by Dr. Yang's team in China, followed by its rediscovery in Korea.

[0004] All existing Japanese encephalitis vaccines are made using Japanese encephalitis virus G3, but they provide protection against G1 through G4. However, regarding Japanese encephalitis virus G5, which had not been reported for 57 years and reappeared a few years ago, it has been reported that only about 50% of people are effective even with existing vaccines. This means that existing vaccines are less effective against Japanese encephalitis virus G5.

[0005] Accordingly, the inventors designed a novel recombinant Japanese encephalitis antigen protein to defend against Japanese encephalitis virus G5. Furthermore, the present invention was completed by confirming that the recombinant Japanese encephalitis antigen protein exhibits excellent protective ability against not only Japanese encephalitis virus G5 but also other genotypes.

[0006] Therefore, the object of the present invention is to provide a nucleic acid encoding a Japanese encephalitis recombinant antigen protein represented by the nucleotide sequence of SEQ ID NO. 3 or 5.

[0007] Another object of the present invention is to provide a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6.

[0008] Another object of the present invention is to provide a Japanese encephalitis vaccine composition comprising: a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6; or a nucleic acid encoding the same.

[0009] Another objective of the present invention is to provide an immunogenic composition against Japanese encephalitis virus comprising a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6.

[0010] Another objective of the present invention is to provide a method for preventing or treating Japanese encephalitis, comprising the step of administering a composition comprising a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6; or a nucleic acid encoding the same, to an individual in need thereof.

[0011] Another objective of the present invention is to provide a method for inducing immunity against the Japanese encephalitis virus, comprising the step of administering a composition containing a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6 to an individual in need thereof.

[0012] To achieve the above objective, the present invention provides a nucleic acid encoding a Japanese encephalitis recombinant antigen protein represented by the nucleotide sequence of SEQ ID NO. 3 or 5.

[0013] In addition, the present invention provides a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6.

[0014] In addition, the present invention provides a Japanese encephalitis vaccine composition comprising: a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6; or a nucleic acid encoding the same.

[0015] In addition, the present invention provides an immunogenic composition against Japanese encephalitis virus comprising a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6.

[0016] In addition, the present invention provides a method for preventing or treating Japanese encephalitis, comprising the step of administering a composition comprising a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6; or a nucleic acid encoding the same, to an individual in need thereof.

[0017] In addition, the present invention provides a method for inducing immunity against the Japanese encephalitis virus, comprising the step of administering a composition containing a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6 to an individual in need thereof.

[0018] It has been confirmed that a composition containing the Japanese encephalitis recombinant antigen protein according to the present invention exhibits superior immunogenicity and protective ability against challenge compared to previously approved live vaccines. In particular, it has been experimentally confirmed that the Japanese encephalitis recombinant antigen protein of the present invention demonstrates superior protective ability against Japanese encephalitis virus G5 as well as other genotypes. Therefore, the Japanese encephalitis recombinant antigen protein of the present invention can be utilized in various ways in the field of preventing infection by Japanese encephalitis virus G1 to G5.

[0019] Figure 1 is a figure showing the results of modeling the structures of common sequences TotDS_cons and AllGV_cons through alphafold.

[0020] Figure 2 shows the three-dimensional structures of TotDS and AIIGV, antigen proteins designed based on the TotDS common sequence (TotDS_cons) and AllGV common sequence (AllGV_cons) through an in-silico method.

[0021] Figure 3 shows the results of predicting the RNA secondary structures of the finally derived recombinant antigen protein candidate substances prM-EasC_D8-LD and prM-Eg5C_D8-LD.

[0022] Figure 4 shows the results of producing the final derived prM-E(A) and prM-E(G5) from host cell 293F.

[0023] Figure 5 is a figure showing the experimental schedule and experimental groups for evaluating the immunogenicity of the vaccine.

[0024] Figure 6 is a figure showing the results of analyzing the binding antibody titers of vaccine compositions (KNIID-JEV04, KNIID-JEV06 and KNIID-JEV04 & KNIID-JEV06) containing the finally derived prM-E (A) and prM-E (G5).

[0025] Figure 7 shows the results of measuring the neutralizing antibody titers against Japanese encephalitis virus G1, G3, and G5 of vaccine compositions (KNIID-JEV04, KNIID-JEV06, and KNIID-JEV04 & KNIID-JEV06) containing the finally derived prM-E(A) and prM-E(G5) (**: p<0.01, ***: p<0.001).

[0026] Figure 8 shows an experimental schedule for evaluating the protective ability against vaccine challenge.

[0027] FIG. 9 shows Japanese encephalitis virus type 3 LD in subjects immunized with a vaccine composition (KNIID-JEV04 & KNIID-JEV06) containing the finally derived prM-E(A) and prM-E(G5). 50 This is a diagram showing the results of evaluating the defensive ability after attack inoculation.

[0028] FIG. 10 shows Japanese encephalitis virus type 3 or 5 LD in subjects immunized with a vaccine composition (KNIID-JEV04 & KNIID-JEV06) containing the finally derived prM-E(A) and prM-E(G5). 50 This is a diagram showing the results of evaluating the defensive ability after attack inoculation.

[0029] The present invention will be described in detail below.

[0030] According to an embodiment of the present invention, the present invention provides a nucleic acid encoding a recombinant Japanese encephalitis antigen protein represented by the nucleotide sequence of SEQ ID NO. 3 or 5. The present invention also provides a recombinant Japanese encephalitis antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6.

[0031] The Japanese encephalitis recombinant antigen protein of the present invention is designed by analyzing the prM and E sequences of Japanese encephalitis virus types G1-G5 to collect common sequences, and based on this, by analyzing protein structural stability and adding point mutations.

[0032] The nucleic acid encoding the Japanese encephalitis recombinant antigen protein of the present invention comprises a prM sequence represented by SEQ ID NO. 7. Additionally, the Japanese encephalitis recombinant antigen protein of the present invention comprises a prM sequence represented by the amino acid sequence of SEQ ID NO. 8.

[0033] The scope of the present invention includes variants of the above nucleotide sequence. Specifically, the gene means a sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% sequence homology with the nucleotide sequence of SEQ ID NO. 3 or 5, and exhibiting substantially the same physiological activity as the nucleotide sequence represented by SEQ ID NO. 3 or 5. The “% of sequence homology” for a polynucleotide is determined by comparing two optimally arranged sequences with a comparison region, and a portion of the polynucleotide sequence in the comparison region may include additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for the optimal arrangement of the two sequences.

[0034] In addition, the Japanese encephalitis recombinant antigen protein includes a protein encoded by the nucleotide sequence of SEQ ID NO. 3 or 5 and a functional equivalent of said protein.

[0035] The above "functional equivalent" refers to a protein that has at least 80%, preferably 90%, more preferably 95%, sequence homology (i.e., identity) with the protein encoded by SEQ ID NO. 3 or 5 as a result of the addition, substitution, or deletion of amino acids, for example, having sequence homology of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, and exhibits substantially the same physiological activity as the protein encoded by SEQ ID NO. 3 or 5. In this specification, sequence homology and homology are defined as the percentage of amino acid residues of the candidate sequence relative to the amino acid sequence coded by SEQ ID NO. 3 or 5, after aligning the candidate sequence with the amino acid sequence coded by SEQ ID NO. 3 or 5 and introducing gaps. Where necessary, conservative substitutions are not considered as part of the sequence homology in order to obtain the maximum percentage sequence homology. The N-terminus, C-terminus, or internal extension, deletion, or insertion of the amino acid sequence coded by SEQ ID NO. 3 or 5 is not interpreted as a sequence affecting the sequence homology or homology.

[0036] In addition, the Japanese encephalitis recombinant antigen protein comprises a protein composed of the amino acid sequence of SEQ ID NO. 4 or 6 and a functional equivalent of said protein. The "functional equivalent of said protein" refers to having at least 80%, preferably 90%, more preferably 95%, sequence homology (i.e., identity) with the peptide of SEQ ID NO. 4 or 6 as a result of the addition, substitution, or deletion of amino acids, and includes, for example, having sequence homology of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0037] The sequence homogeneity mentioned above can be determined by common standard methods used to compare similar portions of the amino acid sequences of two proteins. Computer programs such as BLAST or FASTA align two proteins so that their respective amino acids are optimally matched (along the full length of one or two sequences or along a predicted portion of one or two sequences). The program provides a default opening penalty and a default gap penalty, and provides a scoring matrix such as PAM250 (standard scoring matrix) that can be used in conjunction with the computer program. For example, percentage homogeneity can be calculated as follows: multiply the total number of identical matches by 100 and then divide by the sum of the length of the longer sequence within the corresponding matched span and the number of gaps introduced into the longer sequence to align the two sequences.

[0038] In the above, "substantially homogeneous physiological activity" refers to activity that induces immunity against the Japanese encephalitis virus within an individual when applied to that individual. The scope of "functional equivalents" of the present invention includes derivatives in which a portion of the chemical structure of the peptide is modified while maintaining the basic framework of the peptide encoded by SEQ ID NO. 3 or 5 and the activity of inducing immunity against the Japanese encephalitis virus. For example, structural modifications intended to alter the stability, storage properties, volatility, or solubility of the peptide are included here.

[0039] In a specific embodiment of the present invention, the Japanese encephalitis recombinant antigen protein may have improved solubility or cavity volume.

[0040] In a specific embodiment of the present invention, the Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6 may include the prM sequence represented by SEQ ID NO. 8.

[0041]

[0042] In a specific embodiment of the present invention, the Japanese encephalitis recombinant antigen protein preferably has a minimum free energy (MFE) of -1200 to 1300 kcal / mol, and more preferably -1262.3 kcal / mol or -1266.7 kcal / mol, but the scope of the present invention is not limited thereto.

[0043]

[0044] According to another aspect of the present invention, the present invention provides a composition comprising a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6; or a nucleic acid encoding the same. The composition may be a Japanese encephalitis vaccine composition or an immunogenic composition against the Japanese encephalitis virus.

[0045] In a specific embodiment of the present invention, the composition may alone include a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4; or a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 6; or may include both types of proteins. In a preferred embodiment of the present invention, when the composition includes two types of Japanese encephalitis recombinant antigen proteins, they may be mixed in a weight ratio of 1:0.1 to 10, preferably 1:1.

[0046] In a specific embodiment of the present invention, the nucleic acid encoding the Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 may be represented by the nucleotide sequence of SEQ ID NO. 3. The nucleic acid encoding the Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 6 may be represented by the nucleotide sequence of SEQ ID NO. 5. Additionally, the composition may include the nucleic acid represented by the nucleotide sequence of SEQ ID NO. 3; or the nucleic acid represented by the nucleotide sequence of SEQ ID NO. 5; alone, or may include both types of nucleic acids.

[0047] In a specific embodiment of the present invention, the composition may have a protective ability against one or more infections selected from the group consisting of Japanese encephalitis virus genotypes G1 to G5. In an embodiment of the present invention, it was confirmed that a composition prepared by combining two types of Japanese encephalitis recombinant antigen proteins of the present invention, either alone or in combination, has superior immunogenicity against Japanese encephalitis virus G1 to G5 and a protective ability against challenge compared to commercially available live vaccines.

[0048] In the present invention, the vaccine composition and the immunogenic composition are administered for the purpose of inducing specific and active or passive immunity against the Japanese encephalitis virus.

[0049] The vaccine composition of the present invention may include one or more adjuvants suitable for constituting the vaccine composition. An adjuvant that may be included in the composition of the present invention refers to a substance that enhances the immune response of an injected animal, and numerous different adjuvants are known to those skilled in the art. The above-mentioned adjuvant may be one or more selected from the group consisting of Freund complete and incomplete adjuvants, aluminum salts such as aluminum hydroxide and aluminum phosphate, emulsions such as MF 59 and AS03, TLR agonist substances such as AS04 and CpG ODNs, particulates such as AS01, synthetic dsRNA such as Poly ICLC, GLA and derivatives such as GSA-SE and GSL-AF, imidazoquinolines such as imiquimod and 3M-052, CpG ODNs such as CpG ODN 1018, CpG ODN 7909, IC31, and CDNs such as c-di-AMP, but is not limited thereto.

[0050] In a specific embodiment of the present invention, the composition may have a volume ratio of Japanese encephalitis recombinant antigen protein and an adjuvant of 0.5 to 5:1, and preferably a volume ratio of 1:1.

[0051] The composition according to the present invention may be administered in an immunologically effective amount. The "immunologically effective amount" means a sufficient amount to produce a preventive effect against Japanese encephalitis and an amount that does not cause side effects or serious or excessive immune responses. The exact dosage concentration varies depending on the specific immunogen to be administered and can be easily determined by a person skilled in the art based on factors well known in the medical field, such as the age, weight, health, gender, individual sensitivity to drugs, route of administration, and method of administration of the vaccination subject, and may be administered one to several times.

[0052] The composition according to the present invention may include, in addition to the active ingredient, the Japanese encephalitis recombinant antigen protein or the nucleic acid encoding it, one or more carriers suitable for constituting the composition.

[0053] Carriers that may be included in the composition of the present invention are known to those skilled in the art and include, but are not limited to, proteins, sugars, etc. The above carriers may be aqueous solutions or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including saline solution and buffer medium. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactic acid-treated Ringer's, or fixative oils. Intravenous carriers include electrolyte supplements, liquids, and nutritional supplements, such as those based on Ringer's dextrose, for example.

[0054] The composition of the present invention may further include preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, inert gases, etc. The preservatives include formalin, thimerosal, neomycin, polymyxin B, and amphotericin B, etc. The composition of the present invention may include one or more suitable emulsifiers, such as Span or Tween. Additionally, the vaccine composition of the present invention may include a protective agent, and protective agents known in the art may be used without limitation, which may include, but are not limited to, lactose (LPGG) or trehalose (TPGG).

[0055] In a specific embodiment of the present invention, the composition may be administered subcutaneously (SC), intravenously, intramuscularly, intranasally, intra-articularly, intrasynovially, intrathecally, intrahepatically, intralesionally, or intracranially, and preferably may be administered intramuscularly.

[0056]

[0057] According to another aspect of the present invention, the present invention provides a method for preventing or treating Japanese encephalitis, comprising the step of administering a composition comprising a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6; or a nucleic acid encoding the same, to an individual in need thereof. The present invention also provides a method for inducing immunity against the Japanese encephalitis virus, comprising the step of administering a composition comprising a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6 to an individual in need thereof.

[0058] In a specific embodiment of the present invention, the individual may be an individual expected to develop a disease caused by Japanese encephalitis virus infection; an individual that has developed the disease; or an individual that has been determined to be cured, but is not limited thereto.

[0059] The method of the present invention preferably increases the in vivo immune response to a virus through intramuscular administration of the composition according to the present invention, and more specifically, it is preferable to increase the level of neutralizing antibody titers.

[0060]

[0061] Redundant content is omitted out of consideration for the complexity of this specification, and terms not otherwise defined in this specification have the meanings commonly used in the technical field to which this invention belongs.

[0062] The present invention will be described in more detail below through examples. These examples are solely for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0063]

[0064] Example 1. Derivation of Japanese encephalitis recombinant antigen protein candidate substances through protein structural stability analysis

[0065] In this embodiment, the sequences of prM-E of Japanese encephalitis virus of various genotypes were analyzed to identify a consensus sequence, and an antigen sequence capable of responding to various genotypes and mutations was derived. In addition, an antigen with improved stability was designed through structural modeling based on the derived sequence.

[0066]

[0067] 1-1. Analysis of Antigen Structure Stability

[0068] Approximately 6,000 sequences of the target protein (prM-E) were collected from the public database (GenBank). The collected raw data consist of whole-genome sequences, whole-genome E protein sequences, and partial E protein sequences. In the first step, sequences that were not of the whole length were removed to construct a total of 141 datasets (TotDS), including 101 domestic isolate sequences containing the minimum whole-genome E protein sequence and overseas isolate sequences containing the whole-genome sequence.

[0069] Based on the gene annotation information of the whole genome sequence of the reference strain (genbank acc. no.: NC_001437), the prM-E region of the foreign whole genome sequence was extracted.

[0070] The distribution by genotype based on the analysis of the obtained protein sequences is as shown in Table 1.

[0071] GI (Genotype 1) GII (Genotype 2) GIII (Genotype 3) GIV (Genotype 4) GV (Genotype 5) Domestic Sequence 49130019 International Sequence 1561443 Total 64744422

[0072]

[0073] 1-2. Generation of a consensus sequence

[0074] Common sequences were generated using the Emboss package. Common sequences for all sequences of 5 genotypes, common sequences for each genotype, and common sequences from all 141 sequences (TotDS) were derived. In addition, common sequences for each genotype and common sequences generated from all 77 sequences were compared in a dataset of 77 sequences (GDistDS) constructed considering the distribution ratio of genotypes.

[0075] As a result of comparative analysis of various common sequences, 1 to 3 amino acid variations were identified in the GI to GIII common sequences of TotDS. 22 to 41 variations were identified in GIV and GV, and differences in variation patterns were confirmed between the two genotypes. It was also confirmed that GDistDS has a variation pattern similar to that of TotDS.

[0076] The final common sequences selected were sequences derived from the TotDS dataset (TotDS_cons); and sequences derived from the sequence set containing all genotypes 5 (GV) (AllGV_cons). The selected final common sequences, TotDS_cons and AllGV_cons, are represented by the amino acid sequences of SEQ ID NOs 1 and 2, respectively, and the specific details are as shown in Table 2.

[0077]

[0078] 1-3. Antigen Protein Structure Modeling

[0079] Structural modeling of the final common sequences TotDS_cons and AllGV_cons selected in Examples 1-2 above was performed using AlphaFold. The tertiary structures of TotDS_cons and AllGV_cons modeled through AlphaFold are shown in Figure 1.

[0080] As shown in Figure 1, the full-length protein structures of the ED and stem were predicted for TotDS_cons and AllGV_cons, and three domains (DI–DIII) were identified in the ED. DI (light blue) consisted of 8 sheets and 1 helix located between DII and DIII, DII consisted of 8 sheets and 3 helixes, and DIII consisted of 6 sheets. The remaining C-terminus and stem of the ED were shown in light green.

[0081] The tertiary structures of the modeled TotDS_cons and AllGV_cons were 0.07 Å, which is lower than 2 Å, which is typically known to indicate structural similarity. In other words, it was confirmed that TotDS_cons and AllGV_cons are structurally very similar. Additionally, by aligning the tertiary structures of TotDS_cons and AllGV_cons with the Japanese encephalitis virus E protein (PDB: 3p54), for which the tertiary structure has been elucidated, it was confirmed that the tertiary structures of the two modeled proteins are similar (1.64 Å).

[0082]

[0083] 1-4. Design of Structural Stability-Contributing Antigen Proteins Using In-silico Methods

[0084] Proteins were designed based on protein sequences and structures using the PROSS server. Structural stability analysis was performed according to the guidelines provided by the program developers. Specifically, B-cell epitopes were predicted using BepiPred 2.0; for the TotDS common sequence (TotDS_cons), up to eight design sites overlapped with B-cell epitopes, so all of these sites were reverted to their original amino acids. Meanwhile, for the AllGV common sequence (AllGV_cons), up to eight design sites overlapping with B-cell epitopes were also identified, and these were reverted to their original amino acids.

[0085] For the TotDS common sequences, D4, D6, and D8 were selected, which were designed by applying point mutations to 4 to 24 sites. For the AllGV common sequences, since D4 and D5 showed a difference (V / I) only at site 31, four sequences were selected: D4, D5, D6, and D8, which were designed by applying point mutations to 12 to 28 sites.

[0086] Antigen proteins designed based on the TotDS common sequence (TotDS_cons) and AllGV common sequence (AllGV_cons) are shown in Figure 2.

[0087]

[0088] 1-5. Derivation of Antigen Candidates through Computational RNA Secondary Structure Analysis, etc.

[0089] In this example, a final antigen candidate was derived through solubility, cavity volume, and RNA secondary structure prediction.

[0090]

[0091] - Solubility and Cavity Volume Prediction

[0092] Among solubility prediction programs, the Aggrescan 3D (A3D) server, which utilizes structural information, was used. The predicted 3D structure file of the common sequence was input into the A3D server to calculate the solubility results of the common sequence. Design information (sites and variant amino acids) was input and executed on the results to calculate the solubility values ​​for each design.

[0093] There are research reports indicating that cavity volume is associated with protein stability. Accordingly, cavity calculations for each design structure were performed using the Computed Atlas of Surface Topography of proteins (CASTp) 3.0 server.

[0094] Based on the combined results of solubility and cavity volume predictions, candidate substances prM-EasC_D8-LD and prM-Eg5C_D8-LD with excellent results were selected.

[0095]

[0096] - Prediction of RNA secondary structure

[0097] The RNA secondary structures of candidate substances prM-EasC_D8-LD and prM-Eg5C_D8-LD were predicted, and the results are shown in Figure 3.

[0098] As shown in Figure 3, it was confirmed that the Minimum Free Energy (MFE) of prM-EasC_D8-LD and prM-Eg5C_D8-LD were -1262.3 kcal / mol and -1266.7 kcal / mol, respectively.

[0099]

[0100] The nucleic acid and amino acid sequences of the finally derived recombinant antigen protein candidates prM-EasC_D8-LD and prM-Eg5C_D8-LD are shown in Table 3. In the examples described below, prM-EasC_D8-LD and prM-Eg5C_D8-LD are abbreviated as prM-E(A) and prM-E(G5).

[0101]

[0102]

[0103] Example 2. Production of recombinant antigen protein candidate

[0104] In this example, prM-E(A) and prM-E(G5) finally derived in Example 1 were produced. Specifically, Plasmid DNA was prepared using Maxi prep, and this was applied to 293F cells 1x10 6 PEI was transfected into cells / ml. The transfected cells were cultured, and the cultured cells were harvested and centrifuged for 5 minutes (6,000 rpm). The cells were washed with 1x PBS for 3 minutes and centrifuged for 5 minutes (6,000 rpm) to obtain a cell pellet. 1 ml of RIPA buffer was added to the cell pellet to resuspend it, and it was sonicated three times for 10 seconds each. After sonication, the cells were centrifuged for 30 minutes (20,000 g). 10 μg of the cell lysate fraction was taken and Western blotting was performed (antibody: 6xHis Antibody (Cat. no WD_MAB_429)). The Western blotting results of prM-E(A) and prM-E(G5) are shown in Figure 4.

[0105] As shown in Figure 4, it was confirmed that prM-E(A) and prM-E(G5) were successfully expressed in host cells. Subsequently, the expressed prM-E(A) and prM-E(G5) were isolated and purified and used in the experiments described below.

[0106]

[0107] Example 3. Evaluation of the efficacy of vaccines containing prM-E(A) and prM-E(G5)

[0108] 3-1. Evaluation of Vaccine Immunogenicity

[0109] In this example, immunogenicity was analyzed after immunizing 4-6 week old C57BL / 6 mice with a recombinant antigen protein twice at 3-week intervals. The recombinant antigen protein was administered intramuscularly (IM) or subcutaneously (SC) depending on the experimental group. Aluminum salt (Alum) was used as the adjuvant for this experiment, and the positive control serum was the Ministry of Food and Drug Safety positive control serum (Management No.: MFDS-B-17-001, Standard Product Name: Cell Culture Japanese Encephalitis Vaccine (Beijing Supplement)). In addition, the commercial Japanese encephalitis vaccine (i.e., the live vaccine Imojev) was produced based on genotype 3. The experimental schedule and experimental groups for the immunogenicity evaluation test are shown in Figure 5. The vaccine containing prM-E(A) was designated as 'KNIID-JEV04', and the vaccine containing prM-E(G5) was designated as 'KNIID-JEV06'. These were administered as a single dose at a concentration of 20 µg or simultaneously administered at 10 µg each (KNIID-JEV04 & KNIID-JEV06).

[0110] The binding antibody titers were analyzed using ELISA. For the ELISA antigens, an Overlapping Peptide (OLP) was used, comprising a mixture of six types of upstream peptides containing MHC class I and MHC class II sequences, selected separately based on epitope analysis of the Japanese encephalitis virus E protein sequence and predicted to induce an immune response. The absorbance was measured at 450 nm after reacting the plate coated with the OLP mixture with immunized serum. The ELISA test results are shown in Figure 6.

[0111] As shown in Figure 6, the total binding antibody titer specific to Japanese encephalitis was analyzed via ELISA. As a result, the vaccine immunity group (Group 5) containing a mixture of two recombinant antigen protein candidates had a total IgG value (450 nm absorbance value) higher than that of the previously approved Imojev (live vaccine) immunity group.

[0112]

[0113] Neutralizing antibody titers were analyzed using the PRNT (Plaque Reduction Neutralization Test). Specifically, neutralizing antibody titers against Japanese encephalitis virus genotypes 1, 3, and 5 were measured using BHK-21 hamster kidney cells. Specific information regarding the Japanese encephalitis virus is as follows.

[0114]

[0115] - Japanese Encephalitis Virus Type 1 (K05GS): Japanese encephalitis virus isolated from mosquitoes in 2005

[0116] - Japanese Encephalitis Virus Type 3 (K94A071): Japanese encephalitis virus isolated from mosquitoes in 1994

[0117] - Japanese Encephalitis Virus Type 5 (K15P38): Japanese encephalitis virus isolated from humans in 2015

[0118]

[0119] The results of confirming the neutralizing antibody titer using the PRNT test method are shown in Figure 7.

[0120] As shown in Figure 7, the recombinant antigen protein candidate immune groups (groups 3-5) had significantly higher neutralizing antibody titers against Japanese encephalitis virus types 1, 3, and 5 than the approved Imojev (live vaccine) immune group (group 6).

[0121]

[0122] 3-2. Evaluation of protective ability against vaccine challenge

[0123] The protective efficacy against challenge was evaluated for a vaccine immunization group prepared by mixing two recombinant antigen protein candidates. Specifically, after two doses of the vaccine, the Japanese encephalitis virus was challenged intracranially (IC) three weeks after the last dose. The Japanese encephalitis virus was LD 50 and 50LD 50 It was inoculated with. The above LD 50represents the dose at which 50% of the experimental animals die when the substance is administered to the experimental animals. The test schedule for evaluating the protective ability against vaccine challenge is shown in Figure 8.

[0124]

[0125] LD 50 Survival rate upon attack inoculation

[0126] Japanese encephalitis virus type 3 LD to a vaccine immunization group mixing two recombinant antigen protein candidates 50 The results of evaluating the defensive ability after attack inoculation are shown in Figure 9.

[0127] As shown in Figure 9, the vaccine immunity group (Group 3) containing a mixture of two recombinant antigen protein candidates had a higher survival rate of experimental animals than the previously approved Imojev (live vaccine) immunity group (Group 4).

[0128]

[0129] 50LD 50 Survival rate upon attack inoculation

[0130] Japanese encephalitis virus type 3 or 5 in a vaccine immunization group mixing two recombinant antigen protein candidates 50LD 50 The results of evaluating the protective ability after attack inoculation are shown in Figure 10.

[0131] As shown in Figure 10, it was confirmed that the vaccine immunity group (Groups 3, 6) mixed with two recombinant antigen protein candidate substances had a higher survival rate of experimental animals than the Imojev (live vaccine) immunity group (Groups 4, 7) which is already approved for both types 3 and 5.

[0132]

[0133] In summary, the inventors analyzed the prM and E sequences of Japanese encephalitis virus types G1-G5 to collect common sequences, and based on this, designed two novel Japanese encephalitis recombinant antigen proteins (prM-E(A) and prM-E(G5)) through protein structural stability analysis and the addition of point mutations. Subsequently, Japanese encephalitis vaccine compositions were prepared using the Japanese encephalitis recombinant antigen proteins alone or in combination, and it was confirmed that their immunogenicity and protective ability against challenge were superior to that of an approved live vaccine (Imojev). Therefore, the Japanese encephalitis recombinant antigen proteins prM-E(A) and prM-E(G5) of the present invention can be utilized in various ways in the field of preventing infection by Japanese encephalitis virus G1 to 5.

[0134]

[0135] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A nucleic acid encoding a Japanese encephalitis recombinant antigen protein represented by the nucleotide sequence of SEQ ID NO. 3 or 5.

2. Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6.

3. In paragraph 2, the Japanese encephalitis recombinant antigen protein is one having improved solubility or cavity volume.

4. The Japanese encephalitis recombinant antigen protein according to paragraph 2, wherein the Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6 comprises the prM sequence represented by SEQ ID NO.

8.

5. A Japanese encephalitis vaccine composition comprising: a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6; or a nucleic acid encoding the same.

6. A Japanese encephalitis vaccine composition according to claim 5, wherein the vaccine composition has the ability to protect against one or more infections selected from the group consisting of Japanese encephalitis virus genotypes G1 to G5.

7. The Japanese encephalitis vaccine composition according to claim 5, wherein the vaccine composition comprises a Japanese encephalitis recombinant antigen protein represented by the amino acid sequences of SEQ ID NOs. 4 and 6; or a nucleic acid encoding the same.

8. A Japanese encephalitis vaccine composition according to claim 5, wherein the composition is administered subcutaneously (SC), intravenously, intramuscularly, intranasally, intra-articularly, intrasynovially, intrathecally, intrahepatically, intralesionally, or intracranially.

9. An immunogenic composition against Japanese encephalitis virus comprising a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6.

10. An immunogenic composition against Japanese encephalitis virus according to claim 9, wherein the immunogenic composition comprises a Japanese encephalitis recombinant antigen protein represented by the amino acid sequences of SEQ ID NOs. 4 and 6; or a nucleic acid encoding the same.

11. A method for preventing or treating Japanese encephalitis comprising the step of administering a composition comprising a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6; or a nucleic acid encoding the same; to an individual in need thereof.

12. A method for inducing immunity against Japanese encephalitis virus comprising the step of administering a composition containing a Japanese encephalitis recombinant antigen protein represented by the amino acid sequence of SEQ ID NO. 4 or 6 to an individual in need thereof.

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