Recombinant adenovirus comprising a polynucleotide encoding DENV envelope protein and a polynucleotide encoding ferritin heavy chain protein, and uses thereof

Abstract

The present invention relates to a recombinant adenovirus comprising a polynucleotide encoding a DENV envelope protein and a ferritin heavy chain protein and to the use thereof, wherein the present invention provides a recombinant adenovirus comprising a polynucleotide encoding a DENV envelope protein and a polynucleotide encoding a ferritin heavy chain protein, and a vaccine composition for the prevention of DENV infection comprising said recombinant adenovirus as an active ingredient.

Description

Technology Field

[0001] This invention relates to a recombinant adenovirus comprising a polynucleotide encoding the DENV envelope protein and the ferritin heavy chain protein, and its use as a vaccine against DENV. Background Technology

[0003] The dengue virus is a highly contagious flavivirus transmitted by certain mosquitoes, causing viral infection. The dengue virus exists in four serotypes: DENV-1, DENV-2, DENV-3, and DENV-4, each distinguished by various interactions between the virus type and human immunity. According to the World Health Organization (WHO), cases of dengue infection have been increasing over the past five years, necessitating the development of vaccines and antiviral drugs to address this issue. Currently, vaccines available for the dengue virus are very limited, and there is a need for the additional development of vaccines that enhance immune response and safety.

[0004] The genome of the dengue virus contains three major structural proteins—the capsid, premembrane (prM), and envelope—and seven non-structural proteins. Among these, the structural proteins play a crucial role in the process of the virus penetrating host cells; in particular, the envelope protein is a key structural component that interacts with the host cell membrane. Currently, various vaccine strategies against the dengue virus are being studied, including live attenuated vaccines, inactivated virus vaccines, recombinant subunit vaccines, viral vector vaccines, and DNA vaccines. Most vaccine candidates utilize the DENV envelope protein as the primary antigen. Regarding viral vector-based vaccines, adenovirus, alphavirus, and vaccinia virus vectors are being used, and research is underway to clone antigen molecules and deliver them to host cells. Vaccines using recombinant viruses induce a sustained immune response, offer advantages in vaccine stability and large-scale manufacturing, and are effective in inducing potent antibody and cellular immune responses.

[0005] Under this technical background, the inventors produced a recombinant adenovirus comprising a polynucleotide encoding the DENV envelope protein and a polynucleotide encoding the ferritin heavy chain protein by combining the dengue fever virus envelope protein and the human ferritin heavy chain protein through an adenovirus vector system, and completed the present invention by confirming the excellent vaccine efficacy of the recombinant adenovirus against DENV infection. The problem to be solved

[0007] One aspect is to provide a recombinant adenovirus comprising a polynucleotide encoding a DENV (Dengue Virus) envelope protein and a polynucleotide encoding a ferritin heavy chain protein.

[0008] Another aspect is to provide a vaccine composition for the prevention of DENV infection, comprising the above-mentioned recombinant adenovirus as an active ingredient.

[0009] Another aspect provides an isolated nucleic acid molecule comprising a polynucleotide encoding a DENV envelope protein consisting of the nucleotide sequence of SEQ ID NO. 8 and a polynucleotide encoding a ferritin heavy chain protein consisting of the nucleotide sequence of SEQ ID NO. 10, wherein the polynucleotide encoding the DENV envelope protein and the polynucleotide encoding the ferritin heavy chain protein are connected directly or through a linker sequence.

[0011] Other objects and advantages of this application will become more apparent from the following detailed description, together with the appended claims and drawings. Anything not described in this specification is omitted, as it can be sufficiently recognized and inferred by those skilled in the art of this application or a similar art field. means of solving the problem

[0013] Each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0015] One aspect provides a recombinant adenovirus comprising a polynucleotide encoding a DENV (Dengue Virus) envelope protein and a polynucleotide encoding a ferritin heavy chain protein.

[0016] In this specification, the term “envelope protein” refers to a structural protein located on the outer shell of a virus, which exists on the surface of a viral particle and plays a key role in the process of infection of a host cell. It facilitates the delivery of the viral genome into the host cell by mediating viral attachment and fusion with the host cell membrane. Envelope proteins have structures and functions specific to the type of virus. For example, in the dengue virus (DENV), a virus of the Flavivirus genus, the envelope protein is called protein E, which binds to a receptor on the host cell and exerts infectivity through pH-dependent fusion.

[0017] In one embodiment, the polynucleotide encoding the DENV envelope protein and the polynucleotide encoding the ferritin heavy chain protein may be connected directly or through a linker sequence.

[0018] In this specification, the term “identity” refers to the overall correlation between polymer molecules, for example, between nucleic acids (e.g., DNA molecules and / or RNA molecules) and / or between polypeptides. For example, polypeptides are considered “substantially identical” if their amino acid sequences are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. The calculation of the percentage of identity between two nucleic acid or polypeptide sequences may be performed, for example, by aligning the two sequences for optimal comparison purposes (for example, a gap may be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). For example, the length of the sequence aligned for comparison purposes is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. Next, nucleic acid or polypeptide sequences at corresponding positions are compared. The determination of the percentage of identity between the two sequences and the comparison of the sequences can be achieved using mathematical algorithms. As is widely known to those skilled in the art, amino acid or nucleic acid sequences can be compared using any various algorithms available in commercial computer programs, such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSIBLAST for amino acid sequences.

[0019] In one embodiment, the polynucleotide encoding the DENV envelope protein may be composed of the nucleotide sequence of SEQ ID NO. 8. In addition, it may be composed of the nucleotide sequence of SEQ ID NO. 8 as well as a sequence having 80 to 99% sequence identity with said sequence.

[0020] In one embodiment, the DENV envelope protein may include the amino acid sequence of SEQ ID NO. 12. In addition, it may include not only the amino acid sequence of SEQ ID NO. 12 but also a sequence having 80 to 99% sequence identity with said sequence.

[0021] The term "ferritin" as used in this specification refers to an iron-storing protein that is widely present in prokaryotes and eukaryotes. Ferritin has a molecular weight of approximately 500,000 Da, consists of a heavy chain and a light chain, and exhibits the unique characteristic of forming spherical particles due to its self-assembly ability. Ferritin is a protein in which 24 monomers (single monomers or heteromers composed of either the heavy or light chain) assemble to form a large spherical tertiary structure; in the case of human ferritin, the outer diameter is approximately 12 nm and the inner diameter is approximately 8 nm. Depending on pH conditions, ferritin may disperse into monomers or assemble into nanoparticles.

[0022] In one embodiment, the polynucleotide encoding the ferritin heavy chain protein may be composed of the nucleotide sequence of SEQ ID NO. 10. In addition, it may be composed not only of the polynucleotide sequence of SEQ ID NO. 10 but also of a sequence having 80 to 99% sequence identity with said sequence.

[0023] The above ferritin heavy chain protein may be derived from human or non-human ferritin protein and may each include all or part of the amino acid sequence within the heavy chain constituting ferritin.

[0024] In one embodiment, the ferritin heavy chain protein may comprise the amino acid sequence of SEQ ID NO. 14. In addition, it may comprise not only the amino acid sequence of SEQ ID NO. 14 but also a sequence having 80 to 99% sequence identity with said sequence.

[0025] In one embodiment, the polynucleotide encoding the DENV envelope protein may have a polynucleotide encoding a precursor membrane protein (prM) protein linked to its 5' end either directly or through a linker sequence.

[0026] In this specification, the term “prM protein” refers to an essential structural protein of viruses belonging to the Flaviviridae family, such as the dengue fever virus, which is a post-translational modified protein that initially exists in an inactive state but is converted into an active form by cleaving a specific portion during the maturation process of the viral particle. This cleavage process of prM plays a decisive role in converting the viral particle from a non-infectious state to an infectious state, and this acts as a very important regulatory step in the life cycle of the virus.

[0027] The above recombinant adenovirus can improve the expression efficiency of target proteins, namely DENV envelope protein and ferritin heavy chain protein, by additionally including a polynucleotide encoding prM protein.

[0028] According to one embodiment, the polynucleotide encoding the prM protein may be a polynucleotide encoding a part of the prM protein.

[0029] In one embodiment, the polynucleotide encoding the prM protein may be composed of the nucleotide sequence of SEQ ID NO. 7. In addition, it may be composed of the nucleotide sequence of SEQ ID NO. 7 as well as a sequence having 80 to 99% sequence identity with said sequence.

[0030] In one embodiment, the prM protein may include the amino acid sequence of SEQ ID NO. 11. In addition, it may include not only the amino acid sequence of SEQ ID NO. 11 but also a sequence having 80 to 99% sequence identity with said sequence.

[0031] In one embodiment, the polynucleotide encoding the prM protein, the polynucleotide encoding the DENV envelope protein, and the polynucleotide encoding the ferritin heavy chain protein may be sequentially connected from the 5' end directly or through a linker sequence.

[0032] In this specification, the term “recombinant adenovirus” may refer to an envelopeless virus or viral vector having a linear double-stranded DNA genome (34–43 kb). The recombinant adenovirus may be a genetic construct for delivering and stably expressing a target gene, e.g., a polynucleotide encoding a DENV envelope protein and a polynucleotide encoding a ferritin heavy chain protein, to a target tissue or cell. To this end, the recombinant adenovirus may further comprise a nucleic acid construct containing elements necessary to express the target gene / protein, in addition to the target gene, i.e., the polynucleotide sequence encoding the aforementioned DENV envelope protein and the polynucleotide sequence encoding the ferritin heavy chain protein. Specifically, it may further comprise a nucleic acid construct comprising an expression regulatory sequence including at least a promoter and a nucleic acid for expressing the target protein operably linked to the expression regulatory sequence.

[0033] For the purposes of the present invention, the above-mentioned target protein may be a protein in which a DENV envelope protein and a ferritin heavy chain protein are fused or operably linked.

[0034] In this specification, the term “expression regulatory sequence” refers to a nucleic acid sequence essential for expressing a sequence encoding a target protein operably linked in a specific host organism. For example, it is known that promoters, polyadenylation signals, and enhancers are used as regulatory sequences suitable for eukaryotic cells.

[0035] In this specification, the term “promoter” refers to a nucleic acid fragment that regulates the transcription of one or more coding sequences and is located upstream in the reading direction with respect to the transcription direction from the transcription initiation site of the coding sequence, which is structurally identified by the presence of a binding site for a DNA-dependent RNA polymerase, a transcription initiation site and any other nucleic acid sequence, e.g., non-limitingly, a transcription factor binding site, a repressor and activator protein binding site, and any other nucleotide sequence known to those skilled in the art that directly or indirectly regulates the transcription level of the promoter. Examples of the promoter include any promoter functioning in mammalian cells, such as the CMV promoter, SV40 promoter, EF-1α promoter, CAG promoter, PGK promoter, U3 promoter, U6 promoter, H1 promoter, etc. In addition to these promoters, known inducible promoters, tissue / organ-specific promoters, time-specific promoters, or mutant sequences having functional equivalence to them may be used in the present invention.

[0036] In this specification, the term “enhancer” may refer to a nucleic acid sequence located adjacent to the sequence encoding the target protein. The enhancer factor is typically located in the 5’ direction from the promoter factor, or it may be located downstream of or within the coding nucleic acid sequence (e.g., the recombinant product or the DNA sequence to which it is transcribed or translated into the products). That is, the enhancer factor may be located 100 bp (base pairs), 200 bp, or 300 bp or further upstream or downstream from the nucleic acid sequence encoding the target protein.

[0037] In this specification, the term “operably linked” may indicate that nucleotide sequences are linked on a single nucleic acid fragment so that one function is influenced by another.

[0038] In one embodiment, the recombinant adenovirus may comprise the nucleic acid structure shown in FIG. 1b, and, for example, an envelope ferritin heavy chain (EH) clone according to one embodiment may be introduced into the pacAd5 CMV-N-pA vector, but is not limited thereto.

[0039] According to one embodiment, by constructing a recombinant adenovirus into which a polynucleotide encoding the DENV envelope protein and the ferritin heavy chain protein is introduced, it was found that the recombinant adenovirus can stably express the target proteins, the DENV envelope protein and the ferritin heavy chain protein, within target cells.

[0040] In addition, administration of this recombinant adenovirus was able to significantly enhance the IgG-mediated humoral immune response against the DENV antigen in a mouse animal model. Therefore, a recombinant adenovirus according to one aspect can be utilized as an active ingredient in a vaccine composition for the prevention of DENV infection.

[0042] Another aspect provides a vaccine composition for the prevention of DENV infection comprising the above-mentioned recombinant adenovirus as an active ingredient; and provides a medicinal use of the above-mentioned recombinant adenovirus for the prevention of DENV infection.

[0043] Since the above vaccine composition and medicinal use include or utilize the aforementioned recombinant adenovirus as is, the common details among them are omitted to avoid excessive complexity in this specification.

[0044] In this specification, the term “effective ingredient” means an appropriate effective amount of an ingredient that influences beneficial or desirable clinical or biochemical results. Specifically, it may mean an effective amount of a formulation, activator, or recombinant virus. The effective amount may be administered once or more times and may be an appropriate amount for preventing disease, or for the non-limiting alleviation of symptoms, reduction of disease range, stabilization of disease state (i.e., no worsening), delay or reduction of disease progression, or improvement or temporary alleviation and reduction (partial or wholly) of the disease state.

[0045] In this specification, the term "prevention" refers to any act of blocking the occurrence of a disease in advance, suppressing a disease, or delaying its progression. For example, it refers to preventing or hindering the occurrence of obesity or its characteristic properties, or defending against or protecting against the occurrence of DENV infection or its characteristic properties.

[0046] In this specification, the term “effective amount” refers to the meaning generally accepted in the art. The term may generally refer to an amount of a molecule, compound, or component that elicits an intended biological response (e.g., a beneficial response) in a cell, tissue, system, animal, or human being sought by a researcher, veterinarian, physician, or other clinician. Specifically, a “therapeutically effective amount” may refer to an amount of a molecule, compound, or component that elicits a desirable medical response to the extent that a specific clinical treatment can be considered effective, such that there is a therapeutically relevant change in a measurable parameter related to a disease or disorder, for example. The therapeutically effective amount of a drug for the treatment of said disease or disorder may be the amount required to bring about a therapeutically relevant change in said parameter.

[0047] In one embodiment, the present invention relates to a pharmaceutical composition / vaccine composition comprising the recombinant adenovirus of the present invention in a pharmaceutically acceptable carrier or other pharmaceutical material, adjuvant, diluent, etc. The vaccine composition may be selected from the group consisting of compositions for intramuscular administration, subcutaneous administration, intraperitoneal administration, intravenous administration, dermal administration, ocular administration, and cerebral administration. When the vaccine composition is administered by injection or systemic administration, the carrier will typically be a liquid carrier. For other administration methods, the carrier may be a solid or a liquid, for example, sterile pyrogen-free water or sterile pyrogen-free phosphate-buffered saline solution. As the injection medium, it is preferable to use water containing additives common to injection solutions, such as stabilizers, salts or saline and / or buffers.

[0048] The above vaccine composition refers to a composition comprising the recombinant adenovirus of the present invention described above and one or more components selected from the group consisting of pharmaceutically acceptable and pharmacologically appropriate excipients, e.g., fillers, solvents, diluents, carriers, adjuvants, disintegrants, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, and long-term delivery regulators, the selection and proportion thereof are determined according to the type of administration, route, and dosage. The vaccine composition of the present invention and the method of manufacturing the same will undoubtedly be obvious to those skilled in the art. The vaccine composition should preferably be manufactured in accordance with GMP (Good Manufacturing Practice) requirements. The composition may include a buffer composition, a tonicity agent, a stabilizer, and a solubilizer.

[0049] The above vaccine composition may be manufactured, packaged, or widely sold in the form of a ready formulation in the form of a single unit dose or multiple single unit doses. As used herein, the term “single unit dose” refers to a divided amount of the vaccine composition containing a predetermined amount of the active ingredient. The amount of the active ingredient typically corresponds to the dose of the active ingredient to be administered to an individual, or for convenience, a portion of such dose, for example, 1 / 2 or 1 / 3 of the dose.

[0051] Another aspect provides a method for preventing or improving DENV infection, comprising the step of administering the above vaccine composition to an individual.

[0052] Since the method for preventing or improving the above-mentioned DENV infection includes or utilizes the aforementioned recombinant adenovirus or vaccine composition as is, the common details among them are omitted to avoid excessive complexity in this specification.

[0053] In this specification, the term "individual" refers to a subject requiring prevention or treatment of a disease, specifically DENV infection, and more specifically, may include all mammals such as human or non-human primates, mice, dogs, cats, horses, cattle, sheep, pigs, goats, camels, and antelopes.

[0055] Another aspect provides an isolated nucleic acid molecule comprising a polynucleotide encoding a DENV envelope protein consisting of the nucleotide sequence of SEQ ID NO. 8 and a polynucleotide encoding a ferritin heavy chain protein consisting of the nucleotide sequence of SEQ ID NO. 10, wherein the polynucleotide encoding the DENV envelope protein and the polynucleotide encoding the ferritin heavy chain protein are connected directly or through a linker sequence.

[0056] In one embodiment, the polynucleotide encoding the DENV envelope protein composed of the nucleotide sequence of SEQ ID NO. 8 may have a polynucleotide encoding the premembrane (prM) protein composed of the nucleotide sequence of SEQ ID NO. 7 linked at the 5' end either directly or through a linker sequence.

[0057] In one embodiment, the polynucleotide encoding a prM protein consisting of the nucleotide sequence of SEQ ID NO. 7, the polynucleotide encoding a DENV envelope protein consisting of the nucleotide sequence of SEQ ID NO. 8, and the polynucleotide encoding a ferritin heavy chain protein consisting of the nucleotide sequence of SEQ ID NO. 10 may be sequentially connected from the 5' end directly or through a linker sequence.

[0058] Since the isolated nucleic acid molecule above shares a technical composition with the aforementioned recombinant adenovirus or vaccine composition, the common details between them are omitted to avoid excessive complexity in this specification.

[0059] In this specification, the term “nucleic acid” refers to a polymeric material comprising a plurality of nucleotide units, specifically a polymer in which a plurality of nucleotide units are linked together by phosphodiester bonds of a sugar / phosphate basic backbone. The term “nucleic acid” may be used interchangeably with the terms “polynucleotide” and “nucleic acid molecule.” The nucleic acid is a biopolymer essential to living organisms and may be RNA or DNA, preferably DNA, which encodes genetic information through its unique base sequence. The nucleic acid may be isolated, artificially synthesized, or non-naturally occurring or engineered, and “non-naturally occurring or engineered” refers to a state created by applying artificial modifications rather than the state in which it exists naturally. Here, the artificial modification is intended to enhance the expression of the target protein and may include a codon-optimized, DENV envelope protein and / or ferritin heavy chain protein encoding sequence.

[0060] In this specification, the term “isolated” nucleic acid (e.g., “isolated DNA”) means a polynucleotide that is at least partially isolated from at least a portion of a nucleic acid found to be associated with other components of a naturally occurring organism or virus, e.g., cellular or viral structural components or other polypeptides or polynucleotides.

[0061] In one embodiment, the isolated nucleic acid molecule can be inserted into a viral vector, for example, an adenovirus, and delivered to an individual.

[0062] In one embodiment, the isolated nucleic acid molecule can be delivered to an individual via the aforementioned viral vector to induce an immune response to the DENV antigen. Here, the immune response may include an IgG-mediated humoral immune response. Effects of the invention

[0064] According to a vaccine composition according to one aspect, by including a recombinant adenovirus containing a polynucleotide encoding a DENV envelope protein and a ferritin heavy chain protein as an active ingredient, an immune response to the DENV antigen can be enhanced.

[0065] Accordingly, a polynucleotide according to one aspect or a recombinant adenovirus containing the same can be utilized as an active ingredient in a vaccine composition for the prevention of DENV infection. Brief explanation of the drawing

[0067] FIG. 1a is a figure showing a vector map of a packaging vector used to produce a recombinant adenovirus according to one embodiment. FIG. 1b is a diagram showing a vector map of a shuttle vector used to produce a recombinant adenovirus according to one embodiment. FIG. 1c is a figure showing the expression levels of envelope (E) and envelope ferritin heavy chain (EH) proteins confirmed by Western blot using a Myc antibody after transfection of a recombinant plasmid according to one embodiment. FIG. 1d is a figure showing the expression levels of E and EH proteins confirmed by Western blot using a ferritin heavy chain antibody after transfection of a recombinant plasmid according to one embodiment. FIG. 2a is a figure confirming the specific cytopathological effect (CPE) of a recombinant adenovirus according to one embodiment. FIG. 2b is a figure confirming the presence of E and EH genes from DNA derived from the supernatant of a recombinant adenovirus culture according to one embodiment. FIG. 2c is a figure confirming the presence of an adenovirus band in a recombinant adenovirus according to one embodiment. FIG. 2d is a figure showing the results of performing TCID50 analysis to analyze the titer of a recombinant adenovirus according to one embodiment. Figure 3a is a figure showing the expression levels of E and EH proteins confirmed by Western blot using a Myc antibody in cells transduced with a recombinant adenovirus according to one embodiment. Figure 3b is a figure showing the expression levels of E and EH proteins by Western blot using a ferritin heavy chain antibody in cells transduced with a recombinant adenovirus according to one embodiment. FIG. 4a is a schematic diagram illustrating an experimental process using a mouse animal model to evaluate the immune response of a vaccine composition containing a recombinant adenovirus according to one embodiment. FIGS. 4b and 4c are figures showing the results of confirming the humoral immune response induced from the administration of a vaccine composition containing a recombinant adenovirus according to one embodiment through an enzyme-linked immunosorbent assay. Specific details for implementing the invention

[0068] The present invention will be explained in more detail below through experimental examples and embodiments. However, these experimental examples and embodiments are intended to illustrate the present invention, and the scope of the present invention is not limited to these experimental examples and embodiments.

[0070] [Experimental Example]

[0071] Experimental Example 1. Preparation of Cells and Viruses

[0072] Vero cells were maintained in Minimum Essential Medium (MEM) (WELGENE, Korea) supplemented with 10% fetal bovine serum (FBS) (Biowest, Lakewood Ranch, FL, USA), 1% antibiotic-antifungal agent (AA) (Biowest, Lakewood Ranch, FL, USA), and 1% HEPES (Gibco, USA). HEK 293T and 293A cells were maintained in Dulbecco's modified Eagle's Medium (DMEM) (Biowest, Lakewood Ranch, FL, USA) containing 10% FBS, 1% AA, and 1% HEPES. A549 cells were maintained in RPMI 1640 medium (WELGENE, Korea) supplemented with 10% FBS and 1% AA. The DENV-2 / KBPV-VR-29 strain was used as the virus.

[0074] Experimental Example 2. Virus Culture

[0075] DENV2 was inoculated into Vero cells in serum-free MEM supplemented with 2% FBS and 1% AA, and cultured in a CO2 incubator at 37°C for 3–5 days. The virus culture was scaled up by subculturing 4–5 times in the same manner. Finally, the viruses were harvested and centrifuged at 12,000 × g for 10 minutes at 4°C to collect the supernatant. The cell pellet was suspended in a virus stock solution, thawed through three freeze-thaw cycles, frozen in liquid nitrogen (LN2), and thawed in a 37°C water bath to release intracellular virus particles. The cells were then centrifuged at 12,000 × g for 10 minutes to pellet the cell debris, and the supernatant containing the virus was mixed with the virus supernatant. The viruses were purified and concentrated by centrifugation using a sucrose gradient. Briefly, 25 mL of the virus supernatant was carefully transferred to 10 mL of 20% sucrose dissolved in phosphate-buffered saline (PBS) and ultracentrifuged at 72,000 × g for 5 hours at 4°C in a Hitachi CP100NX ultracentrifuge. The supernatant was aspirated, and 200 μL of PBS containing 1% FBS was added to the pellet, followed by overnight incubation at 4°C. The next day, the virus pellet suspended in the solution was dispensed into a microcentrifuge tube and stored at -80°C.

[0077] Experimental Example 3. Viral RNA isolation, cDNA pretreatment, and qPCR

[0078] Viral RNA was isolated from 300 μL of viral supernatant using Ribospin™ vRD (GeneAll, Seoul, South Korea), and cDNA was prepared using TOPscript™ RT DryMIX (dN18 / dN6) (Enzynomics, Daejeon, South Korea). To confirm cultured and purified viruses, 1 μL of cDNA was used for qPCR analysis, and 1 μL of each previously reported primer, Primer 1, Primer 2 (Table 1), and 10 μL of 2X Real-Time PCR Master Mix (BioFACT™, Daejeon, South Korea) were added to the total reaction mixture (20 μL). RT-qPCR was performed using a standard two-step thermal cycling profile with a QuantGene 9600 Real Time Thermal Cycler (Bioer, Binjiang, China). Initial denaturation at 95°C for 5 minutes, denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 20 seconds, and 40 repeated cycles of denaturation and annealing were performed. Relative expression levels were calculated using the cycle threshold (Ct) value.

[0080] Name Sequence (5'→3') SEQ ID NO. Primer 1 CAAACAGCAGGACCTTGG 1 Primer 2 ATCCATCCTCACCTCTGT 2 Primer 3 GGTACCCTCGAGATGGACTACAAAGACGATGACGACAAGGCAGCAATCCTGGCATAC 3 Primer 4 GGAATTCCAGATCCTCTTCTGAGATGAGTTTTTGTTCACGCGTTCCTTTCTTGAACCAGTT 4 Primer 5 GGAATTCAGCAGCGGCACGACCGCGTCCACCTCGCA 5 Primer 6 CGTCTAGATTAGCTTTCATTATCACTGTCTC 6

[0082] Experimental Example 4. Plasmid Composition

[0083] RAPAd to generate rAd for DENV2 viral envelope (E) and envelope ferritin heavy chain (EH) genes ®A CMV adenovirus expression system (Cell Biolabs, San Diego, CA, USA) was used. The pacAd5 CMV KN pA (shuttle vector) and pacAd5 9.2-100 (packaging vector) vectors were used, and each E and EH gene was cloned into the multiple cloning site (MCS) of the shuttle vector. Initially, 93 nucleotides (31 amino acids) from the C-terminus of the prM gene and 1,185 nucleotides (395 amino acids) from the N-terminus of the envelope gene were amplified from the prepared cDNA using Primers 3 and 4 (Table 1), and then inserted into the XhoI and EcoRI sites of the pacAd5 CMV KN pA vector to obtain the pAd E plasmid. To construct the EH plasmid, the ferritin heavy chain gene amplified from cDNA isolated from 293T cells was cloned between the EcoRI and XbaI enzyme sites downstream of the prM 31 envelope gene using Primers 5 and 6 with a linker (SSG) protruding at the 5′ end, and the pAd EH plasmid was obtained.

[0085] Experimental Example 5. Transfection of a recombinant plasmid

[0086] One day before transfection, HEK 293T cells were 1 × 10 6 Cells were cultured in six-well cell culture plates at a cell / well concentration, and when the cells reached 80-90% confluence, 200 μL of a transfection mixture containing 3 μg of plasmid DNA and 9 μL of polyethyleneimine (PEI) was added to the cells. Afterward, the medium was replaced, and cells were collected for expression analysis after incubation in a 37°C CO2 incubator for 48 hours.

[0088] Experimental Example 6. Adenovirus production, amplification, and purification

[0089] For adenovirus production, 2.4 μg of the shuttle vector containing the gene of interest and 1.2 μg of the Ad backbone vector were cleaved with the PacI enzyme for vector linearization. One day prior to transfection, 2 × 10⁶ were placed in antibiotic-free 60 mm cell culture dishes. 6 Canine cells were seeded. When the cells reached 70–80% confluence, the shuttle vector and backbone vector were co-transfected with Lipofectamine® 3000 transfection reagent (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's instructions. The next day, the medium was aspirated and 4 mL of fresh culture medium (DMEM supplemented with 10% FBS and 1% ampicillin) was added. After incubation in a CO2 incubator at 37°C for 7–14 days, plaques were checked intermittently. The rAds produced from pAd E and pAd EH were named rAd E and rAd EH, respectively.

[0090] When an appropriate cytopathic effect (CPE) / plaque appeared (50% of the cells floating), the cells and virus supernatant were harvested and centrifuged at 2,000 rpm for 10 minutes. The cell pellet was suspended in a virus stock solution, and three freeze-thaw cycles were performed to release the intracellular virus, followed by centrifugation at 12,000 rpm for 10 minutes. Then, the supernatant was mixed with the previous supernatant, inoculated into a new 10 cm dish, and incubated in a 37°C CO2 incubator for 1–2 hours. Subsequently, the medium was replaced with 10 mL of DMEM containing 5% FBS and 1% ampicillin, and the samples were stored in the incubator for 2–3 days until all cells were isolated. After repeating this inoculation procedure, 100 mL of virus supernatant was obtained.

[0091] The virus supernatant was mixed with 50 mL of 20% PEG in a 2.5 mM NaCl solution. The virus was then precipitated by incubating overnight in an inverter at 4°C. The precipitated virus pellet was harvested after centrifugation at 7,500 rpm for 30 minutes, and the supernatant was discarded. The pellet was suspended in 2 mL of 20 mM Tris-HCl (pH 8.0) and centrifuged at 12,000 rpm for 10 minutes to remove debris, and the supernatant containing the adenovirus was recovered. For density gradient ultracentrifugation, CsCl solutions with densities of 1.3 g / mL and 1.4 g / mL were prepared in 20 mM Tris-HCl (pH 8). Sequentially, 1.5 mL of 1.4 g / mL solution, 1 mL of 1.3 g / mL solution, and 2 mL of the recovered adenovirus supernatant were added to each layer in an ultracentrifuge tube without shaking. Finally, the mixture was centrifuged at 100,000 × g for 20 hours at 4°C. The obtained UV bands were recovered, placed in a dialysis bag (molecular weight cutoff value 10,000), and dialyzed overnight against 10 mM Tris-HCl (pH 8.0). The virus was filtered through a 0.2 μM filter, dispensed, and stored at -80°C. To determine the titer of rAd, the TCID50 assay was performed on 293A cells, and the TCID50 values ​​were converted to PFU / mL.

[0093] Experimental Example 7. Confirmation of Viral Transduction and Expression

[0094] One day before transduction, A549 cells (1.5 × 10⁶ 6 After seeding cells (in a well) into a six-well cell culture plate, the purified virus was transduced into cells of different MOIs (2.5, 25, and 250) and cultured in a CO2 incubator at 37°C. After 48 hours of transduction, cells were harvested and protein expression was evaluated by Western blotting.

[0096] Experimental Example 8. Western blot

[0097] Cells were lysed using Ottimolyse I lysis buffer (Jubiotech, Daejeon, Republic of Korea), and a similar amount of protein sample prepared in sample buffer was boiled and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The isolated proteins were transferred to a PVDF membrane (MERCK, Darmstadt, Germany), blocked with 5% skim milk, and treated with an anti-Myc antibody (CSB-PA000085, Cusabio, Wuhan, China) or a ferritin heavy chain antibody (GT1149, GeneTex, Hsinchu, Taiwan) and an anti-rabbit secondary antibody (PA489724, Cusabio, Wuhan, China). The antibody-treated membranes were developed using a Western blot enhancer, and the images were analyzed using a luminograph.

[0099] Experimental Example 9. Immunization of a mouse animal model

[0100] Six-week-old female BALB / c mice were managed at the animal facility of Chungnam National University (CNU), South Korea, in accordance with the guidelines of the Institutional Animal Care and Use Committee. The mice were divided into three groups, with five mice in each group. rAd(8 × 10 6 PFU / mL) was prepared in PBS without adjuvants. Mice in each group were immunized by intramuscular injection with 100 μL of rAd E, rAd EH, or PBS. After two doses at a 2-week interval, blood was collected from mice for retro-orbital bleeding at 1 and 4 weeks after the second booster shot.

[0102] Experimental Example 10. Evaluation of Humoral Immune Response (Enzyme-linked Immunosorbent Test)

[0103] 100 times diluted in PBS (10 -1Dengue fever virus was added to each well of an immunoplate (100 μL / well) and incubated overnight at 4°C. The next day, the plates were washed twice with PBS and blocked with 3% skim milk in PBS (blocking buffer) for 1 hour. After additional washing with PBS, the plates were incubated for 2 hours with serum samples (100 μL / well) diluted in blocking buffer. A wash buffer was prepared using 1% Triton X-100 in PBS, and the plates were washed three times. Then, the plates were incubated with HRP-conjugated anti-mouse IgG (CSB-PA644737, Cusabio, Wuhan, China) diluted (1:10,000) in blocking buffer for 1 hour. After washing three times with PBS-T, 100 μL of TMB substrate and stop solution were added sequentially. Absorbance values ​​were measured at 450 nm using a plate reader (PerkinElmer, Hopkinton, MA, USA).

[0105] [Example]

[0106] Example 1. Production of recombinant adenoviruses (rAds) expressing envelope ferritin heavy chain (EH) protein

[0107] Recombinant adenoviruses (rAds) expressing EH protein according to one embodiment were produced in the manner of Experimental Examples 4 to 6 above. FIG. 1a shows the vector map of the packaging vector used to produce the recombinant adenoviruses (rAds) according to one embodiment, and FIG. 1b shows the vector map of the shuttle vector used to produce the recombinant adenoviruses (rAds) according to one embodiment.

[0108] The sequence of the polynucleotide inserted into the adenovirus according to one embodiment was further confirmed (Cosmogenetech Co., Ltd), and the specific sequence information is as follows.

[0110] Name Sequence SEQ ID NO. prM nucleotide (93 bp) ATGGCAGCAATCCTGGCATACACCATAGGAACGACACATTTCCAAAGAGTCCTGATTTTCATCCTACTGACAGCTGTCGCTCCTTCAATGACA 7 DENV envelope ectodomain (79% C-term deleted nucleotide) ATGCGCTGTATAGGAATAGCAAATAGGGACTTCGTGGAAGGGGTTTCAGGAGGAAGCTGGGTTGACATAGTCTTAGAACACGGAAGCTGTGTGACGACGATGGCAAAAAACAAACCAACACTGGACTTTGAACTGATAAAAACAGAAGCCAAACAACCCGCCACTCTAAGGAAGTACTGCATAGAGGCTAAGCTGACCAACACGACAACAGATTCGCGCTGCCCAACACAAGGGGAACCTAGCCTGAATGAAGAGCAGGACAAAAGGTTTGTCTGCAAACATTCTATGGTAGACAGAGGATGGGGAAATGGATGCGGATTGTTTGGAAAAGGAGGCATCGTGACCTGTGCTATGTTCACATGCAAAAAGAATATGAAAGGAAAAATTGTGCAGCCAGAAAACTTGGAATACACCATCGTGATAACACCTCACTCAGGGGAAGAACATGCAGTCGGAAATAACACAGGAAAACATGGCAAGGAAGTCAAAATAACACCACAGAGCTCCATCACAGAAGCGGAACTGACAGGCTATGGCACTGTTACGATGGAGTGCTCTCCGAGAACGGGCCTCGACTTCAATGAGATGGTGTTGCTGCAAATGGAAGATAAAGCTTGGCTGGTGCACAGACAATGGTTCCTAGACCTGCCGTTACCATGGCTGCCCGGAGCAGATACACAAGGATCAAATTGGATACAGAAAGAGACATTGGTCACTTTCAAAAATCCCCATGCAAAAAAACAGGATGTTGTTGTCTTAGGATCCCAAGAGGGGGCCATGCACACAGCACTCACAGGGGCTACGGAAATCCAGATGTCATCAGGAAACCTACTGTTCACAGGTCATCTTAAGTGCAGGCTGAGAATGGACAAACTACAGCTTAAAGGGATGTCATACTCTATGTGTACAGGGAAGTTTAAAGTCGTGAAGGAAATAGCAGAAACACAACATGGAACAATAGTCATTAGAGTACAATATGAAGGAGACGGCTCTCCATGCAAGATCCCTTTTGAGATAATGGATCTGGAAAAAAGACATGTCTTAGGTCGTCTGATTACAGTCAACCCAATTGTAACAGAAAAGGACAGGCCAGTCAACATAGAAGCAGAACCTCCATTCGGAGACAGCTACATTATCATAGGAGTGGAGCCGGGACAACTGAAGCTTAACTGGTTCAAGAAAGGA 8 Myc Tag nucleotide GAACAAAAACTCATCTCAGAAGAGGATCTG 9 Ferritin heavy chain nucleotide ATGACGACCGCGTCCACCTCGCAGGTGCGCCAGAACTACCACCAGGACTCAGAGGCCGCCATCAACCGCCAGATCAACCTGGAGCTCTACGCCTCCTACGTTTACCTGTCCATGTCTTACTACTTTGACCGCGATGATGTGGCTTTGAAGAACTTTGCCAAATACTTTCTTCACCAATCTCATGAGGAGAGGGAACATGCTGAGAAACTGATGAAGCTGCAGAACCAACGAGGTGGCCGAATCTTCCTTCAGGATATCAAGAAACCAGACTGTGATGACTGGGAGAGCGGGCTGAATGCAATGGAGTGTGCATTACATTTGGAAAAAAATGTGAATCAGTCACTACTGGAACTGCACAAACTGGCCACTGACAAAAATGACCCCCATTTGTGTGACTTCATTGAGACACATTACCTGAATGAGCAGGTGAAAGCCATCAAAGAATTGGGTGACCACGTGACCAACTTGCGCAAGATGGGAGCGCCCGAATCTGGCTTGGCGGAATATCTCTTTGACAAGCACACCCTGGGAGACAGTGATAATGAAAGC 10 prM amino acid (31 amino acids) MAAILAYTIGTTHFQRVLIFILLTAVAPSMT 11 DENV envelope ectodomain(79% C-term deleted amino acid) MRCIGIANRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTDSRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTCKKNMKGKIVQPENLEYTIVITPHSGEEHAVGNNTGKHGKEVKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMV LLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKG MSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRITVNPIVTEKDRPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKG 12 Myc Tag amino acid EQKLISEEDL 13 Ferritin heavy chain amino acid MTTASTSQVRQNYHQDSEAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES 14

[0112] Example 2. Evaluation of E and EH protein expression

[0113] In this example, the pAd E plasmid and pAd EH plasmid prepared in Example 1 were used to transiently transfect HEK293 cells in the manner of Experimental Example 5, and then Western blot was performed in the manner of Experimental Example 8 to evaluate the expression levels of E and EH proteins.

[0114] As a result, as shown in Fig. 1c, when the Myc antibody was used, the recombinant E protein (49 kDa) was detected, but the expression level of the recombinant EH protein was relatively low. Meanwhile, as shown in Fig. 1d, when the ferritin heavy chain antibody was used, the recombinant EH protein (68 kDa) was detected at a significant expression level. From these results, it was confirmed that the recombinant adenovirus according to one pattern can transfect the EH protein into cells and stably express it.

[0116] Example 3. Purification and Verification of rAds

[0117] In this example, the rAds prepared in Example 1 were purified and verified. Before transfecting the adenovirus-specific plasmid into 293A cells, the plasmid DNA cleaved with the PacI enzyme was verified by running an agarose gel with the uncleaved DNA. As shown in Figure 2a, the specific cytopathological effect (CPE) of rAds was observed 12 days after transfection with pAd E and pAd EH. The viral replication rate was low at the first passage stage, and from the third passage onwards, significant CPE was observed within 3 days. To confirm the presence of recombinant genes within the virus, viral DNA was isolated, PCR amplification was performed using respective primers, and then the respective DNA bands for the recombinant E gene were identified. As shown in Figure 2b, the EH gene was identified using agarose gel electrophoresis, and no band was observed in the negative control. As shown in Fig. 2c, after isolating the viruses from the culture supernatant, clear adenovirus bands were obtained for rAd E and rAd EH, and unpacked viruses were observed above the clear packaged virus bands. Optimal buffering conditions for the adenoviruses were established using dialysis and filtration to remove contamination. As shown in Fig. 2d, clear CPE was observed after viral titration in 293A cells. The titers for rAd E and rAd EH were calculated to be 4.64 × 10¹⁰ TCID50 / mL and 3.16 × 10⁸ TCID50 / mL, respectively. Converting these TCID50 values ​​to PFU yielded values ​​of 2.60 × 10¹⁰ PFU / mL and 1.77 × 10⁸ PFU / mL, respectively.

[0119] Example 4. Evaluation of rAds transduction efficiency

[0120] In this example, the in vitro production of recombinant E and EH proteins was evaluated by using the recombinant adenovirus prepared in Example 1, transducing the adenovirus into A549 cells in the manner of Experimental Example 7, and then performing a Western blot in the manner of Experimental Example 8.

[0121] As a result, as shown in Figures 3a and 3b, the expression levels of the proteins were significantly differentiated for the transduced MOI values, and the protein expression patterns were identical to those of transfection expression. Specifically, as shown in Figure 3a, the recombinant E protein was detected at an accurate level, while the recombinant EH protein was detected very slightly using an anti-Myc antibody. Meanwhile, as shown in Figure 3b, the expected size band of recombinant EH was detected using a ferritin heavy chain antibody similar to the transfection expression data. Neither E nor EH proteins were detected at low MOI values ​​(2.5 and 25, respectively), whereas 250 MOI transduction induced significant expression of both proteins. From these results, it was confirmed that the recombinant adenovirus according to one aspect could transduce E and EH proteins into cells and stably express them at a high MOI (250).

[0123] Example 5. Evaluation of humoral immune response using a mouse animal model

[0124] In this example, a recombinant adenovirus (EH rAdV) expressing the EH protein was inoculated into mice to evaluate its effect on the humoral immune response to DENV infection. As shown in Fig. 4a, a group of BALB / c mice (n=5) were intramuscularly immunized at 2-week intervals in the manner of Experimental Example 9. Subsequently, the humoral immune response of each mouse group was confirmed via enzyme-linked immunosorbent assay in the manner of Experimental Example 10. The cultured and purified DENV2 virus strain was identified through qPCR analysis of serially diluted virus samples, and this virus was used as a coating antigen for virus-specific ELISA.

[0125] As a result, as shown in Fig. 4b, rAd EH induced a significant virus-specific IgG titer (p=0.0009) compared to rAd E in BALB / c mice one week after final immunization. Similarly, as shown in Fig. 4b, rAd EH showed a significant IgG response (p=0.0003) compared to rAd E even after four weeks had passed since immunization. In addition, as shown in Fig. 4c, when comparing the immune response of the EH immunized group at one week and four weeks after immunization, the IgG titer was higher at four weeks after immunization, but was not significant when compared to the IgG level one week after immunization. From these results, it was found that the vaccine composition according to one embodiment significantly induced an IgG-mediated humoral immune response.

[0127] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

Claim 1 A recombinant adenovirus containing a polynucleotide encoding the DENV (Dengue Virus) envelope protein and a polynucleotide encoding the ferritin heavy chain protein. Claim 2 A recombinant adenovirus according to claim 1, wherein the polynucleotide encoding the DENV envelope protein and the polynucleotide encoding the ferritin heavy chain protein are linked directly or through a linker sequence. Claim 3 A recombinant adenovirus according to claim 1, wherein the polynucleotide encoding the DENV envelope protein is a polynucleotide encoding a premembrane (prM) protein at the 5' end, directly or through a linker sequence. Claim 4 A recombinant adenovirus according to claim 1, wherein the polynucleotide encoding the DENV envelope protein is composed of the nucleotide sequence of SEQ ID NO.

8. Claim 5 A recombinant adenovirus according to claim 1, wherein the polynucleotide encoding the ferritin heavy chain protein is composed of the nucleotide sequence of SEQ ID NO.

10. Claim 6 A recombinant adenovirus according to claim 3, wherein the polynucleotide encoding the premembrane (prM) protein is composed of the nucleotide sequence of SEQ ID NO.

7. Claim 7 A vaccine composition for the prevention of DENV infection comprising a recombinant adenovirus of any one of claims 1 to 6 as an active ingredient. Claim 8 A vaccine composition according to claim 7, wherein the vaccine composition is selected from the group consisting of compositions for intramuscular administration, subcutaneous administration, intraperitoneal administration, intravenous administration, intravenous administration, dermal administration, ocular administration, and brain administration. Claim 9 A vaccine composition according to claim 7, wherein the vaccine composition further comprises a pharmaceutically acceptable excipient, diluent, or carrier. Claim 10 An isolated nucleic acid molecule comprising a polynucleotide encoding a DENV envelope protein consisting of the nucleotide sequence of SEQ ID NO. 8 and a polynucleotide encoding a ferritin heavy chain protein consisting of the nucleotide sequence of SEQ ID NO. 10, wherein the polynucleotide encoding the DENV envelope protein and the polynucleotide encoding the ferritin heavy chain protein are connected directly or through a linker sequence. Claim 11 An isolated nucleic acid molecule according to claim 10, wherein the polynucleotide encoding a DENV envelope protein composed of the nucleotide sequence of SEQ ID NO. 8 is connected at the 5' end to a polynucleotide encoding a premembrane (prM) protein composed of the nucleotide sequence of SEQ ID NO. 7, either directly or through a linker sequence. Claim 12 The isolated nucleic acid molecule of claim 10, wherein the isolated nucleic acid molecule induces an immune response to the DENV antigen.

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