Recombinant adenovirus comprising SARS-COV-2-derived receptor-binding domain and antibody fc region, and use thereof

Abstract

The present invention relates to a recombinant adenovirus comprising a SARS-CoV-2-derived receptor-binding domain and an antibody Fc region, and a use thereof, and provides: a polynucleotide comprising a SARS-CoV-2 spike-derived receptor-binding domain and an antibody Fc region; a recombinant adenovirus comprising the polynucleotide; and a vaccine composition for preventing SARS-COV-2 infection, comprising the recombinant adenovirus as an active ingredient.

Description

Recombinant adenovirus containing SARS-COV-2-derived receptor binding domain and antibody FC region and its uses

[0001] This invention relates to a recombinant adenovirus comprising a SARS-CoV-2-derived receptor binding domain and an antibody Fc region, and the use of the same as a vaccine against SARS-CoV-2. This patent application claims priority to Korean Patent Application No. 10-2025-0024510 filed with the Korean Intellectual Property Office on February 25, 2025, the disclosures of said patent application are incorporated herein by reference.

[0002] Coronavirus disease 2019 (hereinafter referred to as “COVID-19”) is a respiratory infection caused by a coronavirus. It is transmitted when droplets from an infected person penetrate the respiratory tract or the mucous membranes of the eyes, nose, and mouth. After an incubation period of about 2 to 14 days, the main symptoms appear as fever, respiratory symptoms such as cough or shortness of breath, and pneumonia, but the frequency of asymptomatic cases is also high.

[0003] Vaccines exist in various forms depending on their manufacturing methods, including first-generation vaccines such as attenuated and inactivated vaccines, second-generation vaccines such as subunit and toxoid vaccines, and third-generation vaccines such as DNA, RNA, and recombinant virus vaccines. The efficacy of these vaccines varies widely, and the immunogenicity of each form differs significantly. Currently commercialized mRNA vaccines present a problem in that they must be administered in excessive doses and require booster shots after a certain period to maintain immunity, as they disappear after being translated into protein upon injection. Furthermore, with the emergence of COVID-19 variants, existing vaccines are failing to provide immunity against these variants. Additionally, while subunit or viral vaccines offer excellent safety, adjuvants are currently required to enhance their immunogenicity. Therefore, the development of recombinant virus vaccines that exhibit superior immunogenicity can contribute to the commercialization of vaccines.

[0004] Meanwhile, adenoviruses (AdVs) are unenveloped viruses with a linear double-stranded DNA genome (34-43 kb), and adenovirus vectors have been applied to various diseases, including cardiovascular disease, cancer, metabolic syndrome, muscle disease, immunodeficiency syndrome, and neurological disorders. Additionally, AdVs possess the potential for protein expression and high intracellular gene delivery efficiency, making them suitable for use as excellent vaccine carriers for infectious diseases. Furthermore, SARS-CoV-2 is known to infect the body through the binding of the receptor-binding domain (RBD) of its spike protein to the ACE2 receptor of host cells. In other words, since the RBD acts as the initiating point of infection during the SARS-CoV-2 infection process, the RBD region can serve as a primary target for preventing or treating SARS-CoV-2 viral infections.

[0005] Under this technical background, the inventors produced a recombinant adenovirus comprising a SARS-CoV-2-derived receptor binding domain and an antibody Fc region, and completed the present invention by confirming the excellent vaccine efficacy of the recombinant adenovirus against SARS-CoV-2 infection.

[0006] One aspect is to provide a recombinant adenovirus comprising a polynucleotide containing a SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) spike-derived receptor-binding domain (RBD) and an antibody Fc region.

[0007] Another aspect is to provide a vaccine composition for the prevention of SARS-CoV-2 infection comprising the above-mentioned recombinant adenovirus as an active ingredient.

[0008] Another aspect provides an isolated nucleic acid molecule comprising a receptor-binding domain consisting of the nucleotide sequence of SEQ ID NO. 3 and an antibody Fc region consisting of the nucleotide sequence of SEQ ID NO. 5, wherein the receptor-binding domain and the antibody Fc region are connected directly or through a linker sequence.

[0009]

[0010] 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.

[0011] 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.

[0012]

[0013] One aspect provides a recombinant adenovirus comprising a polynucleotide comprising a SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) spike-derived receptor-binding domain (RBD) and an antibody Fc region.

[0014] In this specification, the term "SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2)" refers to SARS-CoV-2 or the provisional name 2019 novel coronavirus (2019-nCoV), which is a positive sense single-stranded RNA coronavirus in terms of DNA sequencing, is infectious to humans, and is known to be the cause of COVID-19.

[0015] In this specification, the term "spike protein" refers to a protruding structure arranged on the surface of a virus, with a length of 10 to 15 nm, which is responsible for binding to protein receptors on the cell membrane to penetrate the body. Therefore, the spike protein plays a crucial role in the entry of a virus into human cells. In particular, the host that a virus infects varies depending on the binding ability of the spike protein. Accordingly, the spike protein can be utilized in the development of antiviral vaccines, antibodies, and diagnostics.

[0016] In this specification, the term "receptor binding domain (RBD)" refers to a structure directly involved in infecting the body by binding to the ACE2 receptor of a host cell, and may be used interchangeably with the term "receptor binding motif (RBM)." Since the receptor binding domain serves as the starting point of infection during the SARS-CoV-2 infection process, it can be a major target for the prevention or treatment of SARS-CoV-2 infection. For the purposes of the present invention, the nucleotide sequence of the receptor binding domain is stably expressed within the administered individual and can contribute to inducing an immune response to the SARS-CoV-2 antigen.

[0017] In one embodiment, the receptor-binding domain may consist of the nucleotide sequence of SEQ ID NO. 3. Additionally, the nucleotide sequence may be within the range of sequences having 80 to 99% sequence identity with the nucleotide sequence in order to enhance the expression of the target protein under an adenovirus-based expression construct.

[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 this specification, the term "antibody Fc region" refers to a fragment crystallizable region present within an antibody or immunoglobulin, meaning the terminal region of the antibody or immunoglobulin that interacts with a cell surface receptor called the Fc receptor and some proteins of the complement system. In the present invention, to enhance the vaccine efficiency of the protein structure, a protein containing the amino acid sequence within the antibody Fc region, i.e., an antibody Fc region protein, was utilized. The antibody Fc region may refer to the human immunoglobulin G (IgG) Fc region. The antibody Fc region protein may contain all or part of the amino acid sequence within the antibody Fc region.

[0020] In one embodiment, the antibody Fc region may consist of the nucleotide sequence of SEQ ID NO. 5. Additionally, in order to enhance the expression of the target protein under an adenovirus-based expression construct, the nucleotide sequence may correspond to a range of sequences having 80 to 99% sequence identity with the nucleotide sequence as described above.

[0021] In one embodiment, the receptor-binding domain and the antibody Fc region may be connected directly or through a linker sequence.

[0022] 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 a target gene, e.g., a receptor-binding domain and an antibody Fc region construct (RBD_hFc), to a target tissue or cell and for stably expressing it. To this end, the recombinant adenovirus may further comprise a nucleic acid construct comprising elements necessary for expressing the target gene, namely the nucleotide sequence of the aforementioned receptor-binding domain and the nucleotide sequence of the antibody Fc region, as well as the target gene / 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 a target protein operably linked to the expression regulatory sequence.

[0023] For the purposes of the present invention, the target protein may be a protein in which a receptor-binding domain and an antibody Fc region are fused or operably connected. For example, the receptor-binding domain may be an amino acid sequence consisting of SEQ ID NO. 6, and the antibody Fc region may be an amino acid sequence consisting of SEQ ID NO. 8.

[0024] 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.

[0025] 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 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.

[0026] 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.

[0027] In this specification, the term “operably linked” may indicate that nucleotide sequences on a single nucleic acid fragment are linked so that one function is influenced by another.

[0028] In one embodiment, the recombinant adenovirus may include the nucleic acid structure shown in FIG. 1, and, for example, the RBM_hFc clone according to one embodiment may be introduced into the pacAd5 CMV-N-pA vector, but is not limited thereto.

[0029] According to one embodiment, by constructing a recombinant adenovirus into which a receptor-binding domain and an antibody Fc region construct / nucleotide sequence (RBM_hFc) are introduced, it was found that the recombinant adenovirus can stably express the target protein, the receptor-binding domain and antibody Fc region protein, within a target cell.

[0030] Furthermore, administration of this recombinant adenovirus was able to significantly enhance IgG-mediated humoral and cellular immunity against SARS-CoV-2 antigens in a mouse animal model. Therefore, the recombinant adenovirus according to one aspect can be utilized as an active ingredient in a vaccine composition for the prevention of SARS-CoV-2 infection.

[0031] A recombinant adenovirus according to one embodiment is designed to deliver a polynucleotide encoding a SARS-CoV-2-derived receptor binding domain and a polynucleotide encoding an antibody Fc region protein into a target cell. Upon administration, the fusion protein expressed including the Fc region protein spontaneously self-assembles in vivo to form a dimeric complex. This complex presents the SARS-CoV-2-derived receptor binding domain on its surface in a multivalent form, for example, as a dimeric antigen, thereby significantly increasing recognition by immune cells compared to a monomeric antigen and inducing a potent immune response. Furthermore, the recombinant adenovirus according to one embodiment can be utilized as a next-generation gene therapy vaccine by employing an in vivo endogenous production method through gene introduction, rather than a simple one-time vaccine antigen administration, thereby optimizing the folding and glycosylation of the antigen protein and maximizing interaction with the immune system.

[0032]

[0033] Another aspect provides a vaccine composition for the prevention of SARS-CoV-2 infection comprising the recombinant adenovirus as an active ingredient; and a medicinal use of the recombinant adenovirus for the prevention of SARS-CoV-2 infection.

[0034] 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.

[0035] 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.

[0036] 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 features, or defending against or protecting against the occurrence of SARS-CoV-2 infection or its characteristic features.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041]

[0042] Another aspect provides a method for treating, preventing, or improving SARS-CoV-2 infection, comprising the step of administering the recombinant adenovirus to an individual.

[0043] Since the above-mentioned methods for treating, preventing, or improving SARS-CoV-2 infection include or utilize the aforementioned recombinant adenovirus or vaccine composition as is, the common details among them are omitted to avoid excessive complexity in this specification.

[0044] In this specification, the term “treatment” means any form of treatment that provides effects to an individual suffering from a disease or at risk of developing a disease, including improvement of the individual’s condition (e.g., one or more symptoms), delay of disease progression, delay of symptom onset, or slowing of symptom progression. The treatment may refer, for example, to the induction of a therapeutic immune response through sustained antigen expression in the body.

[0045] In this specification, the term “individual” refers to a subject requiring prevention or treatment of a disease, specifically SARS-CoV-2 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.

[0046]

[0047] Another aspect provides an isolated nucleic acid molecule comprising a receptor-binding domain consisting of the nucleotide sequence of SEQ ID NO. 3 and an antibody Fc region consisting of the nucleotide sequence of SEQ ID NO. 5, wherein the receptor-binding domain and the antibody Fc region are connected directly or through a linker sequence.

[0048] 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.

[0049] 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 GNE protein and may include a codon-optimized sequence encoding a SARS-CoV-2 spike-derived receptor-binding domain and / or antibody Fc region.

[0050] 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.

[0051] In one embodiment, the isolated nucleic acid molecule can be inserted into a viral vector, for example, an adenovirus, and delivered to an individual.

[0052] 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 SARS-CoV-2 antigen. Here, the immune response may include an IgG-mediated humoral immune response and / or a cellular immune response.

[0053] According to a vaccine composition according to one aspect, by including a recombinant adenovirus as an active ingredient that comprises a polynucleotide comprising a SARS-CoV-2 spike-derived receptor-binding domain and an antibody Fc region, humoral and cellular immune responses to SARS-CoV-2 antigens can be enhanced.

[0054] 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 SARS-CoV-2 infection.

[0055] FIG. 1 is a schematic diagram illustrating the process of producing and constructing a recombinant adenovirus according to one embodiment.

[0056] Figure 2 is the result of confirming the expression of the RBM_hFc structure by Western blot after transfecting HEK293 cells with a plasmid inserted with the RBM_hFc structure according to one embodiment.

[0057] Figure 3 is the result of confirming the expression of the RBM_hFc structure through an immunofluorescence staining assay after transfecting HEK293 cells with a plasmid containing the RBM_hFc structure according to one embodiment.

[0058] Figure 4 is the result of confirming whether viral plaques were formed in HEK293 cells transfected with a recombinant adenovirus according to one embodiment.

[0059] FIG. 5 shows the confirmation of the presence of a target gene from DNA derived from a recombinant adenovirus culture supernatant according to one embodiment, where FIG. 5A is the result of confirming the presence of the RBM gene; FIG. 5B is the result of confirming the presence of the capsid protein, the AdV IX gene.

[0060] FIG. 6 shows the expression of the RBM_hFc construct in cells transduced with a recombinant adenovirus according to one embodiment, confirmed by Western blot, where FIG. 6A is the result of confirming expression in transduced A549 cells; and FIG. 6B is the result of confirming expression in transduced Huh7 cells.

[0061] FIG. 7 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.

[0062] Figure 8 is the result 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.

[0063] FIG. 9 shows the cellular immune response induced from the administration of a vaccine composition containing a recombinant adenovirus according to one embodiment, confirmed through enzyme-linked immunospot analysis, where FIG. 9A is an image of the enzyme-linked immunospot analysis result; and FIG. 9B is a figure showing the enzyme-linked immunospot analysis result quantitatively.

[0064] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0065]

[0066] [Experimental Example]

[0067] Experimental Example 1. Cell Culture

[0068] HEK293, A549, and Huh7 cell lines were obtained from the Korean Cell Line Bank (KCLB: Seoul, South Korea). HEK293 and Huh7 cells were cultured in Dulbecco Modified Eagle Medium (DMEM: Biowest, Nualille, France), and A549 cells were cultured in Roswell Park Memorial Institute Medium (RPMI, Biowest, France). All restriction enzymes (PacI, BamHI, HindIII) were purchased from New England BioLabs Ipswich, MA, USA. All cells were cultured at 37°C and 5% CO2 conditions with the addition of 10% fetal bovine serum (FBS) (Gibco: New York, NY, USA) and antibiotics (Biowest: France).

[0069]

[0070] Experimental Example 2. Confirmation of overexpression of RBM_hFc protein

[0071] 2-1. Western Blot

[0072] Analysis of SARS-CoV-2 RBM_hFc protein expression was performed via Western blot. Samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane at 250 mA for 2 hours. To prevent non-specific binding, the PVDF membrane was blocked with 5% skim milk powder for 1 hour. A Myc-tag antibody (1:5000 dilution, Abcam: Cambridge, UK) was incubated with the PVDF membrane overnight at 4°C. After washing the PVDF membrane with 0.1% TBST, a secondary antibody conjugated with HRP was added and incubated at room temperature for 2 hours. Finally, bands were detected using an enhanced chemiluminescence solution.

[0073]

[0074] 2-2. Immunofluorescence staining assay

[0075] 1 × 10⁶ Vero cells per well in a 12-well plate 5 After seeding at cell densities, cells were transfected with 1.5 μg of pAdS / RBM_hFc DNA per well. After transfection, cells were cultured at 37°C under 5% CO2 conditions for 48 hours, after which the culture medium was removed from the wells. Subsequently, the cultured cells were fixed in a 4% paraformaldehyde solution (GeneAll Biotechnology Co., Ltd.: Seoul, South Korea) for 15 minutes at room temperature. After washing the cells three times with PBS, permeation treatment was performed with PBS-T (PBS containing 0.1% Triton X-100) for 15 minutes at room temperature. The cells were washed again three times with PBS and blocked with PBS containing 3% BSA on a shaking platform for 1 hour at room temperature. In the blocking solution, cells were incubated overnight at 4°C with diluted Myc antibody (1:500 dilution, Abcam: Cambridge, UK). Subsequently, after washing the cells three times with PBS-T (PBS containing Tween-20), the cells were treated with Alexa Fluor ® Incubated for 1 hour with 488-conjugated anti-rabbit IgG antibody (1:1000 dilution, Abcam: Cambridge, UK).

[0076]

[0077] Experimental Example 3. Amplification of recombinant adenovirus

[0078] One day before cell transduction, place HEK293 cells in 100-mm dishes at a rate of 1 × 10⁶ per well. 5Cells were seeded at a cell density. To enhance viral attachment, cells were infected with a 1:1 mixture of virus and serum-free medium and incubated overnight at 37°C under 5% CO2 conditions. After incubation, the inoculum was removed and replaced with fresh medium containing 5% FBS and antibiotics. Subsequently, cytopathic effects (CPE) were monitored under 37°C and 5% CO2 incubation conditions. The supernatant was obtained by centrifuging at 1500×g for 10 minutes at 4°C. Finally, the cell monolayer was washed with PBS and lysed by repeating the freeze-thaw cycle three times. The cell lysate was obtained by centrifuging at 1500×g for 10 minutes at 4°C. The obtained virus was amplified in fresh HEK293 cells and purified by CsCl density gradient centrifugation. Finally, the above virus was resuspended in storage buffer (10 mM Tris-HCl (pH 8.0), 4% sucrose, 2 mM MgCl2) and stored at -80℃.

[0079]

[0080] Experimental Example 4. Quantitative analysis using the Crystal Violet Approach

[0081] 1 × 10⁶ HEK293 cells per well in a 96-well plate 4 Inoculated at a concentration of [number]. Pure adenovirus was inoculated 10 times in a medium containing 5% FBS and antibiotics. 1 From 10 10 Serially diluted tenfold up to [amount]. The serially diluted viruses were inoculated into HEK293 cells and cultured in an incubator at 37°C under 5% CO2 conditions. Once CPE (cytotoxic effect) was confirmed, the supernatant was removed, and 100 μL of crystal violet was dispensed into each well. Subsequently, the cells were stained and washed, CPE was visually confirmed, and the titer was calculated.

[0082]

[0083] Experimental Example 5. Confirmation of recombinant adenovirus via PCR and Western blot

[0084] PCR and Western blot analyses were performed to confirm the expression of the RBM_hFc protein after adenovirus (AdV) administration. Viral DNA was extracted from the supernatant of AdV cultures using the Ribospin vRD kit (Geneall Biotechnology Co., Ltd.: Seoul, South Korea). The extracted viral DNA and purified recombinant plasmid were used as templates for PCR amplification using primers specific to the SARS-CoV-2 RBM region (201 bp) and the human AdV IX gene (120 bp). Under conditions of multiple infection index (MOI) of 1, recombinant AdV was used to infect human lung epithelial cell lines (A549) and human liver cancer cell lines (Huh7). Total cell lysates were obtained 72 hours after infection, followed by Western blot analysis. The lysates were denatured by boiling in 5× sample buffer at 99°C for 10 minutes and separated on an 8% SDS-polyacrylamide gel. Subsequently, the protein was transferred to a PVDF membrane and blocked with 5% skim milk powder in TBST for 1 hour at room temperature. Immunodetection was performed using an anti-human IgG-HRP-Fc antibody (Cusabio: Houston, TX, USA; #PA00540F0Rb) diluted 1:5000, and specific protein bands were visualized using a chemiluminescent detection reagent.

[0085]

[0086] Experimental Example 6. Immunization of a mouse animal model

[0087] Six-week-old female BALB / c mice (Samtako Co., Ltd.: Seoul, South Korea) were randomly divided into two groups. The experimental group was administered RBM_Fc AdV via a single intramuscular injection, and the control group was administered EGFP-expressing AdV via a single intramuscular injection. Serum samples were collected via orbital venous plexus puncture at designated times, and animals were euthanized via cardiac puncture at the end of the experiment.

[0088]

[0089] Experimental Example 7. Evaluation of Humoral Immune Response (Enzyme-linked Immunosorbent Test)

[0090] Enzyme-linked immunosorbent assay (ELISA) was performed to detect anti-SARS-CoV-2 antibodies. RBM_hFc protein purified using the c-Myc-tagged protein purification kit (MBL International: Schaumburg, IL, USA; #011FA) was dispensed onto ELISA plates and incubated overnight at 4°C. After washing with phosphate-buffered saline (PBS-T) containing Tween-20, the plates were blocked with PBS containing 3% skim milk powder for 1 hour at room temperature. Mouse serum was diluted (1:1000) in the blocking solution and incubated overnight at 4°C. After washing five times with PBS-T, anti-mouse IgG conjugated with horseradish peroxidase (HRP) was added and incubated for 1 hour at room temperature. Finally, after washing with PBS-T, a tetramethylbenzidine (TMB) substrate solution (Koma Biotech: Seoul, South Korea) was added for color development. Subsequently, the reaction was stopped with sulfuric acid (Koma Biotech: Seoul, South Korea), and the absorbance was measured at 450 nm using a spectrophotometer.

[0091]

[0092] Experimental Example 8. Evaluation of Cellular Immune Response (Enzyme-linked Immunospot Analysis)

[0093] Cytokine secretion by peripheral blood mononuclear cells (PBMCs) in response to AdV / RBM_hFc vaccine stimulation was quantified using Enzyme-Linked Immunospot (ELISpot) analysis. Immunized mice were euthanized by cardiac puncture, and their spleens were removed. The spleens were mechanically pulverized into a single-cell suspension using a cell filter (SPL Life Sciences Co., Ltd.: Seoul, South Korea) in a 50 mL conical tube. Red blood cells in the single-cell suspension were lysed, and the splenic cells were filtered and purified using RBM_hFc as the stimulating protein, then cultured at 37°C for 48 hours. After cell counting, 1 × 10⁶ cells per well were placed in a 96-well plate for ELISpot analysis. 5 Splenocytes were seeded at cell density. After 15 hours, the plates were washed with PBS-T and incubated with detection antibody diluted to 100 μL at room temperature for 1.5 hours. Subsequently, the plates were washed and incubated with streptavidin-alkali phosphatase conjugate for 1 hour at room temperature. After washing with distilled water, BCIP / NBT substrate was added to visualize spot formation. The plates were washed with distilled water and dried overnight. The number of cytokine-producing cells was quantified by counting spots using an ELISpot reader.

[0094]

[0095] Experimental Example 9. Statistical Analysis

[0096] All experiments were performed in triplicate and analyzed using independent sample t-tests with SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). The mean ± standard deviation of the data was denoted as * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

[0097]

[0098] [Example]

[0099] Example 1. Production of a recombinant adenovirus expressing RBM_hFc

[0100] As illustrated in FIG. 1, a recombinant adenovirus expressing RBM_hFc according to one embodiment was constructed. The receptor binding motif (RBM) consists of 72 amino acids, and the RBM nucleotide was codon-optimized and amplified from the SARS-CoV-2 spike gene commercially synthesized by Genwiz (Suzhou, China). The sequences of the primers for amplifying the RBM are shown in Table 1 below.

[0101] [Table 1]

[0102]

[0103] Gene amplification was performed on a 20 μL PCR mixture using a MiniAmp Thermal Cycler (Thermo Fisher Scientific: Walmart, MA, USA). Amplification of the PCR product was performed as follows: denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds (32 repetitions), annealing at 58°C for 30 seconds, extension at 72°C for 45 seconds, and final extension at 72°C for 10 minutes. The amplified PCR product was extracted using a gel purification kit (Gel Sv) (Geneall Biotechnology Co., Ltd.: Seoul, Republic of Korea) and then inserted into the BamHI (#R0136S) and HindIII (#R0104S) sites of the pacAd5CMV-N-pA shuttle vector (Cell Biolabs Inc.: San Diego, CA, USA).

[0104] Additionally, human Fc (hFc) was isolated from the pINFUSE-hIgG1-Fc1 vector (InvivoGen: San Diego, CA, USA), and a Myc tag was inserted between the RBM and hFc domains. Finally, an RBM_hFc clone was constructed in the pacAd5 CMV-N-pA vector. The sequence of the RBM_hFc construct inserted into an adenovirus according to one embodiment was further confirmed (Cosmogenetech Co., Ltd), and the specific sequence information is as follows.

[0105] [Table 2]

[0106]

[0107] RAPAd ® The CMV adenovirus expression system (Cell Biolabs Inc.: USA) was used to produce recombinant adenoviruses expressing the target gene RBM_hFc. The pacAd5 CMVK-NpA shuttle vector containing the PacAd5 9.2-100 vector and RBM_hFc (Ad / RBM_hFc) was linearized with PacI (New England BioLabs: USA; #R0547S) at 37°C for 6 hours. The linearized vectors were purified using a DNA purification kit (QIAGEN: Hilden, Germany) and then in HEK293 cells (1 × 10⁶ per 60 mm culture dish). 6 Recombinant adenoviruses expressing the target gene (RBM_hFc) were produced by co-transfecting into cells. After plaque formation was observed on day 10 following transfection, the crude viral lysate was recovered from the cells through three freeze-thaw cycles. Finally, the viral supernatant was centrifuged at 3000×g for 10 minutes to remove cell debris, dispensed, and stored at -80°C for further processing. Meanwhile, positive control viruses were produced in a similar manner using the pacAd5 CMV-GFP control vector.

[0108]

[0109] Example 2. Evaluation of RBM_hFc structure expression

[0110] In this example, the expression level of the RBM_hFc construct was evaluated using the pAd5 / RBM_hFc plasmid prepared in Example 1. Specifically, after transiently transfecting HEK293 cells with the pAd5 / RBM_hFc plasmid, the expression level of the RBM_hFc construct was evaluated by performing Western blow and immunofluorescence staining assays in the manner of Experimental Example 2. Meanwhile, in this example, untreated cells were used as the control group, and cells treated with a plasmid containing EGFP were used as the positive control group.

[0111] As shown in Fig. 2, Western blot analysis revealed a protein band with a molecular weight of approximately 40 kDa in cells treated with a plasmid containing the RBM_hFc structure according to one embodiment. Additionally, as shown in Fig. 3, immunofluorescence staining assay analysis revealed fluorescence staining results similar to a positive control in cells treated with the plasmid containing the RBM_hFc structure according to one embodiment. From these results, it was found that the RBM_hFc structure according to one embodiment can be stably expressed when transfected into cells.

[0112]

[0113] Example 3. Evaluation of RBM_hFc expression levels by introduction of recombinant adenovirus

[0114] In this example, the expression level of RBM_hFc was evaluated using the recombinant adenovirus prepared in Example 1. Specifically, both the PacAd5 9.2-100 vector and the Ad / RBM_hFc vector were linearized by PacI restriction enzyme cleavage at 37°C for 6 hours, and then these linearized vectors were co-transfected into HEK293 cells, an E1-complementary cell line, to produce replicable adenoviruses expressing the target protein. Subsequently, on the 10th day from the date of co-transfection in the manner of Experimental Example 4, the formation of viral plaques was checked and the viral titer was calculated. In addition, the recombinant adenovirus was identified through PCR and Western blot in the manner of Experimental Example 5. Specifically, to confirm the completeness of the adenovirus genome, the AdV IX gene, a capsid protein, was identified using DNA derived from the virus, and after transfecting A549 cells and Huh7 cells with the recombinant adenovirus according to one embodiment, the expression of the RBM_hFc construct was confirmed. Meanwhile, in this embodiment, untreated cells were used as the control group.

[0115] As a result, as shown in Fig. 4, viral plaques were observed in transduced HEK293 cells, thereby confirming the cytopathic effect (CPE). In addition, as shown in Fig. 5, PCR was performed on the DNA derived from the obtained recombinant adenovirus, and the RBM gene of 201 bp and the AdV IX gene of 120 bp were identified. Furthermore, as shown in Fig. 6, the expression of a protein band with a molecular weight of approximately 40 kDa, namely the RBM_hFc construct, was confirmed in transduced A549 and Huh7 cells. Considering the molecular weights of RBM and hFC, these experimental results indicate that the RBM_hFc construct, formed by the combination of RBM and hFC, is expressed.

[0116]

[0117] Example 4. Evaluation of humoral immune response using a mouse animal model

[0118] In this example, a recombinant adenovirus expressing RBM_Fc (RBM_hFc AdV) was administered to mice at a dose of 1 × 10⁶ per 1 ml. 8 The effect on the humoral immune response to SARS-CoV-2 infection was evaluated by inoculating with a viral titer. As shown in Fig. 7, a group of BALB / c mice (n=5) were intramuscularly immunized at 2-week intervals in the manner of Experimental Example 6. Subsequently, the humoral immune response of each mouse group was confirmed by enzyme-linked immunosorbent assay in the manner of Experimental Example 7. Meanwhile, in this example, the negative control group was the group administered PBS (NC), and the positive control group (PC) was the group inoculated only with SARS-CoV-2 spike protein.

[0119] As a result, as shown in Fig. 8, the group administered with the recombinant adenovirus (RBM_hFc AdV) according to one embodiment showed a significantly increased IgG titer. From these results, it was found that the vaccine composition according to one embodiment significantly induces an IgG-mediated humoral immune response.

[0120]

[0121] Example 5. Evaluation of cellular immune response using a mouse animal model

[0122] In this example, a recombinant adenovirus expressing RBM_Fc (RBM_hFc AdV) was administered to mice at a dose of 1 × 10⁶ per 1 ml. 8The effect on the cellular immune response to SARS-CoV-2 infection was evaluated by inoculating with a viral titer. Using the same mouse model as in Example 4, the cellular immune response of each mouse group was confirmed by enzyme-linked immunospot analysis in the manner of Experimental Example 8. In this example, the negative control group was the group administered PBS (NC), and the positive control group was the group inoculated only with SARS-CoV-2 spike protein.

[0123] As a result, as shown in Fig. 9, the number of cytokine-secreting cells from the spleen cells of each group was checked, and it was confirmed that the number of such cells increased in the group administered with the recombinant adenovirus (RBM_hFc AdV) according to one embodiment. From these results, it was found that the vaccine composition according to one embodiment significantly induces a cellular immune response.

[0124]

[0125] 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

1. A recombinant adenovirus comprising a polynucleotide containing a SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) spike-derived receptor-binding domain (RBD) and an antibody Fc region.

2. A recombinant adenovirus according to claim 1, wherein the receptor-binding domain and the antibody Fc region are connected directly or through a linker sequence.

3. A recombinant adenovirus according to claim 1, wherein the receptor-binding domain is composed of the nucleotide sequence of SEQ ID NO.

3.

4. A recombinant adenovirus according to claim 1, wherein the antibody Fc region is composed of the nucleotide sequence of SEQ ID NO.

5.

5. A vaccine composition for preventing SARS-CoV-2 infection comprising the recombinant adenovirus of Claim 1 as an active ingredient.

6. The vaccine composition of claim 5, wherein the vaccine composition is selected from the group consisting of compositions for intramuscular administration, subcutaneous administration, intraperitoneal administration, intravenous administration, dermal administration, ocular administration, and brain administration.

7. The vaccine composition of claim 5, wherein the vaccine composition further comprises a pharmaceutically acceptable excipient, diluent, or carrier.

8. An isolated nucleic acid molecule comprising a receptor-binding domain consisting of the nucleotide sequence of SEQ ID NO. 3 and an antibody Fc region consisting of the nucleotide sequence of SEQ ID NO. 5, wherein the receptor-binding domain and the antibody Fc region are connected directly or through a linker sequence.

9. The isolated nucleic acid molecule of claim 8, wherein the isolated nucleic acid molecule induces an immune response to SARS-CoV-2 antigen.

10. A method for treating, preventing, or improving SARS-CoV-2 infection, comprising the step of administering to an individual a recombinant adenovirus comprising a polynucleotide comprising a SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) spike-derived receptor-binding domain (RBD) and an antibody Fc region.

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