Multivalent COVID-19 Vaccine Based on Adenovirus Vector

Abstract

The present invention relates to the construction of adenovirus-based vectors and their use for inducing broad, potent and persistent cellular and humoral immune responses in a subject.

Description

Technical Field

[0001] The present invention relates to the construction of adenovirus-based vectors and their use for inducing broad, cooperative and sustained cellular and humoral immune responses in a subject.

Background Art

[0002] Coronavirus disease 2019 (COVID-19) is a severe infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a type of coronavirus. Since its emergence in Wuhan, China in 2019, the disease has spread worldwide and has led to the ongoing pandemic. Numerous attempts to treat this disease have not yet achieved sufficient results. Despite medical interventions, the mortality rate remains high, especially among the elderly and those with weakened immune systems. Although the development of drugs to suppress the virus is underway, the main treatment is symptomatic therapy. Management includes treatment of symptoms, supportive care, isolation and experimental measures. Several COVID-19 vaccines have been approved and are being distributed in various countries. Among them are mRNA and adenovirus platform-based vaccines that express the ancestral SARS-CoV-2 spike protein and induce a neutralizing antibody response against the Wuhan strain.

[0003] However, since its initial emergence in Wuhan, several concerning viral variants have emerged, particularly those with multiple mutations in the spike protein. Of particular importance are mutations within the receptor-binding domain (RBD), which reduce neutralization by antibodies present in convalescent sera and vaccine-induced sera. In some first-generation vaccines, it has been shown that the protective effect against COVID-19 by such newly concerning variants is reduced. So far, no vaccines adapted to SARS-CoV-2 variants have been approved. As an alternative strategy, it is to develop multivalent vaccines using recombinant viral vectors. Such vaccines may express additional conserved SARS-CoV-2 antigens in addition to the spike protein to broaden the breadth of T cell immunity. Antigenic changes in conserved viral internal proteins, which are the main focus of the T cell response, are less likely to occur in SARS-CoV-2 viruses, including the concerning variants. Therefore, such multivalent vaccines can enhance spike-specific immunity induced by first-generation vaccines while not requiring frequent updates. Thus, these vaccines are expected to be effective against both ancestral and variant forms of SARS-CoV-2.

[0004] However, there remains a need to provide improved vaccine compositions and methods that are therapeutically effective, reduce or prevent viral entry into cells, reduce the direct and indirect toxicity of the virus to patients, and generate an immune response effective at clearing the virus from patients. Advantageously, the vaccines should be able to provide protection against newly emerging viral variants. SUMMARY OF THE INVENTION

[0005] The present invention provides a solution to this problem by disclosing a replication-deficient adenoviral vector comprising: (i) a nucleic acid encoding at least one SARS-CoV-2 spike protein or a derivative thereof and (ii) a nucleic acid encoding at least one SARS-CoV-2 nucleocapsid protein or a derivative thereof, Here, the adenovirus vector displays a polypeptide containing the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein or a derivative thereof via protein IX (pIX) on its capsid.

[0006] SARS-CoV-2 is a type of severe acute respiratory syndrome-related coronavirus (SARS-CoV). It is a positive-sense single-stranded RNA (+ssRNA) virus with a single linear RNA segment. Similar to other coronaviruses, SARS-CoV-2 also has four structural proteins: S (spike), E (envelope), M (membrane), and N (nucleocapsid). The N protein holds the RNA genome, and the S, E, and M proteins together form the viral envelope. The S protein of coronaviruses is a glycoprotein and a type I membrane protein (a membrane containing a single transmembrane domain facing the extracellular side). The S protein is divided into two functional parts (S1 and S2). The spike protein is the protein for the virus to attach to and fuse with the membrane of the host cell. It fuses with the membrane. The N protein forms a complex with the genomic RNA and interacts with the viral membrane proteins during virion assembly, playing an important role in enhancing the efficiency of virus transcription and assembly.

[0007] Adenovirus is one of the most commonly used vectors for introducing genetic material into human cells. Adenovirus has been used as a vaccine delivery vector and for the expression of recombinant proteins in cell lines and tissues. Adenovirus vectors are characterized by inducing a strong cellular response to the encoded transgene compared to recombinant adjuvant proteins that induce a strong antibody response. However, these antibody responses alone may be insufficient for defense against complex pathogens, and a synergistic T cell response has been proven to be beneficial.

[0008] The adenovirus vector of the present invention encodes the spike protein because it may induce antibodies against the RBD in order to neutralize the ability of the virus to bind to host cells. More specifically, the SARS-CoV-2 spike protein is presented in two ways. First, in the form of a nucleic acid encoding at least one SARS-CoV-2 spike protein or a derivative thereof. Second, in the form of a polypeptide containing the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein or a derivative thereof displayed on the adenovirus capsid via protein IX (pIX). Due to this special feature, the adenovirus vector of the present invention combines the advantages of gene- and protein-based vaccination within one vaccine construct. The gene expression and productivity of the vector are not inhibited by pIX-display. The minor capsid protein IX (pIX) allows the fusion of a relatively large functional protein to the surface-exposed C-terminus without dramatically reducing its function. Compared with immunization with soluble proteins, fusing the RBD of the SARS-CoV-2 spike protein or a derivative thereof to pIX may ensure the presentation of the antigen in its native conformation and with the high degree of repeatability characteristic of the virus capsid, both of which have been shown to transmit strong activation signals to cognate B cells.

[0009] The RBD of the SARS-CoV-2 spike protein or a derivative thereof can be conjugated with or without a linker. For example, a flexible glycine linker or spacer can be used. The accessibility of the surface of the adenovirus vector particle to the RBD of the SARS-CoV-2 spike protein or a derivative thereof can be improved by pIX-fusion via a linker and / or spacer compared to direct fusion. The linker acts as a molecular hinge, making the antigen more flexible in its interaction with the antigen-binding domain of the B cell receptor. The spacer molecule can display the antigen over its full length at an optimal distance from the adenovirus capsid surface by lifting the antigen towards the adenovirus capsid surface.

[0010] Another feature of the adenovirus vector of the present invention is that, in addition to the spike protein, an additional antigen capable of eliciting a response is provided. The adenovirus vector contains a nucleic acid encoding at least one SARS-CoV-2 nucleocapsid protein or a derivative thereof. Thereby, the risk of the emergence of new strains of the virus having mutations in the spike can be addressed. Although the nucleocapsid protein is highly conserved, it is known to have mutation regions in emerging virus variants of concern. The terms "nucleocapsid protein", "nucleoprotein" and "nucleocapsid" are used interchangeably throughout the present disclosure. The nucleocapsid associates with viral RNA within the virus and has a role in viral RNA replication, viral particle assembly, and release. The SARS-CoV-2 nucleocapsid has been found to be a highly antigenic protein, and patients infected with SARS-CoV-2 exhibit an antibody response against the nucleocapsid. Furthermore, the nucleocapsid has been found to particularly induce T cell immunity. The T cell-based response is very important considering the finding that the neutralizing antibody titer decreases after about 3 months in COVID-19 patients. T cells are an important part of long-term immunity.

[0011] (i) At least one SARS-CoV-2 spike protein can be selected from the spike proteins of known SARS-CoV-2 viruses derived from the Wuhan variant and its derivatives. Preferably, the spike protein of the original Wuhan coronavirus is not included. In other words, at least one spike protein of (i) preferably relates to any SARS-CoV-2 spike protein excluding the Wuhan coronavirus spike protein.

[0012] As used herein, in accordance with the NCBI taxonomy database, the terms "original Wuhan coronavirus", "Wuhan variant", "2019-nCoV", "COVID-19", "COVID-19 virus", "Wuhan seafood market pneumonia virus" may be used interchangeably herein.

[0013] Five SARS-CoV-2 variants have been designated as variants of concern (VOCs). These are the Alpha, Beta, Gamma, Delta, and Omicron variants. Thus, according to a preferred embodiment, at least one SARS-CoV-2 spike protein of (i) comprises at least one spike protein or a derivative thereof of these variants of concern. Also included are spike proteins of any subtypes of the above-mentioned variants of concern, such as the Delta Plus variant and Omicron subvariants B.1.1.529 and BA.2.

[0014] According to a particularly preferred embodiment, at least one SARS-CoV-2 spike protein of (i) comprises the spike protein of the SARS-CoV-2 Omicron variant or a derivative thereof, the SARS-CoV-2 Delta Plus variant or a derivative thereof, or a combination thereof.

[0015] However, at least one SARS-CoV-2 spike protein of (i) may be the spike protein of other emerging SARS-CoV-2 variants, such as lineages B.1.1.207, B.1.1.317, B.1.616, B.1.618, B.1.640.2, AZ.5, C.1.2, B.1.429, B.1.427, C.37 (Lambda variant), B.1.621 (Mu variant), CAL.20C (Epsilon variant), B.1.617.1 (Kappa variant), B.1.526 (Iota variant), P.2 (Zeta variant), P.3 (Theta variant), B.1.525 (Eta variant), B.1.630, and B.1.640, and derivatives thereof, as well as combinations of spike proteins from two or more different variants.

[0016] The respective sequences of the spike proteins can be obtained from common databases.

[0017] The term "derivative" of each SARS-CoV-2 spike protein is understood herein to mean a protein that deviates from its sequence from each spike protein without losing the desired functionality. Further, for example, deviation of the sequence in the form of substitution, deletion or addition is possible. For example, a derivative can have a shortened sequence compared to the full-length spike protein while preferably retaining the RBD. For example, the sequence identity of the derivative's sequence to the sequence of the full-length spike protein may be at least 50%, at least 75%, at least 85%, at least 90%, at least 95% or at least 99% over the entire length of the sequence.

[0018] At least one nucleocapsid protein of (ii) can be selected from the known nucleocapsid proteins of known SARS-CoV-2 viruses derived from the Wuhan variant and its derivatives, particularly those described above for the spike protein of (i). For example, it can be a nucleocapsid protein derived from a SARS-CoV-2 virus variant of concern, such as the alpha, beta, gamma, delta and omicron variants, or any subtype thereof, for example the Delta Plus variant and Omicron subvariants B.1.1.529 and BA.2. The nucleocapsid protein of the original Wuhan virus is preferably not included. In other words, at least one nucleocapsid protein of (ii) relates to any SARS-CoV-2 nucleocapsid protein preferably excluding the nucleocapsid protein of the Wuhan coronavirus.

[0019] According to a particularly preferred embodiment, at least one SARS-CoV-2 nucleocapsid protein of (ii) comprises the nucleocapsid protein of the SARS-CoV-2 omicron variant or its derivative, the SARS-CoV-2 delta plus variant or its derivative, or a combination thereof.

[0020] The respective sequences of the nucleocapsid proteins can be obtained from common databases.

[0021] The term "derivative" of each SARS-CoV-2 nucleocapsid protein is understood herein to mean a protein that deviates from that sequence from each nucleocapsid protein without losing the desired functionality. Furthermore, deviations in the sequence are possible, for example, in the form of substitutions, deletions or additions. For example, a derivative can have a shortened sequence compared to the complete nucleocapsid protein. For example, the sequence identity of a derivative to the sequence of the complete nucleocapsid protein may be at least 50%, at least 75%, at least 85%, at least 90%, at least 95% or at least 99% over the entire length of the sequence.

[0022] The RBD displayed on the capsid can be selected from the RBDs of known spike proteins of SARS-CoV-2 viruses derived from the Wuhan variant and its derivatives, particularly those described above for the spike protein of (i). For example, it can be the RBD of a spike protein derived from a SARS-CoV-2 virus variant of concern. Such variants of concern as the Alpha, Beta, Gamma, Delta, and Omicron variants, or their subtypes, such as the Delta Plus variant and the Omicron subtypes B.1.1.529 and BA.2. The respective sequences of the RBDs can be obtained from common databases. The RBD of the spike protein of the original Wuhan virus is preferably not included. In other words, the RBD displayed on the capsid can be selected from the RBDs of known spike proteins of SARS-CoV-2 viruses excluding the spike protein of the Wuhan coronavirus.

[0023] According to a particularly preferred embodiment, the RBD displayed on the capsid is the RBD of the SARS-CoV-2 Delta Plus variant or its derivative.

[0024] Adenoviruses have been isolated from many different species, and more than 100 human serotypes have been reported. The overall structure of the adenovirus genome is conserved among serotypes, and specific functions are similarly arranged. Most adults have been exposed to the adenovirus serotypes most commonly used in therapy. Vectors based on human adenovirus 5 (HAdV5) are most frequently used, but their immunogenicity is impaired due to high existing immunity levels. Alternative adenovirus serotypes such as HAdV35, HAdV26, and ChAdV3 are highly immunogenic, have low seroprevalence, and are not affected by existing immunity to HAdV5, making them suitable as vaccine vectors. The adenovirus vector of the present invention can be derived from different species, preferably human or chimpanzee adenoviruses. The adenovirus serotype can be, for example, 5, 26, 35, or 3, particularly human adenovirus serotypes 5, 26, or 35 and chimpanzee adenovirus serotype 3.

[0025] The adenovirus vector of the present invention is replication-deficient. According to a preferred embodiment, it has deletions in at least the E1, and / or E3 gene regions, and optionally one or more additional regions such as the E2b region. The sequences of the wild-type adenovirus genome and its respective E1 and E3 regions are described in common databases. For example, the wild-type adenovirus 5 genome is described in GenBank Accession No. AY339865.1.

[0026] The term "deletion" or "deficiency", when used in the context of a deletion in the E1 or E3 gene region in this specification, refers to a specific DNA sequence that is mutated to interfere with the expression and / or function of at least one E1 or E3 gene product. Thus, in one embodiment, "deletion" is used in relation to a specific DNA sequence that has been deleted (removed) from the Ad genome. An E1 / E3 deletion or "including a deletion within the E1 / E3 region" refers to a deletion of at least one base pair within each region of the Ad genome. Thus, in one embodiment, one or more base pairs are deleted, and in further embodiments, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 base pairs are deleted. In one embodiment, the deletion is a deletion of 150, 160, 170, 180, 190, 200, 250, or 300 base pairs or more within the E1 and / or E3 regions of the Ad genome, respectively. The deletion may be a deletion that interferes with the expression and / or function of at least one gene product, and thus includes deletions within exons of the portion encoding the E1E3-specific protein, as well as deletions within the promoter and leader sequences. In a further embodiment, "deletion" refers to one or more point mutations in the DNA sequence of this region of the Ad genome such that one or more of the encoded proteins do not function. Such mutations include residues substituted with different residues that result in a change in the amino acid sequence that results in a non-functional protein.

[0027] Referring to human adenovirus serotype 5, the deletion in the E1 gene region can be a deletion from positions 454 to 3513 compared to the wild-type adenovirus 5 genome (GenBank: AY339865.1). The deletion in the E3 gene region can be a deletion from positions 28592 to 30465 compared to the wild-type adenovirus 5 genome (GenBank: AY339865.1).

[0028] The nucleic acid sequence of a particularly preferred adenovirus vector of the present invention is shown in SEQ ID NO: 1.

[0029] The adenovirus vector of the present invention is optimized with respect to immunogenicity. It is capable of providing strong and persistent cell-mediated and humoral immunity against SARS-CoV-2 infection. Thus, in one embodiment, the present disclosure provides an adenovirus vector for use in the prevention or treatment of COVID-19 disease.

[0030] According to a preferred embodiment, the adenovirus vector of the present invention further comprises a nucleic acid encoding at least one protein or a derivative thereof of a microorganism different from SARS-CoV-2. Preferably, such a microorganism is a causative agent of an infectious disease. "Derivative" in the meaning of the present invention means any part of a wild-type, mutant, or engineered protein capable of eliciting an immune response that results in immunogenicity. The microorganism may be any of bacteria, protozoa, and viruses.

[0031] When provided prophylactically, the compositions of the present disclosure are administered before the onset or detection of the onset of coronavirus disease to prevent, inhibit, or delay the onset of coronavirus disease; and / or generally for the purpose of preventing or inhibiting the progression of coronavirus disease in an individual. Thus, the prophylactic composition can be administered to an individual who appears to be free of coronavirus disease (a healthy or normal individual), or an individual in whom coronavirus has not yet been detected. Individuals at high risk of developing coronavirus disease can be prophylactically treated with the compositions of the present disclosure.

[0032] When provided therapeutically, the compositions of the present invention are administered to an individual diagnosed with coronavirus disease for the purpose of ameliorating or curing the coronavirus disease; increasing the survival of the individual; preventing, inhibiting, reversing, or delaying the onset of coronavirus disease in the individual.

[0033] The route of administration, frequency of administration, and dosage of the therapeutic compositions described herein may vary from individual to individual, but can be readily established using standard techniques.

[0034] Administration of the adenovirus vector can be via various suitable routes for delivery. One preferred route contemplated herein is by injection such as intramuscular injection, intravenous injection or subcutaneous injection. In one embodiment, intramuscular injection or subcutaneous injection is preferred. Intramuscular administration is particularly preferred for directly delivering the adenovirus vector to the blood supply in the muscle to optimize immunogenicity in order to induce systemic immunity.

[0035] Another possible route is administration to the airway. For defensive immunity against mucosal pathogens, i.e., (SARS coronavirus), instead of a more general approach of tolerance for maintaining mucosal homeostasis, mucosal immunization can be enhanced and better local defense can be achieved by immunological activation in mucosal tissues. Thus, in one aspect of the present invention, an adenovirus vector can be administered to the airway of a subject, particularly to the airway mucosa, to induce mucosal immunity in addition to cell-mediated immunity and humoral immunity. Such vaccination induces both mucosal immunity and systemic immunity.

[0036] Furthermore, the adenovirus vector can be administered to a patient by multiple routes of administration, for example, to induce local and systemic immune responses.

[0037] In a preferred aspect, administration of the adenovirus vector of the present invention is capable of generating long-term T cell and B cell memory.

[0038] The present invention further provides a vaccine composition comprising the adenovirus vector disclosed herein. The vaccine composition can be formulated in a pharmaceutically acceptable excipient suitable for administration to a subject. According to a preferred embodiment, the vaccine can further comprise an additional active agent, i.e., particularly an additional immunogenicity-conferring agent. Such additional immunogenicity-conferring agents can be directed against the same target, the same virus, the same subtype, or other subtypes, and even other viruses.

[0039] The vaccine composition can be adapted to the above-preferred administration methods. Thus, in one embodiment, the present disclosure provides a vaccine composition adapted for injection, particularly intramuscular or subcutaneous injection.

[0040] In another embodiment, the present disclosure provides a vaccine composition adapted for administration to the airways of a subject, particularly the mucosal surface of the airways, to induce mucosal immunity in addition to cell-mediated and humoral immunity. Thus, the vaccine composition can be adapted for oral, intranasal, or sublingual administration.

[0041] In a preferred aspect, the vaccines disclosed herein generate long-term T-cell and B-cell memory.

[0042] The method is also a method for preventing and / or treating coronavirus disease, particularly COVID-19. Preferably, the method encodes the nucleocapsid protein and spike protein of the coronavirus and displays a polypeptide comprising the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein or a derivative thereof via protein IX (pIX) on its capsid, using the adenovirus vector as defined above. The method can induce a broader cellular immune response against SARS-CoV-2. The adenovirus vector can be administered to a subject individual in an immunogenic composition. The viral vaccine thus administered will induce the cellular expression of the SARS-CoV-2 nucleocapsid and spike proteins and will induce a direct immune response against the SARS-CoV-2 spike protein RBD on the virus surface. With this, an immune response against it will occur in the individual and the individual can be vaccinated. Since the spike protein, particularly the nucleocapsid protein, is a relatively conserved polypeptide, it can elicit an immune response against various members of the coronavirus family.

[0043] The vaccine composition described herein can be used as a universal booster vaccine for any anti-SARS-CoV-2 vaccine directed against the SARS-CoV-2 spike (S) and / or nucleocapsid (N) protein. According to one aspect, the present invention provides a method of boosting an antigen-specific immune response in a mammal, comprising administering to the mammal a sufficient amount of the adenoviral vector or vaccine composition disclosed herein. This booster acts even in patients immunized with a vaccine encoding a SARS-CoV-2 spike or nucleoprotein other than those described herein and can broaden the immunogenicity of these patients.

[0044] In one embodiment, as described herein, the booster is administered at least 7 days, such as at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 28 days, at least 35 days, or at least 42 days after the first prime vaccination. Preferably, the booster is administered at least 2 months, at least 3 months, at least 4 months, or later. A booster administration about 3 months later is particularly preferred. As described herein, the booster can effectively improve both antibody production against SARS-CoV-2 and cell-mediated immunity against SARS-CoV-2. The effectiveness of the booster vaccine can be measured by any standard quantification of the immune response.

[0045] Additional booster vaccine administrations are possible, such as a second or third booster administration.

[0046] In particular, in one embodiment, the boost described herein is administered by injection, preferably intramuscular or subcutaneous injection. In another embodiment, the boost described herein is administered to the subject's airway, such as the mucosa of the airway, particularly by nasal or oral administration. According to a particularly preferred embodiment, the boost is administered by injection, preferably intramuscular or subcutaneous injection, approximately 3 months later.

[0047] In a further aspect, the present invention relates to a platform for providing a multivalent vaccine comprising at least one adenovirus vector, as described herein, i.e., a widely applicable technology. The vaccine platform can be understood as a "plug and play" physical framework that can be used when developing vaccines against emerging infectious diseases. According to a preferred embodiment, the adenovirus vector of the present invention further comprises a nucleic acid encoding at least one protein or a derivative thereof of a microorganism that causes an infectious disease.

[0048] The present invention is further illustrated by the following figures and examples, which are not intended to limit the scope of the present invention.

Brief Description of the Drawings

[0049]

Figure 1A

Figure 1B

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Figure 4A

[0050] [Table 1]

[0051]

Figure 4B

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[0052] Sequence Listing

[0053] [Table 2] Examples

[0054] Production of Recombinant Adenovirus from Plasmid Adjustment of Bacterial Culture Prepare 200 ml of bacterial culture according to the following procedure. 1. Streak Escherichia coli on a freshly prepared LB agar plate containing the appropriate selective antibiotic and incubate overnight at 37°C. 2. Inoculate one colony into 4 - 6 ml of LB medium containing the appropriate selective antibiotic and incubate at 37°C with a rotation speed of 225 - 250 rpm for 8 hours to prepare a starter culture.

[0055] [Table 3]

[0056] 3. Incubate overnight using the starter culture. Dilute the starter culture 1:500 - 1:1000 and transfer it into a flask containing at least 3 - 4 times the volume of the medium of the selective medium. Place the flask in a shaking incubator and incubate at 37 °C with vigorous shaking (~250 - 300 rpm) until saturation (12 - 16 hours).

[0057] Preparation of endotoxin-free recombinant adenovirus pDNA To successfully package adenovirus, high-yield and high-quality plasmid DNA (pDNA) is required to achieve optimal DNA transfection.

[0058] The type of plasmid, especially the size and origin of replication, has a decisive impact on the pDNA yield. Generally, the larger the plasmid, the lower the copy number, and thus the lower the expected pDNA yield. Even the SARS-CoV-2 spike protein adenovirus expression vector constructed using a high-copy construct with a pUC origin of replication behaves like a low-copy construct because of the insertion of the 36 kb Ad5 viral genome.

[0059] The host strain of E. coli has the most influence on the quality of plasmid DNA. The pDNA isolated from E. coli exists in various topological forms, such as covalently closed circular or supercoiled, open circular, linear, as well as catenanes and multimers. Among all the isoforms, supercoiled pDNA has intact strands (no breaks) and corresponds to the active form for plasmid applications. Strains such as DH5α and XL1-Blue usually produce high levels of supercoiled pDNA, while strains with high endonuclease activity, such as HB101, may produce low-quality plasmids, resulting in poor outcomes in downstream applications.

[0060] Endotoxins are released during the lysis step of plasmid preparation and are often co-purified with plasmid DNA. Endotoxins, also known as lipopolysaccharides (LPS), are components of the cell membrane of E. coli host strains. LPS consists of a hydrophobic lipid A moiety, a complex array of sugar residues, and negatively charged phosphate groups. The presence of LPS competes with negatively charged pDNA and "free" transfection reagents, significantly reducing transfection efficiency and greatly affecting the viability of transfected cells.

[0061] Isolated adenoviral pDNA is prepared and qualified according to the following procedure. 1. Isolate at least 50 - 100 μg of purified adenoviral pDNA using the NucleoBond Xtra Midi kit. 2. Measure the pDNA concentration using a NanoDrop. 3. Quality control of plasmid DNA by restriction enzyme digestion, PCR, and Sanger sequencing of the inserted sequence.

[0062] Preparation of Pac I - digested recombinant adenoviral pDNA Prior to adenovirus packaging, it is necessary to expose the left and right ITRs in order to properly perform viral replication and packaging. Each adenoviral expression vector contains a Pac I restriction site adjacent to the ITR. By digesting the vector with Pac I, the left and right viral ITRs can be exposed and bacterial sequences (such as the plasmid origin of replication and the ampicillin resistance gene) can be removed.

[0063] Pac I - digested adenoviral pDNA is prepared and qualified according to the following procedure. 1. Digest at least 15 μg of purified adenoviral pDNA with Pac I (New England Biolabs, catalog number R0547S). Follow the manufacturer's instructions. 2. Purify the digested plasmid DNA by phenol / chloroform extraction followed by ethanol precipitation. A. Add 1 / 10 volume of 3 M sodium acetate (pH 5.2) and 2 volumes of ethanol to precipitate DNA. Incubate at -20 °C for at least 30 minutes. B. Pellet the DNA at maximum speed in a microcentrifuge for 15 minutes. Carefully remove the supernatant. C. Add 500 μl of 70% ethanol to wash the pellet and centrifuge at maximum speed for 15 minutes. D. Carefully remove the supernatant. E. Air-dry for 5 - 10 minutes. F. Dissolve the DNA pellet. 3. Dissolve the pDNA digested with Pac I in sterile water or TE Buffer, pH 8.0 to a final concentration of 0.3 - 1.0 μg / μl. 4. DNA qualification by agarose gel analysis 5. DNA qualification by measuring OD260 / 280 and OD260 / 230. 6. DNA identification by sequencing the insert sequence.

[0064] Production of recombinant adenovirus in 293-derived cell lines The main components for producing recombinant adenovirus are as follows: · The E1- and E3-deleted adenovirus expression vector contains the gene of interest under the control of the selected promoter. The vector also contains the elements necessary to enable packaging of the expression construct into virions (e.g., 5′ and 3′ ITR, encapsidation signal, adenovirus late genes). · The E1-expressing cell line is used to facilitate the initial production, amplification, and titration of replication-incompetent adenovirus. The 293-derived cell line stably integrates a copy of E1 that trans-supplies the E1 proteins (E1a and E1b) required for adenovirus production. · DNA transfection reagent. Transfect 293-derived cells using the jetPRIME (registered trademark) transfection reagent according to the following procedure. 1. One day prior to transfection, trypsinize the cells, count them, and subculture at 5 x 10^5 cells per well in a 6-well plate. Plate the cells in 2 ml of normal growth medium containing serum. 2. On the day of transfection, remove the medium from the 293-derived cells and replace it with 1.5 ml of normal growth medium containing serum. 3. For each transfection sample, prepare the DNA-jetPRIME (registered trademark) transfection reagent complex by the following method: · Dilute 3 μg of Pac I-digested adenoviral pDNA in 300 μl of jetPRIME (registered trademark) buffer. Gently vortex to mix. · Gently vortex the jetPRIME (registered trademark) reagent for 5 seconds and briefly spin down before use. · Add 6 μl of jetPRIME (registered trademark) reagent to the diluted pDNA solution. Vortex for 1 - 2 seconds to mix well gently and briefly spin down. · Incubate at room temperature for 10 minutes to form the DNA-transfection reagent complex. 4. Drop 300 μl of the DNA-transfection reagent complex into each well and distribute evenly. 5. Rock the plate back and forth gently to mix. Incubate overnight at 37°C in a CO2 incubator. 6. The next day, remove the medium containing the DNA-transfection reagent complex and replace it with complete medium (i.e., DMEM containing 10% FBS). 7. Replace the medium with fresh complete medium every 2 - 3 days until a visible area of cytopathic effect (CPE) is observed (usually 5 - 10 days after transfection). 8. Supplement the culture fluid and continue the infection until approximately 80% CPE is observed (usually 10 - 15 days after transfection). 9. Aspirate cells from the plate using a 1 ml pipette tip with filter and collect the cells containing adenovirus. Transfer the cells and the medium to a sterile 15 ml capped tube. 10. Place the tube containing the collected cells and medium at -80 °C for at least 30 minutes. Take out the tube and thaw it in a 37 °C water bath for 15 minutes. Repeat the freezing and thawing twice. 11. Centrifuge the cell lysate at 4000 rpm at 4 °C for 10 minutes using a tabletop centrifuge to pellet the cell debris. 12. Transfer 100 μl aliquots of the supernatant containing virus particles to cryovials. Store the virus stock at -80 °C.

[0065] Amplification of P0 adenovirus strain in T175 flask Amplify the P0 adenovirus stock using 293-derived cells according to the following procedure. Ensure that the 293-derived cells are healthy at the time of plating. 1. One day before infection, trypsinize the cells, count them, and plate them in a T175 flask to a density of 9 x 10^6 cells. Plate the cells in 25 ml of normal growth medium containing serum. 2. On the day of infection, confirm that the cells are 80 - 90% confluent before proceeding. Add the required amount of P0 adenovirus stock (e.g., 3 x 100 μl) to the cells. Gently swirl the plate to mix. 3. Incubate the cells at 37 °C in a CO2 incubator and allow the infection to proceed until 80 - 90% of the cells become round and some float or loosely attach to the tissue culture dish (usually 2 - 4 days after infection). This indicates that the adenovirus particles are loaded onto the cells. 4. Aspirate the cells from the plate using a 10 ml tissue culture pipette and collect the cells containing adenovirus. Transfer the cells and the medium to a sterile 50 ml capped tube. 5. Centrifuge at 4000 rpm at 4 °C for 10 minutes using a tabletop centrifuge. Discard the supernatant. 6. Resuspend the cell pellet in 1.5 ml of sterile HBSS buffer. Transfer the cells to a sterile 15-ml capped tube. 7. Place the tube containing the cells at -80 °C for 30 minutes. Remove the tube and thaw it in a 37 °C water bath for 15 minutes. Repeat the freezing and thawing steps twice. 8. Centrifuge the cell lysate at 4000 rpm at 4 °C for 10 minutes using a tabletop centrifuge to pellet the cell debris. 9. Transfer 100 μl aliquots of the supernatant containing the virus particles to cryovials. Store the virus stock at -80 °C.

[0066] Amplification of P1 Adenovirus Stock in a 5-Layer Cell Factory Amplify the P1 adenovirus stock using 293-derived cells according to the following procedure. Ensure that the 293-derived cells are healthy at the time of plating. 1. One day before infection, trypsinize the 293-derived cells, count them, and plate 1×10^8 cells in a 5-layer cell factory. Plate the cells in 700 ml of normal growth medium containing serum. 2. On the day of infection, confirm that the cells are 80-90% confluent before proceeding. Add the required amount of P1 adenovirus stock (e.g., 3x100 μl) to the cells. Gently shake the plate to mix. 3. Incubate the cells at 37 °C in a CO2 incubator and allow the infection to proceed until 80-90% of the cells have aggregated and some are floating or lightly attached to the tissue culture dish (usually 2-4 days after infection). This indicates that the adenovirus particles are loaded onto the cells. 4. Aspirate the trypsinized cells from the surface of the cell factory using a 25-ml tissue culture pipette to collect the cells containing the adenovirus. Transfer the cells and medium to four sterile 250-ml capped bottles. 5. Centrifuge at 4000 rpm at 4 °C for 20 minutes using a centrifuge. Discard the supernatant. 6. Resuspend the cell pellet in 10 ml of sterile HBSS buffer. Transfer the cells to two sterile 15-ml capped tubes. 7. Place the tube containing cells at -80 °C for 30 minutes. Take out the tube and thaw it in a 37 °C water bath for 15 minutes. Repeat the freezing and thawing steps twice. 8. Centrifuge the cell lysate at 4000 rpm at 4 °C for 10 minutes using a tabletop centrifuge to pellet the cell debris. 9. Transfer 10 ml of the supernatant containing virus particles to an ultracentrifugation tube filled with CsCl gradient solution. 10. Proceed to the purification protocol of recombinant adenovirus by CsCl density gradient ultracentrifugation. 11. After desalting and buffer exchange, transfer the CsCl ultra-purified virus particles to cryovials in 100 μl aliquots. Store the virus stock at -80 °C.

[0067] Plasmid quality check before transfection:

[0068]

Table 4

[0069] Virus strain inspection

[0070]

Table 5

[0071] Confirm the integrity of plasmid DNA and virus stock by PCR: PCR target gene and primer sequences

[0072]

Table 6

[0073] The purified plasmids of Vector-1 and Vector-2 and the adenovirus samples were checked with a specially designed target gene panel. These target regions are useful for checking the integrity of the vectors, identifying transgenes, the backbone of the adenovirus, identifying the promoter, and confirming replication-deficient adenoviruses by the vector map (Figures 1A and 1B). As a result of PCR, it was confirmed that both the prepared plasmids and vectors were as designed and had complete integrity (Figures 2 and 3).

[0074] Identification of the E1A gene: The vectors were induced by recombination of the Ad5 genome with a deletion of the E1 gene. These genes are essential for virus replication in host cells. The genomic DNA of these vectors was examined by PCR to confirm the presence of the E1A gene. These viral genomes cannot express the E1A gene from the original Ad5 (Figures 2 and 3).

[0075] Sanger sequencing of the vectors: Sanger sequencing was performed on Vector-1 and Vector-2, and target regions containing both the transgenes of pIX-RBD and the adenovirus backbone were confirmed.

[0076] Restriction enzyme digestion and confirmation of vector integrity Restriction enzyme digestion of adenovirus vector constructs is a rapid and cost-effective method for providing indirect sequence information. Multiple vector constructs can be analyzed simultaneously for the presence or absence of inserts, the direction of inserts, plasmid size, and some site-specific sequence data. Here, the overall genomic constitution was mapped by RE digestion. Since restriction enzymes have very high specificity in recognizing target sites, this strategy is useful for identifying whether major rearrangements of the genome have occurred. In this study, Vector-1 and Vector-2 were mapped, and the presence or absence of the sites of the following restriction enzymes was examined:

[0077]

Table 7

[0078] The RE digestion pattern was as expected, and it was confirmed that there was no genomic rearrangement and the vector was completely integrated (Figure 4).

[0079] Size heterogeneity (virus particle size by NanoSight) The particle size heterogeneity was performed using nanoparticle tracking analysis (NTA analysis), and the hydrodynamic size of virus particles was confirmed by nanoparticle tracking analysis (Nanoparticle Tracking Analysis (NTA) analysis). The NanoSight apparatus was used for NTA, and the particle size distribution in the submicron region (30 - 1000 nm) was measured. In this technique, the particles in the sample are irradiated with a laser beam. The scattered light from the particles is detected and recorded by a camera at an angle of 90° through a 20x objective lens. The camera operates at 30 frames per second and captures a video of the Brownian motion particles. Multiple particles are tracked individually and simultaneously, and the hydrodynamic size of each particle in the population is calculated using the Stokes-Einstein equation. The vector-1 sample was diluted with PBS before the test to a particle concentration suitable for the NanoSight range (1.0E+8 - 2.5E+9 particles / ml). The hydrodynamic diameter of the main particles is approximately 90 - 100 nm (Figure 7).

[0080] Transmission electron microscope With an electron microscope, virus particles can be directly visualized. First, the grid was inverted and placed on a 10 μL droplet of vector-1 attached to parafilm for 30 seconds to place the vector on a 400 mesh glow-discharged carbon grid. The excess sample was blotted off by gently touching the edge of the grid to Whatman filter paper. Then, the grid was washed twice with two 20 μL droplets of ddH2O. Next, the grid containing the sample was placed on a 20 μL droplet of 1.5% uranyl acetate for 10 seconds for staining.

[0081] Excess staining was blotted off by gently contacting the edges of the grid with Whatman filter paper. The size and shape of the assembled virus particles were confirmed by negative staining transmission electron microscopy (TEM). Images of the virus particles showed an expected icosahedral shape with a diameter of approximately 90 nm. TEM also showed the morphological differences between empty capsids and complete capsids.

[0082] Complete particles were observed as bright spheres, and empty particles were spheres with dark spots. As a result of manually counting these particles, more than 95% of the particles were full capsids. Figure 8

[0083] Western blot analysis of purified virus samples Western blot analysis of purified vector-1 was performed. Gradient SDS-PAGE was carried out, and after visualizing the proteins (Figure 5), the protein bands from other gels were transferred to a nitrocellulose membrane. The blot was blocked with BSA in PBS (PBST) containing 0.5% Tween-20 at 37 °C for 2 hours and further incubated with a SARS-CoV-2 RBD-specific antibody overnight at 4 °C. Subsequently, the blot was incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG at room temperature for 1 hour and visualized with a substrate reagent (Figure 9).

[0084] Cesium chloride density gradient ultracentrifugation of vector-1 Cesium chloride (CsCl) density gradient and ultracentrifugation are the gold standard methods for separating fully formed adenovirus particles and partially formed adenovirus particles on a small scale. 1. Prepare three concentrations (1.25 g / cm3, 1.35 g / cm3, 1.45) of cesium chloride in 0.01 M Tris buffer (pH 7.5). 2. Cell lysis: Add lysis buffer to infected cells and lyse for 2 hours with benzonase treatment. 3. After lysis, centrifuge the lysate at 1500 rpm for 5 minutes and collect the supernatant. 4. Add 21 ml of cesium chloride solution and 8 - 9 ml of cell lysate to a 30 ml tube. First, add 7 ml of CsCl at 1.25 g / cm3 to the bottom of the tube using a syringe. Next, add 7 ml of CsCl at 1.35 g / cm3 to the bottom of the tube and slowly build up the layer. Add CsCl at 1.45 g / cm3 to the very bottom of the tube. 5. Make sure there are no bubbles so that the density boundaries do not mix. Carefully overlay 8 - 9 ml of cell lysate on top of the gradient. 6. Balance the centrifuge and run it at 28000 rpm, 4 °C for 90 minutes. 7. After centrifugation, observe the two layers of fully formed adenovirus particles (full capsids) and partially formed adenovirus particles (empty capsids). 8. The lower buffy coat layer containing the complete virus particles was used for further processing and characterization (Figure 10).

[0085] Analytical ultracentrifugation (AUC) for vector - 1 sample Analytical ultracentrifugation is a powerful and versatile method for quantitative analysis of purified macromolecules and supramolecular assemblies in solution. In AUC, it is possible to distinguish between complete particles, partially filled particles, and empty virus particles, which facilitates the analysis of purification efficiency and aggregates. It is also used to evaluate the homogeneity of preparations of adenovirus vectors used in gene therapy and vaccines under formulation conditions. Adenovirus samples were purified and in - process samples were analyzed by SDS - PAGE analysis and silver staining. The purification efficiency was confirmed by silver - stained gels (Figure 6). These purified final elution samples were used for AUC analysis, which showed that most virus particles were full capsids with a sedimentation coefficient of 597S and a mass of 83.4 MDa (Figure 11).

Claims

1. A replication-deficient adenovirus vector, (i) A nucleic acid encoding at least one SARS-CoV-2 spike protein or a derivative thereof (ii) comprising a nucleic acid encoding at least one SARS-CoV-2 nucleocapsid protein or a derivative thereof, The adenovirus vector is an adenovirus vector that displays a polypeptide containing a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein or a derivative thereof on its capsid via protein IX (pIX).

2. The adenovirus vector according to claim 1, wherein the at least one SARS-CoV-2 spike protein in (i) is selected from known SARS-CoV virus spike proteins derived from Wuhan variants and their derivatives, particularly from spike proteins of SARS-CoV-2 variants of concern such as alpha, beta, gamma, delta, and omicron variants, and more specifically, the at least one SARS-CoV-2 spike protein in (i) comprises the spike protein of the SARS-CoV-2 omicron variant or its derivative, the SARS-CoV-2 delta-plus variant or its derivative, or a combination thereof.

3. The adenovirus vector according to claim 2, wherein at least one SARS-CoV-2 spike protein of (i) is encoded by a nucleic acid sequence comprising nucleotides 1131 to 4931 as shown in SEQ ID NO:

1.

4. The adenovirus vector according to claim 1, wherein the at least one nucleocapsid protein of (ii) above is selected from known SARS-CoV virus nucleocapsid proteins derived from Wuhan variants and their derivatives, particularly nucleocapsid proteins of SARS-CoV-2 variants of concern such as alpha, beta, gamma, delta, and omicron variants, and more specifically, the at least one nucleocapsid protein of (ii) above comprises a nucleocapsid protein of SARS-CoV-2 omicron variant or its derivative, SARS-CoV-2 delta-plus variant or its derivative, or a combination thereof.

5. An adenovirus vector according to claim 4, wherein at least one nucleocapsid protein of (ii) is encoded by a nucleic acid sequence comprising nucleotides 4995 to 6242 of SEQ ID NO:

1.

6. The adenovirus vector according to claim 1, wherein the RBD displayed on the capsid is selected from known SARS-CoV virus RBDs derived from Wuhan variants and their derivatives, in particular from RBDs of SARS-CoV-2 variants of concern such as alpha, beta, gamma, delta, and omicron variants, and more specifically, the RBD displayed on the capsid is an RBD of SARS-CoV-2 delta-plus variant or its derivative.

7. The adenovirus vector according to claim 6, wherein the RBD displayed on the capsid comprises a protein encoded by a nucleic acid sequence including nucleotides 7031 to 7699 of SEQ ID NO:

1.

8. The adenovirus vector according to claim 1, wherein the adenovirus is derived from human or chimpanzee serotype 5, 26, 35, or 3, particularly human adenovirus serotype 5.

9. The adenovirus vector according to claim 1, comprising deletions of the E1 and E3 regions.

10. Unlike SARS-CoV-2, the adenovirus vector according to claim 1 further comprises a nucleic acid encoding at least one protein or derivative thereof of a microorganism that causes an infectious disease.

11. A prophylactic or therapeutic agent for COVID-19 disease comprising the adenovirus vector according to any one of claims 1 to 10.

12. The agent according to claim 11, wherein the vector is administered by injection, particularly by intramuscular or subcutaneous injection, and / or the vector is administered to the target airway, preferably to the mucous membrane of the airway, particularly by intranasal or oral administration.

13. A vaccine composition comprising an adenovirus vector according to any one of claims 1 to 10, and optionally one or more further activators, i.e., further immunogenic agents.

14. (i) Suitable for administration by injection, particularly intramuscular or subcutaneous injection, and / or (ii) The vaccine composition according to claim 13, which is suitable for administration to the target airway, preferably to the airway mucosa, and particularly for intranasal or oral administration.

15. An antigen-specific immune response enhancer in a target, comprising an adenovirus vector according to any one of claims 1 to 10, or a vaccine composition comprising the virus vector and optionally one or more further activators, i.e., further immunogenic agents.

16. The agent according to claim 15, wherein the vector or the vaccine composition is administered by injection, particularly by intramuscular or subcutaneous injection, and / or the vector or the vaccine composition is administered to a target airway, preferably the mucous membrane of the airway, particularly by intranasal or oral administration.

17. A platform for providing a polyvalent vaccine comprising an adenovirus vector according to any one of claims 1 to 10.

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