Manipulated AAV vector

Abstract

The present invention relates to adeno-associated viruses (AAV) or adeno-associated virus-like particles (AAVLP) containing an insert of approximately 75 to 400 amino acids in viral proteins (VP) VP1, VP2, and / or VP3 (the insert is an immunogenic protein or portion thereof, and / or a protein containing a binding domain (e.g., an antigen-binding domain specific for a target antigen)) at insertion site (I) at the apex of variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP. The present invention also relates to pharmaceutical compositions containing the AAV or AAVLP, and to pharmaceutical compositions, or AAVs, or AAVLPs, for use in therapy, particularly for use as vaccines, for use in the treatment or prevention of disease, and / or for use in gene therapy. Methods for producing the AAVs or AAVLPs of the present invention are also described.

Description

Technical Field

[0001] The present invention relates to an adeno-associated virus (AAV) or adeno-associated virus-like particle (AAVLP) comprising an insert consisting of about 75 to 400 amino acids in virus protein (VP) VP1, VP2, and / or VP3, which insert is an immunogenic protein or a part thereof and / or a protein comprising a binding domain (such as an antigen-binding domain specific for a target antigen) at a position of an insertion site (I) at the top of the variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP. The present invention also relates to a pharmaceutical composition comprising the AAV or AAVLP, and to a pharmaceutical composition, or an AAV, or an AAVLP for use in the treatment or prevention of diseases, particularly for use as a vaccine, and / or for use in gene therapy for use in treatment. The present invention also relates to a method for producing the AAV or AAVLP of the present invention.

Background Art

[0002] Recombinant adeno-associated virus (AAV) vectors have proven to be a very suitable delivery system for efficiently and long-term introducing genes into humans (Li, C., Samulski, R. J., Nat Rev Genet, 2020, 21: 255-272). AAV is a non-pathogenic virus belonging to the family Parvoviridae and the genus Dependovirus, and replicates only in the presence of adenovirus, papillomavirus, or herpesvirus. AAV has a single-stranded DNA genome of about 5 kb (Trapani et al., Progress in retinal and eye research, 2014, Volume 43: 108-128).

[0003] The safety and long-term expression of transgenes have been extensively investigated in large animal models, non-human primates, and humans in a number of clinical trials with AAV.

[0004] AAV is a small (25 nm) virus structurally without an envelope, possessing an icosahedral capsid. The AAV capsid typically consists of 60 distinct structural viral proteins (VPs), particularly 5 VP1 proteins, 5 VP2 proteins, and 50 VP3 proteins. The capsid contains a single-stranded DNA genome of approximately 4.7 kb, containing two genes, Rep and Cap, between two reverse-ended repeats (ITRs). Rep encodes many non-structural Rep proteins essential for viral genome replication and packaging. Cap contains an open reading frame (ORF) that generates structural proteins VP1, VP2, and VP3 in a ratio of approximately 1:1:10 through alternative splicing and the use of separate start codons. All three VPs (VP1, VP2, and VP3) share one common sequence, with only VP1 containing a unique sequence (approximately 138 amino acids) at its N-terminus. Therefore, the position within the VP proteins is typically given relative to VP1. In addition, a second +1 frameshifted ORF produces a non-structural assembly activating protein (AAP), which acts as a chaperone facilitating the assembly of the three VPs into an icosahedral capsid structure without being involved in the capsid structure itself. Many variants of the native AAV sequence exist with distinctly different anti-AAV antibody profiles (hence the names serotypes) (Gao et al., Curr Gene Ther., 2005, 5(3): 285-297). These numerous serotypes differ in the composition and structure of their capsid proteins, resulting in different efficiency (i.e., tropism) of transduction into different cell types (Srivastava, A., Curr Opin Virol., 2016, 21: 75-80). Recombinant AAV (rAAV) can be produced and purified at higher titers, making it easier to use clinically (e.g., in gene therapy or vaccination).

[0005] Recombinant AAVs can be produced by transfecting cell lines such as HEK293 cells or HEK293-derived cells (e.g., HEK293 cells) with a DNA plasmid encoding a genome with flanking Cap and Rep sequences, AAV reverse end repeats (ITRs), and adenovirus helper sequences required for AAV replication (Grimm et al., Human Gene Therapy, 1998, 9:18: 2745-2760). Empty AAV particles (AAVLPs) can be produced similarly by excluding the genomic plasmid with flanking AAV ITRs during production (Gao et al., Mol Ther Methods Clin Dev. 2014; 1(9)).

[0006] AAV capsids allow the insertion of peptides (up to approximately 34 amino acids in size) at specific surface-exposed locations without losing structural integrity or major function (see, e.g., WO 2012 / 031760 A1, WO 2998 / 1145401 A2 and EP 3 527 223, WO 2016 / 054554 A1). The VP has nine so-called variable regions (VRs), of which loops in the form of VR-IV, VR-V, and VR-VIII are located at the apex of the protrusion. Known insertion sites for AAV2 are I-587 (e.g., insertion between the amino acid residues asparagine (N) 587 and arginine (R) 588 in AAV2 VP1) and I-453 (e.g., insertion between the amino acid residues glycine (G) 453 and threonine (T) 454 in AAV2 VP1). Such manipulations of AAV capsids have been explored to alter AAV targeting and redirect AAV to specific cell types different from those typically infected by native AAV serotypes (Buning, H and Srivastava, A., Mol Ther Methods Clin Dev., 2019, 12: 248-265). Thus, AAV targeting has been altered using small peptides inserted into the surface-exposed location of the AAV capsid. Alternatively, larger peptides have been introduced into the N-terminus of the capsid protein, potentially resulting in loss of VP3 expression and reduced infectivity (Warrington et al., Journal of Virology (2004), 78(12): 6595-6609). Therefore, the need remains for new strategies involving the introduction of larger inserts, and possibly redirecting AAV to potential target cell types.

[0007] In December 2019, a new highly pathogenic coronavirus caused an outbreak in Wuhan, China, and rapidly spread to other countries around the world. Coronaviruses are positive-sense single-stranded RNA viruses belonging to the Coronaviridae family. These viruses mostly infect animals (including birds and mammals). In humans, coronaviruses typically cause mild respiratory infections. Since 2003, two highly pathogenic human coronaviruses, namely Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), have already led to global epidemics with high morbidity and mortality rates. Both endemics were caused by zoonotic coronaviruses belonging to the Betacoronavirus genus within the Coronaviridae family.

[0008] Like SARS-CoV and MERS-CoV, the novel SARS-CoV-2 belongs to the genus Betacoronavirus. As reported by Zhou et al. (Cell Discovery (2020) 6:14), SARS-CoV-2 has the best nucleotide sequence match with SARS-CoV (79.7%). Specifically, the envelope protein and nucleocapsid protein of SARS-CoV-2 are two evolutionarily conserved regions, with 96% and 89.6% sequence match with SARS-CoV, respectively. The spike protein has been reported to have the least sequence conservation between SARS-CoV-2 and SARS-CoV (77% sequence match), while the SARS-CoV-2 spike protein has only 31.9% sequence match with the MERS-CoV spike protein. The S protein is the most exposed protein, and an antibody response against the SARS-CoV S protein has been shown to protect against SARS-CoV infection in mouse models.

[0009] While several domestic and international research groups are working on developing new vaccines for the prevention and treatment of diseases, particularly Covid-19, the development of effective vaccines for preventing or treating infectious diseases (such as HIV or cancer vaccines) remains challenging. Therefore, new strategies are needed to develop effective therapeutic and / or prophylactic vaccines that can prevent and / or treat infectious diseases (such as Covid-19, other coronavirus-borne diseases, and / or zoonotic diseases) or cancer. [Overview of the project]

[0010] The AAV capsid consists of 60 distinct structural viral proteins (VPs): 5 VP1 proteins, 5 VP2 proteins, and 50 VP3 proteins. Therefore, by inserting an immunogenic protein or a portion of it into the structural proteins VP1, VP2, and VP3 (especially VP3), the immunogenic protein or a portion of it will be presented multiple times on the surface of the complete AAV capsid due to the capsid's repeating structure. This provides a high density of immunogenic protein, mimicking the appearance of a virus (such as SARS-CoV-2), with viral structural proteins (e.g., spike proteins) regularly and repeatedly presented on the surface. This foundational technology can also be used to insert a target molecule, i.e., a protein containing a binding domain (e.g., an antigen-binding domain in an antibody-derived protein or antibody mimetic), as one binding unit of a binding pair, thereby binding to the other binding unit (antigen) of that binding pair.

[0011] This invention relates to repurposing AAV vectors as vaccines by inserting a large immunogenic protein or a portion thereof (such as the major antigenic portion or its immunogenic portion of an infectious medium) into the surface-exposed position of an AAV capsid. In particular, adeno-associated virus (AAV) or adeno-associated virus-like particles (AAVLP) are converted into carrier vehicles for length-variable immunogenic amino acid sequences encoded within the capsid VP sequence, and thus into carriers for subunit vaccines. These vaccines can be used for immunization to induce an immune response (including an antibody response) to treat or prevent disease, or as research tools for antibody production / induction, due to their exceptionally strong antigenic properties.

[0012] The insert is inserted into each of the 60 building blocks of the AAV capsid, either at the I-587 or I-453 position of AAV2 VP2, or at any other surface exposure position that allows insertion within a common sequence shared by all three VPs (VP1, VP2, and VP3), thus being presented 60 times on the surface of a single AAV particle. As demonstrated herein, the surface exposure positions at the apex of variable region VIII (VR-VIII) and / or variable region IV (VR-IV) surprisingly accommodate large inserts. When inserted simultaneously into two insertion sites (one at the apex of VR-VIII and one at the apex of VR-IV (e.g., I-587 and I-453 of the AAV, respectively)), the immunogenic sequence is presented 120 times on the surface of a single AAV particle, or two different immunogenic sequences are presented 60 times each on the surface of a single AAV particle. This repeating structure allows other proteins (such as proteins / target molecules containing binding domains (e.g., antigen-binding domains in antibody-derived proteins or antibody mimetics)) to be inserted into the apical position of VR-VIII and / or VR-IV, redirecting AAV to a modified target, i.e., giving it altered cell-targeting properties.

[0013] In one aspect, the present invention relates to an adeno-associated virus (AAV) or adeno-associated virus-like particle (AAVLP) wherein an insert consisting of approximately 75 to 400 amino acids in a capsid-forming viral protein (VP) is included at the insertion site (I) at the apex of the variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, and optionally flanked on one or both sides of the insert is a linker containing one or more amino acids preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof. The insert can be any protein of any length, and in particular, the insert can be an immunogenic protein or a portion thereof, and / or a protein containing a binding domain.

[0014] In one embodiment, the present invention relates to an adeno-associated virus (AAV) or adeno-associated virus-like particle (AAVLP) wherein an insert consisting of about 75 to 400 amino acids (preferably about 75 to 300 amino acids) in a capsid-forming viral protein (VP) is included at the insertion site (I) at the apex of variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, the insert being an immunogenic protein or a part thereof, and optionally flanking one or both sides of the insert is a linker containing one or more amino acids preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof.

[0015] In one embodiment, the present invention relates to an adeno-associated virus (AAV) or adeno-associated virus-like particle (AAVLP) in which an insert consisting of about 75 to 400 amino acids is included in a capsid-forming viral protein (VP) at the position of an insertion site (I) at the apex of the variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, wherein the insert is a protein containing a binding domain, and optionally, on one or both sides of the insert, there is a linker containing one or more amino acids, preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof. In one embodiment, the protein containing the binding domain is a receptor-binding domain, a ligand-binding domain, or an antigen-binding domain, and preferably a protein containing an antigen-binding domain.

[0016] The top of VR-VIII corresponds to amino acids 585-592 (I-585-I-592) of VP1, more specifically, to approximately amino acids 585-592 (I-585-I-592) of VP1 AAV1 with the amino acid sequence of SEQ ID NO: 1, VP1 AAV2 with the amino acid sequence of SEQ ID NO: 2, VP1 AAV3 with the amino acid sequence of SEQ ID NO: 3, VP1 AAV6 with the amino acid sequence of SEQ ID NO: 6, VP1 AAV7 with the amino acid sequence of SEQ ID NO: 7, VP1 AAV8 with the amino acid sequence of SEQ ID NO: 8, VP1 AAV9 with the amino acid sequence of SEQ ID NO: 9, or VP1 AAV10 with the amino acid sequence of SEQ ID NO: 10, or to approximately amino acids 583-589 of VP1 AAV4 with the amino acid sequence of SEQ ID NO: 4, or to approximately amino acids 574-580 of VP1 AAV5 with the amino acid sequence of SEQ ID NO: 5. Alternatively, the apex of VR-VIII can be defined as the eight downstream amino acids of conserved glutamine corresponding to Q584 of VP1 AAV2, which has the amino acid sequence of SEQ ID NO: 2.

[0017] The top of VR-IV corresponds to amino acids 450-460 (I-450-I-460) of VP1, more specifically, to approximately amino acids 450-460 (I-450-I-460) of VP1 AAV1 with the amino acid sequence of SEQ ID NO: 1, VP1 AAV2 with the amino acid sequence of SEQ ID NO: 2, VP1 AAV3 with the amino acid sequence of SEQ ID NO: 3, VP1 AAV6 with the amino acid sequence of SEQ ID NO: 6, VP1 AAV7 with the amino acid sequence of SEQ ID NO: 7, VP1 AAV8 with the amino acid sequence of SEQ ID NO: 8, VP1 AAV9 with the amino acid sequence of SEQ ID NO: 9, or VP1 AAV10 with the amino acid sequence of SEQ ID NO: 10; or to approximately amino acids 445-455 (I-445-I-455) of VP1 AAV4 with the amino acid sequence of SEQ ID NO: 4; or to approximately amino acids 439-449 (I-439-I-449) of VP1 AAV5 with the amino acid sequence of SEQ ID NO: 5. Alternatively, the apex of VR-IV can be defined as the 12 to 5 amino acids upstream of the conserved phenylalanine corresponding to F462 of VP1 AAV2, which has the amino acid sequence of SEQ ID NO: 2.

[0018] The AAV or AAVLP is preferably derived from AAV serotype 1 (AAV1), 2 (AAV2), 8 (AAV8), or 9 (AAV9). In one embodiment, the AAV or AAVLP is derived from AAV2, and the insertion site is located between two amino acids corresponding to amino acid positions 587 and 588 of AAV2 VP1 having the amino acid sequence of SEQ ID NO: 2 (AAV2 I-587), or between 588 and 589 (AAV2 I-588), and / or between 452 and 453 (AAV2 I-452), between 453 and 454 (AAV2 I-453), or between 454 and 455 (AAV2 I-454), preferably at AAV2 I-587, AAV2 I-588, or AAV2 I-453, and more preferably at AAV2 I-587 or AAV2 I-588.

[0019] In another embodiment, AAV or AAVLP is derived from AAV1, and the insertion site is located between two amino acids corresponding to amino acid positions 587 and 588 in the amino acid sequence of SEQ ID NO: 1 (AAV1 I-587), between 588 and 589 (AAV1 I-588), or between 589 and 590 (AAV1 I-589), and / or between 454 and 455 (AAV1 I-454), between 455 and 456 (AAV1 I-455), or between 456 and 457 (AAV1 I-456). In another embodiment, AAV or AAVLP is derived from AAV8, and the insertion site is located between two amino acids corresponding to amino acid positions 588 and 589 of AAV8 VP1 having the amino acid sequence of SEQ ID NO: 8 (AAV8 I-588), or between 589 and 590 (AAV8 I-589), or between 509 and 591 (AAV8 I-590), and / or between 455 and 456 (I-455), between 456 and 457 (I-456), or between 457 and 458 (I-457). In yet another embodiment, AAV or AAVLP is derived from AAV9, and the insertion site is located between two amino acids corresponding to amino acid positions 587 and 588 of AAV8 VP1 having the amino acid sequence of SEQ ID NO: 9 (AAV9 I-587), or between 588 and 589 (AAV9 I-588), or between 589 and 590 (AAV9 I-589), and / or between 454 and 455 (I-454), between 455 and 456 (I-455), or between 456 and 457 (I-456).

[0020] In one embodiment, AAV or AAVLP includes an insert in the viral protein consisting of about 75 to 300 amino acids, preferably about 75 to 260 amino acids, more preferably about 75 to 250 amino acids, and even more preferably about 80 to 220 amino acids. The linker, if present, may contain 1 to 7 amino acids, preferably 1 to 3 amino acids, more preferably 3 amino acids, at the N-terminal end of the immunogenic protein or a portion thereof, and / or 1 to 3 amino acids, preferably 2 to 3 amino acids, at the C-terminal end.

[0021] In one embodiment, AAV or AAVLP is an AAV containing an ITR flanked genome, which is preferably infectious. The ITR flanked genome may contain a transgene (such as a transgene encoding a further immunogenic protein or a portion thereof). In another embodiment, AAV or AAVLP is an AAVLP that does not contain an ITR flanked genome. In some embodiments, the insert is an immunogenic protein or a portion thereof, which is inserted into the apex of VR-VIII and the apex of VR-IV, and the immunogenic protein or a portion inserted into the apex of VR-VIII and the immunogenic protein or a portion inserted into the apex of VR-IV are the same or different; and / or AAV or AAVLP is formed by two or more viral proteins containing different inserts consisting of at least about 75 to 400 amino acids (preferably about 75 to 300 amino acids), each of which is an immunogenic protein or a portion thereof, which is either an immunogenic protein or immunogenic moiety from a different protein, or different immunogenic moieties from the same protein.

[0022] The AAV VPs forming the capsid can be VP1, VP2, and VP3, preferably in a ratio of 1:1:10. The capsid can also be formed by VP1 and VP3 alone, or by VP3 alone. Therefore, the AAV VPs forming the capsid can also be VP1 and VP3, or VP3 alone. In any variant, it is preferable that the AAV or AAVLP has a capsid consisting of approximately 60 VPs.

[0023] In some embodiments, the AAV or AAVLP according to the present invention is immunogenic to an inserted immunogenic protein or a portion thereof. In some embodiments, the immunogenic protein or a portion thereof can be a viral, bacterial, or parasitic protein or a portion thereof. In the case of a viral protein, the immunogenic protein or a portion thereof is the AAV protein or a portion thereof. In one embodiment, the immunogenic protein or a portion thereof is a portion of the coronavirus spike (S) protein (such as a portion of the SARS-CoV-2 S protein). The portion of the coronavirus S protein may include a portion comprising the coronavirus S protein receptor-binding domain (RBD) or a portion thereof (such as the SARS-CoV-2 S protein receptor-binding domain (RBD) or a portion thereof), preferably a receptor-binding motif (RBM). In one embodiment, a portion of the SARS-CoV-2 S protein contains the amino acid sequence of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69, preferably SEQ ID NOs: 11, 12, 34, 35, 36, 37, 38, 42, or 69, or contains an amino acid sequence that is at least 90% identical in sequence to the amino acid sequence of SEQ ID NOs: 11, 12, 34, 35, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69, preferably SEQ ID NOs: 11, 12, 34, 35, 36, 37, 38, 42, or 69. In other embodiments, the immunogenic protein or a portion thereof is a tumor antigen.

[0024] In one embodiment, the insert is a protein containing a binding domain (antigen-binding domain, such as a single-domain antibody (sdAb), a single-strand variable fragment (scFv), or an antibody mimetic (such as antikalin)). The AAV or AAVLP is preferably an AAV containing a genome flanked by an ITR, is infectious, and the genome flanked by the ITR may contain a transgene.

[0025] In yet another aspect, the present invention relates to a pharmaceutical composition comprising AAV or AAVLP according to the present invention, preferably further comprising at least one excipient acceptable as a medicament.

[0026] In yet another aspect, the present invention relates to the AAV or AAVLP of the present invention for use in therapy, or the pharmaceutical composition of the present invention.

[0027] In yet another aspect, the present invention relates to the AAV or AAVLP or pharmaceutical composition according to the present invention, wherein the insert is an immunogenic protein or a part thereof for use as a vaccine.

[0028] In yet another aspect, the present invention relates to an AAV or AAVLP or pharmaceutical composition according to the present invention, which is used for the treatment or prevention of diseases induced by viruses, bacteria, or parasites, and the insert is an immunogenic protein or a part thereof of each of the aforementioned virus, bacteria, or parasite. In one embodiment, the disease is coronavirus respiratory syndrome, and the immunogenic protein or a part thereof is a part of the coronavirus spike (S) protein. In certain embodiments, the disease is coronavirus disease 2019 (COVID- 19), and the immunogenic protein or a part thereof is a part of the SARS-CoV-2 spike (S) protein. In a particular aspect, the immunogenic protein or a part thereof in the AAV or AAVLP according to the present invention includes a part of the SARS-CoV-2 spike (S) protein, and the AAV or AAVLP is used to induce an immune response against SARS-CoV-2. In certain embodiments of the AAV or AAVLP for use in accordance with the present invention, the aforementioned part of the SARS-CoV-2 spike (S) protein, preferably the aforementioned part of the SARS-CoV-2 spike (S) protein, comprises the amino acid sequence of SEQ ID NO: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69, preferably SEQ ID NO: 11, 12, 34, 35, 36, 37, 38, 42, or 69, or comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69, preferably SEQ ID NO: 11, 12, 34, 35, 36, 37, 38, 42, or 69. Those skilled in the art will appreciate that the SARS-CoV-2 spike (S) protein or a part thereof is a viral entry protein that binds to cell receptors and is thus also a binding protein containing a binding domain, more specifically a receptor binding domain. The S protein binds to the cell receptor ACE-2. Thus, as an insert, it is also possible to have an immunogenic protein or a part thereof and a protein containing a binding domain.

[0029] In yet another aspect, the present invention relates to AAV or AAVLP or pharmaceutical composition according to the present invention for use in the treatment or prevention of cancer, wherein the immunogenic protein or a part thereof is a tumor antigen or a part thereof. Those skilled in the art will see that certain viral immunogenic proteins can also function as tumor antigens (antigens derived from HCV or HPV).

[0030] In yet another aspect, the present invention relates to an AAV or AAVLP of the present invention, or a pharmaceutical composition according to the present invention, for use in gene therapy, wherein the insert is a protein comprising a binding domain (antigen-binding domain, such as a single-domain antibody (sdAb), a single-stranded variable fragment (scFv), or an antibody mimetic). The AAV or AAVLP is preferably an AAV containing an ITR flanked genome, is infectious, and more preferably the ITR flanked genome contains a transgene.

[0031] AAV or AAVLP for use in accordance with the present invention can be administered via the nasal mucosa, sublingual, oral, oral, intravenous, intramuscular, intraperitoneal, or subcutaneous routes. In one embodiment, AAV or AAVLP can be administered by inhalation via the nasal, oral, and / or mucosal routes.

[0032] In yet another aspect, the present invention relates to a method for producing AAV or AAVLP, the method comprising (i) preparing a cell comprising at least one DNA sequence comprising a cap gene and a rep gene, at least one DNA sequence comprising an adenovirus helper sequence, and optionally an ITR comprising at least one DNA sequence comprising a genome adjacent to the cap gene (wherein the cap gene is an insert consisting of about 75 to 400 amino acids (preferably about 75 to 300 amino acids) in the viral protein (VP) that forms the capsid, in the variable region VII of the VP The method comprises (ii) a step of culturing the cells under conditions that enable the production of the AAV or AAVLP; and (iii) a step of purifying the AAV or AAVLP. In some embodiments, the method further comprises (iv) a step of formulating the AAV or AAVLP into a pharmaceutical composition by adding at least one pharmaceutically acceptable excipient to produce the AAV or AAVLP. The insert may contain any protein of any length, and in particular may contain an immunogenic protein or a portion thereof, and / or a protein containing a binding domain.

[0033] The genome adjacent to the ITR may further contain transgenes. In particular, if the insert is an immunogenic protein or a part thereof, the genome adjacent to the ITR may further contain transgenes encoding further immunogenic proteins or parts thereof. In another embodiment, the AAV or AAVLP is an AAVLP that does not contain a genome adjacent to the ITR. As the AAV VPs that form the capsid, VP1, VP2, and VP3 are possible, preferably in a ratio of 1:1:10. The capsid may also be formed by VP1 and VP3 alone, or by VP alone. Thus, as the AAV VPs that form the capsid, VP1 and VP3 are also possible, or VP3 is possible. In any variant, the AAV or AAVLP preferably has a capsid consisting of about 60 VPs. As the immunogenic protein or a part thereof, and / or the protein containing the binding domain and insertion site, those disclosed herein with respect to the AAV or AAVLP according to the present invention are possible. [Brief explanation of the drawing]

[0034] [Figure 1]Structural models of the AAV capsid and AAV viral protein, and schematic diagrams of the cap ORF. (A) Schematic diagram of the ORF reading frame including VP1, VP2, and VP3. The position of the representative insertion site including the insert, I-587, is shown at the bottom. (B~C)(C) Based on the publicly available structure of AAV2 WT (database: Protein Databank (PDB) / ID:6ih9), (B) comparative structural modeling using Robetta from known structures was performed using Robetta (https: / / robetta.bakerlab.org / ) for VP3 of AAV2 (HtW2_S1.1) containing the SARS-CoV-2 S protein domain at position I-587 as an insert, and processed in the same orientation using Chimera software (https: / / www.cgl.ucsf.edu / chimera). The VP portion of AAV is colored gray, and the S protein portion is colored black. (D~E) Using RoseTTAFold (https: / / robetta.bakerlab.org / ), comparative structural modeling from sequence information was performed by comparing (D) the VP3 of AAV2 (HtW2_S1.1) containing the SARS-CoV-2 S protein domain at position I-587 as an insert with (E) the publicly available structure of AAV2 WT (database: Protein Databank (PDB) / ID:6ih9), and processed in the same orientation using Chimera software (3). The VP portion of AAV is colored gray, and the S protein portion is colored black. (F~G) Comparative structural modeling based on sequence information of the corresponding 60-mer capsid structure of HtW2_S1.1 using RoseTTAFold is shown at two different angles (F) and (G), respectively. The VP portion of AAV is colored gray, and the S protein portion is colored black. Therefore, the insertion of the large S protein of more than 200 amino acids did not impair the major giant capsid structure. The scale bars in (F, G) represent 100 angstroms.

[0035] [Figure 2]AAVx affinity purification chromatography of AAV vectors. (AC) This chromatogram shows the elution of (A) AAV2 WT particles, (B) HtW2_S1.1 particles (= filled AAV particles) generated in the presence of a pTransgene plasmid with an adjacent ITR sc-CMV-eGFP expression cassette, and (C) HtW2_S1.1 particles (= empty AAV particles; AAVLP) generated in the absence of the pTransgene plasmid. HtW2_S1.1 filled particles and HtW2_S1.1 empty particles bind to the AAVx affinity purification column and elute similarly but slightly later after the start of the elution process (indicated by the ml value of elution buffer on the x-axis).

[0036] [Figure 3]Transduction assay of AAV vectors into HeLa cells. (A) Representative bright-field epifluorescence images from HeLa cell cultures 24 hours (left) and 48 hours (right) after transduction at MOI 1,000 with either AAV-sc-CMV-eGFP (row 1) packaged with AAV2 WT, or a novel AAV variant HtW2_S1.1 (rows 2-4) containing a 202-amino acid insertion including a portion of the SARS-CoV-2 S1 spike protein with a linker amino acid adjacent to RBD (sequence number 11). Images in rows 3 and 4 show representative images of HeLa cell cultures transduced with HtW2_S1.1 after transduction at MOI 500 and 250, respectively, with AAV-sc-CMV-eGFP packaged with HtW2_S1.1. Scale bars represent 400 μm. (B) Graphs showing the percentage of eGFP-positive cells (in %) measured using a Countess II FL automated cell counter in HeLa cell cultures 48 hours after transduction of AAV-sc-CMV-eGFP packaged with AAV2 WT at MOI 1,000 (upper figure), or AAV-sc-CMV-eGFP packaged with a novel AAV variant HtW2_S1.1 having a 202-amino acid insertion including a SARS-CoV-2 S1 spike RBD portion with adjacent linker amino acids, at MOI 1,000, 500, and 250. Despite the large insertion of over 200 amino acids, HtW2_S1.1 still retained its ability to infect and transduce human cells, even at a very low MOI of 250.

[0037] [Figure 4]Transduction assay of the HtW2_S1.1 vector into HeLa cells. (A) Representative epifluorescence images from natural (left column, -ACE2) HeLa cell cultures or ACE2-transduced (right column, +ACE2) HeLa cell cultures 24 hours (left) and 48 hours (right), after transduction of AAV-sc-CMV-eGFP, packaged with the novel AAV variant HtW2_S1.1 having a 202-amino acid insertion containing the SARS-CoV-2 S1 spike protein moiety including RBD (SEQ ID NO: 11), at MOI 250 (top row) or MOI 500 (bottom row). Scale bars represent 400 μm. (B) Graphs showing the percentage (in %) of eGFP-positive cells measured using a Countess II FL automated cell counter 48 hours after transduction at MOI 250 and 500 of AAV-sc-CMV-eGFP, packaged with a novel AAV variant HtW2_S1.1 having a 202-amino acid insertion including the SARS-CoV-2 S1 spike RBD portion adjacent to the linker amino acids, in natural HeLa cell cultures or ACE2-transduced HeLa cell cultures. One-way ANOVA, Sidac multiple comparison test:**, p<0.01;***, p<0.001.

[0038] [Figure 5]Transduction assay of the HtW2_S1.2 vector into HEK293T cells. (A) Representative epifluorescence images from natural (left column, -ACE2) HEK293T cell cultures or stable ACE2-overexpressing (right column, +ACE2) HEK293T cell cultures 48 hours after transduction (right panel) of AAV-sc-CMV-eGFP packaged with the novel AAV variant HtW2_S1.2, which has a 211-amino acid insertion including a linker amino acid-adjacent portion of the SARS-CoV-2 S1 spike protein (SEQ ID NO: 69), at MOI 250 (top row), MOI 500 (middle row), or MOI 1000 (bottom row). Scale bars represent 200 μm. (B) Graphs showing the percentage (in %) of eGFP-positive cells measured using a Countess II FL automated cell counter in natural HEK293T cell cultures or HEK293T cell cultures overexpressing stable ACE2, 48 hours after transduction at MOI 250, 500, and 1000 with AAV-sc-CMV-eGFP packaged with a novel AAV variant HtW2_S1.2 having a 206-amino acid insertion including the SARS-CoV-2 S1 spike RBD portion adjacent to the linker amino acids. One-way ANOVA, Sidaq multiple comparison test: ***, p<0.001;****, p<0.0001.

[0039] [Figure 6]Humoral response to HtW capsid. (A) Immunization scheme in rabbits (12-week-old females). (B) Immunogenicity of HtW capsid was evaluated by ELISA in serum from rabbits immunized with the indicated AAV empty capsid. IgG endpoint titers against SARS-CoV-2 wild-type RBD are shown. Rabbits were immunized with wild-type AAV empty capsid (AAV2 WT, AAV9 WT) or HtW empty capsid (HtW2_S1.1, HtW2_S1.2, or HtW9_S1.1). (C) Rabbit serum was collected 10 days after the first (blood sample 1), second (blood sample 2), and third (blood sample 3) booster injections using empty AAV capsid and analyzed by ELISA using SARS-CoV-2 RBD as the antigen. Endpoint titers of SARS-CoV-2 RBD-specific IgG and IgM antibodies are shown.

[0040] [Figure 7] Analysis using dot blot analysis of rabbit serum induced against HtW capsids. (A) Schematic representation of the dot blot assay of AAV vectors spotted on the surface of a polyvinylidene fluoride (PVDF) membrane and stained with the indicated antibody or serum. (B) Pipetting scheme showing the capsids used to prepare each AAV vector and the amount of AAV vector spotted on each dot of the blot shown in (C~F) (total vector genome). (C) Dot blot labeled with commercially available rabbit monoclonal anti-SARS-CoV-2 spike S1 antibody at a dilution of 1:500. (D) Dot blot labeled with a 1:10000 dilution of serum (αHtW2_S1.1) from rabbits immunized with HtW2_S1.1 empty capsids. (E) Dot blot labeled with a 1:10000 dilution of serum (αHtW2_S1.2) from rabbits immunized with HtW2_S1.2 empty capsids. (F) Dot blot labeled with a 1:10000 dilution of serum (αHtW9_S1.1) from rabbits immunized with HtW9_S1.1 empty capsids.

[0041] [Figure 8]Dot blot assay of AAV vectors spotted on the surface of a PVDF membrane and stained with serum from human individuals inoculated with Comirnaty and rabbits inoculated with Htw9_S1.1. (A) Pipetting scheme showing the capsid used to prepare each AAV vector and the amount of spotted AAV vector on each dot of the blot shown in (B~C) (genome of the entire vector). (B) Dot blot labeled with a 1:500 dilution of serum collected from patients one week after the second vaccination using Comirnaty (BNT162b2, Biontech / Pfizer). (C) The same dot blot after detachment and relabeling with a 1:10000 dilution of serum (αHtW9_S1.1) from rabbits immunized with empty capsids.

[0042] [Figure 9] Neutralization efficiency of serum from HtW-immunized rabbits. (A) Neutralization assay for the transduction efficiency of stably ACE2-expressing HEK293T cells (HEK293T+ACE2) using serum from HtW9_S1.1-immunized rabbits. HtW2_S1.1 or HtW2_S1.2 vectors containing the sc-CMV-eGFP genome were pre-incubated at 37°C with αHtW9_S1.1 serum at different dilutions (1:1000, 1:5000, 1:10000) and used to transduce HEK293T+ACE2 cells at a MOI of 250. Images of epifluorescence microscopy 48 hours after transduction are shown. (B) 48 hours after transduction, cells were harvested and the percentage of eGFP-positive cells was analyzed using a Countess II FL automated cell counter. The percentage of eGFP-positive cells after transduction using HtW2_S1.1 or HtW2_S1.2 pre-incubated with the indicated dilutions of αHtW9_S1.1 is shown, normalized to the corresponding control transduction using each vector, but in the absence of serum.

[0043] [Figure 10]Structural models of AAV virus proteins (VP) for serotypes AAV2(A), AAV9(B), AAV1(C), and AAV8(D). (A-D) The top of variable region VIII, corresponding to amino acids 585-592, and the top of variable region IV, corresponding to amino acids 450-460, are highlighted in black. (A) Amino acids 585-592 (SEQ ID NO: 17) and 450-460 (SEQ ID NO: 16) of AAV2 WT (PDB 6ih9) are marked. (B) Amino acids 585-592 (SEQ ID NO: 19) and 450-460 (SEQ ID NO: 18) of AAV9 WT (3ux1) are marked. (C) Amino acids 585-592 (SEQ ID NO: 21) and 450-460 (SEQ ID NO: 20) of AAV1 WT (6jcr) are marked. (D) AAV8 Comparative structural modeling of AAV2, AAV9, AAV8, and VP3 of AAV9, with amino acids 585-592 (SEQ ID NO: 23) and 450-460 (SEQ ID NO: 22) of WT(2qa0) marked (https: / / robetta.bakerlab.org / ), was processed using Chimera software.

[0044] [Figure 11] Multiple sequence alignment of the Cap sequences of AAV1, AAV2, AAV8, and AAV9 VP1, each containing the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, and SEQ ID NO: 9, respectively. Sequences were aligned using Clustal O(1.2.4). Shaded regions indicate insertion sites within VR-IV (I-450~I-460) and VR-VIII (I-585~I-592).

[0045] [Figure 12] Multiple sequence alignment of the Cap sequences of AAV1-10 VP1, each containing the amino acid sequences of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, respectively. The sequences were aligned using Clustal O(1.2.4).

[0046] [Figure 13]A vector map of pHtW2_S1.1(AAV2) that expresses AAV2-Rep protein and AAV2-CAP protein, which have an insertion of 202 amino acids (SEQ ID NO: 13) containing a SARS-CoV-2 S1 spike protein with a linker amino acid adjacent to it.

[0047] [Figure 14] A vector map of pHtW2_S1.2(AAV2) that expresses AAV2-Rep protein and AAV2-CAP protein, which have an insertion of 211 amino acids (SEQ ID NO: 70) containing a SARS-CoV-2 S1 spike protein with a linker amino acid adjacent to it.

[0048] [Figure 15] A vector map of pHtW9_S1.1(AAV9) that expresses AAV2-Rep protein and AAV2-CAP protein, which have an insertion of 202 amino acids (SEQ ID NO: 14) containing a SARS-CoV-2 S1 spike protein with a linker amino acid adjacent to it.

[0049] [Figure 16] Structural models of the AAV capsid and AAV viral protein. (A-B) A novel prediction of the protein structure of VP3 of AAV2 (AAV2-aGFP) with an anti-GFP scFv antibody fragment (SEQ ID NO: 73) inserted at position I-587, based on RoseTTAFold (https: / / robetta.bakerlab.org / ). (A) The corresponding predicted 60-mer capsid structure of AAV2-aGFP is shown in (B). The VP portion of AAV is colored gray, and the anti-GFP scFv portion is colored black. Thus, the large 200+ amino acid anti-GFP scFv did not impair the major giant capsid structure. The scale bars in (B) represent 100 angstroms. [Modes for carrying out the invention]

[0050] The terms “contains” or “includes” mean “contains, but is not limited to.” This term is intended to be unrestricted in its number of enumerated items, clearly indicating the presence of any feature, element, integer, antibody, or component listed, but not excluding the presence or addition of one or more other features, elements, integers, antibodies, components, or groups thereof. Thus, the term “contains” encompasses the more restrictive terms “consisting of” and “primarily consisting of.” The term “contains” can be individually replaced by the term “consisting of.” With respect to sequences, the expressions “having the amino acid sequence of” and “containing the amino acid sequence of” are interchangeable and can encompass embodiments “consisting of the amino acid sequence of.” The term “one” in this specification may include multiple, and therefore one is included, but is not limited to one. The term “about” in this specification means ±10% of the specified value.

[0051] The term "expression cassette" as used herein means a nucleic acid unit comprising at least one open reading frame (ORF) under the control of a regulatory sequence (such as a promoter) that controls expression. Preferably, the expression cassette also includes a transcription termination signal.

[0052] The term “protein” is used interchangeably with “amino acid sequence” or “polypeptide” and refers to a polymer of amino acids of any length. These terms also include proteins that are post-translationally modified through reactions (non-limiting examples include glycosylation, acetylation, phosphorylation, glycation, or protein processing). Modifications and changes (e.g., substitution, deletion, or insertion of amino acid sequences) can be realized within the structure of a polypeptide while the molecule maintains its biological functional activity. For example, a substitution of an amino acid sequence can be realized in a polypeptide or the nucleic acid coding sequence from which it originates, resulting in a protein with the same properties. The term “protein” typically refers to a sequence of more than 30 amino acids, typically having a secondary and tertiary structure. The term “peptide” typically refers to a sequence of up to 30 amino acids in length. Peptides are typically characterized by their primary amino acid sequence. The term “immunogenicity” as used herein refers to a substance that can evoke an antigen-specific response in the body of an object upon receiving it. Such immune responses include humoral and / or cell-mediated immune responses.

[0053] The term “immunogenic protein or portion thereof” means, as herein, an antigenic protein or immunogenic protein (such as a viral structural protein or tumor antigen) of a pathogen or tumor cell, for example, or its immunogenic portion. The immunogenic portion of an immunogenic protein typically includes one or more domains of that immunogenic protein (in the case of a transmembrane protein, typically one or more domains of the ectodomain of the immunogenic protein). However, the present invention also includes cases where the immunogenic portion includes only the immunogenic portion of a domain (such as a receptor-binding domain of a viral entry protein or a ligand-binding domain of a tumor antigen). An immunogenic protein or portion thereof may function as a subunit vaccine against a pathogen or tumor cell expressing that immunogenic protein. An immunogenic protein or portion thereof may also function as a vaccine or antigen that induces or produces antibodies after vaccination due to the exceptionally potent antigenic properties of AAV or AAVLP containing up to 60 viral proteins, including the immunogenic protein or portion thereof.

[0054] The term “subunit vaccine” as used herein refers to a vaccine containing only a portion, rather than the whole, of a pathogen (virus, bacterium, or parasite), and can also be used in the context of tumor antigens. Because these vaccines contain only essential antigens and not all other molecules that make up the pathogen or tumor cell, they generally have fewer side effects. The pertussis component of the DTaP vaccine is an example of a subunit vaccine.

[0055] The term "domain" as used herein refers to a single folded protein structure having a tertiary structure, independent of the rest of the protein. Generally, domains are responsible for distinct functional properties of a protein and can often be attached to or transferred to other proteins without loss of function or immunogenicity.

[0056] The term “insert” as used herein means the insertion, rather than substitution, of at least one amino acid or nucleotide into a sequence of amino acids or nucleotides. In the context of the present invention, this term is used primarily in the context of amino acid sequences. An insert can be introduced between two amino acids or replace a sequence of amino acids, resulting in an extended amino acid sequence as a whole. According to the present invention, an insert has at least about 75 amino acids, preferably about 75 to 400 amino acids, and more preferably about 75 to 300 amino acids. The resulting protein is a chimeric protein having an insert of a different origin than VP. Thus, the “insert” in AAV or AAVLP according to the present invention is a protein or polypeptide of about 75 to 400 amino acids, more preferably about 75 to 300 amino acids, fused to the viral protein (VP) at the entry site where the N-terminus and C-terminus are located as specified herein. Linkers containing one or more amino acids can be flanked to one or both sides of the insert.

[0057] The term "transgene" refers to a gene that has been artificially introduced into the genome of another organism. Transgenes can also be called heterologous genes. In the case of AAV, the transgene is typically located between two ITRs in the genome, such as in a pTransgene plasmid.

[0058] In this specification, "protein containing a binding domain" means one binding unit of a binding pair (such as a protein containing an antigen-binding domain specific to a target antigen (e.g., an antibody-derived protein or antibody mimetic)). Possible binding domains are receptor-binding domains, ligand-binding domains, or antigen-binding domains (also called antigen-recognition domains). Thus, a protein containing a binding domain, i.e., one binding unit of a binding pair, determines the directivity of AAV or AAVLP to target cells expressing the binding partner for that protein containing a binding domain, i.e., the other binding unit of the binding pair (e.g., a receptor, ligand, or antigen) on its surface. Non-limiting examples of suitable binding pairs include antigen-binding domains and their antigens (e.g., single-domain antibodies (sdAb), single-strand variable fragments (scFv) and their antigens or antibody mimetic (e.g., antikalin and its antigen)), proteins containing receptor-binding domains and their receptors (e.g., coronavirus spike (S) protein and ACE receptor; antibody Fc region (e.g., scFc) and Fc receptor), and ligand-binding domains and their ligands (e.g., PD-1 and PD-L1).

[0059] The terms “directivity” or “cell-directivity” as used herein refer to the ability of a virus to deliver genes into a particular type of cell. Thus, AAVs or AAVLPs with varying directivity have the ability to deliver genes into different types of cells (e.g., different types of retinal cells). The directivity of AAVs or AAVLPs can be altered by recombinant techniques (genetic engineering), resulting in AAVs or AAVLPs that direct towards a modified target, i.e., AAVs or AAVLPs that are redirected to a specific type of cell other than the type that the native AAV serotype normally infects. While the directivity of AAVs has been altered by inserting small peptides into the surface-exposed position of the AAV capsid presented on the surface of AAV particles, according to the present invention, this change in directivity can be achieved by an insert consisting of approximately 75 to 400, preferably 75 to 300 amino acids in the viral protein, which is a protein containing a binding domain (such as an antibody-binding domain) in an antibody-derived protein (such as sdAb or single-stranded Fv or an antibody mimetic (such as antikalin)). AAV or AAVLP contains an insert consisting of a protein or a portion thereof within the viral protein.

[0060] The present invention relates to an adeno-associated virus (AAV) or adeno-associated virus-like particle (AAVLP) wherein an insert consisting of at least about 75 amino acids (about 75 to 400, preferably about 75 to 300 amino acids) in a capsid-forming viral protein (VP) is included at the insertion site (I) at the apex of the variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, and optionally, one or both sides of the insert are adjacent to a linker containing one or more amino acids preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof. The insert can be any protein or a portion thereof. In some embodiments, the insert is (a) an immunogenic protein or a portion thereof, and / or (b) a protein containing a binding domain. The insert inserted at the apex of VR-VIII and the insert inserted at the apex of VR-IV may be the same or different. In one embodiment, the first insert inserted at the apex of VR-VIII and the second insert inserted at the apex of VR-IV are different. In other embodiments, or additional embodiments, the AAV or AAVLP according to the present invention may also be formed by two or more viral proteins, each containing a different insert consisting of at least about 75 to 400 amino acids. The insert in each of the two or more viral proteins containing the insert is a protein or a portion thereof, and is preferably selected from the group consisting of (a) an immunogenic protein or a portion thereof and (b) a protein containing a binding domain. In a preferred embodiment, each of the capsid-forming viral proteins (VPs) contains an insert at the insertion site located at the apex of VR-VIII or VR-IV of the VP.

[0061] Accordingly, in one embodiment, the present invention relates to an adeno-associated virus (AAV) or adeno-associated virus-like particle (AAVLP) wherein an insert consisting of at least about 75 amino acids (about 75 to 400, preferably about 75 to 300 amino acids) in a capsid-forming viral protein (VP) is included at the insertion site (I) at the apex of the variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, the insert being an immunogenic protein or a portion thereof, and optionally flanking one or both sides of the insert is a linker containing one or more amino acids preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof. The immunogenic protein or portion thereof inserted at the apex of VR-VIII and the immunogenic protein or portion thereof inserted at the apex of VR-IV may be the same or different. However, "different" can mean immunogenic proteins or immunogenic moieties from different proteins, or different immunogenic moieties from the same protein. In the case of pathogens, immunogenic proteins or immunogenic moieties from different proteins originate from the same pathogen (e.g., the same bacterium, the same virus, or the same parasite). In one embodiment, the immunogenic protein or portion inserted at the top of VR-VIII and the immunogenic protein or portion inserted at the top of VR-IV are different (e.g., immunogenic proteins or immunogenic moieties from different proteins, or different immunogenic moieties from the same protein). In other embodiments, or additional embodiments, the AAV or AAVLP according to the present invention may also be formed by two or more viral proteins containing different inserts consisting of at least about 75 to 400 amino acids. Each of these different inserts is an immunogenic protein or portion thereof, and is either an immunogenic protein or immunogenic moiety from different proteins, or different immunogenic moieties from the same protein. In a preferred embodiment, each of the capsid-forming viral proteins (VPs) includes an insert at the insertion site located at the apex of VR-VIII or VR-IV of the VP.

[0062] The present invention also relates to adeno-associated virus (AAV) or adeno-associated virus-like particles (AAVLP), wherein an insert consisting of at least about 75 amino acids (about 75 to 400, preferably about 75 to 300 amino acids) in a capsid-forming viral protein (VP) is included at the insertion site (I) at the apex of variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, the insert being a protein containing a binding domain, and optionally flanking one or both sides of the insert is a linker containing one or more amino acids preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof. In some embodiments, the protein containing the binding domain is a protein containing a receptor-binding domain, a ligand-binding domain, or an antigen-binding domain, preferably a protein containing an antigen-binding domain. The binding domain-containing protein inserted at the apex of VR-VIII and the binding domain-containing protein inserted at the apex of VR-IV may be the same or different, where different means proteins containing binding domains with different binding specificities (e.g., different antigen binding specificities). Alternatively, only one of the inserts may be a binding domain-containing protein. In other embodiments, or additional embodiments, the AAV or AAVLP according to the present invention may also be formed by two or more viral proteins containing different inserts consisting of at least about 75 to 400 amino acids. Each of these different inserts may be a protein containing a binding domain with preferably different binding specificities, or at least one of these inserts may be a protein containing a binding domain. In a preferred embodiment, each of the capsid-forming viral proteins (VPs) contains an insert at the insertion site (i) at the apex of VR-VIII or VR-IV of the VP.

[0063] The VP3 region of AAV (and therefore VP1 and VP2 as well) contains a well-conserved region common to all serotypes: a core eight-stranded β-barrel (βB-βI) and a small α-helix. The loop regions inserted between the β-chains consist of a distinct HI loop between β-chain H and I, a DE loop between β-chain D and E, and nine variable regions (VRs) that form the apex of these loops. These VRs are found on the surface of the capsid and can be associated with specific functional roles in the AAV lifecycle, including receptor binding, gene delivery, and antigen specificity (Drouin and Agbandje-McKenna, Future Virol., 2013, 8(12):1183-1199).

[0064] At least 13 different human and non-human primate AAV serotypes (AAV1-AAV13) have been sequenced to date. Based on antigen activity and sequence comparison, AAVs are classified into six gene clusters (clades A-f) and two clonal isolates (AAV4 and AAV5). While AAV serotypes show only about 65-99% sequence agreement when compared, their structures are remarkably similar at 95-99% (percentage of superimposed Cα positions) (Drouin and Agbandje-McKenna, Future Virol., 2013, 8(12):1183-1199).

[0065] Due to the high degree of structural agreement, those skilled in the art can easily determine the apex of VR-VII or VR-IV with respect to the AAV VP1 sequence. Structural data and 3D diagrams of AAV proteins are available for most serotypes in the Protein Databank, an open-access database of three-dimensional structural data for large biomolecules (https: / / www.rcsb.org / ). For example, the structures of AAV 1, 2, 5, 8, and 9 can be found under the following ID numbers: 6ih9 (AAV2), 6jcr (AAV1), 6jct (AAV5), 2qa0 (AAV8), and 3ux1 (AAV9). Alternatively, the 3D structure of a given sequence can be generated using comparative structure modeling with a protein structure prediction service based on known structures, such as the Robetta protein structure prediction service (https: / / robetta.bakerlab.org / ), or a more recently available modeling service based on deep learning improvements to protein structure prediction from sequence information, such as the RoseTTAFold protein structure prediction service (https: / / robetta.bakerlab.org / ). The apex of VR-VIII and VR-IV can be identified by identifying the amino acids at the outermost tips of VR-VIII and VR-IV, and approximately 2-5 amino acids upstream and downstream of them.

[0066] The AAV or AAVLP according to the present invention can be derived from any AAV serotype. Non-limiting examples of AAVs include AAV-1, AAV-2, AAV-3, AAV-3B, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, rh10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and sheep AAV. "Primate AAV" means AAV that infects primates, "non-primate AAV" means AAV that infects non-primate mammals, "bovine AAV" means AAV that infects bovine mammals, and so on. The AAV or AAVLP is preferably derived from AAV serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), or 10 (AAV10), and more preferably from 1 (AAV1), 2 (AAV2), 8 (AAV8), or 9 (AAV9). The corresponding insertion sites of these AAVs can be moved from the specific insertion sites disclosed herein with respect to AAV1-AAV10 and / or their respective 3D structures, as described above.

[0067] The term “viral protein,” abbreviated as VP, refers herein to the viral proteins VP1, VP2, and VP3, which interact to form a capsid and are therefore AAV structural proteins. Viral proteins can also be called capsid proteins. The AAV capsid is an envelopeless icosahedral 60-mer with three repeat monomers VP1, VP2, and VP3 in a stoichiometric ratio of 1:1:10. This icosahedral capsid has a diameter of approximately 260 Å. The three structural proteins VP1, VP2, and VP3 are generated from a cap ORF containing a single cap gene using a P40 promoter, alternative splicing, and the use of an alternative non-standard ACG translation start codon for VP2, resulting in three separate protein products sharing the same C-terminus as VP3 (Figure 1A). VP3 alone, or a capsid consisting of VP1 and VP3, can be assembled and packaged together with the genome. However, particles consisting solely of VP3 are non-infectious because they lack the PLA2 domain encoded within the VP1-specific region of the cap ORF.

[0068] Those skilled in the art will see that specific insertion sites are provided that correspond to amino acid positions in the VP1 protein, having a given sequence and also specifying the insertion site in a sequence common to VP2 and VP3 (especially VP3). The start of VP3 corresponds to amino acid position M203 of VP1 for AAV1 (SEQ ID NO: 1), AAV2 (SEQ ID NO: 2), AAV3 (SEQ ID NO: 3), AAV6 (SEQ ID NO: 6), and AAV9 (SEQ ID NO: 9); to amino acid position M204 of VP1 for AAV8 (SEQ ID NO: 8) and AAV10 (SEQ ID NO: 10); to amino acid position M197 of VP1 for AAV4 (SEQ ID NO: 4); and to amino acid position M193 of VP1 for AAV5 (SEQ ID NO: 5). For AAV7, it is noted that the start of VP3 corresponds to position 204 of VP1 (SEQ ID NO: 7) (AAV7 VP3 uses an unusual GTG start codon, see paragraph

[29] of EP 1 456 419 B1). In this specification, AAV3 includes AAV3A and AAV3B. The amino acid sequence of SEQ ID NO: 3 provided for the AAV3 cap protein relates to AAV3B. However, AAV3A can be used similarly in the context of this invention, and those skilled in the art will be able to identify their respective integration sites.

[0069] The term “insertion site” as used herein means a position in an amino acid sequence defined by an amino acid position, and includes the possibility of introducing a polypeptide insert between two adjacent amino acids. However, those skilled in the art will see that a polypeptide insert may also be introduced between two amino acids that are not directly adjacent to each other, resulting in the substitution of short-chain amino acids (2, 3, 4, 5, or more amino acids, preferably 2, 3, or 4 amino acids) within the apex of VR-VIII or VR-IV. It is preferable that the insert be inserted between two adjacent amino acids. As used herein, insertion site (I) may be referred to, for example, I-587 or AAV2 I-587. This means the insertion site between two amino acids corresponding to amino acid positions 587 and 588 within the apex of VR-VIII, defined by their respective amino acid positions, i.e., between amino acid position 587 and the next amino acid.

[0070] In this specification, "adeno-associated virus," abbreviated as AAV, refers to the assembled capsid of a natural or recombinant AAV packaged with a DNA genome. The original (or natural) AAV is a single-stranded DNA parvovirus. In the context of this invention, AAV refers to recombinant AAV (rAAV), i.e., genetically modified AAV, which may include those containing a modified capsid protein and / or heterologous polynucleotide sequences. rAAV can be prepared as particles containing a single-stranded DNA (ssDNA) genome or a double-stranded DNA (dsDNA) genome. rAAV packaged with an ssDNA or dsDNA genome may also be called a complete AAV (or rAAV) particle. An ssDNA genome is characterized by two functional reverse end repeat (ITR) sequences adjacent to each other at the 5' (5' ITR) and 3' (3' ITR) positions, while a dsDNA genome is characterized by one functional ITR (located at either the 5' or 3' end) and a second mutated ITR with a deletion covering the terminal segregation site (trs), resulting in a double-stranded or self-complementary DNA genome (scDNA). Both single ssDNA and dsDNA genomes are referred to herein as ITR-adjacent genomes. Typically, such AAVs (or rAAVs) containing ITR-adjacent genomes are infectious, but as described above, in the case of a VP3-only capsid, AAVs (or rAAVs) may also be non-infectious.

[0071] AAVLP, abbreviated as “adeno-associated virus-like particle,” refers herein to an assembled capsid in which the ITR is not packaged together with the flanking genome (ssDNA genome or dsDNA genome). AAVLP can package several DNA molecules but does not contain a genome (whose genome (ssDNA or dsDNA) is characterized by a 5' ITR and a 3' ITR). Therefore, AAVLP is non-infectious. In the examples, AAVLP is constructed in the absence of a pTransgene plasmid having an expression cassette with a flanking ITR.

[0072] The term “reverse terminal repeat” (ITR) is used herein to include any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as a reverse terminal repeat (i.e., mediates a desired function (replication, viral packaging, incorporation, and / or proviral rescue)). An ITR can be an AAV ITR sequence or a non-AAV ITR sequence (a sequence of another parvovirus (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19)), or any other suitable viral sequence. For example, an SV40 hairpin that functions as an origin for SV40 replication can be used as an ITR. ITR sequences can be further modified by cleavage, substitution, deletion, insertion, and / or addition. Furthermore, an ITR can be partially or entirely synthetic (e.g., a “double D sequence”), as described in United States Patent No. 5,478,745. An "AAV terminal repeat" or "AAV ITR" can come from any AAV, and non-limiting examples include serotypes 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, or any other AAV. An AAV terminal repeat does not need to have a native terminal repeat sequence, as long as the terminal repeat mediates the desired function (e.g., replication, viral packaging, integration, and / or proviral rescue) to a region of the AAV genome of approximately 145 nucleotides in length that contains all the elements involved in genome rescue, replication, and packaging (for example, native AAV ITR sequences may be altered by insertions, deletions, cleavage, and / or missense mutations). Thus, ITR, as used herein, means at least the cis elements required for genome packaging.

[0073] In this specification, the term “genome” as used in the context of AAV means a DNA sequence containing 5' and 3' reverse end repeats (ITRs). Typically, a genome is a single-stranded DNA genome (ssDNA genome). However, with mutated ITRs, a genome can also be a packaged double-stranded DNA genome (dsDNA genome) or a self-complementary DNA genome. In nature, an AAV genome contains rep genes and cap genes. In the context of recombinant AAV, a genome often contains ITRs encoding adjacent transgenes (including expression cassettes encoding transgenes), and the rep and cap genes are provided in trans. In this specification, a genome may contain coding sequences (transgenes, or rep genes and / or cap genes). Alternatively, a genome may contain non-coding sequences with immunostimulatory effects (such as CpG motifs). Furthermore, ssDNA itself can have immunostimulatory effects.

[0074] AAV capsids are known to allow the insertion of small peptides at specific surface exposure locations without losing structural integrity or major function. The most commonly used insertion sites in AAV2 are I-587 (e.g., insertion between the amino acid residues asparagine (N) 587 and arginine (R) 588 in AAV2 VP1) and I-453 (e.g., insertion between the amino acid residues glycine (G) 453 and threonine (T) 454 in AAV2 VP1). Such manipulations of AAV capsids have been explored to alter the targeting of AAVs and redirect them to specific types of cells different from those normally infected by native AAV serotypes (Buning, H and Srivastava, A., Mol Ther Methods Clin Dev., 2019, 12: 248-265).

[0075] This invention extends these previous applications by inserting a large immunogenic protein or a portion thereof (such as the major antigenic portion of the infection medium, or the immunogenic domain or a portion thereof) containing approximately 75 or more amino acids (e.g., 75-400 amino acids, or 75-300 amino acids) into the surface-exposed position of the AAV capsid, thereby repurposing the AAV vector as a vaccine. In particular, adeno-associated virus (AAV) or adeno-associated virus-like particle (AAVLP) is converted into a carrier vehicle for immunogenic amino acid sequences of varying lengths encoded within the capsid VP sequence, and thus into a carrier for a subunit vaccine. By thus inserting into a common portion shared by all three VPs (VP1, VP2, and VP3), for example I-587 or I-453, or any other insertable surface-exposed position, the immunogenic protein or a portion thereof is inserted into each of the 60 building blocks of the AAV capsid and thus presented 60 times on the surface of a single AAV particle. As demonstrated herein, the surface exposure sites that surprisingly allow for large insertions are located at the apex of variable region VIII (VR-VIII) and / or variable region IV (VR-IV). When inserting simultaneously into two insertion sites (i.e., one at the apex of VR-VIII and one at the apex of VR-IV (e.g., I-587 and I-453, respectively, of AAV2)), the immunogenic protein or a portion thereof is presented 120 times on the surface of a single AAV particle. Alternatively, when inserting one immunogenic protein or a portion thereof into the insertion site at the apex of VR-VIII and another immunogenic protein or a portion thereof into the insertion site at the apex of VR-IV, these two immunogenic proteins or portions thereof are presented 60 times each on the surface of a single AAV particle. It is preferable that each VP contains an insert at the apex of VR-VIII or VR-IV. When using two or more viral proteins containing different inserts, different insertion sites within each viral protein can be used.Insertion of AAV2 into I-587 disrupts the innate heparan sulfate proteoglycan (HSPG) binding site that defines AAV2's directivity (Opie et al., J Virol., 2003, 77:6995-7006; Kern et al., J Virol., 2003, 77:11072-11081). Therefore, inserts in AAV or AAVLP according to the present invention may also alter the directivity of virions (Figures 4 and 5), facilitating optimal exposure of the inserted protein or a portion thereof according to its sequence-determined biological characteristics, thereby potentially facilitating the induction of a stronger immune response, for example, to immunogenic sequences.

[0076] The present invention further extends these previous applications by inserting a protein containing approximately 75 or more amino acids (e.g., 75-400 amino acids, or 75-300 amino acids) (here, the protein can be any protein, particularly a protein containing a binding domain (such as an antigen-binding domain) (e.g., sdAb or scFv or an antibody mimetic (such as antikalin))) into the surface-exposed position of the AAV capsid, thereby directing the AAV vector to a modified target. In particular, adeno-associated virus (AAV) or adeno-associated virus-like particles (AAVLP), together with the protein containing the binding domain encoded within the capsid VP sequence, are converted into a vehicle with altered cell-targeting properties, and thus, for example, a vehicle for gene therapy. By inserting the binding domain into, for example, I-587 or I-453, or any other arbitrary insertion-permissible surface exposure site within the common region shared by all three VPs (VP1, VP2, and VP3), the protein containing the binding domain is inserted into each of the 60 building blocks of the AAV capsid and thus presented 60 times on the surface of a single AAV particle. As demonstrated herein, the surface exposure sites that remarkably tolerate large insertions are located at the apex of variable region VIII (VR-VIII) and / or variable region IV (VR-IV). When inserting into two insertion sites simultaneously (i.e., one at the apex of VR-VIII and the other at the apex of VR-IV (e.g., I-587 and I-453, respectively, of AAV2)), the immunogenic protein containing the binding domain is presented 120 times on the surface of a single AAV particle. Alternatively, if one protein (such as a first protein containing a binding domain (e.g., a first sdAb or scFv)) is inserted into the apical insertion site of VR-VIII and another protein (such as a second protein containing a binding domain (e.g., a second sdAb or scFv)) is inserted into the apical insertion site of VR-IV) simultaneously, these two proteins will each be presented 60 times on the surface of a single AAV particle. It is preferable that each VP contains an insert at the apex of either VR-VIII or VR-IV.As described above, the AAV or AAVLP according to the present invention may also be formed by two or more viral proteins containing different inserts consisting of at least about 75 to 300 amino acids, in which case the first insert may be a first protein (e.g., a first sdAb or scFv) containing a binding domain (e.g., an antigen-binding domain), and the second insert may be a further protein containing a binding domain (e.g., a further protein containing an antigen-binding domain (e.g., a second sdAb or scFv)). Alternatively, the second protein may be an immunogenic protein or a part thereof. Those skilled in the art will further see that the insert may be a protein (i.e., a bispecific protein) containing two or more binding domains located at the apex of VR-VIII and / or VR-IV, preferably at the apex of VR-VIII or VR-IV. These embodiments can be further combined with embodiments in which the insert is an immunogenic protein or a part thereof (e.g., a viral protein (e.g., coronavirus spike (S) protein or a part thereof), or a tumor antigen). Those skilled in the art will further see that, in certain embodiments, a protein containing a binding domain is also an immunogenic protein or part thereof, and vice versa. For example, the receptor-binding domain of a viral entry protein (such as the receptor-binding domain of the coronavirus spike (S) protein) is an immunogenic protein or part thereof, in addition to the protein containing the binding domain. When using two or more viral proteins (VPs) containing different inserts, different insertion sites of each viral protein can be utilized.

[0077] In one embodiment, the top of VR-VIII corresponds to approximately amino acids 585-592 (I-585-I-592) of VP1 AAV1 having the amino acid sequence of SEQ ID NO: 1, VP1 AAV2 having the amino acid sequence of SEQ ID NO: 2, VP1 AAV3 having the amino acid sequence of SEQ ID NO: 3, VP1 AAV6 having the amino acid sequence of SEQ ID NO: 6, VP1 AAV7 having the amino acid sequence of SEQ ID NO: 7, VP1 AAV8 having the amino acid sequence of SEQ ID NO: 8, VP1 AAV9 having the amino acid sequence of SEQ ID NO: 9, or VP1 AAV10 having the amino acid sequence of SEQ ID NO: 10; or approximately amino acids 583-589 (I-583-I-589) of VP1 AAV4 having the amino acid sequence of SEQ ID NO: 4; or approximately amino acids 574-580 (I-574-I-580) of VP1 AAV5 having the amino acid sequence of SEQ ID NO: 5. Alternatively, the apex of VR-VIII can be defined as the eight downstream amino acids of the conserved glutamine corresponding to Q584 of VP1 AAV2, which has the amino acid sequence of SEQ ID NO: 2.

[0078] The top of VR-IV corresponds to approximately amino acids 450-460 (I-450-I-460) of VP1 AAV1 with the amino acid sequence of SEQ ID NO: 1, VP1 AAV2 with the amino acid sequence of SEQ ID NO: 2, VP1 AAV3 with the amino acid sequence of SEQ ID NO: 3, VP1 AAV6 with the amino acid sequence of SEQ ID NO: 6, VP1 AAV7 with the amino acid sequence of SEQ ID NO: 7, VP1 AAV8 with the amino acid sequence of SEQ ID NO: 8, VP1 AAV9 with the amino acid sequence of SEQ ID NO: 9, or VP1 AAV10 with the amino acid sequence of SEQ ID NO: 10; or approximately amino acids 445-455 (I-445-I-455) of VP1 AAV4 with the amino acid sequence of SEQ ID NO: 4; or approximately amino acids 439-449 (I-439-I-449) of VP1 AAV5 with the amino acid sequence of SEQ ID NO: 5. Alternatively, the apex of VR-IV can be defined as the 12 to 5 amino acids upstream of the conserved phenylalanine corresponding to F462 of VP1 AAV2, which has the amino acid sequence of SEQ ID NO: 2.

[0079] For AAV1 to AAV10, suitable insertion sites at the apex of VR-VIII and / or VR-IV are further disclosed in Table 1 (VR-VIII) and Table 2 (VR-IV) below. [Table 1] [Table 2]

[0080] AAV or AAVLP is preferably derived from AAV serotype 1 (AAV1), 2 (AAV2), 3 (AAV3), 4 (AAV4), 5 (AAV5), 6 (AAV6), 7 (AAV7), 8 (AAV8), 9 (AAV9), or 10 (AAV10), and more preferably from 1 (AAV1), 2 (AAV2), 8 (AAV8), or 9 (AAV9). In one embodiment, AAV or AAVLP is derived from AAV2, and the insertion site is between two amino acids corresponding to amino acid positions 587 and 588 of AAV2 VP1 having the amino acid sequence of SEQ ID NO: 2 (AAV2 I-587), or between 588 and 589 (AAV2 I-588), and / or between 453 and 454 (AAV2 I-453), between 454 and 455 (AAV2 I-454), or between 455 and 456 (AAV2 I-455), preferably AAV2 I-587 or AAV2 I-588 or AAV2 I-453, more preferably AAV2 I-587 or AAV2 I-588. In another embodiment, AAV or AAVLP is derived from AAV1, and the insertion site is between two amino acids corresponding to amino acid positions 587 and 588 of AAV1 VP1 having the amino acid sequence of SEQ ID NO: 1 (AAV1 I-587), between 588 and 589 (AAV1 I-588), or between 589 and 590 (AAV1 I-589), and / or between 454 and 455 (AAV1 I-454), between 455 and 456 (AAV1 I-455), or between 456 and 457 (AAV1 I-456). In another embodiment, AAV or AAVLP is derived from AAV8, and the insertion site is between two amino acids corresponding to amino acid positions 588 and 589 of AAV8 VP1 having the amino acid sequence of SEQ ID NO: 8 (AAV8 I-588), or between 589 and 590 (AAV8 I-589), and / or between 455 and 456 (I-455), between 456 and 457 (I-456), or between 457 and 458 (I-457).In yet another embodiment, AAV or AAVLP is derived from AAV9, and the insertion site is between two amino acids corresponding to amino acid positions 588 and 589 of AAV9 VP1 in the amino acid sequence of SEQ ID NO: 9 (AAV9 I-588), or between 589 and 590 (AAV9 I-589), and / or between 454 and 455 (I-454), between 455 and 456 (I-455), or between 456 and 457 (I-456).

[0081] In one embodiment, AAV or AAVLP contains an insert in the viral protein consisting of about 75 to 400 amino acids, preferably about 75 to 350 amino acids, about 75 to 300 amino acids, about 75 to 260 amino acids, about 75 to 250 amino acids, or about 80 to 220 amino acids.

[0082] In some embodiments, the AAV or AAVLP has approximately 75-390, 75-380, 75-370, 75-360, 75-350, 75-340, 75-330, 75-320, 75-310, 75-300, 75-290, 75-280, 75-270, 75-260, 75-250, 75-240, 75-230, and 75-220 A The insert contains amino acids, preferably approximately 80-390, 80-380, 80-370, 80-360, 80-350, 80-340, 80-330, 80-320, 80-310, 80-300, 80-290, 80-280, 80-270, 80-260, 80-250, 80-240, 80-230, and 80-220 amino acids. In one embodiment, the insert contains approximately 90-390, 90-380, 90-370, 90-360, 90-350, 90-340, 90-330, 90-320, 90-310, 90-300, 90-290, 90-280, 90-270, 90-260, 90-250, 90-240, 90-230, and 90-220 amino acids, preferably. It has approximately 100-390, 100-380, 100-370, 100-360, 100-350, 100-340, 100-330, 100-320, 100-310, 100-300, 100-290, 100-280, 100-270, 100-260, 100-250, 100-240, 100-230, and 100-220 amino acids. Most preferably, it has approximately 75-300 amino acids, 75-260 amino acids, approximately 75-250 amino acids, or approximately 80-220 amino acids.

[0083] The insert may be flanked on one or both sides by linkers containing one or more amino acids selected from the group consisting of A(Ala), G(Gly), S(Ser), T(Thr), L(Leu), and combinations thereof. Each amino acid of the linker is independently selected from the group consisting of A(Ala), G(Gly), S(Ser), T(Thr), L(Leu), and combinations thereof, preferably selected from the group consisting of A(Ala), G(Gly), and S(Ser). Those skilled in the art will see that the linker contains small amino acids, if present. In one preferred embodiment, the linker contains A and / or G, and preferably the linker consists of A and / or G. The insert independently contains linkers on the N-terminal and / or C-terminal sides of the insert. Therefore, in one embodiment, the linker contains about 1 to 7 amino acids, preferably about 1 to 3 amino acids, more preferably about 3 amino acids, on the N-terminal side of the immunogenic protein or a portion thereof, or a protein containing a binding domain, and / or about 1 to 7 amino acids, preferably about 1 to 3 amino acids, more preferably about 2 to 3 amino acids, on the C-terminal side. In one embodiment, the linker contains about 1 to 7 amino acids on the N-terminal side of the insert, and / or about 1 to 7 amino acids on the C-terminal side. In a further embodiment, the linker contains about 2 to 3 amino acids on the N-terminal side, and / or about 2 to 3 amino acids on the C-terminal side. In this context, the term "about" means ±1 amino acid. In a special embodiment, the linker contains 1, 2, 3, 4, 5, 6, or 7 amino acids on the N-terminal side of the insert, and / or 1, 2, 3, 4, 5, 6, or 7 amino acids on the C-terminal side, or any combination thereof.

[0084] In some embodiments, AAV or AAVLP is AAV and includes a genome flanked by an ITR. In some embodiments, the AAV of the present invention includes a genome flanked by an ITR and is preferably infectious. The genome flanked by an ITR may contain a transgene. For example, the genome flanked by an ITR may contain a transgene encoding an immunogenic protein or a portion thereof. In particular, if the insert is an immunogenic protein or a portion thereof, the transgene may encode a further immunogenic protein or a portion thereof. Alternatively, the genome flanked by an ITR may contain a cap gene encoding a viral protein (VP) (including an insert of about 75 to 400 amino acids at the insertion site (I) at the apex of variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP). The genome may also contain non-coding sequences (such as CpG motifs) that have immunostimulatory effects. Furthermore, ssDNA itself may have immunostimulatory effects. In other embodiments, AAV or AAVLP is AAVLP and does not include a genome flanked by an ITR. Those skilled in the art will see that AAVLP is not infectious. Possible AAV VPs forming the capsid are VP1, VP2, and VP3, preferably in a ratio of 1:1:10. The capsid can also be formed by VP1 and VP3 alone, or by VP3 alone. Therefore, VP1 and VP3, or VP3 alone, are possible AAV VPs forming the capsid. However, particles consisting only of VP3 are non-infectious because they lack the PLA2 domain encoded within the VP1-only region of the cap ORF. In any variant, it is preferable that the AAV or AAVLP has a capsid consisting of approximately 60 VPs.

[0085] The AAV or AAVLP according to the present invention (for embodiments in which the insert is an immunogenic protein or a portion thereof) is immunogenic to the inserted immunogenic protein or a portion thereof. Any immunogenic protein and its immunogenic portion can be the immunogenic protein or a portion thereof. In some embodiments, the immunogenic protein or a portion thereof can be a viral, bacterial, or parasitic protein or a portion thereof. However, the present invention also encompasses embodiments in which the immunogenic protein is a mammalian (particularly human) protein or a portion thereof. Such AAV or AAVLP can be used to induce antibody production against mammalian or human antigens or epitopes (particularly target antigens that are difficult to induce antibody production against).

[0086] Non-limiting examples of appropriate representative immunogenic proteins originating from viruses, bacteria, or parasites include, for tuberculosis (Mycobacterium tuberculosis), e.g., fatty acid synthase fas; galactofuranosyltransferase glfT2, or isoniazid-inducible gene protein iniB; for influenza (influenza viruses A, B, or C, including subtypes), e.g., hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP); for dengue fever (dengue virus; DENV1-4), e.g., envelope protein E, especially ectodomain III of E (EDIII); for yellow fever (YFV), e.g., envelope protein E, especially ectodomain III of E (EDIII); for West Nile fever (WNV), For example, envelope protein E, especially ectodomain III of E (EDIII); for congenital Zika syndrome (Zika virus (ZIKV)), for example, envelope protein E, especially ectodomain III of E (EDIII); for malaria (Platyplein malariae), for example, perisporozoite proteins (CSP), erythrocyte membrane protein 1 (PfEMP1), apical membrane antigen 1 (AMA1), merozoite surface protein 1 (MSP1), merozoite surface protein 2 (MSP2), erythrocyte-binding antigen-175 (EBA175), thrombispondin-associated anonymous proteins (TRAP), hepatocyte phase antigens 1 and 3 (LSA1-LSA3), PfROM1, PfROM3, PfROM4, and PfROM6; for AIDS (HIV), for example, env gp160, nef p27, gag p55, or pol; for pertussis (Bordetella pertussis), e.g., pertussis toxin (PT), filamentous hemagglutinin (FHA), partactin (PRN), and cilia (FIM 2 / 3); for pneumonia: respiratory polynuclear virus (RSV), e.g., fusion (F) glycoprotein; and for toxoplasmosis encephalitis (Toxoplasma), e.g., apical membrane antigen 1 (AMA1); enolase 2 (ENO2); high-density granular proteins GRA1, GRA2, GRA4, GRA6, GRA8, GRA14, GRA15, GRA10, GRA12, GRA16, and GRA24; heat shock protein HSP70; microname proteins MIC1, MIC3, MIC4, MIC5, MIC13;These include rhomboid proteases ROM1, ROM4, and ROM5; Loptory proteins ROP2, ROP5, ROP16, ROP17, ROP18, and ROP38; Loptory-neck proteins RON2, RON4, and RON5; and surface antigen proteins SAG1, SAG3, and SAG5D.

[0087] Those skilled in the art will understand that, in the case of viral proteins, an immunogenic protein or a portion thereof is not an AAV protein or a portion thereof. Therefore, a heterologous viral protein or a portion thereof is possible as an immunogenic protein or a portion thereof. The term "heterologous" as used herein means that the protein or protein fragment is from a different host organism / virus.

[0088] In one embodiment, the immunogenic protein or a portion thereof is a coronavirus protein or a portion thereof. Suitable coronavirus proteins include the coronavirus spike (S) protein or a portion thereof (for SARS-CoV-2, etc., having the amino acid sequence of SEQ ID NO: 15), the coronavirus envelope protein (E protein) or a portion thereof (for SARS-CoV-2, etc., having the amino acid sequence of SEQ ID NO: 52), the membrane glycoprotein (M protein) or a portion thereof (for SARS-CoV-2, etc., having the amino acid sequence of SEQ ID NO: 53), the nucleocapsidrin protein (N protein) or a portion thereof (for SARS-CoV-2, etc., having the amino acid sequence of SEQ ID NO: 51), or the ORF1ab polypeptide (replicase complex) or (for SARS-CoV-2, etc., having the amino acid sequence of SEQ ID NO: 68). It is a part thereof, preferably a part of the S protein (particularly a portion having any one amino acid sequence of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69), a part of the E protein, M protein, or N protein (particularly amino acids 179-419 or 212-411 of SEQ ID NO: 51), or a part of the ORF1ab polyprotein (replicase complex) (particularly a portion having any one amino acid sequence of SEQ ID NOs: 62, 63, 64, 65, 66, or 67, or any portion of 75-300 amino acids of SEQ ID NO: 68, which includes at least one sequence of SEQ ID NOs: 54, 55, 56, 57, 58, 59, 60, or 61). The immunogenic protein is preferably a portion of the coronavirus S protein that includes the S1 domain, S2 domain, or receptor-binding domain, more preferably a portion that includes the coronavirus S protein receptor-binding domain (RBD). It is more preferable that the immunogenic protein is derived from SARS-CoV-2 and is a portion of the SARS-CoV-2 protein (such as a portion containing the RBD (amino acids 319-529 of SEQ ID NO: 15) or a portion thereof).The RBD contains a core and receptor-binding motif (RBM; amino acids 437-507 of SEQ ID NO: 15) (Shang et al, Nature, 2020, 581(7807): 221-224 and Supplement). Since an additional T cell epitope has been identified as being between amino acids 300 and 333, the immunogenic portion of the SARS-CoV-2 protein can have amino acids 300-507 of SEQ ID NO: 15 (SEQ ID NO: 38) or amino acids 300-505 of SEQ ID NO: 15 (SEQ ID NO: 69). Therefore, in some embodiments, the immunogenic protein or a portion thereof is a portion of the SARS-CoV-2 protein containing the RBM (preferably the amino acid sequence of SEQ ID NO: 11, 12, 36, 37, 38, or 69). In one embodiment, a portion of the SARS-CoV S protein contains the amino acid sequence of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69 (preferably the amino acid sequence of SEQ ID NOs: 11, 12, 34, 35, 36, 37, 38, 42, or 69), or contains an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69 (preferably the amino acid sequence of SEQ ID NOs: 11, 12, 34, 35, 36, 37, 38, 42, or 69). In one embodiment, the immunogenic portion of the SARS-CoV S protein contains an amino acid sequence that is at least about 95%, at least about 98%, at least about 99%, preferably 100%, identical to the amino acid sequence of SEQ ID NOs. 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69 (preferably the amino acid sequence of 11, 12, 34, 35, 36, 37, 38, 42, or 69). For example, a protein having the amino acid sequence of SEQ ID NOs. 69 contains amino acids 1 to 206 of SEQ ID NOs. 38, and therefore its sequence is about 99% identical to SEQ ID NOs. 38.In one embodiment, SARS-CoV The immunogenic portion of the S protein contains an amino acid sequence consisting of at least 75 amino acids of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69 (preferably 11, 12, 34, 35, 36, 37, 38, 42, or 69), or contains an amino acid sequence that is at least about 90% identical to at least 75 amino acids of the amino acid sequence of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69 (preferably 11, 12, 34, 35, 36, 37, 38, 42, or 69). In one embodiment, the immunogenic portion of the SARS-CoV S protein comprises at least about 95%, at least about 98%, at least about 99%, preferably 100%, of at least 75, at least 80, at least 100, at least 125, at least 150, at least 190, or at least 195 amino acids from sequence numbers 11, 12, 31, 32, 33, 34, 35, 36, 37, 48, 49, 50, or 69 (preferably 11, 12, 34, 35, 36, 37, 38, 42, or 69). In one embodiment, the immunogenic portion of the SARS-CoV S protein comprises an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, preferably 100%, of at least 200, at least 225, or at least 253 amino acids from the amino acid sequence of SEQ ID NO: 12; or an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, preferably 100%, of at least 175, at least 190, or at least 196 amino acids from the amino acid sequence of SEQ ID NO: 11; or an amino acid sequence having at least about 95%, at least about 98%, at least about 99%, preferably 100%, of at least 175, at least 200, or at least 205 amino acids from the amino acid sequence of SEQ ID NO: 69.

[0089] In one embodiment, the AAV or AAVLP according to the present invention includes an insert consisting of approximately 75 to 400 amino acids (preferably 75 to 300 amino acids) in a capsid-forming viral protein (VP) at insertion sites (I) located at the apex of variable region VIII (VR-VIII) and variable region IV (VR-IV), wherein the insert is an immunogenic protein or a portion thereof, and the immunogenic protein or portion inserted at the apex of variable region VIII and the immunogenic protein or portion inserted at the apex of variable region IV may be the same or different, where different means different immunogenic proteins or immunogenic moieties from different proteins, or different immunogenic moieties from the same protein. In the case of pathogens, it is preferable that the immunogenic proteins or immunogenic moieties from different proteins originate from the same pathogen (e.g., the same bacterium, the same virus, or the same parasite). In one embodiment, the immunogenic protein or portion thereof inserted at the apex of VR-VIII and the immunogenic protein or portion thereof inserted at the apex of VR-IV are different (e.g., an immunogenic protein or immunogenic moiety from a different protein, or different immunogenic moieties from the same protein). Thus, the AAV or AAVLP may include a first insert, which is a first immunogenic protein or portion thereof, at the insertion site at the apex of VR-VIII, and a second (or further) insert, which is a second immunogenic protein or portion thereof, at the insertion site at the apex of VR-IV. In other embodiments or additional embodiments, the AAV or AAVLP according to the present invention may also be formed by two or more (preferably two) viral proteins, each containing different inserts consisting of at least about 75 to 400 amino acids (preferably at least about 75 to 300 amino acids), where each of these different inserts is an immunogenic protein or portion thereof, and is either an immunogenic protein or immunogenic moiety from a different protein, or different immunogenic moieties from the same protein.Therefore, an AAV or AAVLP is formed by two or more (preferably two) viral proteins, each containing a first and second (or further) insert consisting of at least about 75 to 300 amino acids, located at the same or different insertion sites (where the first insert is a first immunogenic protein or a portion thereof, and the second (or further) insert is a second immunogenic protein or a portion thereof). In yet another embodiment, the AAV includes a genome in which the ITR is adjacent and contains a transgene encoding a further immunogenic protein or a portion thereof. That immunogenic protein or a portion thereof may be the same as or different from the immunogenic protein or a portion thereof inserted at the insertion site at the apex of VR-VIII and / or VR-IV. Also in this context, different means different immunogenic proteins or immunogenic portions from different proteins, or different immunogenic portions from the same protein. These embodiments can be combined, and thus AAV can be formed by two or more viral proteins comprising an immunogenic protein or a portion thereof inserted at the apical positions of VR-VIII and VR-IV, and / or a genome encoding a different immunogenic protein or a portion thereof, and / or a different insert. These embodiments can further be combined with embodiments in which the insert is a protein (such as an antibody or antibody fragment) containing a binding domain.

[0090] For all the embodiments described above, an immunogenic protein or part thereof may be a coronavirus protein or a part thereof. Suitable coronavirus proteins include the coronavirus spike (S) protein or a part thereof (for SARS-CoV-2, etc., having the amino acid sequence of SEQ ID NO: 15), the coronavirus envelope protein (E protein) or a part thereof (for SARS-CoV-2, etc., having the amino acid sequence of SEQ ID NO: 52), the membrane glycoprotein (M protein) or a part thereof (for SARS-CoV-2, etc., having the amino acid sequence of SEQ ID NO: 53), the nucleocapsidrin protein (N protein) or a part thereof (for SARS-CoV-2, etc., having the amino acid sequence of SEQ ID NO: 51), or the ORF1ab polypeptide (replicase complex) or a part thereof (for SARS-CoV-2, etc., having the amino acid sequence of SEQ ID NO: 68). In one embodiment, the selection of one or more (or first and further) immunogenic proteins or a portion thereof is a portion of the S protein (particularly a portion having any one amino acid sequence of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69, preferably a portion having any one amino acid sequence of SEQ ID NOs: 11, 12, 34, 35, 36, 37, 38, 42, or 69), the E protein, the M protein, the N protein It can consist of a portion of the protein (particularly amino acids 179-419 or 212-411 of SEQ ID NO: 51), a portion of the ORF1ab polyprotein (replicase complex) (particularly a portion having any one amino acid sequence of SEQ ID NOs. 62, 63, 64, 65, 66, or 67, or any portion of the 75-300 amino acids of SEQ ID NO: 68 that includes at least one sequence of SEQ ID NOs. 54, 55, 56, 57, 58, 59, 60, or 61), or any one combination thereof. If AAV or AAVLP contains two or more immunogenic proteins or portions thereof (inserted within a capsid, or inserted within a capsid and encoded by the genome), it is preferable that the first and further immunogenic proteins or portions thereof are different.However, "different" means different immunogenic proteins or immunogenic moieties from different proteins, or different immunogenic moieties from the same protein.

[0091] Preferably, the immunogenic protein is a part of the coronavirus S protein and includes an S1 domain, an S2 domain, or a receptor-binding domain, preferably a coronavirus S protein receptor-binding domain (RBD) and / or receptor-binding motif (RBM). More preferably, the immunogenic protein is derived from SARS-CoV-2 and is a part of the SARS-CoV-2 protein and includes the RBD and / or RBM. In one embodiment, a portion of the SARS-CoV S protein contains an amino acid sequence that is at least about 90% identical to the amino acid sequences of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 69, or a combination thereof (preferably 11, 12, 34, 35, 36, 37, 38, 42, or 69), or an amino acid sequence that is at least about 90% identical to the amino acid sequences of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 69, or a combination thereof (preferably 11, 12, 34, 35, 36, 37, 38, 42, or 69).

[0092] Various S proteins of SARS-CoV-2 can be obtained from GenBank, for example, GenBank accession numbers (protein IDs): MN_908947 (QHD434616.1), MN_988668 (QHQ62107.1), NC_045512 (YP_009724390.1), MN_938384.1 (QHN73795.1), MN Examples include _975262.1(QHN73810.1), MN_985325.1(QHQ60594.1), MN_988713.1(QHQ62877.1), MN_994467.1(QHQ71963.1), MN_994468.1(QHQ71973.1), and MN_997409.1(QHQ82464.1), which show 100% sequence agreement. However, slight variations have been previously reported in the SARS-CoV-2 S protein. For example, the following substitutions have been described by Wrapp et al. (Science, 2020, 367: 1260-1263) in clinical isolates F32I, H49Y, S247R, N354D, D364Y, V367F, D614G, V1129L, and E1262G. Furthermore, substitutions H49Y and V860Q have been reported by Wang et al. (J. Med. Virol. March 13, 2020: 1-8). Further homology analysis of publicly available SARS-CoV-2 sequences by the same authors revealed that the nucleotide homology of the S protein is 99.82% to 100%, and the amino acid homology of the S protein is 99.53% to 100%. However, further substitutions have been identified and are likely to be identified in the future (e.g., N349K and E484K). As multiple SARS-CoV-2 cell lineages, particularly concerning variants, continue to emerge, are being monitored and sequenced, those skilled in the art know how to access the latest sequences that have been identified or assigned for each cell lineage or variant.

[0093] In the context of this invention, the expression "sequence matches at least 90%" means a protein that has at least 90% of a particular amino acid sequence and therefore may differ by less than 10% from the amino acid sequence of a reference sequence (such as the amino acid sequence of SEQ ID NOs. 11, 12, or 69) and / or the nucleic acid sequence encoding the amino acid sequence, and sequence matching can be readily determined by sequence alignment. For example, a variant protein or part of a variant protein from an S protein can be of natural origin (e.g., a variant version or variation of a part of the SARS-CoV-2 S protein with the amino acid sequence of SEQ ID NOs. 11, 12, or 69), or an engineered protein (e.g., an engineered glycoprotein derivative modified by site-directed mutagenesis or cloning), or a combination of these. Codon utilization is known to differ between species. Therefore, when expressing a nucleic acid sequence in target cells, it is necessary, or at least potentially useful, to adapt the nucleic acid sequence to the codon utilization of the target cells. Methods for designing and constructing derivatives of a given protein are well known to those skilled in the art. Adapting a nucleic acid sequence to the codon utilization of a target cell is also known as codon optimization.

[0094] Another SARS-CoV-2 protein or a portion thereof, preferably a SARS-CoV-2 N protein or a portion thereof, may also be used as an immunogenic protein or part thereof. In a preferred embodiment, the SARS-CoV-2 N protein or a portion thereof contains 75 to 400 or 75 to 300 amino acids of the sequence of SEQ ID NO: 51, or a sequence having a sequence that is at least 95% identical to 75 to 400 or 75 to 300 amino acids of SEQ ID NO: 51. Preferably, the portion of the SARS-CoV-2 N protein contains amino acid amino acids 179 to 419 or 212 to 411 of amino acid SEQ ID NO: 51, or a sequence having a sequence that is at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to amino acid amino acids 179 to 419 or 212 to 411 of the sequence of SEQ ID NO: 51. In one embodiment, a portion of the SARS-CoV-2 N protein has an amino acid sequence that is at least 98% to 100% identical to the sequence of amino acids 179-419 or 212-411 of SEQ ID NO: 51. In one embodiment, the AAV or AAVLP according to the present invention is an AAV, and the immunogenic protein or a portion thereof is a coronavirus protein or a portion thereof (coronavirus spike (S) protein or a portion thereof, or coronavirus E protein, M protein, or N protein, or a portion thereof), and the AAV further comprises a genome containing a transgene flanked by the ITR that encodes a further immunogenic protein or a portion thereof (wherein the further immunogenic protein or a portion thereof is selected from the group consisting of a portion of the coronavirus S protein, E protein, M protein, or N protein). In one embodiment, the further immunogenic protein or a portion thereof encoded by the genome flanked by the ITR is different from the immunogenic protein or a portion thereof inserted into the VP of the AAV according to the present invention (wherein different means different proteins or different portions of the same protein).In one embodiment, the immunogenic protein is a portion of the coronavirus S protein containing a receptor-binding domain selected from a portion of the S protein (particularly a portion containing any one amino acid sequence of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69), a portion of the E protein, M protein, or N protein (particularly containing amino acids 179-419 or 212-411 of SEQ ID NOs: 51), or a portion of the ORF1ab polyprotein (replicase complex) (particularly any portion containing any one amino acid sequence of SEQ ID NOs: 62, 63, 64, 65, 66, or 67, or any portion containing at least one sequence of SEQ ID NOs: 54, 55, 56, 57, 58, 59, 60, or 61 among the 75-300 amino acids of SEQ ID NOs: 68). In one embodiment, the AAV or AAVLP according to the present invention is an AAV, and the immunogenic protein or a portion thereof is a portion of a coronavirus spike (S) protein comprising an amino acid sequence selected from the group consisting of portions having the amino acid sequences of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50, and the AAV further comprises an ITR adjacent to further The genome includes a transgene encoding an immunogenic protein or a portion thereof (however, the further immunogenic protein or portion thereof includes the amino acid sequence of the group consisting of amino acids 179-419 or 212-411 of SEQ ID NO: 51). Since the N protein (particularly the portion of the N protein containing amino acids 179-419 or 212-411 of SEQ ID NO: 51) is considered to primarily induce a T cell response, it may be particularly suitable for expressing the genome encoded by the transgene in host cells. Unbound by theory, it is expected that the immunogenic protein or a portion thereof inserted into the VP and exposed on the surface will primarily induce a humoral immune response.

[0095] For vaccines against pathogens, it may be beneficial for the vaccine to target multiple immunogenic proteins or a portion of them, preferably additional structural proteins (such as the N protein). This is because it reduces the risk of immune evasion, for example, due to mutations in the S protein.

[0096] In other embodiments, the immunogenic protein or a portion thereof is a tumor antigen. Representative, non-limiting examples of suitable tumor antigens include carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), folate-binding protein (FBP), GD2, GD3, human epidermal growth factor receptor 2 (HER2, erb-B2), melanoma antigen A1 (MAGE-A1), mesothelin (MSLN), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), mucin-1 (MUC1), glypican-3 (GPC3), Wilms tumor protein (WT1), epithelial cell adhesion molecule (EpCAM), and B cell maturation antigen (B These include CMAs, tyrosine-protein kinase transmembrane receptors (ROR1), or minor or major histocompatibility complex-associated tumor-specific (TSAs), and tumor-associated antigens (TAAs) (such as BCR-ABL fusion, melanoma-associated antigen 3 (MAGE-A3), glycoprotein 100 (gp100), cancer / testicular antigen 1 (LAGE2 or NY-ESO-1), Epstein-Barr virus latent membrane protein 1 (LMP1), P2X purine receptor 7 (P2RX7), and diphthamide biosynthesis protein 1 (DPH1)).

[0097] The AAV or AAVLP according to the present invention is a foundational technology that can be used to insert a target molecule (e.g., an antibody-derived protein or antibody mimetic) as one binding unit of a binding pair, thereby binding it to the other binding unit of that binding pair (e.g., an antigen). The AAV or AAVLP according to the present invention (referring to embodiments in which the insert is a protein containing a binding domain) has cell-directivity provided by the protein containing the binding domain. The protein containing the binding domain is one binding unit of a binding pair (e.g., a protein containing an antigen-binding domain specific to the target antigen). Thus, the insert is a protein containing a binding domain specific to the binding target, and the protein containing the binding domain, i.e., one binding unit of the binding pair, determines the directivity of the AAV or AAVLP to target cells expressing the binding target (e.g., a ligand or receptor or antigen) on its surface, i.e., the other binding unit of the binding pair (e.g., a target antigen that binds to a protein containing an antigen-binding domain). Non-limiting examples of suitable binding pairs include antibody-derived proteins containing an antigen-binding domain (such as nanobodies or single-chain antibodies) and antigens, preferably single-domain antibodies (sdAbs) or single-chain variable fragments (scFv), or antibody mimetic products (such as antikalin, afibodies, adonectin, monobodies, DARPin, afimers, or afitins). Non-limiting examples of suitable binding pairs include proteins containing an antigen-binding domain and their antigens (e.g., single-domain antibodies (sdAbs), single-chain variable fragments (scFv), or antibody mimetic products and their antigens), proteins containing a receptor-binding domain and receptors (e.g., coronavirus spike (S) protein and ACE-2 receptor; antibody Fc region (e.g., scFc) and Fc receptor), and ligand-binding domains and ligands (e.g., PD-1 and PD-L1). Therefore, in one embodiment, the directivity of AAV is determined by an insert in the viral protein consisting of approximately 75 to 400, preferably 75 to 300, amino acids, and this insert is a protein containing a binding domain (such as a protein containing an antigen-binding domain).

[0098] Non-limiting examples of appropriate target antigens include tumor antigens that target cancer cells (e.g., carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), folate-binding protein (FBP), GD2, GD3, human epidermal growth factor receptor 2 (HER2, erb-B2), melanoma antigen A1 (MAGE-A1), mesothelin (MSLN), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), mucin-1 (MUC1), glypican-3 (GPC3), Wilms tumor protein (WT1), epithelial cell adhesion molecule (EpCAM), B cell maturation antigen (BCMA), tyrofoam Syn-protein kinase transmembrane receptor (ROR1), or minor or major histocompatibility complex-associated tumor-specific (TSA), and tumor-associated antigens (TAAs) (such as BCR-ABL fusion, melanoma-associated antigen 3 (MAGE-A3), glycoprotein 100 (gp100), cancer / testicular antigen 1 (LAGE2 or NY-ESO-1), Epstein-Barr virus latent membrane protein 1 (LMP1), P2X purine receptor 7 (P2RX7), diphthamide biosynthesis protein 1 (DPH1)) and / or AAV or AAVLP are modified targets of the said antigens. These are cell type-specific antigens that are directed towards cells expressing surface antigens (such as surface receptors) (cells that are not sensitive to wild-type AAV serotypes, such as endothelial cells or certain types of nerve cells). Non-limited examples of antigen targets that mediate cell specificity include CD4, CD8, CD11b, CD16, CD19, CD133 (prominin), CD105 (endoglin), CD146 (melanoma cell adhesion molecule), CD30, CD32, CD33, CD34, CD36, CD40, CD64, CD68, CD80, CD86, CD163, CD206, CD209, C D301 is an excitatory amino acid transporter 1 (SLC1A3), an excitatory amino acid transporter 2 (SLC1A2), a neuronal / glial antigen 2 (NG2), an EGF-like module-containing mucin-like hormone receptor 1 (EMR1), a folate receptor 1 (FOLR1), a dopamine-activating transporter (DAT or SLC6A3), a platelet-derived growth factor receptor (PDGFR), a vesicular acetylcholine transporter (VAChT), a vesicular inhibitory amino acid transporter (SLC32A1), vesicular glutamate transporters 1 and 2 (SLC17A7 and SLC17A6), or a serotonin transporter (SLC6A4).

[0099] The expression "protein containing an antigen-binding domain" refers to a protein that contains an antigen-binding site capable of selectively binding to a target antigen and thus binds to a specific antigen (including antibody-derived proteins and antibody mimetic proteins). Antibody mimetic proteins are proteins that bind to a specific antigen in a similar manner to antibodies, but whose structure is unrelated to that of antibodies. Proteins containing an antigen-binding domain and consisting of approximately 75 to 400 amino acids, preferably 75 to 300 amino acids, can take any form, and non-limiting examples include sdAbs, single-strand variable fragments (scFv), antikalin, affibodies, adonectin, monobodies, DARPin, affimers, or afitins, preferably antigen-binding domain-containing antibody-derived proteins selected from the group consisting of sdAbs and single-strand variable fragments (scFv), or antigen-binding domain-containing antibody mimetic proteins selected from the group consisting of antikalin, affibodies, adonectin, monobodies, DARPin, affimers, and afitins. Single-domain antibodies (sdAbs) can also be called nanobodies. Those skilled in the art will see that a protein can contain two or more antigen-binding domains, and therefore be multispecific, preferably bispecific (e.g., a bivalent sdAb or bivalent antikalin or any other bivalent antibody mimetic). Furthermore, a protein can be multispecific, preferably bispecific, i.e., specific to two different antigens (e.g., a bispecific sdAb or bispecific antikalin or any other bivalent antibody mimetic).

[0100] In one embodiment, the insert is a protein containing a binding domain (such as an antigen-binding domain), and the AAV or AAVLP is preferably an AAV containing a genome flanked by an ITR, and is infectious. The genome flanked by the ITR more preferably contains a transgene. The AAV according to the present invention (in which the insert is a protein containing a binding domain, and this AAV contains a genome flanked by an ITR, is infectious, and the genome contains a transgene) is particularly useful for use in gene therapy. In gene therapy, a gene is delivered to a specific type of cell, and its expression leads to a therapeutic effect. Non-limiting examples of typical gene therapies include gene augmentation, gene supplementation, gene addition, or gene editing (including CRISPR-Cas or other technologies). Generally, the AAV according to the present invention, directed toward a modified target, is suitable for in vitro, in vivo, and in-situ gene therapy. In in vitro gene therapy (also called in vitro gene therapy), target cells are removed from the patient's body and manipulated by adding a therapeutic gene that allows for correction of the disease phenotype, or by other genetic manipulation, and the manipulated cells are then infused back into the patient. This is particularly applicable to hematological disorders (including chimeric antigen receptor (CAR)-based technologies such as CAR T cells and CAR NK cells). In in vivo gene therapy, the AAV according to the present invention, directed toward the altered target, is systemically administered into the patient's bloodstream or cerebrospinal fluid and enters the brain, spinal canal, or liver, depending on disease target-specific cells. In situ gene therapy, the AAV according to the present invention, directed toward the altered target, is administered in situ, i.e., by direct injection into a specific organ or region of the patient's body (e.g., a tumor (e.g., melanoma) or an appropriate brain region (e.g., neuropathy)) or by insertion of a catheter (e.g., if the organ to be treated is the heart). Gene therapy is preferably in vivo or situ gene therapy. In one embodiment, the protein containing the binding domain is a protein containing an antigen-binding domain specific to a tumor antigen, and the genome flanked by the ITR contains a suicide gene which is preferable for use in the treatment of cancer.

[0101] Those skilled in the art will see that the coronavirus spike (S) protein or a portion thereof is also a binding protein containing a binding domain. The S protein binds to the cell receptor ACE-2. Therefore, possible inserts include an immunogenic protein or a portion thereof, and a protein containing a binding domain.

[0102] In one further aspect, the present invention relates to a pharmaceutical composition comprising an AAV or AAVLP according to the present invention, which preferably further comprises at least one pharmaceutically acceptable excipient. In the context of the present invention, the term “excipient” means a natural or synthetic substance formulated together with the active ingredient of the pharmaceutical. Suitable excipients include anti-adhesives, binders, coatings, disintegrants, flavoring agents, colorants, lubricants, flow enhancers, adsorbents, preservatives, and sweeteners. Excipients may also include adjuvants. In one embodiment, a pharmaceutical composition comprising an AAV or AAVLP according to the present invention further comprises at least one adjuvant and at least one further pharmaceutically acceptable excipient. A pharmaceutical composition according to the present invention may contain two or more AAVs or AAVLPs according to the present invention, or one or more AAVs and one or more AAVLPs according to the present invention (hereinafter referred to as two or more AAVs and / or AAVLPs). However, two or more AAVs and / or AAVLPs may exist in a fixed dosage form (i.e., physically mixed) and / or be provided in separate dosage forms. Each of the two or more AAVs and / or AAVLPs contains a different immunogenic protein or a portion thereof, and / or a protein containing a binding domain (wherein "different" can be an immunogenic protein or immunogenic moiety from a different protein, or a different immunogenic moiety from the same protein), or an immunogenic protein or a portion thereof, and a protein containing a binding domain, or two different proteins containing binding domains (i.e., two different targets (with specificity for antigens, ligands, and / or receptors, etc.)). The immunogenic protein, or immunogenic moiety from a different protein, is preferably derived from the same pathogen (the same bacterium, the same virus, or the same parasite) in the case of a pathogen.

[0103] In the context of this invention, the expression “pharmaceutically acceptable” means that the molecule and other components of the pharmaceutical composition are physiologically acceptable and typically do not produce undesirable reactions when administered to mammals (e.g., humans). The expression “pharmaceutically acceptable” may also mean that it is approved by a federal or state regulatory authority, or that it is listed in the United States Pharmacopeia, or any other generally accepted pharmacopoeia for use in mammals, more specifically, humans.

[0104] In the context of the present invention, AAV or AAVLP, or a pharmaceutical composition containing the AAV or AAVLP, can be adapted for administration via intranasal, mucosal, sublingual, oral, oral, intravenous, intramuscular, intraperitoneal, or subcutaneous routes, preferably via intranasal mucosa, sublingual, intravenous, or subcutaneous routes. In one embodiment, AAV or AAVLP, or a pharmaceutical composition containing the AAV or AAVLP, can be adapted for administration by inhalation via intranasal, oral, and / or mucosal routes. Therapeutic use of AAV or AAVLP according to the present invention

[0105] In a further aspect, the AAV or AAVLP or pharmaceutical composition according to the present invention is therapeutic. In one embodiment, the AAV or AAVLP or pharmaceutical composition according to the present invention is preferably for use as a vaccine in humans. In this context, insert is an immunogenic protein or a part thereof as described herein. The term “vaccine” as herein means a drug that, when administered, can induce an immune response in a target. The vaccine is preferably able to prevent, improve, or treat a disease. In the context of the present invention, the vaccine can be, for example, a protective or prophylactic vaccine to prevent infection with a pathogen, or the vaccine can be a therapeutic vaccine to treat, for example, cancer. However, those skilled in the art will see that in the case of infection with a virus, bacteria, or parasite, the vaccine can also be used therapeutically, for example, to improve the disease or the symptoms after the onset of the disease. The vaccine comprising the AAV or AAVLP of the present invention is a subunit vaccine that uses the AAV or AAVLP as a carrier. The carrier can be inactive or function as an adjuvant by providing an immune stimulus (e.g., ssDNA, or antigenic epitopes and immunogenic epitopes).

[0106] In one particular aspect, the present invention relates to AAV or AAVLP or pharmaceutical composition according to the present invention for use in the treatment or prevention of diseases induced by viruses, bacteria, or parasites, wherein the immunogenic protein or a portion thereof is the immunogenic protein of the virus, bacteria, or parasite, as defined above with respect to the AAV or AAVLP according to the present invention. In one embodiment, the disease is coronavirus respiratory syndrome, and the immunogenic protein or a portion thereof is a portion of the coronavirus spike (S) protein. Preferably, the disease is coronavirus disease 2019 (COVID-19), and the immunogenic protein or a portion thereof is a portion of the SARS-CoV-2 spike (S) protein. In one particular aspect, the immunogenic protein or a portion within the AAV or AAVLP according to the present invention comprises a portion of the SARS-CoV-2 spike (S) protein, and the AAV or AAVLP is used to induce an immune response to SARS-CoV-2. In some embodiments of AAV or AAVLP for use according to the present invention, a portion of the SARS-CoV-2 spike (S) protein comprises a SARS-CoV-2 S protein receptor-binding domain (RBD) or a portion thereof (preferably a portion comprising a receptor-binding motif (RBM)). In one embodiment, a portion of the SARS-CoV-2 S protein contains an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69 (preferably SEQ ID NOs: 11, 12, 34, 35, 36, 37, 38, 42, or 69), or an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69 (preferably SEQ ID NOs: 11, 12, 34, 35, 36, 37, 38, 42, or 69).

[0107] In one further special aspect, the AAV or AAVLP or pharmaceutical composition according to the present invention is for the treatment or prevention of cancer, and the insert is an immunogenic protein or a portion thereof, and that immunogenic protein or a portion thereof is a tumor antigen or a portion thereof. Alternatively, or in addition thereto, the insert may be a protein containing a binding domain (such as an antigen-binding domain) specific to the tumor antigen as the target antigen. Non-limiting examples of tumor antigens suitable as immunogenic proteins and / or target antigens include carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), folate-binding protein (FBP), GD2, GD3, human epidermal growth factor receptor 2 (HER2, erb-B2), melanoma antigen A1 (MAGE-A1), mesothelin (MSLN), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), mucin-1 (MUC1), glypican-3 (GPC3), Wilms' tumor protein (WT1), epithelial cell adhesion molecule (EpCAM), and B cell maturation antigen (BC). The group can be selected from MAs, tyrosine-protein kinase transmembrane receptors (ROR1), or minor or major histocompatibility complex-associated tumor-specific (TSAs), and tumor-associated antigens (TAAs) (such as BCR-ABL fusion, melanoma-associated antigen 3 (MAGE-A3), glycoprotein 100 (gp100), cancer / testicular antigen 1 (LAGE2 or NY-ESO-1), Epstein-Barr virus latent membrane protein 1 (LMP1), P2X purine receptor 7 (P2RX7), diphthamide biosynthesis protein 1 (DPH1)). The cancers to be treated can be solid tumors or hematopoietic malignancies. Non-limiting examples of cancers suitable for treatment include colorectal cancer, breast cancer, hepatocellular carcinoma, glioma, lung cancer (especially small cell lung cancer), ovarian cancer, neuroblastoma, melanoma, head and neck squamous cell carcinoma, gastric cancer, pancreatic cancer, mesothelioma, prostate cancer, hepatocellular carcinoma, AML, or CML. Those skilled in the art will further understand that certain viral immunogenic proteins can also function as tumor antigens (such as antigens derived from HCV or HPV).

[0108] In another aspect, the AAV or AAVLP or pharmaceutical composition according to the present invention is intended for use in therapy (particularly gene therapy). In this context, the insert is a protein containing a binding domain, preferably an antigen-binding domain, a receptor-binding domain, or a ligand-binding domain, more preferably a protein containing an antigen-binding domain. Thus, the AAV or AAVLP is directed to a modification target. For example, the antigen-binding domain is specific to a cell type-specific antigen (particularly a surface antigen such as a surface receptor), and the AAV or AAVLP can be directed to the cell type that is the modification target (such as cells that are not sensitive to wild-type AAV serotypes (e.g., endothelial cells or certain types of nerve cells)). In one particular embodiment, the antigen-binding domain is specific to a tumor antigen, and the AAV or AAVLP can be directed to tumor cells expressing the tumor antigen, which is the modification target. More preferably, the AAV or AAVLP is an AAV, the AAV containing an ITR-facing genome, and is infectious. In one embodiment, the ITR-facing genome contains a transgene. If the modification target of the AAV is a tumor, a suicide gene can also be the transgene.

[0109] The therapeutic use of the present invention may also include two or more AAVs or AAVLPs according to the present invention to be administered, or one or more AAVs and one or more AAVLPs according to the present invention to be administered (hereinafter referred to as two or more AAVs and / or AAVLPs to be administered). However, the two or more AAVs and / or AAVLPs to be administered may exist in a fixed dosage form (i.e., physically mixed) and / or be provided in separate dosage forms. These may be administered simultaneously or at different time points. However, the two or more AAVs and / or AAVLPs each include different inserts (such as different immunogenic proteins or parts thereof, and / or proteins containing binding domains) inserted into the apex of VR-VIII and / or VR-IV. For immunogenic proteins or parts thereof, different may be immunogenic proteins or immunogenic moieties from different proteins, or different immunogenic moieties from the same protein. The immunogenic proteins or immunogenic moieties from different proteins are preferably derived from the same pathogen (such as the same bacterium, the same virus, or the same parasite) in the case of a pathogen, or from the same tumor in the case of cancer. For primary and booster vaccinations, the AAV or AAVLP according to the present invention is based on different serotypes having the same or at least overlapping immunogenic proteins or portions thereof. For proteins containing binding domains, different means proteins with different binding specificities.

[0110] AAV or AAVLP for use according to the present invention can be administered via the nasal mucosa, sublingual, oral, oral, intravenous, intramuscular, intraperitoneal, or subcutaneous routes, preferably via the nasal mucosa, sublingual, intravenous, or subcutaneous routes. In one embodiment, AAV or AAVLP can be administered by inhalation via the nasal, oral, and / or mucosal routes. Method for producing AAV or AAVLP according to the present invention

[0111] In another aspect, the present invention relates to a method for producing AAV or AAVLP, the method comprising: (i) preparing cells comprising at least one DNA sequence comprising a cap gene and a rep gene, at least one DNA sequence comprising an adenovirus helper sequence, and optionally, at least one DNA sequence comprising a genome flanked by an ITR (wherein the cap gene encodes a protein containing an insert of about 75 to 400, preferably about 75 to 300 amino acids in a capsid-forming viral protein (VP) at the insertion site (I) at the apex of variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, and optionally flanking one or both sides of the insert is a linker preferably comprising one or more amino acids selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof); (ii) culturing the cells under conditions that enable the production of AAV or AAVLP; and (iii) purifying the AAV or AAVLP. In one embodiment, the method is a method for producing a pharmaceutical composition, comprising (iv) the steps of the method of the present invention, and further comprising the step of formulating AAV or AAVLP into a pharmaceutical composition by adding at least one pharmaceutically acceptable excipient. Those skilled in the art will understand that the cap gene and rep gene may contain a natural AAV promoter (such as the respective wild-type AAV promoter), and that the expression levels can be regulated and improved by replacing or supplementing the natural rep promoter and / or cap promoter (particularly the cap promoter) with a different eukaryotic promoter, preferably a potent eukaryotic promoter (a potent mammalian promoter, e.g., CMV, RSV, or SV40). The method according to the present invention is an in vitro method. The terms “eukaryotic promoter” and “mammalian promoter” mean, as herein, any promoter (including viral promoters) that drives gene expression in a eukaryotic or mammalian cell.

[0112] In one embodiment, a method for producing AAV or AAVLP includes the steps of (i) preparing a cell comprising at least one DNA sequence comprising a cap gene and a rep gene, at least one DNA sequence comprising an adenovirus helper sequence, and optionally, an ITR comprising at least one DNA sequence comprising an adjacent genome (wherein the cap gene encodes a protein that contains an insert consisting of about 75 to 400, preferably about 75 to 300 amino acids in a capsid-forming viral protein (VP) at the insertion site (I) at the top of variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, The insert is (a) an immunogenic protein or a portion thereof, and / or (b) a protein containing a binding domain, wherein one or both sides of the insert are adjacent to a linker containing one or more amino acids preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof; (ii) a step of culturing cells under conditions that enable the production of AAV or AAVLP; (iii) a step of purifying the AAV or AAVLP; optionally a step of formulating the AAV or AAVLP into a pharmaceutical composition by adding at least one pharmaceutically acceptable excipient. The method according to the present invention is an in vitro method.

[0113] The adenovirus helper sequences are E2A, E4, and VA RNA, and E1A / E1B if they are not already expressed in the producing cell (e.g., HEK293 cells). In some cases, further plasmids encode genomes flanked by ITRs (such as genomes flanked by ITRs containing transgene expression cassettes).

[0114] In one embodiment, at least one DNA sequence containing a cap gene and a rep gene, at least one DNA sequence containing an adenovirus helper sequence, and / or optionally, at least one DNA sequence containing a genome adjacent to an ITR, can be independently, stably, or transiently expressed. In a particular embodiment, the method of the present invention comprises (a) transfecting mammalian cells with at least one DNA molecule containing a cap gene and a rep gene, at least one DNA molecule containing an adenovirus helper sequence, and optionally, at least one DNA molecule containing an adjacent genome with an ITR (preferably, the DNA molecules are plasmids or linear DNA); or (b) transducing mammalian cells using at least one vector containing a cap gene and a rep gene, at least one vector containing an adenovirus helper sequence, and optionally, at least one vector containing an adjacent genome with an ITR (the vectors are preferably viral vectors selected from baculovirus and herpes simplex virus); or (c) providing cells stably expressing at least one DNA sequence containing a cap gene and a rep gene, at least one DNA sequence containing an adenovirus helper sequence, and / or optionally, at least one DNA sequence containing an adjacent genome with an ITR, and providing a DNA sequence that was transiently and stably expressed by transfection and / or transduction. At least one DNA sequence containing a cap gene and a rep gene, at least one DNA sequence containing an adenovirus helper sequence, and / or optionally, at least one DNA sequence containing a genome adjacent to an ITR, can be independently, stably, or transiently expressed.

[0115] Specifically, the AAV according to the present invention was prepared as follows. To produce the AAV, 20 μg of DNA was simultaneously transfected into 15 150-mm petri dishes of HEK293T cells concentrated to 80% density, with each petri dish containing 20 μg of DNA. In this manner, the AAV Rep / Cap plasmid, pHtW2_S1.1 or pHtW9_S1.1, was simultaneously transfected in the same molar ratio as the adenovirus helper plasmid (e.g., pXX6 from J. Samulski, Chapel Hill, NC; Xiao, Li and Samulski (1998) "Construction of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus" J. Virol. 72: 2224-2232), and, in the case of producing a full capsid, in the same molar amount as the pTransgene plasmid containing a CMV-eGFP cassette with an adjacent ITR. After 48 hours, AAV was isolated from a HEK293 cell pellet resuspended in 150 mM NaCl and 50 mM Tris-HCl (pH 8.5), subjected to several freeze-thaw cycles, and treated with benzonase (50 U / ml) at 37°C for 30 minutes. Cell debris was removed by centrifugation, and the supernatant was further treated for an iodixanol gradient. Alternatively, AAV was isolated from the cell culture supernatant after sedimentation with 8% polyethylene glycol (PEG) 8000 at 4°C. The PEG-AAV precipitate was then treated with benzonase (50 U / ml) at 37°C for 30 minutes, followed by further treatment for an iodixanol gradient. Iodixanol gradient ultracentrifugation was performed at 18°C ​​at 70,000 rpm for 1 hour and 45 minutes as described (Zolotukhin et al. (1999) "Recombinant adeno-associated virus purification using a novel method improves infectivity titer and yield" Gene Ther. 6: 973-985). Subsequently, virions were recovered from the 40% iodixanol phase and titrated by DNA dot-blot hybridization using a rep probe (Girod et al. (1999) "Genetic capsid modification enables efficient targeting of adeno-associated virus type 2 to modified targets" Nat. Med. 5: 1052-1056).

[0116] In addition to the approaches described above, rAAV can be produced using alternative approaches. For example, another transient co-transfection method can be used that employs mini-circular DNA or closed linear DNA (e.g., doggybone DNA) lacking a bacterial plasmid backbone instead of conventional plasmids. Alternatively, a stable mammalian-producing cell line (e.g., HeLa) transformed with plasmids encoding rep and cap genes and, optionally, a transgene flanked by an AAV ITR can be used. Such cells can then be infected with wild-type adenovirus (e.g., Ad 5) or with an adenovirus / AAV hybrid virus containing a genome flanked by an AAV ITR to produce selected rAAV particles containing a transgene packaged within an rAAV capsid modified according to the present invention. AAVLP is produced in essentially the same manner as AAV according to the present invention, but differs in that mammalian cells are transformed with a plasmid completely lacking a genome flanked by an ITR, or that mammalian cells are infected with wild-type adenovirus instead of an adenovirus / AAV hybrid virus containing a genome flanked by an AAV ITR. Another suitable approach is to use a baculovirus / Sf8 system that employs two to three separate viruses (Rep baculovirus, VP baculovirus, and optionally, a transgene baculovirus flanked by the AAV ITR) for infection. The resulting rAAV vector contains the transgene packaged within an rAAV capsid modified according to the present invention. AAVLP is produced essentially according to the same method as AAV according to the present invention, but differs in that it is transfected into mammalian cells without a plasmid containing the genome with the ITR flanked, or infects mammalian cells without a genome baculovirus with the AAV ITR flanked. Alternatively, mammalian cells (e.g., hamster BHK21 cells, HEK293 cells, or derivatives) can be infected with one or two recombinant herpes simplex virus (rHSV) expressing the AAV rep gene, a modified cap gene, and optionally, a transgene flanked by the AAV ITR. Any additional helper functions are provided by the rHSV gene.The resulting rAAV vector contains a transgene packaged within an rAAV capsid modified according to the present invention. AAVLP is basically produced according to the same method as AAV according to the present invention, but differs in that mammalian cells are infected only with rHSV expressing the AAV rep gene and the modified cap gene, and rHSV with a transgene adjacent to AAV ITR are not infected.

[0117] The genome adjacent to the ITR may further contain transgenes encoding further immunogenic proteins or parts thereof. In another embodiment, the AAV or AAVLP is an AAVLP that does not contain a genome adjacent to the ITR. Possible AAV VPs forming the capsid are VP1, VP2, and VP3 in a 1:1:10 ratio. The capsid may also be formed by VP1 and VP3 alone, or by VP alone. Thus, possible AAV VPs forming the capsid are VP1 and VP3, or VP3 alone. In any variant, it is preferable that the AAV or AAVLP has a capsid consisting of about 60 VPs. The immunogenic protein or part thereof and the insertion site may be those disclosed above with respect to the AAV or AAVLP according to the present invention. In the context of the methods of the present invention, the genome, in addition to the immunogenic protein or part thereof and the insertion site, may be defined as described above with respect to the AAV or AAVLP of the present invention.

[0118] In light of the above, it will be understood that the present invention also includes the following items. 1. Adeno-associated virus (AAV) or adeno-associated virus-like particle (AAVLP) comprising an insert consisting of approximately 75 to 400 amino acids, preferably approximately 75 to 300 amino acids, within a capsid-forming viral protein (VP) at the insertion site (I) located at the apex of variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, wherein the insert is an immunogenic protein or part thereof, and optionally flanked on one or both sides of the insert are linkers containing one or more amino acids, preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof. 2. (a) The apex of VR-VIII corresponds to amino acids 585-592 (I-585-I-592) of VP1 of AAV 1, 2, 3, 6, 7, 8, 9, or 10 having the amino acid sequence of SEQ ID NOs. 1, 2, 3, 6, 7, 8, 9, or 10, respectively, amino acids 583-589 of VP1 of AAV 4 having the amino acid sequence of SEQ ID NOs. 4, or amino acids 574-580 of VP1 of AAV 5 having the amino acid sequence of SEQ ID NOs. 5, and / or (b) The apex of VR-IV corresponds to amino acids 450-460 (I-450-I-460) of VP1 of AAV 1, 2, 3, 6, 7, 8, 9, or 10 having the amino acid sequence of SEQ ID NOs. 4, respectively, and AAV 5 having the amino acid sequence of SEQ ID NOs. 4 An AAV or AAVLP as described in item 1, corresponding to amino acids 445-455 (I-445-I-455) of VP1 of AAV 4, or amino acids 439-449 (I-439-I-449) of VP1 of AAV 5 having the amino acid sequence of SEQ ID NO: 5. 3. Derived from AAV serotype 1 (AAV1), 2 (AAV2), 8 (AAV8), or 9 (AAV9), preferably, (a) The insertion site is located between two amino acids corresponding to amino acid positions 587 and 588 of AAV2 VP1 having the amino acid sequence of SEQ ID NO: 2 (AAV2 I-587), or between 588 and 589 (AAV2 I-588), and / or between 453 and 454 (AAV2 I-453), preferably AAV2 I-587, or AAV2 I-588, or AAV2 I-453, more preferably AAV2 I-587 or AAV2 I-588; (b) The insertion site is located between two amino acids corresponding to amino acid positions 587 and 588 having the amino acid sequence of SEQ ID NO: 1 (AAV1 I-587), between 588 and 589 (AAV1 I-588), or between 589 and 590 (AAV1 I-589) and / or between 454 and 455 (AAV1 I-454), between 455 and 456 (AAV1 I-455), or between 456 and 457 (AAV1 I-456); (c) The insertion site is located between two amino acids corresponding to amino acid positions 588 and 589 of AAV8 VP1 having the amino acid sequence of SEQ ID NO: 8 (AAV8 I-588), or between 589 and 590 (AAV8 I-589), and / or between 455 and 456 (I-455), between 456 and 457 (I-456), or between 457 and 458 (I-457), (d) The AAV or AAVLP described in any one of claims 1 to 2, wherein the insertion site is located between two amino acids corresponding to amino acid positions 588 and 589 of AAV9 VP1 having the amino acid sequence of SEQ ID NO: 9 (AAV9 I-588), or between 589 and 590 (AAV9 I-589), and / or between 454 and 455 (I-454), between 455 and 456 (I-455), or between 456 and 457 (I-456). 4. (a) The AAV includes an ITR adjacent genome, is infectious, and optionally includes a transgene encoding a further immunogenic protein or a portion thereof; (b) An immunogenic protein or a portion thereof is inserted at the apex of VR-VIII and the apex of VR-IV, and the immunogenic protein or portion inserted at the apex of VR-VIII and the immunogenic protein or portion inserted at the apex of VR-IV are the same or different; and / or (c) The AAV or AAVLP according to any one of claims 1 to 3, wherein the AAV or AAVLP is formed by two or more viral proteins comprising different inserts consisting of at least about 75 to 400 amino acids, preferably about 75 to 300 amino acids, each of which is an immunogenic protein or a part thereof, and is either an immunogenic protein or immunogenic moiety from different proteins, or different immunogenic moieties from the same protein. 5. It has a capsid consisting of approximately 60 VPs, and the VPs are (a) VP3; (b) VP1 and VP3; or (c) AAV or AAVLP as described in any one of items 1 to 4, wherein the VP1, VP2, and VP3 proteins are preferably in a ratio of 1:1:10. 6. AAV or AAVLP as described in any one of items 1 to 5, wherein the immunogenic protein or a portion thereof is a virus, bacterium, or parasite protein or a portion thereof. 7. The immunogenic protein or a part thereof (a) It is part of the coronavirus spike (S) protein; (b) a portion of the SARS-CoV-2 spike (S) protein, preferably the portion of the SARS-CoV-2 spike (S) protein comprising the SARS-CoV-2 S protein receptor-binding domain (RBD) or a portion thereof; and / or (c) AAV or AAVLP as described in item 6, which is part of the SARS-CoV-2 S protein comprising the amino acid sequence of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69, preferably 11, 12, 34, 35, 36, 37, 38, 42, or 69. 8. AAV or AAVLP as described in any one of items 1 to 5, wherein the immunogenic protein or a portion thereof is a tumor antigen. 9. A pharmaceutical composition comprising an AAV or AAVLP as described in any one of paragraphs 1 to 8, further comprising at least one pharmaceutically acceptable excipient. 10. AAV or AAVLP as described in any one of claims 1 to 8, or the pharmaceutical composition of claim 9, for use as a vaccine. 11. An AAV or AAVLP as described in any one of paragraphs 1 to 7, used for the treatment or prevention of a disease induced by a virus, bacterium, or parasite, wherein the immunogenic protein or any part thereof is the immunogenic protein of the respective virus, bacterium, or parasite. 12. AAV or AAVLP for use in accordance with item 11, wherein the disease is coronavirus respiratory syndrome and the immunogenic protein or a portion thereof is a portion of the coronavirus spike (S) protein, preferably AAV or AAVLP for use in accordance with item 11, wherein the disease is coronavirus disease 2019 (COVID-19) and the immunogenic protein or a portion thereof is a portion of the SARS-CoV-2 spike (S) protein. 13. An AAV or AAVLP as described in item 8, used for the treatment of cancer, wherein the immunogenic protein or a portion thereof is a tumor antigen or a portion thereof. 14. AAV or AAVLP for use as described in any one of paragraphs 10 to 13, administered via the nasal mucosa, sublingual, oral, oral, intravenous, intramuscular, intraperitoneal, or subcutaneous routes, preferably by inhalation via the nasal, oral, and / or mucosal routes. 15. A method for producing AAV or AAVLP, (i) A step of preparing cells comprising at least one DNA sequence containing a cap gene and a rep gene, at least one DNA sequence containing an adenovirus helper sequence, and optionally, at least one DNA sequence containing a genome adjacent to an ITR (wherein the cap gene encodes a protein containing an insert consisting of about 75 to 400 amino acids, preferably about 75 to 300 amino acids, in a capsid-forming viral protein (VP) at the insertion site (I) at the apex of variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, wherein the insert is an immunogenic protein or a part thereof, and optionally, one or both sides of the insert are adjacent to a linker containing one or more amino acids preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof); (ii) A step of culturing the cells under conditions that enable the production of AAV or AAVLP; and (iii) A method comprising the step of purifying the AAV or AAVLP. 16. A method for producing a pharmaceutical composition containing AAV or AAVLP, (i) A step of preparing cells comprising at least one DNA sequence containing a cap gene and a rep gene, at least one DNA sequence containing an adenovirus helper sequence, and optionally, at least one DNA sequence containing a genome adjacent to an ITR (wherein the cap gene encodes a protein containing an insert consisting of about 75 to 400 amino acids, preferably about 75 to 300 amino acids, in a capsid-forming viral protein (VP) at the insertion site (I) at the apex of variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, wherein the insert is an immunogenic protein or a part thereof, and optionally, one or both sides of the insert are adjacent to a linker containing one or more amino acids preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof); (ii) A step of culturing the cells under conditions that enable the production of AAV or AAVLP; (iii) a step of purifying the AAV or AAVLP; and (iv) A method comprising the step of formulating the AAV or AAVLP into a pharmaceutical composition by adding at least one pharmaceutically acceptable excipient. 17. Adeno-associated virus (AAV) or adeno-associated virus-like particle (AAVLP), wherein an insert consisting of approximately 75 to 400 amino acids, preferably approximately 75 to 300 amino acids, in a capsid-forming viral protein (VP) is included at the insertion site (I) at the apex of the variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, and optionally, one or both sides of the insert are adjacent to a linker containing one or more amino acids, preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof. 18. The AAV or AAVLP as described in Section 17, wherein the insert is (a) an immunogenic protein or a portion thereof, and / or (b) a protein comprising a binding domain. 19. The AAV or AAVLP according to item 17, wherein the insert is a protein comprising a binding domain, preferably an antigen-binding domain. 20. The AAV or AAVLP according to any one of claims 17 to 19, wherein the AAV includes a genome flanked by an ITR, is infectious, and preferably the genome flanked by the ITR includes a transgene. 21. (a) The apex of VR-VIII corresponds to amino acids 585-592 (I-585-I-592) of VP1 of AAV 1, 2, 3, 6, 7, 8, 9, or 10 having the amino acid sequence of SEQ ID NOs. 1, 2, 3, 6, 7, 8, 9, or 10, respectively, amino acids 583-589 of VP1 of AAV 4 having the amino acid sequence of SEQ ID NOs. 4, or amino acids 574-580 of VP1 of AAV 5 having the amino acid sequence of SEQ ID NOs. 5, and / or (b) The apex of VR-IV corresponds to amino acids 450-460 (I-450-I-460) of VP1 of AAV 1, 2, 3, 6, 7, 8, 9, or 10 having the amino acid sequence of SEQ ID NOs. 4, respectively, and AAV 5 having the amino acid sequence of SEQ ID NOs. 4 An AAV or AAVLP described in any one of items 17 to 20, corresponding to amino acids 445 to 455 (I-445 to I-455) of VP1 of AAV 4, or amino acids 439 to 449 (I-439 to I-449) of VP1 of AAV 5 having the amino acid sequence of SEQ ID NO: 5. 22. Derived from AAV serotype 1 (AAV1), 2 (AAV2), 8 (AAV8), or 9 (AAV9), preferably, (a) The insertion site is located between two amino acids corresponding to amino acid positions 587 and 588 of AAV2 VP1 having the amino acid sequence of SEQ ID NO: 2 (AAV2 I-587), or between 588 and 589 (AAV2 I-588), and / or between 453 and 454 (AAV2 I-453), preferably AAV2 I-587, or AAV2 I-588, or AAV2 I-453, more preferably AAV2 I-587 or AAV2 I-588; (b) The insertion site is located between two amino acids corresponding to amino acid positions 587 and 588 having the amino acid sequence of SEQ ID NO: 1 (AAV1 I-587), between 588 and 589 (AAV1 I-588), or between 589 and 590 (AAV1 I-589), and / or between 454 and 455 (AAV1 I-454), between 455 and 456 (AAV1 I-455), or between 456 and 457 (AAV1 I-456); (c) The insertion site is located between two amino acids corresponding to amino acid positions 588 and 589 of AAV8 VP1 having the amino acid sequence of SEQ ID NO: 8 (AAV8 I-588), or between 589 and 590 (AAV8 I-589), and / or between 455 and 456 (I-455), between 456 and 457 (I-456), or between 457 and 458 (I-457), (d) The AAV or AAVLP described in any one of the claims 17 to 21, wherein the insertion site is located between two amino acids corresponding to amino acid positions 588 and 589 of AAV9 VP1 having the amino acid sequence of SEQ ID NO: 9 (AAV9 I-588), or between 589 and 590 (AAV9 I-589), and / or between 454 and 455 (I-454), between 455 and 456 (I-455), or between 456 and 457 (I-456). 23. It has a capsid consisting of approximately 60 VPs, and the VPs are (a) VP3; (b) VP1 and VP3; or (c) AAV or AAVLP as described in any one of sections 17 to 22, wherein the VP1, VP2, and VP3 proteins are preferably in a ratio of 1:1:10. 24. The AAV or AAVLP according to any one of claims 17 to 23, wherein the insert is a protein comprising a binding domain, the AAV comprises a genome adjacent to the ITR, is infectious, and preferably the genome adjacent to the ITR comprises a transgene. 25. The AAV or AAVLP according to any one of paragraphs 17 to 24, wherein the insert is a protein comprising a binding domain specific to the binding target, and the protein comprising the binding domain determines the directivity of the AAV or AAVLP to target cells expressing the binding target on its surface. 26. An AAV or AAVLP directed at a change target, as described in any one of paragraphs 17-25. 27. AAV or AAVLP as described in any one of items 17 to 26, formed by two or more viral proteins comprising different inserts consisting of at least about 75 to 400 amino acids, preferably about 75 to 300 amino acids, wherein the first insert comprises a first protein comprising a binding domain, and at least one further insert comprises a further protein comprising a binding domain, and / or an immunogenic protein or a portion thereof. 28. The AAV or AAVLP described in any one of items 17 to 27, wherein the insert is a protein containing an antigen-binding domain. 29. The AAV or AAVLP according to paragraph 28, wherein the insert is a protein comprising an antigen-binding domain specific to a target antigen, and the antigen-binding domain determines the directivity of the AAV or AAVLP to a target cell expressing the target antigen on its surface. 30. An AAV or AAVLP according to any one of claims 25 to 27, formed by two or more viral proteins comprising different inserts consisting of at least about 75 to 400 amino acids, preferably about 75 to 300 amino acids, wherein the first insert is a protein comprising an antigen-binding domain specific to a first target antigen, and at least one further insert is a protein comprising an antigen-binding domain specific to a further target antigen. 31. An AAV or AAVLP according to any one of items 28-30, wherein the protein comprising the antigen-binding domain is a single-domain antibody (sdAb) single-strand variable fragment (scFv) or an antibody mimetic (e.g., antikalin, afibody, adonectin, monobody, DARPin, afimer, or afitin). 32. A pharmaceutical composition comprising an AAV or AAVLP as described in any one of claims 17 to 31, further comprising at least one pharmaceutically acceptable excipient. 33. AAV or AAVLP as described in any one of sections 17-31, for use in treatment. 34. AAV or AAVLP as described in any one of sections 17-31, for use in gene therapy. 35. A pharmaceutical composition according to item 32 for use in treatment, preferably gene therapy. 36. The AAV or AAVLP described in item 33 or 34, or the pharmaceutical composition for use described in item 35, wherein the AAV or AAVLP, or the pharmaceutical composition, is administered via a route such as the nasal mucosa, sublingual, oral, oral, intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably the AAV or AAVLP is administered by inhalation via a route such as the nasal, oral, and / or mucosal route. 37. A method for producing AAV or AAVLP, (i) A step of preparing a cell comprising at least one DNA sequence containing a cap gene and a rep gene, at least one DNA sequence containing an adenovirus helper sequence, and optionally, at least one DNA sequence containing a genome adjacent to an ITR (wherein the cap gene encodes a protein containing an insert consisting of about 75 to 400 amino acids, preferably about 75 to 300 amino acids, in a capsid-forming viral protein (VP) at the insertion site (I) at the apex of variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, and optionally, one or both sides of the insert are adjacent to a linker containing one or more amino acids preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof); (ii) A step of culturing the cells under conditions that enable the production of AAV or AAVLP; (iii) a step of purifying the AAV or AAVLP; and A method comprising, depending on the circumstances, the step of formulating the AAV or AAVLP into a pharmaceutical composition by adding at least one pharmaceutically acceptable excipient. 38. A method for producing a pharmaceutical composition containing AAV or AAVLP, (i) A step of preparing a cell comprising at least one DNA sequence containing a cap gene and a rep gene, at least one DNA sequence containing an adenovirus helper sequence, and optionally, at least one DNA sequence containing a genome adjacent to an ITR (wherein the cap gene encodes a protein containing an insert consisting of about 75 to 400 amino acids, preferably about 75 to 300 amino acids, in a capsid-forming viral protein (VP) at the insertion site (I) at the apex of variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, and optionally, one or both sides of the insert are adjacent to a linker containing one or more amino acids preferably selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof); (ii) A step of culturing the cells under conditions that enable the production of AAV or AAVLP; (iii) a step of purifying the AAV or AAVLP; and (iv) A method comprising the step of formulating the AAV or AAVLP into a pharmaceutical composition by adding at least one pharmaceutically acceptable excipient. 39. The method according to item 37 or 38, wherein the insert is a protein comprising a binding domain, preferably a protein comprising an antigen-binding domain. [Examples]

[0119] Example 1: Structural model of AAV capsid and AAV viral protein (VP) To analyze whether large proteins (such as the SARS-CoV-2 spike (S) protein) can be introduced into AAV's VP1, we modeled the structures of AAV2 VP3 (SEQ ID NO: 2) and the obtained AAV capsid (insert: SEQ ID NO: 11, AAV2 VP1 with insert: SEQ ID NO: 13) of AAV2 (HtW2_S1.1) containing the spike receptor binding domain of the SARS-CoV-2 S protein. Comparative structural modeling using the Robetta protein structure prediction service (https: / / robetta.bakerlab.org / ) was performed on HtW2_S1.1 VP3 (Figure 1B) based on AAV2 WT (PDB 6ih9) (Figure 1C), and processed using Chimera software (https: / / www.cgl.ucsf.edu / chimera / ). Based on a novel prediction of the protein structure of VP3 in AAV2(HtW2_S1.1) containing the SARS-CoV-2 S protein spike receptor-binding domain as an insert at position I-587, the same protein was further modeled using RoseTTAFold (https: / / robetta.bakerlab.org / ) (Figure 1D). The published structure based on AAV2 WT (PDB 6ih9) is shown in the same orientation in Figure 1E. The corresponding predicted 60-mer capsid structure of HtW2_S1.1 (a novel AAV variant HtW2_S1.1 with a 202-amino acid insertion containing part of the SARS-CoV-2 S1 spike with the linker amino acids adjacent) was analyzed from two different angles, as shown in Figures 1F and 1E. As can be seen from Figures 1B, D, E, and G, the large 200+ amino acid insert did not impair the major giant VP1 or capsid structure. Example 2: Cloning of AAV Rep / Cap plasmids pHtW2_S1.1 and pHtW9_S1.1, and preparation of HtW2_S1.1 filled particles and HtW2_S1.1 empty particles.

[0120] The RBD-spike sequence with sequence number 11 was amplified by PCR using primers pHtW2_S1.1_fw and pHtW2_S1.1_rv (see Table 3). The PCR product was subjected to agarose gel electrophoresis, and the 567 bp amplicon was excised and purified (QiaQuick gel extraction kit, Qiagen). This amplicon was then used in a Gibson assembly reaction with a 7590 bp pRC'99 plasmid (containing the Rep and Cap sequences of AAV2) that had been double digested with MluI / SgsI and purified on an agarose gel. The Gibson assembly was successful, yielding the 8198 bp AAV2 Rep / HtW2_S1.1 Cap pHtW2_S1.1 helper plasmid shown in Figure 13.

[0121] To clone the pHtW9_S1.1 version, the pAAV2 / 9 Cap plasmid (7390 bp) was used as a template for a PCR reaction using primers pHtW9_S1.1_BB_fw and pHtW9_S1.1_BB_rv (see Table 3). The 7390 bp amplicon was purified on an agarose gel and used as a linear skeleton for the Gibson assembly reaction.

[0122] The RBD-spike sequence with sequence number 11 was amplified by PCR using primers pHtW9_S1.1_fw and pHtW9_S1.1_rv (see Table 3). The resulting 654 bp amplicon was purified on an agarose gel (QiaQuick gel extraction kit, Qiagen) and used in a Gibson assembly reaction to obtain the 7993 bp AAV2 Rep / HtW9_S1.1 Cap pHtW9_S1.1 plasmid shown in Figure 15. The plasmid was confirmed to be correctly assembled by Sanger sequencing.

[0123] The RBD-spike sequence encoding SEQ ID NO: 69 was amplified by PCR using primers HtW2_Var_S1.2_fw and HtW2_Var_S1.2_rv (see Table 3). The PCR product was subjected to agarose gel electrophoresis, and the 685 bp amplicon was excised and purified (QiaQuick gel extraction kit, Qiagen). This amplicon was then used in a Gibson assembly reaction with a 7590 bp pRC'99 plasmid (containing the Rep and Cap sequences of AAV2) that had been double digested with MluI / SgsI and purified on an agarose gel. The Gibson assembly was successful, yielding the 8222 bp AAV2 Rep / HtW2_S1.2 Cap helper plasmid pHtW2_S1.2, shown in Figure 14. [Table 3]

[0124] Using plasmids pHtW2_S1.1 or pHtW9_S1.1, AAV particles were generated containing a novel AAV capsid variant with an RBD-spike sequence having the amino acid sequence of SEQ ID NO: 11. The resulting novel HtW2_S1.1 cap protein has the amino acid sequence of SEQ ID NO: 13. Using plasmids pHtW2_S1.2, AAV particles were generated containing a novel AAV capsid variant with a spike sequence (containing a binding domain and an additional T cell epitope at the N-terminus) having the amino acid sequence of SEQ ID NO: 69. The resulting novel HtW2_S1.2 cap protein has the amino acid sequence of SEQ ID NO: 70. AAV generation was performed using the standard technique described by Michalakis et al. (Mol. Ther. (2010); 18(12): 2057-2063). In short, it involves a self-complementary (sc)AAV cis-plasmid (pTransgene plasmid) containing pHtW2_S1.1, pHtW2_1.2, or pHtW9_S1.1, and in some cases a CMV-eGFP expression cassette (AAV-sc-CMV-eGFP) (Hacker et al. (2005) "Improvement of gene transfer and transduction efficiency toward tumor cells mediated by adeno-associated virus serotypes 1-5" J Gene Med 7(11):1429-38) and an adenovirus helper plasmid for packaging (e.g., J. Samulski, Chapel Hill, NC; Xiao, Li and Samulski (1998) "Construction of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus" J. Virol. 72: AAVs were constructed by transfecting HEK293T cells with equal molar amounts of pXX6 from 2224-2232. HtW2_S1.1 empty particles were constructed in the absence of the pTransgene plasmid (which has an ITR adjacent to the sc-CMV-eGFP expression cassette). To construct AAVs, HEK293T cells were simultaneously transfected with 20 μg of DNA per petri dish into 15 150-mm petri dishes at 80% concentration.By doing so, the AAV Rep / Cap plasmids, namely pHtW2_S1.1, pHtW2_S1.2, or pHtW9_S1.1, were simultaneously transfected in the same molar ratio as the adenovirus helper plasmid (e.g., pXX6 from J. Samulski, Chapel Hill, NC; Xiao, Li and Samulski (1998) "Construction of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus" J. Virol. 72: 2224-2232), and, in the case of full-capsid construction, in the same molar amount as the pTransgene plasmid containing the CMV-eGFP cassette adjacent to the ITR. After 48 hours, AAV was isolated from a HEK293 cell pellet resuspended in 150 mM NaCl and 50 mM Tris-HCl (pH 8.5), subjected to several freeze-thaw cycles, and treated with benzonase (50 U / ml) at 37°C for 30 minutes. Cell debris was removed by centrifugation, and the supernatant was further treated for an iodixanol gradient. Alternatively, AAV was isolated from the cell culture supernatant after sedimentation with 8% polyethylene glycol (PEG) 8000 at 4°C. The PEG-AAV precipitate was then treated with benzonase (50 U / ml) at 37°C for 30 minutes, followed by further treatment for an iodixanol gradient. Iodixanol gradient ultracentrifugation was performed at 18°C ​​at 70,000 rpm for 1 hour and 45 minutes as described (Zolotukhin et al. (1999) "Recombinant adeno-associated virus purification using a novel method improves infectivity titer and yield" Gene Ther. 6: 973-985). Subsequently, virions were recovered from the 40% iodixanol phase and titrated by real-time PCR. Real-time PCR was performed using the AAV2 ITR-free qPCR assay described in D'Costa et al., (2016) Practical use of recombinant AAV vector reference standards: Focusing on vector genome titration by ITR-free qPCR, 5:16019, using a Step one Plus (Thermo Fisher Scientific, Germany). Example 3: AAVx affinity purification chromatography of AAV vectors

[0125] AAV2 WT particles, HtW2_S1.1 filled particles (i.e., filled AAV particles (HtW2_S1.1 particles loaded with the sc-CMV-eGFP genome)), and HtW2_S1.1 empty particles (i.e., empty AAV particles (HtW2_S1.1 particles produced in the absence of a pTransgene plasmid (having an ITR adjacent to the sc-CMV-eGFP expression cassette))) were purified using a Poros Capture Select AAVx affinity purification column (obtained from Thermo Fisher Scientific) according to the manufacturer's instructions. The chromatograms in Figure 2 show the elution of AAV2 WT particles (Figure 2A), HtW2_S1.1 filled particles (Figure 2B), and HtW2_S1.1 empty particles (Figure 2C). HtW2_S1.1-filled particles and HtW2_S1.1-empty particles bound to an AAVx affinity purification column eluted similarly, but slightly later, after the start of the elution process (indicated by the ml value of elution buffer on the x-axis). Therefore, despite the large insertion of over 200 amino acids, both HtW2_S1.1-filled particles and HtW2_S1.1-empty particles still retained their ability to bind to the AAVx affinity purification column. HtW2_S1.2 and HtW9_S1.1 were purified similarly. Example 4: Transduction assay of AAV vector in HeLa cells

[0126] Innate HeLa cells were transduced with AAV-sc-CMV-eGFP, either packaged with AAV2 WT or with a novel AAV variant HtW2_S1.1 containing a 202-amino acid insertion including the SARS-CoV-2 S1 spike RBD portion with the amino acid sequence of SEQ ID NO: 11 adjacent to the linker amino acids, at different infection multiplicities ((MOI): 250 MOI, 500 MOI, and 1000 MOI). Cell images were acquired at 24 and 48 hours and analyzed using bright-field epifluorescence microscopy (Evos FL, Thermo Fisher Scientific). After imaging at 48 hours, cells were harvested and the percentage of eGPF-positive cells was analyzed using a Countess II FL automated cell counter (Thermo Fisher Scientific).

[0127] Surprisingly, as can be seen in Figure 3, despite the large insertion of over 200 amino acids, HtW2_S1.1 still retained its ability to infect and transduce human cells, even at a very low MOI of 250.

[0128] To overexpress the ACE2 receptor in heLa cells, native heLa cells were transiently transfected with a plasmid containing the amino acid sequence of SEQ ID NO: 30, under the control of a CMV promoter. After 48 hours, heLa cells were transduced with AAV-sc-CMV-eGFP packaged in a novel AAV variant, HtW2_S1.1, which contains a 202-amino acid insertion including the SARS-CoV-2 S-spike RBD portion with the amino acid sequence of SEQ ID NO: 11 adjacent to a linker amino acid, at two different MOIs (250 MOI and 500 MOI). Cell images were acquired at 24 and 48 hours using epifluorescence microscopy (Evos FL, Thermo Fisher Scientific) (Figure 4A). After acquiring images at 48 hours, cells were harvested and the percentage of eGPF-positive cells was analyzed using a Countess II FL automated cell counter (Thermo Fisher Scientific) (Figure 4B).

[0129] Interestingly, as can be seen in Figure 4, the insertion of 202 amino acids, including the SARS-CoV-2 S-spike RBD portion with the amino acid sequence of SEQ ID NO: 11, gave the particles higher infectivity and higher transduction efficiency into ACE2-transfected HeLa cells. This confirms that AAV has been successfully repurposed and behaves like SARS-CoV-2 in terms of cell targeting. This means that the incorporated SARS-CoV-2-derived protein sequence folds correctly, giving the AAV particles the immunogenic properties of SARS-CoV-2. Therefore, these data suggest that HtW2_S1.1 has SARS-CoV-2-like targeting and exhibits greater infectivity towards human cells overexpressing ACE2. Example 5: Transduction assay of AAV vector in HEK293T cells stably transfected with ACE2.

[0130] After stably transfecting HEK293T cells with ACE2, the cells were transduced using the HtW2_S1.2 vector. Figure 5A shows representative epifluorescence images from natural (left column, -ACE2) HEK293T cell cultures or stable ACE2-overexpressing (right column, +ACE2) HEK293T cell cultures 48 hours after transduction using AAV-sc-CMV-eGFP packaged with the novel AAV variant HtW2_S1.2, which has an insertion of 206 amino acids (SEQ ID NO: 69) containing a portion of the SARS-CoV-2 S1 spike protein, at MOI 250 (top row), MOI 500 (middle row), or MOI 1000 (bottom row) (right panel). Figure 5B shows the percentage of eGFP-positive cells, measured by a Countess II FL automated cell counter, 48 hours after transduction of AAV-sc-CMV-eGFP, packaged with the novel AAV variant HtW2_S1.2, which contains a 206-amino acid insertion including a linker amino acid-adjacent SARS-CoV-2 S1 spike protein binding domain, using MOI 250, 500, and 1000 in natural HEK293T cell cultures or HEK293T cell cultures overexpressing stable ACE2. Data were analyzed using one-way ANOVA and Sidaq multiple comparison tests. The data shown in Figure 5 confirm that HtW2_S1.2 also exhibits SARS-CoV-2-like targeting and higher infectivity towards human cells overexpressing ACE2. Example 6: Immunogenicity of an AAV vector containing a portion of the SARS-CoV-2 spike protein as an insert in rabbits.

[0131] Humoral responses to HtW were evaluated in rabbits. Rabbits (Zika, 12 weeks old, female) were given wild-type AAV empty capsids (AAV2 WT, AAV9 WT) or HtW empty capsids (HtW2_S1.1, HtW2_S1.2, or HtW9_S1.1) for approximately 7.5 × 10⁻¹⁰ days. 815 μl of the capsid particles (cp) / μl were administered subcutaneously. All animal studies were conducted by private service providers in accordance with European and national regulations concerning animal experimentation (European Directive 2010 / 63 / EU; German Animal Welfare Act). For primary immunization, the supernatant was emulsified in Freund's complete adjuvant (Sigma-Aldrich, #344289), and booster injections were administered subcutaneously at 4-week intervals (days 30, 60, 90, and 120) using Freund's incomplete adjuvant (Sigma-Aldrich, #F5506). Blood was collected 10 days after each booster injection (i.e., after the first injection, on days 40, 70, 100, and 130), with the final blood collection performed 150 days after the first injection (Figure 6A).

[0132] The immunogenicity of HtW capsids was evaluated by ELISA from blood collected 10 days after the second booster injection in rabbits immunized with wild-type AAV empty capsids (AAV2 WT, AAV9 WT) or HtW empty capsids (HtW2_S1.1, HtW2_S1.2, or HtW9_S1.1). Serum was separated by centrifugation at 1,200 g for 20 minutes, and SARS-CoV-2 specific IgG titers were determined by ELISA using recombinant RBD (Acro Biosystems, # SPD-C52H2) as the antigen. Antiserum titers were determined as described in Frey A et al., J Immunol Methods, 1998, 221(1-2):35-41. The IgG endpoint titers against SARS-CoV-2 wild-type RBD are shown in Figure 6B. The smallest amount of HtW empty capsid, below the detection limit of silver staining, induced a strong immune response in rabbits. SARS-CoV-2 RBD-specific IgG signaling was not generated in AAV2 WT or AAV9 WT (Figure 6B). Furthermore, rabbit serum collected 10 days after the first (blood sample 1), second (blood sample 2), and third (blood sample 3) booster injections using an empty AAV vector was analyzed for endpoint antibody titers. Titers were determined by ELISA using SARS-CoV-2 RBD (Acro Biosystems, #SPD-C52H2) as the antigen. Antibody responses were examined using peroxidase-labeled anti-IgG (Abcam, #ab6721) and anti-IgM secondary antibodies (Abcam, #97195). Endpoint titers of SARS-CoV-2 RBD-specific IgG and IgM antibodies are shown in Figure 6C. SARS-CoV-2 wild-type RBD expressing HtW2 S1.2 already induces a strong and sustained humoral IgG response after the first booster injection. IgM titers are weaker but increase with booster injection.

[0133] To further evaluate the immunogenicity of HtW2_S1.1, HtW2_S1.2, and HtW9_S1.1, dot blot assays (final blood collection) were performed on the surface of polyfluorinated (PVDF) membranes, stained with commercially available antibodies and rabbit serum, and using AAV vectors of various titers. The method is schematically shown in Figure 7A. The PVDF membranes were activated with 100% MeOH and incubated in TBS-T buffer. The AAV vector spots were 3 × 10⁶. 7 ~3×10 5The entire vector genome / dot (Figure 7B) was formed on the surface of a PVDF membrane and air-dried. The membrane was then blocked in TBST with 5% milk powder, washed, and incubated with the corresponding serum diluent in TBST containing 1% milk powder at room temperature (RT) for 1 hour. After washing in TBST, the membrane was incubated with HRP-labeled secondary antibody at room temperature for another 1 hour, followed by washing, standard luminescence reaction, and detection. Specifically, dot blots were labeled with rabbit monoclonal anti-SARS-CoV-2 spike S1 antibody (αSARS-CoV-2 spike S1, catalog number: 40150-R007) from Sino Biological at a dilution of 1:500 (Figure 7C), or with anti-HtW2_S1.1 serum, anti-HtW2_S1.2 serum, or anti-HtW9_S1.1 serum (αHtW2_S1.1, αHtW2_S1.2, or αHtW9_S1.1) at a dilution of 10,000 (Figures 7D-E).

[0134] When using commercially available control antibodies, no signal was obtained for AAV2 wild-type (WT) and AAV9 WT, whereas HtW2_S1.1, HtW2_S1.2, and HtW9_S1.1 all showed a signal with the second smallest amount of vector spotted on the surface of the PVDF membrane (e.g., 1.5 × 10⁻⁶). 6Strong immune signals were generated up to the dots (1.5E6) of the entire vector genome (Figure 7C). All rabbit serum immunized with HtW2_S1.1, HtW2_S1.2, or HtW9_S1.1 could be used at high dilutions. Figure 7D shows the results from dot blots labeled with a 1:10000 dilution of serum (αHtW2_S1.1) from rabbits immunized with HtW2_S1.1 empty capsids. Signals were obtained in spot-like dots of HtW2_S1.1, HtW2_S1.2, or HtW9_S1.1. No signal was obtained in AAV9 WT, and only a weak signal was observed in AAV2 WT. Figure 7E shows the results from dot blots labeled with a 1:10,000 dilution of serum (αHtW2_S1.2) from rabbits immunized with HtW2_S1.2 empty capsids. Very strong signals were obtained for both HtW2_S1.1 and HtW2_S1.2. A weaker signal was observed for HtW9_S1.1 at a higher dilution. No signal was obtained for AAV2 WT and AAV9 WT. Figure 7F shows the results from dot blots labeled with a 1:10,000 dilution of serum (αHtW9_S1.1) from rabbits immunized with HtW9_S1.1 empty capsids. Very strong signals were obtained for both HtW2_S1.1, HtW2_S1.2, and HtW9_S1.1. No signal was obtained for AAV9 WT, and only a weak signal was observed for AAV2. These results confirm the presence of the SARS-CoV-2 spike S1 sequence on the novel HtW capsid and demonstrate that all HtW variants induce a strong SARS-CoV-2 specific humoral immune response. Clear cross-reactivity was observed between variants, confirming that the immune response was directed towards the inserted sequence rather than the AAV capsid skeleton. Example 7: Evaluation of serum from HtW-modified capsids immunized with Comirnaty

[0135] As shown in Figure 8A, 3 × 10 7 ~3×10 5 In addition to the individual, 1 x 10 8AAV vectors, consisting of whole vector genomes, were spotted on the surface of a PVDF membrane and stained with serum from Cormirnaty-vaccinated patients in a dot blot. The dot blots were labeled with a 1:500 dilution of serum collected from patients one week after the second vaccination with Cormirnaty (BNT162b2, Biontech / Pfizer). Special signals were obtained in dots that spotted with HtW2_S1.1, HtW2_S1.2, or HtW9_S1.1. The signal was strongest with HtW9_S1.1. No signal was obtained in AAV9 WT, and only a very weak signal was seen in AAV2 WT. This is not surprising, as up to 80% of the total population is serologically positive for AAV2. (C) The same dot blot relabeled with a 1:10000 dilution of serum (αHtW9_S1.1) from rabbits immunized with empty capsids after detachment. Strong to very strong signals were obtained for all three HtW variants (HtW2_S1.2, HtW9_S1.1, and the weaker HtW2_S1.1). No signal was obtained in the AAV9 WT, and only a weak signal was observed in the AAV2 WT. These results confirm that antibodies induced in humans in response to the approved mRNA vaccine Comirnaty (BNT162b2) cross-react with all three HtW-modified capsids.

[0136] In independent experiments, human PBMCs stimulated with HtW were shown to activate a variety of immune cells (including T cells (CD3+, CD4+, and CD8+), B cells (CD19+), and NK cells (CD56+)), as indicated by the activation marker CD69 and an increase in the number of activated subtype cells. This further confirms that HtW is highly immunogenic and has the potential to be used as a vaccine (primary and / or booster) to prevent or treat SARS-CoV-2 infection. Example 8: Neutralization assay using rabbit serum immunized with HtW9_S1.1 in HEK293T cells (HEK293T+ACE2) that stably express ACE2.

[0137] HtW2_S1.1 and HtW2_S1.2 vectors containing the sc-CMV-eGFP genome were pre-incubated at 37°C with different dilutions (1:1000, 1:5000, 1:10000) of serum obtained by immunizing rabbits with HtW9_S1.1 empty capsids. These pre-incubated HtW vector / serum dilutions were then transduced into HEK293T (+ACE2 cells) stably expressing ACE2 at an MOI of 250. Cell images were acquired after 48 hours and analyzed using bright-field epifluorescence microscopy (EvosFL, Thermo Fisher Scientific) (Figure 9A). After acquiring images at 48 hours, cells were harvested and the percentage of eGFP-positive cells was analyzed using a Countess II FL automated cell counter (Thermo Fisher Scientific). At a serum dilution of 1:1000, potent or complete neutralization of both the HtW_S1.1 and HtW_S1.2 vectors was achieved, as evidenced by the absence of eGFP signaling. Neutralization was stronger for the HtW_S1.2 vector. Example 9: Structural model of AAV capsid containing scFv and AAV viral protein (VP1)

[0138] In addition to a portion of the SARS-CoV-2 spike (S) protein, which functions as both an immunogenic protein and a protein containing a binding domain, an anti-GFP scFv antibody fragment (SEQ ID NO: 73) was cloned into AAV2 VP1 at position I-587. The structures of AAV2 VP3 (SEQ ID NO: 2) and the resulting AAV capsid containing the anti-GFP scFv antibody fragment (AAV2_αGFP scFv) (insert: SEQ ID NO: 73, AAV2 VP1 with insert: SEQ ID NO: 74) were modeled using RoseTTAFold (https: / / robetta.bakerlab.org / ) based on new predictions of protein structure (Figure 16A). The corresponding predicted 60-mer capsid structure of AAV2-αGFP is shown in Figure 16B. As can be seen from this model, the large 200+ amino acid anti-GFP scFv did not impair the major giant capsid structure. This indicates that, in addition to the binding portion of the SARS-CoV-2 spike protein, proteins containing antigen-binding domains (such as scFv) can also be introduced into the AAV capsid, specifically directing the vector to its target. [Table 4-1] [Table 4-2] [Table 4-3]

Claims

1. Adeno-associated virus (AAV) or adeno-associated virus-like particles (AAVLP), In each of the approximately 60 viral proteins (VPs) that form the capsid, an insert having approximately 200 to 400 amino acids is inserted into an insertion site (I) at the top of the variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, and a linker containing one or more amino acids is adjacent to one or both sides of the insert, and the AAV or AAVLP has a capsid consisting of the approximately 60 VPs, and the VPs are, (a) VP3; (b) VP1 and VP3; or (c) The AAV or AAVLP, which are VP1, VP2, and VP3 proteins.

2. (a) The apex of VR-VIII corresponds to amino acids 585-592 (I-585-I-592) of VP1 of AAV 1, 2, 3, 6, 7, 8, 9, or 10 having the amino acid sequence of SEQ ID NOs. 1, 2, 3, 6, 7, 8, 9, or 10, respectively, amino acids 583-589 of VP1 of AAV 4 having the amino acid sequence of SEQ ID NOs. 4, or amino acids 574-580 of VP1 of AAV 5 having the amino acid sequence of SEQ ID NOs. 5, and / or (b) The apex of VR-IV corresponds to amino acids 450-460 (I-450-I-460) of VP1 of AAV 1, 2, 3, 6, 7, 8, 9, or 10 having the amino acid sequence of SEQ ID NOs. 4, respectively, and AAV 5 having the amino acid sequence of SEQ ID NOs. 4 The AAV or AAVLP according to claim 1, corresponding to amino acids 445-455 (I-445-I-455) of VP1 of AAV 4, or amino acids 439-449 (I-439-I-449) of VP1 of AAV 5 having the amino acid sequence of SEQ ID NO:

5.

3. Derived from AAV serotype 1 (AAV1), 2 (AAV2), 8 (AAV8), or 9 (AAV9), (a) The insertion site is located between two amino acids corresponding to amino acid positions 587 and 588 of AAV2 VP1 having the amino acid sequence of SEQ ID NO: 2 (AAV2 I-587), or between 588 and 589 (AAV2 I-588), and / or between 453 and 454 (AAV2 I-453); (b) The insertion site is located between two amino acids corresponding to amino acid positions 587 and 588 of AAV1 VP1 having the amino acid sequence of SEQ ID NO: 1 (AAV1 I-587), between 588 and 589 (AAV1 I-588), or between 589 and 590 (AAV1 I-589), and / or between 454 and 455 (AAV1 I-454), between 455 and 456 (AAV1 I-455), or between 456 and 457 (AAV1 I-456); (c) The insertion site is located between two amino acids corresponding to amino acid positions 588 and 589 of AAV8 VP1 having the amino acid sequence of SEQ ID NO: 8 (AAV8 I-588), or between 589 and 590 (AAV8 I-589), and / or between 455 and 456 (I-455), between 456 and 457 (I-456), or between 457 and 458 (I-457), or (d) The AAV or AAVLP according to claim 1 or 2, wherein the insertion site is located between two amino acids corresponding to amino acid positions 588 and 589 of AAV9 VP1 having the amino acid sequence of SEQ ID NO: 9 (AAV9 I-588), or between 589 and 590 (AAV9 I-589), and / or between 454 and 455 (I-454), between 455 and 456 (I-455), or between 456 and 457 (I-456).

4. Having a capsid consisting of about 60 VPs, the VPs are AAV or AAVLP according to any one of claims 1 to 3, wherein the VP1, VP2, and VP3 proteins have a ratio of 1:1:

10.

5. An AAV or AAVLP according to any one of claims 1 to 4, wherein the ITR contains an adjacent genome and is infectious.

6. The AAV or AAVLP according to any one of claims 1 to 5, wherein the insert is (a) an immunogenic protein or a portion thereof, and / or (b) a protein comprising a binding domain.

7. The insert is an immunogenic protein or a part thereof. (a) The AAV contains an ITR adjacent to a genome and is infectious; (b) An immunogenic protein or a portion thereof is inserted at the apex of VR-VIII and the apex of VR-IV, and the immunogenic protein or a portion thereof inserted at the apex of VR-VIII and the immunogenic protein or a portion thereof inserted at the apex of VR-IV are the same or different; and / or (c) The AAV or AAVLP according to claim 6, wherein the AAV or AAVLP is formed by two or more viral proteins comprising different inserts having at least about 75 to 300 amino acids, each of which is an immunogenic protein or a part thereof, and is either an immunogenic protein or immunogenic moiety from different proteins, or different immunogenic moieties from the same protein.

8. The AAV or AAVLP according to claim 6 or 7, wherein the immunogenic protein or a portion thereof is a viral, bacterial, or parasitic protein or a portion thereof, and / or the immunogenic protein or a portion thereof is a tumor antigen.

9. The immunogenic protein or a part thereof (a) It is part of the coronavirus spike (S) protein; (b) being part of the SARS-CoV-2 spike (S) protein; and / or (c) AAV or AAVLP according to claim 8, which is part of the SARS-CoV-2 S protein comprising the amino acid sequence of SEQ ID NOs: 11, 12, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or 69.

10. The AAV or AAVLP according to any one of claims 1 to 6, wherein the insert is a protein comprising a binding domain, the AAV comprises a genome adjacent to the ITR, is infectious, and the genome adjacent to the ITR comprises a transgene.

11. The AAV or AAVLP according to any one of claims 1 to 6 and 10, wherein the insert is a protein containing an antigen-binding domain.

12. A pharmaceutical composition comprising AAV or AAVLP as described in any one of claims 1 to 11, further comprising at least one pharmaceutically acceptable excipient.

13. A pharmaceutical composition comprising AAV or AAVLP according to any one of claims 1 to 10, wherein the insert is an immunogenic protein or a part thereof.

14. A pharmaceutical composition comprising AAV or AAVLP as described in any one of claims 1 to 10, The aforementioned composition, (a) A pharmaceutical composition for the treatment or prevention of a disease induced by a virus, bacterium, or parasite, wherein the immunogenic protein or a portion thereof is an immunogenic protein of the virus, bacterium, or parasite; or, (b) A pharmaceutical composition for the treatment or prevention of cancer, wherein the immunogenic protein or a portion thereof is a tumor antigen or a portion thereof; Pharmaceutical composition.

15. A pharmaceutical composition comprising AAV or AAVLP according to claim 10 or 11 for therapeutic purposes.

16. A method for producing AAV or AAVLP, (i) A step of preparing a cell comprising at least one DNA sequence including a cap gene and a rep gene, and at least one DNA sequence including an adenovirus helper sequence, wherein the cap gene encodes a protein in which each of about 60 viral proteins (VPs) that form a capsid contains an insert having about 200 to 400 amino acids at an insertion site (I) at the top of the variable region VIII and / or variable region IV (VR-VIII and / or VR-IV) of the VP, and one or more linkers containing one or more amino acids are adjacent to one or both sides of the insert, and the AAV or AAVLP has a capsid consisting of the about 60 VPs; (ii) A step of culturing the cells under conditions that enable the production of the AAV or AAVLP; and (iii) A step of purifying the AAV or AAVLP; A method that includes this.

17. AAV or AAVLP according to any one of claims 1 to 11, wherein a linker comprising one or more amino acids selected from the group consisting of A (Ala), G (Gly), S (Ser), T (Thr), L (Leu), and combinations thereof is adjacent to one or both sides of the insert.

18. The AAV or AAVLP according to claim 5, wherein the genome adjacent to the ITR contains a transgene.

19. The AAV or AAVLP according to claim 11, wherein the insert is a protein comprising an antigen-binding domain that is a single-domain antibody (sdAb), a single-strand variable fragment (scFv), or an antibody mimetic.

20. The method according to claim 16, wherein the cell of step (i) further comprises at least one DNA sequence in which the ITR is adjacent to a genome.

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