Adenovirus vector and its use

Abstract

Provided herein are chimeric adenoviral vectors that can be used to induce a protective immune response and / or express a transgene in a subject in need thereof.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 909,853, filed on October 3, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] The present invention relates to biotechnology. More particularly, it relates to the field and use of adenoviral vectors, such as replication - defective adenoviral vectors, for delivering antigens to a host and inducing an immune response.

[0003] Reference to Electronically Submitted Sequence Listing This application includes a sequence listing submitted electronically via EFS - Web as an ASCII - formatted sequence listing having a file name of "004852.120WO1 Sequence Listing", a creation date of August 13, 2020, and a size of 262 kb. The sequence listing submitted through EFS - Web is part of this specification and is hereby incorporated by reference in its entirety.

Background Art

[0004] Recombinant adenovirus vectors are widely applied in gene therapy applications and vaccines. AdV-5 vector-based vaccines have been shown to induce strong and protective immune responses in various animal models (see, for example, WO 2001 / 02607; WO 2002 / 22080; Shiver et al., Nature 415:331 (2002); Letvin et al., Ann. Rev. Immunol. 20:73 (2002); Shiver and Emini, Ann. Rev. Med. 55:355 (2004)). However, the usefulness of recombinant AdV-5 vector-based vaccines may be limited by the high seroprevalence of AdV-5-specific neutralizing antibodies (NAbs) in the human population. The presence of anti-AdV-5 immunity has been shown to substantially suppress the immunogenicity of AdV-5-based vaccines in studies in mice, rhesus monkeys, and humans.

[0005] One promising strategy to avoid the presence of existing immunity in individuals previously infected with or treated by the most common human adenoviruses, such as AdV-5, involves the development of recombinant vectors from adenovirus serotypes that do not encounter such existing immunity. One such strategy involves the use of chimeric adenoviruses that involve replacement of native capsid protein sequences (e.g., hexon and / or fiber protein sequences) with capsid protein sequences from adenoviruses with low (or no) seroprevalence. SUMMARY OF THE INVENTION

[0006] Accordingly, there is a need in the art for alternative adenovirus vectors that can be produced in large quantities, do not encounter existing immunity in the host, are still immunogenic, and can induce a strong immune response against antigens encoded by heterologous nucleic acids inserted into the vector.

[0007] Provided herein is an isolated nucleic acid sequence encoding a chimeric adenovirus capsid or a functional derivative thereof. The chimeric adenovirus capsid or a functional derivative thereof can include, for example, a fiber polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 11, a hexon polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 12, and a penton polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the fiber polypeptide sequence includes the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the hexon polypeptide sequence includes the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the penton polypeptide includes the amino acid sequence of SEQ ID NO: 13.

[0008] Also provided herein are vectors comprising the isolated nucleic acids described herein. In certain embodiments, the vector is an adenovirus vector.

[0009] In certain embodiments, the adenovirus vector further comprises an E1 deletion. In certain embodiments, the adenovirus vector further comprises an E3 deletion. In certain embodiments, the adenovirus vector is a chimeric adenovirus vector comprising one or more adenovirus nucleic acid sequences from at least one of human adenovirus-4, human adenovirus-5, human adenovirus-26, or human adenovirus-35. The adenovirus vector can include, for example, human adenovirus-5 (HAdV-5) E4orf6. In certain embodiments, the adenovirus vector can include, for example, a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0010] In certain embodiments, the adenovirus vector further comprises at least one transgene. In certain embodiments, the at least one transgene is located in an E1 deletion, an E3 deletion, and / or adjacent to a right inverted terminal repeat (rITR).

[0011] Also provided are recombinant cells comprising the adenovirus vectors described herein. Also provided is a method of producing an adenovirus vector. The method comprises: (a) culturing the recombinant cells described herein under conditions for producing an adenovirus vector; and (b) isolating the adenovirus vector from the recombinant cells.

[0012] Also provided is a pharmaceutical composition comprising the adenovirus vector described herein and a pharmaceutically acceptable carrier.

[0013] Also provided is a method of inducing an immune response in a subject in need thereof. The method comprises administering to the subject the pharmaceutical composition described herein.

[0014] Also provided is a method of manufacturing a pharmaceutical composition, the method comprising combining the adenovirus vector described herein with a pharmaceutically acceptable carrier.

[0015] Also provided is a method of expressing a transgene in a subject in need thereof. The method comprises: (a) identifying a subject in need of the transgene to be expressed; (b) contacting the subject with an adenovirus vector comprising the transgene described herein; and (c) expressing the transgene in the subject. In certain embodiments, expression of the transgene in a subject in need thereof treats or prevents a disease or disorder. In certain embodiments, contacting the subject with the vector can include, for example, isolating cells from the subject and contacting the cells with the vector. The subject can be, for example, a human subject.

[0016] The foregoing summary, and the following detailed description of the preferred embodiments of the present application, will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present application is not limited to the exact embodiments shown in the drawings.

Brief Description of the Drawings

[0017]

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[0018] The present disclosure is based, at least in part, on the isolation of chimeric adenovirus vectors comprising a chimeric capsid polypeptide or a functional derivative thereof, wherein the chimeric capsid polypeptide comprises a fiber polypeptide and a hexon polypeptide from a first adenovirus (e.g., human adenovirus - 42) and a penton polypeptide from a second adenovirus (e.g., human adenovirus - 20). The adenovirus vector can induce an immune response while maintaining a low seroprevalence. The adenovirus vector can be formulated for use as a vaccine to induce protective immunity against a specific antigen of interest. The adenovirus vector can also be constructed to express a transgene of interest in a subject in need thereof.

[0019] Various publications, papers, and patents are cited or described throughout the background and the specification; each of these documents is hereby incorporated by reference in its entirety. The discussion of documents, acts, materials, devices, articles, etc. contained herein is for the purpose of providing context for the present invention. Such discussion is not to be construed as an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed herein.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, specific terms used herein have the meaning as defined herein.

[0021] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include references to the plural unless the context clearly dictates otherwise.

[0022] Unless otherwise specified, any numerical values such as concentrations or concentration ranges described herein should be understood to be modified in all cases by the term "about". Thus, the numerical values typically include ±10% of the recited value. For example, a concentration of 1 mg / mL includes from 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes from 0.9% (w / v) to 11% (w / v). As used herein, the use of a numerical range explicitly includes all possible sub-ranges, all individual numerical values within such range including integers and fractional values, unless the context clearly dictates otherwise.

[0023] Unless otherwise indicated, the term "at least" before a series of elements is to be understood as referring to all the elements of that series. One of ordinary skill in the art will be able to recognize or ascertain many equivalents to the particular embodiments of the invention described herein using only routine experimentation. Such equivalents are intended to be encompassed by the present invention.

[0024] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are meant to cover a non-exclusive inclusion, such that a composition, mixture, process, method, article, or apparatus that comprises a list of elements does not necessarily consist of only those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive disjunction and not an exclusive disjunction. For example, the condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0025] As used herein, the conjunction “and / or” between a plurality of recited elements is understood to encompass both alternative and combined alternatives. For example, when two elements are joined by “and / or”, the first alternative refers to the applicability of the first element in the absence of the second element. The second alternative refers to the applicability of the second element in the absence of the first element. The third alternative refers to the applicability of both the first and second elements together. Any of these alternatives is within the scope of the meaning of the term “and / or” as used herein and is thus understood to meet the requirement. The simultaneous applicability of multiple alternatives is also within the scope of the meaning of the term “and / or” and is thus understood to meet the requirement.

[0026] As used herein, the term “consists of” as used throughout this specification and the claims, or variations such as “consist of” or “consisting of” indicate that a recited integer or group of integers includes, but does not allow the addition of any additional integer or group of integers to the specified method, structure, or composition.

[0027] As used herein, the term “consists essentially of” as used throughout this specification and the claims, or variations such as “consist essentially of” or “consisting essentially of” indicate that a recited integer or group of integers includes, and optionally includes any recited integer or group of integers that do not substantially change the basic or novel characteristics of the recited method, structure, or composition. See M.P.E.P. Section 2111.03.

[0028] As used herein, "subject" means any animal, preferably a mammal, and most preferably a human. The term "mammal" as used herein encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and more preferably include humans.

[0029] Also, when referring to the dimensions or properties of components of the preferred invention, the terms "about", "approximately", "generally", "substantially", and similar terms used herein indicate that the recited dimensions / characteristics are not strict boundaries or parameters and do not exclude minor differences therefrom that would be understood by one of ordinary skill in the art as being functionally identical or similar. At a minimum, such references, including numerical parameters, include variations that do not vary the least significant digit using mathematical and industrial principles (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.) accepted in the art.

[0030] The terms "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences (e.g., hexon and fiber polypeptides and the polynucleotides encoding them) refer to two or more sequences or subsequences that are identical or have a specified percentage of amino acid residues or nucleotides that are identical when compared and aligned for maximum correspondence using any of the following sequence comparison algorithms or by visual inspection.

[0031] In a sequence comparison, typically one sequence functions as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, coordinates of subsequences are designated as necessary, and the program parameters of the sequence algorithm are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters.

[0032] Optimal alignment of arrays for comparison can be done, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0033] Examples of algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. In this algorithm, high-scoring sequence pairs (HSPs) are identified by first identifying short words of length W in the query sequence that either match or satisfy a positive-valued threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating a search to find longer HSPs that contain them. Next, word hits are extended in both directions along each sequence as far as possible while the cumulative alignment score can be increased.

[0034] The cumulative score for a nucleotide sequence is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatched residue; always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of a word hit in each direction stops when the cumulative alignment score drops by an amount X from the maximum value achieved; when the cumulative score becomes zero or less due to the accumulation of one or more negatively scored residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) by default uses a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program by default uses a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0035] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences occurred by chance. For example, a nucleic acid is considered similar to a reference sequence if the minimum sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0036] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide if, for example, the two polypeptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0037] As used herein, the term "polynucleotide," which is used interchangeably with "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to unmodified RNA or DNA, or any polyribonucleotide or polydeoxyribonucleotide that can be either modified RNA or DNA. "Polynucleotide" includes single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA that is a mixture of single-stranded and double-stranded regions, and hybrid molecules that can be either single-stranded or, more generally, double-stranded or a mixture of single-stranded and double-stranded regions, comprising DNA and RNA. Further, "polynucleotide" refers to triple-stranded regions containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contains one or more modified bases, and DNA or RNA having modified backbones, for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. Various modifications can be made to DNA and RNA; thus, "polynucleotide" includes polynucleotides in chemically, enzymatically, or metabolically modified forms, as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also includes relatively short nucleic acid chains often referred to as oligonucleotides.

[0038] As used herein, the term "vector" is a replicon into which another nucleic acid segment can be operably inserted to effect replication or expression of the segment.

[0039] As used herein, the term "host cell" refers to a cell that contains a nucleic acid molecule of the invention. A "host cell" can be any type of cell, e.g., a primary cell, a cell in culture, or a cell derived from a cell line. In one embodiment, a "host cell" is a cell transfected with a nucleic acid molecule of the invention. In another embodiment, a "host cell" is a progeny or potential progeny of such a transfected cell. The progeny of a cell may or may not be identical to the parent cell due to, for example, mutations that occur in subsequent generations or environmental influences, or due to the incorporation of the nucleic acid molecule into the host cell genome.

[0040] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term encompasses transcription of a gene into RNA. The term also encompasses translation of the RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications. An expressed polypeptide can be present within the cytoplasm of a host cell, in an extracellular environment such as the growth medium of a cell culture, or can be immobilized on a cell membrane.

[0041] As used herein, the terms "peptide", "polypeptide", or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by those skilled in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term includes naturally or artificially modified amino acid polymers, such as other operations or modifications such as formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids), as well as other modifications known in the art.

[0042] The peptide sequences described herein are written according to the usual convention that the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although amino acid isomers are known, unless otherwise specified, the L-form of the amino acid is represented.

[0043] As used herein, the term "protective immunity" or "protective immune response" means that a vaccinated subject can control infection by the pathogen against which vaccination was performed. The pathogen can be, for example, an antigenic gene product or antigenic protein, or a fragment thereof. Usually, a subject that has acquired a "protective immune response" develops only mild to moderate clinical symptoms or no symptoms at all. Usually, a subject having a "protective immune response" or "protective immunity" against a specific pathogen will not die as a result of infection by that pathogen.

[0044] The term "adjuvant" is defined as one or more substances that cause stimulation of the immune system. In this context, an adjuvant is used to enhance the immune response against the adenoviral vector of the present invention.

[0045] As used herein, the term "antigenic gene product or fragment thereof" or "antigenic protein" can include bacterial, viral, parasitic, or fungal proteins, or fragments thereof. Preferably, the antigenic protein or antigenic gene product can raise a defensive immune response in a host, for example, induce an immune response against a disease or infection (e.g., a bacterial, viral, parasitic, or fungal disease or infection), and / or produce in a subject an immunity that protects the subject from the disease or infection (i.e., vaccination) against the disease or infection.

[0046] As used herein, the term "chimeric" means a gene, nucleic acid, protein, peptide, or polypeptide that contains two or more genes, nucleic acids, proteins, peptides, or polypeptides that are not normally joined together. A "chimeric" gene, nucleic acid, or protein can be a fusion between two or more unrelated sequences (e.g., two or more separate nucleic acids encoding two or more separate proteins). A "chimeric" gene, nucleic acid, or protein can be a fusion between two or more related sequences (e.g., the nucleic acids encode the same protein, but the nucleic acids are from different source materials, i.e., one nucleic acid is derived from one human adenovirus and the other nucleic acid is derived from a second unrelated human adenovirus).

[0047] Adenoviral vector Exposure to a specific adenovirus results in an immune response against that specific adenovirus serotype, which can affect the efficacy of an adenovirus vector. Since infection with human adenoviruses is common in humans, the prevalence of neutralizing antibodies against human adenoviruses in the human population is high. The presence of such neutralizing antibodies in an individual can be expected to reduce the efficacy of gene transfer vectors based on the human adenovirus backbone. One way to avoid this reduction in efficacy is to replace the epitopes of the adenovirus capsid protein that are targets of neutralizing antibodies. The target sequences on the capsid protein can be replaced with protein sequences from other adenoviruses (e.g., chimeric adenoviruses of multiple human adenoviruses) that have a low prevalence and thus neutralizing antibodies are rare in the human population.

[0048] "Capsid protein" refers to a protein or a functional fragment or derivative thereof on the capsid of an adenovirus (e.g., AD20 and / or AD42) that is involved in determining the serotype and / or tropism of the adenovirus. Capsid proteins typically include fiber, penton, and / or hexon proteins. In certain embodiments, the capsid protein is the whole or full-length of the adenovirus capsid protein. In other embodiments, the capsid protein is a fragment or derivative of the full-length adenovirus capsid protein. In certain embodiments, the hexon, penton, and fiber encoded by the adenovirus vector of the present invention are derived from different adenovirus backgrounds.

[0049] As used herein, "chimeric adenovirus capsid" refers to a capsid of adenovirus origin that includes fiber, penton, and / or hexon polypeptides, where the fiber, penton, and / or hexon polypeptides are derived from different adenovirus origins (e.g., the fiber and hexon polypeptides of Ad42 and the penton polypeptide of Ad20).

[0050] "Hexon polypeptide" refers to an adenovirus hexon coat protein, its functional fragments, and derivatives.

[0051] "Fiber polypeptide" refers to an adenovirus fiber protein, its functional fragments, and derivatives.

[0052] "Penton polypeptide" refers to an adenovirus penton protein, its functional fragments, and derivatives.

[0053] One target of neutralizing antibodies against adenovirus is the hexon protein, which is a major coat protein. Replacing the hexon protein or the variable sequences within the hexon protein that define the serotype and bind to neutralizing antibodies with those from rare adenoviruses in the human population may enable the construction of adenovirus vectors that are less susceptible to neutralization by antibodies commonly found in humans.

[0054] The hexon hypervariable region (HVR) is the region of the hexon polypeptide that exhibits the highest variability among different adenovirus serotypes. In general, these HVRs are thought to correspond to the surfaces of the hexon protein trimers that are exposed to the solvent (in the context of intact virus particles), and, accordingly, are predicted to be important determinants of adenovirus neutralization via antibodies (Roberts et al., Nature 441:239-43 (2006)). Thus, replacing the hexon HVR of a given adenovirus vector with that of an adenovirus with low (or no) seroprevalence in humans represents a possible means of avoiding existing anti-vector humoral immunity in the human target population. As a result, multiple studies have explored the concept of hexon chimerism, mainly involving the replacement of the hexon sequence within HAdV-5-based vectors (Roy et al., J Virol. 72:6875-9 (1998); Gall et al., J Virol. 72:10260-4 (1998); Youil et al., Hum. Gene Ther. 13:311-20 (2002); Wu et al. J Virol. 76:12775-82 (2002); Roy et al., Virology. 333:207-14 (2005); Roberts et al., Nature 441:239-43 (2006); Bradley et al., J Virol. 86:1267-72 (2012); Yu et al., Biochem Biophys Res Commun. 421:170-6 (2012); Bruder et al, PLoS One. 7(4):e33920 (2012)).

[0055] A second target for neutralizing antibodies against adenovirus is the fiber protein. Replacing the fiber protein with a fiber sequence from a rare human - origin adenovirus, and more preferably replacing the variable sequence within the fiber protein, may also enable the construction of adenovirus vectors that are less susceptible to neutralization by antibodies commonly found in humans. The combination of the above - mentioned fiber substitution and hexon substitution can provide further resistance to neutralization by antibodies generally present in the human population.

[0056] A third target for neutralizing antibodies against adenovirus is the penton protein. Replacing the penton protein with the penton sequence of a rare human - origin adenovirus can also enable the construction of adenovirus vectors that are less susceptible to neutralization by antibodies commonly found in humans. The combination of the above - mentioned hexon substitution, fiber substitution, and penton substitution can provide further resistance to neutralization by antibodies generally present in the human population.

[0057] The present disclosure provides an isolated nucleic acid sequence encoding a chimeric adenovirus capsid or a functional derivative thereof. The chimeric adenovirus capsid or a functional derivative thereof can include, for example, a fiber polypeptide, a hexon polypeptide, and a penton polypeptide. The fiber and hexon polypeptides can be derived from, for example, a first adenovirus (e.g., human adenovirus - 42), and the penton polypeptide can be derived from, for example, a second adenovirus (e.g., human - adenovirus - 20).

[0058] A "functional derivative" of a polypeptide appropriately refers to a modified version of the polypeptide in which, for example, one or more amino acids of the polypeptide may be deleted, inserted, modified, and / or substituted. A derivative of an unmodified adenovirus capsid protein is, for example, (a) when an adenovirus containing the derivative capsid protein in its capsid retains substantially the same or a lower serum sickness rate compared to an adenovirus containing the unmodified capsid protein; and / or (b) when an adenovirus containing the derivative capsid protein in its capsid retains substantially the same or a higher host cell infectivity compared to an adenovirus containing the unmodified capsid protein; and / or (c) when an adenovirus containing the derivative capsid protein in its capsid retains substantially the same or a higher immunogenicity compared to an adenovirus containing the unmodified capsid protein; and / or (d) when an adenovirus containing the derivative capsid protein in its capsid retains substantially the same or a higher level of transgene productivity compared to an adenovirus containing the unmodified capsid protein, it is considered to be functional.

[0059] An "adenovirus vector" refers to a recombinant vector that is derived from or contains at least a part of the adenovirus genome.

[0060] In a preferred embodiment, the chimeric adenovirus capsid comprises, for example, a fiber polypeptide having an amino acid sequence that exhibits at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 11; a hexon polypeptide having an amino acid sequence that exhibits at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 12; and a penton polypeptide having an amino acid sequence that exhibits at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the fiber polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the hexon polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the penton polypeptide comprises the amino acid sequence of SEQ ID NO: 13.

[0061] In a preferred embodiment, there is provided a vector, preferably an adenovirus vector, comprising the isolated nucleic acid disclosed herein. The adenovirus vector comprises an isolated nucleic acid encoding a chimeric adenovirus capsid or a functional derivative thereof, wherein the chimeric adenovirus capsid or a functional derivative thereof comprises a fiber polypeptide having an amino acid sequence that has at least 95% identity to the amino acid sequence of SEQ ID NO: 11, a hexon polypeptide having an amino acid sequence that has at least 95% identity to the amino acid sequence of SEQ ID NO: 12, and a penton polypeptide having an amino acid sequence that has at least 95% identity to the amino acid sequence of SEQ ID NO: 13.

[0062] Typically, the adenovirus vector of the present invention contains the entire recombinant adenovirus genome, for example, on a plasmid, cosmid, or baculovirus vector. The nucleic acid molecule of the present invention can be obtained by cloning or can be in the form of RNA or DNA produced synthetically. The DNA can be double-stranded or single-stranded.

[0063] One of ordinary skill in the art will recognize that elements from multiple serotypes can be combined in a single adenovirus vector, for example, a human or simian adenovirus. Thus, chimeric adenovirus vectors can be generated that combine desirable properties from different serotypes. Thus, in some embodiments, the chimeric adenovirus vector of the present invention can combine the absence of existing immunity to chimeric hexon and / or fiber polypeptide sequences with the high-level antigen and / or transgene delivery and presentation capabilities of existing adenovirus vectors such as rAd4, rAd5, rAd26, or rAd35.

[0064] The advantages of adenoviral vectors for use as vaccines and / or as vehicles for transgene expression can include, but are not limited to, ease of manipulation, good manufacturability on a large scale, and an excellent safety record based on years of experience in the research, development, manufacture, and clinical testing of numerous adenoviral vectors. Adenoviral vectors used as vaccines generally provide a good immune response, including a cellular immune response, against the protein encoded by the transgene or the antigenic gene product encoded by the transgene. The adenoviral vectors according to the invention can be based on any type of adenovirus and, in certain embodiments, are human adenoviruses which can be of any group or serotype. In a preferred embodiment, the recombinant adenovirus is based on a human adenovirus from group A, B, C, D, E, F, or G. In other preferred embodiments, the recombinant adenovirus is based on human adenovirus serotype 5, 11, 26, 34, 35, 48, 49, or 50. In other embodiments, it is a simian adenovirus such as a chimpanzee or gorilla adenovirus and can be of any serotype. In certain embodiments, the recombinant adenovirus is based on chimpanzee adenovirus type 1, 3, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33, 34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50, 67, or SA7P.

[0065] In a more preferred embodiment, the chimpanzee adenovirus vector of the second composition is ChAdV3. Recombinant chimpanzee adenovirus serotype 3 (ChAd3 or cAd3) is an adenovirus of subgroup C with properties similar to human adenovirus serotype 5 (Ad5). ChAd3 has been shown to be safe and immunogenic in human trials evaluating a candidate vaccine against hepatitis C virus (HCV) (Barnes E, et al. 2012 Science translational medicine 4: 115ra1). ChAd3-based vaccines have been reported to be able to induce an immune response comparable to that of human Ad5 vector vaccines. See, for example, Peruzzi D, et al. 2009 Vaccine 27: 1293-300 and Quinn KM, et al. 2013 J Immunol 190: 2720-35; WO 2005 / 071093 pamphlet: WO 2011 / 0130627 pamphlet, etc.

[0066] Adenoviral vectors, methods for their construction, and methods for their propagation are well known in the art and are described, for example, in U.S. Patent Nos. 5,559,099, 5,837,511, 5,846,782, 5,851,806, 5,994,106, 5,994,128, 5,965,541, 5,981,225, 6,040,174, 6,020,191, and 6,113,913, as well as in Thomas Shenk, “Adenoviridae and their Replication” and M. S. Horwitz, “Adenoviruses” in Chapters 67 and 68, respectively, of Virology, B. N. Fields et al., eds., 3d ed., Raven Press, Ltd., New York (1996), and in other references described herein. Typically, the construction of adenoviral vectors involves standard molecular biological techniques as described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), Watson et al., Recombinant DNA, 2d ed., Scientific American Books (1992), and Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, NY (1995), and in other references described herein.

[0067] In certain embodiments, the adenoviral vector contains a deletion in E1 and / or E3. The deletion of E1 or E3 can include, for example, a complete deletion of the gene or a partial deletion that functionally inactivates the E1 or E3 gene product. Thus, in certain embodiments, the adenovirus is replication-deficient, for example, because it contains a deletion in the E1 region of the genome. As is known to those skilled in the art, when essential regions are deleted from the adenoviral genome, the functions encoded by these regions must be provided in trans, preferably by producer cells, i.e., if part or all of the E1, E2, and / or E4 regions are deleted from the adenovirus, they must be present in producer cells, for example, integrated into their genome or in the form of a so-called helper adenovirus or helper plasmid. The adenovirus may also have a deletion in the E3 region that is not required for replication, and thus such a deletion need not be complemented. One or more of the E1, E2, E3, and E4 regions can also be inactivated by other means, such as by inserting the transgene of interest (usually linked to a promoter) into a region to be inactivated.

[0068] Producer cells that can be used (sometimes referred to in the art and herein as "packaging cells" or "complementing cells") can be any producer cells in which the desired adenovirus can be propagated. For example, the propagation of recombinant adenovirus vectors is carried out in producer cells that complement the deficiencies of adenovirus. Such producer cells preferably have at least the adenovirus E1 sequence in their genomes, thereby being able to complement recombinant adenoviruses having deletions in the E1 region. Any E1-complementing producer cells such as human retinal cells immortalized by E1, such as 911 or PER.C6 cells (see U.S. Patent No. 5,994,128), E1-transformed amniotic cells (see European Patent No. 1230354), E1-transformed A549 cells (e.g., see WO 98 / 39411 pamphlet, U.S. Patent No. 5,891,690), GH329:HeLa (Gao et al, 2000, Hum Gene Ther 11: 213-19), 293, etc. can be used. In certain embodiments, the producer cells are, for example, HEK293 cells, or PER.C6 cells, or 911 cells, or IT293SF cells, etc. The production of adenovirus vectors in producer cells is outlined in (Kovesdi et al., 2010, Viruses 2: 1681-703).

[0069] In certain embodiments, the adenoviral vector is a chimeric adenoviral vector comprising one or more human adenoviral nucleic acid sequences. The nucleic acid of the human adenovirus can be selected, for example, from human adenovirus-4 (Ad-4), human adenovirus-5 (Ad-5), human adenovirus-26 (Ad-26), or human adenovirus-35 (Ad-35). In certain embodiments, the E1-deleted adenoviral vector comprises the E4-orf6 coding sequence of the adenovirus of human Ad5. This enables the growth of such adenoviruses in well-known complementing cell lines that express the E1 gene of Ad5, such as, for example, 293 cells or PER.C6 cells (see, for example, Fallaux et al., 1998, Hum Gene Ther 9: 1909-17, Havenga et al., 2006, J Gen Virol 87: 2135-43; see International Publication No. 03 / 104467 pamphlet, which are incorporated herein by reference in their entirety).

[0070] In certain embodiments, the adenoviral vector comprises a transgene. A "transgene" refers to a heterologous nucleic acid that is a nucleic acid not naturally present in the vector, and according to the present invention, the transgene can encode an antigenic gene product or antigenic protein that induces an immune response in a subject. The transgene can also encode a therapeutic protein for treating or preventing a disease in a subject in need thereof. The transgene can be introduced into the vector, for example, by standard molecular biological techniques. The transgene can be cloned, for example, into the deleted E1 or E3 region of the adenoviral vector, or into the region between the E4 region and the rITR. The transgene is generally operably linked to an expression control sequence. In a preferred embodiment, the transgene is inserted at the transgene insertion site.

[0071] Optionally, the chimeric adenovirus capsid sequences, and / or transgenes, comprising the fiber, hexon, and penton polypeptide sequences according to embodiments of the invention can be codon-optimized to ensure proper expression in the host (e.g., human) being treated. Codon optimization is a technique widely applied in the art.

[0072] The transgene may be under the control (i.e., operably linked) of an adenovirus-derived promoter (e.g., the major late promoter), or under the control of a heterologous promoter. Examples of suitable heterologous promoters include the CMV promoter and the RSV promoter. Preferably, the promoter is located upstream of the heterologous gene of interest within the expression cassette.

[0073] In a preferred embodiment, the adenovirus vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0074] Pharmaceutical and / or immunogenic compositions In another general aspect, there is provided a pharmaceutical composition comprising an isolated polynucleotide of the invention, an isolated polypeptide of the invention, a vector of the invention, an adenovirus vector of the invention, and / or a host cell of the invention, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" means a product comprising an isolated polynucleotide of the invention, an isolated polypeptide of the invention, an isolated vector (e.g., an adenovirus vector) of the invention, and / or a host cell of the invention, together with a pharmaceutically acceptable carrier. The polynucleotides, polypeptides, vectors, and / or host cells of the invention, and compositions containing them, are also useful in the manufacture of medicaments for the therapeutic uses recited herein.

[0075] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, vesicle containing lipid, microsphere, liposomal encapsulation, or other substance well known in the art for use in pharmaceutical formulations. It will be understood that the properties of the carrier, excipient or diluent depend on the route of administration for a particular use. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic substance that does not interfere with the effectiveness or biological activity of the compositions according to the invention. According to certain embodiments, any pharmaceutically acceptable carrier suitable for use in polynucleotide, polypeptide, vector, and / or host cell pharmaceutical compositions, in view of the present disclosure, may be used in the present invention.

[0076] Formulating a pharmaceutically active ingredient with a pharmaceutically acceptable carrier is known in the art, for example, in Remington: The Science and Practice of Pharmacy (e.g., 21st edition (2005) and subsequent editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, tonicity adjusters, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers may be used in formulating the pharmaceutical compositions of the present invention.

[0077] Pharmaceutical compositions can be formulated, for example, for the expression of a transgene in a subject in need thereof (i.e., a pharmaceutical composition designed for the expression of a transgene in a subject in need thereof). Pharmaceutical compositions can be formulated, for example, for the expression of an antigenic polypeptide or an antigenic fragment thereof (e.g., a pharmaceutical composition for inducing an immune response in a subject in need thereof).

[0078] A pharmaceutical composition designed to induce an immune response in a subject in need thereof can be referred to as, for example, an immunogenic composition. An immunogenic composition is a composition containing an immunologically effective amount of a purified or partially purified adenoviral vector for use in the present invention. Said composition can be formulated as a vaccine (also referred to as an "immunogenic composition") according to methods well known in the art. Such a composition can include an adjuvant for enhancing the immune response. The optimal ratio of each component in the formulation can be determined by techniques well known to those skilled in the art in view of the present disclosure.

[0079] The immunogenic composition according to an embodiment of the present invention can be produced using methods known to those skilled in the art in view of the present disclosure. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oils. Saline, dextrose or other sugar solutions, glycols such as ethylene glycol, propylene glycol, polyethylene glycol can be included.

[0080] The immunogenic composition useful in the present invention can include an adjuvant. Adjuvants suitable for co-administration according to the present invention include QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, adjumax, PG-026, GSK-I, AS01, AS03, AS04, AS15, GcMAF, beta-arrestin, MPC-026, adjuvax, CpG ODN, beta-fectin, Alum, and MF59, and should be potentially safe, highly tolerable and effective in a subject.

[0081] Other adjuvants that can be administered include lectins, growth factors, cytokines and lymphokines, such as alpha interferon, gamma interferon, platelet-derived growth factor (PDGF), granulocyte colony-stimulating factor (gCSF), granulocyte macrophage colony-stimulating factor (gMCSF), tumor necrosis factor (TNF), epidermal growth factor (EGF), IL-I, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, or nucleic acids encoding these.

[0082] The compositions of the present invention may contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances well known to those skilled in the art. Such substances should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other substances may depend on the route of administration, for example, intramuscular, subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., intestinal), intranasal or intraperitoneal routes.

[0083] Method for inducing protective immunity and / or expressing an introduced gene Another general aspect of the present invention relates to a method for inducing an immune response and / or expressing an introduced gene in a subject in need thereof. The method may include, for example, identifying a subject in need thereof; contacting the subject in need thereof with the medicaments and / or immunogenic compositions described herein; and inducing an immune response and / or expressing an introduced gene in the subject in need thereof. In certain embodiments, the method may include, for example, administering to the subject a vaccine comprising an adenovirus vector described herein and a pharmaceutically acceptable carrier.

[0084] Also provided herein is a method for manufacturing a vaccine. The method includes combining an adenovirus vector described herein with a pharmaceutically acceptable carrier.

[0085] Any immunogenic composition according to embodiments of the invention, including but not limited to those described herein, can be used in the methods of the invention as a vaccine. Any pharmaceutical composition according to embodiments of the invention, including but not limited to those described herein, can be used in the methods of the invention for treating or preventing a disease in a subject in need thereof by expressing the desired transgene.

[0086] Administration of an immunogenic composition / vaccine / pharmaceutical composition containing a vector is typically intramuscular or subcutaneous. However, other modes of administration such as intravenous, dermal, intradermal, intranasal, etc. are equally contemplated. Intramuscular administration of an immunogenic composition can be achieved by injecting a suspension of the adenoviral vector using a needle. Alternative methods include the use of a needleless injection device (e.g., using a BIOJECTOR®) for administering the composition, or the use of a lyophilized powder containing the vaccine.

[0087] In the case of intravenous injection, dermal injection, subcutaneous injection, or injection into the affected area, the vector will be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has an appropriate pH, isotonicity, and stability. One skilled in the art can readily prepare an appropriate solution using an isotonic vehicle such as, for example, sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc. If desired, preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included. It is also possible to employ sustained release formulations.

[0088] Typically, administration will have a prophylactic purpose of generating an immune response against the antigen of interest (e.g., bacterial, viral, parasitic, and / or fungal pathogens) before infection or the onset of symptoms. Administration of an adenoviral vector expressing the transgene of interest may also have a prophylactic purpose in a subject in need thereof. For example, the subject in need thereof may have a reduced or absent endogenous expression of the gene corresponding to the transgene of interest. Diseases and disorders that can be treated or prevented according to the present invention include those in which an immune response can play a defensive or therapeutic role and / or in which the corrective expression of a transgene results in the normal function of cells in a subject in need thereof. In other embodiments, the adenoviral vector can be administered for post-exposure prophylaxis.

[0089] An immunogenic composition comprising a chimeric human adenoviral vector is administered to a subject to generate an immune response against the antigen of interest in the subject. The amount of the composition sufficient to induce a detectable immune response is defined as the "immunologically effective amount" or "effective amount" of the composition. The immunogenic compositions of the present invention can induce humoral as well as cellular immune responses. In typical embodiments, the immune response is a defensive immune response.

[0090] A pharmaceutical composition can be administered to a subject in need thereof in a therapeutically effective amount for treating or preventing a disease. A therapeutically effective amount means the amount of an adenoviral vector expressing a transgene of interest that results in the treatment of a disease, disorder, or condition; the amount that prevents or delays the progression of a disease, disorder, or condition; or the amount that reduces or completely alleviates the symptoms associated with a disease, disorder, or condition.

[0091] According to certain embodiments, a therapeutically effective amount means an amount of a therapeutic agent sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or ameliorating the severity of the disease, disorder, or condition being treated, or a symptom associated therewith; (ii) shortening the duration of the disease, disorder, or condition being treated, or a symptom associated therewith; (iii) preventing the progression of the disease, disorder, or condition being treated, or a symptom associated therewith; (iv) causing regression of the disease, disorder, or condition being treated, or a symptom associated therewith; (v) preventing the onset or development of the disease, disorder, or condition being treated, or a symptom associated therewith; (vi) preventing recurrence of the disease, disorder, or condition being treated, or a symptom associated therewith; (vii) reducing hospitalization of a subject having the disease, disorder, or condition being treated, or a symptom associated therewith; (viii) shortening the length of hospitalization of a subject having the disease, disorder, or condition being treated, or a symptom associated therewith; (ix) extending the survival period of a subject having the disease, disorder, condition, or a symptom associated therewith; (xi) suppressing or alleviating in a subject the disease, disorder, condition, or a symptom associated therewith; and / or (xii) enhancing or improving the prophylactic or therapeutic effect of another therapy.

[0092] As used herein, the terms "treat", "treating", and "treatment" are all intended to refer to an improvement or reversal of at least one measurable physical parameter related to a disease, disorder, or condition that may not necessarily be distinguishable in a subject but may become distinguishable in the subject. The terms "treat", "treating", and "treatment" may also refer to causing regression of, preventing progression of, or at least delaying progression of a disease, disorder, or condition. In certain embodiments, "treat", "treating", and "treatment" refer to alleviation of one or more symptoms related to a disease, disorder, or condition, prevention of onset or development, or shortening of duration. In certain embodiments, "treat", "treating", and "treatment" refer to preventing recurrence of a disease, disorder, or condition. In certain embodiments, "treat", "treating", and "treatment" refer to extending the survival period of a subject having a disease, disorder, or condition. In certain embodiments, "treat", "treating", and "treatment" refer to eliminating a disease, disorder, or condition in a subject.

[0093] The actual amount administered, and the rate and time course of administration will depend on the nature and severity of what is being treated. Decisions regarding treatment regimens, such as dosage, etc., are within the responsibility of the general practitioner and other physicians, or in the veterinary context the veterinarian, and will typically take into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the above techniques and protocols are described in Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. ed., 1980.

[0094] After production of the adenovirus vector and optional formulation into a composition of such particles, the vector can be administered to an individual, particularly a human or another primate. Administration can be performed on a human, or other mammal, such as a mouse, rat, hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, monkey, dog, or cat. Delivery to a non-human mammal need not be for therapeutic purposes, but can be used, for example, in an experimental context in investigating the mechanisms of the immune response to an adenovirus vector.

[0095] In one exemplary regimen, the adenovirus vector is administered (e.g., intramuscularly) in a volume ranging from about 100 μl to about 10 ml, containing a concentration of from about 10 4 to 10 12 virus particles / ml. Preferably, the adenovirus vector is administered in a volume ranging from 0.1 to 2.0 ml. For example, the adenovirus vector can be administered at 100 μl, 500 μl, 1 ml, 2 ml. More preferably, the adenovirus vector is administered at a volume of 0.5 ml. Optionally, the adenovirus vector can be administered at a concentration of about 10 7 vp / ml, 10 8 vp / ml, 10 9 vp / ml, 10 10 vp / ml, 5×10 10 vp / ml, 10 11 vp / ml, or 10 12 vp / ml. Typically, the adenovirus vector is administered to a human subject in an amount of from about 10 9 to about 10 12 virus particles (vp), more typically in an amount of from about 10 10 to about 10 12 vp in a single administration. For example, after an initial administration, a boost as described above can be performed.

[0096] Following the initial administration, a boost or kick can be performed from a vaccine / composition containing the same adenovirus vector encoding the antigen of interest and / or the transgene of interest, or from a vaccine / composition containing different adenovirus vectors encoding the same antigen of interest and / or the transgene of interest.

[0097] The composition can be presented in a kit, pack or dispenser which may contain one or more unit dosage forms containing the active ingredient, as desired. The kit can include, for example, a metal or plastic foil such as a blister pack. Instructions for administration can be attached to the kit, pack, or dispenser.

[0098] The compositions of the present invention can be administered alone or in combination with other therapies, either simultaneously or sequentially, depending on the condition being treated.

[0099] Embodiments The present invention also provides the following non-limiting embodiments.

[0100] Embodiment 1 is an isolated nucleic acid sequence encoding a chimeric adenovirus capsid or a functional derivative thereof, wherein the chimeric adenovirus capsid or a functional derivative thereof has a fiber polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 11, a hexon polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 12, and a penton polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 13, and is an isolated nucleic acid sequence.

[0101] Embodiment 2 is the isolated nucleic acid sequence according to Embodiment 1, wherein the fiber polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 11.

[0102] Embodiment 3 is the isolated nucleic acid according to Embodiment 1 or 2, wherein the hexon polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 12.

[0103] Embodiment 4 is the isolated nucleic acid according to any one of Embodiments 1 to 3, wherein the penton polypeptide contains the amino acid sequence of SEQ ID NO: 13.

[0104] Embodiment 5 is a vector containing the isolated nucleic acid according to any one of Embodiments 1 to 4.

[0105] Embodiment 6 is the vector according to Embodiment 5, wherein the vector is an adenovirus vector.

[0106] Embodiment 7 is the vector according to Embodiment 6, wherein the adenovirus vector further contains a transgene, and optionally, the transgene is a therapeutic transgene.

[0107] Embodiment 8 is the vector according to Embodiment 6 or 7, wherein the adenovirus vector further contains an E1 deletion.

[0108] Embodiment 9 is the vector according to any one of Embodiments 6 to 8, wherein the adenovirus vector further contains an E3 deletion.

[0109] Embodiment 10 is the vector according to any one of Embodiments 6 to 9, which is a chimeric adenovirus vector, wherein the adenovirus vector contains one or more adenovirus nucleic acid sequences from at least one of human adenovirus-4, human adenovirus-5, human adenovirus-26, or human adenovirus-35.

[0110] Embodiment 11 is the vector according to Embodiment 10, wherein the adenovirus vector contains human adenovirus-5 (HAdV-5) E4orf6.

[0111] Embodiment 12 is the vector according to any one of Embodiments 6 to 10, wherein the adenovirus vector contains a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0112] Embodiment 13 is the vector according to any one of Embodiments 6 to 12, wherein the transgene is located in the E1 deletion, the E3 deletion, and / or adjacent to the right inverted terminal repeat (rITR).

[0113] Embodiment 14 is a recombinant cell comprising the vector according to any one of Embodiments 5 to 12.

[0114] Embodiment 15 is a method for producing a vector, comprising: (a) growing the recombinant cell according to Embodiment 14 under conditions for the production of an adenovirus vector; and (b) isolating the vector from the recombinant cell.

[0115] Embodiment 16 is an immunogenic composition comprising the adenovirus vector according to any one of Embodiments 6 to 13 and a pharmaceutically acceptable carrier.

[0116] Embodiment 17 is a method for inducing an immune response in a subject in need thereof, the method comprising administering the immunogenic composition according to Embodiment 16 to the subject.

[0117] Embodiment 18 is a method for producing a vaccine, the method comprising combining the adenovirus vector according to any one of Embodiments 6 to 13 with a pharmaceutically acceptable carrier.

[0118] Embodiment 19 is an adenovirus vector comprising (a) at least one transgene; and (b) a nucleic acid encoding a chimeric adenovirus capsid or a functional derivative thereof, wherein the chimeric adenovirus capsid has a fiber polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 11, a hexon polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 12, and a penton polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 13.

[0119] Embodiment 20 is the adenovirus vector according to Embodiment 19, wherein the fiber polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 11.

[0120] Embodiment 21 is the adenovirus vector according to Embodiment 19 or 20, wherein the hexon polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 12.

[0121] Embodiment 22 is the adenovirus vector according to any one of Embodiments 19 to 21, wherein the penton polypeptide comprises the amino acid sequence of SEQ ID NO: 13.

[0122] Embodiment 23 is the adenovirus vector according to any one of Embodiments 19 to 22, wherein the adenovirus vector further comprises an E1 deletion.

[0123] Embodiment 24 is the adenovirus vector according to any one of Embodiments 19 to 23, wherein the adenovirus vector further comprises an E3 deletion.

[0124] Embodiment 25 is the adenovirus vector according to any one of Embodiments 19 to 24, which is a chimeric adenovirus vector in which the adenovirus vector contains one or more adenovirus nucleic acid sequences from at least one of human adenovirus-4, human adenovirus-5, human adenovirus-26, or human adenovirus-35.

[0125] Embodiment 26 is the adenovirus vector according to Embodiment 25, in which the adenovirus vector contains human adenovirus-5 (HAdV-5) E4orf6.

[0126] Embodiment 27 is the adenovirus vector according to any one of Embodiments 19 to 26, in which the adenovirus vector contains a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0127] Embodiment 28 is the adenovirus vector according to any one of Embodiments 19 to 27, in which the transgene is located in E1 deletion, E3 deletion, and / or adjacent to the right inverted terminal repeat (rITR).

[0128] Embodiment 29 is the adenovirus vector according to any one of Embodiments 19 to 28, in which the transgene is a therapeutic transgene.

[0129] Embodiment 30 is a recombinant cell containing the adenovirus vector according to any one of Embodiments 19 to 29.

[0130] Embodiment 31 is a method for producing an adenovirus vector, including: (a) growing the recombinant cell according to Embodiment 30 under conditions for the production of the adenovirus vector; and (b) isolating the adenovirus vector from the recombinant cell.

[0131] Embodiment 32 is a pharmaceutical composition comprising the adenovirus vector according to any one of Embodiments 19 to 29 and a pharmaceutically acceptable carrier.

[0132] Embodiment 33 is a method of inducing an immune response in a subject in need thereof, the method comprising administering the pharmaceutical composition according to Embodiment 32 to the subject.

[0133] Embodiment 34 is a method of manufacturing a vaccine, the method comprising combining the adenovirus vector according to any one of Embodiments 19 to 29 with a pharmaceutically acceptable carrier.

[0134] Embodiment 35 is a method of expressing a transgene in a subject in need thereof, the method comprising: a. Identifying a subject in need of the transgene to be expressed; b. Contacting the subject with the vector according to Embodiment 7 or the adenovirus vector according to any one of Embodiments 19 to 29; and c. Expressing the transgene in the subject.

[0135] Embodiment 36 is the method according to Embodiment 35, wherein the expression of the transgene in a subject in need thereof treats or prevents a disease or disorder.

[0136] Embodiment 37 is the method according to Embodiment 35, wherein contacting the subject with the vector comprises isolating cells from the subject and contacting the cells with the vector.

[0137] Embodiment 38 is the method according to any one of Embodiments 35 to 37, wherein the subject in need thereof is a human subject.

[0138] Embodiment 39 is the use of the vector according to Embodiment 7 for treating or preventing a disease or disorder in a subject in need thereof, the method comprising contacting the subject in need thereof with the vector, and contacting the subject in need thereof results in the expression of a therapeutic transgene.

[0139] Embodiment 40 is the use according to Embodiment 39, wherein contacting the subject with the vector comprises isolating cells from the subject and contacting the cells with the vector.

[0140] Embodiment 41 is the use of the adenovirus vector according to any one of Embodiments 19 to 29 for treating or preventing a disease or disorder in a subject in need thereof, the method comprising contacting the subject in need thereof with the adenovirus vector, and contacting the subject in need thereof results in the expression of a therapeutic transgene.

[0141] Embodiment 42 is the use according to Embodiment 41, wherein contacting the subject with the adenovirus vector comprises isolating cells from the subject and contacting the cells with the adenovirus vector.

Examples

[0142] [Example 1] Generation of an E1 and E3 deletion vector based on the novel adenovirus isolate Ad20-42-42. A novel human adenovirus isolate, Ad20-42-42 (SEQ ID NO: 1), was identified and sequenced. This human adenovirus isolate was found to be phylogenetically belong to the human adenovirus species D (HAdV-D), which is a natural chimera of HAdV-20 and HAdV-42. The penton gene is derived from HAdV-20, and the hexon and fiber genes are derived from HAdV-42. Figure 1 shows the genomic map of the Ad20-42-42 human adenovirus isolate.

[0143] Description of the three - plasmid system used for the generation of Ad20 - 42 - 42 - based Ad vectors An Ad20 - 42 - 42 - based recombinant adenovirus vector was generated using a three - plasmid system. This plasmid system consists of an "adapter plasmid" that covers the 5' end of the adenovirus genome, where the E1 region is deleted and replaced with an expression cassette retaining the gene of interest (LacZ, Luc+ or eGFP). The second "intermediate" plasmid covers the central part of the adenovirus genome and has no modifications. The 3' end of the adenovirus sequence is retained by the "right - end" plasmid. In the right - end construct, the E3 region is deleted and the native ORF6 / 7 in the E4 region is replaced with the ORF6 / 7 of HAdV - 5.

[0144]

Table 1

[0145] The plasmids carry viral vector sequences that overlap with each other at approximately 2000 nucleotides, enabling homologous recombination between these sequences in HEK293 or PER.C6® cells (Figure 2).

[0146] Design of the Ad20 - 42 - 42 - based Ad vector genome: The plasmid system was constructed by several steps of standard molecular cloning procedures. The Ad20 - 42 - 42 - based Ad vector genomes were each designed to contain an E1 deletion, an E3 deletion, different transgene insertion sites, and substitution of the native E4 open reading frames (orfs) 6 and orf6 / 7 with those of human adenovirus - 5 (HAdV - 5) (base pairs 32966 - 34077 of GenBank sequence AC_000008).

[0147] Adapter plasmid First, an E1-deleted adapter plasmid "pAdApt20-42-42.Empty" was constructed. The expression cassette was placed at the original position of the deleted E1 region. This cassette was driven by the major immediate-early promoter of cytomegalovirus (i.e., the "CMV promoter") and contained a polyadenylation signal derived from SV40 ("SV40 polyA"). The expression cassette was equipped with a multiple cloning site that facilitated the insertion of various genes of interest (LacZ, Luc+ and eGFP). The empty adapter plasmid pAdApt20-42-42 was constructed as follows.

[0148] Fragment 1 covering the wild-type adenovirus sequence of nucleotides 1 to 461 was generated by the 5'-adjacent PacI restriction enzyme site and 3'-adjacent AvrII site introduced by PCR primers. The PCR product of fragment 1 was double-digested with PacI and AvrII.

[0149] Fragment 2, containing nucleotides 3361 - 5908 of the SV40 polyA and wild-type adenovirus sequence, was generated by preparing two PCR amplicons and subsequently performing assembly PCR on these two products. The first PCR product containing "SV40 polyA" was amplified from a previously constructed plasmid (pAdApt26.Empty; Abbink et al., J. Virol. 81(9):4654 - 63 (2007)). An XbaI restriction site was introduced at the 5' end of the PCR product, while an Ad20 - 42 - 42 homologous fragment was introduced into the PCR product by the reverse primer. This overlap contained a natural XbaI recognition site (present in the Ad20 - 42 - 42 genome), which was intentionally disrupted by changing one base of the recognition site. This PCR fragment was 174 bp in length. The second PCR covered the Ad20 - 42 - 42 genome from the start of pIX (including pIX) to approximately the middle of the polymerase gene. The forward primer of this PCR contained an overlap with PCR product 1 containing SV40 polyA (XbaI recognition was disrupted). A PacI recognition site was introduced into this PCR fragment using the reverse primer. This PCR fragment was 2574 bp in length. Subsequently, assembly PCR was performed using PCR products 1 and 2 as templates to join these fragments. The product size of the assembly PCR was 2710 bp. Subsequently, this assembly PCR product was double-digested with XbaI and PacI.

[0150] A previously constructed plasmid (Abbink et al., J. Virol. 81(9):4654 - 63 (2007)) was digested with XbaI, AvrII, and PacI to obtain the plasmid backbone and the CMV promoter (fragments 2108 and 880). Subsequently, a four-point ligation was performed using the fragments of pAdApt26.Empty (fragments 2108 and 880), fragment 1, and fragment 2. As a result, the pAdApt20 - 42 - 42.Empty plasmid was obtained.

[0151] The reporter gene was inserted into the MCS by using unique KpnI, HIndIII, or BamHI sites together with the XbaI site and then ligating.

[0152] Intermediate plasmid The intermediate plasmid harbored the wild-type adenovirus genome from nucleotide positions 2088 to 18494 without modification. First, two PCR fragments were created. One covered the 5'-end of the intermediate fragment and had a PacI site incorporated into the forward primer, and the reverse primer was designed slightly downstream of the natural SbfI site in the adenovirus genome (product size: 2273 bp). The second PCR fragment created with a forward primer designed slightly upstream from another natural SbfI site in the adenovirus genome had the PacI site incorporated into the reverse primer (product size: 2407 bp). Both PCR fragments were digested with PacI and SbfI restriction enzymes.

[0153] The pBR322 subclone backbone plasmid with adjacent PacI sites (Abbink et al., J. Virol. 81(9):4654 - 63 (2007)) was digested with PacI (2086 bp). A three-point ligation was performed using the two double-digested PCR products and the PacI-digested, dephosphorylated pBr backbone plasmid. As a result, the plasmid pBR.Ad20-42-42.PacI-SbfI was obtained. The pBR.Ad20-42-42.PacI-SbfI plasmid obtained in the previous step was cut open with the SbfI restriction enzyme. Wild-type Ad20-42-42 genomic DNA was digested with the SbfI restriction enzyme, and an 11858-nucleotide-long fragment covering the wild-type genome from the polymerase gene to almost the middle of the pVI gene was ligated into the pBR.Ad20-42-42.PacI-SbfI plasmid. As a result, the "pBR.Ad20-42-42.SbfI final interm" plasmid was obtained.

[0154] Right-end plasmid Construction of pBrAd20-42-42.SrfI-rITR: The 5'-end of the right end of the Ad-20-42-42 genome was amplified by PCR (Fragment 1, 2098 bp) such that the natural SrfI-SbfI fragment was included in the PCR product, and the PacI recognition site was supplemented in the forward primer. Another fragment covering the 3'-end of the right end of the Ad-20-42-42 genome was prepared by introducing the SbfI recognition site with the forward primer and incorporating the PacI recognition site into the reverse primer. There was a natural MluI site near the 5'-end of the PCR product. Subsequently, both PCR products were digested with PacI and SbfI enzymes.

[0155] The pBR322 subclone backbone plasmid with adjacent PacI sites (Abbink et al., J. Virol. 81(9):4654-63 (2007)) was digested with PacI (fragment size: 2108 bp). The two PacI and SbfI digested PCR fragments obtained in the previous step were cloned into the pBR backbone to obtain the pBrSrfI-SbfI / MluI-rITR plasmid. Next, this plasmid was digested with SbfI and MluI. Ad20-42-42 wild-type genomic DNA was digested with SbfI and MluI, and the fragment between genomic nucleotide positions 17742 and 33714 was isolated and cloned into the above plasmid. As a result, pBrAd20-42-42 SrfI-rITR carrying the wild-type Ad20-42-42 genome from position 15373 to the last nucleotide of rITR (35187) was obtained.

[0156] Deletion of the E3 region: For the purpose of deleting the E3 region, two PCRs were performed using the pBrAd20-42-42 SrfI-rITR plasmid as a template. The first was designed upstream of the region to be deleted; the forward primer covered the natural AscI site, and the reverse primer incorporated the SpeI site. The second PCR was designed downstream of the E3 region. The reverse primer covered the natural EcoRI site of the Ad20-42-42 genome, while the forward primer incorporated the SpeI site. Subsequently, the first product was double-digested with SpeI and AscI, and the second product was double-digested with SpeI and EcoRI. The pBrAd20-42-42 Srf-rITR plasmid was digested with AscI and EcoRI, and a three-point ligation was performed using the digested plasmid and the PCR products. Therefore, the fragment between nucleotide positions 26673 and 30753 of the wild-type Ad20-42-42 genome was dropped out of the genome, and the sequences were ligated by the introduced SpeI site. As a result, the pBrAd20-42-42.SrfI-rITR.dE3 plasmid was obtained.

[0157] Replacement of the natural ORF6 / 7 with the homologous region of HAdV-5: To replace ORF6 / 7, three fragments were amplified by PCR. Two were designed to cover the regions upstream and downstream of the ORF6 / 7 to be replaced. The forward primer of the first PCR carried the AscI site, and the reverse primer of the second PCR carried the EcoRI site. The third PCR was performed to obtain the HAdV-5 ORF6 / 7 from a previously constructed plasmid (pBr.Ad26.SrfI-rITR.dE3.5orf6 (Abbink et al., J. Virol. 81(9):4654-63 (2007)), which partially overlapped with the other two PCR products. Next, these three PCR products were subjected to fusion PCR and subsequently double-digested with AscI and EcoRI. The pBrAd20-42-42.SrfI-rITR.dE3 plasmid was also digested with AscI and EcoRI, and the digested fusion PCR product was inserted by two-point ligation to obtain the final right-end plasmid: pBrAd20-42-42 Srf-rITR.dE3.5orf6.

[0158] Generation and production of an Ad20-42-42-based adenovirus vector Adenovirus vectors Ad20-42-42.LacZ.5ORF6, Ad20-42-42.Luc+.5ORF6, and Ad20-42-42.eGFP.5ORF6, each containing the adenovirus vector genome sequence, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively, were generated by transfection of the corresponding plasmids: (1) adapter plasmid "pAdApt20-42-42.LacZ" (SEQ ID NO: 3) or pAdApt20-42-42.Luc+ (SEQ ID NO: 4) or pAdApt20-42-42.eGFP (SEQ ID NO: 5); (2) intermediate plasmid "pBR.Ad20-42-42.SbfI final interm" (SEQ ID NO: 6; Figure 5); (3) right-end plasmid "pBrAd20-42-42 SrfI-rITR.dE3.5orf6" (SEQ ID NO: 7; Figure 6).

[0159] In E1-complemented HEK293 cells, transfection was performed using a three-plasmid system. Before transfection of HEK293 cells grown as adherent cultures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), the Ad vector genome plasmid was digested with PacI to release each adenoviral vector genome fragment from the plasmid. Transfection was carried out using Lipofectamine transfection reagent (Invitrogen; Carlsbad, CA) according to standard procedures. After rescue transfection, the virus was further amplified by several consecutive infections of HEK293 cell cultures. The virus was purified from the crude virus harvest using a two-step cesium chloride (CsCl) density gradient ultracentrifugation method as previously described (Havenga et al., “Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells,” J. Gen. Virol. 87(8): 2135-43 (2006)). The titer of virus particles (VP) was measured by a spectrophotometry-based procedure as previously described (Maizel et al., “The polypeptides of adenovirus: I. Evidence for multiple protein components in the virion and a comparison of types 2, 7A, and 12,” Virology, 36(1):115-25 (1968)).

[0160] [Example 2] Cellular and humoral immune responses induced by novel adenoviral vectors In this example, the experiments conducted to evaluate the immunogenicity of the novel Ad20-42-42-based adenovirus vector generated in this specification will be described. In these experiments, in mice after intramuscular immunization, the novel vector was evaluated for its ability to induce humoral and cellular immune responses against the (model) antigen encoded by the vector. The vectors tested were those expressing firefly luciferase (FLuc) as a model antigen. The vectors were compared side by side with a benchmark vector based on human adenovirus type 26 (HAdV-26, also referred to as Ad26 in this specification). The immune responses against each antigen were measured using well-known immunological assays such as the enzyme-linked immunospot assay (ELISPOT) and the enzyme-linked immunosorbent assay (ELISA).

[0161] Cellular immune response induced by Ad20-42-42.FLuc The immunogenicity of the novel adenovirus vector Ad20-42-42 was evaluated in Balb / C mice immunized by intramuscular injection with Ad26.FLuc (benchmark control), an Ad20-42-42 vector expressing firefly luciferase (Ad20-42-42.FLuc), or an adenovirus vector lacking the transgene (Ad26.E). Except that 10 10 vp was used in the Ad26.E group, two vector doses of 10 9 and 10 10 viral particles (vp) per mouse were used for the administration test. Two weeks after immunization, the animals were sacrificed and serum and splenocytes were sampled (Figure 7A).

[0162] The cellular immune response against the antigen encoded by the vector was evaluated by the FLuc-specific IFN-γ ELISPOT assay. For this purpose, splenocytes sampled at the time of sacrifice from the immunized mice were stimulated overnight with a pool of 15-mer overlapping FLuc peptides. The antigen-specific immune response was determined by measuring the relative number of IFN-γ-secreting cells (Figure 7B). The results showed that the cellular immune response induced by Ad20-42-42 was the highest at the highest immunization dose (10 10It is shown to be comparable to the response seen for Ad26.Fluc. As expected, no FLuc-specific response was detected for the adenovirus lacking firefly luciferase (Ad26.E). Overall, the data show that Ad20-42-42 induces a strong cellular response.

[0163] [Example 3] Evaluation of serological cross-neutralization between novel and existing adenovirus vectors For their potential usefulness as novel adenovirus vaccine vectors, the novel Ad20-42-42 adenovirus vectors created herein are preferably serologically distinct from existing adenovirus vectors currently under development as vaccine vectors, such as vectors based on human adenovirus serotype HAdV-26. Thus, a cross-neutralization test was performed between the novel Ad20-42-42 adenovirus vector and an existing vector based on HAdV-26 (Ad26). For this purpose, in an adenovirus neutralization assay, a cross-test of mouse antisera produced against these vectors was performed against both vectors. The mouse antisera used in this assay were 10 per mouse 10Two weeks after immunization with individual vector particles, samples were taken from Balb / C mice. Adenovirus neutralization assays were performed as previously described (Spangers et al 2003. J.Clin. Microbiol. 41:5046-5052). Briefly, starting from a 1:16 dilution, sera were serially diluted two-fold, pre-mixed with an adenovirus vector expressing firefly luciferase (FLuc), and subsequently incubated overnight with A549 cells (at a multiplicity of infection (MOI) of 500 virus particles per cell). Luciferase activity levels in the infected cell lysates measured 24 hours post-infection represented the vector infection efficiency. The neutralization titer against a given vector was defined as the highest serum dilution rate that could reduce the vector infection efficiency by 90%. Neutralization titers were arbitrarily classified into the following categories: <16 (no neutralization), 16.1 - 200 (slight cross-neutralization), 201 - 2,000 (cross-neutralization), >2,001 (strong cross-neutralization). The results did not show major cross-neutralization between the tested vectors (Figure 8). Only slight one-way cross-neutralization was observed between vectors Ad20-42-42 and Ad26, where Ad26 antiserum showed a neutralization titer of 23 against Ad20-42-42, while Ad20-42-42 antiserum showed no neutralization against Ad26. Thus, the novel adenovirus vector Ad20-42-42 was shown to have low or no cross-neutralization with the human adenovirus vector Ad26.

[0164] [Example 4] Seroprevalence of novel adenovirus vectors in the human population High levels of existing anti-vector humoral immunity in the vaccine target population can potentially hinder the use of novel adenovirus vectors as effective vaccine platforms. Therefore, the Ad20-42-42 vector was evaluated for seroprevalence in 103 human serum samples. Vectors were tested for neutralization by human serum samples by performing a CPE-based assay (for wild-type (wt) virus) and a reporter assay (for Luc+ expressing virus).

[0165] As described in Example 3, a standard adenovirus neutralization assay was performed as previously described (Spangers et al 2003. J.Clin. Microbiol. 41:5046-5052). In the wild-type adenovirus neutralization assay, sera were heat-inactivated at 55°C for 15 minutes and diluted (1 / 2, 1 / 4, 1 / 8, 1 / 16, or 1 / 32). Next, 50 μl of adenovirus stock diluted to 200 50% cell culture infectious doses (CCID 50 ) was added to the wells containing sera. On days 5 - 6, plates were analyzed for inhibition of CPE by MTT assay (Promega). Sera were evaluated as positive for neutralization if protection from CPE was >90%. The percentage of replication inhibition was calculated by comparison to positive and negative controls.

[0166] Briefly, as described above, starting from a 1:16 dilution, sera were serially diluted two-fold, pre-mixed with an adenovirus vector expressing firefly luciferase (FLuc), and subsequently incubated overnight with A549 cells (at a multiplicity of infection of 500 virus particles per cell). At 24 hours post-infection, the level of luciferase activity in the measured infected cell lysates represented the vector infection efficiency. The neutralization titer against a given vector was defined as the highest serum dilution rate that could reduce the vector infection efficiency by 90%. Neutralization titers were arbitrarily classified into the following categories: <16 (no neutralization), 16 - 50, 50 - 200, 200 - 500, 500 - 1000, >1000.

[0167] The results indicate that the Ad20-42-42 adenovirus vector has a considerably lower seroprevalence (seropositivity rate of 20 - 35%) in the tested human subjects compared to the HAdV-5 vector tested in the same assay (Figure 9). Furthermore, the positive neutralizing titers seen against the novel Ad20-42-42 vector were generally low, with most being below 200. In contrast, the positive neutralizing titers seen against HAdV-5 were in a higher range and exceeded 1000. The vector HAdV-35 showed a similar % seropositivity rate as Ad20-42-42, but the titers observed with HAdV-35 were in a higher range than those of Ad20-42-42.

[0168] In summary, the above data indicate that the existing humoral anti-vector immunity against the Ad20-42-42 vector can be considered low in the vaccine target population evaluated, suggesting that this vector has the potential to be an effective vaccine vector in this human population.

[0169] [Example 5] Transduction ability of the Ad20-42-42 vector in human vascular cells The ability to transduce target cells and express the proteins they encode is an essential feature of vectors used in gene therapy. The novel adenovirus vector Ad20-42-42 was tested for its transduction ability in vascular cells using a luciferase assay.

[0170] Briefly, HSVEC (human saphenous vein endothelial cells) cells were transduced in a dose-dependent manner with an Ad20-42-42 vector expressing luciferase and LacZ in the presence or absence of blood coagulation factor X (FX), and the presence or absence of sensitivity to FX-mediated tropism modification was confirmed. HAd5 and HAd35 were used as control vectors. The cells were plated in 96-well plates at a density of 10,000 cells / well. With and without the addition of FX, they were infected with Ad5Luc, Ad35Luc, Ad20-42-42Luc+ expressing luciferase at 1000, 5000, 10,000 viral particles (vp) per cell at 37°C for 3 hours. After 3 hours of incubation, the medium was removed, and the cells were cultured in complete medium for 48 hours, followed by analysis of luciferase transgene expression, which was expressed as relative light units (RLU) per milligram (mg) of protein.

[0171] Statistical significance between groups was calculated using a two-sample two-sided Student's t-test, and p < 0.05 was considered statistically significant (p < 0.05*, p < 0.01**). All results represent mean data from several experiments and were repeated 4 times for each condition (Figure 10).

[0172] The data obtained indicate that Ad20-42-42 has significantly higher transduction potential than HAd5 and HAd35 in the presence of FX. The highest luciferase expression was observed when a dose of 10,000 vp / cell was applied, reaching a level of 1.5×10 9 in the presence of FX and approximately 1×10 8 even in the absence of FX. Therefore, these results indicate that the transduction of Ad20-42-42 is significantly enhanced by FX, and this vector has stronger transduction characteristics than the two control vectors in the presence of FX.

[0173] An excellent transduction profile superior to the HAd5 control vector of Ad20-42-42 was confirmed by LacZ staining of HSVEC cells using three different vector doses (1000, 5000, and 10,000 vp / cell) in the presence of FX. The staining was observed with a simple optical microscope (Figure 11).

[0174] As expected, the results showed a gradual increase in staining as the dose increased. The staining of 1000, 5000, 10,000 vp / cell is shown from right to left in Figure 11. Cells transduced with Ad20-42-42 showed stronger staining compared to cells transduced with HAd5.

[0175] Overall, the data showed that Ad20-42-42 was able to transduce vascular cells, which was higher than the control vector in the presence of FX. This makes Ad20-42-42 an excellent gene therapy vector candidate for use in the treatment of diseases characterized by endothelial cells, such as cardiovascular diseases and cancer.

[0176] [Example 6] Candidate receptors for AdV20-42-42 An important feature of Ad20-42-42 that distinguishes it among many other vectors is its potential to bind to both the coxsackievirus and adenovirus receptor (CAR) and the CD46 cell receptor, and its sensitivity to the enhancement of transduction by FX. Thus, the range of human cells and tissues for which gene therapy using the Ad20-42-42 vector is possible is expanded.

[0177] To examine the ability of Ad20-42-42 to bind to multiple receptors and use them as tools for cell entry, several indicator cell types were used. Chinese hamster ovary (CHO) cells expressing or lacking CAR; CHO cells expressing or lacking sialic acid; TC1 cells expressing or lacking desmoglein 2 (DSG2) were transduced with Ad20-42-42 and HAd5 used as a control vector (Figures 12A and 12B). Cells were transduced with 10,000 vp / cell and luciferase assays were performed as described above.

[0178] The results obtained from these experiments defined CAR, to which Ad20-42-42 binds, as a potentially dominant transmembrane receptor compared to sialic acid and DSG2 in cells having all three receptors on their surface.

[0179] Similar experiments were performed to examine the ability of this vector to bind to CD46, another receptor commonly used by many adenovirus types for cell entry. For this purpose, CHO cells containing or lacking different isoforms (BC1, BC2, C1, and C2) of this receptor were transduced with Ad20-42-42 and HAd5 used as a control vector. Ad20-42-42 was shown to appear to mainly use the C2 receptor isoform during the process of cell infection. On the other hand, K1 cells lacking CD46 were hardly transduced (Figures 12C and 12D).

[0180] Statistical significance between groups was calculated using a two-sample two-sided Student's t-test, and p < 0.05 was considered statistically significant (p < 0.05*, p < 0.01**). All results represent mean data from experiments performed several times and were repeated 4 times for each condition. Error bars are shown as the standard error of the mean (SEM).

[0181] These findings indicate that the novel adenoviral vector Ad20-42-42 binds to both receptors, CAR and CD46, which are present on many cell types. This expands the range of cell types that can be transduced by the Ad20-42-42 vector, which can be beneficial for use in gene therapy.

[0182] [Example 7] In vivo distribution of Ad20-42-42 in mice In vivo experiments were conducted to examine the in vivo distribution of Ad20-42-42, its effect on the cardiovascular system (CVS), and its role as a gene delivery vector.

[0183] Immunocompetent male mice aged 8 - 10 weeks were used. Six groups of animals were formed (each virus test group contained 5 animals, and the control PBS group contained 3 animals). To deplete circulating macrophages and more efficiently evaluate virus passage at the whole organism level, 200 μl of clodronate liposomes (CL) was administered intravenously (iv) to the corresponding groups 48 hours prior to virus administration.

[0184] The treatment groups were infected i.v. with a single virus dose (10x10 11 viral particles (VP)) of Ad20-42-42Luc+ or HAd5Luc used as a control vector. The control groups were injected with 100 ml of PBS at the same time point instead.

[0185] Forty-eight hours after virus delivery, 0.5 ml of luciferin was injected into the animals, and then bioluminescence imaging was used to read out luciferase activity. The animals were maintained under inhaled anesthesia. The detected luciferase expression levels are shown in Figure 13.

[0186] After imaging was completed, the animals were sacrificed and their organs (liver, heart, spleen, kidney, intestine, pancreas, lung) were harvested. The vector genome was quantified by qPCR.

[0187] In the control group of animals infected with HAd5, in the group not treated with CL, the virus was mainly distributed in the liver and spleen at levels of 2×10 5 and approximately 3×10 5 bioluminescence units, respectively. However, in the group pretreated with CL, both organs showed a distribution closer to 5×10 5 (Figures 14A - 14B).

[0188] On the other hand, Ad20 - 42 - 42 appeared to have only spleen tropism and was not detected in other organs. As expected, the total DNA copy number was significantly higher when CL was added (approximately 2.5×10 5 ) compared to the group without CL pretreatment (1×10 6 )(Figures 14C - 14D).

[0189] In summary, these data indicate that Ad20 - 42 - 42 has a favorable safety profile with only the spleen tropism found in the study, and DNA copy numbers are hardly detected in other organs tested. Since the Ad20 - 42 - 42 vector did not show liver tropism, this makes the Ad20 - 42 - 42 vector a more suitable vector for gene therapy where liver availability and toxicity are low and thus gene transfer to the liver does not interfere with the effectiveness in the target tissue.

[0190] It will be understood by those skilled in the art that modifications can be made to the above - described embodiments without departing from the broad inventive concept thereof. Accordingly, it is understood that the present invention is not limited to the specific embodiments disclosed and is intended to cover modifications within the spirit and scope of the invention as defined by this description. It may include the following aspects. [1] An isolated nucleic acid sequence encoding a chimeric adenovirus capsid or a functional derivative thereof, wherein the chimeric adenovirus capsid or a functional derivative thereof has a fiber polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 11, a hexon polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 12, and a penton polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 13. An isolated nucleic acid sequence. [2] The isolated nucleic acid sequence according to [1] above, wherein the fiber polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 11. [3] The isolated nucleic acid according to [1] or [2] above, wherein the hexon polypeptide sequence comprises the amino acid sequence of SEQ ID NO: 12. [4] The isolated nucleic acid according to any one of [1] to [3] above, wherein the penton polypeptide comprises the amino acid sequence of SEQ ID NO: 13. [5] A vector comprising the isolated nucleic acid according to any one of [1] to [4] above. [6] The vector according to [5] above, wherein the vector is an adenovirus vector. [7] The vector according to [6] above, wherein the adenovirus vector further comprises a transgene. [8] The vector according to [6] or [7] above, wherein the adenovirus vector further comprises an E1 deletion. [9] The vector according to any one of [6] to [8] above, wherein the adenovirus vector further comprises an E3 deletion.

[10] The vector according to any one of [6] to [9] above, wherein the adenovirus vector is a chimeric adenovirus vector comprising one or more adenovirus nucleic acid sequences from at least one of human adenovirus - 4, human adenovirus - 5, human adenovirus - 26, or human adenovirus - 35.

[11] The vector according to

[10] above, wherein the adenovirus vector comprises human adenovirus - 5 (HAdV - 5) E4orf6.

[12] The vector according to any one of [6] to

[11] above, wherein the adenovirus vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[13] The vector according to any one of [7] to

[12] above, wherein the transgene is located in E1 deletion, E3 deletion, and / or adjacent to the right inverted terminal repeat (rITR).

[14] A recombinant cell comprising the vector according to any one of [5] to

[12] above.

[15] A method for producing a vector, comprising: (a) growing the recombinant cell according to

[14] above under conditions for the production of an adenovirus vector; and (b) isolating the vector from the recombinant cell.

[16] An immunogenic composition comprising the adenovirus vector according to any one of [6] to

[13] above and a pharmaceutically acceptable carrier.

[17] A method for inducing an immune response in a subject in need thereof, comprising administering the immunogenic composition according to

[16] above to the subject.

[18] A method for producing a vaccine, comprising combining the adenovirus vector according to any one of [6] to

[13] above with a pharmaceutically acceptable carrier.

[19] A method for expressing a transgene in a subject in need thereof, comprising: a. identifying a subject in need of the transgene to be expressed; b. contacting the subject with the vector according to [6] above; and c. expressing the transgene in the subject.

[20] The method according to

[19] above, wherein the expression of the transgene in the subject in need thereof treats or prevents a disease or disorder.

[21] The method according to

[19] above, wherein contacting the subject with the vector comprises isolating cells from the subject and contacting the cells with the vector.

[22] The method according to any one of

[19] to

[21] above, wherein the subject in need thereof is a human subject.

Claims

1. An isolated nucleic acid encoding a chimeric adenovirus capsid or a functional derivative thereof, wherein the chimeric adenovirus capsid or its functional derivative has a fiber polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 11, a hexon polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO: 12, and a penton polypeptide having an amino acid sequence with at least 95% identity to the amino acid sequence of SEQ ID NO:

13. An isolated nucleic acid.

2. The isolated nucleic acid according to claim 1, wherein the fiber polypeptide comprises the amino acid sequence of SEQ ID NO:

11.

3. The isolated nucleic acid according to claim 1 or 2, wherein the hexon polypeptide comprises the amino acid sequence of SEQ ID NO:

12.

4. The isolated nucleic acid according to any one of claims 1 to 3, wherein the penton polypeptide comprises the amino acid sequence of SEQ ID NO:

13.

5. A vector comprising the isolated nucleic acid according to any one of claims 1 to 4.

6. The vector according to claim 5, wherein the vector is an adenovirus vector.

7. The vector according to claim 6, wherein the adenovirus vector further comprises a transgene.

8. The vector according to claim 6 or 7, wherein the adenovirus vector further comprises an E1 deletion.

9. The vector according to any one of claims 6 to 8, wherein the adenovirus vector further comprises an E3 deletion.

10. The vector according to any one of claims 6 to 9, wherein the adenovirus vector is a chimeric adenovirus vector comprising one or more adenovirus nucleic acid sequences from at least one of human adenovirus - 4, human adenovirus - 5, human adenovirus - 26, or human adenovirus - 35.

11. The vector according to claim 10, wherein the adenovirus vector comprises human adenovirus - 5 (HAdV - 5) E4orf6.

12. The vector according to any one of claims 6 to 11, wherein the adenovirus vector comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:

10. **Claim 13**: The adenovirus vector according to any one of claims 7 to 12, wherein the adenovirus vector contains a transgene, the transgene is located in E1 deletion and E3 deletion, and / or is adjacent to the right inverted terminal repeat sequence (rITR). **Claim 14** A recombinant cell comprising the vector according to any one of claims 5 to 12. **Claim 15** A method for producing a vector, comprising: (a) growing the recombinant cell according to claim 14 under conditions for the production of an adenovirus vector; and (b) isolating the vector from the recombinant cell. **Claim 16** An immunogenic composition comprising the adenovirus vector according to any one of claims 6 to 13 and a pharmaceutically acceptable carrier. **Claim 17** The immunogenic composition according to claim 16 for use in a method of inducing an immune response in a subject in need thereof, the method comprising administering the immunogenic composition to the subject. **Claim 18** A method for producing a vaccine, comprising combining the adenovirus vector according to any one of claims 6 to 13 with a pharmaceutically acceptable carrier. **Claim 19** A pharmaceutical composition for use in a method of expressing a transgene in a subject in need thereof, the pharmaceutical composition comprising the adenovirus vector according to any one of claims 6 to 13, the method comprising: a. identifying a subject in need of the transgene to be expressed; b. contacting the subject with the vector; and c. expressing the transgene in the subject. **Claim 20** The pharmaceutical composition according to claim 19, wherein the expression of the transgene in the subject in need thereof treats or prevents a disease or disorder. **Claim 21** The pharmaceutical composition according to claim 19, wherein contacting the subject with the vector comprises isolating cells from the subject and contacting the cells with the vector. **Claim 22** The pharmaceutical composition according to any one of claims 19 to 21, wherein the subject in need thereof is a human subject.

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