阅读说明：本技术 能够复制的腺病毒载体 (Replication-competent adenovirus vectors ) 是由 V.阿门多拉 S.科罗卡 A.维特利 于 2018-10-16 设计创作，主要内容包括：提供了用于递送外源免疫原的能够复制的猿猴腺病毒载体。本发明的载体展示外源免疫原的优异复制和表达。它们可用作预防性和治疗性疫苗以及可用于基因疗法中。(Replication-competent simian adenovirus vectors for the delivery of foreign immunogens are provided. The vectors of the invention exhibit excellent replication and expression of foreign immunogens. They are useful as prophylactic and therapeutic vaccines and in gene therapy.)

1. A replicable simian adenovirus vector comprising an expression cassette comprising a promoter and a transgene, wherein the expression cassette is inserted into the E3 region, the HE1 site, or the HE2 site of the vector.

2. A replicable simian adenoviral vector according to claim 1 wherein the simian is a chimpanzee.

3. The replicating chimpanzee adenovirus vector according to claim 1, further comprising a nucleotide sequence encoding a chimpanzee adenovirus fiber polypeptide or a functional derivative thereof and/or the chimpanzee adenovirus E4 region.

4. A replicable simian adenovirus vector according to claim 1 wherein the vector is an adenovirus having a low seroprevalence in humans.

5. A replicable simian adenovirus vector according to claim 4 wherein the vector is ChAd 155.

6. A replicable simian adenovirus vector according to claim 4 wherein the vector is ChAd 83.

7. A replicable simian adenovirus vector as claimed in claim 1 wherein the promoter is selected from the group consisting of a CASI promoter and an enhanced cytomegalovirus promoter.

8. A replicable simian adenovirus vector according to claim 7 wherein the promoter is a CASI promoter.

9. A replicable simian adenovirus vector according to claim 7 wherein the promoter is an enhanced cytomegalovirus promoter.

10. The replicable simian adenovirus vector of claim 1 wherein the expression cassette further comprises a post-transcriptional regulatory element.

11. A replicable simian adenovirus vector according to claim 10 wherein the post-transcriptional regulatory element is a woodchuck hepatitis post-transcriptional regulatory element.

12. A replicable simian adenovirus vector according to claim 1 wherein the transgene is an antigen.

13. A replicable simian adenovirus vector according to claim 12 wherein the antigen is selected from the group consisting of rabies virus antigens, respiratory syncytial virus antigens, human immunodeficiency virus antigens, tuberculosis antigens, malaria antigens, hepatitis c virus antigens, chikungunya virus antigens and hepatitis b virus antigens.

14. A replicable simian adenovirus vector according to claim 1 wherein the expression cassette is inserted in the region of E3.

15. A replicable simian adenovirus vector according to claim 1 wherein the expression cassette is inserted into the HE1 region.

16. A replicable simian adenovirus vector according to claim 1 wherein the expression cassette is inserted into the HE2 region.

17. A method of inducing an immune response against a disease caused by a pathogen in a subject in need thereof using a replicable simian adenovirus vector according to any one of claims 1 to 16.

18. Use of a replicable simian adenovirus vector according to any one of claims 1 to 16 for the prevention or treatment of disease.

19. A replicable simian adenovirus vector according to any one of claims 1 to 16 wherein the vector is administered by intramuscular injection.

20. A replicable simian adenovirus vector according to any one of claims 1 to 16 wherein the vector is administered orally.

21. A method of using a replicable simian adenovirus vector according to any one of claims 1 to 16 wherein the vector is administered by intramuscular injection.

22. A method of using a replicable simian adenoviral vector according to any one of claims 1 to 16, wherein the vector is administered orally.

Technical Field

The present invention is in the field of recombinant adenoviruses. Isolated replication-competent adenoviral vectors, recombinant polynucleotides, polypeptides, vectors, and compositions comprising polynucleotide and polypeptide sequences are provided.

Background

Human adenoviruses have been widely used for gene transfer applications due to their large transgene capacity and ability to achieve highly efficient gene transfer in various target tissues. Recombinant adenoviruses are useful in gene therapy and as vaccines. Simian adenovirus-based viral vectors may provide an alternative to the use of human-derived adenoviral vectors for the development of nucleic acid-based vaccines.

Most people are exposed to and develop immunity to human adenovirus. There is a need for vectors that efficiently deliver molecules to a target and minimize the pre-existing effects on immunity to human adenovirus serotypes. Simian adenoviruses are effective in this regard; they are sufficiently closely related to human viruses to effectively induce immunity to delivered exogenous antigens to which humans have little or no preexisting immunity.

Replication-defective adenoviruses deliver their genome to the interior of the cell and, because they do not replicate, do not amplify the transgene payload. Typically, the E1 gene is replaced by a transgene cassette comprising a selected promoter and a nucleic acid sequence corresponding to one or more genes of interest, resulting in a replication-defective recombinant virus.

Unlike replication-defective adenoviruses, replication-competent adenoviruses replicate their DNA and their transgenes, thus amplifying their transgene expression to a much greater extent. Replication-competent adenoviruses have the potential to have greater efficacy, but they pose the risk of transmission and infection to family members or healthcare workers. Despite potential safety issues, replication-competent human adenoviruses have been successfully used to immunize against respiratory diseases. Hundreds of thousands of U.S. trainees were effectively and safely vaccinated against acute respiratory illness with live, non-attenuated isolates of the whole viruses human Ad4, Ad7, and Ad21 formulated as enteric-coated capsules or tablets (Cancer Gene Therapy (2004) 11: 819).

Human and canine replication-competent vectors have been described (Vaccine (2002) 20:3485), however, simian replication-competent adenoviral vectors have not been found to be capable of delivering immunogens or therapeutics for the prevention or treatment of disease. Such vectors combine the advantages of efficient replicable vectors with the advantages of simian adenoviruses. Also, although simian vectors have the ability to replicate in human cells, their replication is not as good as in simian cells, and thus their efficacy is reduced compared to that in simians. Thus, there is a need in the art for vectors that combine the advantages of efficient replication and lack of pre-existing immunity in humans.

Summary of The Invention

The replicable simian adenovirus vectors of the invention generate a stronger gene-based vaccine response than replication-defective simian adenovirus vectors. The vectors of the invention have been optimized to provide improved in vivo efficacy while maintaining a safety profile suitable for human immunization. They have an inherently strong immunomodulatory backbone and promoters capable of driving strong and sustained transgene expression. The replicable vectors of the invention are useful as components of immunogenic compositions for inducing an immune response in a subject, methods of use and methods of manufacture thereof in therapy.

The present invention provides a replicable simian adenovirus vector comprising an expression cassette comprising a promoter and a transgene, wherein the expression cassette is inserted into the E3 region, the HE1 site or the HE2 site of the vector.

The invention also provides methods of inducing an immune response against a disease caused by a pathogen in a subject in need thereof using such a simian adenovirus vector capable of replication.

In one embodiment, the simian is a chimpanzee. The vector may be ChAd155 or ChAd 83.

The chimpanzee adenovirus capable of replication may further comprise a nucleotide sequence encoding a chimpanzee adenovirus fiber polypeptide or a functional derivative thereof and/or the region of chimpanzee adenovirus E4.

The promoter may be selected from the group consisting of the CASI promoter and the enhanced cytomegalovirus promoter. In some embodiments, the expression cassette may further comprise a post-transcriptional regulatory element. In one embodiment, the post-transcriptional regulatory element is a woodchuck hepatitis post-transcriptional regulatory element.

The transgene may be an antigen. The antigen may be selected from rabies virus antigens, respiratory syncytial virus antigens, human immunodeficiency virus antigens, tuberculosis antigens, malaria antigens, hepatitis c virus antigens, chikungunya disease antigens and hepatitis b virus antigens.

Description of the drawings

FIG. 1: simian adenovirus constructs capable of replication. The Inverted Terminal Repeat (ITR) is flanked by 3 'and 5' termini; e1 is early gene 1; CMV is a cytomegalovirus promoter; CASI is the CASI promoter; RG is a model antigen, WPRE is a posttranscriptional regulatory element of woodchuck hepatitis, and E3 indicates that early gene 3 is deleted; fiber represents the adenovirus gene encoding fiber protein and E4 is early gene 4.

Replication-competent simian adenoviral vectors were constructed by inserting a transgene expression cassette in place of the E3 region ("RC 1") (top panel) of the adenoviral genome, inserting the transgene expression cassette in the HE1 region, i.e., between the stop codon and the E4 region of the fiber gene (middle panel), or by inserting the transgene expression cassette in the HE2 region, i.e., downstream of the right ITR ("RC 2") (bottom panel).

FIG. 2: replication-competent ChAd155 and ChAd83 expressing RC1 and RC2 vectors were generated in primary human cell lines. Bars represent the number of viral particles expressed per cell.

FIG. 3: in primary human cell lines, total viral genome copy numbers of replication-competent ChAd155 and ChAd83 of RC1 and RC2 vectors were expressed. Bars represent vector genome copy number per cell.

FIG. 4: expression levels of ChAd155 replication-deficient (RD) and replication-competent (RC1 and RC2) vectors by primary human cell lines at multiplicity of infection of 250 and 1250. The vector expresses the rabies glycoprotein transgene (51 kDa), as demonstrated by western blot. The left panel shows expression at day 2 post infection, and the right panel shows expression at day 7 post infection.

FIG. 5: expression levels of ChAd83 replication-deficient (RD) and replication-competent (RC1 and RC2) vectors by primary human cell lines at multiplicity of infection of 250 and 1250. The vector expresses the rabies glycoprotein transgene (51 kDa), as demonstrated by western blot. The right panel shows expression at day 2 post infection, and the bottom panel shows expression at day 7 post infection.

FIG. 6: replication-competent ChAd155 expressing RC1 and RC2 vectors and replication-competent ChAd83 expressing RC1 and RC2 vectors in murine cell line NMuLi (top panel) and Vero non-human primate cell line (bottom panel). Cells were infected at a multiplicity of infection of 50 and 250.

FIG. 7: comparison of the expression levels of ChAd155RC1 and RC2 vectors expressing the model Rabies Glycoprotein (RG) transgene in murine cell lines, as demonstrated by western blots two and five days post infection (top panel). Comparison of the expression levels of the ChAd155RC1 and RC2 vectors and ChAd83RC1 and RC2 vectors expressing the model Rabies Glycoprotein (RG) transgene in murine cell lines, as evidenced by western blots two and five days post infection (bottom panel). Cells were infected at a multiplicity of infection of 50, 250 and 1250.

FIG. 8: immunogenicity in mice of replication-defective ChAd155(RD), ChAd155RC1 and ChAd83RC1 vectors expressing model protein transgenes, measured by IFN- γ ELISpot and expressed every 10⁶Spots of individual splenocytes formed cells.

FIG. 9: neutralizing antibodies (top panel) and T cells (bottom panel) responses in mice against oral and Intramuscular (IM) delivered ChAd155 RD and ChAd155RC1 expressing the model rabies glycoprotein transgenes. The top panel shows neutralizing antibody protection against rabies infection, measured with a fluorescent antibody virus neutralization assay (FAVN). The dashed line indicates the protection threshold. The bottom panel shows rabies-specific T cell responses, measured by interferon gamma ELIspot assay.

Annotation of sequences

1-polynucleotide sequence encoding wild type ChAd155

2-polynucleotide sequence encoding wild type ChAd83

3-polynucleotide sequence encoding a CASI promoter

4-polynucleotide sequence encoding an enhanced hCMV promoter.

Detailed Description

Adenoviral vectors

Adenoviruses are non-enveloped icosahedral viruses with a linear double-stranded DNA genome of approximately 36 kb. Adenoviruses can transduce many cell types of several mammalian species, including both dividing and non-dividing cells, without integrating into the genome of the host cell. They have been widely used in gene transfer applications due to their demonstrated safety, ability to achieve highly efficient gene transfer in various target tissues, and large transgene capacity. Human adenovirus vectors are currently used in gene therapy and vaccines, but have the disadvantage of being highly prevalent worldwide of pre-existing immunity following prior exposure to common human adenovirus.

Adenoviruses have a characteristic morphology with an icosahedral capsid that contains three major proteins: hexon (II), penton matrix (III) and desmosomal (IV), along with many other minor proteins VI, VIII, IX, IIIa and IVa 2. The hexon comprises the majority of the structural components of the capsid, which consists of 240 trimeric hexon capsomeres and 12 penton matrices. The hexon has three conserved double barrels (barrel) and three towers (tower) at the top, each tower containing a loop from each subunit that forms the majority of the capsid. The stroma of the hexon is highly conserved among adenovirus serotypes, while the surface loops are variable. Penton is another adenoviral capsid protein; which forms a pentameric matrix to which the neurites are attached. Trimeric spike proteins project from the penton matrix at each of the 12 vertices of the capsid and are rod-like structures with nodes. The major role of the spike protein is to tether the viral capsid to the cell surface via interaction of the nodal region with cellular receptors. The variation in the flexible axis of the fiber and in the nodal region is characteristic of different adenovirus serotypes.

The adenovirus genome has been well characterized. Linear, double-stranded DNA associates with a highly basic protein VII and a small peptide pX (also referred to as mu). Another protein V is packaged with the DNA-protein complex and provides structural attachment to the capsid via protein VI. With respect to specific open reading frames similarly located (e.g., the location of the E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4, and L5 genes of each virus), there is general conservation in the overall organization of the adenoviral genome. Each end of the adenoviral genome contains a sequence called an Inverted Terminal Repeat (ITR), which is essential for viral replication. The 5 'end of the adenovirus genome contains the 5' cis elements necessary for packaging and replication; i.e., a 5 'ITR sequence (which may function as an origin of replication) and a native 5' packaging enhancer domain (which contains sequences necessary for packaging the linear adenovirus genome and enhancer elements of the E1 promoter). The 3 'end of the adenovirus genome includes the 3' cis elements (including the ITRs) necessary for packaging and encapsidation. The virus also contains virally encoded proteases, which are necessary for processing some of the structural proteins required for the production of infectious viral particles.

The structure of the adenoviral genome is described based on the order in which the viral genes are expressed after host cell transduction. More specifically, the viral genes are referred to as early (E) or late (L) genes, depending on whether transcription occurs before or after the onset of DNA replication. In the early stages of transduction, the E1A, E1B, E2A, E2B, E3 and E4 genes of adenovirus are expressed to prepare the host cell for viral replication. The E1 gene is considered to be the main switch, which acts as a transcriptional activator and is involved in both early and late gene transcription. E2 is involved in DNA replication; e3 is involved in immune regulation, and E4 regulates viral mRNA metabolism. During the late stages of infection, expression of late genes L1-L5 encoding the structural components of the viral particle are activated. Late genes are transcribed from the Major Late Promoter (MLP) and undergo alternative splicing.

Adenovirus capsid protein and polynucleotides encoding same

As outlined above, the adenoviral capsid comprises three main proteins: hexon, penton, and fiber knob. The hexon comprises the majority of the structural components of the capsid, which consists of 240 trimeric hexon capsomeres and 12 penton matrices. The hexon has three conserved double barrels (barrel) and the top has three towers (tower), each tower containing a loop from each subunit that forms the majority of the capsid. The stroma of the hexon is highly conserved among adenovirus serotypes, while the surface loops are variable.

Pentons are another adenoviral capsid protein that forms the matrix for the pentamer to which the fiber is attached. Trimeric spike proteins project from the penton matrix at each of the 12 vertices of the capsid and are rod-like structures with nodes. A significant difference in the surface of the adenoviral capsid relative to the surface of most other icosahedral viruses is the presence of elongated spike proteins. The major role of the spike protein is to tether the viral capsid to the cell surface via its interaction with cellular receptors.

The fiber proteins of many adenovirus serotypes share a common architecture: an N-terminal tail, a central axis consisting of a repetitive sequence, and a C-terminal knob domain (or "head"). The central axial domain consists of a variable number of beta-repeats. The β -repeats are connected to form an elongated structure of three intertwined helical strands that is highly rigid and stable. The shaft connects the N-terminal tail with a spherical segment structure responsible for interaction with the target cell receptor. The spherical nature of the adenovirus knob domain presents a large surface for binding the receptor laterally and at the apex. The effect of this configuration is to protrude the receptor binding site away from the viral capsid, thus freeing the virus from the spatial constraints presented by the relatively flat capsid surface.

Although fibers of many adenovirus serotypes have the same overall structure, they have variable amino acid sequences that affect their function and structure. For example, many exposed areas on the surface of the fiber knob present receptor binding sites that can be easily accommodated. The spherical shape of the fiber knob allows receptors to bind at the sides of the knob or on the top of the fiber knob. These binding sites are usually located on surface exposed loops that are linked to poorly conserved beta-strands between human adenoviruses. The exposed side chains on these rings impart various surface features to the segments while retaining tertiary and quaternary structures. For example, the electrostatic potential and charge distribution at the knob surface can vary due to a wide range of isoelectric points in the fiber knob sequence (varying from a pI of approximately 9 for adenoviruses "Ad" 8, Ad19, and Ad37 to a pI of approximately 5 for subgroup B adenoviruses). As a structurally complex viral ligand, the fiber protein allows the presentation of various binding surfaces (segments) in many orientations and distances (axes) from the viral capsid.

One of the most significant variations between some serotypes is fiber process length. Studies have shown that the length of the fiber axis strongly influences the interaction of the knob and the virus with its target receptor. Furthermore, the spike proteins between serotypes may also differ in their ability to bend. Although the β -repeats in the shaft form a highly stable and regular structure, Electron Microscopy (EM) studies have shown unique hinges in the fiber process. Analysis of protein sequences from several adenovirus serotype fibers accurately localized a disruption in the repeat sequence of the shaft at the third β -repeat from the N-terminal tail, which was strongly associated with one of the hinges in the shaft, as seen by EM. The hinges in the fiber allow the segments to assume various orientations relative to the viral capsid, which can circumvent steric hindrance of receptor engagement, which requires proper presentation of receptor binding sites on the segments. For example, the rigid fibers of subgroup D adenoviruses require a flexible receptor or one predisposed for viral attachment, since they cannot bend themselves.

The identification of specific cellular receptors for different Ad serotypes and the knowledge of how they contribute to tissue tropism has been achieved by using the technique of fiber pseudotyping (pseudotyping). Although some subgroups of ads use coxsackie virus and adenovirus receptors ("CARs") as primary receptors, it is becoming increasingly clear that many ads use alternative primary receptors, leading to very different tropisms in vitro and in vivo. Fibers of these serotypes highlight significant differences in their primary and tertiary structures, such as fiber axis stiffness, fiber axis length, and lack of CAR binding sites and/or putative HSPG binding motifs, as well as differences in net charge within the fiber knob. Thus, pseudotyping Ad5 particles with alternative fiber shafts and knots provides an opportunity to remove important cell binding domains and, in addition, may allow for more efficient (and potentially more cell-selective) delivery of transgenes to defined cell types than achieved with Ad 5. If the fiber used is from an Ad with a lower seroprevalence in human or experimental models (in favor of a situation of successful administration of the vector), neutralization of the fiber-pseudotyped Ad particles can also be reduced. In addition, full-length fiber as well as isolated fiber knob regions (rather than hexons or pentons alone) are capable of inducing dendritic cell maturation and are associated with the induction of an effective CD8+ T cell response. In summary, the adenoviral fiber protein plays an important role at least in receptor binding and immunogenicity of adenoviral vectors.

Adenovirus replication

Historically, adenoviral vaccine development has focused on defective, non-replicating vectors. They were made replication-defective by deleting the gene of the E1 region essential for replication. Typically, the non-essential E3 region gene is also deleted to make room for the exogenous transgene. An expression cassette containing the transgene under the control of the exogenous promoter is then inserted. These replication-defective viruses are then produced in E1-complementing cells.

The term "replication-defective" or "replication-incompetent" adenovirus refers to an adenovirus that is incapable of replication, in that it has been engineered to contain at least a loss-of-function (or "loss-of-function" mutation), i.e., deletions or mutations that impair the function of the gene without completely removing the gene, such as the introduction of artificial stop codons, deletions or mutations of active sites or interaction domains, mutations or deletions of the regulatory sequences of the gene, and the like, or complete removal of a gene encoding a gene product essential for viral replication, such as complete removal of one or more of the adenoviral genes selected from the group consisting of E1A, E1B, E2A, E2B, E3 and E4 (such as E3ORF 1, E3ORF 2, E3ORF 3, E3ORF4, E3ORF 5, E3ORF 6, E3ORF 7, E3ORF 8, E3ORF 9, E4 ORF7, E4 ORF6, E4 ORF4, E4 ORF3, E4 ORF2 and/or E4 ORF 1). Suitably, E1 and optionally E3 and/or E4 are deleted. If deleted, the previously mentioned deleted gene region will suitably not be considered in the alignment when determining the percent identity relative to another sequence.

The term "replication-competent" adenovirus refers to an adenovirus that can replicate in a host cell without any recombinant helper proteins being contained in the cell. Suitably, a "replication-competent" adenovirus comprises the complete structural gene and the following complete or functionally essential early genes: E1A, E1B, E2A, E2B and E4. Wild-type adenovirus isolated from a particular animal will be capable of replication in that animal.

Vectors of the invention

Non-human simian adenovirus based viral vectors represent an alternative to the use of human derived vectors for gene therapy and gene vaccines. Certain adenoviruses isolated from non-human simians are closely related to adenoviruses isolated from humans, as evidenced by their efficient propagation in cells of human origin. Since humans develop little or no immunity to simian adenoviruses, they are expected to provide an improved alternative to human adenovirus use.

The term "vector" refers to at least one polynucleotide or a mixture of at least one polynucleotide and at least one polypeptide capable of introducing the polynucleotide into a cell. By "low seroprevalence" can be meant having a reduced level of pre-existing neutralizing antibodies as compared to human adenovirus 5 (Ad 5). Similarly or alternatively, "low sero-positive rate" may mean less than about 35% sero-positive rate, less than about 30% sero-positive rate, less than about 20% sero-positive rate, less than about 15% sero-positive rate, less than about 10% sero-positive rate, less than about 5% sero-positive rate, less than about 4% sero-positive rate, less than about 3% sero-positive rate, less than about 2% sero-positive rate, less than about 1% sero-positive rate, or no detectable sero-positive rate. Seropositivity can be measured as the percentage of individuals with clinically relevant neutralization titers (defined as 50% neutralization titer >200) using methods as described in hum, genether. (2004) 15: 293.

In one embodiment, the adenoviral vector of the invention is derived from a non-human simian adenovirus, also referred to as a "simian adenovirus". Many adenoviruses have been isolated from non-human apes, such as chimpanzees, bonobo, macaques, orangutans, and gorillas. Vectors derived from these adenoviruses can induce strong immune responses against the transgenes encoded by these vectors. Certain advantages of non-human simian adenovirus-based vectors include the relative lack of cross-neutralizing antibodies to these adenoviruses in the human target population, and thus their use overcomes pre-existing immunity to human adenoviruses. For example, some simian adenoviruses do not have cross-reactivity with pre-existing human neutralizing antibodies, and cross-reactivity of certain chimpanzee adenoviruses with pre-existing human neutralizing antibodies is only present in 2% of the target population, compared to 35% in the case of certain candidate human adenovirus vectors (sci. trans. med. (2012) 4: 1).

The adenoviral vectors of the invention can be derived from a non-human adenovirus, such as a simian adenovirus, e.g., from a chimpanzee (chimpanzee: (a)Pan troglodytes) Bonobo (bonobo)Pan paniscus) A large orangutan (a large orangutan), (b), (c) and (d)Gorilla gorilla) And orangutan (sumanshan chimpanzee), (a)Pongo abelii) And veronica chimpanzee (Pongo pygnaeus)). They include adenoviruses from groups B, C, D, E and G. Chimpanzee adenoviruses include, but are not limited to, ChAd3, ChAd15, ChAd19, ChAd25.2, ChAd26, ChAd27, ChAd29, ChAd30, ChAd31, ChAd32, ChAd33, ChAd34, ChAd35, ChAd37, ChAd38, ChAd39, ChAd40, ChAd63, ChAd83, ChAd155, ChAd157, chaadox 1, chaadox 2, and SadV 41. Alternatively, the adenoviral vector can be derived from a non-human simian adenovirus isolated from bonobo, such as PanAd1, PanAd2, PanAd3, Pan 5, Pan 6, Pan 7 (also referred to as C7), and Pan 9. The vector may include, in whole or in part, nucleotides encoding the fiber, penton or hexon of a non-human adenovirus.

In a preferred embodiment of the invention, the ape is a chimpanzee. In some embodiments of the invention, the chimpanzee adenovirus vector capable of replication further comprises a nucleotide sequence encoding a chimpanzee adenovirus fiber polypeptide or a functional derivative thereof and/or the chimpanzee adenovirus E4 region.

In one embodiment of the invention, the vector is an adenovirus with a low seroprevalence in humans, wherein the "low seroprevalence" is less than 30% in human subjects. In one embodiment of the adenoviral vector of the invention, the adenovirus has a seroprevalence in a human subject of less than 30%, preferably no seroprevalence in a human subject, and more preferably no seroprevalence in a human subject that has not been previously contacted with chimpanzee adenovirus.

The choice of insertion sites for gene expression cassettes for replication-defective vectors has been focused primarily on replacing regions known to be involved in viral replication. The choice of the insertion site of the gene expression cassette of the vector capable of replication must preserve the replication mechanism. Viruses maximize their coding capacity by producing highly complex transcription units controlled by multiple promoters and alternative splicing. Thus, a viral vector capable of replication must retain the sequences necessary for replication while leaving room for a functional expression cassette.

In a preferred embodiment, the simian adenoviral vector of the invention is ChAd155 or ChAd 83.

In an embodiment of the adenoviral vector of the invention, the adenoviral DNA is capable of entering a mammalian target cell, i.e. it is infectious. The infectious recombinant adenoviruses of the invention can be used as prophylactic or therapeutic vaccines and in gene therapy. Thus, in one embodiment, the recombinant adenovirus comprises an endogenous molecule for delivery into a target cell. The target cell is a mammalian cell, such as a bovine cell, a canine cell, a goat cell, a deer cell, a chimpanzee cell, a pterodactyla cell, an equine cell, a feline cell, a human cell, a wolfram cell, a ovine cell, a porcine cell, a rodent cell, a bear cell, or a fox cell. For example, the endogenous molecule for delivery into the target cell may be an expression cassette.

According to the invention, there is a replicable simian adenovirus vector comprising an expression cassette comprising a promoter and a transgene, wherein the expression cassette is inserted into the E3 region, the HE1 site or the HE2 site of the vector. The vector comprises the E1 region or a fragment thereof necessary for replication.

In one embodiment, the promoter is selected from the group consisting of a CASI promoter and an enhanced cytomegalovirus promoter.

In a further embodiment, the expression cassette may further comprise a post-transcriptional regulatory element, and the post-transcriptional regulatory element may be a woodchuck hepatitis post-transcriptional regulatory element.

In another embodiment, the transgene is an antigen. The antigen may be selected from rabies virus antigens, respiratory syncytial virus antigens, human immunodeficiency virus antigens, tuberculosis antigens, malaria antigens, hepatitis c virus antigens, chikungunya disease antigens and hepatitis b virus antigens.

In an embodiment of the invention, the E1 region or fragment thereof necessary for replication is present, and the exogenous sequence of interest is inserted into the completely or partially deleted E3 region. In one embodiment, the vector comprises a left ITR region, followed by an E1 region, then an E3 region, which is replaced by an expression cassette comprising a promoter, an antigen of interest, and optionally additional enhancer elements; these are followed by the fiber knob region, the E4 region and the right ITR; translation occurs in the right direction. In a further embodiment, the promoter is a CMV promoter. In yet a further embodiment, the enhancer element is a hepatitis b posttranslational regulatory element (HPRE) or woodchuck hepatitis posttranslational element (WPRE).

In other embodiments, the vector comprises a left ITR region; followed by the E1 region; the E3 region deleted in whole or in part; a fiber-process region; an E4 region; an expression cassette comprising a promoter, an antigen of interest, and, optionally, one or more enhancer elements, inserted at the HE1 site, i.e., between the stop codon of the fiber gene and the E4 region ("HE 1 site"); followed by the right ITR. The ChAd155 HE1 insertion site was between bp 34611 and 34612 of the wild type ChAd155 sequence. The ChAd83 HE1 insertion site is between bp 33535 and 33536 of the wild type ChAd83 sequence. Translation occurs in the right direction. In a further embodiment, the promoter is a CASI promoter. In yet a further embodiment, the enhancer element is HPRE or WPRE.

In further embodiments, the vector comprises a left ITR region; followed by the E1 region; the E3 region deleted in whole or in part; a fiber-process region; an E4 region; an expression cassette comprising a promoter, an antigen of interest and, optionally, one or more enhancer elements, inserted at the HE2 site, i.e., between the end that acts as an ITR and the cap site of the E4 mRNA ("HE 2 site"); followed by the right ITR. The ChAd155 HE2 insertion site is between bp 37662 and 37663 of the wild type ChAd155 sequence. The ChAd83HE2 insertion site was between bp 36387 and 36388 of the wild type ChAd83 sequence. Translation occurs in the left direction. In a further embodiment, the promoter is a CASI promoter. In yet a further embodiment, the enhancer element is HPRE or WPRE.

The HE1 and HE2 sites were identified as insertion sites for transgenes because insertion at these specific points did not disrupt the coding or regulatory sequences of ChAd155 and ChAd 83. Thus, insertion of the expression cassette into the HE1 or HE2 site of the ChAd genome does not affect the viral replication cycle.

In one embodiment of the invention, the vector is a functional or immunogenic derivative of an adenoviral vector. By "derivative of an adenoviral vector" is meant a modified form of the vector, e.g., one or more nucleotides of the vector are deleted, inserted, modified or substituted.

Adjusting element

Regulatory elements, i.e., expression control sequences, including appropriate transcription initiation, termination, promoter and enhancer sequences; useful RNA processing signals such as splicing and polyadenylation (poly a) signals, including rabbit β -globin poly a; tetracycline-regulated systems, micrornas, post-transcriptional regulatory elements, such as WPRE, post-transcriptional regulatory elements of woodchuck hepatitis virus); sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and, when desired, a sequence that enhances secretion of the encoded product.

A "promoter" is a nucleotide sequence that allows for the binding of RNA polymerase and directs the transcription of a gene. Typically, a promoter is located in a non-coding region of a gene near the transcription start site. Sequence elements within a promoter that function in the initiation of transcription are often characterized by a consensus nucleotide sequence. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals, including simians and humans. A wide variety of expression control sequences (including internal, native, constitutive, inducible and/or tissue-specific promoters) are known in the art and can be utilized.

The promoter of the invention will generally be a heterologous promoter. By "heterologous" is meant derived from an entity that is genotypically different from the remaining entities being compared. The promoters of the invention may be constitutive or inducible. Constitutive promoters initiate RNA synthesis independent of regulatory influences. Inducible promoters allow for the regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature or the presence of particular physiological states.

Promoters of the present invention include, but are not limited to, CMV promoter, β -actin promoter, such as chicken β actin (CAG) promoter, CASI promoter, human phosphoglycerate kinase-1 (PGK) promoter, TBG promoter, retroviral rous sarcoma virus LTR promoter, SV40 promoter, dihydrofolate reductase promoter, phosphoglycerate kinase (PGK) promoter, EF1a promoter, zinc-inducible sheep Metallothionein (MT) promoter, dexamethasone (Dex) -inducible Mouse Mammary Tumor Virus (MMTV) promoter, T7 polymerase promoter system, ecdysone insect promoter, tetracycline-inhibiting system, tetracycline-inducible system, RU 486-inducible system, and rapamycin-inducible system.

The transgene may be operably linked to a tissue-specific promoter. For example, if expression in skeletal muscle is desired, a promoter active in muscle should be used. These include promoters from genes encoding skeletal beta-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, as well as synthetic muscle promoters with higher activity than naturally occurring promoters. Examples of tissue-specific promoters are for liver (e.g., albumin, hepatitis b virus core, alpha-fetoprotein (AFP)); bone (e.g., osteocalcin, bone sialoprotein); lymphocytes (e.g., CD2, immunoglobulin heavy chains, and T cell receptor chains); and neurons (e.g., neuron-specific enolase (NSE)) are known.

Optionally, the vector carrying the transgene encoding the therapeutically useful or immunogenic product may also include a selectable marker or reporter gene. The reporter gene may be selected from those known in the art. Suitable reporter genes include, but are not limited to, enhanced green fluorescent protein, red fluorescent protein, luciferase, and secreted embryonic alkaline phosphatase (seAP), which may include sequences encoding geneticin, hygromycin, or puromycin resistance, among others. Such selection reporter or marker genes (which may or may not be located outside the viral genome to be packaged into viral particles) can be used to signal the presence of the plasmid in the bacterial cell, such as ampicillin resistance. Other components of the vector may include an origin of replication.

Suitable promoters include the Cytomegalovirus (CMV) promoter and the CASI promoter. The CMV promoter is strongly and universally active. It has the ability to drive high levels of transgene expression in many tissue types and is well known in the art. The CMV promoter may be used in the vectors of the invention with or without a CMV enhancer.

The CASI promoter is a synthetic promoter described as a combination of CMV enhancer, chicken β -actin promoter, and splice donor and splice acceptor flanked by Ubiquitin (UBC) enhancers (US 8865881).

In some embodiments, the CASI promoter may include a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity to SEQ ID NO. 3. In some embodiments, the promoter comprises or consists of the nucleic acid sequence of SEQ ID NO 3. In some embodiments, the enhanced hCMV promoter can include a nucleic acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to SEQ ID No. 4. In some embodiments, the promoter comprises or consists of the nucleic acid sequence of SEQ ID NO. 4.

Suitable promoters also include, but are not limited to, the chimpanzee elongation factor 1 promoter (chEF1), a strong active and ubiquitous promoter that produces persistent transgene expression in vivo. In one embodiment, the promoter is a human ferritin light chain promoter with a CMV enhancer. In this embodiment, the 5 'UTRs of the ferritin heavy and light chains are replaced with the 5' UTRs of chimpanzee elongation factor 1 α to eliminate iron regulation by ferritin. In one embodiment, the promoter is a chicken β actin promoter with a CMV enhancer. In one embodiment, the promoter is a hybrid promoter. In one embodiment, the hybrid promoter is a CMV promoter having a CMV enhancer and a ubiquitin gene enhancer, and is a stronger promoter than a conventional CMV promoter.

As used herein, a "post-transcriptional regulatory element" is a DNA sequence that, when transcribed, enhances expression of one or more transgenes or fragments thereof delivered by the viral vectors of the invention. Post-transcriptional regulatory elements include, but are not limited to, hepatitis b virus post-transcriptional regulatory element (HPRE) and woodchuck hepatitis post-transcriptional regulatory element (WPRE). WPRE is a three-part cis-acting element that has been shown to enhance transgene expression driven by some (but not all) promoters.

In embodiments of the invention, the ChAd155 vector may comprise one or more of a promoter, an enhancer, and a reporter gene. For example, a vector of the invention may comprise a ChAd 155-enhanced hCMV-SeAP, a ChAd155-CASI-SeAP and a ChAd155-hCMV-SeAP (which optionally has tetracycline on/off transcriptional control), and a ChAd 155-CMV-hFerL-chEF 1-SeAP (which has tetracycline on/off transcriptional control).

In embodiments of the invention, the ChAd83 vector may comprise one or more of a promoter, an enhancer, and a reporter gene. For example, a vector of the invention may comprise ChAd 83-enhanced hCMV SeAP, ChAd 83-enhanced hCMV SeAP, ChAd83-CASI-SeAP and ChAd83-hCMV-SeAP (which optionally has tetracycline on/off transcriptional control), and ChAd 83-CMV-hFerL-chEF 1-SeAP (which has tetracycline on/off transcriptional control).

Vectors of the invention are generated using the techniques provided herein, in combination with techniques known to those skilled in the art. Such techniques include conventional cloning techniques for cDNA (such as those described in textbooks), the use of overlapping oligonucleotide sequences of the adenovirus genome, polymerase chain reaction, and any suitable method of providing the desired nucleotide sequence.

Transgenosis

Adenoviral vectors can be used to deliver desired RNA or protein sequences, such as heterologous sequences, for expression in vivo. The vectors of the invention may comprise any genetic element, including naked DNA, phage, transposon, cosmid, episome, plasmid or viral component. The vectors of the invention may contain simian adenovirus DNA and an expression cassette. An "expression cassette" comprises a transgene and regulatory elements necessary for translation, transcription and/or expression of the transgene in a host cell.

A "transgene" is a nucleic acid sequence encoding a polypeptide of interest that is heterologous to the vector sequences flanking the transgene. The nucleic acid coding sequence is operably linked to regulatory components in a manner that allows for transcription, translation, and/or expression of the transgene in the host cell. In an embodiment of the invention, the vector expresses the transgene at a therapeutic or prophylactic level. A "functional derivative" of a transgenic polypeptide is a modified form of the polypeptide, for example, in which one or more amino acids have been deleted, inserted, modified or substituted.

The transgenes may be used prophylactically or therapeutically, for example, as vaccines for inducing immune responses, to correct genetic defects by correcting or replacing defective or missing genes, or as cancer therapeutics. As used herein, induction of an immune response refers to the ability of a protein to induce a T cell and/or humoral antibody immune response against the protein.

The immune response elicited by the transgene may be an antigen-specific B cell response, which produces neutralizing antibodies. The immune response elicited may be an antigen-specific T cell response, which may be a systemic response and/or a local response. The antigen-specific T cell response may comprise a CD4+ T cell response, such as a response involving CD4+ T cells expressing cytokines, e.g., interferon gamma (IFN γ), tumor necrosis factor alpha (TNF α), and/or interleukin 2(IL 2). Alternatively or additionally, the antigen-specific T cell response comprises a CD8+ T cell response, such as a response involving CD8+ T cells expressing cytokines (e.g., IFN γ, TNF α, and/or IL 2).

Transgenes of the invention include, but are not limited to, rabies virus antigens, e.g., Rabies Glycoprotein (RG), Respiratory Syncytial Virus (RSV) antigens, Human Immunodeficiency Virus (HIV) antigens, tuberculosis antigens, malaria antigens, Hepatitis C Virus (HCV) antigens, chikungunya disease antigens, and Hepatitis B (HBV) antigens.

The composition of the transgene sequence will depend on the use to which the resulting vector is put. In one embodiment, the transgene is a sequence encoding a product useful in biology and medicine, such as a prophylactic transgene, a therapeutic transgene, or an immunogenic transgene, e.g., a protein or RNA. The protein transgene includes an antigen. The antigenic transgenes of the invention induce an immunogenic response against the disease-causing organism. RNA transgenes include tRNA, dsRNA, ribosomal RNA, catalytic RNA, and antisense RNA. An example of a useful RNA sequence is one that eliminates expression of a targeted nucleic acid sequence in the treated animal.

Alternatively, the transgene sequence may include a reporter sequence that produces a detectable signal upon expression. Such reporter sequences include, but are not limited to, DNA sequences encoding: beta-lactamase, beta-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, Green Fluorescent Protein (GFP), Chloramphenicol Acetyl Transferase (CAT), luciferase, membrane-bound proteins (including, for example, CD2, CD4, CD8, influenza hemagglutinin protein, and other proteins well known in the art for which high affinity antibodies exist or which can be produced by conventional means)) and fusion proteins comprising a membrane-bound protein fused appropriately to an antigen-tag domain, particularly from hemagglutinin or Myc. These coding sequences, when associated with regulatory elements that drive their expression, provide signals that can be detected by conventional means, including enzymatic assays, radiographic assays, colorimetric assays, fluorescent or other spectroscopic assays, fluorescence activated cell sorting assays, and immunological assays, including enzyme-linked immunosorbent assays (ELISAs), Radioimmunoassays (RIA), and immunohistochemistry.

As a result of redundancy in the genetic code, polypeptides may be encoded by a variety of different nucleic acid sequences. The coding bias favors the use of some synonymous codons (i.e., codons encoding the same amino acid) more than others. By "codon-optimized" is meant that the codon composition of a recombinant nucleic acid is modified without altering the amino acid sequence. Codon optimization has been used to increase mRNA expression in different organisms by using organism-specific codon usage frequencies.

In addition to, and regardless of, codon preferences, some synonymous codon pairs are used more frequently than others. This codon pair bias means that some codon pairs are present in excess, while others are present in deficiency. Codon pair de-optimization has been used to reduce viral virulence. For example, polioviruses modified to contain under-represented codon pairs have been reported to exhibit reduced translation efficiency and to be attenuated compared to wild-type polioviruses (Science (2008) 320: 1784). Engineering a synthetic attenuated virus by codon pair de-optimization can produce a different pair-wise arranged virus that encodes the same amino acid sequence as the wild type, but uses synonymous codons. De-optimization of attenuated viruses by codon pairs produces up to 1000-fold less plaques compared to wild-type, produces fewer viral particles, and requires about 100-fold more viral particles to form plaques.

In contrast, polioviruses modified to contain codon pairs that occur in excess in the human genome act in a manner similar to wild-type RNA and generate plaques of the same size as the wild-type RNA (Coleman et al (2008) Science 320: 1784). This occurs despite the fact that: viruses with an excessive number of codon pairs present contain a similar number of mutations as viruses with an under-represented codon pair and demonstrate enhanced translation compared to the wild type. This observation demonstrates that the codon pair optimized constructs are expected to function in a manner similar to their non-codon pair optimized counterparts and are not expected to provide a functional advantage.

The constructs of the invention may comprise codon optimized nucleic acid sequences. Alternatively or additionally, the vector of the invention comprises a codon optimised sequence of the transgene or an immunogenic derivative or fragment thereof. The constructs of the invention may comprise a codon pair optimized nucleic acid sequence. Alternatively or additionally, the vector of the invention comprises or consists of a codon pair optimized sequence of the transgene or an immunogenic derivative or fragment thereof.

Delivery of replication-competent adenovirus vectors

In some embodiments, the recombinant adenovirus of the invention is administered to a subject by epidermal administration, intradermal administration, intramuscular injection, intraperitoneal injection, intravenous injection, mucosal administration, nasal administration, oral administration, rectal administration, subcutaneous injection, transdermal administration, or intravaginal administration.

If the treatment regimen involves co-administration of one or more adenoviral vectors and additional components, each formulated in a different composition, they are advantageously co-locally administered at or near the same site. For example, the components may be administered (e.g., via a route of administration selected from intramuscular, transdermal, intradermal, subcutaneous) to the same side or limb (the "ipsilateral" administration) or to the opposite side or limb (the "contralateral" administration).

In one embodiment of the invention, the vector may be administered Intramuscularly (IM), i.e. directly injected into a muscle. Muscle vascularization is good and is usually taken rapidly.

In one embodiment of the invention, the carrier may be administered orally. Oral vaccine delivery offers several advantages over intramuscular delivery, including elimination of pain at the injection site, ease of delivery and convenience. It allows less qualified health care workers to effectively administer vaccines and circumvent the possibility of infection of needles and syringes in areas with high prevalence of, for example, HIV, hepatitis b and hepatitis c.

The oral mucosa consists of an outer layer of stratified squamous epithelium (mainly non-keratinized) and a bottom layer of dense connective tissue (lamina propria). The lamina propria contains many immune cells and is the site where the immune response occurs as a barrier to protect internal tissues from pathogenic organisms. Administration via the oral/gastrointestinal route provides access for the antigen to a large surface area through a single cell layer of a single cylindrical epithelium, where it targets peyer's patches and induces a systemic response.

Live replication-capable adenoviruses have been successfully administered orally for decades, but administration of viral vectors encoding antigenic transgenes is more challenging. The mechanisms of immune recognition do not readily enter the luminal side of the intestine; this protects the body from producing an immune response against ingested proteins in the food. Thus, the constructs of the invention face a barrier to the generation of an immune response against the protein antigen when delivered to the intestine via the oral route. For example, in the phase 1 study, human subjects were orally administered with a viable human Ad4 vaccine capable of replication, which had influenza hemagglutinin antigen as the transgene. They respond with a cellular immune response, but do not produce a humoral antibody response until intramuscular boosting (Lancet infection Dis (2013) 13: 238). Similarly, conventional pigs are administered orally or subcutaneously with a live, recombinant porcine adenovirus vaccine capable of replication, which has a classical swine fever virus antigen as the transgene. None of the orally administered pigs produced, but 75% of the subcutaneously administered pigs produced antibodies against the transgenic antigen (Vaccine (2001) 146: 1787).

In one embodiment of the invention, the carrier may be administered mucosally. Mucosal vaccine delivery also provides several advantages for intramuscular delivery of vaccines. Since the mucosa is connected to the outside of the human body, mucosal vaccines can be effective and safe at somewhat lower purities than parenteral vaccines, so they are easier to produce. They are generally effective at low doses and are therefore cost effective.

As used herein, "mucosal" delivery encompasses all mucosal membranes. The mucosa generally lines body cavities and passageways that contain the epithelium and lamina propria. The mucosa may or may not be keratinized. Mucosal tissues include, but are not limited to, alveolar, bronchial, buccal, dermal, endometrial, gastric, intestinal, buccal (jungal), lining, chewing, nasal, olfactory, buccal, ear, palatal, rectal, specialized (lingual), sublingual, tracheal, and vaginal mucosa.

The mucosa provides a highly specialized immune system, which is composed of lymphoid compartments such as peyer's patches, mesenteric lymph nodes, the appendix, tonsils, and adenoids. Antigens taken up by absorptive epithelial cells of the mucosa may be shuttled to antigen presenting cells, or presented directly to antigen presenting cells and presented to T cells. The immune response in mucosal tissues is determined by the nature of the antigen, the type of antigen presenting cell and the local microenvironment. Primed mucosal B and T cells leave the site where the original antigens meet, cross the lymph and enter the circulation. Mucosal delivery can be, for example, buccal, genital, e.g., vaginal, intranasal, ocular, e.g., ocular conjunctiva, otic, e.g., inner ear, rectal, or sublingual.

In one embodiment of the invention, the carrier may be administered sublingually. Vaccine delivery via the sublingual route provides a rapid pathway for antigens through a very thin, layered, squamous, non-keratinizing epithelial layer, where they target langerhans cells and induce a systemic response. The antigen delivered under the tongue becomes available to the dense network of dendritic cells in the sublingual mucosa. Sublingually delivered replicable vectors bypass the liver, thus avoiding first-pass metabolism, increasing its persistence and thus potentially generating a stronger immune response.

In one embodiment of the invention, the vector may be administered buccally. Vaccine delivery via the buccal route also provides a pathway for antigens through a stratified, squamous, non-keratinized epithelial layer that is a little thicker than the sublingual layer. Buccal delivery also targets langerhans cells and induces a systemic response.

Adjuvant

A method of establishing strong and durable immunity to a particular pathogen involves adding an adjuvant to the vaccine. By "adjuvant" is meant an agent that enhances, stimulates, activates, potentiates or modulates an immune response to an active ingredient of a composition. The adjuvant effect may occur at the cellular or humoral level or both. Adjuvants stimulate the immune system's response to the actual antigen, but do not have an immunological effect on their own. Alternatively or additionally, the adjuvanted composition of the invention may comprise one or more immunostimulants. By "immunostimulant" is meant an agent that induces an overall temporary increase in the immune response of a subject, whether administered with an antigen or separately.

The compositions of the invention may be administered with or without an adjuvant. Alternatively or additionally, the composition may comprise or be administered in conjunction with one or more adjuvants (e.g. vaccine adjuvants), in particular, the composition comprises an immunologically effective amount of a vector of the invention encoding a transgene.

Method of use/use

Methods for inducing an immune response against a disease caused by a pathogen in a subject in need thereof are provided, comprising the step of administering an immunologically effective amount of a construct or composition as disclosed herein. In some embodiments, there is provided use of a construct or composition disclosed herein for inducing an immune response against a transgenic antigen in a subject in need thereof. The vector of the present invention can be applied to the prevention, treatment or amelioration of diseases caused by infection.

The method of the invention comprises the use of the vector of the invention in medicine. They include the use of the vectors of the invention for the treatment of diseases caused by pathogens. The vectors of the invention are useful for the preparation of medicaments for the treatment of diseases caused by pathogens.

Effective immunization with adenoviral vectors depends on the intrinsic immunomodulatory capacity of the adenoviral vector backbone. Adenoviruses with lower immunological potency induce less antigen expression. Effective immunity also depends on the ability of the promoter to drive strong and sustained transgene expression. For example, adenoviral vectors driven by the viral promoter CMV-IE do not maintain long-term transgene expression because they induce cytokines that reduce expression.

A "subject" is intended to be a vertebrate, such as a mammal, e.g., a human or veterinary mammal. In some embodiments, the subject is a human.

General purpose

Vectors of the invention are generated using the techniques and sequences provided herein, in combination with techniques known to those skilled in the art. Such techniques include conventional cloning techniques for cDNA (such as those described in textbooks), the use of overlapping oligonucleotide sequences of the adenovirus genome, polymerase chain reaction, and any suitable method of providing the desired nucleotide sequence.

Unless defined otherwise, 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 disclosure belongs. The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "plurality" means two or more. Furthermore, the numerical limits given with respect to the concentration or level of a substance (such as the concentration of a solution component or the ratio thereof, and the reaction conditions such as temperature, pressure, and cycle time) are intended to be approximate. The term "about" as used herein means an amount ± 10%.

The invention will now be further described by means of the following non-limiting examples.

Examples