WO2011057254A2 - Simian adenoviral vector-based vaccines - Google Patents

Abstract

The invention relates to a replication-deficient simian adenoviral vector construct comprising a nucleic acid sequence which encodes an antigen of a pathogen, such as an HIV antigen or an influenza virus antigen. The invention also provides a method of inducing an immune response against a pathogen, such as HIV or influenza, in a mammal using a composition comprising the simian adenoviral vector construct. The invention also provides a monkey adenovirus which is capable of propagation in a human cell.

Description

SIMIAN ADENOVIRAL VECTOR-BASED VACCINES

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0001] This invention was made with Government support under Cooperative Research and Development Agreement (CRADA) numbers AI-0134 (NIAID CRADA 2001-0041) and 5102 (NIAID CRADA 2001-0609). The Government has certain rights in this invention.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] Incorporated by reference in its entirety herein is a computer-readable

nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 1 ,444,667 Byte ASCII (Text) file named "7071 14_ST25.TXT," created on November 2, 2010.

BACKGROUND OF THE INVENTION

[0003] The Centers for Disease Control and Prevention (CDC) estimate that in the United States, over one million people are living with HIV infection and approximately 21% are unaware of their infection (CDC, Morb. Mortal. Wkly. Rep., 57(39): 1073-1076 (2008)).

Worldwide, the rate of new HIV infections continues to increase at an unacceptably high level. Although new AIDS diagnoses and deaths have fallen significantly in developed countries since the advent of highly active antiretro viral therapy (HAART), in the developing world the HIV/AIDS epidemic continues to accelerate. The global impact of the epidemic is considerable. According to the Joint United Nations Programme on HIV/AIDS and the World Health

Organization, as of 2008, 33.4 million people were estimated to be living with HIV/AIDS. In addition, the total number of people living with the virus in 2008 was more than 20% higher than the number in 2000, and the prevalence was roughly threefold higher than in 1990

(UNAIDS/WHO, AIDS Epidemic Update December 2009). Worldwide there were an estimated 2 million deaths due to HIV/AIDS in 2008 (UNAIDS/WHO, AIDS Epidemic Update December 2009). Beyond the human tragedy of HIV/AIDS, the costs of the epidemic pose a significant impediment to the economic growth and political stability of many countries. In developing countries and in segments of the U.S. population, anti-HIV therapies are frequently beyond financial reach. Accordingly, effective, low-cost tools for HIV prevention, such as a vaccine, are urgently needed to bring the HIV epidemic under control.

[0004] Influenza A and B viruses cause a highly contagious respiratory disease in humans resulting in approximately 36,000 deaths in the United States annually (see, e.g., Wright, P. F., and Webster, R. G., Orthomyxoviruses, In: Fields Virology, eds. D. M. Knipe and P. M.

Howley, Lippincott Williams & Wilkins, Philadelphia, pp. 1533-79 (2001)). These annual epidemics also have a large economic impact, and cause more than 100,000 hospitalizations per year in the United States alone. Influenza A viruses, which infect a wide number of avian and mammalian species, are responsible for the periodic widespread epidemics, or pandemics, that have caused high mortality rates (Wright and Webster, supra). The most devastating pandemic - occurred in 1918, which caused an estimated 20 to 40 million deaths worldwide (see, e.g., Reid et al., Microbiol. Infect. , 3: 81-87 (2001)). Less devastating pandemics occurred in 1957 and 1968. Influenza B virus infections comprise about 20% of the yearly cases, but influenza B virus, which appears to infect only humans, does not cause pandemics (Wright and Webster, supra).

[0005] Several anti-influenza agents have been developed, including inactivated influenza virus vaccines, cold adapted influenza virus vaccines, genetically engineered live influenza virus vaccines, and other antiviral agents (e.g., amantidine, rimantidine, zanamivir and oseltamivir). However, the widespread use of currently available antiviral agents is limited by concerns over side effects, patient compliance, and the possible emergence of drug-resistant variants.

[0006] Delivery of proteins as therapeutics or for inducing an immune response in biologically relevant forms and amounts has been an obstacle to drug and vaccine development for decades. One solution that has proven to be a successful alternative to traditional antigen delivery approaches is the delivery of exogenous nucleic acid sequences for production of antigenic molecules in vivo. Gene transfer vectors ideally enter a wide variety of cell types, have the capacity to accept large nucleic acid sequences, are safe, and can be produced in quantities required for treating patients. Viral vectors have these advantageous properties and are used in a variety of protocols to treat or prevent biological disorders. [0007] Despite their advantageous properties, widespread use of viral gene transfer vectors is hindered by several factors. In this regard, certain cells are not readily amenable to gene delivery by currently available viral vectors. For example, lymphocytes are impaired in the uptake of adenoviruses (Silver et al., Virology, 165, 377-387 (1988); Horvath et al., J. Virology, 62{\), 341-345 (1988)).

[0008] The use of viral gene transfer vectors also is impeded by the immunogenicity of viral vectors. A majority of the U.S. population has been exposed to wild-type forms of many of the viruses currently under development as gene transfer vectors (e.g., adenovirus). As a result, much of the U.S. population has developed pre-existing immunity to certain virus-based gene transfer vectors. Such vectors are quickly cleared from the bloodstream, thereby reducing the effectiveness of the vector in delivering biologically relevant amounts of a gene product.

Moreover, the immunogenicity of certain viral vectors prevents efficient repeat dosing, which can be advantageous for "boosting" the immune system against pathogens, and results in only a small fraction of a dose of the viral vector delivering its payload to host cells.

[0009] Thus, there remains a need for improved methods and compositions for inducing immune responses against a variety of pathogens, including HIV and influenza virus. The invention provides such methods and compositions.

BRIEF SUMMARY OF THE INVENTION

[0010] The invention provides a replication-deficient simian adenoviral vector construct comprising at least one nucleic acid sequence which encodes an antigen of a pathogen. The simian adenoviral vector construct requires at most complementation of a deficiency in the El A region, the E1B region, and/or the E4 region of the adenoviral genome for propagation. The invention also provides a method of inducing an immune response against HIV or influenza in a mammal using a composition comprising the simian adenoviral vector construct and a pharmaceutically acceptable carrier.

[0011] The invention also provides a monkey adenovirus which is capable of propagation in a human cell. The monkey adenovirus comprises (a) at least one nucleic acid sequence of the El region of a human adenovirus and (b) a nucleic acid sequence encoding the E4 ORF6 gene product of a human adenovirus, or the human cell comprises (a) at least one nucleic acid sequence of the El region of a human adenovirus and (b) a nucleic acid sequence encoding the E4 ORF6 gene product of a human adenovirus.

[0012] The invention provides a monkey adenovirus which is capable of propagation in a cell which expresses one or more gene products of a human adenovirus.

[0013] The invention provides a method of inducing an immune response against a pathogen in a mammal. The method comprises administering to the mammal a composition comprising a replication-deficient simian adenoviral vector construct comprising at least one nucleic acid sequence which encodes an antigen of a pathogen and a pharmaceutically acceptable carrier, whereupon the nucleic acid sequence encoding an antigen is expressed in the mammal to produce the antigen and thereby induce an immune response against the pathogen.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0014] Figure 1 is a diagram comparing the sequences of hexon proteins from different adenoviruses, and demonstrates that hexon proteins from simian adenoviruses are distantly related to hexon proteins of other common serotypes (including human serotype 5).

[0015] Figure 2A is a graph illustrating the CD8⁺ T cell response induced by adenoviral vector constructs encoding a clade B HIV gpl40 ("gpl40B") protein based human serotype 5 and simian serotypes 11 (SEQ ID NO: 28) and 16 (SEQ ID NO: 25) in mice. The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). The adenoviral vector constructs were administered intramuscularly at the indicated dosage to mice, and immune responses at 3 weeks post-injection were measured as HIV Env specific tetramer positive (PA9+) CD8⁺ T cells in PBMC.

[0016] Figure 2B is a graph illustrating the anti-HIV Env IgG response induced by adenoviral vector constructs encoding an HIV gpl40B protein based human serotype 5 and simian serotypes 1 1 (SEQ ID NO: 28) and 16 (SEQ ID NO: 25) in mice. The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). The adenoviral vector constructs were administered intramuscularly at the indicated dosage to mice, and immune responses at 3 weeks post-injection were measured as titers of anti- HIV Env IgG (OD) in the serum at a 1 : 1000 dilution.

[0017] Figure 3A is a graph illustrating the CD8⁺ T cell response induced in mice following administration of a human serotype 5 adenoviral vector construct (Ad5) or a simian serotype 7 adenoviral vector construct encoding an HIV gpHOB protein (simian serotype 7) (SEQ ID NO: 27). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). The adenoviral vector constructs were administered intramuscularly at the indicated dosage to mice, and immune responses at 3 weeks post-injection were measured as HIV Env specific tetramer positive (PA9+) CD8⁺ T cells in PBMC.

[0018] Figure 3B is a graph illustrating the anti-HIV Env IgG response induced in mice following administration of a human serotype 5 adenoviral vector construct (Ad5) or a simian adenoviral vector construct encoding an HIV gpl40B protein (simian serotype 7) (SEQ ID NO: 27). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). The adenoviral vector constructs were administered intramuscularly at the indicated dosage to mice, and immune responses at 3 weeks post-injection were measured as titers of anti-HIV Env IgG (OD) in the serum at a 1 : 1000 dilution.

[0019] Figure 4A is a graph illustrating the CD8⁺ T cell response induced in mice using a DNA-prime/adenoviral vector construct-boost immunization regimen in mice. Adenoviral vector constructs encoding an HIV gpl40B protein based on human serotype 5 and on simian serotypes 11 (SEQ ID NO: 28) and 16 (SEQ ID NO: 25) were administered intramuscularly at the indicated dosage to mice, and immune responses at 3 weeks post-injection were measured as HIV Env-specific tetramer positive (PA9+) CD8⁺ T cells in PBMC. The gpHOB protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0020] Figure 4B is a graph illustrating the anti-HIV Env IgG response induced in mice using a DNA-prime/adenoviral vector construct-boost immunization regimen. Adenoviral vector constructs encoding an HIV gpl40B protein based on human serotype 5 and on simian serotypes 1 1 (SEQ ID NO: 28) and 16 (SEQ ID NO: 25) were administered intramuscularly at the indicated dosage to mice, and immune responses at 3 weeks post-injection were measured as titers of anti-HIV Env IgG (OD) in the serum at a 1 : 1000 dilution. The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0021] Figure 5 is a graph illustrating the humoral immune response (measured as antibody titers (OD)) induced in mice by an immunization regimen utilizing a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7) (SEQ ID NO: 27) as a prime, and a human adenoviral vector construct (serotype 5 or 28) or a LCMV construct encoding an HIV gpl40B vector as a boost. The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0022] Figure 6A is a graph illustrating the CD4⁺ T cell response induced in mice by an immunization regimen utilizing, as a prime, a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7 (s7) (SEQ ID NO: 27), 1 1 (si 1) (SEQ ID NO: 28), or 16 (sl 6) (SEQ ID NO: 25)) or a human adenoviral vector construct (serotype 5, 26, 28, 35, or 41) encoding an HIV gpl40B protein, and a serotype 5 human adenoviral vector construct or a LCMV vector construct encoding an HIV gpl40B as a boost (* p < 0.05 vs. rAd28/rAd5, ** p < 0.05 vs. rAd28/rLCMV). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0023] Figure 6B is a graph illustrating the CD8⁺ T cell response induced in mice by an immunization regimen utilizing, as a prime, a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7 (s7) (SEQ ID NO: 27), 11 (si 1) (SEQ ID NO: 28), or 16 (si 6) (SEQ ID NO: 25)) or a human adenoviral vector construct (serotype 5, 26, 28, 35, or 41) encoding an HIV gpl40B protein, and a serotype 5 human adenoviral vector construct or a LCMV vector construct encoding an HIV gpl40B protein as a boost (* p < 0.05 vs.

rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0024] Figure 6C is a graph illustrating HIV Env-specific tetramer positive (PA9+) CD8⁺ T cell levels in PBMC of mice following an immunization regimen utilizing, as a prime, a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7 (s7) (SEQ ID NO: 27), 1 1 (si 1) (SEQ ID NO: 28), or 16 (si 6) (SEQ ID NO: 25)) or a human adenoviral vector construct (serotype 5, 26, 28, 35, or 41) encoding an HIV gpl40B protein, and a serotype 5 human adenoviral vector construct or a LCMV encoding an HIV gp 140B vector construct as a boost (** p < 0.05 vs. rAd28/rLCMV). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl 2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0025] Figure 6D is a graph illustrating HIV Env-specific tetramer positive (PA9+) CD8⁺ T cell levels in the spleens of mice following an immunization regimen utilizing, as a prime, a simian adenoviral vector construct encoding an HIV gpl40B protein(serotype 7 (s7) (SEQ ID NO: 27), 1 1 (si 1) (SEQ ID NO: 28), or 16 (si 6) (SEQ ID NO: 25)) or a human adenoviral vector construct (serotype 5, 26, 28, 35, or 41) encoding an HIV gpl40B protein, and a serotype 5 human adenoviral vector construct or a LCMV vector encoding an HIV gpl40B protein as a boost (* p < 0.05 vs. rAd28/rAd5, ** p < 0.05 vs. rAd28/rLCMV). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0026] Figure 6E is a graph illustrating the humoral immune response (measured as IgG titers) induced in mice by an immunization regimen utilizing, as a prime, a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7 (s7) (SEQ ID NO: 27), 1 1 (si 1) (SEQ ID NO: 28), or 16 (si 6) (SEQ ID NO: 25)) or a human adenoviral vector construct (serotype 5, 26, 28, 35, or 41) encoding an HIV gpl40B protein, and a serotype 5 human adenoviral vector construct or a LCMV vector construct encoding an HIV gpl40B protein as a boost (* p < 0.05 vs. rAd28/rAd5, ** p < 0.05 vs. rAd28/rLCMV). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0027] Figure 7A is a graph illustrating the CD4⁺ T cell response induced in mice by an immunization regimen utilizing, as a prime, a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7 (s7) (SEQ ID NO: 27), 1 1 (si 1) (SEQ ID NO: 28), or 16 (si 6) (SEQ ID NO: 25)) or a human adenoviral vector construct (serotype 5 or 28) encoding an HIV gpl40B protein, and a human serotype 5 (5) adenoviral vector construct encoding an HIV gpl40B protein as a boost. The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0028] Figure 7B is a graph illustrating the CD8⁺ T cell response induced in mice by an immunization regimen utilizing, as a prime, a simian adenoviral vector construct encoding an HIV gpHOB protein (serotype 7 (s7) (SEQ ID NO: 27), 1 1 (si 1) (SEQ ID NO: 28), or 16 (si 6) (SEQ ID NO: 25)) or human adenoviral vector construct (serotype 5 or 28) encoding an HIV gpl40B protein, and a human serotype 5 (5) adenoviral vector construct encoding an HIV gpl40B protein as a boost (* p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0029] Figure 7C is a graph illustrating the CD8⁺ T cell levels in PBMC of mice following an immunization regimen utilizing, as a prime, a simian adenoviral vector construct encoding an HIV gpHOB protein (serotype 7 (s7) (SEQ ID NO: 27), 1 1 (si 1) (SEQ ID NO: 28), or 16 (si 6) (SEQ ID NO: 25)) or a human adenoviral vector construct (serotype 5 or 28) encoding an HIV gpl40B protein, and a human serotype 5 (5) adenoviral vector construct encoding an HIV gpl40B protein as a boost (* p < 0.05 vs. rAd28/rAd5). The gpHOB protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0030] Figure 7D is a graph illustrating the CD8⁺ T cell levels in the spleens of mice following an immunization regimen utilizing, as a prime, a simian adenoviral vector construct encoding an HIV gpHOB protein (serotype 7 (s7) (SEQ ID NO: 27), 1 1 (si 1) (SEQ ID NO: 28), or 16 (si 6) (SEQ ID NO: 25)) or a human adenoviral vector construct (serotype 5 or 28) encoding an HIV gpl40B protein, and a human serotype 5 (5) adenoviral vector construct encoding an HIV gpl40B protein as a boost (* p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0031] Figure 7E is a graph illustrating the humoral immune response (measured as IgG titers) induced in mice by an immunization regimen utilizing, as a prime, a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7 (s7) (SEQ ID NO: 27), 1 1 (si 1) (SEQ ID NO: 28), or 16 (sl6) (SEQ ID NO: 25)) or a human adenoviral vector construct (serotype 5 or 28) encoding an HIV gpl40B protein, and a human serotype 5 (5) adenoviral vector construct encoding an HIV gpl40B protein as a boost (* p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (d l2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0032] Figure 8A is a graph illustrating the CD4⁺ T cell response induced in mice by an immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein as a prime, and, as a boost, a human adenoviral vector construct encoding an HIV gpl40B protein (serotype 5, 26, 28, 35, or 41) or a simian adenoviral vector construct encoding gpl40B (serotype 7 (SEQ ID NO: 27), 1 1 (SEQ ID NO: 28), or 16 (SEQ ID NO: 25)) (* p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0033] Figure 8B is a graph illustrating the CD8⁺ T cell response induced in mice by an immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein as a prime, and, as a boost, a human adenoviral vector construct encoding an HIV gpl40B protein (serotype 5, 26, 28, 35, or 41) or simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7 (SEQ ID NO: 27), 1 1 (SEQ ID NO: 28), or 16 (SEQ ID NO: 25)) (* p < 0.05 vs. rAd28/rAd5). The gpHOB protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). [0034] Figure 8C is a graph illustrating HIV Env-specific tetramer positive (PA9+) CD8⁺ T cell levels in PBMC of mice following an immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein as a prime, and, as a boost, a human adenoviral vector construct encoding an HIV gpl40B protein (serotype 5, 26, 28, 35, or 41) or a simian adenoviral vector construct encoding gpl40B (serotype 7 (SEQ ID NO: 27), 1 1 (SEQ ID NO: 28), or 16 (SEQ ID NO: 25)) (* p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0035] Figure 8D is a graph illustrating HIV Env-specific tetramer positive (PA9+) CD8⁺ T cell levels in the spleens of mice following an immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein as a prime, and as a boost, a human adenoviral vector construct encoding an HIV gpl40B protein (serotype 5, 26, 28, 35, or 41) or a simian adenoviral vector construct encoding gpl40B (serotype 7 (SEQ ID NO: 27), 1 1 (SEQ ID NO: 28), or 16 (SEQ ID NO: 25)) (* p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0036] Figure 8E is a graph illustrating the humoral immune response (measured as IgG titers) induced in mice by an immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein as a prime, and, as a boost, a human adenoviral vector construct encoding an HIV gpl40B protein (serotype 5, 26, 28, 35, or 41) or a simian adenoviral vector construct encoding gpl40B (serotype 7 (SEQ ID NO: 27), 1 1 (SEQ ID NO: 28), or 16 (SEQ ID NO: 25)) (* p < 0.05 vs. rAd28/rAd5). The gpHOB protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0037] Figure 9A includes graphs illustrating the CD4⁺ (left panels) and CD8⁺ (right panels) T cell responses induced in mice by an immunization regimen utilizing 10⁷ viral particles of a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7 (SEQ ID NO: 27), 1 1 (SEQ ID NO: 28), or 16 (SEQ ID NO: 25)) as a prime and as a boost (* p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0038] Figure 9B includes graphs illustrating the CD4⁺ (left panels) and CD8⁺ (right panels) T cell responses induced in mice by an immunization regimen utilizing 10 viral particles of a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7 (SEQ ID NO: 27), 1 1 (SEQ ID NO: 28), or 16 (SEQ ID NO: 25)) as a prime and as a boost (* p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0039] Figure 9C includes graphs illustrating HIV Env-specific tetramer positive (PA9+) CD8 T cell levels in PBMC of mice following an immunization regimen utilizing 10 viral particles (left panel) or 10 viral particles (right panel) of a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7 (SEQ ID NO: 27), 11 (SEQ ID NO: 28), or 16 (SEQ ID NO: 25)) as a prime and as a boost (* p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0040] Figure 9D includes graphs illustrating HIV Env-specific tetramer positive (PA9+) CD8⁺ T cell levels in the spleens of mice following an immunization regimen utilizing 10⁷ viral particles (left panel) or 10 viral particles (right panel) of a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7 (SEQ ID NO: 27), 1 1 (SEQ ID NO; 28), or 16 (SEQ ID NO: 25)) as a prime and as a boost (* p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0041] Figure 9E includes graphs illustrating the humoral immune response (measured as IgG titers) induced in mice by an immunization regimen utilizing 10⁷ viral particles (left panel) or 10 viral particles (right panel) of a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 7 (SEQ ID NO: 27), 1 1 (SEQ ID NO: 28), or 16 (SEQ ID NO: 25)) as a prime and as a boost (* p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0042] Figure 1 OA is a graph illustrating the CD4⁺ T cell response induced in mice by a homologous or heterologous prime/boost immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein and a simian serotype 18 adenoviral vector construct encoding an HIV gpl40B protein (SEQ ID NO: 29) ((*p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0043] Figure 10B is a graph illustrating the CD8⁺ T cell response induced in mice by a homologous or heterologous prime/boost immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein and a simian serotype 18 adenoviral vector encoding an HIV gpl40B protein (SEQ ID NO: 29) (*p < 0.05 vs.

rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0044] Figure IOC is a graph illustrating HIV Env-specific tetramer positive (PA9+) CD8⁺ T cell levels in PBMC of mice by a homologous or heterologous prime/boost immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein and a simian serotype 18 adenoviral vector construct encoding an HIV gpl40B protein (SEQ ID NO: 29) (*p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0045] Figure 10D is a graph illustrating HIV Env-specific tetramer positive (PA9+) CD8⁺ T cell levels in the spleens of mice by a homologous or heterologous prime/boost immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein and a simian serotype 18 adenoviral vector construct encoding an HIV gpl40B protein (SEQ ID NO: 29) (*p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0046] Figure 10E is a graph illustrating the humoral immune response (measured as IgG titers) induced in mice by a homologous or heterologous prime/boost immunization^' regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein and a simian serotype 18 adenoviral vector construct encoding an HIV gpl40B protein (SEQ ID NO: 29) (*p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0047] Figure 1 1 A is a graph illustrating the CD4⁺ T cell response induced in mice by a homologous or heterologous prime/boost immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein and a simian serotype 38 adenoviral vector construct encoding an HIV gpl40B protein (SEQ ID NO: 26) (*p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0048] Figure 1 IB is a graph illustrating the CD8⁺ T cell response induced in mice by a homologous or heterologous prime/boost immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein and a simian serotype 38 adenoviral vector construct encoding an HIV gpHOB protein (SEQ ID NO: 26) (*p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0049] Figure 1 1C is a graph illustrating HIV Env-specific tetramer positive (PA9+) CD8⁺ T cell levels in PBMC of mice by a homologous or heterologous prime/boost immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein and a simian serotype 38 adenoviral vector construct encoding an HIV gpHOB protein (SEQ ID NO: 26) (* p < 0.05 vs. rAd28/rAd5). The gpHOB protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). [0050] Figure 1 ID is a graph illustrating HIV Env-specific tetramer positive (PA9+) CD8⁺ T cell levels in the spleens of mice by a homologous or heterologous prime/boost immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein and a simian serotype 38 adenoviral vector construct encoding an HIV gpl40B protein (SEQ ID NO: 26) (* p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0051] Figure 1 IE is a graph illustrating the humoral immune response (measured as IgG titers) induced in mice by a homologous or heterologous prime/boost immunization regimen utilizing a human serotype 5 or 28 adenoviral vector construct encoding an HIV gpl40B protein and a simian serotype 38 adenoviral vector construct encoding an HIV gpl40B protein (SEQ ID NO: 26) (*p < 0.05 vs. rAd28/rAd5). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0052] Figure 12 is a graph illustrating the CD4⁺ T cell response, the CD8⁺ T cell response, and IgG titers induced in mice by a single IM administration of 10 viral particles a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 18 (SEQ ID NO: 29), 16 (SEQ ID NO: 25), 38 (SEQ ID NO: 26), 7 (SEQ ID NO: 27), or 1 1 (SEQ ID NO: 28)). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl 2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0053] Figure 13A is a graph illustrating the CD4⁺ T cell response, the CD8⁺ T cell response, and IgG titers induced in mice by a homologous prime/boost immunization regimen utilizing 10 viral particles of a simian adenoviral vector construct encoding an HIV gpl40B protein (serotype 16 (SEQ ID NO: 25), 7 (SEQ ID NO: 27), or 1 1 (SEQ ID NO: 28)). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0054] Figure 13B is a graph illustrating the CD4⁺ T cell response, the CD8⁺ T cell response, and IgG titers induced in mice by a heterologous prime/boost immunization regimen 7

utilizing various combinations of a simian adenoviral vector construct (10 viral particles) encoding an HIV gpl40B protein (serotype 16 (SEQ ID NO: 25), 7 (SEQ ID NO: 27), or 1 1 (SEQ ID NO: 28)). The gpl40B protein encoded by these constructs comprises deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI).

[0055] Figure 14A is a graph illustrating the humoral immune response (measured as IC50 titers) induced in mice by a heterologous or homologous prime/boost immunization regimen utilizing 10⁸ viral particles of a simian adenoviral vector construct encoding a codon-optimized influenza HA protein (serotype 16 (SEQ ID NO: 32), serotype 7 (SEQ ID NO: 30), and serotype 1 1 (SEQ ID NO: 31)), and/or a human adenoviral vector construct based on serotype 28 encoding a codon-optimized influenza HA protein.

[0056] Figure 14B is a graph illustrating anti-influenza neutralizing antibody activity (measured as IC₅₀ titers) in mice following a heterologous prime/boost immunization regimen utilizing 10⁸ viral particles of a simian adenoviral vector construct (serotype 16 (SEQ ID NO: 32), serotype 7 (SEQ ID NO: 30), and serotype 1 1 (SEQ ID NO: 31)) encoding a codon- optimized influenza HA protein as a prime, and a human adenoviral vector construct based on serotype 5 encoding a codon-optimized influenza HA protein as a boost.

[0057] Figure 15 A is a graph illustrating anti-influenza neutralizing antibody activity (measured as IC50 titers) in mice following a heterologous prime/boost immunization regimen utilizing a human adenoviral vector construct encoding a codon-optimized influenza HA protein and a simian adenoviral vector construct encoding a codon-optimized influenza HA protein (serotype 7 (SEQ ID NO: 30), serotype 1 1 (SEQ ID NO: 31), and serotype 16 (SEQ ID NO: 32)).

[0058] Figure 15B is a graph illustrating anti-influenza neutralizing antibody activity (measured as ICgo titers) in mice following a heterologous prime/boost immunization regimen utilizing a human adenoviral vector construct encoding a codon-optimized influenza HA protein and a simian adenoviral vector construct encoding a codon-optimized influenza HA protein (serotype 7 (SEQ ID NO: 30), serotype 1 1 (SEQ ID NO: 31), and serotype 16 (SEQ ID NO: 32)). [0059] Figure 16A and Figure 16B are graphs which illustrate the production of simian adenovirus progeny under single-burst conditions. Figure 16A depicts production in human cell lines differing in expressed Ad5 factors (A549 = no Ad5 factors, A549+Ad5 E40RF6 = Ad5 E40RF6 factor, 293-ORF6 = Ad5 El and E4 ORF6 factors). Figure 16B depicts production in human cell lines with Ad5 El (293 cells), Ad5 El + Ad5 E4 ORF6, or in a monkey cell line (BSC-1). Data are mean +/- standard deviation.

[0060] Figure 17 is a diagram which illustrates a method of constructing simian adenoviral vector constructs with an expression cassette replacing El . Monkey adenovirus genome (SV) with major regions identified (not to scale): the stippled boxes = ITR, Ψ = packaging signal, TATA = Ela promoter's TATAA box, and coding regions for El a, Elb, pIX, E3 and E4. B is the recipient plasmid which comprises an expression cassette comprised of a CMV promoter (arrow), HIV envelope gene (Env), and SV40 polyadenylation signal (SV) linearized between the pIX coding sequence and ITR, bacterial origin of replication (Ori) and a gene that encodes Kanamycin (Kan) drug resistance. Homologous recombination (X) between the SV genome and plasmid B results in replacement of the El promoter and El A and E1B coding sequences with the CMV-Env expression cassette. Homologous recombination is generated by transforming recombination competent bacteria BJ5183 with the two DNAs resulting in plasmid C. Bacteria that are Kan resistant are screened, and plasmid C is identified by restriction digest and sequencing. Before the viral genome is transfected into 293-ORF6 cells to generate virus particles, plasmid C is restricted with an endonuclease that recognizes a site (R) out side of the viral genome.

[0061] Figure 18A is a set of graphs depicting CD8+ T cell responses in mice treated with

7 8 adenoviral vector constructs encoding HIV gpl40B. Mice were injected i.m. with 10', 10°, or 10⁹ pu of Ad5, SVl 1 , or SVl 6 vectors expressing HIV gpl40B. Two weeks post-immunization gpl40B-specific responses were assessed by intracellular cytokine staining (ICS) in splenocytes stimulated with V3 gpl20 HIV (JR-FL) peptide. Two independent experiments were performed with a total of n = 12 per group. For each individual animal, the fraction of CD8+IFNy+ T cells responding to HIV gpl40B is shown together with the mean response. Statistical analyses were performed in OriginPro version 8. Immune responses between vaccine groups were compared using a non-parametric method (Mann- Whitney test). Significance levels were adjusted by the number of group comparisons made using Bonferroni corrections, resulting in a significance threshold of p < 0.005. Groups that were significantly different from each other are marked with asterisks. Responses in mock treated (final formulation buffer (FFB) only) animals were <0.1%.

[0062] Figure 18B is a graph depicting antibody responses in mice treated with adenoviral vector constructs encoding HIV gpl40B. Mice were injected i.m. with 10 , 10 , or 10 pu of Ad5, SVl 1 , or SVl 6 vector constructs expressing HIV gpl40B. Two weeks post-immunization gpl40B-specific antibody responses were assessed by ELISA of mouse sera at a 1 : 1 ,000 dilution. The results shown are from one out of the two experiments conducted.

[0063] Figure 19 is a schematic illustrating the classification of the order of Primates.

DETAILED DESCRIPTION OF THE INVENTION

[0064] The invention provides a replication-deficient simian adenoviral vector construct comprising at least one nucleic acid sequence which encodes an antigen. A "vector" is a molecule, such as plasmid, phage, cosmid, liposome, molecular conjugate (e.g., transferrin), or virus, into which another nucleic acid sequence may be introduced so as to bring about the replication of the inserted sequence. The term "construct," as used herein, refers to a vector (e.g., a plasmid or adenoviral vector) containing a nucleic acid sequence inserted therein. Thus, the invention also provides a construct comprising a vector (e.g., a plasmid vector or an adenoviral vector) having inserted therein a nucleic acid sequence which encodes an antigen. Such constructs are referred to herein as "plasmid constructs," "plasmid vector constructs," or "adenoviral vector constructs." An "empty" or "null" vector is a vector that does not contain a nucleic acid sequence encoding an antigen inserted therein.

[0065] Adenoviruses belong to the family Adenoviridae, which is divided into five genera, Mastadenovirus, Atadenovims, Siadenovirus, Aviadenovirus, and Ichtadenovirus. The adenoviruses in the genus Mastadenovirus infect only mammals and include the human, chimpanzee, and monkey adenoviruses. Adenoviruses isolated from humans and chimpanzees have been used extensively in the art as gene transfer vectors. Humans and chimpanzees are very closely related and are grouped together as hominids. In contrast, there is a significantly greater evolutionary distance between monkeys and hominids. In this respect, the monkeys diverged between 25 and 35 million years ago from the hominids, whereas the humans and chimpanzees diverged about 7 million years ago (Samonte et al., Nature Reviews Genetics, 3, 65- 72 (2002)). These similarities and differences between humans, chimpanzees, and monkeys are consistent with documented host range restrictions of adenoviruses. The taxonomy of the order of Primates is illustrated in Figure 19.

[0066] The term "simian," as used herein, refers to both new world and old world monkeys, and does not include any member of the family Hominidae (e.g., humans, chimpanzees, gorillas, and orangutans, which are also referred to as the "great apes"). Therefore, the simian adenoviral vector construct of the invention preferably is derived from an adenovirus isolated from a new world monkey or an old world monkey (collectively referred to herein as "monkeys"). New world monkeys include the families Callitrichidae (e.g., marmosets and tamarins), Cebidae (e.g., capuchins and squirrel monkeys), Aotidae (e.g., night or owl monkeys (douroucoulis)),

Pitheciidae (e.g., titis, sakis and uakaris), and Atelidae (e.g., howler, spider, and woolly · monkeys) (see, e.g., Hershkovitz (ed.), Living New World Monkeys (Platyrrhini), Volume 1, University of Chicago Press (1977)). Old world monkeys include animals in the family

Cercopithecinae, such as, for example, macaques, baboons, and mangabeys (see, e.g.,

Whitehead, ed., Old World Monkeys, Cambridge University Press (2002)). The terms "simian adenoviral vector," "simian adenoviral vector construct," and "simian adenovirus," as used herein, also are synonymous with the terms "monkey adenoviral vector," "monkey adenoviral vector construct," or "monkey adenovirus," respectively, and are used interchangeably herein.

[0067] Thus, the invention also provides a monkey adenovirus that is capable of propagation in a cell comprising one or more gene products of a human adenovirus. The cell can be any suitable cell described herein, but preferably the cell comprises at least one nucleic acid sequence encoding a species (or serotype) C human adenovirus gene product (e.g., gene products encoded by the El A region, the E1B region, and/or the E4 region (e.g., E4 ORF6) of a human

adenovirus). Such cells include, for example, an HEK-293 cell or a PerC.6 cells, which are described herein. The invention also provides a method of propagating the monkey adenovirus. In one embodiment, the invention provides a method of propagating a monkey adenovirus in a human cell, wherein (i) the monkey adenovirus comprises (a) at least one nucleic acid sequence of the El region of a human adenovirus and (b) at least one nucleic acid sequence of the E4 region of a human adenovirus, or (ii) the human cell comprises (a) at least one nucleic acid sequence of the El region of a human adenovirus and (b) at least one nucleic acid sequence of the E4 region of a human adenovirus.

[0068] Adenovirus serotypes are differentiated on the basis of neutralization assays. A serotype is defined as one which either exhibits no or limited cross-reaction with other types (see, Fauquet et al., (eds.), Virus Taxonomy: The Eighth Report of the International Committee on Taxonomy of Viruses, Academic Press, p. 216 (2005)). The serologically distinguishable serotypes (also referred to herein as adenovirus "types") are grouped into species. Classically, the species name has reflected the first described host. The lack of cross neutralization combined with a calculated phylogenetic distance of more than 10% separates two serotypes into different species. In addition, species designation depends on other characteristics that differ between serotypes of adenovirus, including host range, DNA hybridization, RFLP analysis, percentage of GC in the genome, oncogenicity in rodents, growth characteristics, possibility of recombination, number of VA RNA genes, hemagglutination, genetic organization of the E3 region, and host range. Simian adenoviruses isolated from monkeys are more distant from both human adenoviruses and chimpanzee adenoviruses. The chimpanzee adenoviruses are closely related to common human adenoviruses of species B and E, so similar that the chimpanzee adenoviruses are grouped within the human species B and E. The limited phylogenic reconstructions for the simian adenoviruses reveal that the simian adenoviruses are quite distinct from the common chimpanzee and human adenoviruses {Virus Taxonomy: VHIth Report of the International Committee on Taxonomy of Viruses (2005)). The phylogeny of adenoviruses that infect primates is disclosed in, e.g., Roy et al., PLoS Pathog. , 5(7): el00050. doi:10.1371/journal.ppat.l000503 (2009).

[0069] Various origins, serotypes, or mixtures of serotypes can be used as the source of the viral genome for the simian adenoviral vector construct (such as those described in, e.g., U.S. Patents 7,247,472 and 7,491 ,508). For instance, a simian adenovirus can be of serotype 1 , 3, 7, 1 1, 16, 18, 19, 20, 27, 33, 38, 39, 48, 49, 50, or any other simian adenoviral serotype. A simian adenovirus can be referred to by using any suitable abbreviation known in the art, such as, for example, SV, SAdV, or SAV. Preferably, the simian adenoviral vector construct is a simian adenoviral vector construct of serotype 3, 7, 1 1 , 16, 18, 19, 20, 27, 33, 38, or 39. More preferably, the simian adenoviral vector construct is of serotype 7, 1 1 , 16, 18, or 38. These simian adenoviruses, isolated from monkeys, have low sequence homology to human serotype 5 adenovirus and are more closely related, though quite distinct, from the enteric F and G serotype adenoviruses. They contain two different fiber genes (long and short fibers) instead of one fiber gene, which suggests that they may target the gut mucosa, similar to gut-tropic human adenoviruses (e.g., serotypes 40 and 41), where they are expected to stimulate mucosal immune responses. In addition, comparisons between viral hexon proteins suggest that simian adenovirus serotypes 7, 1 1 , 16, and 38 are distantly related to human adenoviruses, and are categorized more closely to gut-tropic adenoviruses (human Ad40, 41, and 52) than to other groups (see Figure 1). As demonstrated herein (see Examples), the immune stimulatory activities of simian adenoviral vector constructs varies depending on the simian adenovirus serotype used to generate the vector construct, as well as the antigen-encoding nucleic acid sequence incorporated into the vector construct.

[0070] Wild-type simian adenoviruses of any serotype can be isolated using any suitable method. For example, simian adenoviruses can be isolated from monkey biopsy and body secretions, including intestine biopsy, fecal washes, nose washes, lung washes, and other body secretions using standard methods known in the art. Wild-type simian adenoviruses also are available from commercial sources, such as the American Type Culture Collection (ATCC, Manassas, Virginia).

[0071] The simian adenoviral vector construct of the invention can be replication-competent. For example, the simian adenoviral vector construct can have a mutation (e.g., a deletion, an insertion, or a substitution) in the adenoviral genome that does not inhibit viral replication in host cells. Preferably, however, the simian adenoviral vector construct is replication-deficient. By "replication-deficient" is meant that the simian adenoviral vector construct comprises an adenoviral genome that lacks at least one replication-essential gene function (i.e., such that the adenoviral vector construct does not replicate in typical host cells, especially those in a human patient that could be infected by the simian adenoviral vector construct in the course of the inventive method). A deficiency in a gene, gene function, or gene or genomic region, as used herein, is defined as a deletion of sufficient genetic material of the viral genome to impair or obliterate the function of the gene whose nucleic acid sequence was deleted in whole or in part. While deletion of genetic material is preferred, mutation of genetic material by addition or substitution also is appropriate for disrupting gene function. Replication-essential gene functions are those gene functions that are required for replication (e.g., propagation) and are encoded by, for example, the adenoviral early regions (e.g., the El , E2, and E4 regions), late regions (e.g., the L1-L5 regions), genes involved in viral packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g., VA-RNA1 and/or VA-RNA-2). More preferably, the replication-deficient simian adenoviral vector construct comprises an adenoviral genome deficient in at least one replication- essential gene function of one or more regions of the adenoviral genome. Preferably, the simian adenoviral vector construct is deficient in at least one gene function of the El A region, the El B region, or the E4 region of the adenoviral genome required for viral replication (denoted an El- deficient or E4-deficient adenoviral vector construct). In addition to a deficiency in the El region, the simian adenoviral vector construct also can have a mutation in the major late promoter (MLP), as discussed in International Patent Application WO 00/00628. Most preferably, the simian adenoviral vector construct is deficient in at least one replication-essential gene function (desirably all replication-essential gene functions) of the El region and at least one gene function of the nonessential E3 region (e.g., an Xba I deletion of the E3 region) (denoted an E1/E3 -deficient adenoviral vector). With respect to the El region, the simian adenoviral vector construct can be deficient in all or part of the El A region and all or part of the E1B region.

When the simian adenoviral vector construct is deficient in at least one replication-essential gene function in only one region of the adenoviral genome (e.g., an El - or E1/E3 -deficient adenoviral vector), the adenoviral vector construct is referred to as "singly replication-deficient." A particularly preferred singly replication-deficient adenoviral vector construct is, for example, a replication-deficient adenoviral vector construct requiring, at most, complementation of the El A and E1B regions of the adenoviral genome, so as to propagate the adenoviral vector construct (e.g., to form adenoviral vector particles).

[0072] The simian adenoviral vector construct of the invention can be "multiply replication- deficient," meaning that the simian adenoviral vector construct is deficient in one or more replication-essential gene functions in each of two or more regions of the adenoviral genome. For example, the aforementioned El-deficient or E1/E3 -deficient simian adenoviral vector construct can be further deficient in at least one replication-essential gene function of the E4 region (denoted an E1/E4- or El/E3/E4-deficient adenoviral vector construct), and/or the E2 region (denoted an E1/E2- or E1/E2/E3 -deficient adenoviral vector construct), preferably the E2A region (denoted an E1/E2A- or E1/E2A/E3 -deficient adenoviral vector construct).

[0073] By removing all or part of, for example, the El , E3, and E4 regions of the simian adenoviral genome, the resulting simian adenoviral vector construct is able to accept inserts of exogenous nucleic acid sequences while retaining the ability to be packaged into adenoviral capsids. The exogenous (or "heterologous") nucleic acid sequence can be positioned in the El region, the E3 region, or the E4 region of the adenoviral genome. Indeed, the nucleic acid sequence can be inserted anywhere in the simian adenoviral genome so long as the position does not prevent expression of the nucleic acid sequence or interfere with packaging of the simian adenoviral vector construct. The simian adenoviral vector construct also can comprise multiple (i.e., two or more) nucleic acid sequences encoding the same antigen. Alternatively, the adenoviral vector construct can comprise multiple nucleic acid sequences encoding two or more different antigens. Each nucleic acid sequence can be operably linked to the same promoter, or to different promoters depending on the expression profile desired by the practitioner, and can be inserted in the same region of the adenoviral genome (e.g., the E4 region) or in different regions of the adenoviral genome. For example, the simian adenoviral vector construct can comprise a nucleic acid sequence that encodes two or more different antigens. Alternatively, the simian adenoviral vector construct can comprise two or more nucleic acid sequences that each encode a different antigen.

[0074] The simian adenoviral vector construct, when multiply replication-deficient, especially in replication-essential gene functions of the El and E4 regions, preferably includes a spacer sequence to provide for viral growth in a complementing cell line similar to that achieved by singly replication-deficient adenoviral vector constructs, particularly an El -deficient adenoviral vector construct. The spacer sequence can contain any nucleotide sequence or sequences which are of a desired length, such as sequences at least about 15 base pairs (e.g., between about 15 base pairs and about 12,000 base pairs), preferably about 100 base pairs to about 10,000 base pairs, more preferably about 500 base pairs to about 8,000 base pairs, even more preferably about 1,500 base pairs to about 6,000 base pairs, and most preferably about 2,000 to about 3,000 base pairs in length. The spacer element sequence can be coding or non- coding and native or non-native with respect to the adenoviral genome, but does not restore the replication-essential function to the deficient region. The spacer element preferably is located in the E4 region of the adenoviral genome. The use of a spacer in an adenoviral vector is described in U.S. Patent 5,851 ,806.

[0075] Desirably, the simian adenoviral vector construct requires, at most, complementation of replication-essential gene functions of the El A, E1B, E2A, and/or E4 regions of the adenoviral genome for replication (i.e., propagation). However, the adenoviral genome can be modified to disrupt one or more replication-essential gene functions as desired by the

practitioner, so long as the simian adenoviral vector construct remains deficient and can be propagated using, for example, complementing cells and/or exogenous DNA (e.g., helper adenovirus) encoding the disrupted replication-essential gene functions. The simian adenoviral vector construct can be deficient in replication-essential gene functions of only the early regions of the adenoviral genome, only the late regions of the adenoviral genome, and both the early and late regions of the adenoviral genome. The simian adenoviral vector construct also can have essentially the entire adenoviral genome removed, in which case it is preferred that at least either the viral inverted terminal repeats (ITRs) and one or more promoters or the viral ITRs and a packaging signal are left intact (i.e., an adenoviral amplicon). In one embodiment, the simian adenoviral vector construct of the invention comprises an adenoviral genome that lacks native nucleic acid sequences which encode adenoviral proteins. Adenoviral genomic elements required for replication and packaging of the adenoviral genome into adenoviral capsid proteins can be retained. Minimal adenoviral vector constructs lacking adenoviral protein coding sequences are termed "helper-dependent" adenoviral vectors, and often require complementation by helper adenovirus for efficient propagation. Examples of replication-deficient adenoviral vector constructs, including multiply replication-deficient adenoviral vector constructs, are disclosed in U.S. Patents 5,837,51 1 ; 5,851 ,806; 5,994,106; 6,127,175; 6,482,616; and 7,195,896, and International Patent Applications WO 94/28152, WO 95/02697, WO 95/16772, WO

95/34671 , WO 96/22378, WO 97/12986, WO 97/21826, and WO 03/02231 1.

[0076] The known host range restrictions of adenoviruses are the result of a variety of factors. For example, wild-type human adenoviruses do not grow productively on monkey cells. In monkey cells infected with wild-type human adenovirus, the viral early genes are properly expressed (see, e.g., Feldman et al., J. Bacteriol, 91: 813-8 (1966); and Van der Vliet and Levine, Nature, 246: 170-4 (1973)) and viral DNA replication occurs normally (see, e.g., Rapp et al., J. BacterioL, 92: 931 -6 (1966); Reich et al., PNAS, 55: 336-41 (1966); and Friedman et al., J. Virol., 5: 586-97 (1970)). However, the expression of several late viral proteins is reduced. The block to late gene expression appears to be due to abnormal production of the viral late mRNAs (see, e.g., Klessig and Anderson, J. Virol. , 16: 1650-68 (1975)), and this block can be overcome by a single mutation of the adenovirus DNA Binding Protein (DBP) (see, e.g., Klessig and Grodzicker, Cell, 17: 957-66 (1979)). Human adenovirus that contain this single mutation in the DBP grow productively on monkey cells, suggesting that that the key to the monkey/human block is centered on the roles of the DBP during the life cycle of the adenovirus.

[0077] In contrast to the monkey/human block are the observations that adenoviruses isolated from chimpanzees do not have a restriction in human cells and can be propagated efficiently therein (see, e.g., W. P. Rowe et al., Proc. Soc. Exp. Biol. Med. , 97(2): 465-470 (1958); W. D. Hillis et al., American Journal of Epidemiology, 90(4): 344-353 (1969); and N. Rogers et al., Nature, 216: 446-449 (1967)). In fact, replication of some chimpanzee adenovirus isolates was found to be more efficient in human than in monkey cells (see, e.g., M. Basnight et al., American Journal of Epidemiology, 94(2): \ββ-\Ί\ (1971)). Adenoviruses isolated from other great apes species, such as gorillas and bonobos, have also recently been shown to grow in human cells (see, e.g., S. Roy et al, PLoS Pathogens, 5(1): el 000503 (2009)). Wild-type chimpanzee adenovirus replication in human cells does not require the expression of human adenovirus complementing factors as both El -expressing cell lines (e.g., human embryonic kidney 293 cells, human retina PER.C6 cells ) have been used for their propagation (see, e.g., U.S. Patent 6,083,716; S. F. Farina et al., Journal of Virology, 74(23): 1 1603-1 1613 (2001); S. Roy et al, Virology, 324: 361-372 (2004); S. Roy et al., Human Gene Therapy, 15: 519-530 (2004); and E. Fattori et al, Gene Therapy, i3(14): 1088-1096 (2006)), as well as cell lines that do not express El (e.g., A549 human lung epithelial carcinoma cells) (J. Skog et al., Molecular Therapy, 15(12): 2140-2145 (2007); and D. Peruzzi et al., Vaccine, 27(9): 1293-1300 (2009)). This absence of a replication block is consistent with the close evolutionary distance between the human and chimpanzee lineages, which diverged only about 7 million years ago (Samonte & Eichler, Nature Reviews Genetics, 3: 65-72 (2002)). [0078] Consistent with this greater divergence of host, a host range restriction of monkey adenoviruses for growth on human cells has been described (see, e.g., Am. J. Hyg. , 68: 31 (1958); Virology, 35: 248 (1968); Savitskaya et al., Doklady Biochemistry, 375: 242 (2000); Alstein et al., J Virol. , 2: 488 (1968); and Genetika, 39(6): 725-31 (June 2003)), and it has been hypothesized that the determinants are partially E4 and possibly E2. There was no growth of the monkey adenovirus SV7(C8) (now known as SV16 (ICTV 8th Report, p. 220)) on human embryonic kidney (HEK) cell line 293 (Savitskaya et al., supra). Thus, an El region from a human adenovirus was not sufficient to alleviate the block to replication. The virus could grow on HEK-293 cells with Ad5 E4 region inserted (VK- 10-9 cells). However, the VK-10-9 cells provided only partial alleviation of the replication block since replication was 40-fold lower than on CV1 cells (continuous line of green monkey kidney). This showed there was still a block to monkey virus replication in VK-10-9 cells. E4 expression was concluded to be too low, based on E4 ORF3 protein level (Krougliak and Graham, Hum. Gene Ther. , 6: 1575 (1995)) and a virus specific product was likely required (Savitskaya et al., supra). The product was proposed to be encoded by the E2A gene. Thus, adenovirus El and E4 regions are likely not sufficient for alleviating the species block, and that other regions, in particular that encoding the DBP (E2A), are important (Savitskaya et al., supra). The level of E4 expression in the VK-10-9 cells was reported to be sufficient for replication of an E4-deleted human adenovirus type 5 virus to the same level as wild type human Ad5 (Krougliak and Graham, supra). Thus, it is apparent that the Ad5 E4 function required for virus growth is separate from that required to overcome host range restriction of monkey adenoviruses on human cells because the E4 requirement for growth is not the same for host range determination. In addition, an adenovirus-adenovirus hybrid of human Ad2 and SA7(C8) was defective for replication, suggesting that human El and monkey E4, are not compatible and that human adenovirus El is not sufficient for overcoming the host range restriction, consistent with the above described results where human El expressed from the cell did not change host range (see, e.g., Alstein et al., J. Virol , 2: 488 (1968); and Savitskaya et al., supra). A different adenovirus-adenovirus hybrid between Ad2 and SA7(C8) was generated by growth of the two viruses under selection conditions to prevent Ad2 propagation (Grinenko et al., Molecular Genetics, Microbiology and Virology, 5: 25 (2004)). Growth and selection of the hybrid virus on human cells (HEK-293) yielded a defective virus that had incorporated only the L3 region of SA7(C8). Both Ad2 E4 and E2A (encoding the DNA binding protein) were present and intact in the defective hybrid, and the E4 gene and possibly the E2A gene are involved in the determination of species-specific host range, consistent with the earlier conclusions that more than E4 was required for alleviating the host range restriction. In fact, these results only show that 10% of the Ad2 genome could be removed in order for a monkey/human adenovirus hybrid to grow on human cells, leaving 90% of the Ad2 genome to contain host range determining factors. Therefore, this hybrid did not provide further delineation of human adenovirus products required for growth of monkey adenovirus on human cells. Taken together, these reports demonstrate that E4 plays a role in host range determination but other adenovirus genes also are involved. Furthermore, the E4 region is comprised of at least five known protein products and, despite these studies, the component or components of E4 that may have been necessary for the partial alleviation of the host range block have not been identified.

[0079] The invention provides improved monkey adenovirus (or simian adenovirus) replication in human cells with complete alleviation of host range block. Monkey adenoviruses do not grow on human cells; however, equal or even superior growth of monkey adenoviruses occurs on human cells with human adenovirus components compared to monkey cells, as demonstrated herein. The host range restriction can be removed by, for example, propagating a monkey adenovirus on human embryonic kidney cell line 293 (HEK-293) along with human adenovirus E4 ORF6 protein (34K) expressed during adenovirus infection. In accordance with the invention, in order to overcome the host range restriction of simian adenoviruses to grow on human cells, a subset of human adenovirus E4 must be expressed during viral replication in the cell instead of the whole E4 region, as the function of E4 that is necessary and sufficient lies within ORF6. The host range determinant does not include all of E4, and does not include E2A. Rather, the host range determinant is E4 ORF6, and not one of the other factors singly or in combination encoded within E4.

[0080] The identification of the human adenovirus components necessary and sufficient for replication of simian adenovirus on human cells has many advantages, such as, for example, the feasible manufacture of products based on simian adenovirus. In addition, the ability to reduce the components of E4 required to alleviate the host range block to monkey adenoviruses on human cells has clear advantages compared to requiring all of E4. The simplification of the E4 requirement allows for easier manipulation of the DNA sequences and proper regulation of expression of the E4 sequences. This allows for easier design of systems to allow propagation of simian adenoviruses on human cells. For example, the subset of human adenovirus E4 sequences can be included in a simian adenovirus genome, integrated into the genome of a cell, or exist extra chromosomally as neither part of the human cell nor the simian adenovirus.

Working with only a subset of E4 sequences that comprise E4 ORF6 allows for easier and more reliable regulation of expression of these sequences. This enhanced control will lead to higher yields of simian adenoviruses which will allow for reduction in cost of goods and expand the commercial and scientific applications that simian adenoviruses can be used for.

[0081] Another advantage of the identification of the human adenovirus components necessary and sufficient for replication of monkey adenovirus on human cells is the ability to generate conditionally-replicating adenoviruses. The inclusion of human adenovirus El and E4 ORF6 sequences in the simian adenovirus, under expression control elements that are specific to a given disease, syndrome, condition, tissue, or cell type, allow for replication of the simian virus in a controlled fashion only where desired. Applications for conditionally-replication

adenoviruses are numerous and include, for example, lysis of tumor cells, expression of a therapeutic gene only under conditions of viral vector replication, and limited-replication vaccines.

[0082] Another advantage of the identification of the human adenovirus components necessary and sufficient for replication of simian adenovirus on human cells is the ability to generate adenovirus gene transfer vectors where a transgene expression cassette is incorporated into the simian adenovirus genome. Adenovirus vectors derived from simian adenoviruses propagated on a human cell line-human adenovirus system have the following advantages: (1 ) absence of pre-existing immunity in human populations to the simian adenoviruses, (2) species- specific block to replication for enhanced safety to human populations, (3) avoids risk of adventitious xenogeneic pathogens from manufacturing on a non-human cell line. The monkey polyoma virus S V40 was found to contaminate batches of human vaccine product manufactured on monkey cells.

[0083] Complementing cell lines for producing the simian adenoviral vector construct include, but are not limited to, VERO cells (described in, e.g., Yasumura and Kawakita, Nippon Rinsho, 21 (6): 1201-1219 (1963)), 293 cells (described in, e.g., Graham et al., J. Gen. Virol. , 36: 59-72 (1977)), PER.C6 cells (described in, e.g., International Patent Application Publication WO 97/00326, and U.S. Patents 5,994,128 and 6,033,908), and 293-ORF6 cells (described in, e.g., International Patent Application Publication WO 95/34671 and Brough et al., J Virol, 71: 9206- 9213 (1997)). Additional complementing cells are described in, for example, U.S. Patents 6,677,156 and 6,682,929, and International Patent Application Publication WO 03/20879. In some instances, the cellular genome need not comprise nucleic acid sequences, the gene products of which complement for all of the deficiencies of a replication-deficient adenoviral vector construct. One or more replication-essential gene functions lacking in a replication-deficient adenoviral vector construct can be supplied by a helper virus, e.g., an adenoviral vector construct that supplies in trans one or more essential gene functions required for replication of the desired adenoviral vector construct. Helper virus is often engineered to prevent packaging of infectious helper virus. For example, one or more replication-essential gene functions of the El region of the adenoviral genome are provided by the complementing cell, while one or more replication- essential gene functions of the E4 region of the adenoviral genome are provided by a helper virus.

[0084] Ideally, the replication-deficient simian adenoviral vector construct is present in a composition, e.g., a pharmaceutical composition, substantially free of replication-competent adenovirus (RCA) contamination (e.g., the composition comprises less than about 1% of replication-competent adenovirus on the basis of the total adenoviruses in the composition). Most desirably, the composition is RCA-free. Adenoviral vector compositions and stocks that are RCA-free are described in U.S. Patent 5,944,106, and International Patent Application WO 95/34671.

[0085] If the simian adenoviral vector construct is not replication-deficient, ideally the simian adenoviral vector construct is manipulated to limit replication of the vector construct to within a target tissue. For example, the simian adenoviral vector construct can be a

conditionally-replicating adenoviral vector construct which is engineered to replicate under conditions pre-determined by the practitioner. For example, replication-essential gene functions, e.g., gene functions encoded by the adenoviral early regions, can be operably linked to an inducible, repressible, or tissue-specific transcription control sequence, e.g., promoter. In this embodiment, replication requires the presence or absence of specific factors that interact with the transcription control sequence. In the treatment of viral infections, for example, it can be advantageous to control adenoviral vector replication in, for instance, lymph nodes, to obtain continual antigen production and control immune cell production. Conditionally-replicating adenoviral vectors are described further in U.S. Patent 5,998,205.

[0086] The simian adenoviral vector construct can be manipulated to alter the binding specificity or recognition of the adenovirus for a receptor on a potential host cell. For adenovirus, such manipulations can include deletion of regions of adenovirus coat proteins (e.g., fiber, penton, or hexon), insertions of various native or non-native ligands into portions of a coat protein, and the like. Manipulation of the coat protein can broaden the range of cells infected by the simian adenoviral vector construct or enable targeting of the simian adenoviral vector construct to a specific cell type.

[0087] Any suitable technique for altering native binding to a host cell, such as native binding of the fiber protein to its cellular receptor, can be employed. For example, differing fiber lengths can be exploited to ablate native binding to cells. This optionally can be

accomplished via the addition of a binding sequence to the penton base or fiber knob. This addition of a binding sequence can be done either directly or indirectly via a bispecific or multispecific binding sequence. In an alternative embodiment, the adenoviral fiber protein can be modified to reduce the number of amino acids in the fiber shaft, thereby creating a "short- shafted" fiber (as described in, for example, U.S. Patent 5,962,31 1). Use of an adenovirus comprising a short-shafted adenoviral fiber gene reduces the level or efficiency of adenoviral fiber binding to its cell-surface receptor and increases adenoviral penton base binding to its cell- surface receptor, thereby increasing the specificity of binding of the adenovirus to a given cell. Alternatively, use of a simian adenoviral vector construct comprising a short-shafted fiber enables targeting of the simian adenoviral vector construct to a desired cell-surface receptor by the introduction of a nonnative amino acid sequence either into the penton base or the fiber knob.

[0088] In yet another embodiment, the nucleic acid residues encoding amino acid residues associated with native substrate binding can be changed, supplemented, or deleted (see, e.g., International Patent Application Publication WO 00/15823, Einfeld et al., J. Virol., 75(23):

1 1284-1 1291 (2001), and van Beusechem et al., J. Virol. , 76(6): 2753-2762 (2002)) such that the simian adenoviral vector construct incorporating the mutated nucleic acid residues (or having the fiber protein encoded thereby) is less able to bind its native substrate.

[0089] Any suitable amino acid residue(s) of a fiber protein that mediates or assists in the interaction between the knob and the native cellular receptor can be mutated or removed, so long as the fiber protein is able to trimerize. Similarly, amino acids can be added to the fiber knob as long as the fiber protein retains the ability to trimerize. Suitable residues include amino acids within the exposed loops of the fiber knob domain, such as, for example, the AB loop, the DE loop, the FG loop, and the HI loop.

[0090] Any suitable amino acid residue(s) of a penton base protein that mediates or assists in the interaction between the penton base and integrins can be mutated or removed. Suitable residues include, for example, an RGD amino acid sequence motif located in the hypervariable region of the simian adenovirus penton base protein. The native integrin binding sites on the penton base protein also can be disrupted by modifying the nucleic acid sequence encoding the native RGD motif such that the native RGD amino acid sequence is conformational ly

inaccessible for binding to an integrin receptor, such as by inserting a DNA sequence into or adjacent to the nucleic acid sequence encoding the adenoviral penton base protein.

[0091] The simian adenoviral vector construct can comprise a fiber protein and a penton base protein that do not bind to their respective native cellular binding sites. Alternatively, the simian adenoviral vector construct comprises fiber protein and a penton base protein that bind to their respective native cellular binding sites, but with less affinity than the corresponding wild-type coat proteins. The simian adenoviral vector construct exhibits reduced binding to native cellular binding sites if a modified adenoviral fiber protein and penton base protein binds to their respective native cellular binding sites with at least about 5-fold, 10-fold, 20-fold, 30-fold, 50- fold, or 100-fold less affinity than a non-modified adenoviral fiber protein and penton base protein of the same serotype.

[0092] The simian adenoviral vector construct also can comprise a chimeric coat protein comprising a non-native amino acid sequence that binds a substrate (i.e., a ligand), such as a cellular receptor other than a native cellular receptor. The non-native amino acid sequence of the chimeric adenoviral coat protein allows the simian adenoviral vector construct comprising the chimeric coat protein to bind and, desirably, infect host cells not naturally infected by a corresponding adenovirus without the non-native amino acid sequence (i.e., host cells not infected by the corresponding wild-type adenovirus), to bind to host cells naturally infected by the corresponding wild-type adenovirus with greater affinity than the corresponding adenovirus without the non-native amino acid sequence, or to bind to particular target cells with greater affinity than non-target cells. A "non-native" amino acid sequence can comprise an amino acid sequence not naturally present in the adenoviral coat protein or an amino acid sequence found in the adenoviral coat but located in a non-native position within the capsid. By "preferentially binds" is meant that the non-native amino acid sequence binds a receptor, such as, for instance, ανβ3 integrin, with at least about 3-fold greater affinity (e.g., at least about 5-fold, 10-fold, 15- fold, 20-fold, 25-fold, 35-fold, 45-fold, or 50-fold greater affinity) than the non-native ligand binds a different receptor, such as, for instance, ανβΐ integrin.

[0093] The simian adenoviral vector construct can comprise a chimeric coat protein comprising a non-native amino acid sequence that confers to the chimeric coat protein the ability to bind to an immune cell more efficiently than a wild-type adenoviral coat protein. In particular, the simian adenoviral vector construct can comprise a chimeric adenoviral fiber protein comprising a non-native amino acid sequence which facilitates uptake of the simian adenoviral vector construct by immune cells, preferably antigen presenting cells, such as dendritic cells, monocytes, and macrophages. In a preferred embodiment, the simian adenoviral vector construct comprises a chimeric fiber protein comprising an amino acid sequence (e.g., a non-native amino acid sequence) comprising an RGD motif, which increases transduction efficiency of the simian adenoviral vector construct into dendritic cells. The RGD-motif, or any non-native amino acid sequence, preferably is inserted into the adenoviral fiber knob region, ideally in an exposed loop of the adenoviral knob, such as the HI loop. A non-native amino acid sequence also can be appended to the C-terminus of the adenoviral fiber protein, optionally via a spacer sequence. The spacer sequence preferably comprises between one and two-hundred amino acids, and can (but need not) have an intended function.

[0094] In another embodiment, the simian adenoviral vector construct can comprise a chimeric virus coat protein that is not selective for a specific type of eukaryotic cell. The chimeric coat protein differs from a wild-type coat protein by an insertion of a non-native amino acid sequence into or in place of an internal coat protein sequence, or attachment of a non-native amino acid sequence to the N- or C- terminus of the coat protein. For example, a ligand comprising about five to about nine lysine residues (preferably seven lysine residues) can be attached to the C-terminus of the adenoviral fiber protein via a non-functional spacer sequence. In this embodiment, the chimeric virus coat protein efficiently binds to a broader range of eukaryotic cells than a wild-type virus coat, such as described in U.S. Patent 6,465,253 and International Patent Application Publication WO 97/20051.

[0095] The ability of the simian adenoviral vector construct to recognize a potential host cell can be modulated without genetic manipulation of the coat protein, i.e., through use of a bi- specific molecule. For instance, complexing an adenovirus with a bispecific molecule comprising a penton base-binding domain and a domain that selectively binds a particular cell surface binding site enables the targeting of the simian adenoviral vector construct to a particular cell type. Likewise, an antigen can be conjugated to the surface of the adenoviral particle through non-genetic means.

[0096] A non-native amino acid sequence can be conjugated to any of the adenoviral coat proteins to form a chimeric adenoviral coat protein. Therefore, for example, a non-native amino acid sequence can be conjugated to, inserted into, or attached to a fiber protein, a penton base protein, a hexon protein, protein IX, VI, or Ilia, etc. Methods for employing such proteins are well known in the art (see, e.g., U.S. Patents 5,543,328; 5,559,099; 5,712,136; 5,731 ,190;

5,756,086; 5,770,442; 5,846,782; 5,962,31 1 ; 5,965,541 ; 5,846,782; 6,057,155; 6,127,525;

6,153,435; 6,329,190; 6,455,314; 6,465,253; 6,576,456; 6,649,407; 6,740,525; and 6,951 ,755, and International Patent Application Publications WO 96/07734, WO 96/26281 , WO 97/20051 , WO 98/07877, WO 98/07865, WO 98/40509, WO 98/54346, WO 00/15823, WO 01/58940, and WO 01/92549). The chimeric adenoviral coat protein can be generated using standard recombinant DNA techniques known in the art. Preferably, the nucleic acid sequence encoding the chimeric adenoviral coat protein is located within the adenoviral genome and is operably linked to a promoter that regulates expression of the coat protein in a wild-type adenovirus. Alternatively, the nucleic acid sequence encoding the chimeric adenoviral coat protein is located within the adenoviral genome and is part of an expression cassette which comprises genetic elements required for efficient expression of the chimeric coat protein. [0097] The coat protein portion of the chimeric adenovirus coat protein can be a full-length adenoviral coat protein to which the non-native amino acid sequence is appended, or it can be truncated, e.g., internally or at the C- and/or N- terminus. However modified (including the presence of the non-native amino acid), the chimeric coat protein preferably is able to incorporate into an adenoviral capsid. Where the non-native amino acid sequence is attached to the fiber protein, preferably it does not disturb the interaction between viral proteins or fiber monomers. Thus, the non-native amino acid sequence preferably is not itself an oligomerization domain, as such can adversely interact with the trimerization domain of the adenovirus fiber. Preferably the non-native amino acid sequence is added to the virion protein, and is incorporated in such a manner as to be readily exposed to a substrate, cell surface-receptor, or immune cell (e.g., at the N- or C- terminus of the adenoviral protein, attached to a residue facing a substrate, positioned on a peptide spacer, etc.) to maximally expose the non-native amino acid sequence. Ideally, the non-native amino acid sequence is incorporated into an adenoviral fiber protein at the C-terminus of the fiber protein (and attached via a spacer) or incorporated into an exposed loop (e.g., the HI loop) of the fiber to create a chimeric coat protein. Where the non-native amino acid sequence is attached to or replaces a portion of the penton base, preferably it is within the hypervariable regions to ensure that it contacts the substrate, cell surface receptor, or immune cell. Where the non-native amino acid sequence is attached to the hexon, preferably it is within a hypervariable region (Crawford-Miksza et al., J. Virol , 70(3): 1836-44 (1996)). Where the non- native amino acid is attached to or replaces a portion of pIX, preferably it is within the C- terminus of pIX. Use of a spacer sequence to extend the non-native amino acid sequence away from the surface of the adenoviral particle can be advantageous in that the non-native amino acid sequence can be more available for binding to a receptor, and any steric interactions between the non-native amino acid sequence and the adenoviral fiber monomers can be reduced.

[0098] In other embodiments (e.g., to facilitate purification or propagation within a specific engineered cell type), a non-native amino acid (e.g., ligand) can bind a compound other than a cell-surface protein. Thus, the ligand can bind blood- and/or lymph-borne proteins (e.g., albumin), synthetic peptide sequences such as polyamino acids (e.g., polylysine, polyhistidine, etc.), artificial peptide sequences (e.g., FLAG), and RGD peptide fragments (Pasqualini et al., J Cell. Biol. , 130: 1 189 (199.5)). A ligand can even bind non-peptide substrates, such as plastic (e.g., Adey et al., Gene, 156: 27 (1995)), biotin (Saggio et al., Biochem. J , 293: 613 (1993)), a DNA sequence (Cheng et al., Gene, 171 : 1 (1996), and Krook et al., Biochem. Biophys., Res. Commun. , 204: 849 (1994)), streptavidin (Geibel et al., Biochemistry, 34: 15430 (1995), and Katz, Biochemistry, 34: 15421 (1995)), nitrostreptavidin (Balass et al., Anal. Biochem., 243: 264 (1996)), heparin (Wickham et al., Nature Biotechnol., 14: 1570-73 (1996)), and other substrates.

[0099] Disruption of native binding of adenoviral coat proteins to a cell surface receptor can also render it less able to interact with the innate or acquired host immune system. Aside from pre-existing immunity (in the case of human adenoviruses), adenoviral vector administration induces inflammation and activates both innate and acquired immune mechanisms. Adenoviral vector constructs activate antigen-specific (e.g., T-cell dependent) immune responses, which limit the duration of transgene expression following an initial administration of the vector. In addition, exposure to adenoviral vector constructs stimulates production of neutralizing antibodies by B cells, which can preclude gene expression from subsequent doses of adenoviral vector construct (Wilson & Kay, Nat. Med., 3(9): 887-889 (1995)). Indeed, the effectiveness of repeated administration of the vector can be severely limited by host immunity. In addition to stimulation of humoral immunity, cell-mediated immune functions are responsible for clearance of the virus from the body. Rapid clearance of the virus is attributed to innate immune mechanisms (see, e.g., Worgall et al., Human Gene Therapy, 8: 37-44 (1997)), and likely involves Kupffer cells found within the liver. Thus, by ablating native binding of an adenovirus fiber protein and penton base protein, immune system recognition of an adenoviral vector is diminished, thereby increasing vector tolerance by the host.

[0100] Another method for altering host immunity to adenovirus involves modifying an adenoviral coat protein such that it exhibits reduced recognition by the host immune system. Thus, the inventive simian adenoviral vector construct can comprise such a modified coat protein. The modified coat protein preferably is a penton, fiber, or hexon protein. Most preferably, the modified coat protein is a hexon protein. The coat protein can be modified in any suitable manner, but is preferably modified by generating diversity in the coat protein.

Preferably, such coat protein variants are not recognized by pre-existing host (e.g., human) adenovirus-specific neutralizing antibodies. Diversity can be generated using any suitable method known in the art, including, for example, directed evolution (i.e., polynucleotide shuffling) and error-prone PCR (see, e.g., Cadwell, PCR Meth. Appl, 2: 28-33 (1991), Leung et al., Technique, 7: 1 1-15 (1989), and Pritchard et al., J. Theoretical Biol, 234: 497-509 (2005)). Preferably, coat protein diversity is generated through directed evolution techniques, such as those described in, e.g., Stemmer, Nature, 370: 389-91 (1994), Cherry et al., Nat. Biotechnol., 17: 379-84 (1999), and Schmidt-Dannert et al., Nat Biotechnol., 75(7): 750-53 (2000).

[0101] An adenoviral coat protein also can be modified to evade pre-existing host immunity by deleting a region of a coat protein and replacing it with a corresponding region from the coat protein of another adenovirus serotype, particularly a serotype which is less immunogenic in humans. In this regard, amino acid sequences within the fiber protein, the penton base protein, and/or the hexon protein can be removed and replaced with corresponding sequences from a different adenovirus serotype. Thus, for example, when the fiber protein is modified to evade pre-existing host immunity, amino acid residues from the knob region of a simian adenovirus fiber protein can be deleted and replaced with corresponding amino acid residues from an a simian adenovirus of a different serotype, such as those serotypes described herein. Likewise, when the penton base protein is modified to evade pre-existing host immunity, amino acid residues within the hypervariable region of a simian adenovirus penton base protein can be deleted and replaced with corresponding amino acid residues from a simian adenovirus of a different serotype, such as those serotypes described herein. Preferably, the hexon protein of the simian adenoviral vector construct is modified in this manner to evade pre-existing host immunity. In this respect, amino acid residues within one or more of the hypervariable regions, which occur in loops of the hexon protein, are removed and replaced with corresponding amino acid residues from a simian adenovirus of a different serotype. An entire loop region can be removed from the hexon protein and replaced with the corresponding loop region of another simian adenovirus serotype. Alternatively, portions of a loop region can be removed from the simian adenoviral vector hexon protein and replaced with the corresponding portion of a hexon loop of another adenovirus serotype (simian or human). One or more hexon loops, or portions thereof, of a simian adenoviral vector can be removed and replaced with the corresponding sequences from any other adenovirus serotype (simian or human), such as those described herein. Methods of modifying hexon proteins are disclosed in, for example, Rux et al., J Virol. , 77: 9553-9566 (2003), and U.S. Patent 6,127,525. The hypervariable regions of a hexon protein also can be replaced with random peptide sequences, or peptide sequences derived from a disease-causing pathogen (e.g., HIV).

[0102] Modifications to adenovirus coat proteins are described in, for example, U.S. Patents 5,543,328; 5,559,099; 5,712,136; 5,731 ,190; 5,756,086; 5,770,442; 5,846,782; 5,871 ,727;

5,885,808; 5,922,315; 5,962,31 1 ; 5,965,541 ; 6,057,155; 6,127,525; 6,153,435; 6,329,190;

6,455,314; 6,465,253; 6,576,456; 6,649,407; 6,740,525; and 6,951 ,755; and International Patent Applications WO 96/07734, WO 96/26281 , WO 97/20051 , WO 98/07865, WO 98/07877, WO 98/40509, WO 98/54346, WO 00/15823, WO 01/58940, and WO 01/92549.

[0103] The simian adenoviral vector construct comprises at least one nucleic acid that encodes an antigen. A "nucleic acid" is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain non- natural or altered nucleotides. Nucleic acids are typically linked via phosphate bonds to form nucleic acids or polynucleotides, though many other linkages are known in the art (e.g., phosphorothioates, boranophosphates, and the like). Any nucleic acid sequence that is inserted into the simian adenovirus genome also is referred to herein as a "heterologous" or "exogenous" nucleic acid sequence. The nucleic acid sequence is not limited to a type of nucleic acid sequence of any particular origin. In this respect, the nucleic acid sequence can be recombinant DNA or genomic DNA. The nucleic acid sequence can be obtained from a DNA library of potential antigenic epitopes, or can be synthetically generated. If the nucleic acid is RNA, it desirably has a function (e.g., a regulatory function). Examples of such RNA molecules include siRNA, shRNA, microRNA, antisense RNA, and VA RNA. The RNA can also encode a polypeptide such as a protein.

[0104] In a preferred embodiment of the invention, the nucleic acid sequence encoding an antigen comprises codons expressed more frequently in humans than in the pathogen. While the genetic code is generally universal across species, the choice among synonymous codons is often species-dependent. One of ordinary skill in the art would appreciate that, to achieve maximum protection against infection by a pathogen, the adenoviral vector construct must be capable of expressing high levels of antigens in a mammalian, preferably a human, host. In this respect, the nucleic acid sequence preferably encodes the native amino acid sequence of an antigen, but comprises codons that are expressed more frequently in mammals (e.g., humans) than in the pathogen. Changing all native pathogen codons to the most frequently used in mammals will increase expression of the antigen in a mammal (e.g., a human). Such modified nucleic acid sequences are commonly described in the art as "humanized," as "codon-optimized," or as utilizing "mammalian-preferred" or "human-preferred" codons. In the context of the invention, a nucleic acid sequence is said to be "codon-optimized" if at least about 60% (e.g., at least about 70%, at least about 80%, or at least about 90%) of the wild-type codons in the nucleic acid sequence are modified to encode mammalian-preferred codons. That is, a nucleic acid sequence is codon-optimized if at least about 60% of the codons encoded therein are mammalian-preferred codons.

[0105] An "antigen" is a molecule that triggers an immune response in a mammal. An "immune response" can entail, for example, antibody production and/or the activation of immune effector cells. An antigen in the context of the invention can comprise any subunit, fragment, or epitope of any proteinaceous or non-proteinaceous (e.g., carbohydrate or lipid) molecule which provokes an immune response in mammal. By "epitope" is meant a sequence of an antigen that is recognized by an antibody or an antigen receptor. Epitopes also are referred to in the art as "antigenic determinants." The antigen can be a protein or peptide of viral, bacterial, parasitic, fungal, protozoan, prion, cellular, or extracellular origin, which provokes an immune response in a mammal, preferably leading to protective immunity. The antigen also can be a self antigen, i.e., an autologous protein which the body has mistakenly identified as a foreign invader.

[0106] In a preferred embodiment, the heterologous nucleic acid sequence preferably encodes an antigen of a pathogen. The pathogen can be a virus, but not limited to, a virus from any of the following viral families: Arenaviridae, Arterivirus, Astroviridae, Bacidoviridae, Badnavirus, Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae,

Capillovirus, Carlaviriis, Caulimovirus, Circoviridae, Closterovirus, Comoviridae,

Coronaviridae (e.g., Coronavirus, such as severe acute respiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae, Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virus and Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)), Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1 , Dengue virus 2, Dengue virus 3, and Dengue virus 4), Hepadnaviridae (e.g., Hepatitis B virus), Herpesviridae (e.g., Human herpesvirus 1 , 3, 4, 5, and 6, and Cytomegalovirus), Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae, Orthomyxoviridae (e.g., influenza virus A and B), Papovaviridae, Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytial virus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus, hepatovirus, and aphthovirus (e.g., foot and mouth disease virus)), Poxviridae (e.g., vaccinia virus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus, such as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae, and Totiviridae. In one embodiment, the pathogen is a Respiratory Syncytial Virus (RSV), and the antigen can be, for example, an RSV strain A or strain B antigen, such as all or part of the F, G, M, Ml , M2, SH, or NS1 , or NS2 proteins, or a fusion of all or part of more than one of these proteins. In another embodiment, the pathogen is Herpes Simplex Virus 2 (HSV-2). In yet another embodiment, the pathogen is a SARS virus, and the antigen can be, for example, all or part of the UL19, UL47, or gD proteins.

[0107] The pathogen also can be a parasite, such as, for example, a Plasmodium parasite, which causes malaria (e.g., Plasmodium falciparum). Alternatively, the heterologous nucleic acid sequence can encode, for example, an atonal homolog protein (e.g., HATH1 or MATH1), TNF- , or pigment epithelium-derived factor (PEDF).

[0108] The pathogen also can be a bacterium, including, for example, Actinomyces,

Anabaena, Bacillus, Bacteroides, Bdellovibrio, Caulobacter, Chlamydia, Chlorobium,

Chromatium, Clostridium, Cytophaga, Deinococcus, Escherichia, Halobacterium, Heliobacter, Hyphomicrobium, Methanobacterium, Micrococcus, Myobacterium, Mycobacterium (e.g., Mycobacterium tuberculosis), Mycoplasma, Myxococcus, Neisseria, Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus, Thermoplasma,

Thiobacillus, and Treponema. In one embodiment, at least one antigen encoded by the nucleic acid sequence is a Pseudomonas antigen or a Heliobacter antigen.

[0109] An antigen in the context of the invention can comprise any proteinaceous pathogen molecule (e.g., an HIV molecule) or portion thereof that provokes an immune response in a mammal (e.g., a humoral or cellular immune response). A "pathogen molecule" is a molecule that is a part of a particular pathogen, is encoded by a nucleic acid sequence of a particular pathogen, or is derived from or synthetically based upon any such molecule. Administration of an antigen that provokes an immune response in accordance with the invention preferably leads to protective immunity against the pathogen. In this regard, an "immune response" to a pathogen is an immune response to any one or more antigens of the pathogen.

[0110] In one embodiment, the simian adenoviral vector construct comprises a nucleic acid sequence that encodes an HIV antigen. Examples of suitable HIV antigens include all or part of HIV Gag, Env, Pol, Tat, Reverse Transcriptase (RT), Vif, Vpr, Vpu, Vpo, Integrase, or Nef proteins. Preferably, the HIV antigen comprises all or part of an HIV Gag, Env, and/or Pol protein. Suitable Env proteins are known in the art and include, for example, gpl60, gpl20, gp41 , gpl45, gpl40, and gpl40dvl2. The gpl40dvl2 protein comprises deletions of the VI and V2 loops of the Env gpl40 protein (see, e.g., U.S. Patent Application Publication No.

2010/01 1 1998 Al). In addition, an HIV antigen can be modified such that it exhibits enhanced immunogenicity in vivo. For example, the antigen can be an Env protein comprising mutations in the cleavage site, fusion peptide, or interhelical coiled-coil domains of the Env protein (ACFI Env proteins) (see, e.g., Cao et al., J. Virol., 71, 9808-9812 (1997), and Yang et al., J. Virol, 78, 4029-4036 (2004)). The antigen also can be a monomeric or trimeric HIV polypeptide (e.g., Env) which has been modified to increase its stability in vivo, such as those described in, e.g., U.S. Patent Application Publication Nos. 2009/0191235 Al and 2009/01 10690 Al . In addition, the HIV antigen can be synthetically generated. Synthetically generated antigen sequences include, for example, consensus HIV antigens, mosaic HIV antigens, and other bioinformatically generated antigens. "Consensus HIV antigens" are generated by comparing the amino acid sequences of a plurality of naturally-occurring HIV antigens to identify common sequences within them and generating a synthetic HIV antigen in which every amino acid is present in a plurality of sequences. Methods for the generation of "consensus" sequences for the HIV-1 Env protein are described in, for example, Weaver et al., J. Virol , 80: 6745-6756 (2006).

[0111] "Mosaic HIV sequences" are generated using natural sequences as input to algorithms, such as genetic algorithms, which maximize the diversity of potential T-cell epitopes present in the natural sequences. The genetic algorithm identifies potential T-cell epitopes within the input sequences, generates potential recombinants between the input sequences, and identifies those recombinants which have the greatest diversity of T cell epitopes. Epitopes which occur infrequently may be omitted from the mosaic sequences while those which provide enhanced coverage relative to a sequence lacking that epitope may be incorporated into the mosaic sequence. Methods for generating mosaic sequences are described in, e.g., Fischer et al., Nature Medicine, 13(1): 100-106 (2007); and international Patent Application Publications WO 2007/024941 and WO 2010/042817. Other bioinformatic algorithms known in the art can also be employed in the context of the invention to generate HIV-1 antigen sequences having enhanced immunogenicity relative to naturally-occurring sequences.

[0112] Any clade of HIV is appropriate for antigen selection, including HIV clades A, B, C, D, E, MN, and the like. Thus, it will be appreciated that the following HIV antigens can be used in the invention: HIV clade A gpl40, Gag, Env, and/or Pol; HIV clade B gpl40, Gag, Env, and/or Pol proteins; HIV clade C gpl40, Gag, Env, and/or Pol proteins; and HIV clade MN gpl40, Gag, Env, and/or Pol proteins. While it is preferred that the antigen is a Gag, Env, and/or Pol protein, any HIV protein or portion thereof capable of inducing an immune response in a mammal can be used in connection with the invention. HIV Gag, Env, and Pol proteins from the different HIV clades (e.g., HIV clades A, B, C, MN, etc.), as well as nucleic acid sequences encoding such proteins and methods for the manipulation and insertion of such nucleic acid sequences into vectors, are known (see, e.g., HIV Sequence Compendium, Division of AIDS, National Institute of Allergy and Infectious Diseases (2003), HIV Sequence Database (http://hiv- web. lanl.gov/content/hiv-db/mainpage.html), Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)).

[0113] It will be appreciated that an entire, intact HIV protein is not required to produce an immune response. Indeed, most antigenic epitopes of HIV proteins are relatively small in size. Thus, fragments (e.g., epitopes or other antigenic fragments) of an HIV protein, such as any of the HIV proteins described herein, can be used as an HIV antigen. Antigenic fragments and epitopes of the HIV Gag, Env, and Pol proteins, as well as nucleic acid sequences encoding such antigenic fragments and epitopes, are known (see, e.g., HIV Immunology and HIV/SIV Vaccine Databases, Vol. 1 , Division of AIDS, National Institute of Allergy and Infectious Diseases (2003)).

[0114] HIV antigens also include fusion proteins and polyproteins. A fusion protein can comprise one or more antigenic HIV protein fragments (e.g., epitopes) fused to one another, or fused to all or part of a different HIV protein or other polypeptide. The fusion protein can comprise all or part of any of the HIV antigens described herein. For example, all or part of an HIV Env protein (e.g., gpl20 or gp 160), can be fused to all or part of the HIV Pol protein, or all or part of HIV Gag protein can be fused to all or part of the HIV Pol protein. Such fusion proteins effectively provide multiple HIV antigens in the context of the invention, and can be used to generate a more complete immune response against a given HIV pathogen as compared to that generated by a single HIV antigen. Similarly, polyproteins also can provide multiple HIV antigens. Polyproteins useful in conjunction with the invention include those that provide two or more HIV antigens, such as two or more of any of the HIV antigens described herein. Delivery of fusion proteins or polyproteins via adenoviral vector to a mammal allows exposure of an immune system to multiple antigens using a single nucleic acid sequence and, thus, conveniently allows a single composition to provide immunity against multiple HIV antigens or multiple epitopes of a single antigen. Nucleic acid sequences encoding fusion proteins and polyproteins of HIV antigens can be prepared and inserted into vectors using known methods (see, e.g., U.S. Patents 5,130,247 and 5, 130,248, Sambrook et al., supra, and Ausubel et al., supra).

[0115] In addition to the nucleic acid encoding an antigen, the simian adenoviral vector construct preferably comprises expression control sequences, such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), and the like, that provide for the expression of the nucleic acid sequence in a host cell. Exemplary expression control sequences are known in the art and are described in, for example, Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990). Ideally, the HIV antigen-encoding nucleic acid sequence is operably linked to a promoter and a polyadenylation sequence. A large number of promoters, including constitutive, inducible, and repressible promoters, from a variety of different sources are well known in the art. Representative sources of promoters include for example, virus, mammal, insect, plant, yeast, and bacteria, and suitable promoters from these sources are readily available, or can be made synthetically, based on sequences publicly available, for example, from depositories such as the ATCC as well as other commercial or individual sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bi-directional (i.e., initiate transcription in either a 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, pBAD (araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include, for example, the Tet system (U.S. Patents 5,464,758 and 5,814,618), the Ecdysone inducible system (No et al., Proc. Natl. Acad. Sci , 93: 3346-3351 (1996)), the T-RExTM system (Invitrogen, Carlsbad, CA), LACSWITCH™ System (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen inducible recombinase system (Indra et al., Nuc. Acid. Res., 27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); U.S. Patent 7,1 12,715; and Kramer & Fussenegger, Methods Mol. Biol, 308: 123- 144 (2005)).

[0116] A promoter can be selected by matching its particular pattern of activity with the desired pattern and level of expression of an antigen(s). For example, the simian adenoviral vector can comprise two or more nucleic acid sequences that encode different antigens and are operably linked to different promoters displaying distinct expression profiles. In this regard, a first promoter can be selected to mediate an initial peak of antigen production, thereby priming the immune system against an encoded antigen. A second promoter can be selected to drive production of the same or different antigen such that expression peaks several days after that of the first promoter, thereby "boosting" the immune system against the antigen. Alternatively, a hybrid promoter can be constructed which combines the desirable aspects of multiple promoters. For example, a CMV-RSV hybrid promoter combining the CMV promoter's initial rush of activity with the RSV promoter's high maintenance level of activity can be employed. In that antigens can be toxic to eukaryotic cells, it may be advantageous to modify the promoter to decrease activity in complementing cell lines used to propagate the simian adenoviral vector.

[0117] To optimize protein production, preferably the antigen-encoding nucleic acid sequence further comprises a polyadenylation site following the coding sequence. Any suitable polyadenylation sequence can be used, including a synthetic optimized sequence, as well as the polyadenylation sequence of BGH (Bovine Growth Hormone), polyoma virus, TK (Thymidine Kinase), EBV (Epstein Barr Virus), and the papillomaviruses, including human papillomaviruses and BPV (Bovine Papilloma Virus). A preferred polyadenylation sequence is the SV40 (Human Sarcoma Virus-40) polyadenylation sequence. Also, preferably all the proper transcription signals (and translation signals, where appropriate) are correctly arranged such that the nucleic acid sequence is properly expressed in the cells into which it is introduced. If desired, the nucleic acid sequence also can incorporate splice sites (i.e., splice acceptor and splice donor sites) to facilitate mRNA production.

[0118] If the antigen-encoding nucleic acid sequence encodes a processed or secreted protein or peptide, or a protein that acts intracellularly, preferably the antigen-encoding nucleic acid sequence further comprises the appropriate sequences for processing, secretion, intracellular localization, and the like. The antigen-encoding nucleic acid sequence can be operably linked to a signal sequence, which targets a protein to cellular machinery for secretion. Appropriate signal sequences include, but are not limited to, leader sequences for immunoglobulin heavy chains and cytokines (see, for example, Ladunga et al., Current Opinions in Biotechnology, 11: 13-18 (2000)). Other protein modifications can be required to secrete a protein from a host cell, which can be determined using routine laboratory techniques. Preparing expression constructs encoding antigens and signal sequences is further described in, for example, U.S. Patent 6,500,641.

Methods of secreting non-secretable proteins are further described in, for example, U.S. Patent 6,472,176, and International Patent Application Publication WO 02/48377.

[0119] An antigen encoded by the nucleic acid sequence of the simian adenoviral vector construct also can be modified to attach or incorporate the antigen on a host cell surface. In this respect, the antigen can comprise a membrane anchor, such as a gpi-anchor, for conjugation onto a cell surface. A transmembrane domain can be fused to the antigen to incorporate a terminus of the antigen protein into the cell membrane. Other strategies for displaying peptides on a cell surface are known in the art and are appropriate for use in the context of the invention.

[0120] In accordance with the invention, the simian adenoviral vector construct can be a serotype 7 simian adenoviral vector (SAV7) encoding an HIV antigen (e.g., an Env protein or a Gag protein) which comprises, for example, the nucleic acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 27. In another embodiment, the simian adenoviral vector construct can be a serotype 1 1 simian adenoviral vector (SAV1 1) encoding an HIV antigen (e.g., an Env protein or a Gag protein) which comprises, for example, the nucleic acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 28. The simian adenoviral vector construct can be a serotype 16 simian adenoviral vector (SAV16) encoding an HIV antigen (e.g., an Env protein or a Gag protein) which comprises, for example, the nucleic acid sequence of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , or SEQ ID NO: 25. The simian adenoviral vector construct can be a serotype 18 simian adenoviral vector (SAV18) encoding an HIV antigen (e.g., an Env protein or a Gag protein) which comprises, for example, the nucleic acid sequence of SEQ ID NO: 29. The simian adenoviral vector construct can be a serotype 38 simian adenoviral vector (SAV38) encoding an HIV antigen (e.g., an Env protein or a Gag protein) which comprises, for example, the nucleic acid sequence of SEQ ID NO: 1 , SEQ ID NO: 1 1 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, or SEQ ID NO: 26. Further details regarding the HIV antigen-encoding simian adenoviral vectors disclosed herein are set forth in Table 1.

Table 1

[0121] In another embodiment, the virus is influenza virus. Influenza viruses are classified as type A, B, or C viruses. The influenza virus contains a segmented, single negative-strand RNA genome which encodes ten polypeptides that are required for the life cycle of the virus. Each of the eight RNA segments of a complete influenza virus genome is encapsidated with multiple subunits of the nucleocapsid protein (NP) and associated with a few molecules of the trimeric polymerase, which consists of PB1 , PB2, and PA subunits, thereby forming the ribonucleoprotein complex (RNP) (see, e.g., Lamb, R.A., In: The Influenza Viruses, R.M. Krug, ed., Plenum Press, pp. 1-87 (1989)). Surrounding the RNP is a layer of the matrix protein (Ml), which serves as a nexus between the core and the viral envelope. This host cell-derived envelope is studded with the two major virally-encoded surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA), and a much smaller amount of a nonglycosylated small protein M2 (see, e.g., Lamb, supra, and Lamb et al., Cell, 40: 627-633 (1985)). The HA glycoprotein is cleaved by a protease to form HAI and HA2.

[0122] The antigen can be from any influenza subtype. Influenza type A viruses infect humans, birds, pigs, horses, seals, whales, and other animals, but wild birds are their natural hosts. Influenza type A viruses are divided into subtypes based on the hemagglutinin (HA) and neuraminidase (NA) proteins on the virus surface. There are 15 different HA subtypes and 9 different NA subtypes. Thus, specific subtypes of influenza type A are named according to their HA and NA surface proteins, and many different combinations of HA and NA proteins are possible. For example, an "H7N2 virus" designates an influenza A subtype that has an HA 7 protein and an NA 2 protein. Similarly, an "H5N 1 " virus has an HA 5 protein and an NA 1 protein. Only some influenza A subtypes (i.e., H1N1 , H1N2, and H3N2) are currently in general circulation among humans. Other subtypes are found most commonly in other animal species. For example, H7N7 and H3N8 viruses cause illness in horses. Influenza type B viruses are normally found only in humans. Unlike influenza type A viruses, type B viruses are not classified according to subtype. Although influenza type B viruses can cause human epidemics, they have not caused pandemics. Influenza type C viruses cause mild illness in humans and do not cause epidemics or pandemics. Influenza type C viruses are not classified according to subtype.

[0123] In a preferred embodiment, the antigen is from a type A influenza virus. As discussed above, the three known major influenza A subtypes that infect humans are H1N1 , H1N2, and H3N2. Within each of these subtypes are often numerous strains. The invention is not limited to these specific subtypes and strains. One of ordinary skill in the art will appreciate that new strains of influenza virus can emerge each year. Thus, the invention encompasses antigens from all influenza virus subtypes and strains, including subtypes and strains yet to emerge and subtypes and strains that currently do not infect humans.

[0124] While the influenza antigen preferably is from an influenza virus that typically infects humans, the influenza antigen alternatively can be derived from an influenza virus that does not normally infect humans. Such viruses include, for example, influenza viruses which infect birds (i.e., avian influenza). Avian flu is caused by three type A viruses, H5, H7, and H9. Each of these three avian influenza type A viruses can be partnered with any one of nine neuraminidase surface proteins. Thus, there are potentially nine different forms of each avian flu subtype (e.g., H5N1 , H5N2, H5N3, and H5N9). Examples of other suitable animal influenza subtypes are known in the art and include those that infect pigs, cows, horses, seals, whales, and the like.

[0125] In accordance with the invention, the simian adenoviral vector construct can be a serotype 7 simian adenoviral vector (SAV7) encoding a codon-optimized influenza virus antigen (e.g., an HA protein) which comprises, for example, the nucleic acid sequence of SEQ ID NO: 30. In another embodiment, the simian adenoviral vector construct can be a serotype 1 1 simian adenoviral vector (SAV1 1 ) encoding a codon-optimized influenza virus antigen (e.g., an HA protein) which comprises, for example, the nucleic acid sequence of SEQ ID NO: 31. The simian adenoviral vector construct can be a serotype 16 simian adenoviral vector (SAV16) encoding a codon-optimized influenza virus antigen (e.g., an HA protein) which comprises, for example, the nucleic acid sequence of SEQ ID NO: 32. Further details regarding the influenza virus antigen-encoding simian adenoviral vectors disclosed herein are set forth in Table 2.

Table 2

Simian

SEQ Influenza

Vector Name Adenovirus

ID NO Antigen Insert

Serotype

30 SAV7 expressing codon-optimized Influenza H1N1 7 HA (codon- A/New Caledonia/20/1999 Sequence optimized)

31 SAV1 1 expressing codon-optimized Influenza H1N1 1 1 HA (codon- A/New Caledonia/20/1999 Sequence optimized)

32 SAV16 expressing codon-optimized Influenza H1N1 16 HA (codon- A/New Caledonia/20/1999 Sequence optimized) [0126] The invention further provides a method of inducing an immune response against a human immunodeficiency virus (HIV) in a mammal, and a method of inducing an immune response against an influenza virus in a mammal. The method comprises administering to the mammal a composition comprising the aforementioned simian adenoviral vector construct (encoding, for example, an HIV antigen or an influenza antigen) and a pharmaceutically acceptable carrier, whereupon the nucleic acid sequence encoding the antigen is expressed in the mammal to produce the antigen and thereby induce an immune response against a pathogen.

[0127] Widespread use of human adenoviral vector constructs is hindered, at least in part, by the immunogenicity of the vector. A majority of the U.S. population has been exposed to wild- type human adenovirus and developed pre-existing immunity to human adenovirus-based gene transfer vectors. As a result, human adenoviral vector constructs are inactivated by the pre- existing host immune response, thereby reducing the effectiveness of the vector. The neutralization and/or clearance of adenoviral vector constructs in the body complicates use of these vector constructs as DNA vaccines. DNA vaccines employ gene transfer vectors to deliver antigen-encoding DNA to host cells. By producing antigenic proteins in vivo, the humoral and cell-mediated arms of the immune system are activated, thereby generating a more complete immune response against the antigen as compared to traditional vaccines wherein foreign proteins are injected into the body. Despite the advantageous characteristics of human adenoviral vector constructs as gene delivery vehicles, the immunogenicity of the vector prevents efficient repeat dosing, which can be advantageous for "boosting" the immune system against pathogens, and results in only a small fraction of a dose of adenoviral vector construct delivering its payload to host cells.

[0128] The simian adenoviral vector construct described herein is less susceptible (or, in some cases, not susceptible) to neutralization and/or clearance mediated by pre-existing immunity to human adenovirus. Also, the combination of two or more simian adenoviral vector constructs may circumvent the inhibition seen with repeated administration of human adenovirus vector constructs, making it possible to boost the immune system against pathogens.

[0129] The simian adenoviral vector construct desirably is administered in a

pharmaceutically acceptable (e.g., physiologically acceptable) composition, which comprises a carrier, preferably a physiologically (e.g., pharmaceutically) acceptable carrier, and the simian adenoviral vector. Any suitable carrier can be used within the context of the invention, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition is to be administered and the particular method used to administer the composition. Ideally, in the context of adenoviral vectors, the pharmaceutical composition preferably is free of replication-competent adenovirus. The pharmaceutical composition can optionally be sterile.

[0130] In accordance with the invention, the composition is administered to an animal, preferably a mammal, and most preferably a human, wherein the antigen-encoding nucleic acid sequence is expressed to induce an immune response against the pathogen (e.g., HIV or influenza). The immune response can be a humoral immune response, a cell-mediated immune response (e.g., a CD4+ T cell-biased response, a CD8+ T cell-biased response, or a response that is balanced between CD4+ and CD8+ T cells), or, desirably, a combination of humoral and cell- mediated immunity. In this respect, certain serotypes of simian adenoviruses preferentially induce specific immune responses in vivo. For example, adenoviral vector constructs based on simian serotypes 7 and 1 1 induce balanced CD4+ and CD8+ T cell responses, and prime humoral and CD8+ T cell responses in vivo. Adenoviral vector constructs based on simian serotype 16 induce potent CD8+ T cell-biased immune responses. Accordingly, an appropriate simian adenoviral vector can be selected based on the type of immune response that is desired or preferred for a given pathogen. For example, in the context of influenza, a humoral immune response is preferred, while a combination of both humoral and cell-mediated immune responses are desirable in the context of HIV and malaria. Ideally, the immune response provides protection upon subsequent challenge with the pathogen. However, protective immunity is not required in the context of the invention. The inventive method also can be used for antibody production and harvesting.

[0131] To enhance the immune response generated against an antigen, the composition also can comprise a nucleic acid sequence that encodes an immune stimulator, such as a cytokine, a chemokine, or a chaperone. Cytokines include, for example, Macrophage Colony Stimulating Factor (e.g., GM-CSF), Interferon Alpha (IFN-a), Interferon Beta (IFN-β), Interferon Gamma (IFN-γ), interleukins (IL-1 , IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IL-16, and IL-18), the TNF family of proteins, Intercellular Adhesion Molecule- 1 (ICAM-1), Lymphocyte Function-Associated antigen-3 (LFA-3), B7-1 , B7-2, FMS-related tyrosine kinase 3 ligand, (Flt3L), vasoactive intestinal peptide (VIP), and CD40 ligand. Chemokines include, for example, B Cell- Attracting chemokine-1 (BCA-1), Fractalkine, Melanoma Growth Stimulatory Activity protein (MGSA), Hemofiltrate CC chemokine 1 (HCC-1), Interleukin 8 (IL-8), Interferon-stimulated T-cell alpha chemoattractant (I-TAC), Lymphotactin, Monocyte

Chemotactic Protein 1 (MCP-1), Monocyte Chemotactic Protein 3 (MCP-3), Monocyte

Chemotactic Protein 4 (CP-4), Macrophage-Derived Chemokine (MDC), a macrophage inflammatory protein (MIP), Platelet Factor 4 (PF4), RANTES, BRAK, eotaxin, exodus 1 -3, and the like. Chaperones include, for example, the heat shock proteins Hspl 70, Hsc70, and Hsp40.

[0132] Suitable formulations for the composition include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain anti-oxidants, buffers, and bacteriostats, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water, immediately prior to use. Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets. Preferably, the carrier is a buffered saline solution. More preferably, the simian adenoviral vector construct is administered in a composition formulated to protect the simian adenoviral vector construct from damage prior to administration. For example, the composition can be formulated to reduce loss of the adenoviral vector construct on devices used to prepare, store, or administer the simian adenoviral vector construct, such as glassware, syringes, or needles. The composition can be formulated to decrease the light sensitivity and/or temperature sensitivity of the simian adenoviral vector construct. To this end, the composition preferably comprises a pharmaceutically acceptable liquid carrier, such as, for example, those described above, and a stabilizing agent selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof. Use of such a composition will extend the shelf life of the construct, facilitate administration, and increase the efficiency of the inventive method. Formulations for adenoviral vector construct-containing compositions are further described in, for example, U.S. Patent 6,225,289, U.S. Patent 6,514,943, and International Patent Application Publication WO 00/34444.

[0133] A composition also can be formulated to enhance transduction efficiency. In addition, one of ordinary skill in the art will appreciate that the adenoviral vector construct can be present in a composition with other therapeutic or biologically-active agents. For example, factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of the adenoviral vector construct. As discussed herein, immune system stimulators or adjuvants, e.g., interleukins, lipopolysaccharide, and double-stranded RNA, can be administered to enhance or modify any immune response to the antigen. Antibiotics, i.e., microbicides and fungicides, can be present to treat existing infection and/or reduce the risk of future infection, such as infection associated with gene transfer procedures.

[0134] Any route of administration can be used to deliver the composition to the mammal. Indeed, although more than one route can be used to administer the composition, a particular route can provide a more immediate and more effective reaction than another route. Preferably, the composition is administered via intramuscular injection. The composition also can be applied or instilled into body cavities, absorbed through the skin (e.g., via a transdermal patch), inhaled, ingested, topically applied to tissue, or administered parenterally via, for instance, intravenous, peritoneal, or intraarterial administration.

[0135] The composition can be administered in or on a device that allows controlled or sustained release, such as a sponge, biocompatible meshwork, mechanical reservoir, or mechanical implant. Implants (see, e.g., U.S. Patent 5,443,505), devices (see, e.g., U.S. Patent 4,863,457), such as an implantable device, e.g., a mechanical reservoir or an implant or a device comprised of a polymeric composition, are particularly useful for administration of the composition. The composition also can be administered in the form of sustained-release formulations (see, e.g., U.S. Patent 5,378,475) comprising, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), and/or a polylactic-glycolic acid.

[0136] The dose of the composition administered to the mammal will depend on a number of factors, including the extent of any side-effects, the particular route of administration, and the like. The dose ideally comprises an "effective amount" of the simian adenoviral vector construct, i.e., a dose of simian adenoviral vector construct which provokes a desired immune response in the mammal. Desirably, the composition comprises a single dose of adenoviral vector construct comprising at least about lxl O³ particles (which also is referred to as particle units) of adenoviral vector construct. The dose preferably is at least about lxl 0⁶ particles (e.g., about lxlO⁶- 1x10¹² particles), more preferably at least about l xl O⁷ particles, more preferably at

^Q 8 1 1 8 12

least about 1x10 particles (e.g., about 1 x10 -1x10 particles or about 1 x10 -1x10 particles), and most preferably at least about lxl 0⁹ particles (e.g., about Ixl0⁹-l l 0¹⁰ particles or about Ixl 0⁹-lxl0¹² particles), or even at least about l xl O¹⁰ particles (e.g., about Ix l 0¹⁰-lxl0¹² particles) of the adenoviral vector construct. The dose desirably comprises no more than about lxl O¹⁴ particles, preferably no more than about lxlO^1'' particles, even more preferably no more than about lxlO¹² particles, even more preferably no more than about lxlO¹ ' particles, and most preferably no more than about lxl 0¹⁰ particles (e.g., no more than about lxlO⁹ particles). In other words, a single dose of adenoviral vector construct can comprise, for example, about lxl 0⁶ particle units (pu), 2xl0⁶ pu, 4xl0⁶ pu, lxl 0⁷ pu, 2xl0⁷ pu, 4xl0⁷ pu, lxl 0^s pu, 2xl0⁸ pu, 4x10^s pu, lxl0⁹ pu, 2xl 0⁹ pu, 4xl0⁹ pu, lxl 0^{l 0} pu, 2xl 0¹⁰ pu, 4xl 0^{, 0} pu, lxl O^{1 1} pu, 2xlOⁿ pu, 4xlOⁿ pu, 1x10 12 pu, 2x101 pu, or 4x1012 pu of the simian adenovi *ral vector construct.

[0137] Administration of the composition containing the simian adenoviral vector construct can be one component of a multistep regimen for inducing an immune response against a pathogen in a mammal. In this respect, the inventive method further comprises administering to the mammal a priming composition to the mammal prior to administering the simian adenoviral vector construct to the mammal. In this embodiment, therefore, the immune response is

"primed" upon administration of the priming composition, and is "boosted" upon administration of the composition containing the simian adenoviral vector construct. Alternatively, the method further comprises administering to the mammal a boosting composition after administering the simian adenoviral vector construct to the mammal. In this embodiment, therefore, the immune response is "primed" upon administration of the composition containing the simian adenoviral vector construct, and is "boosted" upon administration of the boosting composition.

[0138] Each of the priming composition and the boosting composition desirably is a gene transfer vector that comprises a nucleic acid sequence encoding an antigen. Any gene transfer vector can be employed, including viral and non-viral gene transfer vectors. Examples of suitable viral gene transfer vectors include, but are not limited to, retroviral vectors, adeno- associated virus vectors, vaccinia virus vectors, herpesvirus vectors, lymphocytic

choriomeningitis virus vectors (LCMV), and adenoviral vectors. Examples of suitable non-viral vectors include, but are not limited to, plasmids, liposomes, and molecular conjugates (e.g., transferrin). Preferably, the priming composition or the boosting composition is a plasmid construct or an adenoviral vector construct. Alternatively, an immune response can be primed or boosted by administration of the antigen itself, e.g., an antigenic protein, inactivated pathogen, and the like. When the priming composition and/or the boosting composition is an adenoviral vector construct, it can be an adenoviral vector construct derived from any human or non-human animal as described herein. In a preferred embodiment, the priming composition and/or the boosting composition comprises a human adenoviral vector construct (e.g., serotype 5, 26, 28, or 35) or a simian adenoviral vector construct. For example, a priming composition containing a human serotype 5 adenoviral vector construct can be administered to a human, followed by administration of a boosting composition containing the simian adenoviral vector. Alternatively, a priming composition containing the simian adenoviral vector construct described herein can be administered to a human, followed by a second administration of the same composition. In this manner, the priming composition and the boosting composition are the same. One of ordinary skill in the art will appreciate that any combination of human and/or simian adenoviral vector constructs encoding one or more antigens can be employed as the priming or boosting composition in conjunction with a composition comprising the simian adenoviral vector construct of the present invention.

[0139] The gene transfer vector of the priming composition and the boosting composition comprises at least one nucleic acid sequence encoding an antigen. The antigen encoded by the nucleic acid sequence of the priming composition and/or the boosting composition can be the same as the antigen encoded by the inventive simian adenoviral vector construct. Alternatively, the antigen encoded by the nucleic acid sequence of the priming composition and/or the boosting composition can be different from the antigen encoded by the inventive simian adenoviral vector construct. In one embodiment, the gene transfer vector of the priming composition and/or the boosting composition comprises multiple (i.e., two or more) nucleic acid sequences encoding the same antigen, as described herein. In another embodiment, the gene transfer vector of the priming composition and/or the boosting composition can comprise multiple nucleic acid sequences encoding two or more different antigens, as described herein.

[0140] Administration of the priming composition and the boosting composition can be separated by any suitable timeframe (e.g., at least about 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 16 weeks, or a range defined by any two of the foregoing values). The boosting composition preferably is administered to a mammal (e.g., a human) at least about 2 weeks (e.g., 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 20 weeks, 24 weeks, 28 weeks, 35 weeks, 40 weeks, 50 weeks, 52 weeks, or a range defined by any two of the foregoing values) following administration of the priming composition. More than one dose of priming composition and/or boosting composition can be provided in any suitable timeframe. The dose of the priming composition and boosting composition administered to the mammal depends on a number of factors, including the extent of any side-effects, the particular route of administration, and the like. In embodiments where the priming composition or the boosting composition comprises an adenoviral vector construct (e.g., a human or simian adenoviral vector construct), the priming composition and/or the boosting composition comprises a single dose of adenoviral vector construct as described herein.

[0141] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0142] This example describes a method of inducing an immune response against HIV in a mammal using simian adenoviral vector constructs.

[0143] Several recombinant simian adenoviral vector constructs (rAd) were generated and examined for use as a platform for HIV vaccines. A single intramuscular (IM) injection of a serotype 1 1 , 16, or 7 simian adenoviral vector construct encoding an HIV gpl40B protein in mice generated potent T-cell and humoral responses (see Figures 2A-2B and Figures 3A-3B). The gpl40B inserts in these constructs comprise deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). These constructs generated detectable T-cell responses at the low dosages of 10⁷ and 10 viral particles. Such high immune responses were not observed for any other constructs except those based on human serotype 5 (rAd5). Simian rAdl 1 and 16 also generated potent immune responses in a DNA prime/rAd boost regimen (see Figures 4A-4B). The combination regimen using a simian rAd7 vector construct encoding gpl40B in a priming composition and a human serotype 5 vector construct or a replication-deficient lymphocytic choriomeningitis virus (rLCMV) encoding gpl40B in a boosting composition generated potent IgG responses (see Figure 5).

[0144] The results of this example demonstrate that simian adenoviral vector constructs can be used to generate an immune response against HIV whether administered alone, as a priming construct, or as a boosting construct.

EXAMPLE 2

[0145] This example describes the use of a recombinant simian adenoviral vector construct to prime an immune response against HIV in a mammal.

[0146] Balb/c mice were intramuscularly primed with 10 viral particles (VP) of one of the following adenoviral vector constructs, each of which comprises a nucleic acid sequence encoding an HIV Env protein (gpl40B) (rAd-HIV Env): human serotype 5 (5), human serotype 28 (28), human serotype 26 (26), human serotype (35), human serotype 41 (41), simian serotype 7 (s7), simian serotype 1 1 (si 1), or simian serotype 16 (si 6). The gpl40B inserts in these constructs comprise deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). Three weeks after priming, the mice received 10 VP of rAd5-HIV Env or 2x10 VP of a replication-deficient lymphocytic choriomeningitis virus (rLCMV) encoding an HIV Env (gpl40B) (rLCMV-HIV Env) by intramuscular or intravenous injection, respectively. Two weeks later, the mice were sacrificed and the immune responses were analyzed. The results of these experiments are shown in Figures 6A-6E.

[0147] In a separate experiment, Balb/c mice were intramuscularly primed with 10⁷ VP of one of the following adenoviral vector constructs, each of which comprises a nucleic acid sequence encoding an HIV gpl40B protein (rAd-HIV Env): human serotype 5 (5), human serotype 28 (28), simian serotype 7 (s7), simian serotype 1 1 (si 1), or simian serotype 16 (si 6). Three weeks after priming, the mice received 10⁷ VP of rAd5-HIV Env. The gpl40B inserts in these constructs comprise deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). Two weeks later, the mice were sacrificed and the immune responses were analyzed. The results of these experiments are shown in Figures 7A-7E.

[0148] For all of the above experiments, immune responses were analyzed by (i) examining the frequencies of effector cytokine-producing T cells by intracellular cytokine staining after the in vitro stimulation with an HIV Env peptide pool, (ii) measuring the numbers of HIV Env- specific PA9⁺CD8⁺ T cells in the PBMC and spleen, and (iii) measuring the levels of HIV Env- specific IgG in serum.

[0149] The results of this example demonstrate that compositions comprising simian adenoviral vector constructs can be used to prime an immune response against HIV in a mammal.

EXAMPLE 3

[0150] This example describes the use of a recombinant simian adenoviral vector construct to boost an immune response against HIV in a mammal.

[0151] Balb/c mice were intramuscularly primed with 10⁸ VP of a human serotype 28 adenoviral vector construct encoding an HIV gpl40B protein (rAd28-HIV Env). Three weeks following administration of the priming composition, mice were boosted with 10⁸ VP of one of the following rAd-HIV Env constructs: human serotype 28 (28), human serotype 5 (5), human serotype 26 (26), human serotype 35 (35), human serotype 41 (41), simian serotype 7 (s7), simian serotype 1 1 (si 1), or simian serotype 16 (s i 6). The gpl40B inserts in these constructs comprise deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). Two weeks later, mice were sacrificed and immune responses were analyzed. Specifically, the frequencies of effector cytokine-producing T cells were examined by intracellular cytokine staining after the in vitro stimulation with an HIV Env peptide pool. The numbers of HIV Env-specific PA9⁺CD8⁺ T cells were measured in the PBMC and spleen. The levels of HIV Env-specific IgG were examined in serum. The results of these experiments are shown in Figures 8A-8E.

[0152] The results of this example demonstrate that compositions comprising simian adenoviral vector constructs can be used to boost an immune response against HIV in a mammal.

EXAMPLE 4

[0153] This example describes a heterologous prime/boost HIV immunization regimen using simian adenoviral vector constructs.

7 8

[0154] Balb/c mice were intramuscularly primed with 10 or 10 VP of one of the following simian adenoviral vector constructs encoding an HIV gpl40B protein (rSAV-HIV Env):

serotype 7 (s7), serotype 1 1 (si 1), or serotype 16 (sl6). The gpl40B inserts in these constructs comprise deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). Three weeks after the

7 8

administration of the priming composition, mice received 10 or 10 VP of the aforementioned s7, si 1 , or si 6 rSAV-HIV Env constructs. Control mice received a dose of rAd5-HIV Env alone or preceded by a dose of rAd28-HIV Env. The levels of HIV Env-specific IgG were examined in serum. The results of these experiments are shown in Figures 9A-9E.

[0155] In a separate experiment, Balb/c mice were intramuscularly primed with 10 VP of one of the following rAd-HIV Env (gpl40B) constructs: human serotype 5 (5), human serotype 28 (28), or simian serotype 18 (si 8). The gpl40B inserts in these constructs comprise deletions in the VI and V2 loops of Env (dvl 2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). Three weeks after the administration of the priming composition, mice received 10⁸ VP of the aforementioned hAd5, hAd28, or si 8 vector constructs, as shown in Figures 1 OA- IOC. Two weeks later, the mice were sacrificed and the immune responses were analyzed. The results of this experiment are shown in Figures 10A-10E.

[0156] In yet another experiment, Balb/c mice were intramuscularly primed with 10 VP of one of the following rAd-HIV (gpl40B) Env constructs: human serotype 5 (5), human serotype 28 (28), or simian serotype 38 (s38). The gpl40B inserts in these constructs comprise deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). Three weeks after the administration of the priming composition, mice received 10 VP of the aforementioned hAd5, hAd28, or s38 vector constructs. Two weeks later, the mice were sacrificed, and the immune responses were analyzed. The administration protocols and results of this experiment are shown in Figures 1 lA-1 IE.

[0157] For all of these experiments, immune responses were analyzed by (i) examining the frequencies of effector cytokine-producing T cells by intracellular cytokine staining after the in vitro stimulation with an HIV Env peptide pool, (ii) measuring the numbers of HIV Env-specific PA9⁺CD8⁺ T cells in the PBMC and spleen, and (iii) measuring the levels of HIV Env-specific IgG in serum.

[0158] The results of this example demonstrate that simian adenoviral vector constructs can be used in heterologous prime/boost immunization regimens to induce immune responses against HIV in mammals.

EXAMPLE 5

[0159] This example describes the immunogenicity of simian adenoviral vector constructs of different serotypes in mice.

[0160] Balb/C mice were vaccinated with a single intramuscular (IM) injection of 10 viral particles - 10¹⁰ viral particles (VP) of a serotype 7 (sAd7), 1 1 (sAdl 1), 16 (sAdl6), 18 (sAdl 8), or 38 (sAd38) simian adenoviral vector construct encoding an HIV gpl40B protein. The gpl40B inserts in these constructs comprise deletions in the VI and V2 loops of Env (dvl2), as well as mutations in the cleavage site, fusion peptide, and interhelical coiled-coil domains of Env (ACFI). At three weeks post vaccination, cellular and humoral immune responses to gpl40 were measured as described previously. The relative strength of immunogenicity was compared to the immune responses generated by the construct with the lowest potency, which was set at 1.

Administration of a human Ad5 vector construct served as a control. The results of the single IM vaccination experiment are illustrated in Figure 12 and in Table 3. Table 3

For IgG response: +: normalized to 1 -fold; ++: 5-fold; +++: 10-fold; ++++: 20-fold compared to the response with sAd l 8 .

For CD4, CD8 response: +: normalized to 1 -fold; ++: 2-fold; +++: 3-fold; ++++: 4-fold compared to the response with sAdl 8

[0161] The above constructs also were administered to Balb/C mice in homologous and heterologous prime/boost combinations via IM injection. At three weeks post vaccination, cellular and humoral immune responses to gpl40 were measured as described previously.

Administration of a human Ad5 vector construct served as a control. The results of the prime/boost regimens are illustrated in Figures 13A and 13B. sAdl 1 constructs generated the highest humoral IgG response, while sAd7 and sAdl6 constructs generated the highest T-cell responses.

[0162] The results of this example demonstrate that simian adenoviral vector constructs can induce immune responses against HIV in mammals.

EXAMPLE 6

[0163] This example describes homologous and heterologous prime/boost immunization regimens against influenza virus using simian and human adenoviral vector constructs.

Q

[0164] Balb/C mice were vaccinated with an intramuscular (IM) injection of 10 viral particles (VP) of a simian adenoviral vector construct encoding a codon-optimized influenza hemagglutinin (HA) protein or human adenoviral vector construct encoding a codon-optimized influenza HA protein as a prime, followed three weeks later by an IM injection of 10 VP of an adenoviral vector construct of the same or different serotype as shown in Figure 14A. In a separate experiment, an Ad5 vector construct encoding a codon-optimized HA protein was administered as a boost (see Figure 14B). At three weeks post vaccination, humoral immune responses to HA were measured as IC50 titers. The results of these experiments are illustrated in Figures 14A and 14B. [0165] The results of this example demonstrate that different combinations of simian and human adenoviral vector constructs induce different levels of immunogenicity against influenza HA protein in mice.

EXAMPLE 7

[0166] This example describes homologous and heterologous prime/boost immunization regimens against influenza virus using simian and human adenoviral vector constructs.

[0167] Groups of mice (n=5) were vaccinated with an intramuscular (IM) injection of 10 viral particles (VP) of a simian adenoviral vector construct encoding a codon-optimized influenza HA protein or a human adenoviral vector construct encoding a codon-optimized influenza HA protein from H1N1 A/New Caledonia/20/1999 as a prime, followed three weeks later by an IM injection of 10 VP of an adenoviral vector construct of the same or different serotype encoding the same HA protein. The simian adenoviral vector constructs tested were based on serotype 7 (SEQ ID NO: 30), serotype 1 1 (SEQ ID NO: 31), and serotype 16 (SEQ ID NO: 32). The human adenoviral vector constructs tested were based on serotype 5 and serotype 28. Sera were collected 14 days after the second immunization, and the neutralization of the antisera was tested against the homologous HA using a pseudotyped lentiviral reporter. IC₅0 (Figure 15 A) and IC₈₀ (Figure 15B) titers of neutralizing antibodies were measured.

[0168] The results of this experiment are shown in Figures 15A and 15B, which demonstrate that prime/boost regimens incorporating different adenoviral vector construct combinations produce neutralizing activity against influenza. The highest neutralizing activity was generated when serotype 7 or 1 1 simian adenoviral vector constructs were used as a prime and/or boost.

[0169] The results of this example demonstrate that different combinations of simian and human adenoviral vector constructs induce different levels of immunogenicity against influenza HA protein in mice.

EXAMPLE 8

[0170] This example describes homologous and heterologous prime/boost immunization regimens against influenza virus using simian and human adenoviral vector constructs in non- human primates. [0171] Six groups of Indian rhesus macaques (four monkeys per group) were subject to six different immunization regimens utilizing a simian adenoviral vector construct encoding a codon-optimized influenza HA protein, a human adenoviral vector construct encoding a codon- optimized influenza HA protein from HlNl A/New Caledonia/20/1999, and a plasmid construct encoding a codon-optimized influenza HA protein from HlNl A/New Caledonia/20/1999 (VRC 9332). The simian adenoviral vector constructs tested were based on serotype 7 (SEQ ID NO: 30) or serotype 1 1 (SEQ ID NO: 31). The human adenoviral vector constructs tested were based on serotype 5 and serotype 28. The immunization regimens are outlined below in Table 4.

Table 4

[0172] Sera were collected 14 days after the final immunization at week 16. A pseudotyped neutralization assay was conducted to measure the neutralizing antibody responses in monkeys immunized with the plasmid or adenoviral vector constructs set forth in Table 4. Neutralization was determined for individual animal at titers ranging from 1 :200 to 1 :25,600. Percent neutralization was calculated by the reduction of luciferase activity relative to the values achieved in the non-immune sera. The results of this experiment are shown in Table 5. IC₈₀ titers for the neutralization activity of individual monkey antiserum were calculated and summarized. The geometric mean titer (GMT) with 95% CI is shown for each group.

Table 5

[0173] The results of this example demonstrate that different combinations of simian and human adenoviral vector constructs induce different levels of humoral immunogenicity against influenza HA protein in Indian rhesus macaques.

EXAMPLE 9

[0174] This example demonstrates that simian adenovirus plaque formation is highly efficient on a human cell line expressing human adenovirus components El and E40RF6. [0175] The simian adenoviruses are restricted from replication on human cells and human adenovirus factors overcome the restriction. Two methodologies were used to define growth of simian adenoviruses in cells expressing human adenovirus genes. Plaque formation was used to determine growth under conditions of very low multiplicity of infection (MOI), and more than one infectious cycle was required to generate a positive result, i.e., a plaque in the cell monolayer. Thus, plaque formation demonstrates continuous growth of a virus: single infectious viral particles infect a single cell, full completion of the viral life cycle occurs, followed by infection of neighboring cells by the viral progeny. The second method assessed the number of infectious viral progeny produced from a single round of viral replication. This method is based on synchronous infection of essentially all the cells in the monolayer, known as single-burst growth assessments.

[0176] Of all the E4 factors, ORF6 was sufficient to propagate monkey adenoviruses on human cells. Human cell lines were superior to simian cell lines in supporting the ability of simian adenoviruses to form plaques, a standard method to measure virus growth. Two simian adenoviruses, SV-1 1 and SV-16, which were isolated from Rhesus and Vervet monkeys, respectively, were plaqued on 293-ORF6, BSC-1 , LLC-MK2 (MK2), Vero, and CV-1 cells. All but the 293-ORF6 cell line are derived from monkeys. BSC-1 and MK-2 are the cell lines recommended by the American Type Culture Collection (ATCC) for simian adenovirus propagation. A serial dilution of both viruses was used to infect each cell line which were 80% confluent in 60 mm culture dishes. The infection conditions were 500 μΐ of virus for 1 hour rocked every 15 minutes. The virus was subsequently removed and cells overlaid with EMEM + 2% FBS and 0.9% agarose. Fourteen days later plaques were counted. 293-ORF6 cells gave the highest plaquing efficiency, which was at least ten times better than any of the other cells lines tested (see Table 6). Table 6

[0177] The results of this example demonstrate that simian adenoviruses can be efficiently grown on human cells that contain ORF6 of the E4 region of a human adenovirus.

EXAMPLE 10

[0178] This example demonstrates the generation of high-titers of simian adenovirus progeny on a human cell line with human adenovirus species C factors.

[0179] Single-burst growth experiments were performed to determine if the combination of human adenovirus El and E4 ORF6 was sufficient to overcome the restriction block to simian adenovirus replication in human cells. Human cells expressing either no Ad5 factors (A549), Ad5 E4 ORF6 (A549+Ad5 E40RF6), Ad5 El (293), or Ad5 El and E40RF6 (293-ORF6) were plated in triplicate in 6-well plates at 1.5 x 10⁶ cells per well. BSC-1 cells derived from African Green Monkey that are permissive for simian adenovirus replication were plated at 1 x 10⁵ cells per well. All cells were kept in DMEM with 10% FCS and grown at 37°C, 5% C0₂. The next day, cells were infected with CsCl₂ gradient-purified monkey adenovirus stocks at an MOI of three or one focus forming unit (FFU)/cell in 300 μΐ per well for two hours, followed by aspiration and overlay with 3 ml of DMEM with 5% FCS and 100 μΜ ZnCl₂ to permit induction of E40RF6 expression in A549+Ad5 E40RF6293-ORF6 cells. Cells were then incubated and harvested at 72 hours post-infection. Virus particles were released from cells by three freeze- thaw cycles consisting of alternating exposures to dry ice and 37° C water bath. The number of progeny virions in the virus-cell lysates was assessed using the FFU assay described in Vaccine, 25: 2074-2084 (2007).

[0180] The generation of infectious progeny with simian adenoviruses on human cell lines was highest with 293-ORF6 cells, compared to A549 and A549 cells expressing Ad5 E4 ORF6 (Figure 16A). Similarly, generation of viral progeny at an MOI of 1 was at least as efficient on 293-ORF6 as on the simian cell line BSC-1 (Figure 16B). In some cases, between 10 to 1,000- fold more viral progeny were produced from 293-ORF6 cells as compared to BSC-1 cells. In contrast, simian adenovirus replication on 293 cells was less efficient than on BSC-1 cells.

Therefore, the presence of El and E40RF6 is both necessary and sufficient to overcome the host replication block in human cells. Finally, generation of infectious progeny on 293-ORF6 cells was 1 ,000-fold higher than on BSC-1 cells, indicating that simian adenovirus growth on human cells expressing El and E40RF6 is superior compared to the native host cell lines recommended for monkey adenovirus growth.

[0181] The results of this example demonstrate that simian adenoviruses can efficiently replicate in 293-ORF6 cells, and that the combined expression of human adenovirus components (Ad5 El and E40RF6) is necessary and sufficient to overcome the human replication block.

EXAMPLE 1 1

[0182] This example demonstrates the construction and propagation of simian adenoviral vector constructs with El deletions on a human cell line containing human adenovirus components.

[0183] Five different monkey adenoviruses had their El region replaced with an expression cassette. The monkey adenoviruses were SV-7 (serotype 7), SV-1 1 (serotype 1 1), SV-16 (serotype 16), SV-18 (serotype 18), and SV-38 (serotype 38). The desired viral genome was generated in bacteria which was then transfected into 293-ORF6 cells to make viral particles (see Figure 17). Before transfecting the 293-ORF6 cells, the plasmid was digested with restriction enzyme to free the viral genome. Active viral particles were detected by their cell cytopathic effect (CPE). If CPE was not detected by 5 days post transfection, the cells were harvested, freeze-thawed three times, and used to infect 293-ORF6 cells again. Full CPE was detected by the end of the 5th passage. The virus was then further expanded followed by purification over three CsCl₂ density gradients generated by centrifugation. The viral preparations were of high quality with an average ratio of total particles to infectious particles of 49 +/- 23 (n=6) and high yields, with up to 37,000 particles per cell post-purification. The identity of each virus was confirmed by partial sequencing and PCR. The functionality of the expression cassette was confirmed by Western blot analysis.

[0184] The results of this example demonstrate that simian adenoviral vector constructs with El deletions can be propagated on a human cell line containing human adenovirus components.

EXAMPLE 12

[0185] This example demonstrates that simian adenoviral vector constructs encoding an antigen induce strong adaptive immune responses to the antigen.

[0186] Groups of 6-8 week old female Balb/c mice (Harlan, USA) were immunized intramuscularly (i.m.) with escalating doses (lxlO⁷, lxl O⁸, and lxlO⁹ particle units (pu)) of purified Ad5, Ad28, SV1 1 , or SV16 vector constructs expressing HIV gpl40B from a CMV promoter-driven cassette inserted into the El region, in 100 μΐ FFB in both quadriceps muscles. Two weeks after vaccination, mice were sacrificed and spleens and whole blood were harvested for analysis of T cell and antibody responses, respectively.

[0187] The magnitude of HIV gpl40B-specific T cell responses was assessed via multiparameter intracellular cytokine staining (ICS) assay. Splenocytes were isolated from mice and 1 x 10⁶ cells were incubated for 13 hours at 37°C, 5% C0₂ with RPMI 1640 containing 10% FCS. For stimulation of transgene-specific T cells responses, cultures contained 2.5 μg/mL V3 gpl20 HIV (JR-FL) peptide (Anaspec, San Jose, CA) and monensin (GolgiStop; BD

Biosciences, San Diego, CA). Cells were stained with pre-titered amounts of anti-CD4-FITC (GK1.5; fluorescein isothiocyanate), anti-CD8a-PerCP-Cy5.5 (53-6.7; Peridinin-chlorophyll- protein Complex Cy5.5), and/or anti-CD8a-FITC (53-6.7; fluorescein isothiocyanate) antibodies and fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences). Cells were then stained with anti-IFN-γ-ΡΕ (XMG1.2; phycoerythrin), anti-T F- PE-Cy7 (MP6-XT22; phycoerythrin Cy7), anti-TNF-APC (MP6-XT22; allophycocyanin), and/or anti-IL-2-APC (JES6-5H4;

allophycocyanin) antibodies and fixed with 0.25% Cytofix (BD Biosciences). Samples were analyzed with a FACSCalibur (BD Biosciences, East Rutherford, New Jersey) and analyzed using FlowJo software (TreeStar, Ashland, OR). Approximately 150,000 to 300,000 events were collected per sample. Media background was removed from individual animals by subtracting the media control from peptide-stimulated samples.

[0188] Figure 18A shows the combined results of two independent experiments. Both SV1 1 and SV16 vector constructs were capable of inducing significant CD8⁺ T cell responses to the encoded transgene product. At the 10⁸ and 10⁹ pu vector doses, SV1 1 and SV16 vector constructs induced responses in all immunized animals, with an average of about four to six percent CD8⁺IFNy⁺ T cells. These responses were similar in magnitude compared to those induced by the control Ad5 vector construct. At the lowest vector dose (10⁷ pu),

immunogenicity of the SV1 1 vector construct was comparable to the Ad5 vector construct. Responses elicited by SV1 1 and Ad5 vector constructs were significantly higher than those induced by the SV16 vector construct at this dose level. T cell responses in immunized mice were also detected in the CD4⁺ T cell compartment, and no differences between the constructs were noted. Together, these results show that the SV11 and SV16 monkey adenovirus vector constructs can induce T cell responses to an expressed transgene. In addition, the SV1 1 vector construct is potently immunogenic with immune responses that are comparable in magnitude to those elicited by the Ad5 vector construct.

[0189] The antibody responses to HIV gpl40B were assessed by ELISA. Briefly, 96-well flat-bottom microtiter plates (Thermo Scientific, Waltham, MA) were coated with Galanthus nivalis (GNA) lectin (Sigma, St. Louis, MO). Plates were blocked, washed, and incubated with supernatant from 293 cells transfected with an HIV gpl40B plasmid construct for one hour at room temperature. Sera from immunized mice were then diluted 1 : 1 ,000 and added to plates. HIV gpl40B-specific serum antibodies were detected by incubation with HRP-conjugated anti- mouse IgG (Jackson ImmunoResearch, West Grove, PA) and developed with substrate

(SIGMAFAST™ OPD, Sigma, St. Louis, MO) and 1(N) H₂S0₄. Absorbance was determined by reading at 450 nm.

[0190] Figure 18B shows the absorbance readings of sera from individual animals at the 1 : 1 ,000 serum dilutions and the corresponding mean for each treatment group. The sera shown are from one out of the two experiments conducted. At the highest dose (10⁹ pu), HIV gpl40B antibodies were detectable in all immunized mice, whereas no significant absorbance was measured in the mock (FFB) group. At lower construct doses (10 and 10 pu), measurable antibodies were detected in all mice immunized with SV1 1 or SV16 constructs. In contrast, not all of the mice immunized with the Ad5 vector construct at the 10^s pu dose levels had

measurable responses. No responses were detected in mice immunized with the Ad5 vector construct at 10⁷ pu.

[0191] The results of this example demonstrate that immunization with SV1 1 and SV16 monkey adenovirus vector constructs can elicit antibody responses to a transgene product. These antibody responses are comparable or higher to those induced by an Ad5 vector construct.

[0192] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0193] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non- claimed element as essential to the practice of the invention.

[0194] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIMS:

1. A replication-deficient simian adenoviral vector construct comprising at least one nucleic acid sequence which encodes an antigen of a pathogen, wherein the simian adenoviral vector construct requires at most complementation of a deficiency in the El A region, the EIB region, and/or the E4 region of the adenoviral genome for propagation.

2. The simian adenoviral vector construct of claim 1 , wherein the simian adenoviral vector construct comprises a deficiency in the El A region and the EIB region of the adenoviral genome, and a deficiency in at least a portion of the E4 region of the adenoviral genome.

3. The simian adenoviral vector construct of claim 1 or claim 2, wherein the simian adenoviral vector construct comprises a deficiency in the E3 region of the adenoviral genome.

4. The simian adenoviral vector construct of any one of claims 1-3, which comprises at least one nucleic acid sequence which encodes an HIV antigen.

5. The simian adenoviral vector construct of claim 4, wherein the simian adenoviral vector construct comprises a nucleic acid sequence that encodes two or more different HIV antigens.

6. The simian adenoviral vector construct of claim 4 or claim 5, wherein the simian adenoviral vector construct comprises two or more nucleic acid sequences that each encode a different HIV antigen.

7. The simian adenoviral vector construct of any one of claims 4-6, wherein the HIV antigen is selected from the group consisting of HIV Gag protein, HIV Pol protein, HIV Env protein, HIV Tat protein, HIV Reverse Transcriptase (RT) protein, HIV Vif protein, HIV Vpr protein, HIV Vpu protein, HIV Vpo protein, HIV Integrase protein, HIV Nef protein, and a fusion protein comprising all or part of an HIV Gag protein, HIV Pol protein, or HIV Env protein.

8. The simian adenoviral vector construct of claim 7, wherein the HIV antigen is an HIV gpl40 Env protein or a gpl40dvl2 Env protein.

9. The simian adenoviral vector construct of claim 7 or claim 8, wherein the HIV antigen is a mosaic antigen.

10. The simian adenoviral vector construct of any one of claims 4-9, wherein the HIV antigen is an HIV clade A antigen, HIV clade B antigen, HIV clade C antigen, or an HIV clade MN antigen.

11. The simian adenoviral vector construct of any one of claims 1 -10, wherein the simian adenoviral vector construct is of serotype 3, 7, 1 1, 16, 18, 19, 20, 27, 33, 38, 39, or combinations thereof.

12. The simian adenoviral vector construct of claim 1 1 , wherein the simian adenoviral vector construct is of serotype 7, 11, 16, or 18.

13. The simian adenoviral vector construct of claim 12, which comprises SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 25, or SEQ ID NO: 29.

14. A method of inducing an immune response against HIV in a mammal, which comprises administering to the mammal a composition comprising the simian adenoviral vector construct of any one of claims 4-13 and a pharmaceutically acceptable carrier, whereupon the nucleic acid sequence encoding the HIV antigen is expressed in the mammal to produce the HIV antigen and thereby induce an immune response against HIV.

15. The method of claim 14, wherein the immune response is a CD4+ T cell response.

16. The method of claim 15, wherein the simian adenoviral vector construct is of serotype 7 or 11.

17. The method of claim 14, wherein the immune response is a CD8+ T cell response.

18. The method of claim 17, wherein the simian adenoviral vector construct is of serotype 7, 1 1, or 16.

19. The method of claim 14, wherein the immune response is a humoral immune response.

20. The method of claim 19, wherein the simian adenoviral vector construct is of serotype 7 or 1 1.

21. The method of any one of claims 14-20, wherein the method comprises administering a priming composition to the mammal prior to administering the composition comprising the simian adenoviral vector construct to the mammal.

22. The method of claim 21 , wherein the priming composition comprises a plasmid construct or a viral vector construct comprising a nucleic acid sequence encoding an HIV antigen.

23. The method of any one of claims 14-20, wherein the method comprises administering a boosting composition to the mammal after administering the composition comprising the simian adenoviral vector construct to the mammal.

24. The method of claim 23, wherein the boosting composition comprises a plasmid construct or a viral vector construct comprising a nucleic acid sequence encoding an HIV antigen.

25. The method of any one of claims 21-24, wherein the priming composition or the boosting composition comprises an adenoviral vector construct.

26. The method of claim 25, wherein the priming composition or the boosting composition comprises a simian adenoviral vector construct or a human adenoviral vector construct.

27. The method of claim 22 or claim 24, wherein the HIV antigen is selected from the group consisting of HIV Gag protein, HIV Pol protein, HIV Env protein, HIV Tat protein, HIV Reverse Transcriptase (RT) protein, HIV Vif protein, HIV Vpr protein, HIV Vpu protein, HIV Vpo protein, HIV Integrase protein, HIV Nef protein, and a fusion protein comprising all or part of an HIV Gag protein, HIV Pol protein, or HIV Env protein.

28. The method of claim 27, wherein the HIV antigen is an HIV gpl40 Env protein or a gpl40dvl2 Env protein.

29. The method of claim 27 or claim 28, wherein the HIV antigen is a mosaic antigen.

30. The method of any one of claims 27-29, wherein the HIV antigen is an HIV clade A antigen, HIV clade B antigen, HIV clade C antigen, or an HIV clade MN antigen.

31. The method of claim 21 or claim 22, wherein administration of the priming composition and the administration of the composition comprising the simian adenoviral vector construct are separated by at least about two weeks.

32. The method of claim 23 or claim 24, wherein administration of the composition comprising the simian adenoviral vector construct and administration of the boosting

composition are separated by at least about two weeks.

33. The simian adenoviral vector construct of any one of claim 1 -3, which comprises at least one nucleic acid sequence which encodes an influenza virus antigen.

34. The simian adenoviral vector construct of claim 33, wherein the simian adenoviral vector construct comprises a nucleic acid sequence that encodes two or more different influenza virus antigens.

35. The simian adenoviral vector construct of claim 34 or claim 35, wherein the simian adenoviral vector construct comprises two or more nucleic acid sequences that each encode a different influenza virus antigen.

36. The simian adenoviral vector construct of any one of claims 33-35, wherein the influenza virus antigen is selected from the group consisting a matrix protein (M), a

hemagglutinin (HA) protein, and a neuraminidase (NA) protein.

37. The simian adenoviral vector construct of any one of claims 33-36, wherein the simian adenoviral vector construct is of serotype 3, 7, 1 1 , 16, 18, 19, 20, 27, 33, 38, 39, or combinations thereof.

38. The simian adenoviral vector construct of claim 37, which comprises SEQ ID NO: 30, SEQ ID NO: 31 , or SEQ ID NO: 32.

39. A method of inducing an immune response against influenza virus in a mammal, which comprises administering to the mammal a composition comprising the simian adenoviral vector construct of any one of claims 33-38 and a pharmaceutically acceptable carrier, whereupon the nucleic acid sequence encoding the influenza virus antigen is expressed in the mammal to produce the influenza virus antigen and thereby induce an immune response against influenza virus.

40. The method of claim 39, wherein the immune response is a CD4+ T cell response.

41. The method of claim 40, wherein the simian adenoviral vector construct is of serotype 7 or 1 1.

42. The method of claim 39, wherein the immune response is a CD8+ T cell response.

43. The method of claim 42, wherein the simian adenoviral vector construct is of serotype 7, 11, or 16.

44. The method of claim 39, wherein the immune response is a humoral immune response.

45. The method of claim 44, wherein the simian adenoviral vector construct is of serotype 7 or 1 1.

46. The method of any one of claims 39-45, wherein the method comprises administering a priming composition to the mammal prior to administering the composition comprising the simian adenoviral vector construct to the mammal.

47. The method of claim 46, wherein the priming composition comprises a plasmid construct or a viral vector construct comprising a nucleic acid sequence encoding an influenza virus antigen.

48. The method of any one of claims 39-45, wherein the method comprises administering a boosting composition to the mammal after administering the composition comprising the simian adenoviral vector construct to the mammal.

49. The method of claim 48, wherein the boosting composition comprises a plasmid construct or a viral vector construct comprising a nucleic acid sequence encoding an influenza virus antigen.

50. The method of any one of claims 46-49, wherein the priming composition or the boosting composition comprises an adenoviral vector construct.

51. The method of claim 50, wherein the priming composition or the boosting composition comprises a simian adenoviral vector construct or a human adenoviral vector construct.

52. The method of claim 47 or claim 49, wherein the influenza virus antigen is selected from the group consisting a matrix protein (M), a hemagglutinin (HA) protein, and a neuraminidase (NA) protein.

53. The method of claim 46 or claim 47, wherein administration of the priming composition and the administration of the composition comprising the simian adenoviral vector construct are separated by at least about two weeks.

54. The method of claim 48 or claim 49, wherein administration of the composition comprising the simian adenoviral vector construct and administration of the boosting

composition are separated by at least about two weeks.

55. The simian adenoviral vector construct of any one of claims 1 -3, which comprises at least one nucleic acid sequence which encodes a Mycobacterium tuberculosis antigen.

56. A monkey adenovirus which is capable of propagation in a human cell, wherein

(i) the monkey adenovirus comprises (a) at least one nucleic acid sequence of the El region of a human adenovirus and (b) a nucleic acid sequence encoding the E4 ORF6 gene product of a human adenovirus, or (ii) the human cell comprises (a) at least one nucleic acid sequence of the El region of a human adenovirus and (b) a nucleic acid sequence encoding the E4 ORF6 gene product of a human adenovirus.

57. A monkey adenovirus which is capable of propagation in a cell which expresses one or more gene products of a human adenovirus.

58. The monkey adenovirus of claim 56 or claim 57, wherein the adenovirus is capable of propagation in a cell expressing one or more gene products of a serotype C human adenovirus.

59. The monkey adenovirus of any one of claims 56-58, wherein the adenovirus is replication-deficient.

60. The monkey adenovirus of any one of claims 56-59, wherein the adenovirus comprises a deficiency in the El region and a deficiency in at least a portion of the E4 region of the adenoviral genome.

61. The monkey adenovirus of any one of claims 56-60, wherein the adenovirus comprises a heterologous nucleic acid sequence.

62. The monkey adenovirus of claim 61 , wherein the adenoviral genome comprises a deletion of the El region and a deletion of at least a portion of the E4 region, and the

heterologous nucleic acid sequence is inserted into the deleted El region or the deleted E4 region of the adenovirus.

63. The monkey adenovirus of claim 61 or claim 62, wherein the heterologous nucleic acid sequence is a human adenovirus nucleic acid sequence.

64. The monkey adenovirus of claim 61 or claim 62, wherein the heterologous nucleic acid sequence encodes an antigen.

65. The monkey adenovirus of claim 64, wherein the antigen is isolated from a virus that is not an adenovirus.

66. The monkey adenovirus of claim 65, wherein the virus is human

immunodeficiency virus (HIV) or influenza virus.

67. The monkey adenovirus of any one of claims 56-66, wherein the adenovirus is a simian adenovirus.

68. The monkey adenovirus of claim 67, wherein the simian adenovirus is of serotype 3, 7, 1 1 , 16, 18, 19, 20, 27, 33, 38, 39, or combinations thereof.

69. The monkey adenovirus of any one of claims 56-68, wherein the cell is a HEK- 293 cell or a PerC.6 cell.

70. A method of inducing an immune response against a pathogen in a mammal, which method comprises administering to the mammal a composition comprising a replication-^" deficient simian adenoviral vector construct comprising at least one nucleic acid sequence which encodes an antigen of a pathogen and a pharmaceutically acceptable carrier, whereupon the nucleic acid sequence encoding an antigen is expressed in the mammal to produce the antigen and thereby induce an immune response against the pathogen.

71. The method of claim 70, wherein the immune response is a CD4+ T cell response.

72. The method of claim 71, wherein the simian adenoviral vector construct is of serotype 7 or 1 1.

73. The method of claim 70, wherein the immune response is a CD8+ T cell response.

74. The method of claim 73, wherein the simian adenoviral vector construct is of serotype 7, 1 1 , or 16.

75. The method of claim 70, wherein the immune response is a humoral immune response.

76. The method of claim 75, wherein the simian adenoviral vector construct is of serotype 7 or 1 1.

77. The method of any one of claims 70-76, wherein the pathogen is a virus that is not an adenovirus.

78. The method of claim 77, wherein the virus is human immunodeficiency virus (HIV) or an influenza virus.

79. The method of any one of claims 70-76, wherein the pathogen is a bacterium.

80. The method of claim 79, wherein the bacterium is Mycobacterium tuberculosis.

81. The method of any one of claims 70-80, wherein the method comprises administering a priming composition to the mammal prior to administering the composition comprising the simian adenoviral vector construct to the mammal, wherein the priming composition comprises an adenoviral vector construct comprising a nucleic acid sequence encoding an antigen of the pathogen.

82. The method of any one of claims 70-80, wherein the method comprises administering a boosting composition to the mammal after administering the composition comprising the simian adenoviral vector construct to the mammal, wherein the boosting composition comprises an adenoviral vector construct comprising a nucleic acid sequence encoding an antigen of the pathogen.

83. The method of claim 81 or claim 82, wherein the priming composition or the boosting composition comprises a monkey adenoviral vector construct or a human adenoviral vector construct.