HK1117743A - Vaccine against pandemic strains of influenza viruses - Google Patents

Description

Vaccine against pandemic influenza virus strains

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority of united states provisional application 60/670,826, filed on 11/4/2005, the specification of which is incorporated herein by reference in its entirety.

Credit government support

Various aspects of the invention are accomplished with U.S. government support from the subsidies of the national institutes of health. The united states government has certain rights in this invention.

FIELD

The present application relates to the field of vaccines. More specifically, the present application relates to recombinant vectors for the preparation of avian influenza virus vaccines

Background

Pandemic outbreaks of highly toxic avian influenza pose a significant risk to human and animal health worldwide. Genetic reassortment between human and avian influenza viruses can result in viruses with a novel Hemagglutinin (HA) of avian origin, to which humans lack immunity. The pandemics of 1918, 1957 and 1968 were the result of this antigenic shift in the 20 th century. Recent outbreaks of avian influenza caused by influenza viruses of the H5N1, H7N7 and H9N2 subtypes, and their infection in humans, all raise potential new precautions against the large-scale prevalence of influenza viruses transmitted in poultry. The economic impact of a pandemic in the united states alone is estimated to be as high as 1650 billion dollars, and as high as 200,000 deaths, 730,000 hospitalizations, 42 outpatients, and 5 million other illnesses.

Under the prevailing circumstances of global bioterrorism threats, individuals who are deliberately infected with virulent influenza strains can be used as weapons of undetected large-scale destructive organisms.

To date, three major approaches have been attempted to develop vaccines that are safe and effective against potentially pandemic strains of avian influenza, but none have been entirely successful (Wood et al, Vaccine 20: S84-S87, 2002; Stephenson et al, The Lancet 4: 499-.

Due to the lethality of these influenza strains in poultry, current vaccine preparation strategies involving virus culture in eggs are not feasible. There are several approaches directed to isolating nonpathogenic or attenuated influenza strains that express immunogenic antigens associated with potentially pandemic influenza strains. For example, naturally occurring, nonpathogenic influenza strains with an antigenic virus subtype H5 are evaluated as vaccine candidates. In general, these viruses have proven difficult to culture using conventional techniques, and protection is dependent on the ability of antibodies raised against the nonpathogenic Vaccine strain to cross-react with virulent strains (Takada et al, J.Virol.73: 8303-8307, 1999; Wood et al, Vaccine 18: 579-80, 2000). A candidate vaccine HAs been developed by deleting a basic amino acid at the cleavage site of the HA antigen of the pathogenic H5N1 virus (A/HK/97) using reverse genetics (Li et al, J.Infect.Dis.179: 1132-1138, 1999).

Another approach utilizes recombinant HA ("H5") produced in a baculovirus expression system. However, obtaining a satisfactory immune response requires high doses of purified protein and the use of adjuvants (Treanor et al, Vaccine 19: 1732-1737, 2001).

There is a continuing need to develop vaccines that protect against infection by avian influenza strains in both human and non-human populations, and which can be efficiently prepared and administered without relying on the growth of the virus in eggs. The present disclosure addresses this need and provides novel compositions and methods useful for preventing infections caused by avian and pandemic influenza strains.

Summary of The Invention

The present disclosure relates to methods of eliciting protective immune responses against potentially pandemic influenza strains, as well as compositions, including nucleic acid vectors and non-infectious viruses useful in the methods disclosed herein.

One aspect of the present disclosure relates to recombinant nucleic acids. The recombinant nucleic acids described herein include adenoviral vectors. The adenoviral vectors, including human and non-human adenoviral vectors, each contain a nucleic acid encoding one or more polypeptides corresponding to an antigen of an avian influenza strain.

In another aspect, the present disclosure provides recombinant adenoviruses, such as replication-defective human or non-human adenoviruses expressing one or more avian influenza antigens.

Pharmaceutical compositions, including vaccine compositions, comprising the adenoviral vectors and/or recombinant adenoviruses disclosed herein are disclosed.

Another feature of the present disclosure relates to methods of generating an immune response against avian and/or pandemic influenza strains. In the methods disclosed herein, immunogenic compositions based on adenoviral vectors encoding at least one avian influenza strain antigen and/or recombinant adenoviruses expressing at least one avian influenza strain antigen can be administered to a subject before or after exposure of the subject to an avian or pandemic influenza strain.

The above and other objects, features and advantages of the present invention will become more apparent from the following detailed description with reference to the accompanying drawings.

Brief Description of Drawings

FIG. 1A schematically illustrates the HAd5-H5HA vector. The Cre recombinase-mediated site-specific recombination system was used to generate HAd5 vectors expressing HA of avian influenza virus (H5N1) A/HK/156/97. The HA gene under the control of the CMV promoter was inserted into the StuI site of a shuttle vector (pDC 311-the left end of HAd5(4kb) containing a3.1 kb E1 deletion, loxP sites for site-specific recombination in the presence of Cre recombinase and complete packaging signal) to prepare pDC 311-H5. A HAd-H5HA vector was generated by co-transfecting 293Cre cells (293 cells expressing Cre recombinase) with pDC311-H5HA and pBHGlox Δ E1, 3Cre (a plasmid containing almost the entire HAd5 genome except for the deletion of the packaging signals E1 and E3 for site-specific recombination of loxP sites in the presence of Cre recombinase). FIG. 1B is a Western blot illustrating expression of Hemagglutinin (HA) encoded by the HAd-H5HA vector. HAd-H5HA efficiently expressed HA in infected cells. Structure of the H5-HA gene cassette in the HAd-H5HA vector (A) and H5HA expression in cells infected with the HAd-H5HA vector (B). The 293Cre cells were mock-infected or infected with HAd- Δ E1E3 or HAd-H5 HA. Cells were collected 24 hours after infection and cell extracts were prepared. Cell extracts were analyzed by Western blotting using rabbit H5 HA-specific serum produced by immunizing rabbits with a DNA vector encoding H5 HA.

FIG. 2 is a line graph demonstrating that the HAd-H5HA vector confers complete protection against challenge with the highly pathogenic homotypic H5N1(A/HK/483/97) virus. 25 (6-8 weeks old) female BALB/c mice were randomly divided into 5 groups (5 animals/group) and intramuscularly inoculated with PBS (□), 10. mu.g of recombinant H5HA (hemagglutinin of avian HK/156/97 influenza virus expressed in baculovirus) without alum at 0 and 28 days (PBS) (□)) 10 μ g of purified H5HA (x), 10 containing alum⁸HAd- Δ E1E3 (. DELTA.), or 10 of p.f.u.⁸HAd-H5HA (■) by p.f.u. Using 100LD in 70 days₅₀The H5N1(A/HK/483/97) virus of (1) challenged animals. Mice were monitored daily for clinical symptoms and body weightChange until 14 days post challenge.

FIGS. 3A-C are whisker box plots showing the mean, IQR and range of neutralizing antibody responses against homologous and heterologous avian influenza virus strains in mice immunized with the HAd-H5HA vaccine. (A) HK/156/97, (B) HK/213/03, and (C) VN/1203/04 strain. # and $: the difference between the labeling data was p 0.001. im ═ muscle immunity. in is intranasal immunization.

FIG. 4 is a scatter plot of a panel of FACS cell assays showing the induction of HA-518-epitope specific CD8T cells in mice immunized with the HAd-H5HA vaccine. Flow cytometric analysis of splenocytes from immunized mice (3/group) was stained with the HA518 pentamer epitope. Pentamer-positive cells (circled) were expressed as a percentage of the CD8T lymphocyte population.

FIG. 5 is a bar graph showing secretion of interferon gamma by HA-518 epitope specific CD8T cells. An ELISpot assay of interferon gamma was performed in splenocytes of immunized mice. Data are mean and SD (error bars). im ═ muscle immunity. in is intranasal immunization. By NP_147-155(diagonal shaded bars), HA (white bars), or phorbol-myristyl-acetate (PMA) + ionomycin (black bars) stimulated cultures.

FIGS. 6A-D are line graphs and FIG. 6E is a bar graph illustrating protection against the recent H5N1 virus. BALB/C mice (15 animals/group) were treated with 10 animals⁸p.f.u. HAd-H5HA muscle (●) or intranasal (. smallcircle) inoculations were performed twice at 4 weeks intervals. HAd- Δ E1E3(■) served as negative controls. Four weeks after the second immunization, 5 animals per group were given a 100-fold 50% Lethal Dose (LD)₅₀) A/HK/483/97(A and B) or A/VN/1203/04(C and D) or 100-fold 50% Mouse Infection Dose (MID)₅₀) The A/HK/213/03(E) of (1) was subjected to the challenge. The percentage of initial body weight (a and C) and survival after challenge (B and D) are shown. Error bars describe the standard deviation of the mean. Mice challenged with A/HK/483/97 or A/VN/1203/04 were monitored daily for clinical symptoms and weight changes until 14 days post challenge. Mice challenged with A/HK/213/03 were euthanized and lungs collected 3 days after challenge. Freezing and thawing the tissue onceThen, homogenization was performed in 1ml of PBS containing antibiotics. Solid residue pellets were pelleted by brief centrifugation before virus infectivity titration of the homogenate in 11 day old eggs.

FIGS. 7A-D are schematic illustrations (A and C) and images (B and D) of Western blots showing expression of avian influenza hemagglutinin from porcine and bovine adenoviral vectors. A PAd vector (PAd-H5HA) was prepared with the avian H5N1 influenza virus (A/HK/156/97) hemagglutinin subtype 5(H5HA) gene under the control of the Cytomegalovirus (CMV) immediate early promoter inserted in PAd genomic early region 1 (E1). (A) Structural schematic representation of the PAd vector: PAd- Δ E1E3 (PAd with E1 and E3 deletions), and PAd-H5HA (PAd- Δ E1E3 with the H5HA gene cassette). ITR, inverted terminal repeat; deletions in Δ E1, E1; deletions in Δ E3, E3; h5, H5 HA; pA, simian virus 40 polyadenylation signal. (B) FPRT HE1-5 cells were mock infected or infected with either PAd-. DELTA.E 1E3 or PAd-H5 HA. 24 hours post infection, cells were harvested and cell extracts were analyzed by Western blotting using rabbit hyperimmune serum directed against H5 HA. BAd vectors (BAd-H5HA) were prepared with the avian H5N1 influenza virus (A/HK/156/97) hemagglutinin subtype 5(H5HA) gene under the control of the Cytomegalovirus (CMV) immediate early promoter inserted in BAd genomic early region 1 (E1). (C) BAd structural schematic representation of the vector: BAd- Δ E1E3 (PAd with E1 and E3 deletions), and BAd-H5HA (BAd- Δ E1E3 with the H5HA gene cassette). ITR, inverted terminal repeat; deletions in Δ E1, E1; deletions in Δ E3, E3; h5, H5 HA; pA, simian virus 40 polyadenylation signal. (D) FBRT HE1-5 cells were mock-infected or infected with BAd- Δ E1E3 or BAd-H5 HA. 24 hours post infection, cells were harvested and cell extracts were analyzed by Western blotting using rabbit hyperimmune serum against H5 HA.

FIGS. 8A and B are graphs showing the expression of the bovine and porcine E1 gene in transfected cells. (A) Expression of the BAd3E1A, E1B-19kDa (E1B-1), and E1B-55kDa (E1B-2) information determined by RT-PCR using specific primer sets. (B) Expression of PAd3E1A, E1B-19kDa (E1B-1), and E1B-55kDa (E1B-2) messages as determined by RT-PCR using specific primer sets. The specific bands are indicated by arrows.

Brief description of the sequence listing

SEQ ID NO: 1 (5'-tccatgagcttcctgatcct-3') is an immunostimulatory oligonucleotide.

SEQ ID NO: 2 (5'-tccatgacgttcctgacgtt-3') is an immunostimulatory oligonucleotide.

SEQ ID NO: 3 (5'-tgactgtgaacgttcgagatga-3') is an immunostimulatory oligonucleotide.

SEQ ID NO: 4 is a nucleotide sequence of HAd5E 1.

SEQ ID NO: 5 and 6 are oligonucleotide primers for amplifying bovine adenovirus E1.

SEQ ID NO: 7 is a nucleotide sequence of BAd3E 1.

SEQ ID NO: 8-13 are oligonucleotide primers that detect BAd3E1 transcripts.

SEQ ID NO: 14 and 15 are oligonucleotide primers for amplifying porcine adenovirus E1.

SEQ ID NO: 16 is the nucleotide sequence of PAd3E 1.

SEQ ID NO: 17-22 are oligonucleotide primers for detecting the PAd3E1 transcript.

Detailed description of the invention

Introduction to the design reside in

Influenza viruses are enveloped negative-sense viruses belonging to the family orthomyxoviridae. Influenza viruses are divided into 3 different types, based on their core protein: a, B and C. Within this broad category, Hemagglutinin (HA) and Neuraminidase (NA) can be further divided into subtypes according to two antigen surface proteins. Although influenza B and C viruses are primarily limited to humans, influenza a viruses are pathogens of a wide variety of species including humans, non-human mammals, and birds. Periodically, non-human viral strains, particularly avian influenza, infect human populations and in some cases can cause serious disease with high lethality. Recombination between such avian and human viral strains within co-infected individuals produces recombinant influenza viruses, which lack immunity to this population, resulting in influenza epidemics. Three such epidemics occurred in the 20 th century, resulting in a large number of deaths worldwide in 1918, 1957 and 1968.

Highly pathogenic avian influenza H5N1 virus is becoming endemic in the poultry industry in south east asia. In the early 2004, increased human infection frequency and high mortality of H5N1 virus in the region were reported. In 1997, it was first recognized that the highly pathogenic H5N1 influenza virus caused human respiratory disease, and 18 documented cases, including 6 deaths, occurred after outbreaks of influenza in chicken farms and markets in hong kong. In 2003, two additional human H5N1 infections were identified in a family in hong kong. Since then, the H5N1 virus has spread to 9 asian countries, and recently its geographical distribution has been expanded to several countries in eastern europe. Since 1 month 2004, more than 120 cases of human infections with laboratory-proven lethality rates above 50% have been reported to the world health organization. To date, most human H5N1 viral infections have been due to direct transmission of the virus from infected poultry, although there are exceptions to human-to-human transmission. Genetic recombination between human and avian influenza viruses and/or mutants in the H5N1 viral genome can lead to the generation of new influenza viruses of the H5 subtype, which may trigger a pandemic if they acquire the ability to tolerate sustained transmission in non-immunized humans. Thus, there is a need for vaccines that are effective against highly pathogenic H5N1 and other strains of avian influenza.

Vaccines developed and evaluated in response to 1997H 5N1 influenza outbreaks have only modest immunogenicity in humans, whereas H5N1 viruses isolated from humans in 2004 differ significantly in genetics and antigenicity from those previously isolated in 1997 and 2003, necessitating the development of new vaccine candidates because the resulting immune response is not protected against antigenically distinct strains.

Non-pathogenic avian influenza viruses, either prepared from naturally occurring, non-pathogenic strains sharing the HA subtype with pathogenic strains, or engineered to be non-pathogenic by deletion of spontaneous protein splice sites, have so far been able to be prepared only in eggs. In a worldwide epidemic, the infection of poultry is likely to spread widely, requiring that the chickens be killed and causing a shortage of eggs available for vaccine preparation. Recombinant HA vaccines have been evaluated, but require potentially harmful adjuvants to exert effective protection. Thus, it would be desirable to include diversification of vaccine production substrates based on cellular and/or recombinant DNA technology, improving vaccine productivity in a marketing situation. In addition, recombinant DNA technology is advantageous in accelerating vaccine availability because once the sequence of the virus is known, cloning and expression of one or more viral genes can begin.

The present disclosure provides novel compositions and methods for the preparation of influenza vaccines and the vaccination of human, non-human mammals and avian populations against avian and/or epidemic influenza virus strains, and overcomes the poor immunogenicity and manufacturing disadvantages of existing influenza vaccines, which have been adapted to elicit immune responses against avian influenza strains. The compositions and methods described herein are based on adenoviral vectors expressing one or more immunogenic avian influenza antigens, optionally in combination with internal proteins that can further enhance immune response, reduce morbidity, and aid recovery after exposure or infection of avian or influenza strains. The present disclosure provides the first evidence that human and non-human adenoviruses are effective vectors for eliciting an immune response against avian (and potentially epidemic) influenza strains. In addition, the compositions and methods described herein also provide several benefits over vaccine strategies previously assessed against potential epidemic strains of avian influenza. For example, the adenoviral vectors and adenoviruses described herein are readily grown under tissue culture conditions and purified on a scale suitable for commercial manufacture. In addition, the immune response elicited is strong and long lasting, and depends on a combination of antigens, involving neutralizing antibody production and T cell responses, and protects against antigenically diverse influenza strains.

Description of the exemplary embodiments

One aspect of the present disclosure relates to a recombinant adenoviral vector comprising a polynucleotide sequence encoding one or more influenza antigens. In particular, the adenoviral vectors described herein comprise a polynucleotide sequence encoding at least one avian influenza strain antigen ("avian influenza antigen"). For example, the adenoviral vector can encode one or more avian hemagglutinin ("HA") antigens. The vector may comprise a sequence encoding a single HA of an avian influenza strain, for example an H5 subtype strain, an H7 subtype strain or an H9 subtype strain, for example selected from the H5N1, H7N7 or H9N2 strains that are prevalent in recent outbreaks of avian influenza. Alternatively, the vector may encode multiple (more than one) avian HA antigens. In this case, the encoded HA antigen may be a variant of one subtype (e.g., a variant of H5HA, or a variant of H7HA, or a variant of H9 HA) or the HA antigen may be a different subtype of HA antigen (i.e., a combination of H5, H7, and/or H9, such as H5 and H7, H5 and H9, H7 and H9, or H5, H7, and H9, including a combination of one or more HA antigens of any one subtype with one or more HA antigens of any other subtype).

The recombinant adenoviral vector can further comprise a polynucleotide sequence encoding an avian influenza neuraminidase ("NA") antigen. The NA antigen may be encoded by the vector alone, or in combination with an avian HA antigen. When the vector comprises a polynucleotide sequence encoding both an avian HA antigen and an avian NA antigen, the HA and NA antigens may be the same influenza strain, or may be selected from different avian influenza strains. For example, the recent outbreaks of avian influenza in asian populations have been caused by influenza a strains of H5N1, H7N7 and H9N 2. The adenoviral vector can encode, for example, NA of subtype N1, NA of subtype N7, or NA of subtype N2. Alternatively, the vectors may encode a different NA subtype, such as N3 (e.g., corresponding to a non-pathogenic strain of H5N3 avian influenza virus). It will be appreciated by those skilled in the art that any avian subtype of HA (most commonly, H5, H7 or H9) may be combined with any subtype of 9NA in an adenoviral vector. As noted above, in the case of HA antigens, the adenoviral vector can encode multiple NA antigens, which can be variants of a single NA subtype or representative of different NA subtypes.

As described above, adenoviral vectors can encode a variety of influenza antigens. The plurality of influenza antigens can encode two or more avian influenza antigens, e.g., a plurality of avian HA antigens, a plurality of avian NA antigens, or a combination of avian HA and NA antigens. Alternatively, the vector is capable of encoding one or more avian influenza antigens in combination with one or more internal proteins of avian or non-avian influenza strains. In the case of an avian influenza internal protein, the internal protein may be selected from the same or different avian influenza strains. The encoded internal protein may also be selected from non-avian influenza strains, such as human influenza strains (typically, influenza a). For example, the one or more internal proteins may be selected from H1N1, H2N2, or H3N2 influenza strains. Any internal protein (M1, M2, NP, PB1, PB2, NS1 and NS2) can be encoded by the adenoviral vector. Combinations of internal proteins may also be encoded by the vector. For example, the vector may encode an M protein (one or more of M1 and/or M2), an NP protein or both an M and NP protein.

Human and non-human adenoviral vectors are well known in the art, and both can be constructed to include one or more of the influenza antigens described above. Human adenovirus vectors include human adenovirus serotype 5 ("HAd 5") vectors. Alternatively, the adenoviral vector is a non-human, e.g., porcine or bovine, adenoviral vector (e.g., BAd3 and PAd3 vector). Typically, the adenoviral vector is a replication-defective adenovirus that is incapable of multiple cycles of transcription and translation of the inserted gene in human and animal cells. The replication-defective adenovirus vector may have a mutation in one or more genes (or regions) involved in replication, including one or more of the E1 region gene, the E3 region gene, the E2 region gene, and/or the E4 region gene. For example, the replication-defective adenovirus vector may have a deletion or a mutation in an E1 region gene (e.g., E1A), an E3 region gene, an E2 region gene, an E4 region gene, or a combination thereof.

Thus, in an exemplary embodiment, the adenovirus vector is a replication-defective human adenovirus vector comprising a polynucleotide sequence encoding an avian influenza HA antigen, an avian influenza NA antigen, or both an avian HA antigen and an avian NA antigen. Optionally, the adenoviral vector encodes a plurality of avian HA antigens which are variants of a single HA subtype or different HA subtypes. In certain embodiments, the adenoviral vector further encodes at least one influenza internal protein, such as M1 protein, M2 protein, NP protein or a combination of M and NP proteins.

In another embodiment, the adenovirus vector is a replication-defective non-human adenovirus vector, such as a porcine or bovine adenovirus vector, comprising a polynucleotide sequence encoding an avian influenza HA antigen, an avian influenza NA antigen, or both an avian HA antigen and an avian NA antigen. Optionally, the adenoviral vector encodes a plurality of avian HA antigens, which may be variants of a single HA subtype or different HA subtypes. In certain embodiments, the adenoviral vector can further encode at least one influenza internal protein, such as M1, M2 protein, NP protein, or any combination thereof.

Another aspect of the disclosure relates to recombinant adenoviruses that express (include) at least one avian influenza strain antigen. Typically, the adenovirus expresses an avian HA antigen and/or an avian NA antigen. Thus, the adenovirus may include an avian HA antigen, e.g., H5HA antigen, H7HA antigen, and/or H9HA antigen. Similarly, the adenovirus may include an avian NA antigen, e.g., H1NA antigen, H7NA antigen, and/or H2NA antigen. In some cases, the adenovirus expresses multiple avian influenza antigens, e.g., multiple avian HA antigens or multiple avian NA antigens or a combination of avian HA and NA antigens. For example, the adenovirus may express two or more variants of a single HA (or NA) subtype. Alternatively, the adenovirus may express two or more HA (or NA) antigens of different subtypes. In some cases, the adenovirus expresses at least one influenza internal protein, such as the M1, M2, and/or NP proteins. When the adenovirus expresses multiple influenza antigens, the multiple antigens may be of the same strain or subtype, or of different strains or subtypes.

The recombinant adenovirus may be a human adenovirus or a non-human adenovirus, such as a porcine or bovine adenovirus. Generally, the adenovirus is a replication-defective human or non-human adenovirus. For example, the replication-deficient adenovirus may have a mutation (e.g., a deletion, an insertion, or a substitution) in the E1 region gene, the E3 region gene, the E2 region gene, and/or the E4 region gene.

Such adenoviral vectors and adenoviruses may be suitable for a variety of purposes. For example, such adenoviruses and adenoviral vectors are suitable for use in the preparation of avian (and other) influenza antigens in vitro and in vivo, including in ovo. Accordingly, a method of making a recombinant avian influenza antigen is a feature of the present disclosure. For example, recombinant avian influenza antigens can be prepared by replicating an adenovirus that includes at least one heterologous polynucleotide sequence encoding an avian influenza virus antigen. In some embodiments, the adenovirus comprises a sequence encoding two or more avian influenza virus antigens, or at least one avian influenza virus antigen and a non-avian influenza virus antigen. For example, the avian influenza antigen may be an HA antigen or an NA antigen, for example selected from the HA or NA antigens of H5, H7 or H9 influenza strains. In certain embodiments, the adenovirus comprises polynucleotide sequences encoding multiple influenza antigens.

In certain embodiments, the adenovirus expressing the recombinant avian influenza virus antigen is prepared by introducing a replication-defective adenovirus vector into a cell capable of supporting replication of the replication-defective vector. Such cells typically include at least one heterologous nucleic acid that provides a complementing replication function, e.g., a heterologous nucleic acid encoding one or more E proteins deleted from a replication-defective adenoviral vector. In certain embodiments, a cell capable of supporting the growth of a replication-defective adenovirus vector is capable of supporting the growth of different strains of adenovirus with different tropisms. Optionally, the recombinant avian influenza virus antigen can be isolated and, for example, used to prepare an immunogenic composition, such as a vaccine.

Another aspect of the disclosure relates to a cell line that supports replication of a plurality of replication-defective adenovirus strains that differ in species tropism. Such multifunctional cell lines include heterologous nucleic acids encoding various E proteins of different strains of adenovirus. In exemplary embodiments, the cell line comprises one or more heterologous nucleic acids comprising at least two distinct polynucleotide sequences, one of which encodes at least one E protein of a first adenovirus strain and the other of which encodes at least one E protein of a different adenovirus strain. The E protein is selected to complement the E protein deleted by the recombinant adenoviral vector to be grown in the cell line. Thus, in some embodiments, when the cell is intended to support the growth of a replication deficient adenovirus that lacks one or more E1 proteins, the polynucleotide sequence encodes the corresponding E1 protein. Similarly, when the cell is intended to support the growth of a replication deficient adenovirus lacking one or more E3 proteins, the polynucleotide sequence encodes the corresponding E3 protein. Typically, the cell comprises nucleic acid encoding E proteins of adenovirus strains with different tropisms, such that the cell is capable of optimally supporting growth of multiple adenovirus strains with different tropisms, e.g., a human adenovirus E gene (or fragment thereof) and a non-human E gene (or fragment thereof). In particular embodiments, the polynucleotide sequence encodes human and bovine or porcine E protein.

The adenoviral vectors and recombinant adenoviruses disclosed herein can be used in the context of immunogenic compositions, including vaccines. Such immunogenic compositions can include an adenoviral vector having a polynucleotide sequence encoding at least one avian influenza antigen as previously described. The immunogenic composition may further comprise a recombinant adenovirus expressing at least one avian influenza antigen as described above. In addition to the adenoviral vector and/or adenovirus, the immunogenic composition further comprises a pharmaceutically acceptable carrier or excipient. Thus, the immunogenic composition can include any adenoviral vector and/or adenovirus that encodes or expresses the avian influenza antigens disclosed herein. Optionally, the immunogenic composition includes an adjuvant, an immunostimulatory molecule, a microparticle, or a nanoparticle.

The disclosure also provides methods of eliciting and generating an immune response against an avian or an influenza (or potentially pandemic) strain. The methods disclosed herein involve administering to a subject at least one adenovirus or adenoviral vector that expresses (or encodes) an avian influenza antigen. Typically, the adenovirus or adenoviral vector is administered to the subject prior to exposure to at least one strain of avian influenza or pandemic influenza. That is, typically, the adenovirus or adenoviral vector expressing the avian influenza antigen is administered prophylactically, e.g., as a vaccine.

Administration of the recombinant adenovirus and adenoviral vector can elicit an immune response that protects a subject from serious disease or death due to infection by one or more avian or pandemic influenza strains. In some cases, the immune response protects against disease caused by more than one influenza strain, e.g., multiple avian influenza strains. Typically, the immune response includes neutralizing antibodies that bind to at least one avian influenza antigen, and in some cases, the neutralizing antibodies can cross-react with multiple avian influenza strains. For example, administration of an adenovirus or adenoviral vector expressing an avian influenza HA antigen can result in neutralizing antibodies against the HA subtype that protect (or partially protect) against infection by influenza of the corresponding HA subtype. Similarly, administration of an adenovirus or adenoviral vector expressing avian NA antigen is capable of eliciting an antibody response against the NA subtype.

In some cases, the administered adenovirus or adenoviral vector expresses (or encodes) two or more avian HA and/or NA antigens. Alternatively, multiple adenoviruses or adenoviral vectors are administered, each of which includes a single avian influenza antigen. Whether administered as a single virus or vector or as multiple viruses or hybrids, the antigens, e.g., HA antigens, may be variants of the same antigenic subtype or may be antigens of different influenza subtypes. Optionally, the adenovirus(s) or vector(s) result in the expression of influenza structural or non-structural proteins such as M1, M2, NP, or NS1 protein. In the case of HA and NA antigens, the internal proteins may be included in one or more adenoviruses or vectors. Inclusion of one or more internal protein antigens is suitable for boosting the immune response generated by a vaccine by raising influenza-specific T cells. Thus, the immune response elicited by the compositions described herein may include a T cell response. In general, the T cell response is specific for epitopes of influenza internal proteins that are conserved across multiple influenza strains, e.g., multiple strains of the same or different influenza subtypes.

For example, in one embodiment, a replication-defective adenovirus expressing a single avian HA antigen, e.g., H5HA antigen, H7HA antigen, or H9HA antigen, is administered. In another embodiment, an adenovirus expressing two or more avian HA antigens is administered. In yet another embodiment, an adenovirus expressing at least one avian HA antigen and at least one avian NA antigen is administered. In yet another embodiment, the adenovirus expresses at least one avian HA antigen, at least one avian NA antigen, and one or more influenza internal proteins (e.g., M1, M2, and/or NP proteins) of an avian or non-avian influenza strain. Exemplary embodiments of such adenoviruses are adenoviruses expressing avian HA H5, avian NA N1, and human M (M1 and/or M2) and/or NP proteins. Various other embodiments can be determined by those skilled in the art depending on the strain against which the immune response is desired.

In other embodiments, a plurality of different human or non-human adenoviruses, each comprising one or more influenza antigens, are administered. For example, multiple adenoviruses can be administered at the same or different times (i.e., one or more immunological compositions delivered simultaneously or sequentially). In some cases, multiple adenoviruses may be administered as a "cocktail". When administered as a cocktail, each adenovirus may express a single influenza antigen, or some or all of the adenoviruses may express multiple influenza antigens. For example, in one effective cocktail formulation, an adenovirus expressing avian HA antigen of H5 is co-administered with an adenovirus expressing M2 protein. In another embodiment, an adenovirus expressing avian H5HA antigen and avian N1 NA antigen is administered in combination with human influenza M2 and/or NP protein. In yet another exemplary embodiment, a first adenovirus expressing multiple avian HA antigens (e.g., variants of a single HA subtype, or corresponding to different HA subtypes) is administered with a second adenovirus expressing avian or human M2 and/or NP proteins. Various other combinations may also be determined by those skilled in the art.

In other embodiments, the adenoviral vector, rather than the recombinant adenovirus, is administered to the subject as a nucleic acid (e.g., DNA) vaccine.

Optionally, the adenovirus or adenoviral vector is administered to the subject in an immunogenic composition comprising at least one additional immunostimulatory component of the subject in addition to a pharmaceutically acceptable carrier or excipient. Such immunostimulatory components include adjuvants such as MF59 and aluminum adjuvants. Alternatively, naturally occurring or synthetic immunostimulatory compositions that bind to or stimulate receptors involved in innate immunity can be co-administered with the adenovirus and/or adenoviral vector. For example, agents that stimulate certain Toll-like receptors (e.g., TLR7, TLR8, and TLR9) can be administered in combination with an adenoviral recombinant that expresses influenza antigen and/or an adenoviral vector. In some embodiments, the adenovirus or vector is administered in combination with an immunostimulatory CpG oligonucleotide. In other embodiments, the adenovirus or vector is administered in combination with other adenovirus vectors that express Toll-like receptors and ligands that result in receptor activation, respectively.

The immunogenic compositions described herein can be administered to a human or non-human (e.g., cat, dog, pig, bird) subject to elicit an influenza-specific immune response. For example, if the subject is a human subject, then a human, bovine, or porcine adenovirus recombinant (or adenoviral vector) comprising one or more avian influenza antigens can be administered to elicit an immune response. In addition, influenza-specific immune responses can be elicited in avian subjects, including poultry, such as chickens, ducks, guinea fowl, turkeys, geese, and the like, using the compositions and methods described herein. In addition, compositions and methods described herein can be used to elicit influenza-specific immune responses in non-human subjects, including cats, dogs, horses, non-human primates, and other domestic and wild animals.

Various routes of administration may be suitable for administration of the immunogenic compositions described herein. These methods include intranasal, buccal, ocular, intravenous, intramuscular, transdermal, intradermal, and subcutaneous delivery of the adenovirus and/or adenoviral vector. In particular embodiments, the immunogenic composition can be administered to poultry in drinking water, by spraying or controlled drip irrigation or in ovo prior to hatch.

Additional technical details are provided below under a particular subject matter heading.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The definition of terms commonly used in molecular biology can be found in Benjamin Lewis, Genes V, Oxford university Press, 1994(ISBN 0-19-854287-9); the encyclopedia of Molecular Biology, Blackwell Science Ltd, published by Kendrew et al, 1994(ISBN 0-632-02182-9); and molecular biology and Biotechnology, edited by Robert a.meyers: a Comprehensive Desk Reference, VCHPublishers, Inc. published, 1995(ISBN 1-56081-.

The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is also intended to include "and" unless the context clearly indicates otherwise. It is further understood that all base sizes or amino acid sizes, as well as all molecular weights or molecular mass values given for a nucleic acid or polypeptide are approximate and are used for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The term "comprising" means "including". The acronym "e.g." is from latin exempli gratia, used herein to indicate a non-limiting example. The abbreviation "e.g." is thus synonymous with the term "e.g.".

To facilitate a reading of the various embodiments of the present disclosure, an explanation of specific terms is provided below.

Adjuvant: a medium for enhancing antigenicity; for example, a suspension of minerals to which the antigen can adsorb (alum, aluminium hydroxide, aluminium phosphate); or water-in-oil emulsions, in which the antigen solution is emulsified into oil (MF-59, freund's incomplete adjuvant), sometimes also containing killed mycobacteria (freund's complete adjuvant) to further enhance antigenicity (inhibit the breakdown of antigens and/or cause the uptake of macrophages).

Antigen: a compound, composition or substance capable of stimulating antibody production or a T cell response in an animal, including a composition that is injected, absorbed, or otherwise introduced into an animal. The term "antigen" includes all relevant epitopes. An "antigenic polypeptide" is a polypeptide capable of stimulating an immune response, such as a T cell response or an antibody response. An "epitope" or "antigenic determinant" refers to a site on an antigen to which B and/or T cells respond. In one embodiment, T cells react with an epitope when present in the context of binding to an MHC molecule. Epitopes can be formed by contiguous amino acids or by non-contiguous amino acids juxtaposed by tertiary folding of the antigenic polypeptide. Epitopes formed from the amino acids in the neighborhood are generally retained when exposed to denaturing solvents, whereas epitopes formed from tertiary folding are generally lost after treatment with denaturing solvents. Epitopes typically comprise at least 3, or more commonly, at least 5, about 9, or about 8-10 amino acids unique to the spatial configuration. Methods for determining spatial configuration of epitopes include, for example, X-ray crystallography and multidimensional nuclear magnetic resonance spectroscopy.

The influenza antigen may be a polymorphic Hemagglutinin (HA) or Neuraminidase (NA) antigen selected from influenza strains or related strains sharing antigenic epitopes against which an immune response is desired. The influenza antigen may also be an influenza internal protein, such as PB1, PB2, PA, M1, M2, NP, NS1 or NS2 protein. Avian influenza antigens are influenza antigens of avian influenza strains. The variants of influenza antigens may be naturally occurring variants or engineered variants. As used herein, the term "variant" refers to a protein having one or more amino acid changes, such as deletions, insertions, or substitutions, relative to a reference protein or relative to other variants.

Antibody: immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoresponses) an antigen. Naturally occurring antibodies (e.g., IgG, IgM, IgD) comprise four polypeptide chains, two heavy (H) chains and two light (L) chains that are cross-linked to each other by disulfide bonds. The phrase "antibody response" is an immune response directed to an antigen, involving the secretion of antibodies specific for the antigen. Antibody responses are B cell-mediated immune responses initiated by the interaction of an antigen (or epitope) with a B cell receptor (membrane-bound IgD) on the surface of a B cell. After binding to a stimulus for the B cell receptor by its cognate antigen, the B cell differentiates into a plasma cell that secretes antigen-specific immunoglobulin to produce an antibody response. A "neutralizing antibody" is an antibody that binds to an epitope on a virus and inhibits infection and/or replication as determined, for example, by a plaque neutralization assay.

Animals: living multicellular vertebrate organisms, a category of which includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and non-human subjects, including birds and non-human mammals, such as non-human primates, companion animals (e.g., dogs and cats), livestock (e.g., pigs, sheep, cattle), and non-domesticated animals, such as big cats. The term object is used irrespective of the phase of the life cycle of the organism. Thus, depending on the organism, the term subject may apply to an organism in the uterus or in the eggs (i.e. whether the organism is a mammal or bird, such as a poultry or wild bird).

cDNA (complementary DNA): a piece of DNA lacking internal non-coding segments (introns) and defining regulatory sequences for transcription. cDNA is typically synthesized in the laboratory by reverse transcription of messenger RNA extracted from cells. In the context of preparing an adenoviral vector comprising a polynucleotide sequence encoding an influenza antigen, a cDNA can be prepared, for example, by reverse transcription or amplification (e.g., by polymerase chain reaction, PCR) of a negative strand influenza RNA genome (or genomic fragment).

Host cell: a cell in which a polynucleotide, e.g., a polynucleotide vector or viral vector, can be amplified and its DNA expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to their parent cell, since mutations may occur during replication. However, when the term "host cell" is used, such progeny are included. Thus, the adenoviral vectors described herein can be introduced into host cells in which their polynucleotide sequences (including sequences encoding influenza antigens) can be expressed, e.g., to produce recombinant adenoviruses and/or influenza antigens.

Immune response: the response of immune system cells, such as B cells, T cells or monocytes, to stimulation. In some cases, the response is specific for a particular antigen (i.e., an "antigen-specific response"). In some cases, the immune response is a T cell response, e.g., CD4⁺Reaction or CD8⁺And (4) reacting. Alternatively, the reaction is a B cell reaction and results in the production of specific antibodies. A "protective immune response" is an immune response that inhibits the deleterious function or activity of a pathogenic influenza virus, reduces infection by a pathogenic influenza virus, or reduces symptoms (including death) resulting from infection by a pathogenic organism. Protective immune responses can be measured, for example, by inhibiting viral replication or plaque formation in a plaque reduction assay or ELISA-neutralization assay (NELISA), or by measuring resistance to viral challenge in vivo. Cell-mediated immune responses can be determined by various immunoassays, such as ELISpot, tetramer labeling, cytotoxicity assays.

Immunogenic composition: a composition comprising at least one influenza virus (or other pathogenic organism) epitope capable of inducing a detectable CTL response, or inducing a detectable B cell response (e.g., production of antibodies that specifically bind the epitope). Still further refers to an isolated nucleic acid encoding an influenza virus (or other pathogen) immune epitope that can be used to express the epitope (and thus can be used to elicit an immune response against such a polypeptide or a related polypeptide expressed by the pathogen). For in vitro use, the immunogenic composition may consist of an isolated nucleic acid, protein or peptide. For in vivo use, the immunogenic composition typically includes a nucleic acid or virus capable of expressing the immune epitope in a pharmaceutically acceptable carrier or excipient, and/or other agents, e.g., adjuvants. The ability of an immunizing polypeptide (e.g., an influenza antigen), or a nucleic acid encoding such a polypeptide, to induce a CTL or antibody response can be conveniently determined by assays well known in the art.

Internal protein: the internal proteins of influenza include all structural and non-structural proteins encoded by the influenza genome, except for the two surface antigens Hemagglutinin (HA) and Neuraminidase (NA). The internal proteins of influenza a are encoded by six genomic RNA segments and include three polymerase components designated PB1, PB2, and PA; nucleocapsid Protein (NP); matrix protein (M1); membrane channel protein (M2); and two non-structural proteins: NS1 and NS 2.

Separating: an "isolated" biological component (e.g., a nucleic acid or protein or organelle) has been substantially separated or purified from other biological components, e.g., other chromosomal or extra-chromosomal DNA and RNA, proteins, and organelles, in the cells of the organism in which the component naturally occurs. Nucleic acids and proteins that are "isolated" include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids and proteins produced by recombinant expression in a host cell and chemically synthesized nucleic acids.

The operable connection is as follows: a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first and second nucleic acid sequences are placed in functional association. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence.

A polynucleotide: the term polynucleotide or nucleic acid sequence refers to a polymeric form of nucleotides of at least 10 bases in length. Recombinant polynucleotides include polynucleotides that are not immediately associated with two coding sequences to which they are immediately associated (one at the 5 'end and the other at the 3' end) in the naturally occurring genome of the organism from which they are derived. The term thus includes recombinant DNA, e.g., incorporated into a vector; integration into an autonomously replicating plasmid or virus; or integrated into the genomic DNA of a prokaryote or eukaryote, or it exists as an isolated molecule (e.g., cDNA) independent of other sequences. The nucleotides may be ribonucleotides, deoxyribonucleotides, or a modified form of either nucleotide. The term also includes single-and double-stranded forms of DNA or RNA.

Polypeptide: any chain of amino acids, regardless of length or post-translationally modified (e.g., glycosylated or phosphorylated), such as a protein or a fragment or subsequence of a protein. The term "peptide" is generally used to refer to a chain of amino acids that is 3-20 amino acids in length. For example, the immune-related peptide can be about 7 to about 25 amino acids in length, e.g., about 8 to about 10 amino acids in length.

Prevention or treatment of diseases: inhibiting infection by influenza virus refers to inhibiting the complete development of disease due to exposure to pathogenic influenza virus. For example, inhibiting influenza infection refers to alleviating the symptoms of an infection caused by the virus, e.g., preventing the development of symptoms in a human known to have been exposed to the virus, or reducing the number of viruses or the infectivity of the virus in a subject exposed to the virus. "treatment" refers to a therapeutic or prophylactic intervention to alleviate or prevent the signs or symptoms of a disease or pathological symptoms associated with viral infection in a subject.

A promoter: a promoter is an arrangement of nucleic acid control sequences that direct the transcription of a nucleic acid. Promoters include the essential nucleic acid sequences in the vicinity of the transcription start site, for example in the case of polymerase II type promoters (TATA elements). Promoters also optionally include distal enhancer or repressor elements that are up to several thousand base pairs from the transcription start site. Both constitutive and inducible promoters are included (see, e.g., Bitter et al, Methods in Enzymology 153: 516-544, 1987).

Specific non-limiting examples of promoters that can be used include promoters derived from the genome of a mammalian cell (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the cytomegalovirus immediate early gene promoter, the retroviral long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter). Promoters prepared by recombinant DNA or synthetic techniques may also be used. Polynucleotides may be inserted into expression vectors containing promoter sequences capable of facilitating efficient transcription of inserted genetic sequences of a host. The expression vector typically contains an origin of replication, a promoter, and specific nucleic acid sequences that allow the transformed cell to be phenotypically screened.

Purification of: the term "purified" (e.g., in the case of an adenoviral vector or a recombinant adenovirus) does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nucleic acid refers to a nucleic acid in which the nucleic acid is more enriched than the nucleic acid within the cell in its natural environment. Similarly, a purified peptide preparation is one in which the peptide or protein is more enriched than the peptide or protein in the cell in its natural environment. In one embodiment, the formulation is purified such that the particular ingredient constitutes at least 50% (e.g., without limitation, 70%, 80%, 90%, 95%, 98%, or 99%) of the total formulation weight or volume.

Recombinant: a recombinant nucleic acid is a nucleic acid that has a sequence that is not naturally occurring or that has been made by the artificial combination of two separate pieces of sequence, such as a polynucleotide encoding a fusion protein. Such artificial combinations are typically accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated nucleic acid fragments, for example, by genetic engineering techniques.

Object: living multicellular vertebrate organisms, the class of which includes human and veterinary subjects, including human and non-human mammals and birds.

T cell: white blood cells that are critical for immune response. T cells include, but are not limited to, CD4⁺Cells and CD8⁺A cell. CD4⁺T lymphocytes are immune cells that carry a marker on their surface called CD 4. These cells, also known as helper T cells, help to coordinate immune responses, including antibody responses as well as killer T cell responses. CD8⁺T cells carry the CD8 marker and include T cells that function as cytotoxic or "killer" effectors.

Transduction or transfection: transduced cells are cells into which nucleic acid molecules have been introduced by molecular biology techniques. As used herein, the term introduction or transduction includes all techniques by which nucleic acid molecules can be introduced into such cells, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

Vaccine: a vaccine is a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Typically, vaccines elicit antigen-specific immune responses against pathogen antigens. In the context of the present disclosure, the vaccine elicits an immune response against avian (or pandemic) influenza. The vaccines described herein include adenoviral vectors or recombinant adenoviruses.

Carrier: introducing into a host cell, thereby producing a nucleic acid molecule that transforms the host cell. The vector may include nucleic acid sequences, such as an origin of replication, which permit its replication in the host cell. The vector may also include one or more selectable marker genes and other genetic elements known in the art. The term vector includes plasmids, linear nucleic acid molecules, and adenoviral vectors and adenoviruses as described throughout. The term adenoviral vector as used herein refers to a nucleic acid comprising one or more adenoviral components that replicate in a host cell to produce (e.g., infectious) viral particles. Adenoviruses include nucleic acids that encode at least a portion of the assembled virus. Thus, in many cases, the terms are used interchangeably. Thus, as used herein, the terms are intended to be used specifically to facilitate understanding and are not intended to limit embodiments in any way.

Influenza virus

Influenza viruses have a fragmented single-stranded (negative-sense or antisense) genome. Influenza virions consist of an inner ribonucleoprotein core containing a single-stranded RNA genome and an outer lipoprotein envelope linked by a matrix protein. The fragmented genome of influenza a consists of 8 linear RNA molecules encoding 10 polypeptides. Two polypeptides, HA and NA, comprise the major antigenic determinants or epitopes necessary for a protective immune response against influenza. Influenza strains can be classified into subtypes based on antigenic characteristics of the HA and NA proteins. For example, avian influenza, which HAs recently exploded in asia, can be classified into H5N1, H7N7, and H9N2, according to its HA and NA phenotype. These subtypes have all been shown to be highly infectious in birds and to be able to skip species barriers to directly infect humans, causing severe morbidity and mortality.

HA is a surface glycoprotein that protrudes from the lipoprotein envelope and mediates adsorption and invasion into cells. The HA protein is about 566 amino acids in length and is encoded by a polynucleotide sequence of about 1780 bases in genomic fragment 4. Polynucleotides and amino acid sequences of HA (and other influenza antigens) isolated from recent and historical strains of avian influenza can be found, for example, in GENEBANKDatabases (available from the world wide web ncbi, nlm, nih, gov/entrez). For example, the recent avian H5 subtype HA sequence includes: AY075033, AY075030, AY818135, AF046097, AF046096 andAF 046088; the recent HA sequence of H7 subtype includes: AJ704813, AJ704812 and Z47199; and the recent avian H9 subtype HA sequence includes: AY862606, AY743216 and AY 664675. It will be appreciated by those skilled in the art that essentially any of the foregoing or newly discovered avian HA antigens can be used in the compositions and methods described herein. Typically, the appropriate HA sequence(s) are selected based on circulating or predicted avian and/or pandemic HA subtypes, e.g., as recommended by the world health organization.

In addition to the HA antigen, which is the primary target of neutralizing antibodies against influenza, the Neuraminidase (NA) envelope glycoprotein is also the target of a protective immune response against influenza. NA is a protein of about 450 amino acids encoded by about 1410 nucleotide sequences of influenza genome segment 6. The most recent pathogenic avian influenza strains belong to subtypes N1, N7 and N2. Exemplary NA polynucleotide and amino acid sequences include, for example, N1: AY651442, AY651447 and AY 651483; n7: AY340077, AY340078 and AY 340079; and N2: AY664713, AF508892, and AF 508588. Additional NA antigens may be selected from previously described or newly discovered NA antigens based on circulating and/or predicted avian and/or pandemic NA subtypes.

The remaining influenza genome segment encodes an internal protein. Although immunization with internal proteins alone does not produce a significant protective neutralizing antibody response, the T cell response to one or more internal proteins can be significantly beneficial in protecting against influenza infection. The internal proteins are more highly conserved between strains, and between subtypes, than are the polymorphic HA and NA antigens. Thus, T cell receptors caused by exposure to internal proteins of avian or human influenza subtypes can also bind to other comparable internal proteins of avian and human subtypes.

PB2 is a 759 amino acid polypeptide, which is one of the three proteins that make up the RNA-dependent RNA polymerase complex. PB2 is encoded by about 2340 nucleotides of influenza genome segment 1. Of the remaining two polymerase proteins, the PB1, 757 amino acid polypeptide, and the PA, 716 amino acid polypeptide, were encoded by 2341 and 2233 nucleotide sequences (fragments 2 and 3), respectively.

Fragment 5 consists of 1565 nucleotides, which encodes a 498 amino acid Nucleoprotein (NP) protein that forms the nucleocapsid. Fragment 7 consists of a 1027 nucleotide sequence encoding 252 amino acids of M1 protein, and 96 amino acids of M2 protein, which is translated from a splice variant of M RNA. Fragment 8 consists of an 890 nucleotide sequence encoding two non-structural proteins, NS1 and NS 2.

Of these proteins, the M (M1 and M2) and NP proteins are most likely to elicit protective humoral and/or cellular T cell responses. Thus, although any internal proteins (e.g., in addition to one or more avian HA and/or NA antigens) may be included in the compositions and methods described herein, adenoviral vectors and adenoviruses may also typically include one or more M1, M2, and/or NP proteins. When the reaction to an internal protein of one strain tends to interact with internal proteins of other influenza strains, the internal protein can be selected from essentially any avian and/or human strain. For example, the internal protein may be selected from avian H5N1, H7N7 and/or H9N2 strains. Alternatively, the internal protein may be selected from human H3N2, H1N1, and/or H2N 2. Exemplary internal protein polynucleotides and amino acid sequences can be found, for example, in GENEBANIs found in (1). For example, H3N2M and NP nucleic acids and proteins are represented by accession numbers AF255370 and CY000756, respectively. Influenza internal proteins are more conserved between strains and tend to elicit cross-reactive T cell responses that are beneficial for protective immune responses against influenza.

It will be understood by those skilled in the art that the particular nucleic acids and proteins identified by the above accession numbers are non-limiting examples and that numerous other influenza antigen sequences will be expressed in the context of the adenoviral vectors disclosed herein. For example, other influenza antigens, and nucleic acids encoding these antigens, are substantially similar in primary structure to the antigens and nucleic acids referenced above. Similarity between amino acid (and polynucleotide) sequences may be expressed in terms of similarity between sequences, or may be referred to as sequence identity. Sequence identity is typically determined as a percentage of identity (or similarity); the higher the percentage, the more similar the primary structure of the two sequences. In general, the more similar the primary structures of two amino acid sequences, the more similar the higher order structures after folding and assembly. Thus, for example, HA antigens of the same influenza subtype typically share a high degree of sequence identity. Variants of the HA antigen (from a particular subtype) may have one or a few amino acid deletions, insertions or substitutions, but still share a high percentage of their amino acid (and typically their polynucleotide sequence) sequence. To the extent that the variants of the subtype differ from each other, their overall antigenic character remains. In contrast, HA antigens of different subtypes share less sequence identity (e.g., in the receptor binding pocket) and/or differ from each other such that their antigenic characteristics are no longer identical.

Methods for determining sequence identity are well known in the art. Various programs and alignment algorithms are described in Smith and Waterman, adv.appl.math.2: 482, 1981; needlemanand Wunsch, J.mol.biol.48: 443, 1970; higgins and Sharp, Gene 73: 237, 1988; higgins and Sharp, cabaos 5: 151, 1989; corpet et al, nucleic acids Research 16: 10881, 1988; and Pearson and Lipman, proc.natl.acad.sci.usa 85: 2444, 1988. Altschul et al, Nature Genet.6: 119, 1994, provides detailed consideration of sequence alignment methods and homology calculations.

NCBIbasic Local Alignment search tool (BLAST) is available from several sources (Altschul et al, J.mol.biol.215: 403, 1990), including the national center for Biotechnology information (NCBI, Bethesda, Md.) and the Internet, for use with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. A description of how to determine sequence identity using this program is available on the Internet at the NCBI's website.

Another indication of sequence similarity between two nucleic acids is hybridization ability. The more similar two nucleic acid sequences are, the more stringent the conditions under which they can hybridize. The stringency of the hybridization conditions is sequence dependent and varies under different environmental parameters. Thus, hybridization conditions that result in a particular degree of stringency will vary depending on the nature of the hybridization method chosen, as well as the composition and length of the hybridizing nucleic acid sequences. In general, the hybridization temperature and the ionic strength of the hybridization buffer (in particular Na)⁺And/or Mg⁺⁺Concentration) will determine the stringency of hybridization, although wash times will also affect stringency. Generally, stringent conditions will be selected to be higher than the thermal melting temperature (T) of a particular sequence under defined ionic strength and pH conditions_m) About 5 ℃ to 20 ℃. T is_mIs the temperature (under defined ionic strength and temperature) at which the target sequence hybridizes 50% to a perfectly matched probe. Conditions for nucleic acid hybridization and calculation of stringency conditions can be found, for example, in Sambrook et al, Molecular Cloning: ALABORT Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; tijssen, Hybridization With Nucleic Acid Probes, Part I: the Laboratory technologies in biochemistry and Molecular Biology, Elsevier Science Ltd, NY, NY, 1993, and Ausubel et al, Short Protocols in Molecular Biology, 4 th edition, John Wiley& Sons，Inc.，1999。

For the purposes of this disclosure, "stringent conditions" include conditions under which hybridization will occur only if the mismatch between the hybridizing molecule and the target sequence is less than 25%. "stringent conditions" can be broken down into specific stringency levels for more precise definition. Thus, as used herein, a "moderately stringent" condition is one under which molecules with sequence mismatches of more than 25% cannot hybridize; "moderately stringent" conditions are conditions under which molecules with more than 15% sequence mismatches do not hybridize, whereas "highly stringent" conditions are conditions under which sequences with more than 10% sequence mismatches do not hybridize. "very highly stringent" conditions are those conditions under which sequences that are more than 6% mismatched will not hybridize. In contrast, nucleic acid molecules that hybridize under "low stringency" conditions include those molecules that have much lower sequence identity, or that have sequence identity only in short subsequences of the nucleic acid.

Thus, the adenoviral vectors disclosed herein can include and/or express any of a number of influenza antigens, such as variants of hemagglutinin antigens of the H5, H7, and/or H9 subtypes (or other antigens discussed herein) that are similar in sequence, as determined by the above-described sequence similarity or hybridization assays.

Adenoviral vectors

The term "adenovirus" as used herein is intended to include all adenoviruses, including mammalian adenoviruses and avian adenoviruses. To date, at least 47 human-serum-type adenoviruses have been identified (see, e.g., FIELDS et al, VIROLOGY, Vol.2, Chapter 67 (third edition)., Lippincont-Raven Publishers). Adenoviruses are linear double-stranded DNA viruses of approximately 36kb in size. The genome includes Inverted Sequences (ITRs), encapsidated sequences, early genes and late genes at each end. The major early genes are contained in the E1, E2, E3 and E4 regions. Among them, genes contained in the region of E1 (particularly E1a and E1b) are essential for viral replication. The E4 and L5 regions, for example, are involved in viral transmission, and the major late genes are contained in the L1-L5 regions. For example, the human Ad5 adenovirus genome has been completely sequenced and its sequences are available on the world Wide Web (see, e.g., GENEBANKRegistration number M73260). Similarly, the human and non-human adenovirus genomes of some or in some cases all of the different serotypes (Ad2, Ad3, Ad7, Ad12, etc.) have also been sequenced.

Once a subject (or host) is infected with a recombinant adenovirus, or a recombinant adenovirus vector is introduced, the exogenous nucleic acid contained in the adenovirus genome can be transcribed by the host cell RNA polymerase and translation system (machinery), as well as translated. Thus, polynucleotide sequences encoding one or more influenza antigens can be integrated into an adenoviral vector and introduced into the cells of a subject, where the polynucleotide sequences are transcribed and translated, thereby producing influenza antigens. Recently, non-replicating adenoviral vectors have also been used to prepare and deliver immunologically effective HA1 strain vaccines (VanKampen et al, Vaccine 23: 1029-1036, 2005). In the context of the compositions and methods described herein, polynucleotide sequences encoding at least one avian influenza antigen (as described above, e.g., hemagglutinin antigens of subtypes H5, H7, or H9) can be incorporated into adenoviral vectors using established molecular biology methods. Nucleic acids encoding influenza antigens (e.g., cDNA) can be manipulated using standard procedures, such as restriction enzyme digestion, DNA polymerase filling, deletion by exonuclease, extension by terminal deoxynucleotidyl transferase, synthetic or cloned DNA sequence ligation, site-directed sequence alteration by single-stranded phage, or using specific oligonucleotides in conjunction with PCR or other in vitro amplification techniques.

Exemplary methods sufficient to direct those skilled in the art throughout the production of recombinant adenoviral vectors comprising polynucleotide sequences encoding one (or more) influenza antigen can be found in Sambrook et al, Molecular Cloning: a Laboratory Manual, 2 nd edition, Spring Harbor Laboratory Press, 1989; sambrook et al, Molecular Cloning: a Laboratory Manual, 3 rd edition, Spring Harbor Laboratory Press, 2001; ausubel et al, Current Protocols in Molecular Biology, Greene publishing associates, 1992(and Supplements to 2003); and Ausubel et al, short protocols in Molecular Biology: a Complex of Methods from Current protocols in Molecular Biology, 4 th edition, Wiley & Sons, 1999.

Typically, the polynucleotide sequence encoding the influenza antigen is operably linked to transcriptional control sequences, including, for example, a promoter and a polyadenylation signal. A promoter is a polynucleotide sequence recognized by the transcription system (or introduced synthesis system) of a host cell, which is involved in the initiation of transcription. Polyadenylation signals are polynucleotide sequences that direct the addition of a series of nucleotides to the ends of an mRNA transcript, which is appropriately processed and transported out of the nucleus into the cytoplasm for translation.

Exemplary promoters known in the art include viral promoters, including cytomegalovirus immediate early gene promoter ("CMV"), herpes simplex virus thymidylate kinase ("tk"), SV40 early transcription unit, polyoma virus, retrovirus, papilloma virus, hepatitis b virus, and human and simian immunodeficiency virus promoters. Other promoters isolated from mammalian genes include immunoglobulin heavy chain, immunoglobulin light chain, T-cell receptor, HLA DQ alpha and DQ beta, interferon beta, interleukin-2 receptor, MHC class II, HLA-DR alpha, beta-actin, muscle creatine kinase, prealbumin (transthyretin), elastase I, metallothionein, collagenase, albumin, fetoprotein, beta-globulin, c-fos, c-HA-ras, insulin, Neuronal Cell Adhesion Molecule (NCAM), alpha 1-antitrypsin, H2B (TH2B) histidine, type I collagen, glucose regulatory proteins (GRP94 and GRP78), rat growth hormone, human Serum Amyloid A (SAA), troponin I (TNI), platelet derived growth factor, and dystrophin, dendritic cell specific promoters such as CD11c, macrophage specific promoters such as CD68, langerhans cell specific promoters such as Langerin, and promoters specific for keratinocytes, epithelial cells of the skin and lung.

Promoters may be inducible or constitutive. Inducible promoters are promoters that are inactive or exhibit low activity unless an inducer is present. Some examples of promoters that may be included as part of the present invention include, but are not limited to, MT II, MMTV, collagenase, stromelysin, SV40, murine MX gene, alpha-2-macroglobulin, MHC class I gene h-2kb, HSP70, doramectin, tumor necrosis factor, or thyroid stimulating hormone gene promoters. Typically, however, the promoter is a constitutive promoter that can result in high levels of transcription upon introduction into a host cell in the absence of other factors. Optionally, the transcriptional control sequence may include one or more enhancer elements that are binding recognition sites for one or more transcription factors that are capable of increasing transcription beyond that observed using the basal promoter alone.

In general, it is also desirable to include a polyadenylation signal so that proper termination and polyadenylation of the gene transcript can be affected. Exemplary polyadenylation signals have been isolated from bovine growth hormone, SV40, and herpes simplex virus adenylate kinase genes. Any of these or other polyadenylation signals may also be used in the context of the adenoviral vectors described herein.

In general, the polynucleotide sequence encoding the influenza antigen can be a full-length open reading frame that includes a translation initiation site. However, it is equally possible to use polynucleotide sequences encoding immunogenic portions (sub-portions) of the antigen. If the polynucleotide sequence lacks a translation initiation site or codon, it may be introduced at an appropriate point prior to the polynucleotide sequence encoding the antigenic sub-portion when the vector is prepared.

Nucleic acid vectors encoding adenoviruses are well known in the art and can be used in gene therapy and vaccine applications. Exemplary adenoviral vectors are described in Berkner, BioTechniques 6: 616-629, 1988; graham, Trend Biotechnol, 8: 85-87, 1990; graham & Prevec, Vaccines: new apuraches to immunological schemes, Ellis (ed.), pp.363-90, Butterworth-Heinemann, Woburn, 1992; mittal et al, Recombinant and Synthetic Vaccines, Talwar et al, (eds) pp.362-366, Springer Verlag, New York, 1994; rasmussen et al, hum. gene ther.16: 2587, 2599, 1999; hitt & Graham, adv. virus res.55: 479 and 505,2000, published U.S. patent application 2002/0192185, which is incorporated herein by reference in its entirety.

In general, vectors can be modified to be replication-deficient, i.e., incapable of autonomous replication in a host cell, and adenoviral vectors and viruses that are conditionally replication-effective and replication-effective can be used in addition to such helper-dependent adenoviral vectors. Typically, the genome of a replication-defective virus lacks at least some of the sequences necessary for the virus to replicate in an infected cell. These regions may be removed (in whole or in part), or rendered non-functional, or replaced by other sequences, and in particular by sequences encoding molecules of therapeutic interest. Typically, the defective virus retains sequences involved in encapsidation of the viral particle.

Replication-defective adenoviruses typically contain mutations, e.g., deletions, in one or more of the E1(E1a and/or E1b), E3 region, E2 region, and/or E4 region. In addition to the ITRs and packaging elements, the entire adenoviral genome can also be deleted and the resulting adenoviral vector is referred to as a helper-dependent or "inactive" vector. In some cases, a heterologous DNA sequence may be inserted at the position of the deleted adenovirus sequence (Levrero et al, Gene 101: 195-202, 1991; Ghosh-Choudhury et al, Gene 50: 161-171, 1986). Other constructs contain deletions of non-essential parts in the E1 region and E4 region (WO 94/12649). Exemplary adenoviral vectors can be found in U.S. Pat. Nos. 6,328,958, 6,669,942, and 6,420,170, which are incorporated herein by reference in their entirety.

These replication-defective recombinant adenoviruses can be prepared in different ways, for example, in competent cell lines capable of complementing all the defective functions necessary for replication of the recombinant adenovirus. For example, the adenoviral vector can be prepared in a complementing cell line (e.g., 293 cells) that incorporates a portion of the adenoviral genome. Such cell lines contain the left-hand end (about 11-12%) of the adenovirus serotype 5(Ad5) genome, including the left-hand ITR, encapsidation region, and the E1 region, the E1 region including E1a, E1b, and a partial region encoding the pIX protein. This line is a recombinant adenovirus that is reverse-complementary to the E1 region deficiency. Generally, E1 supplementation requires expression of E1A and E1B proteins.

Human adenovirus vectors are commonly used to introduce foreign nucleic acids into human and animal cells and organisms. Adenoviruses exhibit a wide host cell range, and adenoviruses can be used to infect humans as well as non-human animals, including birds. Most commonly, the human adenoviral vector is the HAd5 vector derived from adenovirus serotype 5 virus. Due to the large genome size of the intact adenovirus, the insertion of heterologous polynucleotide sequences is most convenient using shuttle plasmids. Sequences such as those of influenza antigens can be cloned into shuttle vectors which will subsequently undergo homologous recombination with all or part of the adenovirus genome in cultured cells. Furthermore, homologous recombination can also be performed in bacteria to produce full-length adenoviral vectors.

In some cases, it is desirable to use non-human adenoviral vectors to avoid pre-existing host immunity to human adenovirus. Infection with human adenovirus is common in the human population, with many or most individuals having circulating antibody titers that will bind and neutralize recombinant human adenovirus. Thus, neutralization of the vector containing the influenza sequence does not result in an effective immune response against influenza in at least a portion of a human population vaccinated with the human adenovirus vector. To avoid this problem, non-human adenovirus vectors can be used to circumvent any pre-existing immunity to human adenovirus.

Adenoviruses of animal origin are also capable of efficiently infecting human, as well as non-human cells, whereas subsequent infection is generally not transmissible in human cells (see, international application WO 94/26914). Thus, adenoviruses of animal origin may be used in the vector and virus categories described herein. The use of animal adenovirus vectors developed for human and animal vaccines is described in Bangari & Mittal, Vaccine 24: 849-862, 2006, which are hereby incorporated by reference. For example, the animal adenoviral vector can be selected from the group consisting of canine, bovine, murine (e.g., MAV1, et al, Virology 75: 81, 1990), ovine, porcine, avian (e.g., chicken) or alternatively simian (e.g., SAV) adenovirus. For example, bovine and porcine adenoviruses can be used to prepare adenoviral vectors expressing influenza antigens including the various bovine serotypes obtained from ATCC (types 1-8) under references ATCC VR-313, 314, 639-642, 768, and 769, as well as porcine adenovirus 5359. In addition, simian adenoviruses of various serotypes may be used, including SAd25, SAd22, SAd23 and SAd24, such as those identified in ATCC by reference numerals VR-591-594, 941-943, 195-203, etc., several avian adenovirus serotypes (1-10) available at the ATCC, such as the strains Phelps (ATCC VR-432), Fontes (ATCC VR-280), P7-A (ATCC VR-827), IBH-2A (ATCC VR-828), J2-A (ATCC VR-829), T8-A (ATCC VR-830), K-11(ATCC VR-921) and the strains referenced ATCC VR-831-835, and murine adenoviruses FL (ATCC VR-550) and E20308(ATCC VR-528), and ovine adenoviruses type 5(ATCC VR-1343) or type 6(ATCC VR-1340).

For example, bovine and porcine adenovirus vectors are capable of infecting human cells and can be used as vectors to express avian influenza antigens. Exemplary bovine and porcine adenovirus vectors are described in published U.S. patent application 2002/0192185, and U.S. patents 6,492,343 and 6,451,319, which are incorporated herein by reference.

The compositions and methods described herein can be applied to any influenza antigen. In particular, the compositions and methods are useful for expressing and generating immune responses against avian influenza strains. For example, the adenoviral vectors, recombinant adenoviruses, and immunological compositions disclosed herein can include HA antigens of any avian or pandemic influenza strain. Numerous avian HA antigens have been identified and their sequences can be obtained, for example, by using the publicly available NCBI database (world wide web NCBI. An exemplary HA antigen from a recent avian influenza outbreak may be represented by AY818135(a/Viet Nam/1203/04); AF084280 (A/HongKong/483/97); AF036356(A/Hong Kong/156/97); AY575870 (A/HongKong/213/03). Nucleic acids comprising these sequences may be obtained by cloning and/or amplifying viral isolates, or may be prepared synthetically. Similarly, a novel HA antigen isolated from a emerging strain or a novel isolate may also be included in the compositions described herein. Similarly, known NA antigens of avian or newly discovered avian strains can be incorporated into the vectors, viruses, and compositions described herein. Optionally, the virus, vector and/or immunogenic composition may include one or more internal proteins of avian or non-avian, e.g. human, influenza strains.

Recombinant adenoviruses expressing avian and/or other influenza antigens can be prepared from such vectors following introduction of the adenoviral vector into an appropriate host cell. In the case of replication-defective vectors, the adenoviral vector can generally be introduced into a cell line that complements the defective function. For example, an E1-deficient virus can be grown in a cell line that supplements the function of E1 due to the expression of an introduced nucleic acid encoding the adenoviral E1 protein. Exemplary cell lines include human and non-human cell lines engineered to express adenovirus E1 (e.g., E1A) protein. For example, 293 cells expressing the adenovirus E1 protein are commonly used to grow recombinant replication-defective adenoviruses with deletions of the E region. Other suitable cell lines include MDBK-221, FBK-34, and embryonic kidney cells of various origins. Specific examples of cell lines suitable for growing recombinant porcine and bovine adenoviruses include FPRT-HE1-5 cells (Bangari & Mittal, Virus Res.105: 127-118, 2004) and FBRT-HE1 cells (van Olphen et al, Virology, 295: 108-118, 2002), respectively. In certain embodiments, the cell expresses the adenovirus E1 gene of more than one strain, e.g., 2 or more strains with different tropisms. For example, the cells can express human and non-human E1 genes (e.g., porcine and/or bovine E1 genes). One skilled in the art can readily select or prepare appropriate additional or alternative cell lines that complement the replication function of the replication-defective adenovirus vector. For example, any of the various mammalian cell lines disclosed herein (or known in the art) can be transfected with the E1 and/or E3 genes of any adenoviral strain, such as the exemplary strains disclosed herein, depending on the particular adenoviral vector to be grown. For example, E1 (and/or E3) corresponding to (i.e., derived from the same or a functionally similar viral strain) the same strain may typically be selected as the adenoviral vector. It will be appreciated by those skilled in the art that functionally similar variants of any of the exemplary adenovirus genes (e.g., variants that share substantial sequence identity, or, for example, variants that hybridize specifically under highly stringent conditions) can be used to prepare cell lines that support the growth of an adenovirus vector encoding an influenza antigen.

In Ng et al, hum. gene ther.10: 2667-2672, 1999 and hum. GeneTher.11: 693-699, 2000, which is incorporated herein by reference in its entirety, describes a common method for preparing replication-defective adenovirus vectors incorporating exogenous nucleic acids. Briefly, to prepare a human adenovirus vector containing an influenza antigen, a polynucleotide sequence encoding an influenza antigen (e.g., one or more avian HA antigens) operably linked to a strong promoter (e.g., CMV immediate early promoter) can be inserted into a shuttle vector, such as pDC 311. The pDC311 shuttle vector is a plasmid containing the 3.1kb E1 deleted HAd5 (about 4kb) left terminus, loxP sites suitable for site-specific recombination in the presence of Cre recombinase, and an intact packaging signal (ψ). The shuttle vector may be co-transfected into an appropriate cell expressing Cre recombinase (e.g., 293Cre cells) with a plasmid comprising a replication-deficient HAd5 genome lacking the packaging signal (e.g., containing a deletion in the E1 and/or E3 region genes) and containing loxP sites. Homologous Cre-mediated recombination results in the generation of an adenovirus vector plasmid encoding a replication-defective adenovirus expressing the inserted influenza antigen.

Cells expressing a gene complementing replication function (e.g., E1 when the replication-defective adenoviral vector lacks E1 function) can be transfected with a recombinant adenoviral vector or with a low infectivity (e.g., between 1-1000 p.f.u./cell) pair of adenoviruses according to standard procedures, e.g., electroporation, calcium phosphate precipitation, lipofection, and the like. In some cases, a monolayer of cells confluent in a 60mm culture dish, for example, may be used. The cells are then cultured (grown) for a period of time sufficient for expression and replication of the adenovirus, and prior to harvesting of the recombinant adenovirus, the cells are divided to maintain viable growth and maximize virus recovery. Typically after several passages (e.g., 2-5 passages), recombinants are collected by lysing the cells to release the virus, and then concentrating the virus. Can be prepared by mixing the above virusesThe lysate of (a) is passed through a density gradient (e.g., CsCl density gradient) to concentrate the recombinant adenovirus. After concentration, a buffer (e.g., 10mM Tris pH 8.0, 2mM MgCl) is typically utilized₂5% sucrose) was dialyzed against the recombinant adenovirus, titrated and stored at-80 ℃ until use. Methods for preparing adenoviruses on a larger scale, e.g., suitable for preparing immunogenic compositions for use as vaccines, can be found, for example, in published U.S. patent application 20030008375, which is incorporated herein by reference.

Preparation of recombinant influenza virus antigens from adenovirus vectors

In addition to their use in preparing immunological compositions comprising adenoviral vector nucleic acids and/or adenoviruses capable of expressing avian influenza antigens, the adenoviral vectors disclosed herein can also be used in the preparation and production of recombinant influenza antigens, such as recombinant HA antigens from highly pathogenic avian influenza strains, as well as other influenza antigens. Methods for preparing recombinant antigens using human and non-human adenoviral vectors are well known in the art, and exemplary compositions and methods can be found, for example, in U.S. Pat. Nos. 5,824,770, 6,319,716 and 6,824,770, which are incorporated herein in their entirety. In addition, commercially available vectors, such as ADEASY from Stratagene (La Jolla, Calif.), may be used^TMAn adenovirus vector system for preparing adenovirus vectors and adenovirus recombinants (recombinant adenoviruses) capable of expressing recombinant influenza proteins.

As described above, the adenoviral vector comprises a heterologous polynucleotide sequence encoding one or more avian influenza virus antigens at a position in the E1 and/or E3 gene region of the adenoviral vector. Optionally, two, or even three or more influenza antigens can be encoded by the heterologous nucleic acid. In contrast, influenza antigen fragments containing immunogenic epitopes can be encoded by polynucleotide sequences inserted into adenoviral vectors. Typically, the polynucleotide sequence encoding the influenza antigen is operably linked to transcriptional regulatory sequences (e.g., promoter and/or enhancer elements, and/or polyadenylation sequences) that enable high levels of expression to be produced. Various eukaryotic promoters and polyadenylation sequences which provide for the successful expression of a foreign gene in mammalian cells, and how to construct expression cassettes, are well known in the art, and can be found, for example, in U.S. Pat. No. 5,151,267, the disclosure of which is incorporated herein by reference. The promoter may be selected to give optimal expression of the immunity protein which then satisfactorily leads to humoral, cell-mediated and mucosal immune responses as described by well-known criteria.

Optionally, the polynucleotide encoding the influenza antigen includes a portion encoding a peptide (e.g., an epitope) or polypeptide tag to facilitate subsequent purification of the recombinant antigen. Typically, the influenza antigen is expressed as a fusion protein, wherein the influenza antigen is linked to one or more peptide (or polypeptide) domains that facilitate expression and/or purification. A variety of suitable tags are known in the art, and expressed proteins comprising such tags can be conveniently isolated using commercially available reagents and kits. If desired, the tag may be removed from the antigen, for example by enzymatic or chemical cleavage, before the recombinant antigen is used in subsequent applications. Exemplary tags include Mys epitope tags, histidine tags, and GST tags.

Recombinant adenoviral vectors containing one or more influenza virus antigens can be expressed in cell lines that have been introduced with expression cassettes encoding the complementing E1 region (and/or E2 region). These recombinant cell lines are capable of allowing a recombinant adenovirus (having a deletion in the region of the E1 gene that is replaced by a heterologous nucleotide sequence encoding one or more influenza antigens or fragments thereof) to replicate and express a desired foreign gene or fragment encoded by the recombinant adenovirus. Optionally, such cell lines may include E1 (and/or E2) genes corresponding to more than one adenovirus strain. For example, suitable cell lines include those containing nucleic acids that express E1 of human adenovirus as well as E1 of, for example, porcine or bovine adenovirus strains.

For use in pharmaceutical compositions, recombinant influenza antigens are typically purified after expression in cultured cells. Methods for isolating recombinant proteins expressed In cultured cells are well known In the art, and specific methods can be found In Sambrook et al, (In Molecular Cloning: Arabidopsis Manual, CSHL, New York, 2001) and Brent et al, Current protocols In Molecular Biology, John Wiley and Sons, New York, 2003). It will be appreciated by those skilled in the art that numerous methods can be used to purify recombinant polypeptides, and that typical methods for purification of these proteins can be used to purify the expressed influenza antigen from an adenoviral vector. Such methods include, for example, protein chromatography including ion exchange, gel filtration, HPLC, monoclonal antibody affinity chromatography and purification of insoluble protein inclusions after preparation. In addition, purification can also be based on an attached tag (as described above), such as a 6-histidine sequence, which can be recombinantly fused to the protein and used to facilitate polypeptide purification using a nickel affinity column (e.g., (nickel-nitrilotriacetic acid (Ni-NTA)) metal affinity chromatography matrix (qiaxpressinst, QIAGEN, 1997).

If desired, the recombinant influenza antigen can be conjugated to a vaccine vector for administration to a subject. Vaccine vectors are well known in the art: for example, Bovine Serum Albumin (BSA), Human Serum Albumin (HSA) Keyhole Limpet Hemocyanin (KLH), and rotavirus VP 6. In some cases, one or more adjuvants, such as alum, can be used in combination with the recombinant influenza antigen for administration to a subject, as described below.

Immunogenic compositions comprising adenoviral vectors and recombinant adenoviruses

Recombinant adenovirus vectors and recombinant adenoviruses that express influenza antigens (e.g., avian influenza antigens) can be administered to cells or subjects in vitro, ex vivo, or in vivo. In general, it is desirable to prepare the vector and virus as a pharmaceutical composition suitable for the intended application. Accordingly, included herein are methods of preparing a medicament or pharmaceutical composition comprising an adenoviral vector or adenovirus as described above. In general, the preparation of pharmaceutical compositions (medicaments) requires the preparation of pharmaceutical compositions that are substantially free of pyrogens and any other impurities that could cause harm to humans or animals. Typically, the pharmaceutical composition contains appropriate salts and buffers to stabilize the components of the composition and allow uptake of the nucleic acid or virus by the target cell.

Pharmaceutical (e.g., immunogenic) compositions generally include an effective amount of adenoviral vector or virus dispersed (e.g., dissolved or suspended) in a pharmaceutically acceptable carrier or excipient. The phrases "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic, or other undesirable reaction when administered to a human or animal subject. A variety of pharmaceutically acceptable carriers and/or pharmaceutically acceptable excipients are well known in the art and can be found, for example, in Remington's Pharmaceutical Sciences, published by e.w. martin, Mack Publishing co., Easton, PA, 15 th edition (1975).

In general, the nature of the carrier will depend on the particular mode of administration to be employed. For example, parenteral formulations typically include injectable liquids comprising a drug and a physiologically acceptable liquid, such as water, saline, balanced salt solutions, aqueous dextrose, glycerol or the like as carriers. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical compositions to be administered may also include minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the immunological compositions is contemplated. Supplementary active ingredients may also be incorporated into the composition. For example, certain pharmaceutical compositions may include a carrier or virus in water, mixed with a suitable surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical compositions (medicaments) prepared can be used in prophylactic dosing regimens (e.g., vaccines) and administered to human or non-human subjects (including birds, such as poultry, e.g., chickens, ducks, guinea fowl, turkeys and geese) to elicit an immune response against an influenza antigen (or antigens). Thus, the pharmaceutical compositions typically contain an immunologically effective amount of an adenoviral vector or adenovirus (or indeed a recombinant influenza antigen produced by expression of an adenoviral vector as disclosed herein). An immunologically effective amount, e.g., a vaccine composition, is an amount sufficient to elicit a desired immune response, e.g., a protective immune response, in an immunocompetent subject when administered in one or more doses. Typically, one or more doses of an immunologically effective amount of the vaccine are administered to a subject prior to exposure (i.e., prophylactically) to an infectious agent (e.g., influenza virus), thereby eliciting a protective immune response against the infectious agent. In the present description, one or more doses of an adenovirus vector and/or adenovirus containing an avian influenza antigen (and/or a recombinant influenza antigen prepared from expression of such adenovirus) may be administered to a subject to elicit a protective response against avian and/or pandemic influenza.

In some cases, the composition may be administered after infection to enhance the immune response, and in such applications, the pharmaceutical composition may be administered in a therapeutically effective amount. A therapeutically effective amount is an amount used to achieve a desired effect in a subject. For example, it may be the amount of the composition that inhibits viral replication or prevents or detectably alters the external symptoms of viral infection. When administered to a subject, a commonly used dose is one that achieves a target tissue concentration (e.g., in lymphocytes) that is shown to achieve an in vitro or in vivo effect.

For example, the compositions described herein can be administered to a human (or non-human) subject to elicit an immune response against avian influenza viruses, such as pathogenic H5N1, H7N7, or H9N2 avian influenza strains. Generally, the adenoviral vectors and/or adenoviruses described herein elicit a protective or partially protective immune response against at least one avian (pandemic) influenza strain or subtype. That is, the pharmaceutical compositions described herein are generally capable of preventing influenza, or reducing the severity of symptoms (e.g., morbidity and/or mortality) in at least a substantial portion of a population administered the composition following subsequent exposure to a pathogenic virus strain. In some cases, the immune response is protective or partially protective against multiple virus strains, or even multiple subtypes of avian (or pandemic) influenza, including administration to both human and non-human subjects (including birds).

In general, protective immune responses against influenza all involve the production of neutralizing antibodies that bind to the HA antigen. Because these antigens are highly polymorphic, and antibodies raised against one subtype are generally not protective against another influenza subtype, immunogenic pharmaceutical compositions described herein may include adenoviral vectors or adenoviruses that express more than one HA antigen, e.g., via one or more vectors or viruses. As previously described, the HA antigen may be a single subtype or a variant of more than one HA subtype. Optionally, an adenovirus expressing the NA antigen (or a vector encoding the NA antigen) can also be administered.

T-cell responses to influenza antigens broaden the immune response to influenza, increasing vaccine efficacy. Internal proteins, such as M2 and NP proteins of influenza a possess suitable B-cell and/or T-cell epitopes, and when administered in combination with antigenic polypeptides such as HA and NA antigens, elicit T cell responses, enhance protection and help reduce morbidity due to influenza infection. Furthermore, such internal protein epitopes are highly conserved among influenza subtypes, providing cross-protection against a variety of avian and human influenza strains. Thus, a pharmaceutical composition comprising one or more adenoviral vectors or adenoviruses expressing an avian influenza internal protein can be used to elicit an influenza-specific immune response.

Therapeutic compositions comprising recombinant adenoviruses expressing influenza antigens, including avian influenza antigens, can be administered by any common means, as long as the target tissue (typically, the respiratory tract) is receptive to that route. This includes oral, intranasal, ocular, buccal, or other mucosal (e.g., rectal, vaginal) or topical administration. Alternatively, administration may be by the in situ, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous routes. Such immunogenic compositions are typically administered as a pharmaceutically acceptable composition comprising a physiologically acceptable carrier, buffer or other excipient.

The immunogenic composition may also be administered in the form of an injectable composition in liquid solution or suspension; solid forms suitable for solution or suspension in a liquid prior to injection can also be prepared. These formulations may also be emulsified. Typical compositions suitable for such purposes include a pharmaceutically acceptable carrier. For example, the composition may contain about 100mg of human serum albumin per ml of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients including salts, preservatives, buffers and the like which may be employed. Examples of water insoluble solvents are propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, ethanol/water solutions, saline solutions, parenteral media such as sodium chloride, Ringer's dextrose, and the like. Intravenous media includes fluids and nutritional supplements. Preservatives include antimicrobials, antioxidants, chelating agents and inert gases. The pH and exact concentration of the various ingredients in the pharmaceutical composition may be adjusted according to well known parameters.

There are other formulations suitable for oral administration. Oral formulations may include excipients such as, for example, pharmaceutically pure mannose, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. The compositions (medicaments) are generally in the form of solutions, suspensions, aerosols or powders. Exemplary formulations can be found in U.S. patent publication 20020031527, the disclosure of which is incorporated herein by reference in its entirety. When the route is topical, the form may be a cream, salve, ointment or spray. Exemplary methods for intramuscular, intranasal, and topical administration of adenoviral vectors and adenoviruses described herein can be found, for example, in USPN 6,716,823, which is incorporated herein by reference.

Optionally, the pharmaceutical composition or medicament may include a suitable adjuvant, thereby enhancing the immune response against the influenza antigen. As used herein, an "adjuvant" is any potentiator or enhancer of an immune response. The term "suitable" is meant to include any substance that can be used in combination with an adenoviral vector or adenovirus to enhance an immune response without producing an adverse reaction in the vaccinated subject. The effective amount of a particular adjuvant can be conveniently determined so as to optimise the enhancing effect of the adjuvant on the immune response of the vaccinated subject. For example, 0.5% -5% (e.g., 2%) aluminum hydroxide (or aluminum phosphate) and MF-59 oil emulsions (0.5% polysorbate 80 and 0.5% sorbitan trioleate. squalene (5.0%) aqueous emulsions) are very suitable adjuvants for influenza vaccines. Other adjuvants include emulsions of mineral, vegetable or fish oils with water, incomplete freund's adjuvant, escherichia coli (e.coli) J5, dextran sulfate, iron oxide, sodium alginate, Bacto-adjuvant, certain synthetic polymers, such as Carbopol (BF Goodrich Company, Cleveland, Ohio), polyamino acids and amino acid copolymers, saponins, carrageenan, REGRESSIN (Vetrepharm, adhens, Ga.), avidine (N, N-dioctadecyl-N ', N' -bis (2-hydroxyethyl) -propylenediamine), long chain polydispersity β (1, 4) linked mannan polymers (e.g., ACEMANNAN) distributed with O-acetylated groups, deproteinized highly purified cell wall extracts from nonpathogenic mycobacterial species (e.g., equimum, Vetrepharm research inc., adhens Ga.), mannitol monooleate, paraffin oil and muramyl dipeptide. Suitable adjuvants may be selected by those skilled in the art.

An effective amount of the immunological composition may be determined according to the intended target, e.g. vaccination of a human or non-human subject. Suitable dosages will vary depending upon the characteristics of the subject (e.g., whether the subject is human or non-human, age, weight, and other health considerations regarding the condition or state of the subject), the mode, route, number of doses administered, and whether the pharmaceutical composition comprises a nucleic acid or virus. Generally, the immunogenic compositions described herein can be administered for the purpose of eliciting an immune response against an influenza antigen (or antigens) or influenza virus. Thus, the dose is typically an immunologically effective amount of the recombinant adenovirus.

Typical doses of recombinant adenoviruses expressing influenza antigens may range from 10p.f.u. -10¹⁵p.f.u./administration. For example, the pharmaceutical composition can include about 100p.f.u. recombinant adenoviruses, e.g., about 1000p.f.u., about 10,000p.f.u., or about 100,000p.f.u., of each recombinant adenovirus in a single dose. Optionally, the pharmaceutical composition can include at least about one million p.f.u. or more per administration. For example, in some cases, it is desirable to administer about 10⁷，10⁸，10⁹Or 10¹⁰p.f.u. recombinant adenoviruses expressing specific influenza antigens. In some cases, for example, when a single adenovirus is administered, a higher dose of virus may be administered to a subject, for example, when a recombinant virus expressing multiple influenza antigens is administered. Alternatively, when several adenoviruses are administered, each expressing a different influenza antigen, less of each virus may be administered, although the total viral dose may still be as high as 10⁸Or higher than 10⁹Or 10¹⁰p.f.u.。

In addition, an adenovirus vector (nucleic acid vector) can be administered. For example, when a nucleic acid vaccine comprising an adenoviral vector encoding an influenza antigen is administered, facilitators of nucleic acid uptake and/or expression, such as bupivacaine, cardiotoxin and sucrose, and transfection facilitating media, such as liposomes or lipid formulations, which are routinely used for delivery of nucleic acid molecules, may also be included. Anionic and neutral Liposomes are widely available and well known for delivery of nucleic acid molecules (see, e.g., Liposomes: APracial Approach, RPC New Ed., IRL Press, 1990). Cationic lipid formulations are also well known for the delivery of nucleic acid moleculesA medium. Suitable lipid formulations include DOTMA (N- [1- (2, 3-dioleyloxy) propyl)]N, N, N-trimethylammonium chloride), under the name LIPOFECTINObtained, and DOTAP (1, 2-bis (oleyloxy) -3- (trimethylammonium) propane), see, e.g., Felgner et al, proc.natl.acad.sci.usa 84: 7413-7416, 1987; malone et al, proc.natl.acad.sci.usa 86: 6077-; us patents 5,283,185 and 5,527,928, and international applications WO 90/11092, WO 91/15501 and WO 95/26356. These cationic lipids can preferably be used in combination with neutral lipids, such as DOPE (dioleylphosphatidylethanolamine). Other transfection facilitating compositions that may be added to the lipid or liposome formulations described above include spermine derivatives (see, e.g., international publication WO 93/18759) and membrane permeant compounds such as GALA, gramicidin S and cationic bile salts (see, e.g., international publication WO 93/19768).

Alternatively, the nucleic acid encoding the avian influenza virus gene (adenoviral vector) can be encapsulated, adsorbed, or associated with a particulate carrier. Suitable particulate carriers include carriers derived from polymethylmethacrylate polymers, and PLG microparticles derived from poly (lactide) and poly (lactide-co-glycolide). See, e.g., Jeffery et al, pharm. res.10: 362-368, 1993. Other particle systems and polymers may also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules.

The formulated vaccine composition thus typically comprises an effective amount of an adenovirus-containing polynucleotide (e.g., a plasmid) that elicits an immune response, the adenovirus comprising a polynucleotide sequence that encodes an avian influenza antigen. An appropriate effective amount can be readily determined by one skilled in the art. Such amounts may fall within a relatively wide range that can be determined by routine experimentation. For example, as little as 1 μ g of DNA may be used to obtain an immune response, while in other administrations, as much as 2mg of DNA may be used. An effective dose of the polynucleotide containing the genomic fragment is generally expected to fall within the range of about 10 μ g to 1mg, however, above and below this range may also be effective.

Adenovirus vector nucleic acids can be coated onto vector particles (e.g., core vectors) using a variety of techniques well known in the art. The carrier particles are selected from materials having a density in the range of particle sizes normally used for intracellular delivery from a suitable particle-mediated delivery device. The optimum particle size of the vector will, of course, depend on the diameter of the target cell. Alternatively, colloidal gold particles can be used, wherein the coated colloidal gold can be administered (e.g., injected) into a tissue (e.g., skin or muscle) and subsequently taken up by immune competent cells.

Tungsten, gold, platinum and iridium support particles may be used. Tungsten and gold particles are preferred. Tungsten particles having an average particle diameter of 0.5 to 2.0 μm are easily obtained. While such particles have the optimum density for particle-accelerated delivery methods and allow for more efficient coating with DNA, tungsten may be potentially toxic to certain cell types. Gold particles or microcrystalline gold (e.g., gold powder a1570 available from Engelhard corp., East Newark, n.j.) have also been found to be useful in the present invention. Gold particles are provided with uniform size (1-3 μm particle size obtained from Alpha Chemicals or from Degussa, South Plainfield, N.J., particle size range including 0.95 μm) and reduced toxicity.

Various methods are known and have been described for coating or precipitating DNA or RNA onto gold or tungsten particles. Most such methods generally involve the addition of a predetermined amount of gold or tungsten to the plasmid DNA, CaCl₂And spermidine. The resulting solution was continuously vortexed during the coating process, thereby ensuring homogeneity of the reaction mixture. Following the nucleic acid precipitation, the coated particles may be transferred to a suitable membrane and allowed to dry prior to use, coated on the surface of a sample module or cassette, or loaded into a delivery cassette for a suitable particle delivery device such as a gene gun. Alternatively, the nucleic acid vaccine may be administered through the mucosa or through the skin, for example using a transdermal patch. Such patches may include moisteningAgents, chemicals and other components that are capable of disrupting the integrity of the skin, allowing the nucleic acid to enter the subject's cells.

In addition to adjuvants that normally potentiate or enhance the immune response in a non-specific manner, e.g., by concentration or slow diffusion of antigens, genetic immunostimulants can be included in the pharmaceutical composition with the adenoviral vector or adenovirus, thereby enhancing the immune response against avian influenza. For example, certain CpG oligonucleotides (e.g., 5'-tccatgagcttcctgatcct-3' (SEQ ID NO: 1); 5'-tccatgacgttcctgacgtt-3' (SEQ ID NO: 2) and 5'-tgactgtgaacgttcgagatga-3' (SEQ ID NO: 3)) have been shown to be potent immunostimulatory molecules when administered with an antigen. exemplary CpG oligonucleotides can be found in U.S. Pat. Nos. 6,610,661; 6,589,940 and 6,514,948, the disclosures of which are incorporated herein by reference for all purposes.

In addition, certain subjects with low TLR expression (e.g., elderly subjects) may benefit from co-expression of TLRs (e.g., TLR5, TLR7, and TLR9) in cells in which an adenoviral vector or adenovirus expressing the influenza antigen is expressed. Thus, the TLR-containing adenoviral vector particle is comprised in the above pharmaceutical composition. The TLR may be integrated into the same adenoviral vector encoding the influenza antigen, or may be included in the pharmaceutical composition (agent) as an isolated adenoviral vector or virus. Optionally, the TLR and its ligand (e.g., CpG oligonucleotide, flagellin) are included together in a pharmaceutical composition.

In certain embodiments, the medicament (e.g., vaccine composition) may be administered to an avian subject, such as poultry, including, but not limited to, chickens, ducks, turkeys and geese. The pharmaceutical composition is administered to the chick and/or the adult and/or the embryo in the egg. Methods for administering vaccine compositions in the poultry industry are well known in the art, and any such method is suitable for administration of an immunological composition, such as an adenovirus containing an avian influenza antigen as disclosed herein. For example, can be atInfluenza vaccine based adenoviruses are administered to poultry in the drinking water. Various methods are available for administering vaccines in poultry drinking water. In one convenient method, a solution containing the immunizing composition may be placed in an intravenous solution bag (e.g., SELECT fieldbab BOOST available from Merial, inc., Lyon, France)^TMSystem) which can be connected to one or more drinking water lines. Optionally, the solution contains a dye (or other visually perceptible indicator, such as skim milk powder) and a diluent to facilitate monitoring of administration of the vaccine. In the case of adenovirus-based vaccines, it is important that the solution be free of disinfectants, such as chlorine or other disinfectants that may be used to clean the delivery system. If desired, the avians may be stimulated to drink more aggressively, thereby increasing the consumption of the vaccine/water solution. For example, by increasing light intensity, delivering food, and/or disturbing birds (e.g., by walking through a flock), birds can be stimulated to increase vaccine-loaded drinking water consumption.

In another method, the vaccine composition may be administered by spraying. Spray applications are commonly available for many birds caged in a common space, for example, 1 day old birds in a shipping container, or for spray administration to conventional caged birds. For example, freshly hatched birds may be vaccinated using a spray delivery system, as described in U.S. Pat. Nos. 6,713,073, 4,674,490 and 4,449,968, the disclosures of which are incorporated herein by reference. In one exemplary method, a1 day-old flock of birds, loaded in a transport box with up to about 150 subjects, can be exposed to a vaccine diluted in an aqueous medium, such as water, delivered by a nozzle method, which forms very small droplets (e.g., in the range of about 100 μ to about 500 μ diameter). 1 day old birds can ingest the vaccine by ocular, intranasal, and oral routes from the surface of the container and other birds. The vaccine may be delivered to larger birds by spray administration, for example using a pressurized spray device or a controlled droplet administration device. Pressurized spray devices typically include a pressure chamber, a spray gun, and a nozzle. The nozzle and operating pressure can be varied to vary the particle size, which is typically in the range of about 10 μ to about 1000 μ. The droplets are either sprayed from a nozzle, either by breathing or by eye or oral route, directly into contact with the bird, or indirectly into contact with the vaccine deposited on the ground or other bird by a spraying device. Devices for spray administration of vaccine compositions are readily available, exemplary devices of which are disclosed in, for example, U.S. patent nos. 5,312,353 and 4,863,443, the disclosures of which are incorporated herein by reference. Controlled droplet applicators developed for horticultural and insect control uses may also be used to deliver adenovirus-based vaccines to poultry. In such devices, a spray may be generated by centrifugal force when the diluted vaccine is delivered to the rotating disc, which forms an atomized vaccine spray having a size range suitable for ingestion by breath. Such a mist vaccine may be distributed by means of a fan, which is wider than the vaccine distribution area achieved by a pressurized spray device. This approach provides the added advantage of requiring a relatively small amount of diluent (e.g., water). For example, up to 30,000 birds can be immunized with about 1 liter of vaccine solution.

The immunogenic compositions disclosed herein can also be administered to poultry using more invasive and/or laborious methods, comprising, in addition to the methods disclosed above: eye drops, punctures and scratches (e.g., subcutaneously in the inside of the wings or feet), injections, and in ovo administration. Automated and semi-automated injection devices suitable for delivering the disclosed vaccines to poultry can be found, for example, in U.S. patents 4,681,565 and 4,515,590, the disclosures of which are incorporated herein by reference.

In ovo administration of adenoviruses containing avian influenza antigens is equally well used to elicit protective immune responses against influenza in poultry. In ovo administration typically involves injecting into ovo, at an appropriate incubation stage of immunocompetence development, prior to hatching, an immunologically effective amount of adenovirus containing one or more avian influenza virus antigens. For example, eggs are typically injected between 17.5-19 days of incubation. The calculated volume is a range that does not substantially destroy the integrity of the egg, and is typically 0.01-0.1ml (e.g., 0.05 ml). Typically, a small hole is made in the egg shell through which a needle is inserted to deliver the immunizing composition. The injection may be performed manually or assisted according to the manufacturer's instructions using commercially available automated equipment (e.g., available from Embrex, TrianglePark, n.c.). Methods and devices for administering in ovo solutions to avian eggs suitable for administration of the vaccine compositions disclosed herein can be found in U.S. Pat. nos. 4,903,635, 5,056,464, 5,136,979, 5,699,751, 5,900,929, 6,032,612, 6,244,214 and 6,981,470, the disclosures of which are incorporated herein by reference in their entirety.

Examples

Example 1: preparation of adenovirus vector expressing avian Hemagglutinin (HA) antigen

Exemplary adenoviral vectors expressing avian H5 Hemagglutinin (HA) antigen can be prepared using homologous recombination. The HAd5 vector comprising the avian HA antigen can be prepared using the Cre recombinase-mediated site-specific recombination system of Ng et al (Human Gene Therapy 10: 2667-2672, 1999). To prepare the HAd5E1 insertion vector expressing the HA antigen of the H5N1 influenza strain, the HA gene of the a/Hong Kong/156/97 strain under the control of the cytomegalovirus immediate early promoter ("CMV" promoter) was inserted into the Stu I site of the shuttle vector (pDC 311). The pDC311 shuttle vector is a plasmid containing the left end of HAd5(4kb) with a3.1 kb E1 deletion, loxP sites for site-specific recombination in the presence of Cre recombinase, and a complete packaging signal (ψ). The resulting vector pDC311-H5 was co-transfected with pBHGlox. DELTA.E 1, 3Cre into 293Cre (293 cells expressing Cre recombinase). The pBHGlox Δ E1, 3Cre plasmid contains almost the entire HAd5 genome except for the packaging signals and deletions in the genes of the E1 and E3 regions. The plasmid also includes loxP sites suitable for Cre recombinase mediated recombination. When introduced into 293Cre cells together, Cre-mediated recombination between the two plasmids produced vector HAd5-HA (FIG. 1A).

Analysis of the cell extracts by Western blot indicated that the introduced avian HA antigen was expressed (fig. 1B).

Example 2: HAd-H5 in immunized animals

To demonstrate expression of avian HAThe immunogenicity of the recombinant adenovirus of (a), a mouse can be vaccinated with the recombinant adenovirus and challenged with a lethal amount of avian influenza virus. 25 female C57BL/6 mice, 6-8 weeks old, were divided into 5 groups of 5 animals per group. The animals were intramuscularly inoculated with PBS on days 1 and 28, 15 μ g of alum-free recombinant H5 (hemagglutinin of avian H5N1 influenza virus expressed in baculovirus) (H5 only), 15 μ g of alum-containing H5(H5+ alum), 10 μ g⁸p.f.u. HAd- Δ E1F3(HAd5 control vector), or 10⁸p.f.u. HAd-H5 (HAd5 vector expressing H5). Serum samples were collected at 21 and 49 days and the development of H5-specific immune responses was monitored by ELISA and microneutralization assays. Use 100LD in 70 days₅₀The H5N1(A/Hong Kong/483/97) virus challenged animals and was monitored daily for weight gain or loss and clinical signs of overt influenza infection.

HAd5-H5 caused over 3.5 log of H5-specific IgG ELISA titers at 21 days, demonstrating that H5 expressed by HAd5-H5 is highly immunogenic. Serum samples were collected at day 49 and the development of H5N1 virus neutralizing antibody responses was monitored by virus neutralization assay. The H5N 1-specific neutralizing antibody response induced by immunization of mice with HAd5-H5 was similar to that obtained with high doses of H5+ alum (table 1).

Mice immunized with HAd5-H5 were fully protected against disease and death after challenge with the pathogenic H5N1 virus. Use 100LD in 70 days₅₀The H5N1(A/HK/483/97) virus of (1) challenged the animals. The level of protection was better than that observed with high dose recombinant H5+ alum (table 1 and figure 2). None of the mice in group HAd5-H5 exhibited any observable discomfort after challenge.

Table 1: serological response in mice immunized with HAd5-H5HA vaccine

Group of	Geometric mean equine HI titer	Geometric mean neutralization titer	Survival rate
				PBS	25	20	0
HAd-5(108p.f.u.)i.m.	25	20	0
				HAd-H5HA(108p.f.u.)i.m.	696.4	2228	100
rH5HA + alum i.m.	696.4	2228	100
				rH5HA i.m.	37.9	60	80

Example 3: protective immune response by intranasal administration of HAd5-HA

HAd5-H5HA vector's ability to elicit HA-specific antibody responses can be determined following intramuscular and intranasal administration. Three groups of 15 (6-to 8-week-old) female BALB/c mice were randomly divided into 3 groups (5 animals/group) and intramuscularly vaccinated on days 0 and 28 for 10⁸HAd- Δ E1E3 of p.f.u. or intramuscular or intranasal administration 10⁸HAd-H5HA, p.f.u.. Sera were collected at 21 and 49 days and the development of H5-specific immune responses against the A/HK/483/97, A/HK/213/03, and A/VN/1203/04 strains was monitored by coagulation inhibition (HI) analysis using equine erythrocytes. ELISA demonstrated that intramuscular or intranasal administration of HAd5-H5HA resulted in strong HA antibody titers, as shown in Table 2.

Table 2: titers against the homologous and recent H5 strain induced by the HAd5-HA vaccine

Similarly, with 1x10⁸p.f.u. HAd-H5HA BALB/c mice (20/group) were immunized intramuscularly or intranasally twice at 4 weeks intervals for virus neutralization assay. The mice of the other groups (20 mice/group) were treated with 10 mice⁸p.f.u. HAd- Δ E1E3 or 3 μ g rH5HA with alum. Serum samples were taken 4 weeks after the second immunization and analyzed by virus neutralization assay to assess their presence and presence of homologous (HK/156/97) or heterologous (A/HongKong/213/2003[ HK/213/03 ] viruses]And A/Vietnam/1203/04[ VN/1203/04]) The ability to carry out the reaction. Compared to HK/156/97, the amino acid homology in the hemagglutinin subunit was 94.8% for HK213/03 and 95.5% for VN/1203/04. Exemplary results are shown in fig. 3. Mice immunized with rH5HA + alum showed high virus-neutralizing antibody titers against the homologous HK/156/97 virus, but were not able to neutralize the K/213/03 or VN/1203/04 viruses. In contrast, mice immunized with HAd-H5HA produced neutralizing antibodies against both the homologous and heterologous viruses, indicating a comparable neutralizing antibody titer against HK/156/97High and lower titers against heterologous HK/213/03 or VN/1203/04 viruses.

Example 4: vaccination with HAd5-H5 elicited HA-specific T cell responses

To evaluate whether HAd-H5HA vaccine induced HA-specific CD8⁺T cell response by intramuscular or intranasal administration 10⁸Had- Δ E1E3 or 10⁸U.HAd-H5HA, and by immunization of BALB/c mice with a vaccine against immunodominant HA518 (HA)_518-526) Kd-specific pentamer staining of epitopes (HA first documented for H1N1 virus, A/Puerto Rico/8/34) to assess splenic T cell responses against influenza epitopes. The epitope is widely conserved among H5N1 viruses, including the currently transmitted avian and human H5N1 viruses, as well as the more diverse viruses, such as the H9N2 strain. Mice receiving the HAd-H5HA vector intranasally or intramuscularly had HA-specific CD8 at least 3-8 fold more frequently than mice immunized with HAd- Δ E1E3⁺T cells (fig. 4). No NP-147 (NP) was observed in animals immunized with the HAd-H5HA vaccine_147-155) Epitope-specific CD8⁺A detectable increase in T cells. In contrast, control mice infected with H5N1 virus showed strong NP147 epitope-specific CD8⁺T cell response. None of the mice vaccinated with HAd- Δ E1E3 or rH5HA + alum showed HA518 epitope-specific CD8⁺The frequency of T cells increases.

1X10 pairs of 1X10 splenocytes irradiated by pulsing with 10. mu.g/ml of the indicated peptides (including MHC class I binding epitopes: NP147 and HA462 and HA518, which appear as the major epitopes in H5N3 infected animals)⁶Splenocytes were stimulated to determine epitope specific T cell responses. PMA + ionomycin was used as a positive control. IFN- γ production was assessed by ELISpot analysis, as shown in FIG. 5. Vaccination with HAd5-H5 elicited HA-specific T cell responses against MHC class I binding epitopes of HA. This T cell response was not observed in animals vaccinated with recombinant H5+ alum.

Example 5: vaccination with HAd5-H5HA produced protection against lethal challenge

Animals vaccinated HAd5-H5HA by the intramuscular or intranasal routes of administration were stimulated with homologous virus strains and the most recent strain of avian influenza. After inoculation, 100LD can be used₅₀Mice were challenged with either the A/HK/156/97 or A/VN/1203/04 strains. As shown in table 3, all vaccinated animals survived a lethal challenge with either the homologous virus or a different strain of H5N 1.

Table 3: HAd-5H5HA provide protection against lethal challenge

Example 6: incidence of challenge of animals with A/HK/483/97 or A/VN/1203/04 viruses

To evaluate the protective efficacy against challenge with the H5N1 variant, mice immunized with HAd-H5HA were challenged with HK/483/97, HK/213/03, or VN/1203/04 viruses. Unlike the virulent HK/483/97 and VN/1203/04 strains, HK/213/03 virus is not highly lethal to mice. Thus, to evaluate the efficacy of the vaccine against HK/213/03, viral titers in the lungs were determined in animals infected with HK/213/03 4 days post challenge. HAd-H5HA mice vaccinated intranasally or intramuscularly exhibited the lowest morbidity and provided complete protection against death following challenge with HK/483/97 virus (FIGS. 6A and B). All mice vaccinated with HAd-H5HA by either vaccination route survived and showed the lowest incidence after lethal challenge with the more recent H5N1 virus, VN/1203/04, as determined by weight loss (fig. 6C and D). In addition, mice vaccinated HAd-H5HA and challenged with HK/213/03 virus on either vaccination route had no detectable virus in the lungs 4 days after infection (FIG. 6E), while mice vaccinated with control vector HAd-. DELTA.E 1E3 had greater than 10⁶EID₅₀Average pneumovirus titer per ml (p < 0.001). Thus, the HAd-H5HA vaccine induced significant protection against the heterologous H5N1 virus, even in the presence of low levels of cross-neutralizing serum antibody titers.

These data confirm the potential for Ad vector-based delivery of avian influenza antigens as pandemic influenza vaccines. Such vectors induce strong humoral and cellular immunity and produce cross-protection against the continuously evolving H5N1 virus without the need for adjuvants.

Example 7: preparation and characterization of non-human vectors expressing H5N1 influenza HA

Infectious clones of the complete genome of non-human adenoviruses (porcine adenovirus type 3, PAd3 or bovine adenovirus type 3, BAd3) with deletions of the E1 and E3 regions and with or without an E1 insertion can be prepared by homologous recombination in E.coli BJ 5183. The HA gene of H5N1 flanked by a CMV promoter, and the BGH polyadenylation signal of bovine growth hormone were cloned into the Avr II site of pDS2((Bangari & Mittal, Virus Research 105: 127-136, 2004) to obtain pDS 2-H5. pPAS-H5 (genomic plasmid with avian HA inserted in the E1A gene region of porcine adenovirus) was prepared in E.coli BJ5183 using homologous recombination, as described in VanOlphen & Mittal, J.Virol.methods 77: 125-129, 1999, for bovine adenovirus, by co-transfecting E.coli with E3 deleted PAd3 genomic DNA and Stu I linearized pDS 2-H5.

For the preparation of HA for H5N1 influenza from PAd3 vector, LIPOFECTIN was used according to the manufacturer's recommendationsMediated transfection, FPRT HE1-5 cells (e.g., Bangari) were transfected with PacI-digested pPAd-H5 (5. mu.g/60-mm dish)&Mittal, Virus Res.105: 127-136, 2004) of the E1 expressing pig cell line. The cytopathic effect induced by the recombinant virus can be observed 2-3 weeks after transfection.

As demonstrated by Western blotting (FIG. 7B), the replication-deficient recombinant PAd3 vector (PAd-H5HA) containing the full-length coding region of the HA gene of the H5N1 virus (HK/156/97) inserted in early region 1(E1) of the PAd3 genome (FIG. 7A) was efficiently expressed in FPRT HE1-5 cells (FIG. 7B). PAd with deletions of the E1 and E3 regions (PAd-. DELTA.E 1E3) served as negative controls.

Similarly, replication-deficient recombinant BAd3 vector (BAd-H5HA) containing the full-length coding region of the HA gene of the H5N1 virus (HK/156/97) inserted in early region 1(E1) of the BAd3 genome (FIG. 7C) was efficiently expressed in FBRT HE1 cells expressing BAd3E 1(van Olphen et al, Virology 295: 108. sup. 118, 2002), as shown in FIG. 7D. Bad3 (BAd-. DELTA.E 1E3) with deletion of E1 and E3 regions served as a negative control.

These non-human adenoviral vectors are suitable as vaccine vectors, which can be administered to both human and non-human subjects to elicit protective immune responses specific to avian (and other) influenza strains.

Example 8: cell lines for expressing adenoviral vectors

Novel cell lines expressing the E1 antigen of adenoviral vectors with different tropisms (human and non-human) were prepared. Such a multifunctional cell line is particularly advantageous for the optimized production of replication-defective adenoviruses, and thus adenoviruses and/or recombinant proteins, from recombinant vectors corresponding to viruses in a variety of viral strains.

Two exemplary multifunctional cell lines were prepared based on the previously described cell line expressing the HAd5E1 gene in bovine or porcine cells. FBRT-HE1 is a fetal bovine kidney cell line expressing the E1 gene of human adenovirus strain HAd5 (SEQ ID NO: 4). Construction and isolation of the FBRT-HE1 cell line is disclosed in van Olphen et al, (Virology 295: 108-118, 2002), which is incorporated herein by reference. Briefly, primary fetal bovine kidney (FBRT) cells were transfected with a plasmid containing HAd5E1 (of which HAd5E1 is under the control of the PGK promoter). Multiple G418 resistant clones were isolated and examined for expression of E1B-19kDa proteins by Western blotting. Cell lines expressing E1B-10kDa were further tested for expression of E1A, E1B-19kDa and E1B-55kDa by immunoprecipitation. A representative cell line, designated FBRT-HE1, expresses all three E1 proteins: E1A, E1B-19kDa and E1B-55 kDa. The FBRT-HE1 cell line supports the growth of both the E1 deleted human adenovirus vector and the E1 deleted bovine adenovirus vector.

To prepare multifunctional cell lines, the fragment containing BAd3E 1(604-3148) was amplified using the BAd E1-F (ccatgaagtacctggtcctc; SEQ ID NO: 5) and BAd E1-R (ccccacctatttatacccctc; SEQ ID NO: 6) primer sets. The PCR fragment containing BAd3E1 gene (SEQ ID NO: 7) can BE cloned into pPGK-puro at the EcoR V site and pCMV-puro at the KpnI-XbaI site to obtain pPGK-BE1 and pCMV-BE1, respectively. These plasmids were then used to transfect FBRT-HE1 cells using Lipofectin (Life technologies). 48 hours after transfection, the cells were grown in selection medium containing 3. mu.g/ml puromycin. Isolated (secreted) cell clones were visible in pPGK-BE1 or pCMV-BE1 transfected FBRT-HE1 cells after approximately 30 days of antibiotic selection. 24 clones (12 clones each in pPGK-BE1 and pCMV-BE1 transfected cells) were picked, and all three BAd3E1 transcripts (E1A, E1B-1 and E1B-2) were amplified and screened for expression by RT-PCR using specific primer sets (Table 4), thereby identifying clones expressing all three BAd E1 transcripts. The FBRT-HE1/PE1 cell clone shown in FIG. 8A demonstrated that all three BAdE1 genes were expressed under the PGK promoter.

Table 4: primers for RT-PCR of bovine E1 transcript

Gene	Primer sequences (5 'to 3')
		BAd E1A (Zheng)	CTGATATCATGAAGTACCTGGCCTC(SEQ ID NO：8)
BAd E1A (reverse)	ATGCAATGGTAGGTTTGG(SEQ ID NO：9)
		BAd E1B-1 (positive)	GATATCATGGATCACTTAAGCGTTC(SEQ ID NO：10)
BAd E1B-1 (reverse)	GTCGACAACTGATGTGCTCGAAACG(SEQ ID NO：11)
		BAd E1B-2 (Zheng)	GATATCGTTCAAGATCACCCAGAG(SEQ ID NO：12)
BAd E1B-2 (reverse)	GTCGACCACTTTTAATCCTGCTC(SEQ ID NO：13)

Similarly, multifunctional porcine cells can be prepared by introducing porcine adenovirus E1(PAd 3E 1) into a cell line expressing HAd5E 1. FPRT-HE1-5 is a fetal porcine kidney cell line constitutively expressing HAd5E1(SEQ ID NO: 4). The preparation of FPRT-HE1-5 is disclosed in Bangari & Mittal (Virus Res.105: 127-136, 2004), which is incorporated herein by reference. Briefly, primary fetal pig kidney (FPRT) cells were transfected with a plasmid containing HAd5E1 under the control of the Cytomegalovirus (CMV) immediate early or phosphoglycerate kinase (PGK) promoter. Transformed cell lines transfected with the HAd5E1 sequence under the control of CMV or PGK promoters were selected and further characterized. FPRT-HE1-5 is an exemplary cell line that efficiently expresses all three E1 genes and can be used to prepare and grow E1-deleted porcine and human adenovirus vectors.

The PAd3 plasmid was constructed for transfection into FPRT-HE1-5 cells in the following manner. The neomycin ORF in pcDNA3.1(Invitrogen) was replaced with the puromycin ORF from pBABE-puro (Addgene) to obtain plasmid pCMV-puro. The CMV promoter sequence in plasmid pcDNA3.1-puro was replaced with the PGK promoter from pGT-N28(NewEngland Biolabs) to obtain plasmid pPGK-puro. Fragments containing PAd3E 1(526-3259) were amplified using the PAdE1-F (TGGATCCTCGACATGGCGAACAGACTT; SEQ ID NO: 14) and PAd E1-R (TCTCGAGTCATCCTCAGTCATCGTCATCG; SEQ ID NO: 15) primer sets. The resulting PCR product including the coding sequence of the PAd3E1 gene (SEQ ID NO: 16) was subsequently cloned into the BamHI-XhoI site of pPGK-puro or pCMV-puro, thereby obtaining pPGK-PE1 and pCMV-PE1, respectively.

These plasmids were then used to transfect the FPRT-HE1 cell line using Lipofectin (Life technologies). 48 hours after transfection, the cells were grown in selection medium containing 2. mu.g/ml puromycin. Isolated cell clones were visible after approximately 30 days of antibiotic selection in pPGK-PE1 and pCMV-PE1 transfected FPRT-HE1 cells. 24 clones (12 clones each of pPGK-PE1 and pCMV-PE1 transfected cells) were picked, and all three PAd3E1 transcripts (E1A, E1B-1 and E1B-2) were amplified and screened for expression by RT-PCR using specific primer sets (Table 5). Multiple clones from pPGK-PE1 transfected cells and pCMV-PE1 transfected cells were found to be positive for expression of all three PAd E1 transcripts. The FBRT-HE1/PE1 cell clone shown in FIG. 8B illustrates the expression of E1 under the PGK promoter.

Table 5: primers for RT-PCR of porcine E1 transcript

Gene	Primer sequences (5 'to 3')
		PAd E1A (Zheng)	AGGTGGAGGTGATTGTGACTGA(SEQ ID NO：17)
PAd E1A (reverse)	GACGCAAGAGGAAGTACTGCTA(SEQ ID NO：18)
		PAd E1B-1 (positive)	CTGGCCAAGCTTACTAACGTGAAC(SEQ ID NO：19)
PAd E1B-1 (reverse)	TTTAAGTCTTCTGGTGCCGCCA(SEQ ID NO：20)
		PAd E1B-2 (positive)	ATGCATGAGCGCTACAGCTTTG(SEQ ID NO：21)
PAd E1B-2 (reverse)	CTGAGTTCCGCAAGAATGTGCT(SEQ ID NO：22)

Example 9: adenovirus vectors expressing multiple influenza antigens

Adenovirus vectors are prepared by incorporating multiple influenza antigen(s). The HAd5 vector containing two or more influenza antigens can be prepared using the Cre recombinase-mediated site-specific recombination system of Ng et al (Human Gene Therapy 10: 2667-2672, 1999). To prepare HAd5E1 insertion vectors that express multiple influenza antigens, the polynucleotide sequence encoding the selected antigen can be cloned under the control of a promoter, such as the giant cell immediate early promoter ("CMV promoter"), and inserted, for example, at the Stu I site of the shuttle vector. The shuttle vector, e.g., pDC311, includes loxP sites for site-specific recombination in the presence of Cre recombinase, and an intact packaging signal (ψ). The resulting vector was co-transfected with pBHGlox. DELTA.E 1, 3Cre into 293Cre (293 cells expressing Cre recombinase). The pBHGlox Δ E1, 3Cre plasmid contained almost the entire HAd5 genome except for the packaging signal and gene deletions in the E1 and E3 regions. The plasmid also contains loxP sites for Cre recombinase mediated recombination. When introduced together into 293Cre cells, Cre-mediated recombination between the two plasmids generates a vector comprising a polynucleotide sequence encoding the selected influenza antigen. Optionally, this method may be used to integrate polynucleotide sequences encoding other polypeptides that enhance immune function, such as the TLRs described above. Exemplary influenza antigen combinations are provided in table 6. The exemplary combinations given in table 6 may be understood as illustrative. Other combinations of antigens can be determined by one skilled in the art. Similarly, non-human adenoviral vectors (e.g., PAd3 or BAd3) can be used to prepare various recombinants expressing exemplary influenza antigen combinations as provided in table 6.

Table 6: exemplary antigen combinations in Multi-antigen adenovirus vectors

Exemplary combination	Hemagglutinin (HA)	Neuraminidase (NA)	Internal proteins(s)
				1	H5	N1	(-)
2	H7	N7	(-)
				3	H9	N2	(-)
4	H5	N1	M*
				5	H7	N7	M
6	H9	N2	M
				7	H5	N1	NP
8	H7	N7	NP
				9	H9	N2	NP
10	H5	N1	M+NP
				11	H7	N7	M+NP
12	H9	N2	M+NP
				13	H5	N1	NS1
14	H7	N7	NS1
				15	H9	N2	NS1
16	H5		M
				17	H5		NP
18	H5		M+NP
				19	H7		M
20	H7		NP
				21	H7		M+NP
22	H9		M
				23	H9		NP
24	H9		M+NP
				25	H51+H52+H53
26	H51+H52+H53	N1
				27	H51+H52+H53		M
28	H51+H52+H53		NP

29	H51+H52+H53		M
				30	H51+H52+H53	N1	NP
31	H51+H52+H53	N1	M+NP
				32	H5+H7+H9
33	H5+H7+H9	N1
				34	H5+H7+H9		M
35	H5+H7+H9		NP
				36	H5+H7+H9		M
37	H5+H7+H9	N1	NP
				38	H5+H7+H9	N1	M+NP

The superscript refers to variants.

^*M refers to M1, M2 or both M1 and M2.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the appended claims. We therefore claim as a whole our invention within the scope and spirit of these claims.

Sequence listing

<110> U.S. government health and human services department, center for disease control and prevention

(Goverment of the United States of America, as presentedbase of the Collection of Health and Human Services, Centers for Disease Control and preservation) Research Foundation (university) Research Foundation

<120> vaccine against pandemic influenza virus strain

(VACCINE AGAINST PANDEMIC STRAINS OF INFLUENZA VIRUSES)

<130>SCT075063-66

<140>PCT/US2006/013384

<141>2006-04-10

<150>60/670,826

<151>2005-04-11

<160>22

<170>PatentIn version 3.3

<210>1

<211>20

<212>DNA

<213>artificial sequence

<220>

<223>immunostimulatory oligonucleotide

<400>1

tccatgagct tcctgatcct 20

<210>2

<211>20

<212>DNA

<213>artificial sequence

<220>

<223>immunostimulatory oligonucleotide

<400>2

tccatgacgt tcctgacgtt 20

<210>3

<211>22

<212>DNA

<213>artificial sequence

<220>

<223>immunostimulatory oligonucleotide

<400>3

tgactgtgaa cgttcgagat ga 22

<210>4

<211>2950

<212>DNA

<213>Human adenovirus type 5

<400>4

atgagacata ttatctgcca cggaggtgtt attaccgaag aaatggccgc cagtcttttg 60

gaccagctga tcgaagaggt actggctgat aatcttccac ctcctagcca ttttgaacca 120

cctacccttc acgaactgta tgatttagac gtgacggccc ccgaagatcc caacgaggag 180

gcggtttcgc agatttttcc cgactctgta atgttggcgg tgcaggaagg gattgactta 240

ctcacttttc cgccggcgcc cggttctccg gagccgcctc acctttcccg gcagcccgag 300

cagccggagc agagagcctt gggtccggtt tctatgccaa accttgtacc ggaggtgatc 360

gatcttacct gccacgaggc tggctttcca cccagtgacg acgaggatga agagggtgag 420

gagtttgtgt tagattatgt ggagcacccc gggcacggtt gcaggtcttg tcattatcac 480

cggaggaata cgggggaccc agatattatg tgttcgcttt gctatatgag gacctgtggc 540

atgtttgtct acagtaagtg aaaattatgg gcagtgggtg atagagtggt gggtttggtg 600

tggtaatttt ttttttaatt tttacagttt tgtggtttaa agaattttgt attgtgattt 660

ttttaaaagg tcctgtgtct gaacctgagc ctgagcccga gccagaaccg gagcctgcaa 720

gacctacccg ccgtcctaaa atggcgcctg ctatcctgag acgcccgaca tcacctgtgt 780

ctagagaatg caatagtagt acggatagct gtgactccgg tccttctaac acacctcctg 840

agatacaccc ggtggtcccg ctgtgcccca ttaaaccagt tgccgtgaga gttggtgggc 900

gtcgccaggc tgtggaatgt atcgaggact tgcttaacga gcctgggcaa cctttggact 960

tgagctgtaa acgccccagg ccataaggtg taaacctgtg attgcgtgtg tggttaacgc 1020

ctttgtttgc tgaatgagtt gatgtaagtt taataaaggg tgagataatg tttaacttgc 1080

atggcgtgtt aaatggggcg gggcttaaag ggtatataat gcgccgtggg ctaatcttgg 1140

ttacatctga cctcatggag gcttgggagt gtttggaaga tttttctgct gtgcgtaact 1200

tgctggaaca gagctctaac agtacctctt ggttttggag gtttctgtgg ggctcatccc 1260

aggcaaagtt agtctgcaga attaaggagg attacaagtg ggaatttgaa gagcttttga 1320

aatcctgtgg tgagctgttt gattctttga atctgggtca ccaggcgctt ttccaagaga 1380

aggtcatcaa gactttggat ttttccacac cggggcgcgc tgcggctgct gttgcttttt 1440

tgagttttat aaaggataaa tggagcgaag aaacccatct gagcgggggg tacctgctgg 1500

attttctggc catgcatctg tggagagcgg ttgtgagaca caagaatcgc ctgctactgt 1560

tgtcttccgt ccgcccggcg ataataccga cggaggagca gcagcagcag caggaggaag 1620

ccaggcggcg gcggcaggag cagagcccat ggaacccgag agccggcctg gaccctcggg 1680

aatgaatgtt gtacaggtgg ctgaactgta tccagaactg agacgcattt tgacaattac 1740

agaggatggg caggggctaa agggggtaaa gagggagcgg ggggcttgtg aggctacaga 1800

ggaggctagg aatctagctt ttagcttaat gaccagacac cgtcctgagt gtattacttt 1860

tcaacagatc aaggataatt gcgctaatga gcttgatctg ctggcgcaga agtattccat 1920

agagcagctg accacttact ggctgcagcc aggggatgat tttgaggagg ctattagggt 1980

atatgcaaag gtggcactta ggccagattg caagtacaag atcagcaaac ttgtaaatat 2040

caggaattgt tgctacattt ctgggaacgg ggccgaggtg gagatagata cggaggatag 2100

ggtggccttt agatgtagca tgataaatat gtggccgggg gtgcttggca tggacggggt 2160

ggttattatg aatgtaaggt ttactggccc caattttagc ggtacggttt tcctggccaa 2220

taccaacctt atcctacacg gtgtaagctt ctatgggttt aacaatacct gtgtggaagc 2280

ctggaccgat gtaagggttc ggggctgtgc cttttactgc tgctggaagg gggtggtgtg 2340

tcgccccaaa agcagggctt caattaagaa atgcctcttt gaaaggtgta ccttgggtat 2400

cctgtctgag ggtaactcca gggtgcgcca caatgtggcc tccgactgtg gttgcttcat 2460

gctagtgaaa agcgtggctg tgattaagca taacatggta tgtggcaact gcgaggacag 2520

ggcctctcag atgctgacct gctcggacgg caactgtcac ctgctgaaga ccattcacgt 2580

agccagccac tctcgcaagg cctggccagt gtttgagcat aacatactga cccgctgttc 2640

cttgcatttg ggtaacagga ggggggtgtt cctaccttac caatgcaatt tgagtcacac 2700

taagatattg cttgagcccg agagcatgtc caaggtgaac ctgaacgggg tgtttgacat 2760

gaccatgaag atctggaagg tgctgaggta cgatgagacc cgcaccaggt gcagaccctg 2820

cgagtgtggc ggtaaacata ttaggaacca gcctgtgatg ctggatgtga ccgaggagct 2880

gaggcccgat cacttggtgc tggcctgcac ccgcgctgag tttggctcta gcgatgaaga 2940

tacagattga 2950

<210>5

<211>20

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>5

ccatgaagta cctggtcctc 20

<210>6

<211>21

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>6

ccccacctat ttatacccct c 21

<210>7

<211>2507

<212>DNA

<213>Bovine adenovirus type 3

<400>7

atgaagtacc tggtcctcgt tctcaacgac ggcatgagtc gaattgaaaa agctctcctg 60

tgcagcgatg gtgaggtgga tttagagtgt catgaggtac ttcccccttc tcccgcgcct 120

gtccccgctt ctgtgtcacc cgtgaggagt cctcctcctc tgtctccggt gtttcctccg 180

tctccgccag ccccgcttgt gaatccagag gcgagttcgc tgctgcagca gtatcggaga 240

gagctgttag agaggagcct gctccgaacg gccgaaggtc agcagcgtgc agtgtgtcca 300

tgtgagcggt tgcccgtgga agaggatgag tgtctgaatg ccgtaaattt gctgtttcct 360

gatccctggc taaatgcagc tgaaaatggg ggtgatattt ttaagtctcc ggctatgtct 420

ccagaaccgt ggatagattt gtctagctac gatagcgatg tagaagaggt gactagtcac 480

ttttttctgg attgccctga agaccccagt cgggagtgtt catcttgtgg gtttcatcag 540

gctcaaagcg gaattccagg cattatgtgc agtttgtgct acatgcgcca aacctaccat 600

tgcatctata gtaagtacat tctgtaaaag aacatcttgg tgatttctag gtattgttta 660

gggattaact gggtggagtg atcttaatcc ggcataacca aatacatgtt ttcacaggtc 720

cagtttctga agaggaaatg tgagtcatgt tgactttggc gcgcaagagg aaatgtgagt 780

catgttgact ttggcgcgcc ctacggtgac tttaaagcaa tttgaggatc acttttttgt 840

tagtcgctat aaagtagtca cggagtcttc atggatcact taagcgttct tttggatttg 900

aagctgcttc gctctatcgt agcgggggct tcaaatcgca ctggagtgtg gaagaggcgg 960

ctgtggctgg gacgcctgac tcaactggtc catgatacct gcgtagagaa cgagagcata 1020

tttctcaatt ctctgccagg gaatgaagct tttttaaggt tgcttcggag cggctatttt 1080

gaagtgtttg acgtgtttgt ggtgcctgag ctgcatctgg acactccggg tcgagtggtc 1140

gccgctcttg ctctgctggt gttcatcctc aacgatttag acgctaattc tgcttcttca 1200

ggctttgatt caggttttct cgtggaccgt ctctgcgtgc cgctatggct gaaggccagg 1260

gcgttcaaga tcacccagag ctccaggagc acttcgcagc cttcctcgtc gcccgacaag 1320

acgacccaga ctaccagcca gtagacgggg acagcccacc ccgggctagc ctggaggagg 1380

ctgaacagag cagcactcgt ttcgagcaca tcagttaccg agacgtggtg gatgacttca 1440

atagatgcca tgatgttttt tatgagaggt acagttttga ggacataaag agctacgagg 1500

ctttgcctga ggacaatttg gagcagctca tagctatgca tgctaaaatc aagctgctgc 1560

ccggtcggga gtatgagttg actcaacctt tgaacataac atcttgcgcc tatgtgctcg 1620

gaaatggggc tactattagg gtaacagggg aagcctcccc ggctattaga gtgggggcca 1680

tggccgtggg tccgtgtgta acaggaatga ctggggtgac ttttgtgaat tgtaggtttg 1740

agagagagtc aacaattagg gggtccctga tacgagcttc aactcacgtg ctgtttcatg 1800

gctgttattt tatgggaatt atgggcactt gtattgaggt gggggcggga gcttacattc 1860

ggggttgtga gtttgtgggc tgttaccggg gaatctgttc tacttctaac agagatatta 1920

aggtgaggca gtgcaacttt gacaaatgct tactgggtat tacttgtaag ggggactatc 1980

gtctttcggg aaatgtgtgt tctgagactt tctgctttgc tcatttagag ggagagggtt 2040

tggttaaaaa caacacagtc aagtccccta gtcgctggac cagcgagtct ggcttttcca 2100

tgataacttg tgcagacggc agggttacgc ctttgggttc cctccacatt gtgggcaacc 2160

gttgtaggcg ttggccaacc atgcagggga atgtgtttat catgtctaaa ctgtatctgg 2220

gcaacagaat agggactgta gccctgcccc agtgtgcttt ctacaagtcc agcatttgtt 2280

tggaggagag ggcgacaaac aagctggtct tggcttgtgc ttttgagaat aatgtactgg 2340

tgtacaaagt gctgagacgg gagagtccct caaccgtgaa aatgtgtgtt tgtgggactt 2400

ctcattatgc aaagcctttg acactggcaa ttatttcttc agatattcgg gctaatcgat 2460

acatgtacac tgtggactca acagagttca cttctgacga ggattaa 2507

<210>8

<211>25

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>8

ctgatatcat gaagtacctg gcctc 25

<210>9

<211>18

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>9

atgcaatggt aggtttgg 18

<210>10

<211>25

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>10

gatatcatgg atcacttaag cgttc 25

<210>11

<211>25

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>11

gtcgacaact gatgtgctcg aaacg 25

<210>12

<211>24

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>12

gatatcgttc aagatcaccc agag 24

<210>13

<211>23

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>13

gtcgaccact tttaatcctg ctc 23

<210>14

<211>27

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>14

tggatcctcg acatggcgaa cagactt 27

<210>15

<211>29

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>15

tctcgagtca tcctcagtca tcgtcatcg 29

<210>16

<211>2722

<212>DNA

<213>Porcine adenovirus 3

<400>16

atggcgaaca gacttcacct ggactgggac ggaaaccccg aggtggtgcc ggtgctggaa 60

tgggacccgg tggatctgcg cgacccctct ccgggggatg agggcttctg tgagccgtgc 120

tgggagagtc tggtcgatgg actgccggac gagtggctgg acagtgtgga cgaggtggag 180

gtgattgtga ctgagggggg tgagtcagag gacagtggtg ggagtgccgc tggtgactca 240

ggtggctctc agggggtctt tgagatggac cccccagaag agggggacag taatgaggag 300

gatatcagcg cggtggctgc ggaggtgctg tctgaactgg ctgatgtggt gtttgaggac 360

ccacttgcgc caccctctcc gtttgtgttg gactgccccg aggtacctgg tgtgaactgc 420

cgctcttgtg attaccatcg ctttcactcc aaggacccca atctgaagtg cagtctgtgc 480

tacatgaggg atgcatgcct ttgctgtcta tggtgagtgt ttttggacat ttgtgggatt 540

atgtggaaaa aaaggaaaaa gtgcttgtaa gaaatctcat gtgctatttc ccattttttg 600

tctttttaga agctgtttct ccagcacctc acaggtcggg ttccccggga cttggagacc 660

tgccaggacg caagaggaag tactgctatg actcatgcag cgaacaacct ttggacctgt 720

ctatgaagcg cccccgcgat taatcattaa cctcaataaa cagcatgtga tgatgactga 780

ttgtctgtgt ctctgcctat atataccctt gtggtttgca gggaagggat gtggtgactg 840

agctattcct cagcatcatc atcgctctgc ttttttctac tgcaggctat ttcttgctag 900

ctcgctgtcc cttttctttt tctgtgggca tggactatca acttctggcc aagcttacta 960

acgtgaacta ccttaggaag gtgatagtac aggggtctca gaactgccct tggtggaaaa 1020

agattttttc ggacaggttt atcaaggtag tagcagaggc caggaggcag tacgggcaag 1080

agttgattga gatttttgtg gagggtgaga ggggctttgg tcctgagttc ctgcgggaag 1140

ggggactgta cgaagaggcc gttctgaaag agttggattt cagcaccttg ggacgcaccg 1200

tagctagtgt ggctctggtc tgcttcattt ttgagaagct tcagaagcac agcgggtgga 1260

ctgacgaggg tattttaagt cttctggtgc cgccactatg ttccctgctg gaggcgcgaa 1320

tgatggcgga gcaggtgcgg caggggctgt gcatcatcag gatgccgagc gcggagcggg 1380

agatgctgtt gcccagtggg tcatccggca gtggcagcgg ggccgggatg cgggaccagg 1440

tggtgcccaa gcgcccgcgg gagcaggaag aggaggagga ggacgaggat gggatggaag 1500

cgagcgggcg caggctcgaa gggccggatc tggtttagat cgccgccggc ccgggggagc 1560

gggtggagag gggagcgggg aggaggcggg ggggtcttcc atggttagct atcagcaggt 1620

gctttctgag tatctggaga gtcctctgga gatgcatgag cgctacagct ttgagcagat 1680

taggccctat atgcttcagc cgggggatga tctgggggag atgatagccc agcacgccaa 1740

ggtggagttg cagccgggca cggtgtacga gctgaggcgc ccgatcacca tccgcagcat 1800

gtgttacatc atcgggaacg gggccaagat caagattcgg gggaattaca cggagtacat 1860

caacatagag ccgcgtaacc acatgtgttc cattgcgggc atgtggtcgg tgactatcac 1920

ggatgtggtt tttgatcggg agctaccggc ccggggtggt ctgattttag ccaacacgca 1980

cttcatcctg cacggctgca acttcctggg ctttctgggc tcggtaataa cggcgaacgc 2040

cgggggggtg gtgcggggat gctacttttt cgcctgctac aaggcgcttg accaccgggg 2100

gcggctgtgg ctgacggtga acgagaacac gtttgaaaag tgtgtgtacg cggtggtctc 2160

tgcggggcgt tgcaggatca agtacaactc ctccctgtcc accttctgct tcttgcacat 2220

gagctatacg ggcaagatag tggggaacag catcatgagc ccttacacgt tcagcgacga 2280

cccctacgtg gacctggtgt gctgccagag cgggatggtg atgcccctga gcacggtgca 2340

catcgctccc tcgtctcgcc tgccctaccc tgagttccgc aagaatgtgc tcctccgcag 2400

caccatgttt gtgggcggcc gcctgggcag cttcagcccc agccgctgct cctacagcta 2460

cagctccctg gtggtggacg agcagtccta ccggggtctg agtgtgacct gctgcttcga 2520

tcagacctgt gagatgtaca agctgctgca gtgtacggag gcggacgaga tggagacgga 2580

tacctctcag cagtacgcct gcctgtgcgg ggacaatcac ccctggccgc aggtgcggca 2640

gatgaaagtg acagacgcgc tgcgggcccc ccggtccctg gtgagctgca actgggggga 2700

gttcagcgat gacgatgact ga 2722

<210>17

<211>22

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>17

aggtggaggt gattgtgact ga 22

<210>18

<211>22

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>18

gacgcaagag gaagtactgc ta 22

<210>19

<211>24

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>19

ctggccaagc ttactaacgt gaac 24

<210>20

<211>22

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>20

tttaagtctt ctggtgccgc ca 22

<210>21

<211>22

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>21

atgcatgagc gctacagctt tg 22

<210>22

<211>22

<212>DNA

<213>artificial sequence

<220>

<223>oligonucleotide primer

<400>22

ctgagttccg caagaatgtg ct 22

Claims (75)

1. A recombinant adenoviral vector comprising a polynucleotide sequence encoding at least one antigen of an avian influenza strain.

2. The recombinant adenoviral vector of claim 1, wherein the polynucleotide sequence encodes an avian influenza HA antigen.

3. The recombinant adenoviral vector of claim 2, wherein the polynucleotide sequence encodes a plurality of avian influenza HA antigens.

4. The recombinant adenoviral vector of claim 3, wherein said polynucleotide sequences encode multiple variants of the same HA antigenic subtype.

5. The recombinant adenoviral vector of claim 3, wherein the polynucleotide sequences encode multiple HA antigens of different HA subtypes.

6. The recombinant adenoviral vector of claim 1, wherein at least one polynucleotide sequence encodes an avian influenza NA antigen.

7. The recombinant adenoviral vector of claim 1, wherein the avian influenza antigen is a H5N1 strain antigen, a H7N7 strain antigen or a H9N2 strain antigen.

8. The recombinant adenoviral vector of claim 1, wherein the polynucleotide sequence encodes a plurality of influenza antigens.

9. The recombinant adenoviral vector of claim 8, wherein the polynucleotide sequence encodes at least one influenza internal protein.

10. The recombinant adenoviral vector of claim 9, wherein the influenza internal protein is M1 protein, M2 protein, NP protein, PB1 protein, PB2 protein, and NS1 protein, and NS2 protein, or a combination thereof.

11. The recombinant adenoviral vector of claim 9, wherein the influenza internal protein is an M1 protein, an M2 protein, an NP protein, or a combination of two or more thereof.

12. The recombinant adenoviral vector of claim 9, wherein the internal protein is an internal protein of a non-avian influenza strain.

13. The recombinant adenoviral vector of claim 9, wherein the internal protein is an internal protein of an influenza strain of H1N1, H2N2 or H3N 2.

14. The recombinant adenoviral vector of claim 1, wherein said adenoviral vector is a human adenoviral vector.

15. The recombinant adenoviral vector of claim 1, wherein said adenoviral vector is a non-human adenoviral vector.

16. The recombinant adenoviral vector of claim 15, wherein the non-human adenoviral vector is a porcine adenoviral vector, a bovine adenoviral vector, a canine adenoviral vector, a murine adenoviral vector, an ovine adenoviral vector, an avian adenoviral vector or a simian adenoviral vector.

17. The recombinant adenoviral vector of claim 1, wherein the adenoviral vector is a replication-defective adenoviral vector.

18. The recombinant adenovirus vector of claim 17, wherein the replication-defective adenovirus comprises a mutation in at least one of the E1 region gene and the E3 region gene.

19. The recombinant adenoviral vector of claim 1, wherein the adenoviral vector comprises a replication-defective human adenoviral vector comprising a polynucleotide sequence encoding HA, NA antigen, or both HA antigen and NA antigen of an avian influenza strain and M1 protein, M2 protein, NP protein, or a combination of two or more of M1 protein, M2 protein, and NP protein of another influenza strain.

20. The recombinant adenoviral vector of claim 1, wherein the adenoviral vector comprises a replication-defective porcine or bovine adenoviral vector comprising a polynucleotide sequence encoding HA, NA antigen, or both HA antigen and NA antigen of an avian influenza strain and two or more combinations of M1 protein, M2 protein, NP protein, or M1 protein, M2 protein, and NP protein of another influenza strain.

21. A recombinant adenovirus comprising at least one avian influenza strain antigen.

22. The recombinant adenovirus of claim 21, comprising at least one of an avian HA antigen and an avian NA antigen.

23. The recombinant adenovirus of claim 22, comprising a plurality of HA antigens.

24. The recombinant adenovirus of claim 23, wherein said adenovirus comprises multiple variants of the same HA antigenic subtype.

25. The recombinant adenovirus of claim 23, wherein said adenovirus comprises multiple HA antigens of different HA subtypes.

26. The recombinant adenovirus of claim 23, comprising a plurality of influenza antigens.

27. The recombinant adenovirus of claim 26, wherein the plurality of influenza antigens comprises at least one influenza internal protein.

28. The recombinant adenovirus of claim 27, wherein the at least one influenza internal protein is from a different strain than the avian influenza antigen.

29. The recombinant adenovirus of claim 27, wherein the at least one influenza internal protein comprises one or more of an NP protein and an M protein.

30. The recombinant adenovirus of claim 21, wherein said adenovirus is a human adenovirus.

31. The recombinant adenovirus of claim 21, wherein said adenovirus is a non-human adenovirus.

32. The recombinant adenovirus of claim 31, wherein the non-human adenovirus is a porcine adenovirus, a bovine adenovirus, a canine adenovirus, a murine adenovirus, an ovine adenovirus, an avian adenovirus, or a simian adenovirus.

33. The recombinant adenovirus of claim 21, wherein the adenovirus is a replication-defective adenovirus.

34. The recombinant adenovirus of claim 33, wherein the adenovirus comprises a mutation in at least one of the E1 region gene and the E3 region gene.

35. An immunogenic composition comprising:

at least one of an adenoviral vector and a recombinant adenovirus, the adenoviral vector or adenovirus comprising one or more polynucleotides encoding at least one avian influenza antigen and an avian influenza antigen; and

a pharmaceutically acceptable carrier.

36. The immunogenic composition of claim 35 comprising the adenoviral vector of any one of claims 1-20, and a pharmaceutically acceptable carrier.

37. The immunogenic composition of claim 35, comprising the recombinant adenovirus of any one of claims 21-34, and a pharmaceutically acceptable carrier.

38. The immunogenic composition of claim 35, further comprising at least one adjuvant.

39. An isolated cell comprising one or more heterologous nucleic acids comprising a first polynucleotide sequence encoding at least a first adenoviral E protein and a second polynucleotide sequence encoding at least a second adenoviral E protein, wherein the first and second adenoviral E proteins are selected from different adenoviral strains, and

wherein the cell supports the growth of a plurality of replication-deficient human and/or non-human adenovirus strains.

40. The cell line of claim 39, wherein said at least first adenoviral E protein and said at least second adenoviral E protein are both E1 proteins.

41. The cell line of claim 39, wherein the at least first and second adenoviral E proteins are from different strains of adenovirus with different tropisms.

42. The cell line of claim 41, wherein said at least a first adenoviral E protein comprises one or more human adenoviral E proteins, and wherein said at least a second adenoviral E protein comprises one or more non-human adenoviral E proteins.

43. The cell line of claim 42, wherein said at least a second adenoviral E protein comprises one or more bovine E proteins.

44. The cell line of claim 43, comprising a plurality of human E1 proteins and a plurality of bovine E1 proteins.

45. The cell line of claim 42, wherein said at least a second adenoviral E protein comprises one or more porcine E proteins.

46. The cell line of claim 45, comprising a plurality of human E1 proteins and a plurality of porcine E1 proteins.

47. A method of generating an immune response against at least one avian or pandemic influenza strain in a subject, the method comprising:

administering to a subject an immunogenic composition comprising at least one of:

a recombinant adenoviral vector comprising a polynucleotide encoding at least one antigen of an avian influenza strain; and

a recombinant adenovirus comprising at least one antigen of an avian influenza strain.

48. The method of claim 47, wherein the immune response is a protective or partially protective immune response against one or more avian or pandemic influenza strains.

49. The method of claim 48, wherein the immune response is a protective or partially protective immune response against a plurality of avian or pandemic influenza strains.

50. The method of claim 47, wherein the immune response comprises production of neutralizing antibodies that bind to an antigen of at least one avian or pandemic influenza strain.

51. The method of claim 47, wherein the immune response comprises production of neutralizing antibodies that bind to antigens of a plurality of avian or pandemic influenza strains.

52. The method of claim 47, wherein said immune response further comprises a T cell response specific for at least one influenza internal protein.

53. The method of claim 52, wherein the at least one internal protein is M1 protein, M2 protein, NP protein, or a combination thereof.

54. The method of claim 53, wherein the T cell response is specific for an epitope of a conserved influenza internal protein in a plurality of influenza strains.

55. The method of claim 54, wherein the plurality of influenza strains are influenza strains of more than one serotype.

56. The method of claim 47, comprising administering the immunogenic composition intranasally, orally, ocularly, intravenously, intramuscularly, transdermally, intradermally, or subcutaneously.

57. The method of claim 47, wherein the subject is a human subject.

58. The method of claim 47, wherein the subject is a non-human mammal.

59. The method of claim 47, wherein the subject is a bird.

60. The method of claim 59, wherein the subject is poultry.

61. The method of claim 60, comprising administering the immunogenic composition in ovo in an egg having an embryo.

62. The method of claim 60, comprising administering the immunogenic composition in the form of a spray or controlled droplets to a plurality of birds in a common space.

63. The method of claim 60, comprising administering the immunogenic composition in a drinking water.

64. The method of claim 47, comprising administering an adenoviral vector having polynucleotide sequences encoding a plurality of influenza antigens or an adenovirus comprising a plurality of influenza antigens.

65. The method of claim 64, wherein the plurality of antigens comprises HA antigen, NA antigen, or both HA antigen and NA antigen of an avian influenza strain.

66. The method of claim 64 wherein said plurality of antigens comprises variants of the same HA antigenic subtype.

67. The method of claim 64, wherein said plurality of antigens comprises a plurality of HA antigens from different avian influenza subtypes.

68. The method of claim 64, wherein said plurality of antigens comprises at least one avian influenza HA antigen and at least one influenza internal protein.

69. The method of claim 68, wherein said influenza internal protein is an M1 protein, an M2 protein, an NP protein, or a combination of two or more thereof.

70. A method of making a recombinant avian influenza virus antigen, the method comprising:

an adenovirus comprising at least one polynucleotide sequence encoding an avian influenza antigen replicates in a cell.

71. The method of claim 70, comprising replicating the adenovirus by introducing a replication defective adenovirus vector into a cell capable of supporting replication of the replication defective vector.

72. The method of claim 71, wherein said cell is a multifunctional cell that expresses at least two different E proteins, wherein said different E proteins are E proteins of at least two different strains of adenovirus with different tropisms.

73. The method of claim 70, wherein the avian influenza antigen is at least one of an HA antigen or an NA antigen.

74. The method of claim 73, wherein said antigen is selected from the group consisting of H5, H7, and H9 influenza strains.

75. The method of claim 70, wherein said at least one polynucleotide sequence encodes a plurality of influenza antigens.