U.S. patent application number 11/911189 was filed with the patent office on 2008-08-07 for vaccine against pandemic strains of influenza viruses.
Invention is credited to Dinesh S. Bangari, Mary Hoelscher, Jacqueline Katz, Suresh K. Mittal, Suryaprakash Sambhara.
Application Number | 20080187557 11/911189 |
Document ID | / |
Family ID | 37011944 |
Filed Date | 2008-08-07 |
United States Patent
Application |
20080187557 |
Kind Code |
A1 |
Sambhara; Suryaprakash ; et
al. |
August 7, 2008 |
Vaccine Against Pandemic Strains Of Influenza Viruses
Abstract
The present disclosure provides compositions and methods for
eliciting an immune response against avian or pandemic influenza.
The compositions include adenovirus vectors comprising avian
influenza antigens, recombinant adenovirus and immunogenic
compositions comprising such recombinant vectors and adenovirus.
Methods for eliciting an immune response against avian or pandemic
influenza involving administering such adenovirus vectors or
recombinant adenovirus are also provided.
Inventors: |
Sambhara; Suryaprakash;
(Atlanta, GA) ; Katz; Jacqueline; (Atlanta,
GA) ; Hoelscher; Mary; (Atlanta, GA) ; Mittal;
Suresh K.; (West Lafayette, IN) ; Bangari; Dinesh
S.; (West Lafayette, IN) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET, SUITE 1600
PORTLAND
OR
97204
US
|
Family ID: |
37011944 |
Appl. No.: |
11/911189 |
Filed: |
April 10, 2006 |
PCT Filed: |
April 10, 2006 |
PCT NO: |
PCT/US06/13384 |
371 Date: |
October 10, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60670826 |
Apr 11, 2005 |
|
|
|
Current U.S.
Class: |
424/233.1 ;
435/320.1; 435/326 |
Current CPC
Class: |
A61K 2039/5256 20130101;
C12N 2760/16134 20130101; C12N 2710/10343 20130101; A61K 39/12
20130101; A61K 39/145 20130101; A61P 31/16 20180101; C12N 15/86
20130101; A61K 2039/543 20130101; C12N 2800/108 20130101; A61K
2039/55505 20130101; C12N 2710/10352 20130101; C12N 2760/16122
20130101; C12N 7/00 20130101; C07K 14/005 20130101 |
Class at
Publication: |
424/233.1 ;
435/320.1; 435/326 |
International
Class: |
A61K 39/235 20060101
A61K039/235; C12N 15/861 20060101 C12N015/861; C12N 5/10 20060101
C12N005/10 |
Goverment Interests
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
[0002] Aspects of this disclosure were made with the support of the
United States Government pursuant to a grant from the National
Institutes of Health. The Government has certain rights in this
invention.
Claims
1. A recombinant adenovirus vector comprising a polynucleotide
sequence that encodes at least one antigen of an avian influenza
strain.
2. The recombinant adenovirus vector of claim 1, wherein the
polynucleotide sequence encodes an avian influenza HA antigen.
3. The recombinant adenovirus vector of claim 2, wherein the
polynucleotide sequence encodes a plurality of avian influenza HA
antigens.
4. The recombinant adenovirus vector of claim 3, wherein the
polynucleotide sequence encodes a plurality of variants of the same
HA antigen subtype.
5. The recombinant adenovirus vector of claim 3, wherein the
polynucleotide sequence encodes a plurality of HA antigens of
different HA subtypes.
6. The recombinant adenovirus vector of claim 1, wherein the at
least one polynucleotide sequence encodes an avian influenza NA
antigen.
7. The recombinant adenovirus vector of claim 1, wherein the avian
influenza antigen is an H.sub.5N.sub.1 strain antigen, an
H.sub.7N.sub.7 strain antigen, or an H.sub.9N.sub.2 strain
antigen.
8. The recombinant adenovirus vector of claim 1, wherein the
polynucleotide sequence encodes a plurality of influenza
antigens.
9. The recombinant adenovirus vector of claim 8, wherein the
polynucleotide sequence encodes at least one influenza internal
protein.
10. The recombinant adenovirus vector of claim 9, wherein the
influenza internal protein is and M1 protein, an M2 protein, an NP
protein, ma PB1 protein, a PB2 protein, and NS1 protein, and NS2
protein, or a combination thereof.
11. The recombinant adenovirus 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 adenovirus vector of claim 9, wherein the
internal protein is of a non-avian influenza strain.
13. The recombinant adenovirus vector of claim 9, wherein the
internal protein is of an H1N1, H2N2 or H3N2 influenza strain.
14. The recombinant adenovirus vector of claim 1, wherein the
adenovirus vector is a human adenovirus vector.
15. The recombinant adenovirus vector of claim 1, wherein the
adenovirus vector is a non-human adenovirus vector.
16. The recombinant adenovirus vector of claim 15, wherein the
non-human adenovirus vector is a porcine adenovirus vector, a
bovine adenovirus vector, a canine adenovirus vector, a murine
adenovirus vector, an ovine adenovirus vector, an avian adenovirus
vector or a simian adenovirus vector.
17. The recombinant adenovirus vector of claim 1, wherein the
adenovirus vector is a replication defective adenovirus vector.
18. The recombinant adenovirus vector of claim 17, wherein the
replication defective adenovirus comprises a mutation in at least
one of an E I region gene and an E3 region gene.
19. The recombinant adenovirus vector of claim 1, wherein the
adenovirus vector comprises a replication defective human
adenovirus vector comprising a polynucleotide sequence that encodes
an HA, an NA antigen, or both an HA antigen and an NA antigen of an
avian influenza strain and an M1 protein, an M2 protein, an NP
protein, or a combination of two or more of an M1 protein, an M2
protein and an NP protein of a second strain of influenza.
20. The recombinant adenovirus vector of claim 1, wherein the
adenovirus vector comprises a replication defective porcine or
bovine adenovirus vector comprising a polynucleotide sequence that
encodes an HA, an NA antigen, or both an HA antigen and an NA
antigen of an avian influenza strain an M1 protein, an M2 protein,
an NP protein, or a combination of two or more of an M1 protein, an
M2 protein and an NP protein of a second strain of influenza.
21. A recombinant adenovirus comprising the adenovirus vector of
claim 1.
22-34. (canceled)
35. An immunogenic composition comprising: the recombinant
adenovirus vector of claim 1 and a pharmaceutically acceptable
carrier.
36-38. (canceled)
39. An isolated cell comprising one or more heterologous nucleic
acids, which one or more heterologous nucleic acids comprises a
first polynucleotide sequence that encodes at least a first
adenovirus E protein and a second polynucleotide sequence that
encodes at least a second adenovirus E protein, wherein the first
and second adenovirus E proteins are selected from different
adenovirus strains, and wherein the cell supports the growth of a
plurality of strains of replication deficient human and/or
non-human adenoviruses.
40. The cell line of claim 39, wherein the at least first
adenovirus E protein and the at least second adenovirus E protein
are both E1 proteins.
41. The cell line of claim 39, wherein the at least first and
second adenovirus E proteins are from different adenovirus strains
with different species tropism.
42. The cell line of claim 41, wherein the at least first
adenovirus E protein comprises one or more human adenovirus E
proteins, and wherein the at least second adenovirus E protein
comprises one or more non-human adenovirus E proteins.
43. The cell line of claim 42, wherein the at least second
adenovirus 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 the at least second
adenovirus 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 producing an immune response against at least one
avian or pandemic strain of influenza in a subject, the method
comprising: administering to a subject an immunogenic
composition_comprising at least one of: a recombinant adenovirus
vector comprising a polynucleotide that encodes 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 strains of influenza.
49. The method of claim 48, wherein the immune response is
protective or partially protective against a plurality of avian or
pandemic strains of influenza.
50. The method of claim 47, wherein the immune response comprises
the production of neutralizing antibodies that bind an antigen of
at least one avian or pandemic strain of influenza.
51. The method of claim 47, wherein the immune response comprises
the production of neutralizing antibodies that bind antigens of a
plurality of avian or pandemic strains of influenza.
52. The method of claim 47, wherein the 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 an M1 protein, an M2 protein, an NP protein, or a
combination thereof.
54. The method of claim 53, wherein the T cell response is specific
for an epitope of the influenza internal protein that is conserved
among a plurality of influenza strains.
55. The method of claim 54, wherein the plurality of influenza
strains are 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 a domesticated
fowl.
61. The method of claim 60, comprising administering the
immunogenic composition in ovo to an embryonated egg.
62. The method of claim 60, comprising administering the
immunogenic composition in a spray or controlled droplet to a
plurality of birds in a common airspace.
63. The method of claim 60, comprising administering the
immunogenic composition in drinking water.
64. The method of claim 47, comprising administering an adenovirus
vector with a polynucleotide sequence that encodes 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 an HA antigen, an NA antigen or both an HA antigen and an
NA antigen of an avian influenza strain
66. The method of claim 64, wherein the plurality of antigens
comprises variants of the same HA antigen subtype.
67. The method of claim 64, wherein the plurality of antigens
comprises a plurality of HA antigens from different avian influenza
subtypes.
68. The method of claim 64, wherein the 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 the 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 producing a recombinant avian influenza virus
antigen, the method comprising: replicating in a cell an adenovirus
comprising at least one polynucleotide sequence that encodes an
avian influenza antigen.
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 the cell is a multifunctional
cell that expresses at least two different E proteins, wherein the
different E proteins are of at least two different strains of
adenovirus with different species tropism.
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 the antigen is selected from an
H5, an H7 or an H9 strain of influenza.
75. The method of claim 70, wherein the at least one polynucleotide
sequence encodes a plurality of influenza antigens.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application No. 60/670,826, filed Apr. 11, 2005, the specification
of which is incorporated by reference in its entirety.
FIELD
[0003] This application relates to the field of vaccines. More
specifically, this application concerns a recombinant vector for
the production of vaccines for avian influenza viruses.
BACKGROUND
[0004] Pandemic outbreaks of highly virulent avian influenza
present a serious risk to human and animal health worldwide.
Genetic reassortment between human and avian influenza viruses can
result in a virus with a novel hemagglutinin (HA) of avian origin,
against which humans lack immunity. In the 20.sup.th century, the
pandemics of 1918, 1957 and 1968 were the result of such antigenic
shifts. The recent outbreaks of avian influenza caused by H5N1,
H7N7 and H9N2 subtype influenza viruses, and their infection of
humans have created a new awareness of the pandemic potential of
influenza viruses that circulate in domestic poultry. The estimated
economic impact of a pandemic would be up to $165 billion in the
United States alone, with as many as 200,000 deaths, 730,000
hospitalizations, 42 outpatient visits, and 50 million additional
illnesses.
[0005] In the context of prevailing threats of global bioterrorism,
individuals deliberately infected with a highly virulent influenza
strain could act as difficult-to-detect biological weapons of mass
destruction.
[0006] To date, three major approaches to developing a safe and
effective vaccine against potentially pandemic avian influenza
strains have been attempted, none of which is entirely successful
(Wood et al., Vaccine 20:S84-S87, 2002; Stephenson et al., The
Lancet 4:499-509, 2004, and references cited therein).
[0007] Due to the lethality of these influenza strains in poultry,
current vaccine production strategies involving growth of virus in
hen's eggs are not feasible. Some approaches have focused on
isolating non-pathogenic or attenuated strains of influenza that
express the relevant immunogenic antigens of the potentially
pandemic influenza strains. For example, naturally occurring,
apathogenic strains of influenza with the H5 subtype antigen virus
have been evaluated as vaccine candidates. In general, these
viruses have proved difficult to grow using conventional
technology, and protection is dependent on the ability of
antibodies raised against the apathogenic vaccine strain to
cross-react with the virulent strain of virus (Takada et al., J.
Virol. 73:8303-8307, 1999; Wood et al., Vaccine 18:579-80, 2000). A
reverse genetics approach has been employed to delete a stretch of
basic amino acids at the cleavage site of the HA antigen of a
pathogenic H5N1 virus (A/HK/97) to develop a candidate vaccine (Li
et al., J. Infect. Dis. 179:1132-1138, 1999).
[0008] Another approach has been to utilize recombinant HA ("H5")
produced in a baculovirus expression system. However, high doses of
purified protein and the use of adjuvants are required to achieve a
satisfactory immune response. (Treanor et al. Vaccine 19:1732-1737,
2001).
[0009] There remains a need to develop vaccines that are protective
against infection by avian influenza strains in both human and
non-human populations, and which can be efficiently produced and
administered without reliance on viral growth in hen's eggs. The
present disclosure addresses this need, and provides novel
compositions and methods useful for preventing infection by avian
and pandemic influenza strains.
SUMMARY
[0010] The present disclosure relates to methods for eliciting a
protective immune response against potentially pandemic strains of
influenza, and to compositions, including nucleic acid vectors and
non-infectious viruses useful in the methods disclosed herein.
[0011] One aspect of the disclosure relates to recombinant nucleic
acids. The recombinant nucleic acids described herein include
adenovirus vectors. The adenovirus vectors, including human and
non-human adenovirus vectors, contain polynucleotide sequences that
encode one or more polypeptides that correspond to antigens of
avian influenza strains.
[0012] In another aspect, the disclosure provides a recombinant
adenovirus, for example, a replication defective human or non-human
adenovirus that expresses one or more avian influenza antigen.
[0013] Pharmaceutical compositions, including vaccine compositions,
are disclosed that contain the adenovirus vectors and/or the
recombinant adenoviruses disclosed herein.
[0014] Another feature of the disclosure relates to methods of
producing an immune response against avian and/or pandemic strains
of influenza. In the methods disclosed herein, immunogenic
compositions based on adenovirus vectors encoding and/or
recombinant adenoviruses expressing at least one antigen of an
avian influenza strain are administered to a subject prior to or
following exposure to an avian or pandemic strain of influenza.
[0015] The foregoing and other objects, features, and advantages of
the invention will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1A schematically illustrates the HAd5-H5HA vector. The
Cre recombinase-mediated site-specific recombination system was
used to generate a HAd5 vector expressing HA of avian influenza
virus (H.sub.5N.sub.1) A/HK/156/97. The HA gene under the control
of the CMV promoter was inserted at the StuI site in a shuttle
vector (pDC311--a plasmid containing the left end of HAd5 (4 kb)
with 3.1 kb E1 deletion, a loxP site for site specific
recombination in the presence of Cre recombinase and an intact
packaging signal to produce pDC311-H5. 293Cre cells (293 cells
expressing Cre recombinase) were cotransfected with pDC311-H5HA and
pBHGlox.DELTA.E1,3Cre (plasmid containing almost the entire HAd5
genome except the packaging signal, E1 and E3 deletions, a loxP
site for site specific recombination in the presence of Cre
recombinase) to generate HAd-H5HA vector. FIG. 1B is a western blot
illustrating expression of hemagglutinin (HA) encoded by the
HAd5-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 HAd-H5 HA vector
(B). 293Cre cells were mock-infected or infected with
HAd-.DELTA.E1E3 or HAd-H5HA. At 24 h post-infection cells were
harvested and cell extracts were prepared. Cell extracts were
analyzed by Western blot using a rabbit H5HA-specific sera
generated by immunizing rabbits with a DNA vector encoding
H5HA.
[0017] FIG. 2 is a line graph illustrating that the HAd-H5HA vector
confers complete protection against challenge with a highly
pathogenic homotypic H.sub.5N1 (A/HK/483/97) virus. Twenty-five
(6-to 8-week-old) female BALB/c mice were randomly divided into 5
groups (5 animals/group) and inoculated intramuscularly on days 0
and 28 with either PBS (.quadrature.), 10 .mu.g of recombinant H5HA
(hemagglutinin of avian HK/156/97 influenza virus expressed in
baculovirus) without alum 10 .mu.g of purified H5HA with alum
(.times.), 10.sup.8 p.f.u. of HAd-.DELTA.E1E3 (.DELTA.), or
10.sup.8 p.f.u. of HAd-H5HA (.box-solid.). The animals were
challenged with 100 LD.sub.50 of H.sub.5N1 (A/HK/483/97) virus on
day 70. The mice were monitored for clinical signs and changes in
body weights daily up to 14 days post-challenge.
[0018] FIGS. 3A-C are Box-whisker plots showing mean, IQRs, and
range of the neutralizing antibody response against homologous and
heterologous avian influenza virus strains in mice immunized with
HAd-H5HA vaccine. (A) HK/156/97, (B) HK/213/03, and (C) VN/1203/04
strains. *, # and $: differences between marked data are p=0001.
im=intramuscular immunization. in=intranasal immunization.
[0019] FIG. 4 is a series of scatter plots of flow cytometric
analysis showing induction of HA-518-epitope-specific CD8 T cells
in mice immunized with HAd-H5HA vaccine. Flow cytometric analysis
of spleen cells from immunized mice (three per group) stained with
HA 518 pentamer epitope. Pentamer-positive cells (circled) are
shown as a percentage of CD8 T lymphocyte population.
[0020] FIG. 5 is a bar graph illustrating interferon gamma
secretion by HA-518-epitope-specific CD8 T cells. ELISpots
measurements of interferon gamma in spleen cells of immunized mice.
Data are mean and SDs (error bars). im=intramuscular immunization.
in=intranasal immunization. Cultures were stimulated with
NP.sub.147-155 (diagonally hatched bars), HA (white bars), or
phorbol myristate acetate (PMA)+ ionomycin (black bars).
[0021] FIGS. 6A-D are line graphs and FIG. 6E is a bar graph
illustrating protection against recent H.sub.5N.sub.1 viruses.
BALB/C mice (15 animals/group) were inoculated intramuscularly ( )
or intranasally (.smallcircle.) twice at a four weeks interval with
10.sup.8 p.f.u. of HAd-H5HA. HAd-.DELTA.E1E3 (.box-solid.) served
as a negative control. Five animals from each group were challenged
four weeks after the second immunization with 100 50% lethal dose
(LD.sub.50) of A/HK/483/97 (A and B) or A/VN/1203/04 (C and D) or
100 50% mouse infectious dose (MID.sub.50) of A/HK/213/03 (E). The
percent initial body weight (A and C) and survival post-challenge
(B and D) are shown. Error bars depict the standard error of the
mean. The mice challenged with A/HK/483197 or A/VN/1203/04 were
monitored for clinical signs and changes in body weights daily up
to 14 days post-challenge. The mice challenged with A/HK/213/03
were euthanized on day 3 post-challenge and lungs were collected.
Tissues were frozen and thawed once, then homogenized in 1 mL PBS
with antibiotics. Solid debris was pelleted by brief centrifugation
before homogenates were titered for virus infectivity in 11-day old
eggs.
[0022] FIGS. 7A-D are schematic illustrations (A and C) and images
of western blots (B and D) demonstrating expression of avian
influenza hemagglutinin from porcine and bovine adenovirus vectors.
PAd vector (PAd-H5HA) carrying the hemagglutinin subtype 5 (H5HA)
gene of avian H.sub.5N1 influenza virus (A/HK/156/97) under the
control of the cytomegalovirus (CMV) immediate early promoter
inserted in the early region 1 (E1) of the PAd genome was
generated. (A) Diagrammatic representation of structures of PAd
vectors: PAd-.DELTA.E1E3 (PAd with E1 and E3 deletions), and
PAd-H5HA (PAd-.DELTA.E1E3 with the H5HA gene cassette). ITR,
inverted terminal repeat; .DELTA.E1, deletion in E1; .DELTA.E3,
deletion in the E3; H5, H5HA; and pA, simian virus 40
polyadenylation signal. (B) FPRT HE1-5 cells were mock-infected or
infected with PAd-.DELTA.E1E3 or PAd-H5HA. At 24 h post-infection,
cells were harvested and cell extracts were analyzed by Western
blot using a rabbit hyperimmune serum against H5HA. BAd vector
(BAd-H5HA) carrying the hemagglutinin subtype 5 (H5HA) gene of
avian H.sub.5N1 influenza virus (A/HK/156/97) under the control of
the cytomegalovirus (CMV) immediate early promoter inserted in the
early region 1 (E1) of the BAd genome was generated. (C)
Diagrammatic representation of structures of BAd vectors:
BAd-.DELTA.E1E3 (PAd with E1 and E3 deletions), and BAd-H5HA
(BAd-.DELTA.E1E3 with the H5HA gene cassette). ITR, inverted
terminal repeat; .DELTA.E1, deletion in E1; .DELTA.E3, deletion in
the E3; H5, H5HA; and pA, simian virus 40 polyadenylation signal.
(D) FBRT-HE1 cells were mock-infected or infected with
BAd-.DELTA.E1E3 or BAd-H5HA. At 24 h post-infection, cells were
harvested and cell extracts were analyzed by Western blot using a
rabbit hyperimmune serum against H5HA.
[0023] FIGS. 8A and B are images showing expression of bovine and
porcine E1 genes in transfected cells. (A) Expression of BAd3 E1A,
E1B-19 kDa (E1B-1), and E1B-55 kDa (E1B-2) messages as detected by
RT-PCR using specific primer sets. (B) Expression of PAd3 E1A,
E1B-19 kDa (E1B-1), and E1B-55 kDa (E1B-2) messages as detected by
RT-PCR using specific primer sets. Specific bands are shown by
arrows.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0024] SEQ ID NO: 1 (5'-tccatgagcttcctgatcct-3') is an
immunostimulatory oligonucleotide.
[0025] SEQ ID NO:2 (5'-tccatgacgttcctgacgtt-3') is an
immunostimulatory oligonucleotide.
[0026] SEQ ID NO:3 (5'-tgactgtgaacgttcgagatga-3)' is an
immunostimulatory oligonucleotide.
[0027] SEQ ID NO:4 is the nucleotide sequence of HAd5 E1.
[0028] SEQ ID NOs:5 and 6 are oligonucleotide primers for the
amplification of bovine adenovirus E1.
[0029] SEQ ID NO:7 is the nucleotide sequence of BAd3 .mu.l.
[0030] SEQ ID NOs:8-13 are oligonucleotide primers for detection of
BAd3 E1 transcripts.
[0031] SEQ ID NOs:14 and 15 are oligonucleotide primers for the
amplification of porcine adenovirus E1.
[0032] SEQ ID NO:16 is the nucleotide sequence of PAd3 E1.
[0033] SEQ ID NOs:17-22 are oligonucleotide primers for the
detection of PAd3 E1 transcripts.
DETAILED DESCRIPTION
Introduction
[0034] Influenza viruses are enveloped negative-sense viruses
belonging to the Orthomyxoviridae family. Influenza viruses are
classified on the basis of their core proteins into three distinct
types: A, B, and C. Within these broad classifications, subtypes
are further divided based on the characterization of two antigenic
surface proteins hemagglutinin (HA) and neuraminidase (NA). While B
and C type influenza viruses are largely restricted to humans,
influenza A viruses are pathogens of a wide variety of species
including humans, non-human mammals, and birds. Periodically,
non-human strains, particularly of avian influenza, have infected
human populations, in some cases causing severe disease with high
mortality. Recombination between such avian strains and human
strains in conflicted individuals has given rise to recombinant
influenza viruses to which immunity is lacking in the human
population, resulting in influenza pandemics. Three such pandemics
occurred during the twentieth century, in 1918, 1957, and 1968,
resulting in numerous deaths world-wide.
[0035] Highly pathogenic avian influenza H.sub.5N.sub.1 viruses
have become endemic in domestic poultry in Southeast Asia. Since
early 2004, human infections with H.sub.5N1 viruses have been
reported in the region with increasing frequency and high mortality
rates. Highly pathogenic H.sub.5N1 influenza viruses were first
recognized to cause respiratory disease in humans in 1997, when 18
documented cases, including 6 deaths, occurred following outbreaks
of influenza in poultry farms and markets in Hong Kong. Two
additional human H.sub.5N.sub.1 infections were identified in a
family in Hong Kong in 2003. Since then, H.sub.5N1 viruses have
spread to 9 Asian countries, and recently have expanded their
geographical distribution to several countries in Eastern Europe.
Over 120 laboratory confirmed cases of human infection with a
fatality rate of greater than 50% have been reported to the World
Health Organization since January 2004. To date, the majority of
human H.sub.5N1 virus infections have been due to direct
transmission of the virus from infected poultry, although
exceptional cases of human-to-human transmission have been
reported. Genetic reassortment between a human and avian influenza
virus and/or mutations in the avian H.sub.5N1 virus genome may
result in the generation of a novel influenza virus of the H5
subtype that may initiate a pandemic if it has acquired the ability
to undergo sustained transmission in an immunologically naive human
population. Therefore, effective vaccines against highly pathogenic
H.sub.5N.sub.1 and other avian influenza strains are urgently
needed.
[0036] Vaccines developed and evaluated in response to the 1997
outbreak of H.sub.5N.sub.1 influenza were only modestly immunogenic
in humans, and the H.sub.5N1 viruses isolated from humans in 2004
were genetically and antigenically distinct from those isolated
previously in 1997 and 2003, necessitating the development of new
vaccine candidates because the elicited immune response was not
protective against antigenically distinct viral strains.
[0037] Non-pathogenic avian influenza viruses, either produced from
naturally occurring apathogenic strains that share an HA subtype
with a pathogenic strain, or that have been engineered to be
apathogenic by deletion of a spontaneous protein cleavage site have
thus far been produced only in hen's eggs. In the event of a
world-wide pandemic, infection of domestic fowl is likely to be
widespread requiring the killing of chickens and resulting in a
shortage of eggs to be used for vaccine production. Recombinant HA
vaccines have been evaluated, but require potentially detrimental
adjuvants for efficient protection. Thus, diversification of
vaccine manufacturing substrates including cell-based and/or
recombinant DNA technologies is desirable to enhance vaccine
production capacity in a pandemic situation. Furthermore,
recombinant DNA technologies are advantageous in accelerating
vaccine availability, as cloning and expression of one or more
viral genes can begin as soon as the viral sequence is known.
[0038] The present disclosure provides novel compositions and
methods for producing influenza vaccines and vaccinating human,
non-human mammals and avian populations against avian and/or
pandemic strains of influenza virus and overcoming the poor
immunogenicity and manufacturing drawbacks of currently available
influenza vaccines, which have been adapted to elicit an immune
response against avian strains of influenza. The compositions and
methods described herein are based on adenovirus vectors that
express one or more immunogenic avian influenza antigen, optionally
in combination with internal proteins that further enhance the
immune response, reduce morbidity and facilitate recovery following
exposure or infection by avian or pandemic influenza strains. This
disclosure provides the first evidence that human and non-human
adenoviruses are effective vectors for eliciting an immune response
against avian (and potentially pandemic) influenza strains.
Additionally, the compositions and methods described herein offer
several benefits over strategies that have previously been
evaluated as vaccines for potentially pandemic strains of avian
influenza virus. For example, the adenovirus vectors and adenovirus
described herein can readily be grown in tissue culture, and
purified at a scale suitable for commercial manufacture.
Furthermore, the immune response elicited is robust and long
lasting, and depending on the combination of antigens, involves
both neutralizing antibody production and T cell responses, and is
protective against antigenically distinct strains of influenza.
DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0039] One aspect of the present disclosure relates to recombinant
adenovirus vectors that include polynucleotide sequences that
encode one or more influenza antigens. In particular, the
adenovirus vectors described herein include a polynucleotide
sequence that encodes at least one antigen of an avian influenza
strain (an "avian influenza antigen"). For example, the adenovirus
vector can encode one or more avian hemagglutinin ("HA") antigens.
The vector can include a sequence that encodes a single HA of an
avian influenza strain, such as an H5 subtype strain, an H7 subtype
strain or an H9 subtype strain, e.g., selected from H.sub.5N1,
H.sub.7N.sub.7 or H.sub.9N.sub.2 strains prevalent in recent
outbreaks of avian influenza. Alternatively, the vector can encode
a plurality (more than one) of avian HA antigens. In this case, the
HA antigens encoded can be variants of one subtype (for example,
variants of H5 HA, or variants of H7 HA, or variants of H9 HA) or
the HA antigens can be HA antigens of different subtypes (that is,
a combination of H5, H7 and/or H9, e.g., H5 and H7, H5 and H9, H7
and H9, or H5, H7 and H9, including one or more than one HA antigen
of any subtype in combination with one or more than one HA antigen
of any other subtype).
[0040] The recombinant adenovirus vector can also include a
polynucleotide sequence that encodes an avian influenza
neuraminidase ("NA") antigen. The NA antigen can be encoded by the
vector alone, or in combination with an avian HA antigen. When a
vector includes a polynucleotide sequence that encodes both an
avian HA antigen and an avian NA antigen, the HA and NA antigens
can be of the same strain of influenza, or can be selected from
different avian strains of influenza. For example, recent outbreaks
of avian influenza in human populations in Asia have been caused by
H.sub.5N.sub.1, H.sub.7N.sub.7 and H.sub.9N.sub.2 strains of
influenza A. An adenovirus vector can encode, e.g., an N1 subtype
NA, an N7 subtype NA, or an N2 subtype of NA. Alternatively, the
vector can encode a different NA subtype, such as N3 (e.g.,
corresponding to an apathogenic H.sub.5N.sub.3 strain avian
influenza virus). One of skill in the art will appreciate that any
avian HA subtype (most commonly, H5, H7 or H9) can be combined with
any of 9 NA subtypes in an adenovirus vector. As described above
with respect to HA antigens, an adenovirus vector can encode a
plurality of NA antigens, which can be variants of a single NA
subtype or representative of different NA subtypes.
[0041] As indicated above, the adenovirus vectors can encode a
plurality of influenza antigens. The plurality of influenza
antigens can encode two or more avian influenza antigens, such as
multiple avian HA antigens, multiple avian NA antigens, or a
combination of avian HA antigens and NA antigens. Alternatively,
the vectors can encode 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 can be selected from the
same or a different strain of avian influenza. The encoded internal
protein can also be selected from a non-avian strain of influenza,
such as a human strain of influenza (typically, influenza A). For
example, the internal protein or proteins can be selected from a
H1N1, H.sub.2N.sub.2 or H.sub.3N.sub.2 influenza strain. Any of the
internal proteins (M1, M2, NP, PB1, PB2, NS1, and NS2) can be
encoded by the adenovirus vector. A combination of internal
proteins can also be encoded by the vector. For example, the vector
can encode an M protein (one or more of M1 and/or M2), an NP
protein or both an M and an NP protein.
[0042] Human and non-human adenovirus 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 ("HAd5") vectors.
Alternatively, the adenovirus vectors are non-human, such as
porcine or bovine, adenovirus vectors (for example, BAd3 and PAd3
vectors). Typically, the adenovirus vector is a replication
defective adenovirus that is incapable of multiple cycles of
transcription and translation of the inserted genes in human or
animal cells. The replication defective adenovirus vectors can have
mutations in one or more gene (or region) involved in replication,
including one or more of an E1 region gene, an E3 region gene, an
E2 region gene, and/or an E4 region gene. For example, a
replication defective adenovirus vector can have a deletion or
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.
[0043] Thus, in one exemplary embodiment, the adenovirus vector is
a replication defective human adenovirus vector that includes a
polynucleotide sequence that encodes an avian influenza HA antigen,
an avian influenza NA antigen, or both an avian HA antigen and an
avian NA antigen. Optionally, the adenovirus vector encodes a
plurality of avian HA antigens, which are variants of a single HA
subtype or are different HA subtypes. In certain embodiments, the
adenovirus vector also encodes at least one influenza internal
protein, such as an M1 protein, an M2 protein, an NP protein or a
combination of M and NP proteins.
[0044] In another embodiment, the adenovirus vector is a
replication defective non-human adenovirus vector, such as a
porcine or bovine adenovirus vector, that includes a polynucleotide
sequence that encodes an avian influenza HA antigen, an avian
influenza NA antigen, or both an avian HA antigen and an avian NA
antigen. Optionally, the adenovirus vector encodes a plurality of
avian HA antigens, which are variants of a single HA subtype or are
different HA subtypes. In certain embodiments, the adenovirus
vector also encodes at least one influenza internal protein, such
as an M1, M2 protein, an NP protein or any combination thereof.
[0045] Another aspect of the disclosure relates to a recombinant
adenovirus that expresses (includes) at least one antigen of an
avian influenza strain. Commonly, the adenovirus expresses an avian
HA antigen and/or an avian NA antigen. Thus, the adenovirus can
include an avian HA antigen, e.g., an H5 HA antigen, an H7 HA
antigen, and/or an H9 HA antigen. Similarly, the adenovirus can
include an avian NA antigen, e.g., an H1 NA antigen, an H.sub.7NA
antigen, and/or an H.sub.2NA antigen. In some instances, the
adenovirus expresses a plurality of avian influenza antigens, such
as a plurality of avian HA antigens or a plurality of avian NA
antigens or a combination of avian HA and NA antigens. For example,
the adenovirus can express two or more variants of a single HA (or
NA) subtype. Alternatively, the adenovirus can 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 an M1, M2 and/or NP protein. Where an adenovirus expresses a
plurality of influenza antigens, the multiple antigens can be of
the same strain or subtype, or a different strains or subtypes.
[0046] The recombinant adenovirus can be either 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 defective
adenovirus can have a mutation (e.g., a deletion, addition or
substitution) in an E1 region gene, an E3 region gene, an E2 region
gene and/or an E4 region gene.
[0047] Such adenovirus vectors and adenovirus are useful for a
variety of purposes. For example, such adenovirus and adenovirus
vectors are useful for producing recombinant avian (and other)
influenza antigens in vitro and in vivo (including in ovo).
Accordingly, methods for producing recombinant avian influenza
antigens are a feature of this disclosure. For example, recombinant
avian influenza antigens can be produced by replicating adenovirus
that include at least one heterologous polynucleotide sequence that
encodes an avian influenza virus antigen. In some embodiments, the
adenovirus includes sequences that encode 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 can be an HA antigen or an NA antigen, such
as an HA or NA antigen selected from an H5, an H7 or an H9 strain
of influenza. In certain embodiments, the adenovirus contain
polynucleotide sequences that encode a plurality of influenza
antigens.
[0048] In certain embodiments, adenovirus expressing recombinant
avian influenza virus antigens are produced 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 complementary replication function, such as a
heterologous nucleic acid that encodes one or more E proteins that
are deleted from the replication defective adenovirus vector. In
certain embodiments, the cells that are capable of supporting
growth of the replication defective adenovirus vector are capable
of supporting growth of different strains of adenovirus with
different species tropism. Optionally, the recombinant avian
influenza virus antigen is isolated, and for example, used to
produce immunogenic compositions, such as vaccines.
[0049] Another aspect of the disclosure relates to cell lines that
support the replication of multiple strains of replication
defective adenovirus having different tropisms. Such
multi-functional cell lines include heterologous nucleic acids that
encode multiple E proteins of different strains of adenovirus. In
exemplary embodiments the cell lines include one or more
heterologous nucleic acids that include 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
proteins are selected to complement those deleted from recombinant
adenovirus vectors that are to be grown in the cell lines. Thus, in
some embodiment, where the cells are intended to support growth of
replication defective adenovirus lacking one or more E1 proteins,
the polynucleotide sequence encodes the corresponding E1
protein(s). Similarly, where the cells are intended to support
growth of replication defective adenovirus lacking one or more E3
proteins, the polynucleotide sequence encodes the corresponding E3
protein(s). Typically, the cells include nucleic acids that encode
E proteins of strains of adenovirus with different species tropism
so that the cells optimally support growth of multiple strains of
adenovirus with different species tropisms, such as a human
adenovirus E gene (or fragment thereof) and a non-human E gene (or
fragment thereof). In specific examples, the polynucleotide
sequences encode human and either bovine or porcine E proteins.
[0050] The adenovirus vectors and recombinant adenoviruses
disclosed herein are useful in the context of immunogenic
compositions, including vaccines. Such immunogenic compositions can
include an adenovirus vector with a polynucleotide sequence that
encodes at least one avian influenza antigen as previously
discussed. The immunogenic compositions can also include
recombinant adenoviruses that express at least one avian influenza
antigen as discussed above. The immunogenic compositions include,
in addition to the adenovirus vectors and/or adenoviruses, a
pharmaceutically acceptable carrier or excipient. Thus, the
immunogenic compositions can include any of the adenovirus vectors
and/or adenoviruses encoding or expressing avian influenza antigens
as disclosed herein. Optionally, the immunogenic compositions
include an adjuvant, immunostimulatory molecule, miroparticle, or
nanoparticile.
[0051] The present disclosure also provides methods for eliciting
or producing an immune response against an avian or pandemic (or
potentially pandemic) strain of influenza. The methods disclosed
herein involve administering at least one adenovirus or adenovirus
vector that expresses (or encodes) an avian influenza antigen to a
subject. Typically, the adenovirus or adenovirus 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 adenovirus vector expressing the avian
influenza antigen is administered prophylactically, e.g., as a
vaccine.
[0052] Administration of the recombinant adenoviruses and
adenovirus vectors can elicit an immune response that protects the
subject from serious disease or death due to infection by one or
more avian or pandemic strains of influenza. In some cases, the
immune response protects against disease caused by more than one
strain of influenza, such as multiple strains of avian influenza.
Typically, the immune response includes neutralizing antibodies
that bind to at least one avian influenza antigen, and can, in some
cases, cross-react with multiple strains of avian influenza. For
example, administration of an adenovirus or adenovirus vector that
expresses an avian influenza HA antigen can elicit neutralizing
antibodies against the subtype of HA that protect (or partially
protect) against infection with influenza of the corresponding HA
subtype. Similarly, administration of an adenovirus or adenovirus
vector that expresses an avian NA antigen can elicit an antibody
response against the subtype of NA.
[0053] In some cases, an adenovirus or adenovirus vector is
administered that expresses (or encodes) two or more avian HA
and/or NA antigens. Alternatively, multiple adenovirus or
adenovirus vectors, each of which includes a single avian influenza
antigen, are administered. Whether administered in a single virus
or vector, or in multiple viruses or vectors, the antigens, e.g.,
the HA antigens, can be variants of the same antigen subtype or can
be antigens of different influenza subtypes. Optionally, the one
(or several) adenoviruses or vectors results in the expression of
an influenza structural or nonstructural protein, such as an M1,
M2, NP, or NS1 protein. As is the case for the HA and NA antigens,
the internal proteins can be included within one or more than one
adenovirus or vector. Inclusion of one or more internal protein
antigens is useful in augmenting the immune response generated by a
vaccine by enhancing the production of influenza specific T cells.
Thus, an immune response elicited by the compositions described
herein can include a T cell response. Generally, the T cell
response is specific for an epitope of the influenza internal
protein that is conserved among a plurality of influenza strains,
such as multiple strains of the same or different subtype of
influenza.
[0054] For example, in one embodiment, a replication defective
adenovirus that expresses a single avian HA antigen, such as an H5
HA antigen, an H7 HA antigen, or an H9 antigen, is administered. In
another embodiment, an adenovirus that expresses two or more avian
HA antigens is administered In another embodiment, an adenovirus
that expresses at least one avian HA antigen and at least one avian
NA antigen is administered. In another embodiment, the adenovirus
expresses at least one avian HA antigen, at least one avian NA
antigen, and one or more influenza internal proteins (such as the
M1, M2 and/or NP proteins) of an avian or non-avian strain of
influenza. An exemplary embodiment of such an adenovirus is an
adenovirus that expresses an H5 avian HA, an N1 avian NA, and human
M (M1 and/or M2) and/or NP proteins. Numerous other examples can be
determined by those of ordinary skill in the art based on the
strain or strains against which an immune response is desired.
[0055] In other embodiments, a plurality of different human or
nonhuman adenoviruses are administered, each of which includes one
or more influenza antigens. For example, multiple adenoviruses can
be administered at the same or different times (that is, in one or
more immunogenic compositions delivered simultaneously or
sequentially). In some cases, multiple adenoviruses are
administered in a "cocktail." When administered in a cocktail each
adenovirus can express a single influenza antigen, or some or all
of the adenoviruses can express multiple influenza antigens. For
example, in one favorable cocktail an adenovirus that expresses an
H5 avian HA antigen is administered along with an adenovirus that
expresses an M2 protein. In another example, an adenovirus that
expresses an H5 avian HA antigen and an N1 avian NA antigen is
administered in combination with a human influenza M2 and/or NP
protein. In another exemplary embodiment, a first adenovirus that
expresses a plurality of avian HA antigens (such as variants of a
single HA subtype, or corresponding to different HA subtypes) is
administered along with a second adenovirus that expresses an avian
or human M2 and/or NP protein. Numerous other combinations can be
determined by those of ordinary skill in the art.
[0056] In other embodiments, adenovirus vectors rather than
recombinant adenoviruses are administered to the subject in the
form of nucleic acid (e.g., DNA) vaccines.
[0057] Optionally, the adenovirus or adenovirus vector is
administered to the subject in an immunogenic composition that
includes, in addition to a pharmaceutically acceptable carrier or
excipient, at least one additional immunostimulatory component.
Such immunostimulatory components include adjuvants, such as MF 59
and aluminum adjuvants. Alternatively, naturally occurring or
synthetic immunostimulatory compositions that bind to and stimulate
receptors involved in innate immunity can be administered along
with the adenoviruses and/or adenovirus vectors. For example,
agents that stimulate certain Toll-like receptors (such as TLR7,
TLR8 and TLR9) can be administered in combination with the
adenovirus recombinants and/or adenovirus vectors that express
influenza antigens. In some embodiments, the adenovirus or vector
is administered in combination with immunostimulatory CpG
oligonucleotides. In other embodiments, the adenovirus or vector is
administered in combination with additional adenovirus vectors that
express a Toll-like receptor and a ligand that results in
activation of the receptor, respectively.
[0058] The immunogenic compositions described herein can be
administered to human or non-human (for example, cats, dogs, pigs,
birds) subjects to elicit an influenza specific immune response.
For example, if the subject is a human subject, a human, bovine or
porcine adenovirus recombinant (or adenovirus vector) including one
or more avian influenza antigens can be administered to elicit an
immune response. Additionally, the compositions and methods
described herein can be utilized to elicit influenza specific
immune responses in avian subjects, including domesticated fowl,
such as chickens, ducks, guinea fowl, turkeys, geese and the like.
Additionally, the compositions and methods described herein can be
utilized to elicit influenza specific immune responses in non-human
subjects, including cats, dogs, horses, non-human primates, and
other domestic and wild mammals.
[0059] A variety of routes of administration are suitable for
administering the immunogenic compositions described herein. Such
methods include intranasal, oral, ocular, intravenous,
intramuscular, transdermal, intradermal and subcutaneous delivery
of the adenoviruses and/or adenovirus vectors. In specific
embodiments, the immunogenic compositions are administered to
domesticated fowl, in their drinking water, by spray or controlled
droplet or in ovo prior to hatching.
[0060] Additional technical details are provided under the specific
topic headings below.
Terms
[0061] Unless otherwise explained, 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.
Definitions of common terms in molecular biology may be found in
Benjamin Lewin, Genes V, published by Oxford University Press, 1994
(ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of
Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN
0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and
Biotechnology: a Comprehensive Desk Reference, published by VCH
Publishers, Inc., 1995 (ISBN 1-56081-569-8).
[0062] The singular terms "a," "an," and "the" include plural
referents unless context clearly indicates otherwise. Similarly,
the word "or" is intended to include "and" unless the context
clearly indicates otherwise. It is further to be understood that
all base sizes or amino acid sizes, and all molecular weight or
molecular mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of this disclosure, suitable
methods and materials are described below. The term "comprises"
means "includes." The abbreviation, "e.g." is derived from the
Latin exempli gratia, and is used herein to indicate a non-limiting
example. Thus, the abbreviation "e.g." is synonymous with the term
"for example."
[0063] In order to facilitate review of the various embodiments of
this disclosure, the following explanations of specific terms are
provided:
[0064] Adjuvant: A vehicle used to enhance antigenicity; such as a
suspension of minerals (alum, aluminum hydroxide, aluminum
phosphate) on which antigen is adsorbed; or water-in-oil emulsion
in which antigen solution is emulsified in oil (MF-59, Freund's
incomplete adjuvant), sometimes with the inclusion of killed
mycobacteria (Freund's complete adjuvant) to further enhance
antigenicity (inhibits degradation of antigen and/or causes influx
of macrophages).
[0065] Antigen: A compound, composition, or substance that can
stimulate the production of antibodies or a T cell response in an
animal, including compositions that are injected, absorbed or
otherwise introduced into an animal. The term "antigen" includes
all related antigenic epitopes. An "antigenic polypeptide" is a
polypeptide to which an immune response, such as a T cell response
or an antibody response, can be stimulated. "Epitope" or "antigenic
determinant" refers to a site on an antigen to which B and/or T
cells respond. In one embodiment, T cells respond to the epitope
when the epitope is presented in conjunction with an MHC molecule.
Epitopes can be formed both from contiguous amino acids or
noncontiguous amino acids juxtaposed by tertiary folding of an
antigenic polypeptide. Epitopes formed from contiguous amino acids
are typically retained on exposure to denaturing solvents whereas
epitopes formed by tertiary folding are typically lost on treatment
with denaturing solvents. An epitope typically includes at least 3,
and more usually, at least 5, about 9, or about 8-10 amino acids in
a unique spatial conformation. Methods of determining spatial
conformation of epitopes include, for example, x-ray
crystallography and multi-dimensional nuclear magnetic resonance
spectroscopy.
[0066] An influenza antigen can be a polymorphic hemagglutinin (HA)
or neuraminidase (NA) antigen selected from an influenza strain to
which an immune response is desirable or a related strain that
shares antigenic epitopes. An influenza antigen can also be an
influenza internal protein, such as a PB1, PB2, PA, M1, M2, NP, NS1
or NS2 protein. An avian influenza antigen is an influenza antigen
of an avian influenza strain. A variant of an influenza antigen can
be a naturally occurring variant or an engineered variant. As used
herein, the term "variant" refers to a protein (e.g. an antigen)
with one or more amino acid alterations, such as deletions,
additions or substitutions, relative to a reference protein or with
respect to another variant.
[0067] Antibody: Immunoglobulin molecules and immunologically
active portions of immunoglobulin molecules, that is, molecules
that contain an antigen binding site that specifically binds
(immunoreacts with) an antigen. A naturally occurring antibody
(e.g., IgG, IgM, IgD) includes four polypeptide chains, two heavy
(H) chains and two light (L) chains interconnected by disulfide
bonds. The phrase "antibody response" refers to an immunological
response against an antigen involving the secretion of antibodies
specific for the antigen. An antibody response is a B cell mediated
immune response initiated through the interaction of an antigen (or
epitope) with a B cell receptor (membrane bound IgD) on the surface
of a B cell. Following binding of the stimulation of 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. "Neutralizing antibodies" are
antibodies that bind to an epitope on a virus inhibiting infection
and/or replication as measured, e.g., in a plaque neutralization
assay.
[0068] Animal: Living multi-cellular vertebrate organisms, a
category that 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 (such as dogs and cats), livestock (such as pigs,
sheep, cows), as well as non-domesticated animals, such as the big
cats. The term subject applies regardless of the stage in the
organism's life-cycle. Thus, the term subject applies to an
organism in utero or in ovo, depending on the organism (that is,
whether the organism is a mammal or a bird, such as a domesticated
or wild fowl).
[0069] cDNA (complementary DNA): A piece of DNA lacking internal,
non-coding segments (introns) and regulatory sequences that
determine transcription. cDNA is typically synthesized in the
laboratory by reverse transcription from messenger RNA extracted
from cells. In the context of preparing adenovirus vectors
including polynucleotide sequences that encode influenza antigen, a
cDNA can be prepared, for example by reverse transcription or
amplification (e.g., by the polymerase chain reaction, PCR) from a
negative stranded influenza RNA genome (or genome segment).
[0070] Host cells: Cells in which a polynucleotide, for example, a
polynucleotide vector or a viral vector, can be propagated 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 the parental
cell since there may be mutations that occur during replication.
However, such progeny are included when the term "host cell" is
used. Thus, the adenovirus vectors described herein can be
introduced into host cells where their polynucleotide sequences
(including those encoding influenza antigen(s)) can be expressed,
e.g., to produce recombinant adenoviruses and/or influenza
antigens.
[0071] Immune response: A response of a cell of the immune system,
such as a B cell, T cell, or monocyte, to a stimulus. In some
cases, the response is specific for a particular antigen (that is,
an "antigen-specific response"). In some cases, an immune response
is a T cell response, such as a CD4+ response or a CD8+ response.
Alternatively, the response is a B cell response, and results in
the production of specific antibodies. A "protective immune
response" is an immune response that inhibits a detrimental
function or activity of a pathogenic influenza virus, reduces
infection by a pathogenic influenza virus, or decreases symptoms
(including death) that result from infection by the pathogenic
organism. A protective immune response can be measured, for
example, by the inhibition of viral replication or plaque formation
in a plaque reduction assay or ELISA-neutralization assay (NELISA),
or by measuring resistance to viral challenge in vivo. A
cell-mediated immune response can be measured by various
immunological assays, e.g., ELISpot, tetramer-labelling,
cytotoxicity assay.
[0072] Immunogenic composition: A composition comprising at least
one epitope of an influenza virus (or other pathogenic organism),
that induces a measurable CTL response, or induces a measurable B
cell response (for example, production of antibodies that
specifically bind the epitope). It further refers to isolated
nucleic acids encoding an immunogenic epitope of an influenza virus
(or other pathogen) that can be used to express the epitope (and
thus be used to elicit an immune response against this polypeptide
or a related polypeptide expressed by the pathogen). For in vitro
use, the immunogenic composition can consist of the isolated
nucleic acid, protein or peptide. For in vivo use, the immunogenic
composition will typically include the nucleic acid or virus that
expresses the immunogenic epitope in pharmaceutically acceptable
carriers or excipients, and/or other agents, for example,
adjuvants. An immunogenic polypeptide (such as an influenza
antigen), or nucleic acid encoding the polypeptide, can be readily
tested for its ability to induce a CTL or antibody response by
art-recognized assays.
[0073] Internal Protein: The internal proteins of influenza include
all of the structural and nonstructural proteins encoded by the
influenza genome, with the exception of 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; the
nucleocapsid protein (NP); the matrix protein (M1); the membrane
channel protein (M2); and two nonstructural proteins: NS1 and
NS2.
[0074] Isolated: An "isolated" biological component (such as a
nucleic acid or protein or organelle) has been substantially
separated or purified away from other biological components in the
cell of the organism in which the component naturally occurs, for
example, other chromosomal and extra-chromosomal DNA and RNA,
proteins and organelles. Nucleic acids and proteins that have been
"isolated" include nucleic acids and proteins purified by standard
purification methods. The term also embraces nucleic acids and
proteins prepared by recombinant expression in a host cell as well
as chemically synthesized nucleic acids.
[0075] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence.
[0076] Polynucleotide: The term polynucleotide or nucleic acid
sequence refers to a polymeric form of nucleotide at least 10 bases
in length. A recombinant polynucleotide includes a polynucleotide
that is not immediately contiguous with both of the coding
sequences with which it is immediately contiguous (one on the 5'
end and one on the 3' end) in the naturally occurring genome of the
organism from which it is derived. The term therefore includes, for
example, a recombinant DNA which is incorporated into a vector;
into an autonomously replicating plasmid or virus; or into the
genomic DNA of a prokaryote or eukaryote, or which exists as a
separate molecule (e.g., a cDNA) independent of other sequences.
The nucleotides can be ribonucleotides, deoxyribonucleotides, or
modified forms of either nucleotide. The term includes single- and
double-stranded forms of DNA or RNA.
[0077] Polypeptide: Any chain of amino acids, regardless of length
or post-translational modification (for example, glycosylation or
phosphorylation), such as a protein or a fragment or subsequence of
a protein. The term "peptide" is typically used to refer to a chain
of amino acids of between 3 and 30 amino acids in length. For
example an immunologically relevant peptide may be between about 7
and about 25 amino acids in length, e.g., between about 8 and about
10 amino acids.
[0078] Preventing or treating a disease: Inhibiting infection by an
influenza virus refers to inhibiting the full development of
disease caused by exposure to a pathogenic influenza virus. For
example, inhibiting an influenza infection refers to lessening
symptoms resulting from infection by the virus, such as preventing
the development of symptoms in a person who is known to have been
exposed to the virus, or to lessening virus number or infectivity
of a virus in a subject exposed to the virus. "Treatment" refers to
a therapeutic or prophylactic intervention that ameliorates or
prevents a sign or symptom of a disease or pathological condition
related to infection of a subject with a virus.
[0079] Promoter: A promoter is an array of nucleic acid control
sequences that directs transcription of a nucleic acid. A promoter
includes necessary nucleic acid sequences near the start site of
transcription, such as in the case of a polymerase II type promoter
(a TATA element). A promoter also optionally includes distal
enhancer or repressor elements which can be located as much as
several thousand base pairs from the start site of transcription.
Both constitutive and inducible promoters are included (see e.g.,
Bitter et al., Methods in Enzymology 153:516-544, 1987).
[0080] Specific, non-limiting examples of promoters include
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
cytomegalovirus immediate early gene promoter, the retrovirus long
terminal repeat; the adenovirus late promoter; the vaccinia virus
7.5K promoter) may be used. Promoters produced by recombinant DNA
or synthetic techniques may also be used. A polynucleotide can be
inserted into an expression vector that contains a promoter
sequence which facilitates the efficient transcription of the
inserted genetic sequence of the host. The expression vector
typically contains an origin of replication, a promoter, as well as
specific nucleic acid sequences that allow phenotypic selection of
the transformed cells.
[0081] Purified: The term "purified" (e.g., with respect to an
adenovirus vector or recombinant adenovirus) does not require
absolute purity; rather, it is intended as a relative term. Thus,
for example, a purified nucleic acid is one in which the nucleic
acid is more enriched than the nucleic acid in its natural
environment within a cell. Similarly, a purified peptide
preparation is one in which the peptide or protein is more enriched
than the peptide or protein is in its natural environment within a
cell. In one embodiment, a preparation is purified such that the
specified component represents at least 50% (such as, but not
limited to, 70%, 80%, 90%, 95%, 98% or 99%) of the total
preparation by weight or volume.
[0082] Recombinant: A recombinant nucleic acid is one that has a
sequence that is not naturally occurring or has a sequence that is
made by an artificial combination of two otherwise separated
segments of sequence, for example, a polynucleotide encoding a
fusion protein. This artificial combination is often accomplished
by chemical synthesis or, more commonly, by the artificial
manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques.
[0083] Subject: Living multi-cellular vertebrate organisms, a
category that includes both human and veterinary subjects,
including human and non-human mammals and birds.
[0084] T Cell: A white blood cell critical to the immune response.
T cells include, but are not limited to, CD4.sup.+ T cells and
CD8.sup.+ T cells. A CD4.sup.+ T lymphocyte is an immune cell that
carries a marker on its surface known as CD4. These cells, also
known as helper T cells, help orchestrate the immune response,
including antibody responses as well as killer T cell responses.
CD8.sup.+ T cells carry the CD8 marker, and include T cells with
cytotoxic or "killer" effector function.
[0085] Transduced or Transfected: A transduced cell is a cell into
which a nucleic acid molecule has been introduced by molecular
biology techniques. As used herein, the term introduction or
transduction encompasses all techniques by which a nucleic acid
molecule might be introduced into such a cell, including
transfection with viral vectors, transformation with plasmid
vectors, and introduction of naked DNA by electroporation,
lipofection, and particle gun acceleration.
[0086] 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, a vaccine elicits an antigen-specific immune response to
an antigen of a pathogen. In the context of this disclosure, the
vaccines elicit an immune response against avian (or pandemic)
influenza. The vaccines described herein include adenovirus vectors
or recombinant adenoviruses.
[0087] Vector: A nucleic acid molecule as introduced into a host
cell, thereby producing a transformed host cell. A vector may
include nucleic acid sequences that permit it to replicate in a
host cell, such as an origin of replication. A vector may also
include one or more selectable marker gene and other genetic
elements known in the art. The term vector includes plasmids,
linear nucleic acid molecules, and as described throughout
adenovirus vectors and adenoviruses. The term adenovirus vector is
utilized herein to refer to nucleic acids including one or more
components of an adenovirus that replicate to generate (e.g.,
infectious) viral particles in host cells. An adenovirus includes
nucleic acids that encode at least a portion of the assembled
virus. Thus, in many circumstances, the terms can be used
interchangeably. Accordingly, as used herein the terms are used
with specificity to facilitate understanding and without the intent
to limit the embodiment in any way.
Influenza Virus
[0088] Influenza viruses have a segmented single-stranded (negative
or antisense) genome. The influenza virion consists of an internal
ribonucleoprotein core containing the single-stranded RNA genome
and an outer lipoprotein envelope lined by a matrix protein. The
segmented genome of influenza A consists of eight linear RNA
molecules that encode ten polypeptides. Two of the polypeptides, HA
and NA include the primary antigenic determinants or epitopes
required for a protective immune response against influenza. Based
on the antigenic characteristics of the HA and NA proteins,
influenza strains are classified into subtypes. For example, recent
outbreaks of avian influenza in Asia have been categorized as
H.sub.5N.sub.1, H.sub.7N.sub.7 and H.sub.9N.sub.2 based on their HA
and NA phenotypes. These subtypes have proven highly infectious in
poultry and have been able to jump the species barrier to directly
infect humans causing significant morbidity and mortality.
[0089] HA is a surface glycoprotein which projects from the
lipoprotein envelope and mediates attachment to and entry into
cells. The HA protein is approximately 566 amino acids in length,
and is encoded by an approximately 1780 base polynucleotide
sequence of segment 4 of the genome. Polynucleotide and amino acid
sequences of HA (and other influenza antigens) isolated from
recent, as well as historic, avian influenza strains can be found,
e.g., in the GENBANK.RTM. database (available on the world wide web
at ncbi.nlm.nih.gov/entrez). For example recent avian H5 subtype HA
sequences include: AY075033, AY075030, AY818135, AF046097,
AF046096, and AF046088; recent H7 subtype HA sequences include:
AJ704813, AJ704812, and Z47199; and, recent avian H9 subtype HA
sequences include: AY862606, AY743216, and AY664675. One of
ordinary skill in the art will appreciate that essentially any
previously described or newly discovered avian HA antigen can be
utilized in the compositions and methods described herein.
Typically, the appropriate HA sequence or sequences are selected
based on circulating or predicted avian and/or pandemic HA
subtypes, e.g., as recommended by the World Health
Organization.
[0090] In addition to the HA antigen, which is the predominant
target of neutralizing antibodies against influenza, the
neuraminidase (NA) envelope glycoprotein is also a target of the
protective immune response against influenza. NA is an
approximately 450 amino acid protein encoded by an approximately
1410 nucleotide sequence of influenza genome segment 6. Recent
pathogenic avian strains of influenza have belonged to the N1, N7
and N2 subtypes. Exemplary NA polynucleotide and amino acid
sequences include, e.g., N1: AY651442, AY651447, and AY651483; N7:
AY340077, AY340078 and AY340079; and, N2: AY664713, AF508892 and
AF508588. Additional NA antigens can be selected from among
previously described or newly discovered NA antigens based on
circulating and/or predicted avian and/or pandemic NA subtypes.
[0091] The remaining segments of the influenza genome encode the
internal proteins. While immunization with internal proteins alone
does not give rise to a substantially protective neutralizing
antibody response, T-cell responses to one or more of the internal
proteins can significantly contribute to protection against
influenza infection. Compared to the polymorphic HA and NA
antigens, the internal proteins are more highly conserved between
strains, and between subtypes. Thus, a T cell receptor elicited by
exposure to an internal protein of an avian or human subtype of
influenza binds to the comparable internal protein of other avian
and human subtypes.
[0092] PB2 is a 759 amino acid polypeptide which is one of the
three proteins which comprise the RNA-dependent RNA polymerase
complex. PB2 is encoded by approximately 2340 nucleotides of the
influenza genome segment 1. The remaining two polymerase proteins,
PB1, a 757 amino acid polypeptide, and PA, a 716 amino acid
polypeptide, are encoded by a 2341 nucleotide sequence and a 2233
nucleotide sequence (segments 2 and 3), respectively.
[0093] Segment 5 consists of 1565 nucleotides encoding a 498 amino
acid nucleoprotein (NP) protein that forms the nucleocapsid.
Segment 7 consists of a 1027 nucleotide sequence encoding a 252
amino acid M1 protein, and a 96 amino acid M2 protein, which is
translated from a spliced variant of the M RNA. Segment 8 consists
of a 890 nucleotide sequence encoding two nonstructural proteins,
NS 1 and NS2.
[0094] Of these proteins, the M (M1 and M2) and NP proteins are
most likely to elicit protective humoral and/or cellular T cell
responses. Accordingly, while any of the internal proteins can be
included (for example, in addition to one or more avian HA and/or
NA antigens) in the compositions and methods described herein,
adenovirus vectors and adenoviruses commonly also include one or
more of M1, M2 and/or NP proteins. As responses against internal
protein(s) of one strain of virus tend to interact with internal
protein(s) of other strains of influenza, the internal protein can
be selected from essentially any avian and/or human strain. For
example, the internal protein(s) can be selected from avian
H.sub.5N.sub.1, H.sub.7N.sub.7 and/or H.sub.9N.sub.2 strains.
Alternatively, the internal protein(s) can be selected from human
H.sub.3N.sub.2, H1N1, and/or H.sub.2N.sub.2. Exemplary internal
protein polynucleotide and amino acid sequences can be found, e.g.,
in GENBANK.RTM.. For example, H.sub.3N.sub.2 M and NP nucleic acids
and proteins are represented by Accession Nos.AF255370 and
CY000756, respectively. The internal proteins of influenza are more
conserved between strains and tend to elicit a cross-reactive T
cell response that contributes to the protective immune response
against influenza.
[0095] It will be appreciated by those of skill in the art that the
specific nucleic acids and proteins indicated above by accession
number are non-exclusive examples, and numerous additional
influenza antigen sequences can be expressed in the context of the
adenovirus vectors disclosed herein. For example, additional
influenza antigens, and the nucleic acids encoding them are
commonly similar in primary structure to the antigens and nucleic
acids referenced above. The similarity between amino acid (and
polynucleotide) sequences is expressed in terms of the similarity
between the sequences, otherwise referred to as sequence identity.
Sequence identity is frequently measured in terms of percentage
identity (or similarity); the higher the percentage, the more
similar are the primary structures of the two sequences. In
general, the more similar the primary structures of two amino acid
sequences, the more similar are the higher order structures
resulting from folding and assembly. Thus, for example, HA antigens
of the same influenza subtype typically share a high degree of
sequence identity. Variants of an HA antigen (of a particular
subtype) can have one or a small number of amino acid deletions,
additions or substitutions but will nonetheless share a very high
percentage of their amino acid (and generally their polynucleotide
sequence). To the extent that variants of a subtype differ from
each other, their overall antigenic characteristics are maintained.
In contrast, HA antigens of different subtypes share less sequence
identity (e.g., at the receptor binding pocket) and/or differ from
each other such that their antigenic characteristics are no longer
identical.
[0096] Methods of 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; Needleman and
Wunsch, J. Mol. Biol. 48:443, 1970; Higgins and Sharp, Gene 73:237,
1988; Higgins and Sharp, CABIOS 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, presents a detailed consideration of sequence alignment
methods and homology calculations.
[0097] The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul
et al., J. Mol. Biol. 215:403, 1990) is available from several
sources, including the National Center for Biotechnology
Information (NCBI, Bethesda, Md.) and on the internet, for use in
connection 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 NCBI
website on the internet.
[0098] Another indicia of sequence similarity between two nucleic
acids is the ability to hybridize. The more similar are the
sequences of the two nucleic acids, the more stringent the
conditions at which they will hybridize. The stringency of
hybridization conditions are sequence-dependent and are different
under different environmental parameters. Thus, hybridization
conditions resulting in particular degrees of stringency will vary
depending upon the nature of the hybridization method of choice and
the composition and length of the hybridizing nucleic acid
sequences. Generally, the temperature of hybridization and the
ionic strength (especially the Na.sup.+ and/or Mg.sup.++
concentration) of the hybridization buffer will determine the
stringency of hybridization, though wash times also influence
stringency. Generally, stringent conditions are selected to be
about 5.degree. C. to 20.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Conditions for nucleic
acid hybridization and calculation of stringencies can be found,
for example, in Sambrook et al., Molecular Cloning: 4A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 2001; Tijssen, Hybridization With Nucleic Acid Probes, Part
I: Theory and Nucleic Acid Preparation, Laboratory Techniques in
Biochemistry and Molecular Biology, Elsevier Science Ltd., NY,
N.Y., 1993.and Ausubel et al. Short Protocols in Molecular Biology,
4.sup.th ed., John Wiley & Sons, Inc., 1999.
[0099] For purposes of the present disclosure, "stringent
conditions" encompass conditions under which hybridization will
only occur if there is less than 25% mismatch between the
hybridization molecule and the target sequence. "Stringent
conditions" may be broken down into particular levels of stringency
for more precise definition. Thus, as used herein, "moderate
stringency" conditions are those under which molecules with more
than 25% sequence mismatch will not hybridize; conditions of
"medium stringency" are those under which molecules with more than
15% mismatch will not hybridize, and conditions of "high
stringency" are those under which sequences with more than 10%
mismatch will not hybridize. Conditions of "very high stringency"
are those under which sequences with more than 6% mismatch will not
hybridize. In contrast nucleic acids that hybridize under "low
stringency conditions include those with much less sequence
identity, or with sequence identity over only short subsequences of
the nucleic acid.
[0100] Thus, the adenovirus vectors disclosed herein can include
and/or express any of numerous influenza antigens, such as variants
of H5, H7 and/or H9 subtype hemagglutinin antigens (or other
antigens discussed herein) that are similar in sequence, as
measured by sequence similarity or hybridization measures indicated
above.
Adenovirus Vectors
[0101] The term "adenovirus" as used herein is intended to
encompass all adenoviruses, including the Mastadenovirus and
Aviadenovirus genera. To date, at least forty-seven human serotypes
of adenoviruses have been identified (see, e.g., FIELDS et al.,
VIROLOGY, volume 2, chapter 67 (3d ed., Lippincont-Raven
Publishers). Adenoviruses are linear double-stranded DNA viruses
approximately 36 kb in size. Their genome includes an inverted
sequence (ITR) at each end, an encapsidation sequence, early genes
and late genes. The main early genes are contained in the E1, E2,
E3 and E4 regions. Among them, the genes contained in the E1 region
(E1a and E1b, in particular) are necessary for viral replication.
The E4 and L5 regions, for example, are involved in viral
propagation, and the main late genes are contained in the L1 to L5
regions. For example, the human Ad5 adenovirus genome has been
sequenced completely and the sequence is available on the world
wide web, (see, for example, GENBANK.RTM. Accession No. M73260).
Similarly, portions, or in some cases the whole, of the genome of
human and non-human adenoviruses of different serotypes (Ad2, Ad3,
Ad7, Ad12, and the like) have also been sequenced.
[0102] Upon infection of a subject (or host) with recombinant
adenoviruses, or introduction of a recombinant adenovirus vector,
exogenous nucleic acids contained within the adenovirus genome are
transcribed, and translated, by the host cell RNA polymerase and
translational machinery. Thus, a polynucleotide sequence that
encodes one or more influenza antigens can be incorporated into an
adenovirus vector and introduced into the cells of a subject where
the polynucleotide sequence is transcribed and translated to
produce the influenza antigen. Recently, non-replicating adenovirus
vectors have been used to produce and deliver an immunologically
effective HA1 strain vaccine (Van Kampen et al., Vaccine
23:1029-1036, 2005). In the context of the compositions and methods
described herein, a polynucleotide sequence that encodes at least
one avian influenza antigen (as described above, such as a
hemagglutinin antigen of H5, H7 or H9 subtype) is incorporated into
an adenovirus vector using established molecular biology
procedures. Nucleic acids (e.g., cDNAs) that encode influenza
antigens can be manipulated with standard procedures such as
restriction enzyme digestion, fill-in with DNA polymerase, deletion
by exonuclease, extension by terminal deoxynucleotide transferase,
ligation of synthetic or cloned DNA sequences, site-directed
sequence-alteration via single-stranded bacteriophage intermediate
or with the use of specific oligonucleotides in combination with
PCR or other in vitro amplification.
[0103] Exemplary procedures sufficient to guide one of ordinary
skill in the art through the production of a recombinant adenovirus
vector that includes a polynucleotide sequence that encodes one (or
more than one) influenza antigen can be found in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor
Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A
Laboratory Manual, 3d ed., Cold Spring Harbor 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 Compendium of Methods from
Current Protocols in Molecular Biology, 4th ed., Wiley & Sons,
1999.
[0104] Typically, the polynucleotide sequence encoding the
influenza antigen(s) is operably linked to transcriptional control
sequences including, e.g., a promoter and a polyadenylation signal.
A promoter is a polynucleotide sequence recognized by the
transcriptional machinery of the host cell (or introduced synthetic
machinery), that is involved in the initiation of transcription. A
polyadenylation signal is a polynucleotide sequence that directs
the addition of a series of nucleotides on the end of the mRNA
transcript for proper processing and trafficking of the transcript
out of the nucleus into the cytoplasm for translation.
[0105] Exemplary promoters known in the art include viral
promoters, including those of the cytomegalovirus immediate early
gene promoter ("CMV"), herpes simplex virus thymidine kinase
("tk"), SV40 early transcription unit, polyoma, retroviruses,
papilloma virus, hepatitis B virus, and human and simian
immunodeficiency viruses. Other promoters are isolated from
mammalian genes, including the immunoglobulin heavy chain,
immunoglobulin light chain, T-cell receptor, HLA DQ .alpha. and DQ
.beta.,.beta.-interferon, interleukin-2, interleukin-2 receptor,
MHC class II, HLA-DR.alpha.,.beta.-actin, muscle creatine kinase,
prealbumin (transthyretin), elastase I, metallothionein,
collagenase, albumin, fetoprotein, .beta.-globin, c-fos, c-HA-ras,
insulin, neural cell adhesion molecule (NCAM),
.alpha.1-antitrypsin, H.sub.2B (TH2B) histone, type I collagen,
glucose-regulated proteins (GRP94 and GRP78), rat growth hormone,
human serum arnyloid 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, and epithelial cells of the skin and
lung.
[0106] The promoter can be either inducible or constitutive. An
inducible promoter is a promoter which is inactive or exhibits low
activity except in the presence of an inducer substance. Some
examples of promoters that may be included as a 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-2 kb, HSP70,
proliferin, tumor necrosis factor, or thyroid stimulating hormone
gene promoter. Typically, however, the promoter is a constitutive
promoter that results in high levels transcription upon
introduction into a host cell in the absence of additional factors.
Optionally, the transcription control sequences include one or more
enhancer elements, which are binding recognition sites binds for
one or more transcription factors that increase transcription above
that observed for the minimal promoter alone.
[0107] Often it is also desirable to include a polyadenylation
signal to effect proper termination and polyadenylation of the gene
transcript. Exemplary polyadenylation signals have been isolated
from bovine growth hormone, SV40 and the herpes simplex virus
thymidine kinase genes. Any of these or other polyadenylation
signals can be utilized in the context of the adenovirus vectors
described herein.
[0108] Typically, a polynucleotide sequence that encodes an
influenza antigen will be a full length open reading frame
including a translation initiation site. However, it is also
possible to use polynucleotide sequences that encode an immunogenic
portion (subportion) of the antigen. If the polynucleotide sequence
lacks a translation initiation site or codon, one can be introduced
at an appropriate site preceding the polynucleotide sequence
encoding an antigen subportion during production of the vector.
[0109] Nucleic acid vectors encoding adenoviruses are well-known in
the art, and have been utilized for gene therapy and vaccine
applications. Exemplary adenovirus vectors are described in
Berkner, BioTechniques 6:616-629, 1988; Graham, Trend Biotechnol,
8:85-87, 1990; Graham & Prevec, in Vaccines: new approaches to
immunological problems, Ellis (ed.), pp. 363-90,
Butterworth-Heinemann, Woburn, 1992; Mittal et al., in 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. 55:479-505,
2000, Published US Patent Application 2002/0192185, which are
incorporated herein in their entirety.
[0110] Generally, the vectors are modified to make them replication
defective, that is, incapable of replicating autonomously in the
host cell, although in addition to such helper dependent adenovirus
vectors, conditional replication competent and replication
competent adenovirus vectors and viruses can also be used.
Generally, the genome of replication defective viruses lack at
least some of the sequences necessary for replication of the virus
in an infected cell. These regions may be either removed (wholly or
partially), or rendered non-functional, or replaced by other
sequences, and in particular by a sequence coding for a molecule of
therapeutic interest. Typically, the defective virus retains the
sequences which are involved in encapsidation of viral
particles.
[0111] Replication defective adenoviruses typically include a
mutation, such as a deletion, in one or more of the E1 (E1a and/or
E1b), E3 region, E2 region and/or E4 region--have been deleted. The
entire adenovirus genome except the ITR and packaging elements can
be deleted and the resultant adenovirus vectors and know as
helper-dependent vectors or "gutless" vectors. In some cases a
heterologous DNA sequences are inserted in place of the deleted
adenovirus sequence (Levrero et al., Gene 101: 195-202, 1991;
Ghosh-Choudhury et al., Gene 50:161-171, 1986). Other constructions
contain a deletion in the E1 region and of a non-essential portion
of the E4 region (WO 94/12649). Exemplary adenovirus vectors are
also described in U.S. Pat. Nos. 6,328,958, 6,669,942 and
6,420,170, which are incorporated herein by reference.
[0112] These replication defective recombinant adenoviruses may be
prepared in different ways, for example, in a competent cell line
capable of complementing all the defective functions essential for
replication of the recombinant adenovirus. For example,
adenoviruses vectors can be produced in a complementation cell line
(such as 293 cells) in which a portion of the adenovirus genome has
been integrated. Such cells lines contains the left-hand end
(approximately 11-12%) of the adenovirus serotype 5 (Ad5) genome,
comprising the left-hand ITR, the encapsidation region and the E1
region, including E1a, E1b and a portion of the region coding for
the pIX protein. This line is capable of trans-complementing
recombinant adenoviruses which are defective for the E1 region.
Typically, expression of both E1A and E1B proteins is needed for E1
complementation.
[0113] Human adenovirus vectors are commonly utilized to introduce
exogenous nucleic acids into human and animal cells and organisms.
Adenoviruses exhibit broad host cell range, and adenovirus vectors
can be utilized to infect human as well as non-human animal,
including birds. Most commonly, the human adenovirus vectors are
HAd5 vectors derived from adenovirus serotype 5 viruses. Due to the
large size of the intact adenovirus genome, insertion of
heterologous polynucleotide sequences is most conveniently
performed using a shuttle plasmid. Sequences, such as those
influenza antigens are cloned into a shuttle vector which then
undergoes homologous recombination with all or part of an
adenovirus genome in cultured cells. In addition, homologous
recombination can also be done in bacteria to generate full length
adenovirus vectors.
[0114] In some cases, it is desirable to use non-human adenovirus
vectors to avoid pre-existing host immunity to human adenoviruses.
Infection with human adenovirus is common in human populations, and
many or most individuals have circulating antibody titers that will
bind and neutralize a recombinant human adenovirus. Thus, in at
least some proportion of the human population vaccination with
human adenovirus vectors will not result in efficient generation of
an immune response against influenza due to neutralization of the
vector containing the influenza sequences. To avoid this problem,
non-human adenovirus vectors can be used to circumvent any
pre-existing immunity against human adenovirus.
[0115] Adenoviruses of animal origin are also capable of infecting
human, as well as non-human, cells efficiently, and following
infection are generally incapable of propagating in human cells
(see, Application WO 94/26914). Thus, adenoviruses of animal origin
can be used in the context of the vectors and viruses described
herein. The use of animal adenovirus vectors for human and animal
vaccine development is discussed in detail in Bangari & Mittal,
Vaccine 24:849-862, 2006, which is incorporated herein by
reference. For example animal adenovirus vectors can be selected
from canine, bovine, murine (for example: MAV1, Beard et al.,
Virology 75:81, 1990), ovine, porcine, avian (e.g., chicken) or
alternatively simian (for example: SAV) adenoviruses. For example,
bovine and porcine adenoviruses can be used to produce adenovirus
vectors that express influenza antigens, including various bovine
serotypes available from the ATCC (types 1 to 8) under the
references ATCC VR-313, 314, 639-642, 768 and 769, and porcine
adenovirus 5359. Additionally, simian adenoviruses of various
serotypes, including SAd25, SAd22, SAd23 and SAd24, such as those
referenced in the ATCC under the numbers VR-591-594, 941-943,
195-203, and the like, several serotypes (1 to 10) of avian
adenovirus which are available in 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 strains referenced as ATCC VR-831 to 835, as well
as murine adenoviruses FL (ATCC VR-550) and E20308 (ATCC VR-528),
and ovine adenovirus type 5 (ATCC VR-1343) or type 6 (ATCC VR-1340)
can be used.
[0116] For example, both 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 US patent application
no. 2002/0192185, and in U.S. Pat. Nos. 6,492,343 and 6,451,319,
which are incorporated herein by reference.
[0117] The compositions and methods described herein are applicable
to any influenza antigens. In particular, the compositions and
methods can be employed to express, and to generate an immune
response against avian strains of influenza. For example, the
adenovirus vectors, recombinant adenoviruses and immunogenic
compositions disclosed herein can include an HA antigen of any
avian or pandemic strain of influenza. Numerous avian HA antigens
have been identified, and the sequences can be obtained, for
example using the publicly available NCBI database (on the world
wide web at ncbi.nlm.nih.gov/entrez/query.fcgi). Exemplary HA
antigens from recent avian flu outbreaks are represented by
AY818135 (A/Viet Nam/1203/04); AF084280 (A/Hong Kong/483/97);
AF036356 (A/Hong Kong/156/97); AY575870 (A/Hong Kong/213/03).
Nucleic acids including these sequences can be obtained by cloning
and/or amplification from virus isolates, or can be produced
synthetically. Similarly, novel HA antigens isolated from newly
emergent strains or newly isolated strains can also be included in
the compositions described herein. Likewise, NA antigens of avian
known or newly discovered avian strains can be incorporated into
the vectors, viruses and compositions described herein. Optionally,
the viruses, vectors and/or immunogenic compositions include one or
more internal proteins of an avian or non-avian, e.g., human
influenza strain.
[0118] Recombinant adenovirus expressing avian and/or other
influenza antigens can be produced from the vectors described above
following introduction of the adenovirus vector into a suitable
host cell. In the case of replication defective vectors, the
adenovirus vector is typically introduced into a cell line that
complements the defective function. For example, E1 deficient virus
can be grown in a cell line that complements E1 function due to
expression of an introduced nucleic acid that encodes adenovirus E1
protein. Exemplary cell lines include both human and non-human cell
lines that have been engineered to express an adenovirus E1 (e.g.,
E1A) proteins. For example, 293 cells that express adenovirus E1
proteins are commonly utilized to grow recombinant
replication-defective adenoviruses that have a deletion of the E1
region. Additional suitable cell lines include MDBK-221, FBK-34,
and fetal retinal cells of various origins. Specific examples of
cell lines suitable for growing porcine and bovine recombinant
adenovirus include FPRT-HE1-5 cells (Bangari & Mittal, Virus
Res. 105:127-136, 2004) and FBRT-HE1 cells (van Olphen et al.,
Virology, 295:108-118, 2002), respectively. In certain embodiments,
the cell express adenovirus E1 genes of more than one strain of
virus, such as 2 or more different strains of virus with different
species tropism. For example, the cells can express E1 genes of a
human and a non-human (e.g., pig and/or cow E1 genes). Those of
ordinary skill in the art will readily be able to select or produce
suitable additional or alternative cell lines that complement the
replication functions of replication-defective adenovirus vectors.
For example, any of the various mammalian cell lines disclosed
herein (or known in the art) can be transfected with E1 and/or E3
genes of any of the strains of adenovirus, such as the exemplary
strains disclosed herein, based on the particular adenovirus vector
to be grown. For example, it is common to select E1 (and/or E3)
genes that correspond to (that is, are from the same or a
functionally similar strain) the same strain as the adenovirus
vector. One of skill in the art will also appreciate that
functionally similar variants (such as variants that share
substantial sequence identity, or that specifically hybridize,
e.g., under high stringency conditions) to any of the exemplary
adenovirus genes, can also be used to produce cell lines that
support the growth of adenovirus vectors that encode influenza
antigens.
[0119] One common method for producing replication defective
adenovirus vectors that incorporate exogenous nucleic acids is
described in Ng et al., Hum. Gene Ther. 10:2667-2672, 1999, and
Hum. Gene Ther. 11:693-699, 2000, which are incorporated herein in
their entirety. Briefly, to produce a human adenovirus vector
containing an influenza antigen, a polynucleotide sequence encoding
an influenza antigen (for example, one or more avian HA antigens)
operably linked to a strong promoter (such as the CMV immediate
early promoter) is inserted into a shuttle vector, such as pDC311.
The pDC311 shuttle vector is a plasmid that contains the left end
of HAd5 (approximately 4 kb) with a 3.1 kb E1 deletion, a loxp site
for site specific recombination in the presence of Cre recombinase
and an intact packaging signal (.psi.). The shuttle vector is
co-transfected into appropriate cells that express the Cre
recombinase (e.g., 293 Cre cells) along with a plasmid that
includes a replication defective HAd5 genome (e.g., containing
deletions in the E1 and/or E3 region genes) that lacks a packaging
signal, and contains a loxp site. Homologous Cre mediated
recombination results in the production of an adenovirus vector
plasmid that encodes a replication defective adenovirus that
expresses the inserted influenza antigen.
[0120] Cells that express complementing replication function (such
as E1 when the replication defective adenovirus vector lacks E1
function) can be transfected with a recombinant adenovirus vector
according to standard procedures, such as electroporation, calcium
phosphate precipitation, lipofection, etc., or infected with
adenovirus at low infectivity (e.g., between 1-1000 p.f.u./cell).
In some cases confluent monolayers of cells are utilized, e.g., in
60 mm dishes. The cells are then incubated (grown) for a period of
time sufficient for expression and replication of adenovirus, and
the cells are divided to maintain active growth and maximize virus
recovery, prior to harvesting of recombinant adenovirus. Typically
following several passages (for example, 2-5 passages), recombinant
is collected by lysing the cells to release the virus, and then
concentrating the virus. Recombinant adenovirus can be concentrated
by passing the lysate containing the virus over a density gradient
(such as a CsCl density gradient). Following concentration the
recombinant adenoviruses are typically dialyzed against a buffer
(such as 10 mM Tris pH 8.0, 2 mM MgCl.sub.2, 5% sucrose), titered
and stored until use at -80.degree. C. Methods for producing
adenovirus at a large scale, e.g., suitable for preparation of
immunogenic compositions to be used as vaccines are described,
e.g., in published US patent application no. 20030008375, which is
incorporated herein by reference.
Production of Recombinant Influenza Virus Antigens from Adenovirus
Vectors.
[0121] In addition to their utility in the production of
immunogenic compositions containing adenovirus vector nucleic acids
and/or adenovirus that are capable of expressing avian influenza
antigens, the adenovirus vectors disclosed herein can be used for
the production and manufacture of recombinant influenza antigens,
such as recombinant HA antigens from highly pathogenic avian
strains of influenza as well as other influenza antigens. Methods
for producing recombinant antigens using human and non-human
adenovirus vectors are well known in the art, and exemplary
compositions and methods are described in, for example, U.S. Pat.
Nos. 5,824,770, 6,319,716, and 6,824,770, which are incorporated
herein in their entirety. Additionally, commercially available
vectors, such as the ADEASY.TM. adenovirus vector system from
Stratagene (La Jolla, Calif.) can be employed to produce adenovirus
vectors and adenovirus recombinants (recombinant adenovirus) that
are capable of expressing recombinant influenza proteins.
[0122] As discussed above, the adenovirus vectors contain a
heterologous polynucleotide sequence that encodes one or more avian
influenza virus antigen(s) in place of the E1 and/or E3 gene region
of the adenovirus vector. Optionally, two, or even three or more
influenza antigens are encoded by the heterologous nucleic acid.
Conversely, fragments of influenza antigens containing immunogenic
epitopes can be encoded by the polynucleotide sequence inserted
into the adenovirus vector. Typically, the polynucleotide sequence
encoding the influenza antigen(s) is operably linked to
transcription regulatory sequences (e.g., a promoter and/or
enhancer elements, and/or polyadenylation sequences) capable of
producing high levels of expression. A number of eukaryotic
promoter and polyadenylation sequences which provide successful
expression of foreign genes in mammalian cells and how to construct
expression cassettes, are known in the art, for example in U.S.
Pat. No. 5,151,267, the disclosures of which are incorporated
herein by reference. The promoter is selected to give optimal
expression of immunogenic protein which in turn satisfactorily
leads to humoral, cell mediated and mucosal immune responses
according to known criteria.
[0123] Optionally, the polynucleotide encoding an influenza antigen
includes a portion that encodes 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 in which the influenza antigen is linked to one
or more peptide (or polypeptide) domains that facilitate expression
and/or purification. Numerous suitable tags are known in the art,
and expressed proteins that include such tags can readily be
isolated using commercially available reagents and kits. If
desired, the tag can be removed from the antigen, for example by
enzymatic or chemical cleavage, before the recombinant antigen is
used in subsequent applications. Exemplary tags include Myc epitope
tags, Histidine tags and GST tags.
[0124] The recombinant adenovirus vector containing one or more
influenza virus antigen(s) can be expressed in cell lines into
which has been introduced an expression cassette encoding a
complementary E1 region (and/or an E2 region). These recombinant
cell lines are capable of allowing a recombinant adenovirus, having
an E1 gene region deletion replaced by heterologous nucleotide
sequence encoding one or more influenza antigen(s) or fragments
thereof, to replicate and express the desired foreign-gene or
fragment, which is encoded by the recombinant adenovirus.
Optionally, such a cell line can include E1 (and/or E2) genes
corresponding to more than one strain of adenovirus. For example,
suitable cell lines include those that contain nucleic acids that
express E1 of a human adenovirus as well as, for example, E1 of a
porcine or bovine adenovirus strain.
[0125] For use in pharmaceutical compositions, recombinant
influenza antigens are typically purified following expression in
cultured cells. Methods for isolated recombinant proteins expressed
in cultured cells are well known in the art, and specific methods
are described in Sambrook et al. (In Molecular Cloning: A
Laboratory Manual, CSHL, New York, 2001) and in Brent et al.,
Current Protocols in Molecular Biology, John Wiley and Sons, New
York, 2003). One skilled in the art will understand that there are
myriad ways to purify recombinant polypeptides, and such typical
methods of protein purification may be used to purify influenza
antigens expressed from adenovirus vectors. Such methods include,
for instance, protein chromatographic methods including ion
exchange, gel filtration, HPLC, monoclonal antibody affinity
chromatography and isolation of insoluble protein inclusion bodies
after over production. In addition, purification can be based on
attached tags (as discussed above), for instance a six-histidine
sequence, may be recombinantly fused to the protein and used to
facilitate polypeptide purification using Nickel affinity columns
(such as, nickel-nitrilotriacetic acid (Ni-NTA) metal affinity
chromatography matrix (The QIAexpressionist, QIAGEN, 1997).
[0126] If desired, the recombinant influenza antigen(s) can be
conjugated to a vaccine carrier for administration to a subject.
Vaccine carriers are well-known in the art: for example, bovine
serum albumin (BSA), human serum albumin (HSA) keyhole limpet
hemocyanin (KLH), and rotavirus VP6. In some cases one or more
adjuvant, such as alum, is combined with the recombinant influenza
antigen(s) for administration to a subject, as discussed below.
Immunogenic Compositions Comprising Adenovirus Vectors and
Recombinant Adenovirus.
[0127] The recombinant adenovirus vectors and recombinant
adenoviruses that express influenza antigens (e.g., avian influenza
antigens) can be administered in vitro, ex vivo or in vivo to a
cell or subject. Generally, it is desirable to prepare the vectors
or viruses as pharmaceutical compositions appropriate for the
intended application. Accordingly, methods for making a medicament
or pharmaceutical composition containing the adenovirus vectors or
adenoviruses described above are included herein. Typically,
preparation of an pharmaceutical composition (medicament) entails
preparing a pharmaceutical composition that is essentially free of
pyrogens, as well as any other impurities that could be harmful to
humans or animals. Typically, the pharmaceutical composition
contains appropriate salts and buffers to render the components of
the composition stable and allow for uptake of nucleic acids or
virus by target cells.
[0128] Pharmaceutical (for example, immunogenic) compositions
typically include an effective amount of the adenovirus vector or
virus dispersed (for example, 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. Numerous pharmaceutically acceptable
carriers and/or pharmaceutically acceptable excipients are known in
the art and are described, e.g., in Remington's Pharmaceutical
Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th
Edition (1975).
[0129] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch or magnesium stearate. In
addition to biologically neutral carriers, pharmaceutical
compositions to be administered can contain 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.
[0130] 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
pharmaceutical 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 immunogenic compositions is
contemplated. Supplementary active ingredients also can be
incorporated into the compositions. For example, certain
pharmaceutical compositions can include the vectors or viruses in
water, mixed with a suitable surfactant, such as
hydroxypropylcellulose. Dispersions also can be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0131] The pharmaceutical compositions (medicaments) can be
prepared for use in prophylactic regimens (e.g., vaccines) and
administered to human or non-human subjects (including birds, such
as domestic fowl, e.g., chickens, ducks, guinea fowl, turkeys and
geese) to elicit an immune response against an influenza antigen
(or a plurality of influenza antigens). Thus, the pharmaceutical
compositions typically contain an immunologically effective amount
of the adenovirus vector or adenovirus (or indeed of recombinant
influenza antigen produced by expressing an adenovirus vector as
disclosed herein). An immunologically effective amount, e.g., of a
vaccine composition, is an amount sufficient to elicit a desired
immune response, such as a protective immune response in an
immunocompetent subject, when administered in one or more doses.
Typically, an immunologically effective amount of a vaccine is
administered to a subject in one or more doses, prior to exposure
(that is, prophylactically) to an infectious agent (such as an
influenza virus) to elicit a protective immune response against the
infectious agent. In this context, adenovirus vectors and/or
adenoviruses containing avian influenza antigens (and/or
recombinant influenza antigens produced by expressing such
adenoviruses) can be administered to a subject in one or more doses
to elicit a protective response against avian and/or pandemic
influenza.
[0132] In some cases the compositions are administered following
infection to enhance the immune response, in such applications, the
pharmaceutical composition is administered in a therapeutically
effective amount. A therapeutically effective amount is a quantity
of a composition used to achieve a desired effect in a subject. For
instance, this can be the amount of the composition necessary to
inhibit viral replication or to prevent or measurably alter outward
symptoms of viral infection. When administered to a subject, a
dosage will generally be used that will achieve target tissue
concentrations (for example, in lymphocytes) that has been shown to
achieve an in vitro or in vivo effect.
[0133] For example, the compositions described herein can be
administered to a human (or non-human) subject to elicit an immune
response against avian influenza virus, e.g., a pathogenic
H.sub.5N.sub.1, H.sub.7N.sub.7 or H.sub.9N.sub.2 strain of avian
influenza virus. Generally, the adenovirus vectors and/or
adenoviruses described herein elicit a protective or partially
protective immune response against at least one strain or subtype
of avian (or pandemic) influenza. That is, the pharmaceutical
compositions described herein are typically capable of preventing
influenza, or of decreasing the severity of symptoms (e.g.,
morbidity and/or mortality) following subsequent exposure to a
pathogenic strain of virus in at least a significant portion of the
population to which the composition is administered. In some cases,
the immune response is protective or partially protective against
multiple strains, or even multiple subtypes of avian (or pandemic)
influenza, including their administration to humans and non-human
subjects (including birds).
[0134] Typically, a protective immune response against influenza
involves the production of neutralizing antibodies that bind to the
HA antigen. Because these antigens are highly polymorphic and
antibodies raised against one subtype are typically not protective
against influenza of another subtype, the immunogenic
pharmaceutical compositions described herein can include adenovirus
vectors or adenoviruses that express more than one HA antigen,
e.g., by one or more vectors or viruses. As previously discussed,
the HA antigens can be variants of a single subtype or of more than
one HA subtype. Optionally, an adenovirus that expresses (or a
vector that encodes) an NA antigen is also administered.
[0135] T-cell responses against influenza antigens broaden the
immune response against influenza, increasing vaccine efficacy.
Internal proteins, such as the 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 a T cell response that increases protection
and contributes to reduced morbidity due to influenza infection.
Furthermore, such internal protein epitopes are more highly
conserved between influenza subtypes offering cross protection
against multiple strains of avian and human influenza. Accordingly,
pharmaceutical compositions that include one or more adenovirus
vectors or adenoviruses that express influenza internal protein(s)
can be administered to elicit an influenza specific immune
response.
[0136] Administration of therapeutic compositions including
recombinant adenovirus that expresses an influenza antigen
(including an avian influenza antigen) can be by any common route
as long as the target tissue (typically, the respiratory tract) is
available via that route. This includes oral, nasal, ocular,
buccal, or other mucosal (e.g., rectal, vaginal) or topical
administration. Alternatively, administration will be by
orthotopic, intradermal subcutaneous, intramuscular,
intraperitoneal, or intravenous injection routes. Such immunogenic
compositions are usually administered as pharmaceutically
acceptable compositions that include physiologically acceptable
carriers, buffers or other excipients.
[0137] The immunogenic compositions can also be administered in the
form of injectable compositions either as liquid solutions or
suspensions; solid forms suitable for solution in, or suspension
in, liquid prior to injection may also be prepared. These
preparations also may be emulsified. A typical composition for such
purpose comprises a pharmaceutically acceptable carrier. For
instance, the composition may contain about 100 mg of human serum
albumin per milliliter of phosphate buffered saline. Other
pharmaceutically acceptable carriers include aqueous solutions,
non-toxic excipients, including salts, preservatives, buffers and
the like may be used. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oil and injectable
organic esters such as ethyloleate. Aqueous carriers include water,
alcoholic/aqueous solutions, saline solutions, parenteral vehicles
such as sodium chloride, Ringer's dextrose, etc. Intravenous
vehicles include fluid and nutrient replenishers. Preservatives
include antimicrobial agents, anti-oxidants, chelating agents and
inert gases. The pH and exact concentration of the various
components the pharmaceutical composition are adjusted according to
well known parameters.
[0138] Additional formulations which are suitable for oral
administration. Oral formulations can include excipients such as,
pharmaceutical grades of mannitol, lactose, starch, magnesium
stearate, sodium saccharine, cellulose, magnesium carbonate and the
like. The compositions (medicaments) typically take the form of
solutions, suspensions, aerosols or powders. Exemplary formulations
can be found in US patent publication no. 20020031527, the
disclosure of which is incorporated herein by reference. When the
route is topical, the form may be a cream, ointment, salve or
spray. Exemplary methods for intramuscular, intranasal and topical
administration of the adenovirus vectors and adenoviruses described
herein can be found, e.g., in U.S. Pat. No. 6,716,823, which is
incorporated herein by reference.
[0139] Optionally, the pharmaceutical compositions or medicaments
can include a suitable adjuvant to increase the immune response
against the influenza antigen(s). As used herein, an "adjuvant" is
any potentiator or enhancer of an immune response. The term
"suitable" is meant to include any substance which can be used in
combination with the adenovirus vector or adenovirus to augment the
immune response, without producing adverse reactions in the
vaccinated subject. Effective amounts of a specific adjuvant may be
readily determined so as to optimize the potentiation effect of the
adjuvant on the immune response of a vaccinated subject. For
example, 0.5%-5% (e.g., 2%) aluminum hydroxide (or aluminum
phosphate) and MF-59 oil emulsion (0.5% polysorbate 80 and 0.5%
sorbitan trioleate. Squalene (5.0%) aqueous emulsion) are adjuvants
which have been favorably utilized in the context of influenza
vaccines. Other adjuvants include mineral, vegetable or fish oil
with water emulsions, incomplete Freund's adjuvant, E. coli J5,
dextran sulfate, iron oxide, sodium alginate, Bacto-Adjuvant,
certain synthetic polymers such as Carbopol (BF Goodrich Company,
Cleveland, Ohio), poly-amino acids and co-polymers of amino acids,
saponin, carrageenan, REGRESSIN (Vetrepharm, Athens, Ga.), AVRIDINE
(N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine), long
chain polydispersed beta. (1,4) linked mannan polymers interspersed
with O-acetylated groups (e.g. ACEMANNAN), deproteinized highly
purified cell wall extracts derived from non-pathogenic strain of
Mycobacterium species (e.g. EQUIMUNE, Vetrepharm Research Inc.,
Athens Ga.), Mannite monooleate, paraffin oil and muramyl
dipeptide. A suitable adjuvant can be selected by one of ordinary
skill in the art.
[0140] An effective amount of the immunogenic composition is
determined based on the intended goal, for example vaccination of a
human or non-human subject. The appropriate dose will vary
depending on the characteristics of the subject, for example,
whether the subject is a human or non-human, the age, weight, and
other health considerations pertaining to the condition or status
of the subject, the mode, route of administration, and number of
doses, and whether the pharmaceutical composition includes nucleic
acids or viruses. Generally, the immunogenic compositions described
herein are administered for the purpose of eliciting an immune
response against an influenza antigen (or antigens) or an influenza
virus. Accordingly, the dose is typically an immunologically
effective amount of the recombinant adenovirus.
[0141] A typical dose of a recombinant adenovirus that expresses an
influenza antigen is from 10 p.f.u. to 10.sup.15
p.f.u./administration. For example, a pharmaceutical composition
can include from about 100 p.f.u. of a recombinant adenovirus, such
as about 1000 p.f.u., about 10,000 p.f.u., or about 100,000 p.f.u.
of each recombinant adenovirus in a single dosage. Optionally, a
pharmaceutical composition can include at least about a million
p.f.u. or more per administration. For example, in some cases it is
desirable to administer about 10.sup.7, 10.sup.8, 10.sup.9 or
10.sup.10 p.f.u. of recombinant adenovirus that expresses a
particular influenza antigen. In some cases, for example, where a
single adenovirus is administered, a higher dosage of virus is
administered to a subject, for example, when a recombinant virus
that expresses multiple influenza antigens is administered.
Alternatively, when several adenovirus, each of which expresses a
different influenza antigen(s), is administered, fewer of each
virus is administered, although the total dose of virus may
nonetheless be upwards of 10.sup.8, or greater than 10.sup.9, or
10.sup.10 p.f.u.
[0142] In addition, adenovirus vectors (nucleic acid vectors) can
be administered For example, when administering a nucleic acid
vaccine including an adenovirus vector that encodes an influenza
antigen, facilitators of nucleic acid uptake and/or expression can
also be included, such as bupivacaine, cardiotoxin and sucrose, and
transfection facilitating vehicles such as liposomal or lipid
preparations that are routinely used to deliver nucleic acid
molecules. Anionic and neutral liposomes are widely available and
well known for delivering nucleic acid molecules (see, e.g.,
Liposomes: A Practical Approach, RPC New Ed., IRL Press, 1990).
Cationic lipid preparations are also well known vehicles for use in
delivery of nucleic acid molecules. Suitable lipid preparations
include DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium
chloride), available under the tradename LIPOFECTIN.RTM.., and
DOTAP (1,2-bis(oleyloxy)-3-(trimethylammonio)propane), see, e.g.,
Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7416, 1987;
Malone et al., Proc. Natl. Acad. Sci. USA 86:6077-6081, 1989; U.S.
Pat. Nos. 5,283,185 and 5,527,928, and International Publication
Nos WO 90/11092, WO 91/15501 and WO 95/26356. These cationic lipids
may preferably be used in association with a neutral lipid, for
example DOPE (dioleyl phosphatidylethanolamine)-. Still further
transfection-facilitating compositions that can be added to the
above lipid or liposome preparations include spermine derivatives
(see, e.g., International Publication No. WO 93/18759) and
membrane-permeabilizing compounds such as GALA, Gramicidine S and
cationic bile salts (see, e.g., International Publication No. WO
93/19768).
[0143] Alternatively, nucleic acids (adenovirus vectors) encoding
avian influenza viral genes can be encapsulated, adsorbed to, or
associated with, particulate carriers. Suitable particulate
carriers include those derived from polymethyl methacrylate
polymers, as well as PLG microparticles derived from poly(lactides)
and poly(lactide-co-glycolides). See, e.g., Jeffery et al., Pharm.
Res. 10:362-368, 1993. Other particulate systems and polymers can
also be used, for example, polymers such as polylysine,
polyarginine, polyornithine, spermine, spermidine, as well as
conjugates of these molecules.
[0144] The formulated vaccine compositions will thus typically
include a polynucleotide (e.g. a plasmid) containing an adenovirus
that includes a polynucleotide sequence encoding an avian influenza
antigen in an amount sufficient to mount an immunological response.
An appropriate effective amount can be readily determined by one of
skill in the art. Such an amount will fall in a relatively broad
range that can be determined through routine trials. For example,
immune responses have been obtained using as little as 1 .mu.g of
DNA, while in other administrations, up to 2 mg of DNA has been
used. It is generally expected that an effective dose of
polynucleotides containing the genomic fragments will fall within a
range of about 10 .mu.g to 1 mg, however, doses above and below
this range may also be found effective.
[0145] Adenovirus vector nucleic acids can be coated onto carrier
particles (e.g., core carriers) using a variety of techniques known
in the art. Carrier particles are selected from materials which
have a suitable density in the range of particle sizes typically
used for intracellular delivery from an appropriate
particle-mediated delivery device. The optimum carrier particle
size will, of course, depend on the diameter of the target cells.
Alternatively, colloidal gold particles can be used wherein the
coated colloidal gold is administered (e.g., injected) into tissue
(e.g., skin or muscle) and subsequently taken-up by
immune-competent cells.
[0146] Tungsten, gold, platinum and iridium carrier particles can
be used. Tungsten and gold particles are preferred. Tungsten
particles are readily available in average sizes of 0.5 to 2.0
.mu.m in diameter. Although such particles have optimal density for
use in particle acceleration delivery methods, and allow highly
efficient coating with DNA, tungsten may potentially be toxic to
certain cell types. Gold particles or microcrystalline gold (e.g.,
gold powder A1570, available from Engelhard Corp., East Newark,
N.J.) will also find use with the present methods. Gold particles
provide uniformity in size (available from Alpha Chemicals in
particle sizes of 1-3 .mu.m, or available from Degussa, South
Plainfield, N.J. in a range of particle sizes including 0.95 .mu.m)
and reduced toxicity.
[0147] A number of methods are known and have been described for
coating or precipitating DNA or RNA onto gold or tungsten
particles. Most such methods generally combine a predetermined
amount of gold or tungsten with plasmid DNA, CaCl.sub.2 and
spermidine. The resulting solution is vortexed continually during
the coating procedure to ensure uniformity of the reaction mixture.
After precipitation of the nucleic acid, the coated particles can
be transferred to suitable membranes and allowed to dry prior to
use, coated onto surfaces of a sample module or cassette, or loaded
into a delivery cassette for use in a suitable particle delivery
instrument, such as a gene gun. Alternatively, nucleic acid
vaccines can be administered via a mucosal membrane or through the
skin, for example, using a transdermal patch. Such patches can
include wetting agents, chemical agents and other components that
breach the integrity of the skin allowing passage of the nucleic
acid into cells of the subject.
[0148] In addition to adjuvants, which typically potentiate or
enhance an immune response in a non-specific manner, for example by
concentrating or slowing diffusion of an antigen, genetic
immunstimulatory agents can be included in the pharmaceutical
compositions along with adenovirus vectors or adenoviruses to
enhance the anti-avian influenza immune response. For example,
certain CpG oligonucleotides (such as 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 in conjunction
with an antigen. Exemplary CpG oligonucleotides are described in
U.S. Pat. Nos. 6,610,661; 6,589,940, and 6,514,948, the disclosures
of which are incorporated herein for all purposes. CpG containing
oligonucleotides interact with Toll-like receptors (such as TLR 7
and TLR9) and result in the production of interferon gamma
(IFN-.gamma.), which potentiates the antiviral immune response.
[0149] Additionally, some subjects (for example, aged subjects) who
have reduced expression of TLRs can benefit from the co-expression
of TLRs (such as TLR5, TLR7 and TLR9) in cells in which the
adenovirus vector or adenovirus expressing the influenza antigen is
expressed. Accordingly, adenovirus vectors containing a TLR can be
included in the pharmaceutical compositions described above. A TLR
can be incorporated into the same adenovirus vector that encodes
the influenza antigen(s) or can be included in the pharmaceutical
composition (medicament) as a separate adenovirus vector or virus.
Optionally, a TLR and its ligand (e.g., a CpG oligonucleotide,
flagellin) are included together in a pharmaceutical
composition.
[0150] In certain embodiments, the pharmaceutical (e.g., vaccine
compositions) are administered to avian subjects, such as
domesticated fowl, including but not limited to, chickens, ducks,
turkeys and geese. The pharmaceutical compositions can be
administered to young and/or adult birds and/or to embryos in ovo.
Methods utilized in the poultry industry for administering vaccine
compositions are well known, and any such methods are suitable for
administering the immunogenic compositions, e.g., adenovirus
containing avian influenza antigen(s) disclosed herein. For
example, adenovirus based influenza vaccines can be administered to
domesticated fowl in drinking water. Various methods are available
for administering vaccines in the drinking water of domesticated
birds. In one convenient method, a solution containing the
immunogenic composition is placed in an intravenous solution bag
(for example, the SELECT FIELD BAG BOOST.TM. system from Merial,
Inc., Lyon, France), which is connected to one or more drinker
lines. Optionally, the solution contains a dye (or other visually
detectable indicator, such as skimmed milk powder) along with the
diluent to facilitate monitoring of vaccine administration. In the
case of adenovirus-based vaccines, it is important that the
solution be free of disinfecting agents, such as chlorine or other
disinfectants that may be used to clean the delivery system. If
desired, drinking activity of the birds can be stimulated to
increase consumption of the vaccine/water solution. For example, by
increasing light intensity, delivering food and/or disturbing the
birds (e.g., by walking through the flock) birds can be stimulated
to increase consumption of vaccine laden drinking water.
[0151] In another method, the vaccine composition is administered
by spraying. Typically spray applications are performed on many
birds housed in a common airspace, for example, spray
administration can be performed on day old birds in delivery boxes,
or to birds in conventional housing. For example, newly hatched
birds can be vaccinated using a spray delivery system as described,
e.g., 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, day old birds contained in delivery boxes in
groups of up to approximately 150 subjects are exposed to vaccine
diluted in an aqueous medium, such as water, delivered by means of
a spray nozzle, which forms very small droplets (for example, in
the range of approximately 100.mu. to about 500.mu. diameter).
Vaccine is taken up by day old birds via the ocular, intranasal, as
well as oral routes from the container surface and other birds.
Vaccine can be delivered to older birds by spray administration,
for example, using a pressurized spray apparatus or controlled
droplet application device. A pressurized spray apparatus typically
includes a pressure chamber, lance and nozzle. The nozzle and the
operating pressure can be varied to alter the particle size, which
is typically within a range of about 10.mu. to about 1000.mu..
Droplets come into contact with the birds either directly from
nozzle emission via inhalation, or via ocular or oral routes, or
indirectly from contact with vaccine deposited on the ground or
other birds by the spray apparatus. Equipment for spray
administration of vaccine compositions are readily available and
exemplary devices are disclosed, e.g., in U.S. Pat. Nos. 5,312,353
and 4,863,443, the disclosures of which are incorporated herein by
reference. Controlled droplet application devices developed for
horticultural and insect control use can also be used to deliver
adenovirus-based vaccine to domesticated fowl. In such devices,
spray is generated by centrifugal force as the diluted vaccine is
delivered to spinning disc which forms a spray of nebulized vaccine
with a size range suitable for uptake by inhalation. This nebulized
vaccine can be distributed by means of a fan which distributes the
vaccine over a broader field than can typically be achieved with a
pressurized spray apparatus. This process offers the added
advantage that relatively small volumes of diluent (for example,
water) are required. For example, up to 30,000 birds can be
vaccinated with approximately one liter of vaccine solution.
[0152] More invasive and/or laborious procedures can also be
employed to administer the immunogenic compositions disclosed
herein to domesticated fowl, including, in addition to the methods
disclosed above: eye drop, transfixion and scarification (e.g., via
a cutaneous route in the wing web or foot), injection and in ovo
administration. Automated and semi-automated injection devices
suitable for delivering the disclosed vaccines to poultry are
described, e.g., in U.S. Pat. Nos. 4,681,565 and 4,515,590, the
disclosures of which are incorporated herein by reference.
[0153] In ovo administration of adenovirus containing avian
influenza antigens is also favorably used to elicit a protective
immune response against influenza in domestic fowl. In ovo
administration typically involves injecting an immunologically
effective dose of adenovirus containing one or more avian influenza
virus antigens into eggs at an appropriate stage of gestation at
which immunological competency has developed, but prior to
hatching. For example, chicken eggs are typically injected at
between 17.5 and 19 days of incubation. The volume is calculated to
not substantially disrupt the integrity of the egg, and is
typically in the range of between 0.01 and 0.1 ml (such as, 0.05
ml). Typically, a small hole is made in the shell of the egg, into
which an injection needle is inserted to deliver the immunogenic
composition. The injections can be performed manually or with the
assistance of commercially-available automated equipment (such as
that available from Embrex, Triangle Park, N.C.) used according to
the manufacturer's instructions. Methods and equipment for in ovo
administration of solutions to poultry eggs suitable for
administering the vaccine compositions disclosed herein are
disclosed 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.
EXAMPLES
Example 1
Generation of Adenovirus Vector Expressing Avian Hemagglutinin (HA)
Antigen
[0154] Homologous recombination was used to produce an exemplary
adenovirus vector that expresses avian H5 hemagglutinin (HA)
antigen. The Cre recombinase-mediated site specific recombination
system of Ng et al. (Human Gene Therapy 10:2667-2672, 1999) was
utilized to generate HAd5 vectors including an avian HA antigen. To
generate a HAd5 E1 insertion vector expressing a HA antigen of a
H.sub.5N1 strain influenza, the HA gene of strain A/Hong
Kong/156/97 under the control of the cytomegalovirus immediate
early promoter ("CMV promoter") was inserted at the StuI site in a
shuttle vector (pDC311). The pDC311 shuttle vector is a plasmid
that contains the left end of HAd5 (4 kb) with a 3.1 kb E1
deletion, a loxP site for site specific recombination in the
presence of Cre recombinase, and an intact packaging signal
(.psi.). The resulting vector pDC311-H5 was cotransfected into 293
Cre (293 cells expressing Cre recombinase), along with
pBHGlox.DELTA.E1,3Cre. The pBHGlox.DELTA.E1,3Cre plasmid contains
almost the entire HAd5 genome with the exception of the packaging
signal and deletions in the E1 and E3 region genes. This plasmid
also includes a loxp site for Cre recombinase mediated
recombination. When introduced together into 293 Cre cells Cre
mediated recombination between the two plasmids generated the
vector HAd5-HA (FIG. 1A).
[0155] Cell extracts analyzed by Western blotting exhibited
expression of the introduced avian HA antigen (FIG. 1B).
Example 2
Immunogenicity and Efficacy of Protection in HAd-H5 Immunized
Animals
[0156] To demonstrate immunogenicity of recombinant adenovirus
expressing avian HA, mice were inoculated with recombinant
adenovirus and challenged with a lethal dose of avian influenza
virus. Twenty-five 6-8 week-old female C57BL/6 mice were randomly
divided into 5 groups with 5 animals per group. The animals were
inoculated intramuscularly on days 1 and 28 with either PBS, 15
.mu.g recombinant H5 (hemagglutinin of avian H.sub.5N.sub.1
influenza virus expressed in baculovirus) without alum (H5 alone),
15 .mu.g H5 with alum (H5+alum), 10.sup.8 p.f.u. of HAd-.DELTA.E1E3
(HAd5 control vector), or 10.sup.8 p.f.u. of HAd-H5 (HAd5 vector
expressing H5). Serum samples were collected on days 21 and 49 to
monitor the development of H5-specific immune response by ELISA and
micro-neutralization assay. Animals were challenged with
100LD.sub.50 of H.sub.5N1 (A/Hong Kong/483/97) virus on day 70 and
were monitored daily for gain or loss in body weight and obvious
clinical signs of influenza infection.
[0157] A single inoculation of HAd-H5 elicited H5-specific IgG
ELISA titers above 3.5 logs on day 21 demonstrating that H5
expressed by HAd5-H5 was highly immunogenic. Serum samples were
collected on day 49 to monitor the development of H.sub.5N1 virus
neutralizing antibody response by virus neutralization assay.
Immunization of mice with HAd-H5 elicited H.sub.5N1-specific
neutralizing antibody response similar to those obtained with a
high dose of H5+alum (Table 1).
[0158] Mice immunized with HAd-H5 were fully protected against
morbidity and mortality following challenge with pathogenic
H.sub.5N1 virus. Animals were challenged with 100LD.sub.50 of
H.sub.5N1 (A/HK/483/97) virus on day 70. The level of protection
was better than that observed with high dose recombinant H5+alum
(Table 1 and FIG. 2). None of the mice in the HAd5-H5 showed any
visual discomfort following challenge.
TABLE-US-00001 TABLE 1 Serological response in mice immunized with
HAd-H5HA vaccines. Geometric Geometric Mean Mean Horse
Neutralization Groups HI titers Titers Survival PBS 25 20 0 HAd-5
(10.sup.8 p.f.u.) i.m. 25 20 0 HAd-H5HA (10.sup.8 p.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
Intranasal Administration of HAd5-HA Elicits Protective Immune
Response
[0159] The ability of the HAd-H5HA vector to elicit an antibody
response specific for HA was measured following intramuscular and
intranasal administration. Three sets of 15 (6- to 8-week old)
female BALB/c mice were randomly divided into 3 groups (5
animal/group) and inoculated intramuscularly on days 0 and 28 with
10.sup.8 p.f.u. of HAd-.DELTA.E1E3 or 10.sup.8 p.f.u. of HAd-H5HA
administered intramuscularly or intranasally. Sera were collected
on days 21 and 49 to monitor the development of H5-specific immune
response against A/HK/483/97, A/HK/213/03 and A/VN/1203/04 strains
by hemagglutination inhibition (HI) assay using horse red blood
cells. Intramuscular or intranasal administration of HAd5-H5HA
elicited strong HA antibody titers by ELISA, as shown in Table
2.
TABLE-US-00002 TABLE 2 Titer against homologous and recent H5
strains induced by HAd5-HA vaccine A/HK/156/97 A/HK/213/03
A/VN/1203/04 1.sup.st 2.sup.nd 1.sup.st 2.sup.nd 1.sup.st 2.sup.nd
Group bleed bleed bleed bleed bleed bleed Vector Control 5 5 5 5 5
5 HAd5-H5 i.m. 95 189 21.4 23.1 14.1 18 HAd5-H5 i.n. 146 218 36.4
36.4 18.2 18
[0160] Similarly, virus neutralization assay BALB/c mice (20 per
group) were immunized either intramuscularly or intranasally with
1.times.10.sup.8 p.f.u. of HAd-H5HA twice every 4 weeks. Other
groups of mice (20 per group) were intramuscularly immunized with
10.sup.8 p.f.u. of HAd-.DELTA.E1E3 or 3 .mu.g of rH5HA with alum.
The serum samples were obtained 4 weeks after the second
immunization and analysed by virus neutralization assays to assess
their ability to react with a homologous virus (HK/156/97), or with
antigenically heterologous viruses (A/Hong Kong/213/2003
[HK/213/03] and A/Vietnam/1203/04 [VN/1203/04]). Compared with
HK/156/97, the amino acid homology in the hemagglutinin subunit is
94-8% for HK/213/03 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 homologous HK/156/97 virus,
but failed to neutralize HK/213/03 or VN/1203/04 virus. In
contrast, mice immunized with HAd-H5HA produced neutralizing
antibodies to both homologous and heterologous viruses, showing
higher neutralizing antibody titers against HK/156/97 and lower
titers against heterologous HK/213/03 or VN/1203/04 virus.
Example 4
Vaccination with HAd5-H5 Elicits HA-Specific T Cell Responses
[0161] To assess whether the HAd-H5HA vaccine induced an
HA-specific CD8.sup.+ T cell response, BALB/c mice immunized with
10.sup.8 p.f.u. of HAd-.DELTA.E1E3 or 10.sup.8 p.f.u. of HAd-H5HA
administered intramuscularly or intranasally and splenic T cell
responses to influenza epitopes were evaluated by staining with a
K.sub.d-specific pentamer for the immunodominant HA 518
(HA.sub.518-526) epitope, originally described for HA of an H1N1
virus, A/Puerto Rico/8/34). This epitope is broadly conserved among
H.sub.5N1 viruses, including currently circulating avian and human
H.sub.5N1 viruses, as well as more divergent viruses, e.g.,
H.sub.9N.sub.2 strains. Mice that received the HAd-H5HA vector
either i.n. or i.m. had at least a 3 to 8 fold higher frequency of
HA-specific CD8.sup.+ T cells compared to the mice immunized with
HAd-.DELTA.E1E3 (FIG. 4). No detectable increase in NP-147
(NP.sub.147-155)-epitope specific CD8.sup.+ T cells was observed in
animals immunized with HAd-H5HA vaccine. In contrast, control mice
infected with H.sub.5N.sub.1 virus exhibited a strong NP 147
epitope-specific CD8.sup.+ T cell response. None or the mice
vaccinated with HAd-.DELTA.E1E3 or rH5HA+alum showed an increase in
HA 518 epitope-specific CD8.sup.+ T cell frequencies.
[0162] Epitope specific T cell responses were determined by
stimulating 1.times.10.sup.6 spleen cells with syngeneic
gamma-irradiated spleen cells pulsed with 10 .mu.g/ml of the
designated peptide (including MHC Class I binding epitopes: NP 147
and HA 462 and HA 518, which have been shown to be dominant
epitopes in H.sub.5N.sub.3 infected animals). PMA+ionomycin was
used as a positive control. IFN-.gamma. production was evaluated by
ELIspot assay, as shown in FIG. 5. Vaccination with HAd5-H5
elicited HA-specific T cell responses against Class I MHC binding
epitopes of HA. This T cell response was not observed in animals
inoculated with recombinant H5+alum.
Example 5
Inoculation with HAd5-H5HA Confers Protection Against Lethal
Challenge
[0163] Animals vaccinated with HAd5-H5HA by either intramuscular or
intranasal administration routes were challenged with the
homologous strain virus and with a recent strain of avian
influenza. Following inoculation, mice were challenged with 100
LD.sub.50 A/HK/156/97 or A/VN/1203/04 strain virus. As shown in
Table 3, all inoculated animals survived lethal challenge with
either the homologous virus or a different H.sub.5N1 virus
strain.
TABLE-US-00003 TABLE 3 HAd-5H5HA confers protection against lethal
challenge % Survival against challenge with Group A/HK/156/97
A/VN/1203/04 Vector Control 0 0 HAd-H5HA i.m. 100 100 HAd-H5HA i.n.
100 100
Example 6
Morbidity of Animal Challenged with A/HK/483197 or A/VN/1203/04
Virus
[0164] To assess the protective efficacy against challenge with the
variant H.sub.5N1 strains, 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 for mice. Therefore, to evaluate the vaccine
efficacy against HK/213/03, virus titers in the lungs were measured
in HK/213/03-infected animals on day 4 post-challenge. Immunization
of mice either i.n. or i.m. with HAd-H5HA exhibited minimal
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 route of inoculation survived
and exhibited minimal morbidity, as measured by weight loss,
following a lethal challenge with more recent H.sub.5N1 virus,
VN/1203/04 (FIGS. 6C and D). Furthermore, mice vaccinated with
HAd-H5HA by either route of inoculation and challenged with
HK/213/03 virus had no detectable virus in the lungs on day 4
post-infection (FIG. 6E), whereas mice vaccinated with the control
vector HAd-.DELTA.E1E3 had a mean lung viral titer of greater than
106 EID.sub.50/ml (p<0.001). Therefore, the HAd-H5HA vaccine
induced significant protection against heterologous H.sub.5N.sub.1
viruses, even in the presence of low levels of cross-neutralizing
serum antibody titers.
[0165] These data confirm the potential of Ad vector-based delivery
of avian influenza antigen(s) as pandemic influenza vaccines. Such
vectors induce strong humoral and cellular immunity and confer
cross-protection against continuously evolving H.sub.5N.sub.1
viruses without the need of an adjuvant.
Example 7
Generation and Characterization of Nonhuman Vectors Expressing HA
of H.sub.5N.sub.1 Influenza Virus
[0166] Infectious clones containing the entire genome of nonhuman
adenovirus (porcine adenovirus type 3, PAd3 or bovine adenovirus
type 3, BAd3) with deletions in E1 and E3 regions with or without
insertion in E1 were generated by homologous recombination in E.
coli BJ5183. The HA gene of H.sub.5N1, flanked by the CMV promoter
and the bovine growth hormone BGH polyadenylation signal was cloned
into pDS2 (Bangari & Mittal, Virus Research 105:127-136, 2004)
at the AvrII site to obtain pDS2-H5. Using homologous recombination
in E. coli BJ5183 as described in van Olphen & Mittal, J.
Virol. Methods 77:125-129, 1999, with respect to bovine adenovirus,
pPAd-H5 (a genomic plasmid with an avian HA insertion into the E1A
gene region of porcine adenovirus) was generated by
cotransformation of E. coli with E3-deleted PAd3 genomic DNA and
StuI linearized pDS2-H5.
[0167] To generate HA of H.sub.5N.sub.1 influenza from the PAd3
vector, monolayer cultures of FPRT HE1-5 cells (an E1 expressing
porcine cell line described in Bangari & Mittal, Virus Res.
105:127-136, 2004) were transfected with PacI-digested pPAd-H5 (5
.mu.g/60-mm dish) using LIPOFECTIN.RTM.-mediated transfection
according to the manufacturer's recommendations. Recombinant
virus-induced cytopathic effect was visible in 2-3 weeks
post-transfection.
[0168] Replication-defective recombinant PAd3 vector (PAd-H5HA)
containing the full-length coding region of the HA gene of
H.sub.5N1 virus (HK/156/97) inserted in the early region 1 (E1) of
PAd3 genome (FIG. 7A) was expressed efficiently in FPRT HE1-5 cells
as demonstrated by western blotting (FIG. 7B). A PAd with deletions
of E1 and E3 regions (PAd-.DELTA.E1E3) served as a negative
control.
[0169] Similarly, a replication-defective recombinant BAd3 vector
(BAd-H5HA) including the full-length coding region of the HA gene
of H.sub.5N1 virus (HK/156/97) inserted in the early region 1 (E1)
of BAd3 genome (FIG. 7C) was expressed efficiently in FBRT-HE1
cells that express BAd3 E1 (van Olphen et al., Virology
295:108-118, 2002) as shown in FIG. 7D. A BAd3 with deletions of E1
and E3 regions (BAd-.DELTA.E1E3) served as a negative control.
[0170] These nonhuman adenoviral vectors are suitable as vaccine
vectors, which can be administered to human and non-human subjects
to elicit a protective immune response specific for avian (and
other) influenza strains.
Example 8
Cell Lines for Expressing Adenovirus Vectors
[0171] Novel cell lines were produced that express E1 antigens of
adenovirus vectors with different species tropism (human and
non-human). Such multi-functionality cell lines are particularly
advantageous for optimizing production of replication deficient
adenovirus from recombinant vectors corresponding to viruses from
multiple strains to produce adenovirus and/or recombinant
protein.
[0172] Two exemplary multi-functionality cell lines were produced,
based on previously described cell lines that express the HAd5 E1
gene in either bovine or porcine cells. FBRT-HE1 is a fetal bovine
retinal cell line that expresses the E1 gene of the 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. In
brief, primary fetal bovine retina (FBRT) cells were transfected
with the plasmid containing HAdS E1 (where HAd5 E1 was under the
control of the PGK promoter). A number of G418-resistant colonies
were isolated and tested for the expression of E1B-19 kDa protein
by Western blot. Cell lines expressing E1B-19 kDa were further
tested for E1A, E1B-19 kDa and E1B-55 kDa expression by
immunoprecipitation. A representative cell line, designated
FBRT-HE1, expressed all three E1 proteins: E1A, E1B-19 kDa and
E1B-55 kDa. The FBRT-HE1 cell line supports the growth of
E1-deleted human adenovirus vectors as well as E1 deleted bovine
adenovirus vectors.
[0173] To produce a multi-functionality cell line, a fragment
containing BAd3 .mu.l (604-3148) was amplified using BAd E1-F
(CCATGAAGTACCTGGTCCTC; SEQ ID NO:5) and BAd E1-R
(CCCCACCTATTTATACCCCTC; SEQ ID NO:6) primer set. The PCR fragment
encompassing the BAd3 E1 gene (SEQ ID NO:7) was cloned in pPGK-puro
at the EcoRV site and in 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).
After 48 hrs of transfection the cells were grown in the selection
media containing 3 .mu.g/ml puromycin. Discreet colonies of cells
could be seen after about 30 days of antibiotic selection in
pPGK-BE1 or pCMV-BE1 transfected FBRT-HE1 cells. 24 colonies (12
colonies each from pPGK-BE1 and pCMV-BE1 transfected cells) were
picked, expanded and screened for expression of all three BAd3 E1
transcripts (E1A, E1B-1 and E1B-2) by RT-PCR using the specific
primer sets (Table 4) to identify clones that expressed all three
BAd E1 transcripts. The FBRT-HE1/PE1 cell clone shown in FIG. 8A
illustrates expression of all three BAd3 E1 genes under the PGK
promoter.
TABLE-US-00004 TABLE 4 Primers for RT PCR of bovine E1 transcripts
Gene Primer sequence (5' to 3') BAd E1A (For)
CTGATATCATGAAGTACCTGGCCTC (SEQ ID NO:8) BAd E1A (Rev)
ATGCAATGGTAGGTTTGG (SEQ ID NO:9) BAd E1B-1 (For)
GATATCATGGATCACTTAAGCGTTC (SEQ ID NO:10) BAd E1B-1 (Rev)
GTCGACAACTGATGTGCTCGAAACG (SEQ ID NO:11) BAd E1B-2 (For)
GATATCGTTCAAGATCACCCAGAG (SEQ ID NO:12) BAd E1B-2 (Rev)
GTCGACCACTTTTAATCCTGCTC (SEQ ID NO:13)
[0174] Similarly, multi-functional porcine cells were produced by
introducing a porcine adenovirus E1 (PAd3 E1) into a cell line that
expressed HAd5 E1. FPRT-HE1-5 is a fetal porcine retinal cell line
that constitutively expresses the HAd5 E1 (SEQ ID NO:4). Production
of FPRT-HE1-5 is disclosed in Bangari & Mittal (Virus Res.
105:127-136, 2004), which is incorporated herein by reference. In
brief, primary fetal porcine retinal (FPRT) cells were transfected
with a plasmid containing HAd5 E1 under the control of either the
cytomegalovirus (CMV) immediate early or phosphoglycerate kinase
(PGK) promoter. Transformed cell lines obtained by transfection
with HAd5 E1 sequences under the control of CMV or PGK promoter
were selected and further characterized. FPRT-HE1-5 is an exemplary
cell line that expresses all three E1 genes efficiently and can be
used to generate and grow E1-deleted porcine and human adenovirus
vectors.
[0175] PAd3 plasmids were constructed for transfection into
FPRT-HE1-5 cells in the following manner. The neomycin ORF in the
pcDNA3.1 (Invitrogen) was replaced with puromycin ORF from
pBABE-puro (Addgene) to obtain the plasmid pCMV-puro. The CMV
promoter sequence in plasmid pcDNA3.1-puro was replaced with PGK
promoter from pGT-N28 (New England Biolabs) to obtain the plasmid
pPGK-puro. The fragments containing PAd3 E1 (526-3259) was
amplified using PAd E1-F (TGGATCCTCGACATGGCGAACAGACTT; SEQ ID
NO:14) and PAd E1-R (TCTCGAGTCATCCTCAGTCATCGTCATCG; SEQ ID NO:15)
primer set. The resulting PCR product including the coding sequence
of the PAd3 E1 gene (SEQ ID NO:16) was subsequently cloned at the
BamHI-XhoI site of pPGK-puro or pCMV-puro to obtain pPGK-PE1 and
pCMV-PE1, respectively.
[0176] These plasmids were then used to transfect FPRT-HE1 cell
line using Lipofectin (Life Technologies). After 48 hrs of
transfection the cells were grown in the selection media containing
2 .mu.g/ml puromycin. Discreet colonies of cells could be seen
after about 30 days of antibiotic selection in both pPGK-PE1 and
pCMV-PE1 transfected FPRT-HE1 cells. Twenty-four colonies (12
colonies each from pPGK-PE1 and pCMV-PE1 transfected cells) were
picked, expanded and screened for expression of all three PAd3 E1
transcripts (E1A, E1B-1 and E1B-2) by RT-PCR using the specific
primer sets (Table 5). Multiple clones from pPGK-PE1 transfected
cells and pCMV-PE1 transfected cells were found positive for
expression of all three PAd3 E1 transcripts. The FPRT-HE1/PE1 cell
clone shown in FIG. 8B illustrates E1 expression under the PGK
promoter.
TABLE-US-00005 TABLE 5 Primers for RT PCR of porcine E1 transcripts
Gene Primer sequence (5' to 3') PAd E1A (For)
AGGTGGAGGTGATTGTGACTGA (SEQ ID NO:17) PAd E1A (Rev)
GACGCAAGAGGAAGTACTGCTA (SEQ ID NO:18) PAd E1B-1 (For)
CTGGCCAAGCTTACTAACGTGAAC (SEQ ID NO:19) PAd E1B-1 (Rev)
TTTAAGTCTTCTGGTGCCGCCA (SEQ ID NO:20) PAd E1B-2 (For)
ATGCATGAGCGCTACAGCTTTG (SEQ ID NO:21) PAd E1B-2 (Rev)
CTGAGTTCCGCAAGAATGTGCT (SEQ ID NO:22)
Example 9
Adenovirus Vectors Expressing Multiple Influenza Antigens
[0177] Adenovirus vectors are generated that incorporate multiple
(a plurality of) influenza antigens. The Cre recombinase-mediated
site specific recombination system of Ng et al., (Human Gene
Therapy 10:2667-2672, 1999) can be utilized to generate HAd5
vectors including two or more influenza antigens. To generate a
HAd5 E1 insertion vector expressing multiple influenza antigens,
polynucleotide sequences that encode the selected antigens are
cloned under the control of a promoter, such as the cytomegalovirus
immediate early promoter ("CMV promoter"), and inserted, e.g., at
the Stul site in a shuttle vector. The shuttle vector, such as
pDC311, includes a loxp site for site specific recombination in the
presence of Cre recombinase, and an intact packaging signal
(.psi.). The resulting vector is cotransfected into 293 Cre (293
cells expressing Cre recombinase), along with
pBHGlox.DELTA.E1,3Cre. The pBHGlox.DELTA.E1,3Cre plasmid contains
almost the entire HAd5 genome with the exception of the packaging
signal and deletions in the E1 and E3 region genes. This plasmid
also includes a loxP site for Cre recombinase mediated
recombination. When introduced together into 293 Cre cells Cre
mediated recombination between the two plasmids generates a vector
that includes the polynucleotide sequence encoding the selected
influenza antigens. Optionally, this procedure can be used to
incorporate a polynucleotide sequence that encodes an additional
polypeptide that augments immune function, such as the TLRs
described above. Exemplary combinations of influenza antigens are
provided in Table 6. The exemplary combinations given in Table 6
are meant to be illustrative. Additional combinations of antigens
can be determined by those of ordinary skill in the art. Similarly,
non-human adenovirus vectors (e.g., PAd3 or BAd3) can be used to
generate various recombinants expressing the exemplary combinations
of influenza antigens are provided in Table 6.
TABLE-US-00006 TABLE 6 Exemplary combinations of antigens in
multi-antigen adenovirus vectors. Exemplary Internal Combination
Hemagglutinin (HA) Neuraminidase (NA) Protein(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
H5.sup.1 + H5.sup.2 + H5.sup.3 26 H5.sup.1 + H5.sup.2 + H5.sup.3 N1
27 H5.sup.1 + H5.sup.2 + H5.sup.3 M 28 H5.sup.1 + H5.sup.2 +
H5.sup.3 NP 29 H5.sup.1 + H5.sup.2 + H5.sup.3 M 30 H5.sup.1 +
H5.sup.2 + H5.sup.3 N1 NP 31 H5.sup.1 + H5.sup.2 + H5.sup.3 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 Superscript designations indicate variants. *M indicates
either M1 M2 or both M1 and M2.
[0178] 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 following claims. We therefore claim as our
invention all that comes within the scope and spirit of these
claims.
Sequence CWU 1
1
22120DNAartificial sequenceimmunostimulatory oligonucleotide
1tccatgagct tcctgatcct 20220DNAartificial sequenceimmunostimulatory
oligonucleotide 2tccatgacgt tcctgacgtt 20322DNAartificial
sequenceimmunostimulatory oligonucleotide 3tgactgtgaa cgttcgagat ga
2242950DNAHuman adenovirus type 5 4atgagacata ttatctgcca cggaggtgtt
attaccgaag aaatggccgc cagtcttttg 60gaccagctga tcgaagaggt actggctgat
aatcttccac ctcctagcca ttttgaacca 120cctacccttc acgaactgta
tgatttagac gtgacggccc ccgaagatcc caacgaggag 180gcggtttcgc
agatttttcc cgactctgta atgttggcgg tgcaggaagg gattgactta
240ctcacttttc cgccggcgcc cggttctccg gagccgcctc acctttcccg
gcagcccgag 300cagccggagc agagagcctt gggtccggtt tctatgccaa
accttgtacc ggaggtgatc 360gatcttacct gccacgaggc tggctttcca
cccagtgacg acgaggatga agagggtgag 420gagtttgtgt tagattatgt
ggagcacccc gggcacggtt gcaggtcttg tcattatcac 480cggaggaata
cgggggaccc agatattatg tgttcgcttt gctatatgag gacctgtggc
540atgtttgtct acagtaagtg aaaattatgg gcagtgggtg atagagtggt
gggtttggtg 600tggtaatttt ttttttaatt tttacagttt tgtggtttaa
agaattttgt attgtgattt 660ttttaaaagg tcctgtgtct gaacctgagc
ctgagcccga gccagaaccg gagcctgcaa 720gacctacccg ccgtcctaaa
atggcgcctg ctatcctgag acgcccgaca tcacctgtgt 780ctagagaatg
caatagtagt acggatagct gtgactccgg tccttctaac acacctcctg
840agatacaccc ggtggtcccg ctgtgcccca ttaaaccagt tgccgtgaga
gttggtgggc 900gtcgccaggc tgtggaatgt atcgaggact tgcttaacga
gcctgggcaa cctttggact 960tgagctgtaa acgccccagg ccataaggtg
taaacctgtg attgcgtgtg tggttaacgc 1020ctttgtttgc tgaatgagtt
gatgtaagtt taataaaggg tgagataatg tttaacttgc 1080atggcgtgtt
aaatggggcg gggcttaaag ggtatataat gcgccgtggg ctaatcttgg
1140ttacatctga cctcatggag gcttgggagt gtttggaaga tttttctgct
gtgcgtaact 1200tgctggaaca gagctctaac agtacctctt ggttttggag
gtttctgtgg ggctcatccc 1260aggcaaagtt agtctgcaga attaaggagg
attacaagtg ggaatttgaa gagcttttga 1320aatcctgtgg tgagctgttt
gattctttga atctgggtca ccaggcgctt ttccaagaga 1380aggtcatcaa
gactttggat ttttccacac cggggcgcgc tgcggctgct gttgcttttt
1440tgagttttat aaaggataaa tggagcgaag aaacccatct gagcgggggg
tacctgctgg 1500attttctggc catgcatctg tggagagcgg ttgtgagaca
caagaatcgc ctgctactgt 1560tgtcttccgt ccgcccggcg ataataccga
cggaggagca gcagcagcag caggaggaag 1620ccaggcggcg gcggcaggag
cagagcccat ggaacccgag agccggcctg gaccctcggg 1680aatgaatgtt
gtacaggtgg ctgaactgta tccagaactg agacgcattt tgacaattac
1740agaggatggg caggggctaa agggggtaaa gagggagcgg ggggcttgtg
aggctacaga 1800ggaggctagg aatctagctt ttagcttaat gaccagacac
cgtcctgagt gtattacttt 1860tcaacagatc aaggataatt gcgctaatga
gcttgatctg ctggcgcaga agtattccat 1920agagcagctg accacttact
ggctgcagcc aggggatgat tttgaggagg ctattagggt 1980atatgcaaag
gtggcactta ggccagattg caagtacaag atcagcaaac ttgtaaatat
2040caggaattgt tgctacattt ctgggaacgg ggccgaggtg gagatagata
cggaggatag 2100ggtggccttt agatgtagca tgataaatat gtggccgggg
gtgcttggca tggacggggt 2160ggttattatg aatgtaaggt ttactggccc
caattttagc ggtacggttt tcctggccaa 2220taccaacctt atcctacacg
gtgtaagctt ctatgggttt aacaatacct gtgtggaagc 2280ctggaccgat
gtaagggttc ggggctgtgc cttttactgc tgctggaagg gggtggtgtg
2340tcgccccaaa agcagggctt caattaagaa atgcctcttt gaaaggtgta
ccttgggtat 2400cctgtctgag ggtaactcca gggtgcgcca caatgtggcc
tccgactgtg gttgcttcat 2460gctagtgaaa agcgtggctg tgattaagca
taacatggta tgtggcaact gcgaggacag 2520ggcctctcag atgctgacct
gctcggacgg caactgtcac ctgctgaaga ccattcacgt 2580agccagccac
tctcgcaagg cctggccagt gtttgagcat aacatactga cccgctgttc
2640cttgcatttg ggtaacagga ggggggtgtt cctaccttac caatgcaatt
tgagtcacac 2700taagatattg cttgagcccg agagcatgtc caaggtgaac
ctgaacgggg tgtttgacat 2760gaccatgaag atctggaagg tgctgaggta
cgatgagacc cgcaccaggt gcagaccctg 2820cgagtgtggc ggtaaacata
ttaggaacca gcctgtgatg ctggatgtga ccgaggagct 2880gaggcccgat
cacttggtgc tggcctgcac ccgcgctgag tttggctcta gcgatgaaga
2940tacagattga 2950520DNAartificial sequenceoligonucleotide primer
5ccatgaagta cctggtcctc 20621DNAartificial sequenceoligonucleotide
primer 6ccccacctat ttatacccct c 2172507DNABovine adenovirus type 3
7atgaagtacc tggtcctcgt tctcaacgac ggcatgagtc gaattgaaaa agctctcctg
60tgcagcgatg gtgaggtgga tttagagtgt catgaggtac ttcccccttc tcccgcgcct
120gtccccgctt ctgtgtcacc cgtgaggagt cctcctcctc tgtctccggt
gtttcctccg 180tctccgccag ccccgcttgt gaatccagag gcgagttcgc
tgctgcagca gtatcggaga 240gagctgttag agaggagcct gctccgaacg
gccgaaggtc agcagcgtgc agtgtgtcca 300tgtgagcggt tgcccgtgga
agaggatgag tgtctgaatg ccgtaaattt gctgtttcct 360gatccctggc
taaatgcagc tgaaaatggg ggtgatattt ttaagtctcc ggctatgtct
420ccagaaccgt ggatagattt gtctagctac gatagcgatg tagaagaggt
gactagtcac 480ttttttctgg attgccctga agaccccagt cgggagtgtt
catcttgtgg gtttcatcag 540gctcaaagcg gaattccagg cattatgtgc
agtttgtgct acatgcgcca aacctaccat 600tgcatctata gtaagtacat
tctgtaaaag aacatcttgg tgatttctag gtattgttta 660gggattaact
gggtggagtg atcttaatcc ggcataacca aatacatgtt ttcacaggtc
720cagtttctga agaggaaatg tgagtcatgt tgactttggc gcgcaagagg
aaatgtgagt 780catgttgact ttggcgcgcc ctacggtgac tttaaagcaa
tttgaggatc acttttttgt 840tagtcgctat aaagtagtca cggagtcttc
atggatcact taagcgttct tttggatttg 900aagctgcttc gctctatcgt
agcgggggct tcaaatcgca ctggagtgtg gaagaggcgg 960ctgtggctgg
gacgcctgac tcaactggtc catgatacct gcgtagagaa cgagagcata
1020tttctcaatt ctctgccagg gaatgaagct tttttaaggt tgcttcggag
cggctatttt 1080gaagtgtttg acgtgtttgt ggtgcctgag ctgcatctgg
acactccggg tcgagtggtc 1140gccgctcttg ctctgctggt gttcatcctc
aacgatttag acgctaattc tgcttcttca 1200ggctttgatt caggttttct
cgtggaccgt ctctgcgtgc cgctatggct gaaggccagg 1260gcgttcaaga
tcacccagag ctccaggagc acttcgcagc cttcctcgtc gcccgacaag
1320acgacccaga ctaccagcca gtagacgggg acagcccacc ccgggctagc
ctggaggagg 1380ctgaacagag cagcactcgt ttcgagcaca tcagttaccg
agacgtggtg gatgacttca 1440atagatgcca tgatgttttt tatgagaggt
acagttttga ggacataaag agctacgagg 1500ctttgcctga ggacaatttg
gagcagctca tagctatgca tgctaaaatc aagctgctgc 1560ccggtcggga
gtatgagttg actcaacctt tgaacataac atcttgcgcc tatgtgctcg
1620gaaatggggc tactattagg gtaacagggg aagcctcccc ggctattaga
gtgggggcca 1680tggccgtggg tccgtgtgta acaggaatga ctggggtgac
ttttgtgaat tgtaggtttg 1740agagagagtc aacaattagg gggtccctga
tacgagcttc aactcacgtg ctgtttcatg 1800gctgttattt tatgggaatt
atgggcactt gtattgaggt gggggcggga gcttacattc 1860ggggttgtga
gtttgtgggc tgttaccggg gaatctgttc tacttctaac agagatatta
1920aggtgaggca gtgcaacttt gacaaatgct tactgggtat tacttgtaag
ggggactatc 1980gtctttcggg aaatgtgtgt tctgagactt tctgctttgc
tcatttagag ggagagggtt 2040tggttaaaaa caacacagtc aagtccccta
gtcgctggac cagcgagtct ggcttttcca 2100tgataacttg tgcagacggc
agggttacgc ctttgggttc cctccacatt gtgggcaacc 2160gttgtaggcg
ttggccaacc atgcagggga atgtgtttat catgtctaaa ctgtatctgg
2220gcaacagaat agggactgta gccctgcccc agtgtgcttt ctacaagtcc
agcatttgtt 2280tggaggagag ggcgacaaac aagctggtct tggcttgtgc
ttttgagaat aatgtactgg 2340tgtacaaagt gctgagacgg gagagtccct
caaccgtgaa aatgtgtgtt tgtgggactt 2400ctcattatgc aaagcctttg
acactggcaa ttatttcttc agatattcgg gctaatcgat 2460acatgtacac
tgtggactca acagagttca cttctgacga ggattaa 2507825DNAartificial
sequenceoligonucleotide primer 8ctgatatcat gaagtacctg gcctc
25918DNAartificial sequenceoligonucleotide primer 9atgcaatggt
aggtttgg 181025DNAartificial sequenceoligonucleotide primer
10gatatcatgg atcacttaag cgttc 251125DNAartificial
sequenceoligonucleotide primer 11gtcgacaact gatgtgctcg aaacg
251224DNAartificial sequenceoligonucleotide primer 12gatatcgttc
aagatcaccc agag 241323DNAartificial sequenceoligonucleotide primer
13gtcgaccact tttaatcctg ctc 231427DNAartificial
sequenceoligonucleotide primer 14tggatcctcg acatggcgaa cagactt
271529DNAartificial sequenceoligonucleotide primer 15tctcgagtca
tcctcagtca tcgtcatcg 29162722DNAPorcine adenovirus 3 16atggcgaaca
gacttcacct ggactgggac ggaaaccccg aggtggtgcc ggtgctggaa 60tgggacccgg
tggatctgcg cgacccctct ccgggggatg agggcttctg tgagccgtgc
120tgggagagtc tggtcgatgg actgccggac gagtggctgg acagtgtgga
cgaggtggag 180gtgattgtga ctgagggggg tgagtcagag gacagtggtg
ggagtgccgc tggtgactca 240ggtggctctc agggggtctt tgagatggac
cccccagaag agggggacag taatgaggag 300gatatcagcg cggtggctgc
ggaggtgctg tctgaactgg ctgatgtggt gtttgaggac 360ccacttgcgc
caccctctcc gtttgtgttg gactgccccg aggtacctgg tgtgaactgc
420cgctcttgtg attaccatcg ctttcactcc aaggacccca atctgaagtg
cagtctgtgc 480tacatgaggg atgcatgcct ttgctgtcta tggtgagtgt
ttttggacat ttgtgggatt 540atgtggaaaa aaaggaaaaa gtgcttgtaa
gaaatctcat gtgctatttc ccattttttg 600tctttttaga agctgtttct
ccagcacctc acaggtcggg ttccccggga cttggagacc 660tgccaggacg
caagaggaag tactgctatg actcatgcag cgaacaacct ttggacctgt
720ctatgaagcg cccccgcgat taatcattaa cctcaataaa cagcatgtga
tgatgactga 780ttgtctgtgt ctctgcctat atataccctt gtggtttgca
gggaagggat gtggtgactg 840agctattcct cagcatcatc atcgctctgc
ttttttctac tgcaggctat ttcttgctag 900ctcgctgtcc cttttctttt
tctgtgggca tggactatca acttctggcc aagcttacta 960acgtgaacta
ccttaggaag gtgatagtac aggggtctca gaactgccct tggtggaaaa
1020agattttttc ggacaggttt atcaaggtag tagcagaggc caggaggcag
tacgggcaag 1080agttgattga gatttttgtg gagggtgaga ggggctttgg
tcctgagttc ctgcgggaag 1140ggggactgta cgaagaggcc gttctgaaag
agttggattt cagcaccttg ggacgcaccg 1200tagctagtgt ggctctggtc
tgcttcattt ttgagaagct tcagaagcac agcgggtgga 1260ctgacgaggg
tattttaagt cttctggtgc cgccactatg ttccctgctg gaggcgcgaa
1320tgatggcgga gcaggtgcgg caggggctgt gcatcatcag gatgccgagc
gcggagcggg 1380agatgctgtt gcccagtggg tcatccggca gtggcagcgg
ggccgggatg cgggaccagg 1440tggtgcccaa gcgcccgcgg gagcaggaag
aggaggagga ggacgaggat gggatggaag 1500cgagcgggcg caggctcgaa
gggccggatc tggtttagat cgccgccggc ccgggggagc 1560gggtggagag
gggagcgggg aggaggcggg ggggtcttcc atggttagct atcagcaggt
1620gctttctgag tatctggaga gtcctctgga gatgcatgag cgctacagct
ttgagcagat 1680taggccctat atgcttcagc cgggggatga tctgggggag
atgatagccc agcacgccaa 1740ggtggagttg cagccgggca cggtgtacga
gctgaggcgc ccgatcacca tccgcagcat 1800gtgttacatc atcgggaacg
gggccaagat caagattcgg gggaattaca cggagtacat 1860caacatagag
ccgcgtaacc acatgtgttc cattgcgggc atgtggtcgg tgactatcac
1920ggatgtggtt tttgatcggg agctaccggc ccggggtggt ctgattttag
ccaacacgca 1980cttcatcctg cacggctgca acttcctggg ctttctgggc
tcggtaataa cggcgaacgc 2040cgggggggtg gtgcggggat gctacttttt
cgcctgctac aaggcgcttg accaccgggg 2100gcggctgtgg ctgacggtga
acgagaacac gtttgaaaag tgtgtgtacg cggtggtctc 2160tgcggggcgt
tgcaggatca agtacaactc ctccctgtcc accttctgct tcttgcacat
2220gagctatacg ggcaagatag tggggaacag catcatgagc ccttacacgt
tcagcgacga 2280cccctacgtg gacctggtgt gctgccagag cgggatggtg
atgcccctga gcacggtgca 2340catcgctccc tcgtctcgcc tgccctaccc
tgagttccgc aagaatgtgc tcctccgcag 2400caccatgttt gtgggcggcc
gcctgggcag cttcagcccc agccgctgct cctacagcta 2460cagctccctg
gtggtggacg agcagtccta ccggggtctg agtgtgacct gctgcttcga
2520tcagacctgt gagatgtaca agctgctgca gtgtacggag gcggacgaga
tggagacgga 2580tacctctcag cagtacgcct gcctgtgcgg ggacaatcac
ccctggccgc aggtgcggca 2640gatgaaagtg acagacgcgc tgcgggcccc
ccggtccctg gtgagctgca actgggggga 2700gttcagcgat gacgatgact ga
27221722DNAartificial sequenceoligonucleotide primer 17aggtggaggt
gattgtgact ga 221822DNAartificial sequenceoligonucleotide primer
18gacgcaagag gaagtactgc ta 221924DNAartificial
sequenceoligonucleotide primer 19ctggccaagc ttactaacgt gaac
242022DNAartificial sequenceoligonucleotide primer 20tttaagtctt
ctggtgccgc ca 222122DNAartificial sequenceoligonucleotide primer
21atgcatgagc gctacagctt tg 222222DNAartificial
sequenceoligonucleotide primer 22ctgagttccg caagaatgtg ct 22
* * * * *