U.S. patent application number 12/509670 was filed with the patent office on 2009-11-26 for influenza nucleic acids, polypeptides, and uses thereof.
This patent application is currently assigned to University of Massachusetts, a Massachusetts corporation. Invention is credited to Shan Lu, Shixia Wang.
Application Number | 20090291472 12/509670 |
Document ID | / |
Family ID | 37053853 |
Filed Date | 2009-11-26 |
United States Patent
Application |
20090291472 |
Kind Code |
A1 |
Lu; Shan ; et al. |
November 26, 2009 |
INFLUENZA NUCLEIC ACIDS, POLYPEPTIDES, AND USES THEREOF
Abstract
Codon-optimized nucleic acids encoding influenza polypeptides
and uses of the nucleic acids and polypeptides for inducing immune
responses are provided herein.
Inventors: |
Lu; Shan; (Franklin, MA)
; Wang; Shixia; (Northborough, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
University of Massachusetts, a
Massachusetts corporation
|
Family ID: |
37053853 |
Appl. No.: |
12/509670 |
Filed: |
July 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11362617 |
Feb 24, 2006 |
7566454 |
|
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12509670 |
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60655979 |
Feb 24, 2005 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 530/350; 530/387.9; 536/23.72 |
Current CPC
Class: |
A61K 39/12 20130101;
A61K 2039/70 20130101; C12N 2760/16134 20130101; C12N 2760/16122
20130101; C12N 2760/16234 20130101; Y10S 514/888 20130101; C07K
2319/00 20130101; C07K 14/005 20130101; A61K 39/145 20130101; C12N
2760/16222 20130101; A61K 2039/545 20130101; A61K 2039/53
20130101 |
Class at
Publication: |
435/69.1 ;
536/23.72; 435/320.1; 530/350; 530/387.9 |
International
Class: |
C12P 21/06 20060101
C12P021/06; C07H 21/04 20060101 C07H021/04; C12N 15/63 20060101
C12N015/63; C07K 14/00 20060101 C07K014/00; C07K 16/00 20060101
C07K016/00 |
Claims
1. An isolated nucleic acid molecule comprising: a sequence
encoding an influenza type hemagglutinin (HA) polypeptide or
antigenic fragment thereof, or an influenza neuraminidase (NA)
polypeptide or antigenic fragment thereof, wherein the sequence has
been codon-optimized for expression in a mammalian cell.
2. The nucleic acid molecule of claim 1, wherein the sequence
encodes an influenza type A HA polypeptide selected from the
following subtypes: H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11,
H12, H13, H14, and H15.
3. The nucleic acid molecule of claim 1, wherein the sequence
encodes an HA polypeptide of the H1 subtype or the H3 subtype, and
wherein the sequence is at least 90% identical to SEQ ID NO:1 or
SEQ ID NO:3, or a fragment thereof containing at least 30
contiguous nucleotides of SEQ ID NO:1or SEQ ID NO:3.
4. The nucleic acid molecule of claim 1, wherein the sequence
further encodes a leader peptide that is not naturally associated
with the influenza HA polypeptide.
5. The nucleic acid molecule of claim 1, wherein the sequence
encodes an extracellular portion of HA.
6. The nucleic acid molecule of claim 1, wherein the sequence
encodes an NA polypeptide, wherein the NA polypeptide is of the N2
subtype, and wherein the sequence is at least 90% identical to SEQ
ID NO:5.
7. A nucleic acid expression vector comprising the nucleic acid
molecule of claim 1.
8. A composition comprising: (a) the nucleic acid molecule of claim
1; (b) a mammalian cytomegalovirus immediate-early promoter
operably linked to the nucleic acid molecule, wherein the promoter
directs transcription of mRNA encoding the influenza polypeptide;
and (c) a mammalian polyadenylation signal derived from a bovine
growth hormone gene operably linked to the nucleic acid
molecule.
9. An isolated polypeptide encoded by the nucleic acid molecule of
claim 1.
10. An isolated antibody or antigen binding fragment thereof that
specifically binds to a polypeptide of claim 9.
11. A nucleic acid composition comprising: (a) a first sequence
encoding a first type of influenza polypeptide of a first influenza
subtype; and (b) a second sequence encoding the first type of
influenza polypeptide of a second influenza subtype.
12. The nucleic acid composition of claim 11, wherein the first
subtype is influenza A H1N1 and the second subtype is influenza A
H3N2.
13. The nucleic acid composition of claim 11, wherein one or both
sequences have been codon-optimized for expression in a mammalian
cell.
14. The nucleic acid composition of claim 13, wherein the
composition comprises a sequence at least 90% identical to SEQ ID
NO:1, SEQ ID NO:3, or SEQ ID NO:5.
15. The nucleic acid composition of claim 11, further comprising an
influenza polypeptide.
16. A nucleic acid composition comprising: (a) a first sequence
encoding a first influenza polypeptide of a first influenza
subtype; and (b) a second sequence encoding a second influenza
polypeptide of the first influenza subtype or a second influenza
subtype; wherein the sequence of (a) and the sequence of (b) have
been codon-optimized for expression in a mammalian cell.
17. The nucleic acid composition of claim 16, further comprising a
sequence encoding a third influenza polypeptide.
18. The nucleic acid composition of claim 16, wherein the first
polypeptide is an influenza HA polypeptide comprising a sequence at
least 90% identical to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID
NO:5.
19. The nucleic acid composition of claim 16, further comprising an
influenza polypeptide.
20. A method for producing an influenza polypeptide, the method
comprising: providing the nucleic acid molecule of claim 1; and
expressing the nucleic acid in a host cell under conditions in
which the influenza polypeptide encoded by the nucleic acid
molecule is produced.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
application Ser. No. 11/362,617, filed on Feb. 24, 2006 and issued
as U.S. Pat. No. 7,566,454 on Jul. 28, 2009, which claims priority
from U.S. Provisional Application No. 60/655,979, filed on Feb. 24,
2005, the entire contents of which are hereby incorporated by
reference.
TECHNICAL FIELD
[0002] This invention relates to viral nucleic acid sequences,
proteins, and subunit (both nucleic acid and recombinant protein)
vaccines and more particularly to viral nucleic acids sequences
that have been optimized for expression in mammalian host
cells.
BACKGROUND
[0003] Influenza virus is a worldwide public health problem.
Influenza causes, on average, 20,000 deaths and many more thousands
of hospitalizations annually in the United States alone (Palese and
Garcia-Sastre, J. Clin. Invest., 110(1): 9-12, 2002). Vaccination
is recommended for nearly half of the population of the United
States (Couch, Ann. Intern. Med., 133: 992-998, 2000). Influenza
also causes the death of thousands of domestic animals
annually.
[0004] The effectiveness of currently available vaccines depends on
the degree to which the vaccine antigens match those of the
circulating influenza strains. Immune responses to an antigen of a
particular type of influenza may be poorly cross-reactive with the
antigen encoded by a second type of influenza. Influenza viruses
have the tendency to undergo antigenic changes, complicating
efforts to produce effective vaccines. Antigenic shift, which
occurs when genes from different influenza types reassort in
infected hosts, is one mechanism by which dramatic antigenic
variation occurs. Antigenic shift occurs in influenza A types,
which circulate among humans and animals. Influenza B types are
more restricted to humans and are not thought to undergo antigenic
shift (Palese and Garcia-Sastre, J. Clin. Invest., 110(1): 9-12,
2002). Antigenic drift is a second, less drastic mechanism, in
which viral genes accumulate mutations over time. Both types of
antigenic variation increase the difficulty of generating vaccines
effective for protection against a broad range of influenza
strains.
SUMMARY
[0005] We have discovered that codon-optimized forms of nucleic
acids encoding influenza polypeptides such as influenza
hemagglutinin (HA), neuraminidase (NA), or membrane ion channel
(M2), are useful for expressing such polypeptides in appropriate
host cells. Codon-optimization permits more efficient expression
than expression achieved using codons native to the virus. Enhanced
expression is useful for producing large quantities of polypeptides
for therapeutic and diagnostic applications. Nucleic acids encoding
influenza antigens that are efficiently expressed in mammalian host
cells are useful, e.g., for inducing immune responses to the
antigens in the host. The nucleic acid sequences described herein
may induce higher levels of specific antibodies to an influenza
antigen when administered to an animal (as compared to nucleic acid
sequences which are not codon-optimized). In various embodiments,
the nucleic acid sequences induce hemagglutination inhibiting,
and/or virus-neutralizing antibodies when expressed in a mammalian
subject.
[0006] Furthermore, viral proteins produced in mammalian cells can
fold properly, oligomerize with natural binding partners, and/or
can possess native post-translational modifications such as
glycosylation. These features can enhance immunogenicity, thereby
increasing protection afforded by vaccination with the proteins (or
with the nucleic acids encoding the proteins). Codon-optimized
nucleic acids can be constructed by synthetic means, obviating the
need to obtain nucleic acids from live virus and/or increasing the
ease of manipulation of sequences.
[0007] We have also discovered novel polyvalent and multi-component
compositions for use in inducing immune responses. Multi-component
compositions include or encode multiple different influenza
polypeptides, or antigenic fragments thereof, e.g., they include or
encode HA, NA, and/or M2. Polyvalent compositions include or encode
multiple forms of a single antigen from different subtypes, such as
HA from subtypes H1, H2, H3, H5, H7, and/or H9.
[0008] Accordingly, in one aspect, the invention features isolated
nucleic acid molecules that include a sequence encoding an
influenza polypeptide or an antigenic fragment thereof, wherein all
or part of the sequence has been codon-optimized for expression in
a host cell (e.g., a eukaryotic cell, e.g., a mammalian cell, such
as a human cell). In various embodiments, more than 5%, 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 85%, 87%, 90%, 92%, 95%, 97%, 98%,
99%, or 100% of the codons in the sequence are mutated, relative to
the codons in a wild-type viral sequence, to codons common to
mammalian genes.
[0009] For example, isolated nucleic acid molecules that include a
codon-optimized sequence encoding an influenza type hemagglutinin
(HA) polypeptide or an antigenic fragment thereof are provided
herein. In one embodiment, the sequence has been codon-optimized
for expression in a human cell. The sequence can encode, e.g., an
influenza type B HA polypeptide or an influenza type A HA
polypeptide, e.g., selected from the following subtypes: H1, H2,
H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, and H15.
[0010] The sequences can encode an HA polypeptide of the H1
subtype, e.g., wherein the sequence is at least 70%, 80%, 85%, 87%,
90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:1 or
to a fragment thereof containing at least 30 contiguous nucleotides
of SEQ ID NO:1. The sequence can encode an HA polypeptide of the H3
subtype, e.g., wherein the sequence is at least 70%, 80%, 85%, 87%,
90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:3 or
to a fragment thereof containing at least 30 contiguous nucleotides
of SEQ ID NO:3. The sequence can further encode a leader peptide,
e.g., a leader peptide that is not naturally associated with the
influenza HA polypeptide, e.g., a mammalian leader peptide, e.g., a
tissue plasminogen activator (tPA) leader peptide.
[0011] The fragment of an HA polypeptide encoded by the sequence
can include, e.g., an HA1 domain of the HA polypeptide, an HA2
domain of the HA polypeptide, or an extracellular portion of
HA.
[0012] The sequences encoding the HA polypeptide differ from
naturally-occurring viral HA sequences. For example, a
codon-optimized sequence encoding H1 HA can differ from SEQ ID NO:7
by at least 5, 10, 15, 20, 25, 50, 100, or 150 nucleotides. In some
embodiments, the codon-optimized sequence encoding H1 HA differs
from SEQ ID NO:7 by fewer than 400, 350, 300, or 250 nucleotides.
In another example, the codon-optimized sequence encoding H3 HA can
differ from SEQ ID NO:8 by at least 5, 10, 15, 20, 25, 50, 100, or
150 nucleotides. In some embodiments, the codon-optimized sequence
encoding H3 HA differs from a SEQ ID NO:8 by fewer than 400, 350,
300, or 250 nucleotides. The codon-optimized sequences can encode
polypeptides that are 95%, 97%, 98%, 99%, or 100% identical to
polypeptides encoded by a naturally-occurring viral sequence.
[0013] In some embodiments, the isolated nucleic acid molecule
encoding an influenza type A HA polypeptide or an antigenic
fragment thereof includes SEQ ID NO:1 and/or SEQ ID NO:3.
[0014] In another aspect, the invention features isolated nucleic
acid molecules that include a sequence encoding an influenza
neuraminidase (NA) polypeptide or an antigenic fragment thereof,
wherein the sequence has been codon-optimized for expression in a
human cell. The sequences encode, e.g., an influenza type B NA
polypeptide or an influenza type A NA polypeptide, e.g., selected
from the following subtypes: N1, N2, N3, N4, N5, N6, N7, N8, and
N9.
[0015] In one embodiment, the sequence encodes an NA polypeptide of
the N2 subtype, e.g., wherein the sequence is at least 70%, 80%,
85%, 87%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID
NO:5. The sequence can further encode a leader peptide, e.g., a
leader peptide that is not naturally associated with the influenza
NA polypeptide, e.g., a mammalian leader peptide, e.g., a tissue
plasminogen activator (tPA) leader peptide.
[0016] The sequences encoding the NA polypeptide differ from
naturally-occurring viral NA sequences. For example, a
codon-optimized sequence encoding NA can differ from SEQ ID NO:9 by
at least 5, 10, 15, 20, 25, 50, 100, or 150 nucleotides. In some
embodiments, the codon-optimized sequence encoding NA differs from
SEQ ID NO:9 by fewer than 350, 300, 250, or 200 nucleotides.
[0017] In one embodiment, the isolated nucleic acid molecule
encoding an NA polypeptide or an antigenic fragment thereof
includes SEQ ID NO:5.
[0018] Also provided herein are isolated nucleic acid molecules
that include a sequence encoding two or more copies of an
extracellular portion of an influenza M2 polypeptide, e.g., wherein
the two or more copies of the extracellular portion of the M2
polypeptide are expressed as a single fusion polypeptide. Also
provided are codon-optimized sequences encoding the M2 polypeptide
and fusions containing two or more copies of the M2 polypeptide.
The M2 sequences can further include a second sequence encoding an
influenza HA or NA polypeptide as a fusion with the two or more
copies of the extracellular portion of the M2 polypeptide.
[0019] The nucleic acid molecules described herein can be operably
linked to a promoter. Also provided herein are nucleic acid
expression vectors that include one or more nucleic acid molecule
described herein. Also provided are compositions that include a
nucleic acid molecule described herein and a mammalian promoter
operably linked to the nucleic acid molecule, wherein the promoter
directs transcription of mRNA encoding the influenza polypeptide
(e.g., a cytomegalovirus immediate-early promoter); and a mammalian
polyadenylation signal (e.g., a polyadenylation signal derived from
a bovine growth hormone gene) operably linked to the nucleic acid
molecule. The compositions can further include an adjuvant and/or a
pharmaceutically acceptable carrier. In some embodiments, the
compositions further include particles to which the isolated
nucleic acid is bound, e.g., wherein the particles are suitable for
gene gun, intradermal, intramuscular, or mucosal
administration.
[0020] Also provided are cells that include one or more of the
nucleic acids described herein. The cells are, e.g., eukaryotic,
e.g., mammalian, e.g., human.
[0021] In another aspect, the invention features polypeptides
encoded by the nucleic acid molecules described herein, e.g.,
wherein the polypeptide is produced in a mammalian cell such as a
human cell. Also provided are isolated antibodies or antigen
binding fragments thereof that specifically bind to the
polypeptides. The antibodies can be polyclonal or monoclonal
antibodies.
[0022] In yet another aspect, the invention features nucleic acid
compositions that include various combinations of sequences. For
example, a composition can include (a) a first sequence encoding a
first type of influenza polypeptide (e.g., HA) of a first influenza
subtype; and (b) a second sequence encoding the first type of
influenza polypeptide of a second influenza subtype. The first and
second sequences encode, for example, HA polypeptides or antigenic
fragments thereof, NA polypeptides or antigenic fragments thereof,
or M2 polypeptides or antigenic fragments thereof. In various
embodiments, both the first and second influenza subtypes are
influenza A subtypes (e.g., the first subtype is influenza A H1N1
and the second subtype is influenza A H3N2); both the first and
second influenza subtypes are influenza B subtypes; or the first
influenza subtype is an influenza A subtype and the second
influenza subtype is an influenza B subtype. One or both sequences
can be codon-optimized for expression in a mammalian cell. For
example, the composition can include a sequence at least 90%
identical to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In one
embodiment, the composition includes a sequence at least 90%, 95%,
97%, or 99% identical to SEQ ID NO:1 and a sequence at least 90%,
95%, 97%, or 99% identical to SEQ ID NO:3. In some embodiments, the
composition further includes a sequence encoding the first type of
influenza polypeptide of a third influenza subtype.
[0023] In some embodiments, the composition further includes a
sequence encoding a second type of influenza polypeptide. For
example, the composition can include a sequence encoding an HA
polypeptide of a first and second subtype (e.g., H1 HA and H3 HA)
and also a sequence encoding an NA polypeptide.
[0024] In some embodiments, the nucleic acid composition further
includes one or more types of influenza polypeptides (e.g., one or
more of HA, NA, and M2). In some embodiments, the composition
further includes a second composition including influenza virions,
e.g., live and/or inactivated virions. In various embodiments, the
second composition includes two or more types of influenza virions,
e.g., three types of influenza virions, e.g., inactivated influenza
A H1N1 virions, inactivated influenza A H3N2 virions, and
inactivated influenza B virions.
[0025] Also provided are pharmaceutical compositions including a
nucleic acid composition described herein. The compositions can
further include an adjuvant.
[0026] In another aspect, the invention features nucleic acid
compositions including (a) a sequence encoding a first influenza
polypeptide of a first influenza subtype; and (b) a sequence
encoding a second influenza polypeptide of the first influenza
subtype or a second influenza subtype; wherein the sequence of (a)
and the sequence of (b) have been codon-optimized for expression in
a mammalian cell. For example, the first and second influenza
polypeptides are selected from a hemagglutinin (HA) polypeptide or
antigenic fragment thereof, an influenza neuraminidase (NA)
polypeptide or antigenic fragment thereof, or an influenza membrane
ion channel (M2) polypeptide. In some embodiments, the composition
further includes a sequence encoding a third influenza polypeptide.
In one embodiment, the first polypeptide is an influenza HA
polypeptide with a sequence at least 90%, e.g., 95%, 97%, 98%, 99%,
or 100% identical to SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.
[0027] The composition can further include one or more types of
influenza polypeptides, e.g., the composition further includes a
composition comprising influenza virions, e.g., live and/or
inactivated virions. The composition can include other features
described herein.
[0028] The invention also features methods for inducing an immune
response to one or more influenza polypeptides in a subject (e.g.,
a subject in need of treatment for, or a subject at risk for
exposure to, influenza). The methods include, for example,
administering to the subject a composition described herein,
wherein the composition is administered in an amount sufficient for
the sequence to express the one or more influenza polypeptides at a
level sufficient to induce an immune response in the subject. The
methods can further include administering to the subject a second
composition comprising an influenza polypeptide. In one embodiment,
the second composition comprises influenza virions, e.g., live
and/or inactivated virions, e.g., live, attenuated virions. In one
embodiment, two or more types of influenza virions, e.g., three
types of, e.g., inactivated, influenza virions, are administered.
In one embodiment, the three types of influenza virions are
inactivated influenza A H1N1 virions, inactivated influenza A H3N2
virions, and inactivated influenza B virions. The second
composition can be administered simultaneously with, before, or
after the first composition.
[0029] Also provided herein are methods for producing an influenza
polypeptide. The methods include providing a nucleic acid molecule
described herein; and expressing the nucleic acid in a host cell
(e.g., a mammalian cell, e.g., a human cell) under conditions in
which the influenza polypeptide encoded by the nucleic acid
molecule is produced. The method can further include isolating a
composition comprising the influenza polypeptide from the
cells.
[0030] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0031] A "subunit" vaccine is a vaccine whose active ingredient
antigen is only part of a pathogen, e.g., one protein or a fragment
of such protein in a pathogen with multiple proteins.
[0032] A "nucleic acid vaccine" is a vaccine whose active
ingredient is at least one isolated nucleic acid that encodes a
polypeptide antigen.
[0033] A "recombinant protein vaccine" is a vaccine whose active
ingredient is at least one protein antigen that is produced by
recombinant expression.
[0034] An "isolated nucleic acid" is a nucleic acid free of the
genes that flank the gene of interest in the genome of the organism
or virus in which the gene of interest naturally occurs. The term
therefore includes a recombinant DNA incorporated into an
autonomously expressing plasmid in mammalian systems. It also
includes a separate molecule such as a cDNA, a genomic fragment, a
fragment produced by polymerase chain reaction, or a restriction
fragment. It also includes a recombinant nucleotide sequence that
is part of a hybrid gene, i.e., a gene encoding a fusion protein.
An isolated nucleic acid is substantially free of other cellular or
viral material (e.g., free from the protein components of a viral
vector), or culture medium when produced by recombinant techniques,
or substantially free of chemical precursors or other chemicals
when chemically synthesized.
[0035] Expression control sequences are "operably linked" to a gene
of interest when they are incorporated into other nucleic acids so
that they effectively control expression of the gene.
[0036] An "adjuvant" is a compound or mixture of compounds that
enhances the ability of a nucleic acid composition and/or a
polypeptide composition to elicit an immune response in a
subject.
[0037] A "mammalian promoter" is any nucleic acid sequence,
regardless of origin, that is capable of driving transcription of
an mRNA coding for a polypeptide within a mammalian cell.
[0038] A "mammalian polyadenylation signal" is any nucleic acid
sequence, regardless of origin, that is capable of terminating
transcription of an mRNA encoding a polypeptide within a mammalian
cell.
[0039] "Protein" is used interchangeably with "polypeptide," and
includes both polypeptides produced in vitro and polypeptides
expressed in vivo after nucleic acid sequences are administered
into the host animals or human subjects. "Polypeptide" refers to
any chain of amino acids, regardless of length or
post-translational modification (e.g., glycosylation or
phosphorylation).
[0040] An "anti-influenza antibody" is an antibody that
specifically interacts with (e.g., specifically binds to) an
influenza antigen, e.g., HA or NA.
[0041] As used herein, the term "treat" or "treatment" is defined
as the application or administration of a nucleic acid encoding an
influenza antigen, or fragment thereof, or anti-influenza
antibodies to a subject, e.g., a patient, or application or
administration to an isolated tissue or cell from a subject, e.g.,
a patient, which is returned to the patient. Treatment also covers
the administration of polypeptides encoded by the nucleic acids, or
antibodies that specifically bind to the polypeptides. The nucleic
acids can be administered alone or in combination with a second
agent. The subject can be a patient having influenza, a symptom of
influenza, a predisposition toward influenza, or a patient who is
at risk for contracting an influenza infection. The treatment can
cure, heal, alleviate, relieve, alter, remedy, ameliorate,
palliate, improve, or affect the infection or symptoms of
influenza.
[0042] As used herein, an amount of a nucleic acid, protein, or an
anti-influenza antibody effective to treat a disorder, or a
"therapeutically effective amount," refers to an amount that is
effective, upon single or multiple dose administration to a
subject, in treating a subject with influenza. As used herein, an
amount of a nucleic acid, protein, or an anti-influenza antibody
effective to prevent or inhibit infection with, and/or disease
caused by influenza, or a "a prophylactically effective amount," of
the antibody refers to an amount which is effective, upon single-
or multiple-dose administration to the subject, in inhibiting or
delaying the occurrence of the onset or recurrence of influenza, or
reducing a symptom (e.g., reducing the severity of a symptom)
thereof.
[0043] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0044] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the claims.
All cited patents, patent applications, and references (including
references to public sequence database entries) are incorporated by
reference in their entireties for all purposes. U.S. Provisional
App. No. 60/655,979 is incorporated by reference in its entirety
for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1A is a representation of a codon optimized influenza A
H1 HA nucleic acid sequence (SEQ ID NO:1). The boundaries for each
domain encoded within the sequences are as follows: nucleotides
1-69 encode the leader peptide; 70-1032 encode the HA1 domain;
1033-1695 encode the HA2 domain; 1585-1653 encode the membrane
domain; 1654-1695 encode the cytoplasmic domain.
[0046] FIG. 1B is a representation of the influenza A H1 HA amino
acid sequence (SEQ ID NO:2) encoded by the nucleic acid sequence in
FIG. 1A. Amino acids 1-23 correspond to the leader sequences;
24-344 correspond to the HA1 domain; 345-565 correspond to the HA2
domain; 529-551 correspond to the transmembrane domain; and 552-565
correspond to the cytoplasmic domain.
[0047] FIG. 2A is a representation of a codon optimized influenza A
H3 HA nucleic acid sequence (SEQ ID NO:3). The boundaries for each
domain encoded within the sequences are as follows: 1-63 encode the
leader peptide; 64-1035 encode the HA1 domain; 1036-1695 encode the
HA2 domain; 1585-1653 encode the transmembrane domain; 1654-1695
encode the cytoplasmic domain.
[0048] FIG. 2B is a representation of the influenza A H3 HA amino
acid sequence (SEQ ID NO:4) encoded by the nucleic acid sequence in
FIG. 2A. Amino acids 1-21 correspond to the leader sequences;
22-345 correspond to the HA1 domain; 346-565 correspond to the HA2
domain; 529-551 correspond to the transmembrane domain; and 552-565
correspond to the cytoplasmic domain.
[0049] FIG. 3A is schematic diagram depicting various influenza H1
HA polypeptides encoded by nucleic acid constructs described
herein. "Wt" refers to a leader sequence that is naturally
associated with the influenza polypeptide. "tPA" refers to the
tissue plasminogen leader sequence. "dTM" refers to a polypeptide
lacks a transmembrane domain and cytoplasmic domain.
[0050] FIG. 3B is a schematic diagram depicting various influenza
H3 HA polypeptides described herein. "dTM" refers to a polypeptide
lacks a transmembrane domain and cytoplasmic.
[0051] FIG. 4A is a representation of SDS-PAGE and Western blot
analysis of H1 HA polypeptides expressed in 293T cells (lane 1) as
compared to a negative control (vector only; lane 2).
[0052] FIG. 4B is a representation of SDS-PAGE and Western blot
analysis of H1 HA polypeptides expressed in 293T cells (lane 1) as
compared to a negative control (vector only; lane 2).
[0053] FIGS. 5A-5I are graphs depicting the results of assays to
determine reactivity of antisera from rabbits immunized with
various codon optimized DNA vectors encoding influenza HA
polypeptides. Rabbits were immunized with the following vectors:
H1-wt.HA0, H1-tPA.HA0, H1-tPA.HA0.dTM (FIG. 5A); H1-tPA.HA1,
H1-tPA.HA2, H1-tPA-HA2.dTM (FIG. 5B); H3-wt.HA0, H3-tPA.HA0,
H3-tPA.HA0.dTM (FIG. 5D); H3-tPA.HA1, H3-tPA.HA2, H3-tPA-HA2.dTM
(FIG. 5E); H1+H3 tPA.HA0.dTM, H1+H3 tPA.HA1, or empty vector (FIGS.
5C and 5F). Levels of HA-specific antibodies in sera at each time
point were examined by ELISA and are plotted in the graphs. FIG. 5G
depicts HA-specific IgG titers in sera from rabbits immunized with
each of the various H1 HA vectors, or empty vector. The sera
analyzed in these assays were collected two weeks after the fourth
immunization. FIG. 5H depicts HA-specific IgG titers in sera from
rabbits immunized with each of the various H3 HA vectors, or empty
vector. The sera analyzed in these assays were collected two weeks
after the fourth immunization. FIG. 5I depicts HA-specific IgG
titers in sera from rabbits immunized with the following two
combinations of vectors: H1-tPA.HA0.dTM and H3-tPA.HA0dTM;
H1-wt.HA0 and H3-tPA.HA0.dTM.
[0054] FIG. 6A is a representation of a codon optimized influenza A
N2 NA nucleic acid sequence (SEQ ID NO:5).
[0055] FIG. 6B is a representation of the influenza A N2 NA amino
acid sequence (SEQ ID NO:6) encoded by the nucleic acid sequence in
FIG. 6A.
[0056] FIG. 7 is a schematic diagram depicting various influenza
ion channel M2 polypeptides described herein.
[0057] FIG. 8 is a schematic diagram depicting various influenza NA
and NA/M2 fusion polypeptide vaccines described herein.
[0058] FIG. 9 is a representation of a sequence encoding H1 HA from
influenza A New Caledonia/20/99 (SEQ ID NO:7; See also Genbank.RTM.
Acc. No. AJ344014.1; GI No. 19849783).
[0059] FIG. 10 is a representation of an influenza viral sequence
encoding an influenza H3 HA polypeptide from Influenza
A/Panama/2007/99 (H3N2) (SEQ ID NO:8).
[0060] FIG. 11 is a representation of a sequence encoding NA from
Influenza A/Panama/2007/99 (H3N2) (SEQ ID NO:9; See GenBank.RTM.
Acc. No. AJ457937.1; GI No. 22859354).
[0061] FIG. 12 is a representation of SDS-PAGE and Western blot
analysis of H1 HA polypeptides expressed in mammalian cells
transfected with a codon-optimized nucleic acid sequence ("opt";
lane 1), a wild-type nucleic acid sequence ("wt"; lane 2) or vector
only (lane 3).
[0062] FIG. 13A is a graph depicting the results of assays to
determine reactivity of antisera from rabbits immunized with
codon-optimized or non-codon-optimized (wild-type) influenza
protein sequences. Levels of HA-specific antibodies in sera at each
time point were examined by ELISA and are plotted in the graph.
Rabbits #316 and #317 (filled-in symbols) received wild-type DNA
encoding H1 HA. Rabbits #381 and #382 (open symbols) received
codon-optimized DNA encoding H1 HA.
[0063] FIG. 13B is a graph depicting the results of assays to
determine reactivity of antisera from rabbits immunized with
codon-optimized or non-codon-optimized (wild-type) influenza
protein sequences. HA-specific IgG titers were detected two weeks
after the fourth immunization with codon-optimized or wild-type DNA
encoding H1 HA. Rabbits #316 and #317 received wild-type DNA
encoding H1 HA. Rabbits #381 and #382 received codon-optimized DNA
encoding H1 HA.
[0064] FIG. 13C is a graph depicting the results of assays to
determine reactivity of antisera from mice immunized with
codon-optimized H1 HA DNA (H1-HA.opt), non-codon-optimized H1 HA
DNA (H1-HA.wt), or an empty DNA vector. HA-specific IgG titers in
sera collected two weeks after the fourth immunization were
measured.
[0065] FIG. 14A is a graph depicting the results of assays to
determine the titers of hemagglutination-inhibiting antibodies
against the H1N1 influenza virus A/NewCaledonia/20/99 strain in
sera from animals immunized with the following codon-optimized H1
HA DNA vectors: H1-wt.HA0, H1-tPA.HA0, H1-tPA.HA0.dTM; H1-tPA.HA1,
H1-tPA.HA2, H1-tPA-HA2.dTM. A sera sample from animals that were
not yet immunized was also tested (pre-bleed).
[0066] FIG. 14B is a graph depicting the results of assays to
determine the titers of hemagglutination-inhibiting antibodies
against the H3N2 influenza virus A/Panama/2007/99 strain in sera
from animals immunized with the following codon-optimized H3 HA DNA
vectors: H3-wt.HA0, H3-tPA.HA0, H3-tPA.HA0.dTM, H3-tPA.HA1,
H3-tPA.HA2, and H3-tPA-HA2.dTM. A sera sample from animals that
were not yet immunized was also tested (pre-bleed).
[0067] FIG. 15A is a graph depicting the results of assays to
determine the titers of neutralizing antibodies against the H1N1
influenza virus A/NewCaledonia/20/99 strain in sera from animals
immunized with the following codon-optimized H1 HA DNA vectors:
H1-wt.HA0, H1-tPA.HA0, H1-tPA.HA0.dTM; H1-tPA.HA1, H1-tPA.HA2,
H1-tPA-HA2.dTM. A sera sample from animals that were not yet
immunized was also tested (pre-bleed).
[0068] FIG. 15B is a graph depicting the results of assays to
determine the titers of neutralizing antibodies against the H3N2
influenza virus A/Moscow/10/99 strain in sera from animals
immunized with the following codon-optimized H3 HA DNA vectors:
H3-wt.HA0, H3-tPA.HA0, H3-tPA.HA0.dTM, H3-tPA.HA1, H3-tPA.HA2, and
H3-tPA-HA2.dTM. A sera sample from animals that were not yet
immunized was also tested (pre-bleed).
[0069] FIGS. 16A and 16B are graphs depicting the results of assays
to determine the titers of hemagglutination-inhibiting antibodies
against the H1N1 influenza virus A/NewCaledonia/20/99 strain (FIG.
16A) and H3N2 influenza virus A/Panama/2007/99 (FIG. 16B) in sera
from animals immunized with one of the following bivalent
combinations of codon-optimized DNAs: H1-tPA.HA0.dTM and
H3-tPA.HA0.dTM; and H1-wt.HA0 and H3-tPA.HA0.dTM. A sera sample
from animals that were not yet immunized was also tested
(pre-bleed).
[0070] FIGS. 16C and 16D are graphs depicting the results of assays
to determine the titers of hemagglutination-inhibiting antibodies
against the H1N1 influenza virus A/NewCaledonia/20/99 strain (FIG.
16C) and H3N2 influenza virus A/Moscow/10/99 (FIG. 16D) in sera
from animals immunized with one of the following bivalent
combinations of codon-optimized DNAs: H1-tPA.HA0.dTM and
H3-tPA.HA0.dTM; and H1-wt.HA0 and H3-tPA.HA0.dTM. A sera sample
from animals that were not yet immunized was also tested
(pre-bleed).
[0071] FIGS. 17A and 17B are graphs depicting the results of assays
to determine HA-specific IgG titers in sera from rabbits immunized
with different combinations of agents. "Fluzone X2" refers to
rabbits administered a prime and boost of Fluzone.RTM., an
influenza vaccine. "DNA+Fluzone" refers to rabbits immunized first
with a bivalent combination codon-optimized DNA encoding H1 HA and
H3 HA antigens at week 0, followed by a boost with Fluzone.RTM. at
week 4.
[0072] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0073] Influenza is a significant cause of human mortality.
Influenza virus infections are particularly dangerous for
immunologically naive and high risk populations, such as the
elderly, the very young, and health care professionals. Continual
antigenic changes arising in circulating influenza strains
complicate efforts to design effective vaccines. Here, we describe
compositions and methods for providing potent and broad-based
immunity to influenza.
[0074] Most human cases of influenza are caused by influenza type A
or B. Influenza types are further categorized by expression of two
surface antigens, HA and NA. One example of a subtype circulating
in humans today is influenza A H1N1 (hemagglutinin 1, neuraminidase
1). Other influenza A subtypes circulating among humans in the last
century include H2N2 and H3N2. There are 15 HA subtypes and 9 NA
subtypes found in influenza A viruses. Many are maintained in
non-human reservoirs and have the potential to cause pandemics in
human populations (Wright et al., Fields Virology, 4.sup.th Ed.,
Knipe and Howley, Eds., Lippincott, Williams & Wilkins, 1:
1533-1579, 2001; Katz, ASM News, 70(9): 412-491, 2004). Subtypes
that have caused human pandemics in the last 120 years include
H2N2, H3N8, H1N1, and H3N2 (Wright et al., Fields Virology,
4.sup.th Ed., Knipe and Howley, Eds., Lippincott, Williams &
Wilkins, 1: 1533-1579, 2001). In recent years, H5N1 and H7N7
subtypes have caused outbreaks in poultry. Spread of these subtypes
has caused limited numbers of fatalities in humans as well (Webby
and Webster, Science, 302(5650):1519-22, 2003). H9N2 subtypes have
been detected in humans, and thus are another cause for concern
(Peiris et al., Lancet, 354(9182): 916-917).
Influenza Antigens
[0075] Hemagglutinin (HA)
[0076] Influenza HA is the major glycoprotein antigen against which
protective immune responses are directed. HA spikes coating
influenza virions mediate attachment to receptors on the surface of
host cells and fusion to host cell membranes prior to viral entry
(reviewed in Shaw et al., Clin. Microbio. Rev., 5(1):74-92, 1992).
Native HA is synthesized as a single polypeptide of approximately
570 amino acids, which undergoes two post-translational cleavages
during maturation. One cleavage removes an amino-terminal leader
peptide. A second cleavage divides the polypeptides into the HA1
and HA2 domains, which remain linked to each other by a disulfide
bond. On native virions, HA spikes are formed from trimers of
HA1-HA2 domains. The external domain of HA is linked to the viral
membrane via a carboxy-terminal hydrophobic membrane domain.
[0077] The receptor binding site is located at the membrane-distal
end of HA1. Regions of antigenic variation are also clustered at
the membrane-distal end of the polypeptide. Within a given subtype,
the amino acid sequence of HA may vary by up to 20%. Amino acid
sequence identity between HA of different subtypes varies by
30%-70% (Skehel and Wiley, Annu. Rev. Biochem., 69:531-569, 2000).
A viral nucleic acid sequence (i.e., a native, non-codon-optimized
influenza viral sequence) encoding the H1 HA polypeptide of
influenza A New Caledonia/20/99 is shown in FIG. 9 (See also SEQ ID
NO:7 and Genbank.RTM. Acc. No. AJ344014. 1; GI No. 19849783). A
viral nucleic acid sequence encoding the H3 HA polyptide of
Influenza A/Panama/2007/99(H3N2) is shown in FIG. 10 (See also SEQ
ID NO:8).
[0078] Neuraminidase (NA)
[0079] Influenza NA is an exoglycosidase that hydrolyzes terminal
sialic residues from glycoproteins. Tetramers of NA polypeptides
are bound to the surface of influenza virions via an amino-terminal
hydrophobic domain. The enzymatic portion of NA is localized to the
membrane-distal carboxy-terminal end of the polypeptide. A viral
nucleic acid sequence encoding the NA polypeptide of Influenza
A/Panama/2007/99 (H3N2) is shown in FIG. 11 (See also SEQ ID NO:9
and GenBank.RTM. Acc. No. AJ457937.1; GI No. 22859354).
[0080] Membrane Ion Channel (M2)
[0081] M2 is an integral membrane protein encoded by influenza A.
M2 contains 23 amino-terminal extracellular amino acid residues, 19
transmembrane residues, and 54 cytoplasmic residues (Lamb et al.,
Cell, 40:627-633, 1985). While abundantly expressed on the surface
of infected host cells, it is only a minor component of influenza
virions. M2 has ion channel activity that is inhibited by the
antiviral drug amantidine (Wang et al., J. Virol., 67(9):5585-5594,
1993). Mutations in M2, typically in the transmembrane region,
cause resistance to amantidine.
[0082] Antigens with Amino Acid Substitutions
[0083] It is understood that the influenza polypeptides and
fragments thereof described herein may have additional conservative
or non-essential amino acid substitutions, which do not have a
substantial effect on the polypeptide functions. Whether or not a
particular substitution will be tolerated, i.e., will not adversely
affect desired biological properties, such as binding activity, can
be determined as described in Bowie et al., (1990) Science,
247:1306-13 10. A "conservative amino acid substitution" is one in
which an amino acid residue is replaced with an amino acid residue
having a similar side chain. Families of amino acid residues having
similar side chains have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., asparagine, glutamine,
serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
glycine, alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
[0084] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of a polypeptide, such as a
binding agent, e.g., an antibody, without substantially altering a
biological activity, whereas an "essential" amino acid residue
results in such a change.
Construction of Optimized Sequences
[0085] Viral proteins and proteins that are naturally expressed at
low levels can provide challenges for efficient expression by
recombinant means. In addition, viral proteins often display a
codon usage that is inefficiently translated in a host cell (e.g.,
a mammalian or avian host cell). Alteration of the codons native to
the viral sequence can facilitate more robust expression of these
proteins. Codon preferences for abundantly expressed proteins have
been determined in a number of species, and can provide guidelines
for codon substitution. Synthesis of codon-optimized sequences can
be achieved by substitution of viral codons in cloned sequences,
e.g., by site-directed mutagenesis, or by construction of
oligonucleotides corresponding to the optimized sequence by
chemical synthesis. See, e.g., Mirzabekov et al., J. Biol. Chem.,
274(40):28745-50, 1999.
[0086] The optimization should also include consideration of other
factors such as the efficiency with which the sequence can be
synthesized in vitro (e.g., as oligonucleotide segments) and the
presence of other features that affect expression of the nucleic
acid in a cell. For example, sequences that result in RNAs
predicted to have a high degree of secondary structure should be
avoided. AT- and GC-rich sequences that interfere with DNA
synthesis should also be avoided. Other motifs that can be
detrimental to expression include internal TATA boxes, chi-sites,
ribosomal entry sites, procarya inhibitory motifs, cryptic splice
donor and acceptor sites, and branch points. These features can be
identified manually or by computer software and they can be
excluded from the optimized sequences.
[0087] An influenza polypeptide (e.g., HA or NA) or antigenic
fragment thereof encoded by a codon-optimized nucleic acid is any
polypeptide sharing an epitope with a naturally occurring influenza
polypeptide, e.g., an HA or NA polypeptide. The influenza
polypeptides provided herein can differ from a wild type sequence
by additions or substitutions within the amino acid sequence, and
may preserve a biological function of the influenza polypeptide
(e.g., receptor binding). Amino acid substitutions may be made on
the basis of similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues involved.
[0088] Nonpolar (hydrophobic) amino acids include alanine, leucine,
isoleucine, valine, proline, phenylalanine, tryptophan, and
methionine. Polar neutral amino acids include glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine.
Positively charged (basic) amino acids include arginine, lysine,
and histidine. Negatively charged (acidic) amino acids include
aspartic acid and glutamic acid.
[0089] Alteration of residues are preferably conservative
alterations, e.g., a basic amino acid is replaced by a different
basic amino acid, as described herein.
Nucleic Acids, Vectors, and Host Cells
[0090] Isolated nucleic acid, vector, and host cell compositions
that can be used, e.g., for recombinant expression of the optimized
influenza nucleic acid sequences (e.g., HA, NA, or M2) and for
vaccines are provided herein.
[0091] Prokaryotic or eukaryotic host cells may be used for
expression of the influenza polypeptides. The terms "host cell" and
"recombinant host cell" are used interchangeably herein. Such terms
refer not only to the particular subject cell, but to the progeny
or potential progeny of such a cell. Because certain modifications
may occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein. A host cell can be any
prokaryotic cells, e.g., bacterial cells such as E. coli, or
eukaryotic cells, e.g., insect cells, yeast, avian cells (e.g.,
chicken cells, duck cells), or mammalian cells (e.g., cultured cell
or a cell line, e.g., a primate cell such as a Vero cell, or a
human cell). Other suitable host cells are known to those skilled
in the art.
[0092] The recombinant expression vectors provided herein can be
designed for expression of the influenza polypeptides (e.g., HA,
NA), anti-influenza antibodies, or antigen-binding fragments
thereof, in prokaryotic or eukaryotic cells. For example, new
polypeptides described herein can be expressed in E. coli, insect
cells (e.g., using baculovirus expression vectors), yeast cells,
avian cells, or mammalian cells. Suitable host cells are discussed
further in Goeddel, Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif., 1990.
Alternatively, the recombinant expression vector can be transcribed
and translated in vitro, for example using T7 promoter regulatory
sequences and T7 polymerase.
[0093] Expression of proteins in prokaryotes is often carried out
in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to protein or
antibody encoded therein, usually to the constant region of a
recombinant antibody.
[0094] A nucleic acid that is codon-optimized for expression in
mammalian cells can be expressed in mammalian cells using a
mammalian expression vector. Examples of mammalian expression
vectors include pCDM8 (Seed, B. Nature 329:840, 1987) and pMT2PC
(Kaufman et al., EMBO J. 6:187-195, 1987). When used in mammalian
cells, the expression vector's control functions are often provided
by viral regulatory elements. For example, commonly used promoters
are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian
Virus 40. For other suitable expression systems for both
prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of
Sambrook, J., Fritsh, E. F., and Maniatis, T., Molecular Cloning: A
Laboratory Manual., 2nd ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0095] In one embodiment, the recombinant expression vector (e.g.,
recombinant mammalian expression vector) is capable of directing
expression of the nucleic acid preferentially in a particular cell
type (e.g., in which tissue-specific regulatory elements are used
to express the nucleic acid). Tissue-specific regulatory elements
are known in the art. Non-limiting examples of suitable
tissue-specific promoters include the albumin promoter
(liver-specific; Pinkert et al., Genes Dev., 1:268-277, 1987),
lymphoid-specific promoters (Calame and Eaton, Adv. Immunol.,
43:235-275, 1988), in particular promoters of T cell receptors
(Winoto and Baltimore, EMBO J., 8:729-733, 1989) and
immunoglobulins (Banerji et al., Cell, 33:729-740, 1983; Queen and
Baltimore, Cell, 33:741-748, 1983), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne and Ruddle, Proc. Natl.
Acad. Sci., USA 86:5473-5477, 1989), pancreas-specific promoters
(Edlund et al., Science, 230:912-916, 1985), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss, Science,
249:374-379, 1990 and the .alpha.-fetoprotein promoter (Campes and
Tilghman, Genes Dev., 3:537-546, 1989).
[0096] In addition to the coding sequences, the new recombinant
expression vectors described herein carry regulatory sequences that
are operatively linked and control the expression of the genes in a
host cell.
[0097] As used herein, the term "substantially identical" (or
"substantially homologous") refers to a first amino acid or
nucleotide sequence that contains a sufficient number of identical
or equivalent (e.g., with a similar side chain, e.g., conserved
amino acid substitutions) amino acid residues or nucleotides to a
second amino acid or nucleotide sequence such that the first and
second amino acid or nucleotide sequences have similar activities.
In the case of antibodies, the second antibody has the same
specificity and has at least 50% of the affinity of the first
antibody.
[0098] Calculations of "homology" or "identity" between two
sequences are performed as follows. The sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in one or
both of a first and a second amino acid or nucleic acid sequence
for optimal alignment and non-homologous sequences can be
disregarded for comparison purposes). In different embodiments, the
length of a reference sequence aligned for comparison purposes is
at least 60%, e.g., at least 70%, 80%, 90%, or 100% of the length
of the reference sequence. The amino acid residues or nucleotides
at corresponding amino acid positions or nucleotide positions are
then compared. When a position in the first sequence is occupied by
the same amino acid residue or nucleotide as the corresponding
position in the second sequence, then the molecules are identical
at that position (as used herein amino acid or nucleic acid
"identity" is equivalent to amino acid or nucleic acid "homology").
The percent identity between the two sequences is a function of the
number of identical positions shared by the sequences, taking into
account the number of gaps, and the length of each gap, which need
to be introduced for optimal alignment of the two sequences.
[0099] The comparison of sequences and determination of percent
homology between two sequences are accomplished using a
mathematical algorithm. The percent homology between two amino acid
sequences is determined using the Needleman and Wunsch, J. Mol.
Biol., 48:444-453, 1970, algorithm which has been incorporated into
the GAP program in the GCG software package, using a Blossum 62
scoring matrix with a gap penalty of 12, a gap extend penalty of 4,
and a frameshift gap penalty of 5.
[0100] As used herein, the term "hybridizes under low stringency,
medium stringency, high stringency, or very high stringency
conditions" describes conditions for hybridization and washing.
Guidance for performing hybridization reactions can be found in
Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y., 1989, 6.3.1-6.3.6, which is incorporated herein by reference.
Aqueous and nonaqueous methods are described in that reference and
either can be used. Specific hybridization conditions referred to
herein are as follows: 1) low stringency hybridization conditions
in 6.times. sodium chloride/sodium citrate (SSC) at about
45.degree. C., followed by two washes in 0.2.times.SSC, 0.1% SDS at
least at 50.degree. C. (the temperature of the washes can be
increased to 55.degree. C. for low stringency conditions); 2)
medium stringency hybridization conditions in 6.times.SSC at about
45.degree. C., followed by one or more washes in 0.2.times.SSC,
0.1% SDS at 60.degree. C.; 3) high stringency hybridization
conditions in 6.times.SSC at about 45.degree. C., followed by one
or more washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C.; and 4)
very high stringency hybridization conditions are 0.5M sodium
phosphate, 7% SDS at 65.degree. C., followed by one or more washes
at 0.2.times.SSC, 1% SDS at 65.degree. C.
Nucleic Acid Vaccines
[0101] The nucleic acids useful for inducing an immune response
include at least three components: (1) a nucleic acid sequence that
begins with a start codon and encodes an influenza polypeptide or
antigenic fragment thereof, (2) a transcriptional promoter
operatively linked to the sequence encoding the influenza
polypeptide or antigenic fragment thereof, and (3) a mammalian
polyadenylation signal operably linked to the coding sequence to
terminate transcription driven by the promoter. In this context, a
"mammalian" promoter or polyadenylation signal is not necessarily a
nucleic acid sequence derived from a mammal. For example, it is
known that mammalian promoters and polyadenylation signals can be
derived from viruses.
[0102] The nucleic acid vector can optionally include additional
sequences such as enhancer elements, splicing signals, termination
and polyadenylation signals, viral replicons, and bacterial plasmid
sequences. Such vectors can be produced by methods known in the
art. For example, a nucleic acid encoding the desired influenza
polypeptide can be inserted into various commercially available
expression vectors. See, e.g., Invitrogen Catalog, 1998. In
addition, vectors specifically constructed for nucleic acid
vaccines are described in Yasutomi et al., J. Virol., 70:678-681,
1996.
[0103] Administration of Nucleic Acids
[0104] The new nucleic acids described herein can be administered
to an individual, e.g., naked, in combination with a carrier, or in
combination with a substance that promotes nucleic acid uptake or
recruits immune system cells to the site of the inoculation. For
example, nucleic acids encapsulated in microparticles have been
shown to promote expression of rotaviral proteins from nucleic acid
vectors in vivo (U.S. Pat. No. 5,620,896).
[0105] A mammal can be inoculated with nucleic acid through any
parenteral route, e.g., intravenous, intraperitoneal, intradermal,
subcutaneous, intrapulmonary, or intramuscular routes. The new
nucleic acid compositions can also be administered orally, by
particle bombardment using a gene gun, or by other needle-free
delivery systems. Muscle is a useful tissue for the delivery of
nucleic acids encoding influenza polypeptides because mammals have
a proportionately large muscle mass which is conveniently accessed
by direct injection through the skin. A comparatively large dose of
nucleic acid can be deposited into muscle by multiple and/or
repetitive injections. Multiple injections can be performed over
extended periods of time.
[0106] Conventional particle bombardment can be used to deliver
nucleic acids that express influenza polypeptides into skin or onto
mucosal surfaces, e.g., using commercial devices. For example, the
Accell II.RTM. (PowderJect.RTM. Vaccines, Inc., Middleton, Wis.)
particle bombardment device, one of several commercially available
"gene guns," can be employed to deliver nucleic acid-coated gold
beads. A Helios Gene Gun.RTM. (Bio-Rad) can also be used to
administer the DNA particles. Information on particle bombardment
devices and methods can be found in sources including the
following: Yang et al., Proc. Natl. Acad. Sci. USA, 87:9568, 1990;
Yang, CRC Crit. Rev. Biotechnol., 12:335, 1992; Richmond et al.,
Virology, 230:265-274, 1997; Mustafa et al., Virology, 229:269-278,
1997; Livingston et al., Infect. Immun., 66:322-329, 1998; and
Cheng et al., Proc. Natl. Acad. Sci. USA, 90:4455, 1993.
[0107] In some embodiments, an individual is inoculated by a
mucosal route. The codon-optimized nucleic acids or compositions
can be administered to a mucosal surface by a variety of methods
including nucleic acid-containing nose-drops, inhalants,
suppositories, or microspheres. Alternatively, nucleic acid vectors
containing the codon-optimized nucleic acids can be encapsulated in
poly(lactide-co-glycolide) (PLG) microparticles by a solvent
extraction technique, such as the ones described in Jones et al.,
Infect. Immun., 64:489, 1996; and Jones et al., Vaccine, 15:814,
1997. For example, the nucleic acids can be emulsified with PLG
dissolved in dichloromethane, and this water-in-oil emulsion is
emulsified with aqueous polyvinyl alcohol (an emulsion stabilizer)
to form a (water-in-oil)-in-water double emulsion. This double
emulsion is added to a large quantity of water to dissipate the
dichloromethane, which results in the microdroplets hardening to
form microparticles. These microdroplets or microparticles are
harvested by centrifugation, washed several times to remove the
polyvinyl alcohol and residual solvent, and finally lyophilized.
The microparticles containing nucleic acid have a mean diameter of
0.5 .mu.m.
[0108] To test for nucleic acid content, the microparticles are
dissolved in 0.1 M NaOH at 100.degree. C. for 10 minutes. The
A.sub.260 is measured, and the amount of nucleic acid calculated
from a standard curve. Incorporation of nucleic acid into
microparticles is in the range of 1.76 g to 2.7 g nucleic acid per
milligram PLG Microparticles containing about 1 to 100 .mu.g of
nucleic acid are suspended in about 0.1 to 1 ml of 0.1 M sodium
bicarbonate, pH 8.5, and orally administered to mice or humans.
[0109] Regardless of the route of administration, an adjuvant can
be administered before, during, or after administration of the
codon-optimized nucleic acid encoding an influenza polypeptide. An
adjuvant can increase the uptake of the nucleic acid into the
cells, increase the expression of the polypeptide from the nucleic
acid within the cell, induce antigen presenting cells to infiltrate
the region of tissue where the polypeptide is being expressed, or
increase the antigen-specific response provided by lymphocytes.
[0110] Evaluating Vaccine Efficacy
[0111] Before administering the nucleic acids, polypeptides, and/or
antibodies described herein to humans, efficacy testing can be
conducted using animals. In an example of efficacy testing, mice
are vaccinated by intramuscular injection. After the initial
vaccination or after optional booster vaccinations, the mice (and
negative controls) are monitored for indications of
vaccine-induced, influenza-specific immune responses. Methods of
measuring immune responses are described in Townsend et al., J.
Virol., 71:3365-3374, 1997; Kuhober et al., J. Immunol., 156:
3687-3695, 1996; Kuhrober et al., Int. Immunol., 9:1203-1212, 1997;
Geissler et al., Gastroenterology, 112:1307-1320, 1997; and
Sallberg et al., J. Virol., 71:5295-5303, 1997.
[0112] Anti-influenza serum antibody levels in vaccinated animals
can be determined by known methods. The concentrations of
antibodies can be standardized against a readily available
reference standard. The functional activity of antibodies can be
measured, e.g., using hemagglutination inhibition assays and/or
virus neutralization assays (described in Example 5, below).
[0113] Cytotoxicity assays can be performed as follows. Spleen
cells from immunized mice are suspended in complete MEM with 10%
fetal calf serum and 5.times.10.sup.-5 M 2-mercapto-ethanol.
Cytotoxic effector lymphocyte populations are harvested after 5
days of culture, and a 5-hour .sup.51Cr release assay is performed
in a 96-well round-bottom plate using target cells. The effector to
target cell ratio is varied. Percent lysis is defined as
(experimental release minus spontaneous release)/(maximum release
minus spontaneous release).times.100.
Antibodies
[0114] This invention also provides, inter alia, antibodies, or
antigen-binding fragments thereof, to influenza polypeptides, e.g.,
HA, NA, M2, and/or antigenic fragments of the polypeptides, e.g.,
portions of the polypeptides that lack transmembrane domains.
[0115] As used herein, "specific binding" or "specifically binds
to" refer to the ability of an antibody to: (1) bind to an
influenza polypeptide as shown by a specific biochemical analysis,
such as a specific band in a Western Blot analysis, or (2) bind to
an influenza polypeptide with a reactivity that is at least
two-fold greater than its reactivity for binding to an antigen
(e.g., BSA, casein) other than an influenza polypeptide.
[0116] As used herein, the term "antibody" refers to a protein
including at least one, and preferably two, heavy (H) chain
variable regions (abbreviated herein as VH), and at least one and
preferably two light (L) chain variable regions (abbreviated herein
as VL). The VH and VL regions can be further subdivided into
regions of hypervariability, termed "complementarity determining
regions" (CDR), interspersed with regions that are more conserved,
termed "framework regions" (FR). Each VH and VL is composed of
three CDRs and four FRs, arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, FR4.
[0117] The VH or VL chain of the antibody can further include all
or part of a heavy or light chain constant region. In one
embodiment, the antibody is a tetramer of two heavy immunoglobulin
chains and two light immunoglobulin chains, wherein the heavy and
light immunoglobulin chains are inter-connected by, e.g., disulfide
bonds. The heavy chain constant region includes three domains, CH1,
CH2 and CH3. The light chain constant region is comprised of one
domain, CL. The variable region of the heavy and light chains
contains a binding domain that interacts with an antigen. The
constant regions of the antibodies typically mediate the binding of
the antibody to host tissues or factors, including various cells of
the immune system (e.g., effector cells) and the first component
(Clq) of the classical complement system. The term "antibody"
includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM
(as well as subtypes thereof), wherein the light chains of the
immunoglobulin may be of types kappa or lambda.
[0118] As used herein, "isotype" refers to the antibody class
(e.g., IgM or IgG1) that is encoded by heavy chain constant region
genes.
[0119] The term "antigen-binding fragment" of an antibody (or
simply "antibody portion," or "fragment"), as used herein, refers
to a portion of an antibody that specifically binds to an influenza
polypeptide (e.g., HA or NA), e.g., a molecule in which one or more
immunoglobulin chains is not full length, but which still
specifically binds to an influenza polypeptide. Examples of
antigen-binding fragments include: (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a
F(ab').sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., Nature 341:544-546,
1989), which consists of a VH domain; and (vi) an isolated
complementarity determining region (CDR) having sufficient
framework to specifically bind to, e.g., an antigen binding portion
of a variable region. An antigen binding portion of a light chain
variable region and an antigen binding portion of a heavy chain
variable region, e.g., the two domains of the Fv fragment, VL and
VH, can be joined, using recombinant methods, by a synthetic linker
that enables them to be made as a single protein chain in which the
VL and VH regions pair to form monovalent molecules (known as
single chain Fv (scFv); see e.g., Bird et al., Science,
242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA,
85:5879-5883, 1988). Such single chain antibodies are also
encompassed within the term "antigen-binding fragment" of an
antibody. These antibody fragments are obtained using conventional
techniques known to those with skill in the art, and are screened
for utility in the same manner as are intact antibodies.
[0120] The term "monospecific antibody" refers to an antibody that
displays a single binding specificity and affinity for a particular
target, e.g., an epitope. This term includes a "monoclonal
antibody" or "monoclonal antibody composition," which as used
herein refer to a preparation of antibodies or fragments thereof of
single molecular composition.
[0121] The term "polyclonal antibody" refers to an antibody
preparation, either as animal or human sera or as prepared by in
vitro production, which can bind to more than one epitope on one
antigen or multiple epitopes on more than one antigen.
[0122] The term "recombinant" antibody, as used herein, refers to
antibodies that are prepared, expressed, created, or isolated by
recombinant means, such as antibodies expressed using a recombinant
expression vector transfected into a host cell, antibodies isolated
from a recombinant, combinatorial antibody library, antibodies
isolated from an animal (e.g., a mouse) that is transgenic for
human immunoglobulin genes or antibodies prepared, expressed,
created or isolated by any other means that involves splicing of
human immunoglobulin gene sequences to other DNA sequences. Such
recombinant antibodies include humanized, CDR grafted, chimeric, in
vitro generated (e.g., by phage display) antibodies, and may
optionally include constant regions derived from human germline
immunoglobulin sequences.
[0123] Many types of anti-influenza antibodies, or antigen-binding
fragments thereof, are useful in the methods described herein. The
antibodies can be of the various isotypes, including: IgG (e.g.,
IgG1, IgG2, IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. Preferably,
the antibody is an IgG isotype, e.g., IgG1. The antibody molecules
can be full-length (e.g., an IgG1 or IgG4 antibody) or can include
only an antigen-binding fragment (e.g., a Fab, F(ab').sub.2, Fv or
a single chain Fv fragment). These include monoclonal antibodies,
recombinant antibodies, chimeric antibodies, human antibodies, and
humanized antibodies, as well as antigen-binding fragments of the
foregoing.
[0124] Monoclonal antibodies can be used in the new methods
described herein. Monoclonal antibodies can be produced by a
variety of techniques, including conventional monoclonal antibody
methodology, e.g., the standard somatic cell hybridization
technique of Kohler and Milstein, Nature 256:495, 1975. Polyclonal
antibodies can be produced by immunization of animal or human
subjects. The advantages of polyclonal antibodies include the broad
antigen specificity against a particular pathogen. See generally,
Harlow, E. and Lane, D. (1988) Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
[0125] Useful immunogens for uses described herein include the
influenza polypeptides described herein, e.g., influenza
polypeptides expressed from codon-optimized nucleic acid
sequences.
[0126] Anti-influenza antibodies or fragments thereof useful in
methods described herein can also be recombinant antibodies
produced by host cells transformed with DNA encoding immunoglobulin
light and heavy chains of a desired antibody. Recombinant
antibodies may be produced by known genetic engineering techniques.
For example, recombinant antibodies can be produced by cloning a
nucleotide sequence, e.g., a cDNA or genomic DNA, encoding the
immunoglobulin light and heavy chains of the desired antibody. The
nucleotide sequences encoding those polypeptides are then inserted
into expression vectors so that both genes are operatively linked
to their own transcriptional and translational expression control
sequences. The expression vector and expression control sequences
are chosen to be compatible with the expression host cell used.
Typically, both genes are inserted into the same expression vector.
Prokaryotic or eukaryotic host cells may be used.
[0127] Expression in eukaryotic host cells is useful because such
cells are more likely than prokaryotic cells to assemble and
secrete a properly folded and immunologically active antibody.
However, any antibody produced that is inactive due to improper
folding may be renatured according to well known methods (Kim and
Baldwin, "Specific Intermediates in the Folding Reactions of Small
Proteins and the Mechanism of Protein Folding," Ann. Rev. Biochem.,
51, pp. 459-89 (1982)). It is possible that the host cells will
produce portions of intact antibodies, such as light chain dimers
or heavy chain dimers, which also are antibody homologs.
[0128] It will be understood that variations on the above procedure
are useful. For example, it may be desired to transform a host cell
with DNA encoding either the light chain or the heavy chain (but
not both) of an antibody. Recombinant DNA technology may also be
used to remove some or all of the DNA encoding either or both of
the light and heavy chains that is not necessary for binding, e.g.,
the constant region may be modified by, for example, deleting
specific amino acids. The molecules expressed from such truncated
DNA molecules are useful in the methods described herein. In
addition, bifunctional antibodies may be produced in which one
heavy and one light chain are anti-influenza antibody and the other
heavy and light chain are specific for an antigen other than the
influenza polypeptide, or another epitope of the same influenza, or
of another influenza polypeptide.
[0129] Chimeric antibodies can be produced by recombinant DNA
techniques known in the art. For example, a gene encoding the Fc
constant region of a murine (or other species) monoclonal antibody
molecule is digested with restriction enzymes to remove the region
encoding the murine Fc, and the equivalent portion of a gene
encoding a human Fc constant region is substituted (see Robinson et
al., International Patent Publication PCT/US86/02269; Akira, et
al., European Patent Application 184,187; Taniguchi, M., European
Patent Application 171,496; Morrison et al., European Patent
Application 173,494; Neuberger et al., International Application WO
86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.,
European Patent Application 125,023; Better et al., Science,
240:1041-1043, 1988; Liu et al., Proc. Natl. Acad. Sci.,
84:3439-3443, 1987; Liu et al., J. Immunol., 139:3521-3526, 1987;
Sun et al., Proc. Natl. Acad. Sci., 84:214-218, 1987; Nishimura et
al., Canc. Res., 47:999-1005, 1987; Wood et al., Nature,
314:446-449, 1985; and Shaw et al., J. Natl Cancer Inst.,
80:1553-1559, 1988).
[0130] An antibody or an immunoglobulin chain can be humanized by
methods known in the art. For example, once murine antibodies are
obtained, variable regions can be sequenced. The location of the
CDRs and framework residues can be determined (see, Kabat et al.,
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242, 1991, and Chothia, C. et al., J. Mol. Biol., 196:901-917,
1987, which are incorporated herein by reference). The light and
heavy chain variable regions can, optionally, be ligated to
corresponding constant regions.
[0131] Murine antibodies can be sequenced using art-recognized
techniques. Humanized or CDR-grafted antibody molecules or
immunoglobulins can be produced by CDR-grafting or CDR
substitution, wherein one, two, or all CDRs of an immunoglobulin
chain can be replaced. See e.g., U.S. Pat. No. 5,225,539; Jones et
al., Nature, 321:552-525, 1986; Verhoeyan et al., Science,
239:1534, 1988; Beidler et al., J. Immunol., 141:4053-4060, 1988;
and Winter, U.S. Pat. No. 5,225,539, the contents of all of which
are hereby expressly incorporated by reference.
[0132] Winter describes a CDR-grafting method that may be used to
prepare the humanized anti-influenza antibodies (Winter U.S. Pat.
No. 5,225,539), the contents of which is expressly incorporated by
reference. All of the CDRs of a particular human antibody may be
replaced with at least a portion of a non-human CDR or only some of
the CDRs may be replaced with non-human CDRs. It is only necessary
to replace the number of CDRs required for binding of the humanized
antibody to a predetermined antigen.
[0133] Humanized antibodies can be generated by replacing sequences
of the Fv variable region that are not directly involved in antigen
binding with equivalent sequences from human Fv variable regions.
General methods for generating humanized antibodies are provided by
Morrison, Science, 229:1202-1207, 1985, by Oi et al.,
BioTechniques, 4:214, 1986, and by Queen et al., U.S. Pat. Nos.
5,585,089; 5,693,761; and 5,693,762, the contents of all of which
are hereby incorporated by reference. Those methods include
isolating, manipulating, and expressing the nucleic acid sequences
that encode all or part of immunoglobulin Fv variable regions from
at least one of a heavy or light chain. Sources of such nucleic
acid are well known to those skilled in the art and, for example,
may be obtained from a hybridoma producing an antibody against a
predetermined target, as described above. The recombinant DNA
encoding the humanized antibody, or fragment thereof, can then be
cloned into an appropriate expression vector.
[0134] Also included herein are humanized antibodies in which
specific amino acids have been substituted, deleted, or added. In
particular, preferred humanized antibodies have amino acid
substitutions in the framework region, such as to improve binding
to the antigen. For example, a selected, small number of acceptor
framework residues of the humanized immunoglobulin chain can be
replaced by the corresponding donor amino acids. Preferred
locations of the substitutions include amino acid residues adjacent
to the CDR, or which are capable of interacting with a CDR (see
e.g., U.S. Pat. No. 5,585,089). Criteria for selecting amino acids
from the donor are described in U.S. Pat. No. 5,585,089 (e.g.,
columns 12-16), the contents of which are hereby incorporated by
reference. The acceptor framework can be a mature human antibody
framework sequence or a consensus sequence.
[0135] As used herein, the term "consensus sequence" refers to the
sequence formed from the most frequently occurring amino acids (or
nucleotides) in a family of related sequences (See e.g., Winnaker,
From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987).
In a family of proteins, each position in the consensus sequence is
occupied by the amino acid occurring most frequently at that
position in the family. If two amino acids occur equally
frequently, either can be included in the consensus sequence. A
"consensus framework" refers to the framework region in the
consensus immunoglobulin sequence. Other techniques for humanizing
antibodies are described in Padlan et al. EP 519596 A1, published
on Dec. 23, 1992.
[0136] Also provided herein are antibodies that are produced in
mice that bear transgenes encoding one or more fragments of an
immunoglobulin heavy or light chain. See, e.g., U.S. Patent
Publication No. 20030138421. Also provided are antibodies that are
fully human (100% human protein sequences) produced in transgenic
mice in which mouse antibody gene expression is suppressed and
effectively replaced with human antibody gene expression (such mice
are available, e.g., from Medarex, Princeton, N.J.). See, e.g.,
U.S. Patent Publication No. 20030031667.
[0137] An antibody, or antigen-binding fragment thereof, can be
derivatized or linked to another functional molecule (e.g., another
peptide or protein). For example, a protein or antibody can be
functionally linked (by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other
molecular entities, such as another antibody, a detectable agent, a
cytotoxic agent, a pharmaceutical agent, and/or a protein or
peptide that can mediate association with another molecule (such as
a streptavidin core region or a polyhistidine tag).
[0138] One type of derivatized protein is produced by crosslinking
two or more proteins (of the same type or of different types).
Suitable crosslinkers include those that are heterobifunctional,
having two distinct reactive groups separated by an appropriate
spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or
homobifunctional (e.g., disuccinimidyl suberate). Such linkers are
available from Pierce Chemical Company, Rockford, Ill.
[0139] Useful detectable agents with which a protein can be
derivatized (or labeled) to include fluorescent compounds, various
enzymes, prosthetic groups, luminescent materials, bioluminescent
materials, and radioactive materials. Exemplary fluorescent
detectable agents include fluorescein, fluorescein isothiocyanate,
rhodamine, and, phycoerythrin. A protein or antibody can also be
derivatized with detectable enzymes, such as alkaline phosphatase,
horseradish peroxidase, .beta.-galactosidase, acetylcholinesterase,
glucose oxidase and the like. When a protein is derivatized with a
detectable enzyme, it is detected by adding additional reagents
that the enzyme uses to produce a detectable reaction product. For
example, when the detectable agent horseradish peroxidase is
present, the addition of hydrogen peroxide and diaminobenzidine
leads to a colored reaction product, which is detectable. A protein
can also be derivatized with a prosthetic group (e.g.,
streptavidin/biotin and avidin/biotin). For example, an antibody
can be derivatized with biotin, and detected through indirect
measurement of avidin or streptavidin binding.
[0140] Labeled proteins and antibodies can be used, for example,
diagnostically and/or experimentally in a number of contexts,
including (i) to isolate a predetermined antigen by standard
techniques, such as affinity chromatography or immunoprecipitation;
(ii) to detect a predetermined antigen (e.g., an influenza virion,
e.g., in a cellular lysate or a serum sample) in order to evaluate
the abundance and pattern of expression of the protein; and (iii)
to monitor protein levels in tissue as part of a clinical testing
procedure, e.g., to determine the efficacy of a given treatment
regimen.
[0141] An anti-influenza antibody or antigen-binding fragment
thereof may be conjugated to another molecular entity, typically a
label or a therapeutic (e.g., a cytotoxic or cytostatic) agent or
moiety.
[0142] Radioactive isotopes can be used in diagnostic or
therapeutic applications. Radioactive isotopes that can be coupled
to proteins and antibodies include, but are not limited to
.alpha.-, .beta.-, or .gamma.-emitters, or .beta.- and
.gamma.-emitters.
Pharmaceutical Compositions
[0143] In another aspect, compositions, e.g., pharmaceutically
acceptable compositions, are provided which include a polypeptide
or antibody molecule described herein, formulated together with a
pharmaceutically acceptable carrier.
[0144] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, isotonic and
absorption delaying agents, and the like that are physiologically
compatible. The carrier can be suitable for intravenous,
intramuscular, subcutaneous, parenteral, rectal, spinal or
epidermal administration (e.g., by injection or infusion).
[0145] The compositions may be in a variety of forms. These
include, for example, liquid, semi-solid and solid dosage forms,
such as liquid solutions (e.g., injectable and infusible
solutions), dispersions or suspensions, liposomes and
suppositories. The preferred form depends on the intended mode of
administration and therapeutic application. Useful compositions are
in the form of injectable or infusible solutions. A useful mode of
administration is parenteral (e.g., intravenous, subcutaneous,
intraperitoneal, intramuscular). For example, the protein or
antibody can be administered by intravenous infusion or injection.
In another embodiment, the protein or antibody is administered by
intramuscular or subcutaneous injection.
[0146] Compositions for administration to animals and humans
typically should be sterile and stable under the conditions of
manufacture and storage. The composition can be formulated as a
solution, microemulsion, dispersion, liposome, or other ordered
structure suitable to high antibody concentration. Sterile
injectable solutions can be prepared by incorporating the active
compound (i.e., codon-optimized nucleic acid or polypeptide) in the
required amount in an appropriate solvent with one or a combination
of ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the active compound into a sterile vehicle that contains a basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum drying and freeze-drying that yields a
powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. The
proper fluidity of a solution can be maintained, for example, by
the use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prolonged absorption of injectable compositions can be
brought about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin.
[0147] The compositions can be administered by a variety of methods
known in the art, although for many therapeutic and prophylactic
applications. As will be appreciated by the skilled artisan, the
route and/or mode of administration will vary depending upon the
desired results.
[0148] In certain embodiments, a composition (e.g., codon-optimized
nucleic acid composition) may be orally administered, for example,
with an inert diluent or an assimilable edible carrier. The
compound (and other ingredients, if desired) may also be enclosed
in a hard or soft shell gelatin capsule, compressed into tablets,
or incorporated directly into the subject's diet. For oral
therapeutic administration, the compounds may be incorporated with
excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. To administer a compound by other than parenteral
administration, it may be necessary to coat the compound with, or
co-administer the compound with, a material to prevent its
inactivation. Therapeutic compositions can be administered with
medical devices known in the art.
[0149] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms are dictated by and
directly dependent on (a) the unique characteristics of the active
compound and the particular therapeutic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0150] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a polypeptide or antigenic
fragment thereof is 0.1-100 mg/kg, e.g., 1-10 mg/kg. It is to be
further understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed composition. The exact
dosage can vary depending on the route of administration. For
intramuscular injection, the dose range can be 100 .mu.g
(microgram) to 10 mg (milligram) per injection. Multiple injections
may be needed.
[0151] Suitable doses of nucleic acid compositions for humans can
range from 1 .mu.g/kg to 1 mg/kg of total nucleic acid, e.g., from
5 .mu.g/kg-500 mg/kg of total DNA, 10 .mu.g/kg-250 .mu.g/kg of
total DNA, or 10 .mu.g/kg-170 .mu.g/kg of total DNA. In one
embodiment, a human subject (18-50 years of age, 45-75 kg) is
administered 1 mg-10 mg of DNA. "Total DNA" and "total nucleic
acid" refers to a pool of nucleic acids encoding distinct antigens.
For example, a dose of 50 mg of total DNA encoding five different
influenza HA antigens can have 1 mg of each antigen. DNA vaccines
can be administered multiple times, e.g., between two-six times,
e.g., three times. In an exemplary method, 100 .mu.g of a DNA
composition is administered to a human subject at 0, 4, and 12
weeks (100 .mu.g per administration).
[0152] The pharmaceutical compositions described herein can include
a therapeutically effective amount or a prophylactically effective
amount of a nucleic acid, polypeptide, antibody, or antibody
portion. A therapeutically effective amount of a codon-optimized
nucleic acid vaccine, polypeptide, or antibody or antibody fragment
varies according to factors such as the disease state, age, sex,
and weight of the individual, and the ability of the composition to
elicit a desired response in the individual. A therapeutically
effective amount is also one in which any toxic or detrimental
effects of the pharmaceutical composition is outweighed by the
therapeutically beneficial effects. The ability of a compound to
inhibit a measurable parameter can be evaluated in an animal model
system predictive of efficacy in the target subject (e.g., a human
subject). Alternatively, this property of a composition can be
evaluated by examining the ability of the compound to modulate,
such modulation in vitro by assays known to the skilled
practitioner.
[0153] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result, i.e., protective immunity against
a subsequent challenge by the influenza virus. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount. Also provided
herein are kits including one or more of a codon-optimized nucleic
acid encoding an influenza polypeptide, the polypeptide encoded by
the nucleic acid, and/or an anti-influenza antibody or
antigen-binding fragment thereof. The kits can include one or more
other elements including: instructions for use; other reagents,
e.g., a label, a therapeutic agent, or an agent useful for
chelating, or otherwise coupling, an antibody to a label or
therapeutic agent, or a radioprotective composition; devices or
other materials for preparing the composition for administration;
pharmaceutically acceptable carriers; and devices or other
materials for administration to a subject.
[0154] Instructions for use can include instructions for diagnostic
applications of the nucleic acid sequence, polypeptides, or
antibodies (or antigen-binding fragment thereof) to detect
influenza, in vitro, e.g., in a sample, e.g., a biopsy or cells
from a patient, or in vivo. The instructions can include
instructions for therapeutic or prophylactic application including
suggested dosages and/or modes of administration, e.g., in a
patient at risk for or suffering from a symptom of influenza.
[0155] The kit can further contain at least one additional reagent,
such as a diagnostic or therapeutic agent, e.g., one or more
additional codon-optimized nucleic acid encoding and influenza
polypeptide, and/or an antiviral agent in one or more separate
pharmaceutical preparations.
Therapeutic Uses
[0156] The new nucleic acid vaccines, polypeptides, and antibodies
described herein have in vitro and in vivo diagnostic, therapeutic,
and prophylactic utilities. For example, the nucleic acid vaccines
can be administered to cells in culture, e.g., in vitro or ex vivo,
or in a subject, e.g., in vivo, to treat, prevent, and/or diagnose
influenza.
[0157] As used herein, the term "subject" is intended to include
humans and non-human animals. The term "non-human animals" includes
all vertebrates, e.g., mammals and non-mammals, such as non-human
primates, pigs, chickens and other birds, mice, dogs, cats, cows,
and horses.
[0158] Methods of administering nucleic acid vaccines, polypeptide,
and antibody compositions are described above. Suitable dosages of
the molecules used will depend on the age and weight of the subject
and the particular drug used. The nucleic acid vaccines can be used
to prevent an influenza infection by inducing a protective immunity
in the inoculated subject, or to treat an existing influenza
infection if improved immune responses can be useful in controlling
the viral infection. The antibody molecules can be used to reduce
or alleviate an acute influenza infection.
[0159] In other embodiments, immunogenic compositions and vaccines
that contain an immunogenically effective amount of an influenza
polypeptide, or antigenic fragments thereof, are provided.
Immunogenic epitopes in a polypeptide sequence can be identified
according to methods known in the art, and proteins, or fragments
containing those epitopes can be delivered by various means, in a
vaccine composition.
[0160] The polypeptide and nucleic acid compositions described
herein can be used in combination with agents used for inducing
immune responses to influenza in humans, such as trivalent
inactivated influenza vaccines (e.g., trivalent vaccines that
include H1N1, H3N2, and influenza B strains). Other compositions
suitable for use in combination with the novel nucleic acid and
polypeptide compositions described herein include live influenza
vaccines such as cold-adapted influenza vaccines (see, e.g.,
Wareing and Tannock, Vaccine, 19(25-26):3320-3330, 2001) and
vaccines generated by reverse genetics (see, e.g., Hoffmann et al.,
Vaccine, 20(25-26):3165-3170, 2002). These compositions can be
administered simultaneously with, before, or after a composition
described herein.
[0161] Suitable compositions can include, for example, lipopeptides
(e.g., Vitiello et al., J. Clin. Invest., 95:341, 1995), peptide
compositions encapsulated in poly(DL-lactide-co-glycolide) ("PLG")
microspheres (see, e.g., Eldridge et al., Molec. Immunol.,
28:287-94, 1991; Alonso et al., Vaccine, 12:299-306, 1994; Jones et
al., Vaccine, 13:675-81, 1995), peptide compositions contained in
immune stimulating complexes (ISCOMS) (see, e.g., Takahashi et al.,
Nature, 344:873-75, 1990; Hu et al., Clin. Exp. Immunol.,
113:235-43, 1998), and multiple antigen peptide systems (MAPs)
(see, e.g., Tam, Proc. Natl. Acad. Sci. U.S.A., 85:5409-13, 1988;
Tam, J. Immunol. Methods, 196:17-32, 1996). Toxin-targeted delivery
technologies, also known as receptor-mediated targeting, such as
those of Avant Immunotherapeutics, Inc. (Needham, Mass.) can also
be used.
[0162] Useful carriers that can be used with immunogenic
compositions and vaccines are well known, and include, for example,
thyroglobulin, albumins such as human serum albumin, tetanus
toxoid, polyamino acids such as poly L-lysine, poly L-glutamic
acid, influenza, hepatitis B virus core protein, and the like. The
compositions and vaccines can contain a physiologically tolerable
(i.e., acceptable) diluent such as water, or saline, typically
phosphate buffered saline. The compositions and vaccines also
typically include an adjuvant. Adjuvants such as incomplete
Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum
are examples of materials well known in the art. Additionally, CTL
responses can be primed by conjugating influenza polypeptides (or
fragments, derivatives or analogs thereof) to lipids, such as
tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P.sub.3CSS).
[0163] Immunization with a composition or vaccine containing a
protein composition, e.g., via injection, aerosol, oral,
transdermal, transmucosal, intrapleural, intrathecal, or other
suitable routes, induces the immune system of the host to respond
to the composition or vaccine by producing large amounts of CTLs,
and/or antibodies specific for the desired antigen. Consequently,
the host typically becomes at least partially immune to later
infection (e.g., with influenza), or at least partially resistant
to developing an ongoing chronic infection, or derives at least
some therapeutic benefit. In other words, the subject is protected
against subsequent infection by the influenza virus.
EXAMPLES
[0164] The invention is further described in the following
examples, which do not limit the scope of the invention described
in the claims.
Example 1
Construction of Codon-Optimized Sequences Encoding Influenza HA and
NA Polypeptides
[0165] To generate DNA for efficient expression of influenza HA and
NA polypeptides and various fragments of these polypeptides,
codon-optimized nucleic acids were constructed. These
codon-optimized nucleic acids were designed to express polypeptides
with amino acid sequences identical, or nearly identical, to
sequences encoded by the native influenza polypeptide but with
codons known to be efficiently translated in mammalian host cells.
Substitution of viral codons for mammalian codons can facilitate
high levels of expression of viral proteins in recombinant
systems.
[0166] The codon usage of HA-encoding sequences from influenza A
H1N1 and H3N2 strains and the NA-encoding sequence from H3N2 was
analyzed by the MacVector software (V. 7.2, Accelrys, San Diego,
Calif.) against that of the Homo sapiens genome. Sequences were
generated in which the codons in the influenza sequences that are
less optimal for mammalian expression were changed to the codons
more preferred in mammalian systems. The sequences were also
designed to avoid unwanted RNA motifs, such as internal TATA-boxes,
chi-sites, ribosomal entry sites, AT-rich or GC-rich sequence
stretches, repeat sequences, sequences likely to encode RNA with
secondary structures, (cryptic) splice donor and acceptor sites, or
branch points.
[0167] The codon-optimized nucleic acids encoding H1 HA, H3 HA, and
N2 NA polypeptides were chemically synthesized. The codon-optimized
nucleic acid sequence and amino acid sequence encoded by the
nucleic acids are shown in FIGS. 1A, 1B, 2A, 2B, 6A, and 6B,
respectively. The viral sequence from which SEQ ID NO:1 was derived
is shown in FIG. 9 as SEQ ID NO:7. The viral sequence from which
SEQ ID NO:3 was derived is shown in FIG. 10 as SEQ ID NO:8. The
viral sequence from which SEQ ID NO:5 was derived is shown in FIG.
11 as SEQ ID NO:9.
[0168] We constructed expression vectors that encode six different
forms of the HA polypeptides, shown schematically in FIGS. 3A and
3B. These forms were: a wild type, full length HA sequence
containing a sequence encoding the native HA leader peptide
(wt.HA0); a full length HA sequence encoding a tPA leader peptide
(tPA.HA0); an HA sequence encoding the external portion of HA with
a tPA leader peptide (tPA.HA0.dTM); a sequence encoding the HA1
domain of HA with a tPA leader peptide (tPA.HA1); a sequence
encoding the HA2 domain of HA with a tPA leader peptide (tPA.HA2);
and a sequence encoding the HA2 domain of the HA polypeptide
lacking the transmembrane region, also with a tPA leader peptide
(tPA.HA2.dTM).
[0169] We designed expression vectors that encode four different
forms of NA polypeptides, shown schematically in FIG. 8. These
forms are a wild type, full length NA sequence containing a
sequence encoding the native NA leader peptide (wt.NA); a full
length NA sequence encoding a tPA leader peptide (tPA.NA); an NA
sequence encoding the external portion of NA with a tPA leader
peptide (tPA.NA.dTM); a sequence encoding a fusion protein of M2
polypeptide extracelluar domain (M2-ex) and the external portion of
NA with a tPA leader peptide (tPA.M2-NA.dTM).
[0170] The sequences described above were subcloned into DNA
vaccine vector pSW3891 (Wang et al., Journal of Virology,
79:1906-1910, 2005) which is a modified form of the pJW4303 vector
(Lu et al., Methods in Molecular Medicine, 29:355-74, 1998). The
pSW3891 vector contains a cytomegalovirus immediate early promoter
(CMV-IE) with its downstream Intron A sequence for initiating
transcription of eukaryotic gene inserts and a bovine growth
hormone (BGH) poly-adenylation signal for termination of
transcription. For certain constructs, a human tissue plasminogen
activator (tPA) leader sequence was included to direct expression
of secreted proteins. The vector also contains the ColE1 origin of
replication for prokaryotic replication and the kanamycin
resistance gene for selective growth in antibiotic containing
media.
[0171] Each individual DNA plasmid was confirmed by DNA sequencing
before large amounts of DNA plasmids were prepared from Escherichia
coli (HB101 strain) with a Mega purification kit (Qiagen, Valencia,
Calif.) for both in vitro transfection and in vivo animal
immunization studies.
Example 2
Expression of Codon-Optimized HA Polypeptides In Vitro and
Immunogenicity In Vivo
[0172] HA expression in vitro. Cells (293T) were transfected with
the codon-optimized wt.HA0 vector encoding full-length, wild-type
H1 HA. Expression of the HA antigen was evaluated by Western
blotting. HA was detected using a commercial anti-HA monoclonal
antibody. As shown in FIGS. 4A and 4B, the HA antigen was expressed
in 293T cells. Cells transfected with empty vectors (as a negative
control) did not express HA antigen.
[0173] Immunization. NZW Rabbits (female, .about.2 kg each) were
purchased from Millbrook Farms (Millbrook, Mass.) and housed in the
Department of Animal Medicine at the University of Massachusetts
Medical School (UMMS) in accordance with IACUC approved protocols.
The animals were immunized with a Helios gene gun (Bio-Rad,
Hercules, Calif.) at the shaved abdominal skin as previously
reported (Wang et al., Methods Mol. Biol., 245:185-96, 2004). A
total of 36 .mu.g of plasmid DNA was administrated to each
individual rabbit for each immunization at 0, 2, 4, and 8 weeks.
Serum samples were taken at 0, 2, 4, 6, 8, and 10 weeks after each
immunization for analyses of HA-specific antibody responses.
Animals were immunized with wt.HA0, tPA.HA0, tPA.HA0.dTM, tPA.HA1,
tPA.HA2, tPA.HA2.dTM vectors encoding H1 or H3 subtype HA antigens.
A subset of animals were immunized with both H1 tPA.HA0 and H3
tPA.HA0 or both H1 tPA.HA1 and H3 tPA.HA1 vectors.
[0174] ELISA to Determine Anti-HA Antibody Responses. ELISA assays
were conducted to measure the anti-HA antibody responses in
immunized rabbits. The ELISA plates were coated with 100 or 200
.mu.l/well of either H1 or H3 HA antigen (1 .mu.g/ml in PBS at pH
7.2) from the supernatant of 293T cells transiently transfected
with tPA.HA0.dTM overnight at 4.degree. C. Plates were washed five
times with PBS containing 0.1% Triton X-100 and blocked with 200
.mu.l/well of blocking buffer (5% non-fat dry milk, 4% whey, 0.5%
Tween-20 in PBS at pH 7.2) for 1 hour. After five washes, 100 .mu.l
of rabbit serum diluted 1:5000 in Whey dilution buffer (4% Whey,
0.5% Tween-20 in PBS) was added in duplicate wells and incubated
for 1 hour at room temperature. After another set of washes, the
plates were incubated for 1 hour at room temperature with 100 .mu.l
of biotinylated anti-rabbit IgG (Vector Laboratories) diluted at
1:1000 in Whey dilution buffer. Then 100 .mu.l of horseradish
peroxidase-conjugated streptavidin (Vector Laboratories) diluted at
1:2000 in Whey buffer was added to each well and incubated for 1
hour. After the final wash, the plates were developed with
3,3',5,5' Tetramethybenzidine solution at 100 .mu.l per well
(Sigma, St. Louis, Mo.) for 3.5 minutes. The reactions were stopped
by adding 25 .mu.l of 2 M H.sub.2SO.sub.4, and the plates were read
at OD 450 nm. The results of these assays are depicted in FIGS.
5A-5I.
[0175] All of the HA-encoding DNA constructs tested induced
antibodies to HA antigens in the rabbits. Thus, the codon-optimized
sequences were expressed in the animals and the polypeptides they
expressed were immunogenic. Rabbits immunized with constructs
encoding both H1 and H3 HA antigens (FIGS. 5C, 5F, and 5I) mounted
responses to both H1 and H3 HA antigens, i.e., it does not appear
that immunization with two DNAs compromised the response to either
one. The ability to induce a robust response to antigens of
multiple subtypes simultaneously can convey broader protection than
would result from vaccination with a monovalent construct.
Example 3
Construction of Influenza M2 DNA Vectors
[0176] The influenza M2 polypeptide contains approximately 23 amino
acids in the extracellular domain. This region of the polypeptide
is a potential target of protective antibodies reported recently
(Neirynck et al., Nature Medicine, 5:1157-1163, 1999; Fan, et al.,
Vaccine, 22:2993-3003, 2005). However, short synthetic peptides,
instead of recombinant protein, have been used in previous studies
due to the difficulty of expressing M2 protein. We can enhance
immunogenicity of M2 by expressing multiple copies of the
extracellular domain of M2 as a fusion, as shown schematically in
FIG. 7. DNA expressing these M2 fusions, or the polypeptides
expressed by the DNA, can be administered to animals to induce
immune responses against this antigen. DNA expressing M2 fused to a
second type of influenza antigen (e.g., HA or NA, or a fragment
thereof), or the fusion polypeptides themselves, can also be used
to induce immune responses in animals. See, e.g., the tPA.M2-NA.dTM
construct depicted in FIG. 8.
Example 4
Comparison of Immune Responses Induced by Codon-Optimized and
Wild-Type Influenza Nucleic Acid Sequences
[0177] HA expression in vitro. To compare expression of
codon-optimized and non-codon-optimized (wild-type) sequences in
vitro, mammalian cells were transfected with either a
codon-optimized nucleic acid encoding H1 HA or a wild-type nucleic
acid encoding H1 HA. Expression of the HA antigen was evaluated by
Western blotting. HA was detected using a commercial anti-HA
monoclonal antibody. As shown in FIG. 12, the H1 HA antigen was
expressed more robustly in cells transfected with the
codon-optimized sequence as compared to cells transfected with the
wild-type sequence. Cells transfected with empty vectors (as a
negative control) did not express HA antigen.
[0178] Antibody responses induced by codon-optimized and wild-type
nucleic acid sequences. Rabbits were immunized with codon-optimized
or wild-type H1 HA nucleic acid sequences and ELISA assays were
conducted with sera from immunized rabbits. Immunization and ELISA
protocols are described above in Example 2. Animals were immunized
at 0, 2, 4, and 8 weeks. Sera samples collected at 0, 2, 4, 6, 8,
10, and 12 weeks were tested by ELISA at 1:5000 serum dilutions.
The results of this experiment are depicted in FIG. 13A. Rabbits
R#316 and R#317 were immunized with the wild-type H1 HA DNA while
rabbits R#381 and R#382 received the codon-optimized H1 HA DNA. The
antibody titers in animals immunized with the codon-optimized H1 HA
DNA were higher than titers in animals immunized with wild-type H1
HA DNA at all time points examined after week 0. FIG. 13B depicts
anti-HA IgG titers in sera from the animals collected two weeks
after the fourth immunization. These data show that codon-optimized
DNA induced anti-HA titers much higher than titers induced by
wild-type influenza DNA sequences, with average titers of
approximately 3,500,000 and 500,000, respectively, indicating that
the codon-optimized DNAs are expressed at a higher level than
non-codon-optimized DNAs.
[0179] A similar immunization experiment was performed in mice.
Mice were immunized four times with codon-optimized H1 HA DNA or
wild-type H1 HA DNA. A set of mice was also immunized with empty
vector as a control. Sera collected two weeks after the fourth
immunization were tested for anti-HA IgG titers. Group mean titers
are plotted in FIG. 13C. The results depicted in FIG. 13C show that
codon-optimized DNA induced anti-HA titers much higher than titers
induced by wild-type influenza DNA sequences, with average titers
of approximately 2,000,000 and 200,000, respectively. Sera from
mice immunized with empty vector did not contain any detectable
HA-reactive IgG.
Example 5
Sera from Animals Immunized with Codon-Optimized DNA Mediate
Hemagglutinin Inhibition and Virus Neutralization
[0180] Hemagglutination Inhibition. Sera from animals immunized
with various codon-optimized DNAs encoding H1 HA or H3 HA were
tested in standard hemagglutination inhibition assays (HAI or HI)
in the presence of the A/NewCaledonia/20/99 (H1N1) influenza strain
and the A/Panama/2007/99 (H3N2) influenza strain.
[0181] Hemagglutination inhibition assays were performed with sera
that had been pre-treated with bacterial neuraminidase/Receptor
Destroying Enzyme (RDE) to remove nonspecific inhibitors of virus
hemagglutination. Briefly, 25 .mu.l of a preparation of influenza
virus (hemagglutination titer=8) was mixed with 25 .mu.l of 2-fold
dilutions of the specific RDE-treated serum in PBS in V-bottom
96-well plates. After 30 minutes incubation at 4 degrees, 50 .mu.l
of 0.5% chicken red blood cells were added to the mixtures. The
plates were incubated at 4 degrees until hemagglutination occurred
in non-serum containing control wells. The HI titer is defined as
the highest dilution of serum that inhibits hemagglutination.
[0182] The HI antibody titers are depicted in FIGS. 14A and 14B.
Sera from animals immunized with a codon-optimized DNA encoding a
full-length H1 HA, wt.HA0, exhibited the highest level of
hemagglutination activity towards the H1N1 A/NewCaledonia/20/99
strain (FIG. 14A).
[0183] Sera from animals immunized with codon-optimized DNA
encoding full-length H3 HA with a tPA leader sequence (tPA-HA0) and
DNA encoding H3 HA lacking the transmembrane region (tPA.HA0.dTM)
exhibited the highest levels of hemagglutination activity (FIG.
14B). Activity was also observed in sera from animals immunized
with DNA encoding full length H3 HA with a wild-type leader
sequence (wt.HA0) and with DNA encoding HA1, HA2, and partial HA2
domains of H3 HA (tPA.HA1, tPA.HA2, and tPA.HA2.dTM,
respectively).
[0184] Neutralizing antibody responses. Neutralizing antibody
responses induced by various codon-optimized DNAs encoding H1 HA or
H3 HA were determined. The assays were performed using viruses
which were able to infect cells and express green fluorescent
protein (GFP), but which do not propagate (replication is
restricted to a single cycle). To generate the
replication-restricted virus for these assays, 293 cells were
transfected with 8 viral RNA expression plasmids and with 5 viral
protein expression plasmids. The HA viral RNA expression plasmids
were replaced by a GFP viral RNA expression plasmid that includes
the 3' and 5' HA-specific regions required for the replication,
transcription and packaging of this RNA into an influenza virus, as
previously described. Transfected 293 cells were co-cultured with
an MDCK cell line expressing the desired influenza virus HA
protein. Viruses in which the HA gene is replaced by the GFP gene
were obtained and propagated in the HA-expressing MDCK cell line.
In the presence of HA neutralizing antibodies, infection and GFP
expression by these viruses is prevented.
[0185] Sera from animals immunized with various codon-optimized H1
HA vectors were tested against H1N1 influenza virus
A/NewCaledonia/20/99 (FIG. 15A). The highest levels of neutralizing
antibody titers were detected in sera from animals immunized with a
vector encoding the full-length H1 HA, wt.HA0.
[0186] Sera from animals immunized with various codon-optimized H3
HA vectors were tested against H3N2 influenza virus A/Moscow/10/99.
Sera from animals immunized with DNA encoding full-length H3 HA
with a tPA leader sequence, tPA.HA0, exhibited the highest levels
of neutralizing activity. Activity was observed in sera from
animals immunized with wt.HA0, tPA.HA0.dTM, tPA.HA1, and tPA.HA2
vectors.
[0187] In summary, these data show that different vectors induce
different levels of functional antibody responses although they
induce similar levels of binding antibody responses when measured
by ELISA.
[0188] Hemagglutination inhibition and bivalent immunization.
Hemagglutination inhibition by sera from animals immunized with two
different codon-optimized DNAs encoding H1 HA or H3 HA were
determined. Sera from animals immunized with the combinations of
codon-optimized H1 HA vectors were tested against H1N1 influenza
virus A/NewCaledonia/20/99 and H3N2 influenza virus
A/Panama/2007/99 (FIGS. 16A and 16B). Animals were immunized with
either H1-tPA.HA0.dTM and H3-tPA.HA0.dTM; or with H1-wt.HA0 and
H3-tPA.HA0.dTM. Sera from animals immunized with the latter
combination showed the highest levels of hemagglutination
inhibition activity against both virus strains. Activity was more
modest in sera from animals immunized with the former combination,
and higher levels of activity were seen against the H3N2 Panama
strain.
[0189] Neutralization and bivalent immunization. Neutralizing
activity induced by bivalent immunization was also examined. The
same bivalent combinations described in the previous paragraph were
tested. In these experiments, neutralizing activity to H1N1
influenza virus A/NewCaledonia/20/99 and H3N2 influenza virus
A/Moscow/10/99 was examined. Sera from animals immunized with the
first combination (H1-tPA.HA0.dTM+H3-tPA.HA0.dTM) exhibited low
titers to the H1N1 New Caledonia strain yet exhibited high titers
to the H3N2 Moscow strain (FIGS. 16C and 16D). Sera from the second
combination (H1-wt.HA0+H3-tPA.HA0.dTM) exhibited high levels of
neutralizing titers against both strains.
[0190] In summary, H1 HA constructs encoding full-length H1 HA (as
opposed to a form lacking the transmembrane region) induced higher
levels of protective antibodies against H1 strains in both
monovalent and bivalent immunization regimens. In contrast,
constructs encoding both full-length H3 HA and forms lacking the
transmembrane region were effective in inducing protective
antibodies.
Example 6
Immunization with Multiple Agents
[0191] The codon-optimized nucleic acids described herein (and
other compositions described herein) may be used in combination
with other agents that induce immune responses to influenza
antigens. In the following experiments, codon-optimized DNAs
encoding H1HA and H3 HA (H1-HA0.wt+H3-HA0.dTM; 250 micrograms/dose
of each DNA) were administered to rabbits at week 0 followed by a
boost with Fluzone.RTM. (Aventis Pasteur), an influenza vaccine
prepared from inactivated influenza virus, at week 4. Another set
of rabbits was administered Fluzone.RTM. alone, at weeks 0 and 4.
Fluzone.RTM. (0.25 ml/dose) was administered by intramuscular
injection. Sera collected at week 8 from both sets of animals were
examined.
[0192] HA-specific IgG responses were determined by ELISA. The
results are depicted in FIGS. 17A and 17B. Sera from animals
administered Fluzone.RTM. alone contained IgG titers of less than
200,000 to H1 HA and titers of less than 1,250,000 to H3 HA. In
contrast, sera from animals administered the bivalent H1 HA, H3 HA
prime and Fluzone.RTM. boost exhibited very high titers of
HA-specific IgG. Titers to H1 HA were approximately 1,200,000.
Titers to H3 HA were approximately 3,500,000. These results show
that the DNA prime, Fluzone.RTM. boost protocol was much more
effective in inducing HA-specific antibodies than the use of
Fluzone.RTM. alone.
OTHER EMBODIMENTS
[0193] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
Sequence CWU 1
1
911698DNAArtificial SequenceSynthetically generated oligonucleotide
1atgaaggcca agctgctggc cctgctgtgc accttcaccg ccacctacgc cgacaccatc
60tgcatcggct accacgccaa caacagcacc gacaccgtgg ataccgtgct ggagaagaac
120gtgacagtga cccacagcgt gaacctgctg gaggacagcc acaacggcaa
gctgtgtctg 180ctgaaaggca tcgcccccct gcagctgggc aactgtagcg
tggccggctg gattctgggc 240aaccccgaat gcgagctgct gatctccaag
gagagctgga gctacatcgt ggagaccccc 300aaccccgaga atggcacctg
ctaccccggc tacttcgccg actacgagga gctgcgggag 360cagctgagca
gcgtgagcag cttcgagaga ttcgagatct tccccaagga aagcagctgg
420cccaaccaca ccgtgaccgg agtgagcgcc agctgcagcc acaatgggaa
gagcagcttc 480tacagaaatc tgctgtggct gaccggcaag aacggcctgt
accccaacct gagcaagtcc 540tacgtgaaca acaaagagaa ggaagtgctg
gtgctgtggg gcgtgcacca cccccctaac 600atcggcaacc agcgggccct
gtaccacacc gagaacgcct atgtgagcgt ggtgagcagc 660cactacagca
gaagattcac ccccgagatc gccaagagac ccaaagtgag agatcaggag
720ggcagaatca actactactg gaccctgctg gagcccggcg acgccatcat
cttcgaggcc 780aacggcaacc tgatcgcccc ctggtacgcc ttcgccctga
gcagaggctt cggcagcggc 840atcatcacca gcaatgcccc catggacgaa
tgcgacgcca agtgtcagac accccagggc 900gccatcaaca gcagcctgcc
cttccagaac gtgcaccccg tgaccatcgg agagtgcccc 960aagtacgtgc
ggagcgccaa gctgcggatg gtgaccggcc tgcggaacat ccccagcatt
1020cagagcagag gcctgttcgg cgccatcgcc ggcttcatcg agggcggctg
gaccggcatg 1080gtggacggct ggtatggcta ccaccaccag aacgagcagg
gatctggcta cgccgccgat 1140cagaagagca cccagaacgc catcaacggc
atcaccaaca aagtgaacag cgtgatcgag 1200aagatgaaca cccagttcac
agccgtgggc aaggagttca acaaactgga gcggcggatg 1260gaaaccctga
acaagaaagt ggacgacggc ttcctggaca tctggaccta caacgccgag
1320ctgctggtgc tgctggagaa tgagcggacc ctggacttcc acgacagcaa
cgtgaagaac 1380ctgtacgaga aagtgaagag ccagctgaag aacaacgcca
aggagatcgg caacggctgc 1440ttcgagttct accacaagtg caacaacgag
tgcatggaga gcgtgaagaa cggcacctac 1500gactacccca agtactccga
ggagagcaag ctgaaccggg agaagatcga cggcgtgaag 1560ctggagagca
tgggcgtgta ccagatcctg gccatctaca gcaccgtggc cagcagcctg
1620gtgctgctgg tgagcctggg cgccatctct ttctggatgt gctccaacgg
cagcctgcag 1680tgcagaatct gcatctga 16982565PRTArtificial
SequenceSynthetically generated peptide 2Met Lys Ala Lys Leu Leu
Ala Leu Leu Cys Thr Phe Thr Ala Thr Tyr1 5 10 15Ala Asp Thr Ile Cys
Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30Val Asp Thr Val
Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45Leu Leu Glu
Asp Ser His Asn Gly Lys Leu Cys Leu Leu Lys Gly Ile 50 55 60Ala Pro
Leu Gln Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu Gly65 70 75
80Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser Trp Ser Tyr Ile
85 90 95Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr
Phe 100 105 110Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val
Ser Ser Phe 115 120 125Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser
Trp Pro Asn His Thr 130 135 140Val Thr Gly Val Ser Ala Ser Cys Ser
His Asn Gly Lys Ser Ser Phe145 150 155 160Tyr Arg Asn Leu Leu Trp
Leu Thr Gly Lys Asn Gly Leu Tyr Pro Asn 165 170 175Leu Ser Lys Ser
Tyr Val Asn Asn Lys Glu Lys Glu Val Leu Val Leu 180 185 190Trp Gly
Val His His Pro Pro Asn Ile Gly Asn Gln Arg Ala Leu Tyr 195 200
205His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg
210 215 220Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asp
Gln Glu225 230 235 240Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu
Pro Gly Asp Ala Ile 245 250 255Ile Phe Glu Ala Asn Gly Asn Leu Ile
Ala Pro Trp Tyr Ala Phe Ala 260 265 270Leu Ser Arg Gly Phe Gly Ser
Gly Ile Ile Thr Ser Asn Ala Pro Met 275 280 285Asp Glu Cys Asp Ala
Lys Cys Gln Thr Pro Gln Gly Ala Ile Asn Ser 290 295 300Ser Leu Pro
Phe Gln Asn Val His Pro Val Thr Ile Gly Glu Cys Pro305 310 315
320Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn
325 330 335Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala
Gly Phe 340 345 350Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp
Tyr Gly Tyr His 355 360 365His Gln Asn Glu Gln Gly Ser Gly Tyr Ala
Ala Asp Gln Lys Ser Thr 370 375 380Gln Asn Ala Ile Asn Gly Ile Thr
Asn Lys Val Asn Ser Val Ile Glu385 390 395 400Lys Met Asn Thr Gln
Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu 405 410 415Glu Arg Arg
Met Glu Thr Leu Asn Lys Lys Val Asp Asp Gly Phe Leu 420 425 430Asp
Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 435 440
445Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys
450 455 460Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn
Gly Cys465 470 475 480Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys
Met Glu Ser Val Lys 485 490 495Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr
Ser Glu Glu Ser Lys Leu Asn 500 505 510Arg Glu Lys Ile Asp Gly Val
Lys Leu Glu Ser Met Gly Val Tyr Gln 515 520 525Ile Leu Ala Ile Tyr
Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val 530 535 540Ser Leu Gly
Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln545 550 555
560Cys Arg Ile Cys Ile 56531698DNAArtificial SequenceSynthetically
generated oligonucleotide 3atgaaaacca tcatcgccct gagctacatc
ctgtgcctgg tgttcgccca gaaactgccc 60ggcaacgaca acagcaccgc caccctgtgt
ctgggccacc acgccgtgag caacggcacc 120ctggtgaaaa ccatcaccaa
tgaccagatc gaagtgacca acgccaccga gctggtgcag 180agcagcagca
ccggcagaat ctgcgacagc cctcaccaga tcctggacgg cgagaactgt
240accctgatcg acgccctgct gggagaccct cactgcgacg gcttccagaa
caaggagtgg 300gacctgttcg tggagcgcag caaggcctac agcaactgct
acccttacga cgtgcccgac 360tacgcctccc tgcggagcct ggtggccagc
tctggcaccc tggagttcaa caacgagagc 420ttcaattgga ccggcgtggc
ccagaacggc accagcagcg cctgcaagcg gagaagcaac 480aagagcttct
tcagcagact gaactggctg caccagctga agtacaagta ccccgccctg
540aacgtgacca tgcccaacaa cgaaaagttc gacaaactgt acatttgggg
cgtgcaccac 600cccagcaccg acagcgacca gatcagcatc tacgcccagg
ccagcggcag agtgaccgtg 660tctaccaaga gaagccagca gaccgtgatc
cccaatatcg gcagcagacc ctgggtgcgg 720ggcgtgtcca gcggaatctc
catctactgg acaatcgtga agcccggcga catcctgctg 780atcaactcca
ccggcaacct gattgcccct cggggctact tcaagatccg gagcggcaaa
840agcagcatca tgcggagcga tgcccccatc ggcaagtgca acagcgagtg
catcaccccc 900aacggcagca tccccaatga caagcccttc cagaacgtga
accggatcac ctacggcgcc 960tgccccagat acgtgaagca gaacaccctg
aagctggcca caggaatgcg gaacgtgccc 1020gagaagcaga cccggggcat
cttcggcgcc atcgccggct tcatcgagaa tggctgggag 1080ggcatggtgg
acggctggta cggcttccgg caccagaaca gcgagggcac cggacaggcc
1140gacctgaaga gcacccaggc cgccatcaac cagatcaacg gcaagctgaa
ccggctgatc 1200gagaaaacca acgagaagtt ccaccagatc gagaaggagt
tcagcgaagt ggaaggcaga 1260atccaggacc tggagaagta cgtggaggac
accaagatcg atctgtggag ctacaacgcc 1320gagctgctgg tcgccctgga
gaaccagcac accatcgacc tgaccgactc cgagatgaac 1380aaactgttcg
agagaaccaa gaagcagctg cgggagaacg ccgaggacat gggcaacggc
1440tgtttcaaga tctaccacaa gtgcgacaac gcctgcatcg gcagcatcag
aaacggcacc 1500tacgaccacg acgtgtacag agatgaggcc ctgaacaacc
ggttccagat caagggcgtg 1560gagctgaaga gcggctacaa ggattggatt
ctgtggatct ccttcgccat cagctgcttc 1620ctgctgtgcg tggtgctgct
gggcttcatc atgtgggcct gtcagaaggg caacatccgg 1680tgcaacatct gcatctga
16984565PRTArtificial SequenceSynthetically generated peptide 4Met
Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala1 5 10
15Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly
20 25 30His His Ala Val Ser Asn Gly Thr Leu Val Lys Thr Ile Thr Asn
Asp 35 40 45Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser
Ser Thr 50 55 60Gly Arg Ile Cys Asp Ser Pro His Gln Ile Leu Asp Gly
Glu Asn Cys65 70 75 80Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His
Cys Asp Gly Phe Gln 85 90 95Asn Lys Glu Trp Asp Leu Phe Val Glu Arg
Ser Lys Ala Tyr Ser Asn 100 105 110Cys Tyr Pro Tyr Asp Val Pro Asp
Tyr Ala Ser Leu Arg Ser Leu Val 115 120 125Ala Ser Ser Gly Thr Leu
Glu Phe Asn Asn Glu Ser Phe Asn Trp Thr 130 135 140Gly Val Ala Gln
Asn Gly Thr Ser Ser Ala Cys Lys Arg Arg Ser Asn145 150 155 160Lys
Ser Phe Phe Ser Arg Leu Asn Trp Leu His Gln Leu Lys Tyr Lys 165 170
175Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu Lys Phe Asp Lys
180 185 190Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asp Ser Asp
Gln Ile 195 200 205Ser Ile Tyr Ala Gln Ala Ser Gly Arg Val Thr Val
Ser Thr Lys Arg 210 215 220Ser Gln Gln Thr Val Ile Pro Asn Ile Gly
Ser Arg Pro Trp Val Arg225 230 235 240Gly Val Ser Ser Gly Ile Ser
Ile Tyr Trp Thr Ile Val Lys Pro Gly 245 250 255Asp Ile Leu Leu Ile
Asn Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly 260 265 270Tyr Phe Lys
Ile Arg Ser Gly Lys Ser Ser Ile Met Arg Ser Asp Ala 275 280 285Pro
Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile 290 295
300Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly
Ala305 310 315 320Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu
Ala Thr Gly Met 325 330 335Arg Asn Val Pro Glu Lys Gln Thr Arg Gly
Ile Phe Gly Ala Ile Ala 340 345 350Gly Phe Ile Glu Asn Gly Trp Glu
Gly Met Val Asp Gly Trp Tyr Gly 355 360 365Phe Arg His Gln Asn Ser
Glu Gly Thr Gly Gln Ala Asp Leu Lys Ser 370 375 380Thr Gln Ala Ala
Ile Asn Gln Ile Asn Gly Lys Leu Asn Arg Leu Ile385 390 395 400Glu
Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser Glu 405 410
415Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys
420 425 430Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu
Glu Asn 435 440 445Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn
Lys Leu Phe Glu 450 455 460Arg Thr Lys Lys Gln Leu Arg Glu Asn Ala
Glu Asp Met Gly Asn Gly465 470 475 480Cys Phe Lys Ile Tyr His Lys
Cys Asp Asn Ala Cys Ile Gly Ser Ile 485 490 495Arg Asn Gly Thr Tyr
Asp His Asp Val Tyr Arg Asp Glu Ala Leu Asn 500 505 510Asn Arg Phe
Gln Ile Lys Gly Val Glu Leu Lys Ser Gly Tyr Lys Asp 515 520 525Trp
Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys Phe Leu Leu Cys Val 530 535
540Val Leu Leu Gly Phe Ile Met Trp Ala Cys Gln Lys Gly Asn Ile
Arg545 550 555 560Cys Asn Ile Cys Ile 56551410DNAArtificial
SequenceSynthetically generated oligonucleotide 5atgaacccca
accagaagat catcaccatc ggcagcgtga gcctcaccat cgccaccgtg 60tgcttcctca
tgcagattgc catcctggtg accaccgtga cactgcactt caagcagtac
120gagtgcgact cccccgccag caaccaggtg atgccctgcg agcccatcat
catcgagcgg 180aacatcaccg agatcgtgta cctgaacaac accaccatcg
agaaagagat ctgccccaag 240gtagtggagt accggaactg gagcaagccc
cagtgccaga tcaccggctt tgcccccttc 300agcaaggaca acagcatccg
gctgagcgct ggcggcgaca tctgggtgac cagagaaccc 360tatgtgagct
gcgaccacgg caagtgctac cagttcgccc tcggccaggg caccacactg
420gacaacaagc acagcaatga caccatccac gacagaatcc ctcaccgaac
cctgctgatg 480aacgagctgg gcgtgccctt ccacctgggc acacggcaag
tgtgcatcgc ctggtccagc 540agcagctgcc acgatggcaa agcctggctg
cacgtgtgca tcacaggcga cgacaagaat 600gccaccgcca gcttcatcta
cgacggccgg ctggtggaca gcattggcag ctggagccag 660aacatcctcc
ggacccagga gagcgagtgc gtgtgcatca atggcacctg caccgtggtg
720atgaccgacg gcagcgccag cggcagagcc gacacaagaa tcctgttcat
cgaggagggc 780aagatcgtcc acatcagccc cctgagcggc agcgcccagc
acgtggaaga gtgctcctgc 840tatccccggt accctggcgt ccggtgcatc
tgtagagaca actggaaggg cagcaaccgg 900cccgtggtgg acatcaacat
ggaggactac agcatcgact ccagctacgt gtgcagcggc 960ctggtgggcg
acacaccccg gaacgacgac cggagcagca acagcaactg ccggaacccc
1020aacaatgaga gaggcaacca aggagtgaag ggctgggcct tcgacaatgg
cgatgacgtg 1080tggatgggcc ggaccatcag caaggacctg cgcagcggct
acgagacctt caaggtgatt 1140ggcggctggt ccacccccaa ctccaagagc
cagatcaaca gacaggtgat cgtggacagc 1200gacaaccgga gcggctacag
cggcatcttc agcgtggagg gcaagagctg catcaaccgg 1260tgcttctacg
tggagctgat ccggggccgg aagcaggaga ccagagtgtg gtggaccagc
1320aacagcatcg tggtgttctg tggcaccagc ggcacctacg gcaccggcag
ctggcctgat 1380ggcgccaaca tcaacttcat gcccatctaa
14106469PRTArtificial SequenceSynthetically generated peptide 6Met
Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Ser Leu Thr1 5 10
15Ile Ala Thr Val Cys Phe Leu Met Gln Ile Ala Ile Leu Val Thr Thr
20 25 30Val Thr Leu His Phe Lys Gln Tyr Glu Cys Asp Ser Pro Ala Ser
Asn 35 40 45Gln Val Met Pro Cys Glu Pro Ile Ile Ile Glu Arg Asn Ile
Thr Glu 50 55 60Ile Val Tyr Leu Asn Asn Thr Thr Ile Glu Lys Glu Ile
Cys Pro Lys65 70 75 80Val Val Glu Tyr Arg Asn Trp Ser Lys Pro Gln
Cys Gln Ile Thr Gly 85 90 95Phe Ala Pro Phe Ser Lys Asp Asn Ser Ile
Arg Leu Ser Ala Gly Gly 100 105 110Asp Ile Trp Val Thr Arg Glu Pro
Tyr Val Ser Cys Asp His Gly Lys 115 120 125Cys Tyr Gln Phe Ala Leu
Gly Gln Gly Thr Thr Leu Asp Asn Lys His 130 135 140Ser Asn Asp Thr
Ile His Asp Arg Ile Pro His Arg Thr Leu Leu Met145 150 155 160Asn
Glu Leu Gly Val Pro Phe His Leu Gly Thr Arg Gln Val Cys Ile 165 170
175Ala Trp Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val
180 185 190Cys Ile Thr Gly Asp Asp Lys Asn Ala Thr Ala Ser Phe Ile
Tyr Asp 195 200 205Gly Arg Leu Val Asp Ser Ile Gly Ser Trp Ser Gln
Asn Ile Leu Arg 210 215 220Thr Gln Glu Ser Glu Cys Val Cys Ile Asn
Gly Thr Cys Thr Val Val225 230 235 240Met Thr Asp Gly Ser Ala Ser
Gly Arg Ala Asp Thr Arg Ile Leu Phe 245 250 255Ile Glu Glu Gly Lys
Ile Val His Ile Ser Pro Leu Ser Gly Ser Ala 260 265 270Gln His Val
Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg 275 280 285Cys
Ile Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Val Asp 290 295
300Ile Asn Met Glu Asp Tyr Ser Ile Asp Ser Ser Tyr Val Cys Ser
Gly305 310 315 320Leu Val Gly Asp Thr Pro Arg Asn Asp Asp Arg Ser
Ser Asn Ser Asn 325 330 335Cys Arg Asn Pro Asn Asn Glu Arg Gly Asn
Gln Gly Val Lys Gly Trp 340 345 350Ala Phe Asp Asn Gly Asp Asp Val
Trp Met Gly Arg Thr Ile Ser Lys 355 360 365Asp Leu Arg Ser Gly Tyr
Glu Thr Phe Lys Val Ile Gly Gly Trp Ser 370 375 380Thr Pro Asn Ser
Lys Ser Gln Ile Asn Arg Gln Val Ile Val Asp Ser385 390 395 400Asp
Asn Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser 405 410
415Cys Ile Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Lys Gln
420 425 430Glu Thr Arg Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe
Cys Gly 435 440 445Thr Ser Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp
Gly Ala Asn Ile 450 455 460Asn Phe Met Pro Ile46571692DNAInfluenza
A virus (A/NewCaledonia/20/1999(H1N1) 7atgaaagcaa aactactggt
cctgttatgt acatttacag ctacatatgc agacacaata 60tgtataggct
accatgccaa caactcaacc gacactgttg acacagtact tgagaagaat
120gtgacagtga cacactctgt caacctactt gaggacagtc acaacggaaa
actatgtcta 180ctaaaaggaa tagccccact acaattgggt aattgcagcg
ttgccggatg gatcttagga 240aacccagaat gcgaattact gatttccaag
gaatcatggt cctacattgt agaaacacca 300aatcctgaga atggaacatg
ttacccaggg tatttcgccg actatgagga actgagggag 360caattgagtt
cagtatcttc atttgagaga ttcgaaatat tccccaaaga aagctcatgg
420cccaaccaca ccgtaaccgg agtatcagca tcatgctccc ataatgggaa
aagcagtttt 480tacagaaatt tgctatggct gacggggaag aatggtttgt
acccaaacct gagcaagtcc 540tatgtaaaca acaaagagaa agaagtcctt
gtactatggg gtgttcatca cccgcctaac 600ataggggacc aaagggccct
ctatcataca gaaaatgctt atgtctctgt agtgtcttca 660cattatagca
gaagattcac cccagaaata gccaaaagac ccaaagtaag agatcaggaa
720ggaagaatca actactactg gactctgctg gaacctgggg atacaataat
atttgaggca 780aatggaaatc taatagcgcc atggtatgct tttgcactga
gtagaggctt tggatcagga 840atcatcacct caaatgcacc aatggatgaa
tgtgatgcga agtgtcaaac acctcaggga 900gctataaaca gcagtcttcc
tttccagaat gtacacccag tcacaatagg agagtgtcca 960aagtatgtca
ggagtgcaaa attaaggatg gttacaggac taaggaacat cccatccatt
1020caatccagag gtttgtttgg agccattgcc ggtttcattg aaggggggtg
gactggaatg 1080gtagatgggt ggtatggtta tcatcatcag aatgagcaag
gatctggcta tgctgcagat 1140caaaaaagta cacaaaatgc cattaacggg
attacaaaca aggtgaattc tgtaattgag 1200aaaatgaaca ctcaattcac
agctgtgggc aaagaattca acaaattgga aagaaggatg 1260gaaaacttaa
ataaaaaagt tgatgatggg tttctagaca tttggacata taatgcagaa
1320ttgttggttc tactggaaaa tgaaaggact ttggatttcc atgactccaa
tgtgaagaat 1380ctgtatgaga aagtaaaaag ccaattaaag aataatgcca
aagaaatagg aaacgggtgt 1440tttgaattct atcacaagtg taacaatgaa
tgcatggaga gtgtgaaaaa tggaacttat 1500gactatccaa aatattccga
agaatcaaag ttaaacaggg agaaaattga tggagtgaaa 1560ttggaatcaa
tgggagtcta tcagattctg gcgatctact caactgtcgc cagttccctg
1620gttcttttgg tctccctggg ggcaatcagc ttctggatgt gttccaatgg
gtctttgcag 1680tgcagaatat gc 169281698DNAInfluenza A virus
(A/Panama/2007/1999(H3N2)) 8atgaagacta tcattgcttt gagctacatt
ttatgtctgg ttttcgctca aaaacttccc 60ggaaatgaca acagcacggc aacgctgtgc
ctggggcacc atgcagtgtc aaacggaacg 120ctagtgaaaa caatcacgaa
tgaccaaatt gaagtgacta atgctactga gctggttcag 180agttcctcaa
caggtagaat atgcgacagt cctcaccaaa tccttgatgg agaaaactgc
240acactaatag atgctctatt gggagaccct cattgtgatg gcttccaaaa
taaggaatgg 300gacctttttg ttgaacgcag caaagcctac agcaactgtt
acccttatga tgtgccggat 360tatgcctccc ttaggtcact agttgcctca
tccggcacac tggagtttaa caatgaaagc 420ttcaattgga ctggagtcgc
tcagaatgga acaagctctg cttgcaaaag gagatctaat 480aaaagtttct
ttagtagatt gaattggttg caccaattaa aatacaaata tccagcactg
540aacgtgacta tgccaaacaa tgaaaaattt gacaaattgt acatttgggg
ggttcaccac 600ccgagtacgg acagtgacca aatcagcata tatgctcaag
catcagggag agtcacagtc 660tctaccaaaa gaagccaaca aactgtaatc
ccgaatatcg gatctagacc ctgggtaagg 720ggtgtctcca gcggaataag
catctattgg acaatagtaa aaccgggaga catacttttg 780attaacagca
cagggaatct aattgctcct cggggttact tcaaaatacg aagtgggaaa
840agctcaataa tgaggtcaga tgcacccatt ggcaaatgca attctgaatg
catcactcca 900aatggaagca ttcccaatga caaaccattt caaaatgtaa
acaggatcac atatggggcc 960tgtcccagat atgttaagca aaacactctg
aaattggcaa cagggatgcg gaatgtacca 1020gagaaacaaa ctagaggcat
attcggcgca atcgcgggtt tcatagaaaa tggttgggag 1080ggaatggtgg
acggttggta cggtttcagg catcaaaatt ctgagggcac aggacaagca
1140gatcttaaaa gcactcaagc agcaatcaac caaatcaacg ggaaactgaa
taggttaatc 1200gagaaaacga acgagaaatt ccatcaaatt gaaaaagaat
tctcagaagt agaagggaga 1260attcaggacc tcgagaaata tgttgaggac
actaaaatag atctctggtc gtacaacgcg 1320gagcttcttg ttgccctgga
gaaccaacat acaattgatc taactgactc agaaatgaac 1380aaactgtttg
aaagaacaaa gaagcaactg agggaaaatg ctgaggatat gggcaatggt
1440tgtttcaaaa tataccacaa atgtgacaat gcctgcatag ggtcaatcag
aaatggaact 1500tatgaccatg atgtatacag agacgaagca ttaaacaacc
ggttccagat caaaggtgtt 1560gagctgaagt caggatacaa agattggatc
ctatggattt cctttgccat atcatgcttt 1620ttgctttgtg ttgttttgct
ggggttcatc atgtgggcct gccaaaaagg caacattagg 1680tgcaacattt gcatttga
169891446DNAInfluenza A virus (A/Panama/2007/1999(H3N2))
9gcaaaagcag gagtgaaaat gaatccaaat caaaagataa taacgattgg ctctgtttct
60ctcactattg ccacaatatg cttccttatg caaatagcca tcctggtaac tactgtaaca
120ttgcatttca agcaatatga atgcaactcc cccccaaaca accaagtaat
gctgtgtgaa 180ccaacaataa tagaaagaaa cataacagag atagtgtatc
tgaccaacac caccatagag 240aaggaaatat gccccaaact agcagaatac
agaaattggt caaagccgca atgtaaaatt 300acaggatttg cacctttttc
taaggataat tcaattcggc tttccgctgg tggggacatt 360tgggtgacaa
gagaacctta tgtgtcatgc gatcctgaca agtgttatca atttgccctt
420ggacagggaa caacactaaa caacaggcat tcaaatgaca cagtacatga
taggacccct 480tatcgaaccc tattgatgaa tgagttgggt gttccatttc
atttgggaac caagcaagtg 540tgtatagcat ggtccagctc aagttgtcac
gatggaaaag catggctgca tgtttgtgta 600actgggcatg atgaaaatgc
aactgctagc ttcatttacg atgggagact tgtagatagt 660attggttcat
ggtccaaaaa aatcctcagg acccaggagt cggaatgcgt ttgtatcaat
720ggaacttgta cagtagtaat gactgatggg agtgcttcag gaagagctga
tactaaaata 780cttttcattg aggaggggaa aatcgttcat actagcaaat
tgtcaggaag tgctcagcat 840gtcgaggagt gctcctgtta tcctcgatat
cctggtgtca gatgtgtctg cagagacaac 900tggaaaggct ccaataggcc
catcgtagat ataaatgtaa aggattatag cattgtttcc 960agttatgtgt
gctcaggact tgttggagac acacccagaa aaaacgacag ctccagcagt
1020agccattgcc tggatcctaa caatgaagaa gggggtcatg gagtgaaagg
ctgggccttt 1080gatgatggaa atgacgtgtg gatgggaaga acgatcagcg
agaagtcacg ctcaggttat 1140gaaaccttca aggtcattga aggctggtcc
aaacctaact ccaaattgca gataaatagg 1200caagtcatag ttgaaagagg
taatatgtcc ggttattctg gtattttctc tgttgaaggc 1260aaaagctgca
tcaatcggtg cttttatgtg gagttgataa ggggaaggaa acaggaaact
1320gaagtctggt ggacctcaaa cagtattgtt gtgttttgtg gcacctcagg
tacatatgga 1380acaggctcat ggcctgatgg ggcggacatc aatctcatgc
ctatataagc tttcgcaatt 1440ttagaa 1446
* * * * *