U.S. patent application number 13/579845 was filed with the patent office on 2017-04-27 for vaccines for use in the prophylaxis and treatment of influenza virus disease.
This patent application is currently assigned to Icahn School of Medicine at Mount Sinai. The applicant listed for this patent is Adolfo Garcia-Sastre, Peter Palese, Gene S. Tan, Taia T. Wang. Invention is credited to Adolfo Garcia-Sastre, Peter Palese, Gene S. Tan, Taia T. Wang.
Application Number | 20170114103 13/579845 |
Document ID | / |
Family ID | 44483593 |
Filed Date | 2017-04-27 |
United States Patent
Application |
20170114103 |
Kind Code |
A9 |
Garcia-Sastre; Adolfo ; et
al. |
April 27, 2017 |
VACCINES FOR USE IN THE PROPHYLAXIS AND TREATMENT OF INFLUENZA
VIRUS DISEASE
Abstract
Provided herein are polypeptides comprising portions of the
influenza virus hemagglutinin, compositions comprising such
polypeptides that can be used as immunogens in vaccines and methods
of their use to generate an immune response against multiple
influenza subtypes in a subject.
Inventors: |
Garcia-Sastre; Adolfo; (New
York, NY) ; Palese; Peter; (New York, NY) ;
Tan; Gene S.; (New York, NY) ; Wang; Taia T.;
(Stanford, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Garcia-Sastre; Adolfo
Palese; Peter
Tan; Gene S.
Wang; Taia T. |
New York
New York
New York
Stanford |
NY
NY
NY
CA |
US
US
US
US |
|
|
Assignee: |
Icahn School of Medicine at Mount
Sinai
New York
NY
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20130209499 A1 |
August 15, 2013 |
|
|
Family ID: |
44483593 |
Appl. No.: |
13/579845 |
Filed: |
February 18, 2011 |
PCT Filed: |
February 18, 2011 |
PCT NO: |
PCT/US11/25467 PCKC 00 |
371 Date: |
December 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12788103 |
May 26, 2010 |
8673314 |
|
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13579845 |
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61305898 |
Feb 18, 2010 |
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61354160 |
Jun 11, 2010 |
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61385083 |
Sep 21, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2760/16122
20130101; C12N 2760/16134 20130101; A61K 2039/53 20130101; A61K
2039/6012 20130101; C07K 14/005 20130101; C07K 14/11 20130101; A61K
2039/6081 20130101; A61K 39/12 20130101; A61K 2039/55566 20130101;
A61P 31/16 20180101; C12N 2760/16111 20130101 |
International
Class: |
C07K 14/11 20060101
C07K014/11 |
Goverment Interests
[0002] This invention was made with United States Government
support under award numbers U01 A1070469-02 and 1RC1 A1086061-01
awarded by the National Institutes of Health (NIH). The United
States Government has certain rights in this invention.
Claims
1. An isolated core polypeptide comprising the formula:
TABLE-US-00030 (a) Formula I: (SEQ ID NO: 3)
X.sub.1-X.sub.2-X.sub.3-X.sub.4-X.sub.5-X.sub.6-X.sub.7-X.sub.8-X.sub.9-X.-
sub.10-D-X.sub.11-X.sub.12-X.sub.13-X.sub.14-X.sub.15-W-X.sub.16-Y-
X.sub.17-A-E-L-L-V- X.sub.18-X.sub.19-E-N-X.sub.20-
X.sub.21-T-X.sub.22-D-X.sub.23-X.sub.24-D-S-X.sub.25-X.sub.26-X.sub.27-X.-
sub.28-L-X.sub.29-X.sub.30-X.sub.31-X.sub.32-X.sub.33-
X.sub.34-Q-L-X.sub.35-X.sub.36-N-X.sub.37,
wherein: X.sub.1 is a hydrophilic amino acid; X.sub.2 is a
hydrophobic amino acid; X.sub.3 is a hydrophilic amino acid;
X.sub.4 is a hydrophilic amino acid; X.sub.5 is a hydrophobic amino
acid; X.sub.6 is N, E or I; X.sub.7 is a hydrophilic amino acid;
X.sub.8 is K, Y or W; X.sub.9 is M, V or T; X.sub.10 is a
hydrophilic residue; X.sub.11 is A, G, T or S; X.sub.12 is F, K, or
M; X.sub.13 is L, I or T; X.sub.14 is a hydrophilic, acidic amino
acid; X.sub.15 is a hydrophobic amino acid; X.sub.16 is a
hydrophilic amino acid; X.sub.18 is a hydrophobic amino acid;
X.sub.19 is a hydrophobic amino acid; X.sub.20 is a hydrophilic
amino acid; X.sub.21 is a hydrophilic, basic amino acid; X.sub.22
is a hydrophobic amino acid; X.sub.23 is a hydrophobic amino acid;
X.sub.24 is H, Tor A; X.sub.25 is a hydrophilic amino acid;
X.sub.26 is a hydrophobic amino acid; X.sub.27 is a hydrophilic
amino acid; X.sub.28 is a hydrophilic amino acid; X.sub.29 is a
hydrophobic amino acid; X.sub.30 is a hydrophilic, acidic amino
acid; X.sub.31 is a hydrophilic, basic amino acid; X.sub.32 is T or
V; X.sub.33 is a hydrophilic, basic amino acid; X.sub.34 is K, L,
M, S or R; X.sub.35 is a hydrophilic, basic amino acid; X.sub.36 is
a hydrophilic amino acid; and X.sub.37 is a hydrophobic amino acid,
and wherein the isolated core polypeptide is not a full length
influenza virus hemagglutinin; TABLE-US-00031 (b) Formula II: (SEQ
ID NO: 4)
RIENLNKKX.sub.1EDGFLDVWTYNAELLVLMENERTLDX.sub.2HDSNVKNLYEKV
RX.sub.3QLRX.sub.4NA,
wherein: X.sub.1 is M, V, T; X.sub.2 is a hydrophobic amino acid;
X.sub.3 is L, M, S, K, R; and X.sub.4 is a hydrophilic amino acid;
TABLE-US-00032 (c) Formula III: (SEQ ID NO: 5)
RX.sub.1ENLNKKX.sub.2X.sub.3DGFLDX.sub.4WTYNAELLVLX.sub.5ENERTLDX.sub.6HDS-
NVKNL YX.sub.7KVRX.sub.8QLX.sub.9X.sub.10NX.sub.11,
wherein: X.sub.1 is a hydrophobic amino acid; X.sub.2 is a
hydrophobic amino acid; X.sub.3 is a hydrophilic amino acid;
X.sub.4 is a hydrophobic amino acid; X.sub.5 is a hydrophobic amino
acid; X.sub.6 is a hydrophobic acidic amino acid; X.sub.7 is a
hydrophilic, acidic amino acid; X.sub.8 is L, M, or S; X.sub.9 is a
hydrophilic, basic amino acid; X.sub.10 is a hydrophilic amino
acid; and X.sub.11 is a hydrophobic amino acid; TABLE-US-00033 (d)
Formula IV: (SEQ ID NO: 6)
X.sub.1IX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9DX.sub.10-
X.sub.11X.sub.12X.sub.13X.sub.14WSYNAELLVAX.sub.15E
NQHTIDLX.sub.16DSEMNKLX.sub.17E X.sub.18 X.sub.19
X.sub.20RQLRENA,
wherein: X.sub.1 is a hydrophilic amino acid; X.sub.2 is a
hydrophilic amino acid; X.sub.3 is a hydrophilic amino acid;
X.sub.4 is a hydrophobic amino acid; X.sub.5 is E or I; X.sub.6 is
a hydrophilic amino acid; X.sub.7 is a hydrophobic amino acid;
X.sub.8 is V or T; X.sub.9 is a hydrophilic amino acid; X.sub.10 is
a hydrophilic amino acid; X.sub.11 is K or M; X.sub.12 is I or T;
X.sub.13 is a hydrophilic, acidic amino acid; X.sub.14 is a
hydrophobic amino acid; X.sub.15 is a hydrophobic amino acid;
X.sub.16 is T or A; X.sub.17 is a hydrophobic amino acid; X.sub.18
is a hydrophilic, basic amino acid; X.sub.19 is T or V; and
X.sub.20 is a hydrophilic, basic amino acid; or TABLE-US-00034 (e)
Formula V: (SEQ ID NO: 7)
RX.sub.1ENLNKKVDDGFX.sub.2DIWTYNAELLVLLENERTLDX.sub.3HDSNVX.sub.4NLYE
KVX.sub.5SQLKNNA,
wherein: X.sub.1 is a hydrophobic amino acid; X.sub.2 is a
hydrophobic amino acid; X.sub.3 is a hydrophobic amino acid;
X.sub.4 is a hydrophilic, basic amino acid; and X.sub.5 is a
hydrophilic, basic amino acid.
2. The isolated core polypeptide of claim 1, wherein: (a) the
polypeptide has the Formula I and wherein: X.sub.1 is R or Q;
X.sub.2 is L, M or I; X.sub.3 is E, D, Q or G; X.sub.4 is D or N;
X.sub.5 is L, M or V; X.sub.6 is N, E or I; X.sub.7 is K or N;
X.sub.8 is K, R, Y or W; X.sub.9 is M, V or T; X.sub.10 is E, D, K
or R; X.sub.11 is A, G, T or S; X.sub.12 is F, K, I, L or M;
X.sub.13 is L, I or T; X.sub.14 is D or E; X.sub.15 is V, I or L;
X.sub.16 is S or T; X.sub.17 is Q or N; X.sub.18 is A or L;
X.sub.19 is L or M; X.sub.20 is E or Q; X.sub.21 is R or H;
X.sub.22 is L or I; X.sub.23 is F, V, M, Y or L; X.sub.24 is H, Tor
A; X.sub.25 is N or E; X.sub.26 is V or M; X.sub.27 is K, N, S or R
X.sub.28 is K, S, or N; X.sub.29 is Y or F; X.sub.30 is D or E;
X.sub.31 is K or R; X.sub.32 is T or V; X.sub.33 is K or R;
X.sub.34 is K, L, M, S or R; X.sub.35 is K or R; X.sub.36 is D, N,
Q or E; and X.sub.37 is A or V and wherein the isolated core
polypeptide is not a full length influenza virus hemagglutinin; (b)
the polypeptide has the Formula II and wherein: X.sub.1 is M, V, T;
X.sub.2 is F, Y or L; X.sub.3 is L, M, S, K, R; and X.sub.4 is D, N
or E; (c) the polypeptide has the Formula III and wherein: X.sub.1
is L or I; X.sub.2 M or V; X.sub.3 is E or D; X.sub.4 V or I;
X.sub.5 is M or L; X.sub.6 is F or Y; X.sub.7 is D or E; X.sub.8 L,
M or S; X.sub.9 R or K; X.sub.10 is D or N and X.sub.11 is A or V;
(d) the polypeptide has the Formula IV and wherein: X.sub.1 is R or
Q; X.sub.2 is Q or G; X.sub.3 is D or N; X.sub.4 is L or V; X.sub.5
is E or X.sub.6 is K or N; Y or W; X.sub.8 V or T; X.sub.9 is E or
R; X.sub.10 is T or S; X.sub.11 is K or M; X.sub.12 is I or T;
X.sub.13 is D or E; X.sub.14 is L or V; X.sub.15 is L or M;
X.sub.16 is T or A; X.sub.17 F or Y; X.sub.18 is K or R; X.sub.19
is T or V and X.sub.20 is K or R; or (e) the polypeptide has the
Formula IV and wherein: X.sub.1 is M or I; X.sub.2 is L or I;
X.sub.1 is F or Y; X.sub.4 is K or R; and X.sub.5 is K or R.
3.-11. (canceled)
12. The isolated core polypeptide of claim 1 having the amino acid
sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 9,
SEQ ID NO: 10 or SEQ ID NO: 11.
13. An isolated core polypeptide comprising the formula:
TABLE-US-00035 (a) Formula I: (SEQ ID NO: 12)
RLENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYXKVRM QLRDNV,
wherein X is a hydrophilic, acidic amino acid; TABLE-US-00036 (b)
Formula II: (SEQ ID NO: 15)
RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEX.sub.1T
X.sub.2X.sub.3QLRENA,
wherein X.sub.1, X.sub.2, and X.sub.3 are hydrophilic, basic amino
acids; TABLE-US-00037 (c) Formula III: (SEQ ID NO: 19)
RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDVTDSEMNKLFERVR X.sub.1QLRENA,
wherein X.sub.1 is a hydrophilic, basic amino acid; TABLE-US-00038
(d) Formula IV: (SEQ ID NO: 22)
RIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRL QLRDNA; (e)
Formula V: (SEQ ID NO: 23)
RIX.sub.1NX.sub.2NKRMEDGFLDVWTYNAELLVLLENX.sub.3RTLDX.sub.4HDANVKX.sub.5L
X.sub.6EKVKSX.sub.7LX.sub.8DNA,
wherein X.sub.1 is G or D; X.sub.2 is a hydrophobic amino acid;
X.sub.3 is a hydrophilic amino acid; X.sub.4 is a hydrophobic amino
acid; X.sub.5 is a hydrophilic amino acid; X.sub.6 is H or Y;
X.sub.7 is Q or L; and X.sub.8 is a hydrophilic, basic amino acid;
TABLE-US-00039 (f) Formula VI: (SEQ ID NO: 30)
QIGNVINWTRDX.sub.1MTEX.sub.2WSYNAELLVAMENQHTIDLADSEMX.sub.3KLYER
VX.sub.4KQLRENA,
wherein X.sub.1 is a hydrophobic amino acid or a hydrophilic amino
acid; X.sub.2 is a hydrophobic amino acid; X.sub.3 is a hydrophilic
amino acid; and X.sub.4 is a hydrophilic, basic amino acid;
TABLE-US-00040 (g) Formula VII: (SEQ ID NO: 36)
RINMINDKIDDQIEX.sub.1LWAYNAELLVLLENQKTLDEHDSNVKNLFDEVK RRLSANA,
wherein X.sub.1 is a hydrophilic amino acid; TABLE-US-00041 (h)
Formula VIII: (SEQ ID NO: 39)
RLNMINNKIDDQIQDX.sub.1WAYNAELLVLLENQKTLDEHDANVNNLYNKVK RALGSNA,
wherein X.sub.1 is a hydrophobic amino acid; TABLE-US-00042 (i)
Formula IX: (SEQ ID NO: 42)
QIGNVINWTKDSITDIWTYX.sub.1AELLVAMENQHTIDMADSEMLNLYERVR KQLRQNA,
wherein X.sub.1 is a hydrophilic amino acid; TABLE-US-00043 (j)
Formula X: (SEQ ID NO: 45)
RINQLSKHVDDSVX.sub.1DIWSYNAQLLVLLENEKTLDLHDSNVRNLHEKVR RMLKDNA,
wherein X.sub.1 is a hydrophobic amino acid; TABLE-US-00044 (k)
Formula XI: (SEQ ID NO: 48)
RINMINSKIDDQITDIWAYNAELLVLLENQKTLDEHDANVRNLHDRVRR
X.sub.1LX.sub.2ENA,
wherein X.sub.1 is a hydrophobic amino acid and X.sub.2 is a
hydrophilic, basic amino acid; TABLE-US-00045 (l) Formula XII: (SEQ
ID NO: 51)
RINMLADRIDDAVTDX.sub.1WSYNAKLLVLLENDKTLDMHDANVRNLHX.sub.2QV
RRX.sub.3LKX.sub.4NA,
wherein X.sub.1 is a hydrophobic amino acid; X.sub.2 is a
hydrophilic, acidic amino acid; X.sub.3 is A, S or E; and X.sub.4
is a hydrophilic amino acid; TABLE-US-00046 (m) Formula XIII: (SEQ
ID NO: 55) RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDVTDSEMNKLFERVRR
QLRENA; (n) Formula XIV: (SEQ ID NO: 56)
QIGNVINWTRDSLTEIWSYNAELLVAMENQHTIDLADSEMNKLYERVRR QLRENA; or (o)
Formula XV: (SEQ ID NO: 57)
RINMLADRVDDAVTDIWSYNAKLLVLX.sub.1ENDRTLDLHDANVX.sub.2NLH
X.sub.3QVKRALKX.sub.4NA,
wherein: X.sub.1 is a hydrophobic amino acid; X.sub.2 is a
hydrophilic, basic amino acid; X.sub.3 is a hydrophilic, acidic
amino acid; and X.sub.4 is a hydrophilic amino acid.
14. The isolated core polypeptide of claim 13, wherein: (a) the
polypeptide has the Formula I and wherein X is D or E; (b) the
polypeptide has the Formula II and wherein X.sub.1 is K or R;
X.sub.2 is K or R, and X.sub.3 is K or R; (c) the polypeptide has
the Formula III and wherein X.sub.1 is R or H; (d) the polypeptide
has the Formula V and wherein X.sub.1 is G or D; X.sub.2 is L or M;
X.sub.3 is E or G; X.sub.4 is L or M; X.sub.5 N or S; X.sub.6 is H
or Y; X.sub.7 is Q or L and X.sub.8 is R or K; (e) the polypeptide
has the Formula VI and wherein X.sub.1 is A or S; X.sub.2 is V or
I; X.sub.3 is N or S; and X.sub.4 is K or R; (f) the polypeptide
has the Formula VII and wherein X.sub.1 is D or N; (g) the
polypeptide has the Formula VIII and wherein X.sub.1 is V or I; (h)
the polypeptide has the Formula IX and wherein X.sub.1 is Q or N;
(i) the polypeptide has the Formula X and wherein X.sub.1 is V or
I; (j) the polypeptide has the Formula XI and wherein X.sub.1 is V
or I and X.sub.2 is R or K; (k) the polypeptide has the Formula XII
and wherein X.sub.1 is V or I; X.sub.2 is D or E; X.sub.3 is A S or
E and X.sub.4 is T or D; or (l) the polypeptide has the Formula XV
and wherein X.sub.1 is L or I; X.sub.2 is K or R; X.sub.3 is D or E
and X.sub.4 is S or N.
15. The isolated core polypeptide of claim 13 having the amino acid
sequence of SEQ ID NO: 13 SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:
17, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 25, SEQ
ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO:
30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ
ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO:
41, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 47, SEQ
ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:
54, SEQ ID NO: 58, SEQ ID NO: 59, or SEQ ID NO: 60.
16.-51. (canceled)
52. A flu polypeptide comprising the formula: X-L-X-L-X, wherein X
is any one of the core polypeptides of claim 1 or 13 or a fragment
thereof; and L is a linker or is absent.
53. The flu polypeptide of claim 52, wherein the core polypeptides
are the same.
54. A flu polypeptide comprising the core polypeptides of claim 1
or 13, wherein (a) the flu polypeptide is acetylated at its
N-terminus; (b) the flu polypeptide is linked to polyethylene
glycol at its N- and/or C-terminus; (c) the core polypeptide is
directly or indirectly linked at its N- and/or C-terminus to one or
more protein tags; (d) the core peptide is directly or indirectly
linked at its N- and/or C-terminus to a T cell epitope; (e) the
core polypeptide is directly or indirectly linked at its N- and/or
C-terminus to a Toll Like Receptor ligand; or (f) the isolated core
polypeptide is directly or indirectly linked at its N- and/or
C-terminus to a T4 foldon domain or a fragment thereof.
55.-64. (canceled)
65. A flu polypeptide comprising a core polypeptide linked at its
N-terminus to a His-tag and linked to its C-terminus to a T4 foldon
domain or a fragment thereof.
66. The flu polypeptide of claim 52, wherein (a) the flu
polypeptide is directly or indirectly linked at its N- or
C-terminus to one or more protein tags; (b) the flu polypeptide is
directly or indirectly linked at its N- or C-terminus to a CD8
T-cell epitope; or (c) the flu polypeptide is directly or
indirectly linked at its N- or C-terminus to a Toll Like Receptor
Ligand; (d) the flu polypeptide is directly or indirectly linked at
its N-terminus to a His-tag, linked at its C-terminus to a
FLAG-tag, and wherein L is glycine residues; (e) the flu
polypeptide is directly or indirectly linked at its N-terminus to a
His-tag and linked at its C-terminus to a Samonella flagellin; or
(f) the flu polypeptide is directly or indirectly linked at its
N-terminus to a Hist-tag and linked at its C-terminus to a
influenza A nuclear protein (NP) or fragment thereof.
67.-75. (canceled)
76. The isolated core polypeptide of any one of claim 1 or 13 that
selectively binds neutralizing antiserum capable of binding an
influenza hemagglutinin.
77. A composition comprising the of claim 1 or 13 linked to a
carrier protein.
78.-79. (canceled)
80. A flu polypeptide comprising a core polypeptide having the
amino acid sequence RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKT
RRQLRENA, wherein the core polypeptide is linked at its C-terminus
to a polypeptide having the sequence DYKDDDDKC.
81. (canceled)
82. An isolated nucleic acid encoding the polypeptide of any one of
claims 1, 13, or 80.
83. An isolated cell expressing the nucleic acid of claim 82.
84. An isolated virus comprising a genome engineered to express the
nucleic acid of claim 82.
85.-90. (canceled)
91. A viral-like particle comprising the polypeptide of any one of
claims 1, 13 or 80.
92. An immunogenic composition comprising the polypeptide of any
one of claims 1, 13, or 80.
93.-100. (canceled)
101. A method of immunizing a subject or preventing an influenza
virus disease or treating an influenza virus infection or an
influenza virus disease in a subject comprising administering to
the subject an effective amount of the immunogenic composition of
claim 92.
102.-107. (canceled)
108. A method of immunizing a subject or preventing influenza
disease or treating an influenza virus infection or an influenza
virus disease in a subject comprising administering to the subject
an effective amount of the virus of claim 84.
109.-111. (canceled)
Description
[0001] This application claims priority benefit of U.S. provisional
application No. 61/305,898, filed Feb. 18, 2010, U.S.
nonprovisional application Ser. No. 12/788,103, filed May 26, 2010,
U.S. provisional application No. 61/354,160, filed Jun. 11, 2010
and U.S. provisional application No. 61/385,083, filed Sep. 21,
2010, each of which is incorporated herein by reference in its
entirety.
1. INTRODUCTION
[0003] Provided herein are polypeptides comprising portions of the
influenza virus hemagglutinin, compositions comprising such
polypeptides that can be used as immunogens in vaccines and methods
of their use to generate an immune response against multiple
influenza subtypes in a subject.
2. BACKGROUND
[0004] Influenza viruses are enveloped RNA viruses that belong to
the family of Orthomyxoviridae (Palese and Shaw (2007)
Orthomyxoviridae: The Viruses and Their Replication, 5th ed.
Fields' Virology, edited by B. N. Fields, D. M. Knipe and P. M.
Howley. Wolters Kluwer Health/Lippincott Williams & Wilkins,
Philadelphia, USA, p1647-1689). The natural host of influenza
viruses are avians, but influenza viruses (including those of avian
origin) also can infect and cause illness in humans and other
animal hosts (canines, pigs, horses, sea mammals, and mustelids).
For example, the H5N1 avian influenza virus circulating in Asia has
been found in pigs in China and Indonesia and has also expanded its
host range to include cats, leopards, and tigers, which generally
have not been considered susceptible to influenza A (CIDRAP--Avian
Influenza: Agricultural and Wildlife Considerations). The
occurrence of influenza virus infections in animals could
potentially give rise to human pandemic influenza strains.
[0005] Influenza A and B viruses are major human pathogens, causing
a respiratory disease that ranges in severity from sub-clinical
infection to primary viral pneumonia which can result in death. The
clinical effects of infection vary with the virulence of the
influenza strain and the exposure, history, age, and immune status
of the host. The cumulative morbidity and mortality caused by
seasonal influenza is substantial due to the relatively high rate
of infection. In a normal season, influenza can cause between 3-5
million cases of severe illness and is associated with 200,000 to
500,000 deaths worldwide (World Health Organization (April, 2009)
Influenza (Seasonal) Fact Sheet 211). In the United States,
influenza viruses infect an estimated 10-15% of the population
(Glezen and Couch RB (1978) Interpandemic influenza in the Houston
area, 1974-76. N Engl J Med 298: 587-592; Fox et al. (1982)
influenza virus infections in Seattle families, 1975-1979. II.
Pattern of infection in invaded households and relation of age and
prior antibody to occurrence of infection and related illness. Am J
Epidemiol 116: 228-242) and are associated with approximately
30,000 deaths each year (Thompson W W et al. (2003) Mortality
Associated with Influenza and Respiratory Syncytial Virus in the
United States. JAMA 289: 179-186; Belshe (2007) Translational
research on vaccines: influenza as an example. Clin Pharmacol Ther
82: 745-749).
[0006] In addition to annual epidemics, influenza viruses are the
cause of infrequent pandemics. For example, influenza A viruses can
cause pandemics such as those that occurred in 1918, 1957 and 1968.
Due to the lack of pre-formed immunity against the major viral
antigen, hemagglutinin (HA), pandemic influenza viruses can affect
greater than 50% of the population in a single year and often cause
more severe disease than seasonal influenza viruses. A stark
example is the pandemic of 1918, in which an estimated 50-100
million people were killed (Johnson and Mueller (2002) Updating the
Accounts: Global Mortality of the 1918-1920 "Spanish" Influenza
Pandemic Bulletin of the History of Medicine 76: 105-115). Since
the emergence of the highly pathogenic avian H5N1 influenza virus
in the late 1990s (Claas et al. (1998) Human Influenza A H5N 1
virus related to a highly pathogenic avian influenza virus. Lancet
351: 472-7), there have been concerns that the virus may become
transmissible between humans and cause a major pandemic.
[0007] An effective way to protect against influenza virus
infection is through vaccination; however, current vaccination
approaches rely on achieving a good match between circulating
strains and the isolates included in the vaccine formulation. Such
a match is often difficult to attain due to a combination of
factors. First, influenza viruses are constantly undergoing change:
every 3-5 years the predominant strain of influenza A virus is
replaced by a variant that has undergone sufficient antigenic drift
to evade existing antibody responses. Isolates to be included in
vaccine preparations must therefore be selected each year based on
the intensive surveillance efforts of the World Health Organization
(WHO) collaborating centers. Second, to allow sufficient time for
vaccine manufacture and distribution, strains must be selected
approximately six months prior to the initiation of the influenza
season. Occasionally, the predictions of the vaccine strain
selection committee are inaccurate, resulting in a substantial drop
in the efficacy of vaccination.
[0008] The possibility of a novel subtype of influenza A virus
entering the human population also presents a significant challenge
to current vaccination strategies. Since it is impossible to
predict what subtype and strain of Influenza virus will cause the
next pandemic, current, strain-specific approaches cannot be used
to prepare a pandemic influenza vaccine.
3. SUMMARY
[0009] Polypeptide compositions ("flu polypeptides") are described
that can be used in a subject (animal subjects, including human
subjects) to generate an immune response that is cross-reactive
with a plurality of influenza virus strains of a particular subtype
or strains from different subtypes. In particular, the flu
polypeptides comprise "core polypeptides" that correspond in amino
acid sequence and/or structure to a region of the long alpha helix
of the HA2 subunit of influenza hemagglutinin described herein, or
modified core polypeptides.
[0010] The invention is based, in part, on the design of flu
polypeptides that mimic the structure and function/activity of the
long alpha helix region of the HA2 subunit of influenza
hemagglutinin. Surprisingly, immunization with a flu polypeptide
corresponding to the HA2 long alpha helix of a particular influenza
subtype induces serum antibodies that cross-react with
hemagglutinin from multiple influenza subtypes. The data described
herein also demonstrate that animals immunized with a flu
polypeptide corresponding to the HA2 long alpha helix of one
particular subtype are protected against lethal influenza viral
challenges with different influenza virus subtypes. Accordingly,
the flu polypeptides provided herein may be used in immunogenic
compositions (e.g., vaccines) capable of generating immune
responses against a plurality of different influenza strains and
subtypes--in other words, a "universal" flu vaccine.
[0011] While not intending to be bound by any particular theory of
operation, it may be that despite the variability of HA in the
different influenza subtypes, the long alpha helix region of the
HA2 subunit of influenza hemagglutinin contains a conserved
epitope(s)/region recognized by rare, cross reactive antibodies
(e.g., such as monoclonal antibody 12D1 which has broad
neutralizing activity against H3 influenza viruses). Flu
polypeptides presented to the immune system in a construct designed
to expose this epitope/region in the proper conformation and confer
enhanced immunogenicity to the cross-reactive or "universal"
epitope/region and which can be used to generate a serum antibody
response in a subject, and preferably a neutralizing response,
against multiple influenza subtypes.
[0012] In other aspects, described herein are nucleic acids
encoding a flu polypeptide(s), viruses and immunogenic compositions
comprising a flu polypeptide(s) and methods of immunization.
[0013] 3.1 Terminology
[0014] The terms "about" or "approximate," when used in reference
to an amino acid position refer to the particular amino acid
position in a sequence or any amino acid that is within five, four,
three, two or one residues of that amino acid position, either in
an N-terminal direction or a C-terminal direction.
[0015] As used herein, the term "about" or "approximately" when
used in conjunction with a number refers to any number within 1, 5
or 10% of the referenced number.
[0016] The term "amino acid" or any reference to a specific amino
acid is meant to include naturally occurring proteogenic amino
acids as well as non-naturally occurring amino acids such as amino
acid analogs. Those skilled in the art would know that this
definition includes, unless otherwise specifically noted, naturally
occurring proteogenic (L)-amino acids, their optical (D)-isomers,
chemically modified amino acids, including amino acid analogs such
as penicillamine (3-mercapto-D-valine), naturally occurring
non-proteogenic amino acids such as norleucine and chemically
synthesized amino acids that have properties known in the art to be
characteristic of an amino acid. Additionally, the term "amino acid
equivalent" refers to compounds that depart from the structure of
the naturally occurring amino acids, but which have substantially
the structure of an amino acid, such that they can be substituted
within a peptide, which retains its biological activity despite the
substitution. Thus, for example, amino acid equivalents can include
amino acids having side chain modifications or substitutions, and
also include related organic acids, amides or the like. The term
"amino acid" is intended to include amino acid equivalents.
[0017] The term "amino acid sequence identity" refers to the degree
of identity or similarity between a pair of aligned amino acid
sequences, usually expressed as a percentage. As used herein, the
terms "percent identity," "percent identical," "% identity," and "%
identical" with respect to amino acid sequence refer to the
percentage of amino acid residues in a candidate sequence that are
identical (i.e., the amino acid residues at a given position in the
alignment are the same residue) to the corresponding amino acid
residue in the peptide after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
homology. As used herein, the terms "percent similarity," "percent
similar," "% similarity," and "% similar" with respect to amino
acid sequence refer to the percentage of amino acid residues in a
candidate sequence that are similar (i.e., the amino acid
substitution at a given position in the alignment is a conservative
substitution, as discussed below), to the corresponding amino acid
residue in the peptide after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
homology. Sequence homology, including percentages of sequence
identity and similarity, are determined using sequence alignment
techniques well-known in the art, preferably computer algorithms
designed for this purpose, using the default parameters of said
computer algorithms or the software packages containing them.
Non-limiting examples of computer algorithms and software packages
incorporating such algorithms include the following. The BLAST
family of programs exemplify a particular, non-limiting example of
a mathematical algorithm utilized for the comparison of two
sequences (e.g., Karlin & Altschul, 1990, Proc. Natl. Acad.
Sci. USA 87:2264-2268 (modified as in Karlin & Altschul, 1993,
Proc. Natl. Acad. Sci. USA 90:5873-5877), Altschul et al., 1990, J.
Mol. Biol. 215:403-410, (describing NBLAST and XBLAST), Altschul et
al., 1997, Nucleic Acids Res. 25:3389-3402 (describing Gapped
BLAST, and PSI-Blast). Another particular example is the algorithm
of Myers and Miller (1988 CABIOS 4:11-17) which is incorporated
into the ALIGN program (version 2.0) and is available as part of
the GCG sequence alignment software package. Also, another
particular example is the FASTA program (Pearson W. R. and Lipman
D. J., Proc. Nat. Acad. Sci. USA, 85:2444-2448, 1988), available as
part of the Wisconsin Sequence Analysis Package. Additional
examples include BESTFIT, which uses the "local homology" algorithm
of Smith and Waterman (Advances in Applied Mathematics, 2:482-489,
1981) to find the best single region of similarity between two
sequences, and which is preferable where the two sequences being
compared are dissimilar in length; and GAP, which aligns two
sequences by finding a "maximum similarity" according to the
algorithm of Neddleman and Wunsch (J. Mol. Biol. 48:443-354, 1970),
and is preferable where the two sequences are approximately the
same length and an alignment is expected over the entire
length.
[0018] "Conservative substitution" refers to replacement of an
amino acid of one class is with another amino acid of the same
class. In particular embodiments, a conservative substitution does
not alter the structure or function, or both, of a polypeptide.
Classes of amino acids for the purposes of conservative
substitution include hydrophobic (Met, Ala, Val, Leu, Ile), neutral
hydrophilic (Cys, Ser, Thr), acidic (Asp, Glu), basic (Asn, Gin,
His, Lys, Arg), conformation disrupters (Gly, Pro) and aromatic
(Trp, Tyr, Phe).
[0019] As used herein, the term "core polypeptide" refers to a
polypeptide segment that corresponds to a region of an influenza
hemagglutinin HA2 polypeptide, i.e., core polypeptides as referred
to herein do not comprise an entire influenza hemagglutinin HA2
polypeptide. In a specific embodiment, the term refers to a
polypeptide segment that corresponds to a region of the long alpha
helix region of an influenza hemagglutinin HA2 polypeptide. See
Section 5.1.1 for examples of core polypeptides.
[0020] As used here, the term "fragment" refers to a portion of a
particular polypeptide. In certain embodiments, a fragment of a
polypeptide (e.g., a core polypeptide) is at least 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 40, 35, 40, 45 or 50 amino
acids in length. In some embodiments, a fragment of a polypeptide
(e.g., a core polypeptide) is between 8 to 15, 8 to 20, 8 to 25, 8
to 30, 8 to 40, 10 to 15, 10 to 20, 10 to 25, 10 to 30, 10 to 40,
10 to 45, 10 to 50, 15 to 20, 15 to 25, 15 to 30, 15 to 35, 15 to
40, 15 to 45, 15 to 50, 25 to 30, 25 to 40, 25 to 45 or 25 to 50
amino acids in length.
[0021] As used herein, the term "modified core polypeptide" refers
to a core polypeptide that has been modified in some manner to
extend or increase the half-life of the core polypeptide in vivo.
Techniques of modifying a polypeptide to extend or increase the
half-life of the core polypeptide are known to those of skill in
the art. In some embodiments, the core polypeptide may be modified
by substitution of terminal L-amino acids with D-amino acids, by
pegylation of the polypeptide, by amidation of the C-terminus of
the polypeptide, or by acetylation of the N-terminus of the
polypeptide. See Section 5.1.2 for examples of modified core
polypeptides.
[0022] As used herein, the term "flu polypeptide" refers to a
polypeptide comprising a core polypeptide or a modified core
polypeptide. In some embodiments, the flu polypeptide consists of a
core polypeptide. In certain embodiments, the flu polypeptide
consists of a modified core polypeptide. In certain embodiments,
the flu polypeptide comprises a peglylated core polypeptide. In
certain embodiments, the flu polypeptide is pegylated at its N-
and/or C-terminus. In certain embodiments, the flu polypeptide
comprises a core polypeptide acetylated at its N- and/or
C-terminus. In certain embodiments, the flu polypeptide is
acetylated at its N- and/or C-terminus. In certain embodiments, the
flu polypeptide comprises a core polypeptide or modified core
polypeptide and a linker. In certain embodiments, the flu
polypeptide comprises a core polypeptide or modified core
polypeptide linked to a carrier.
[0023] In certain embodiments, the flu polypeptide comprises one,
two, three or more core polypeptides and/or modified core
polypeptides and one, two, three or more or all of the following:
1) one, two, or more T cell epitopes (e.g., CD8 T cell epitope); 2)
one, two, or more immunogenic polypeptides; 3) a polypeptide that
facilitates multimerization of the flu polypeptide; 4) one, two, or
more protein tags that facilitate purification and/or solubility of
the flu polypeptide; 5) one, two or more carriers; and 6) one, two
or more linkers.
[0024] As used herein, the term "effective amount" in the context
of administering a therapy to a subject refers to the amount of a
therapy which has a prophylactic and/or therapeutic effect(s). In
certain embodiments, an "effective amount" in the context of
administration of a therapy to a subject refers to the amount of a
therapy which is sufficient to achieve one, two, three, four, or
more of the following effects: (i) reduce or ameliorate the
severity of an influenza virus infection, disease or symptom
associated therewith; ii) reduce the duration of an influenza virus
infection, disease or symptom associated therewith; (iii) prevent
the progression of an influenza virus infection, disease or symptom
associated therewith; (iv) cause regression of an influenza virus
infection, disease or symptom associated therewith; (v) prevent the
development or onset of an influenza virus infection, disease or
symptom associated therewith; (vi) prevent the recurrence of an
influenza virus infection, disease or symptom associated therewith;
(vii) reduce or prevent the spread of an influenza virus from one
cell to another cell, one tissue to another tissue, or one organ to
another organ; (ix) prevent or reduce the spread of an influenza
virus from one subject to another subject; (x) reduce organ failure
associated with an influenza virus infection; (xi) reduce
hospitalization of a subject; (xii) reduce hospitalization length;
(xiii) increase the survival of a subject with an influenza virus
infection or disease associated therewith; (xiv) eliminate an
influenza virus infection or disease associated therewith; (xv)
inhibit or reduce influenza virus replication; (xvi) inhibit or
reduce the entry of an influenza virus into a host cell(s); (xviii)
inhibit or reduce replication of the influenza virus genome; (xix)
inhibit or reduce synthesis of influenza virus proteins; (xx)
inhibit or reduce assembly of influenza virus particles; (xxi)
inhibit or reduce release of influenza virus particles from a host
cell(s); (xxii) reduce influenza virus titer; and/or (xxiii)
enhance or improve the prophylactic or therapeutic effect(s) of
another therapy.
[0025] In certain embodiments, the effective amount does not result
in complete protection from an influenza virus disease, but results
in a lower titer or reduced number of influenza viruses compared to
an untreated subject. In certain embodiments, the effective amount
results in a 0.5 fold, 1 fold, 2 fold, 4 fold, 6 fold, 8 fold, 10
fold, 15 fold, 20 fold, 25 fold, 50 fold, 75 fold, 100 fold, 125
fold, 150 fold, 175 fold, 200 fold, 300 fold, 400 fold, 500 fold,
750 fold, or 1,000 fold or greater reduction in titer of influenza
virus relative to an untreated subject. In some embodiments, the
effective amount results in a reduction in titer of influenza virus
relative to an untreated subject of approximately 1 log or more,
approximately 2 logs or more, approximately 3 logs or more,
approximately 4 logs or more, approximately 5 logs or more,
approximately 6 logs or more, approximately 7 logs or more,
approximately 8 logs or more, approximately 9 logs or more,
approximately 10 logs or more, 1 to 3 logs, 1 to 5 logs, 1 to 8
logs, 1 to 9 logs, 2 to 10 logs, 2 to 5 logs, 2 to 7 logs, 2 logs
to 8 logs, 2 to 9 logs, 2 to 10 logs 3 to 5 logs, 3 to 7 logs, 3 to
8 logs, 3 to 9 logs, 4 to 6 logs, 4 to 8 logs, 4 to 9 logs, 5 to 6
logs, 5 to 7 logs, 5 to 8 logs, 5 to 9 logs, 6 to 7 logs, 6 to 8
logs, 6 to 9 logs, 7 to 8 logs, 7 to 9 logs, or 8 to 9 logs.
Benefits of a reduction in the titer, number or total burden of
influenza virus include, but are not limited to, less severe
symptoms of the infection, fewer symptoms of the infection and a
reduction in the length of the disease associated with the
infection.
[0026] As used herein, "Hemagglutinin" and "HA" refer to any
hemagglutinin known to those of skill in the art. In certain
embodiments, the hemagglutinin is influenza hemagglutinin, such as
an influenza A hemagglutinin, an influenza B hemagglutinin or an
influenza C hemagglutinin. There are currently 16 hemagglutinin
subtypes of influenza viruses that fall into two different groups:
Group 1 and Group 2. A typical hemagglutinin comprises domains
known to those of skill in the art including a signal peptide
(optional herein), a stem domain, a globular head domain, a luminal
domain (optional herein), a transmembrane domain (optional herein)
and a cytoplasmic domain (optional herein). In certain embodiments,
a hemagglutinin consists of a single polypeptide chain, such as
HA0. In certain embodiments, a hemagglutinin consists of more than
one polypeptide chain in quaternary association, e.g., HA1 and HA2.
Those of skill in the art will recognize that an immature HA0 might
be cleaved to release a signal peptide (approximately 20 amino
acids) yielding a mature hemagglutinin HA0. A hemagglutinin HA0
might be cleaved at another site to yield HA1 polypeptide
(approximately 320 amino acids, including the globular head domain
and a portion of the stem domain) and HA2 polypeptide
(approximately 220 amino acids, including the remainder of the stem
domain, a luminal domain, a transmembrane domain and a cytoplasmic
domain). In certain embodiments, a hemagglutinin comprises a signal
peptide, a transmembrane domain and a cytoplasmic domain. In
certain embodiments, a hemagglutinin lacks a signal peptide, i.e.
the hemagglutinin is a mature hemagglutinin. In certain
embodiments, a hemagglutinin lacks a transmembrane domain or
cytoplasmic domain, or both. As used herein, the terms
"hemagglutinin" and "HA" encompass hemagglutinin polypeptides that
are modified by post-translational processing such as signal
peptide cleavage, disulfide bond formation, glycosylation (e.g.,
N-linked glycosylation), protease cleavage and lipid modification
(e.g., S-palmitoylation).
[0027] As used herein, "HA2" refers to a polypeptide domain that
corresponds to the HA2 domain of an influenza hemagglutinin
polypeptide known to those of skill in the art. In certain
embodiments, an HA2 consists of a stem domain, a luminal domain, a
transmembrane domain and a cytoplasmic domain (see, e.g., Scheiffle
et al., 2007, EMBO J. 16(18):5501-5508, the contents of which are
incorporated by reference in their entirety). In certain
embodiments, an HA2 consists of a stem domain, a luminal domain and
a transmembrane domain. In certain embodiments, an HA2 consists of
a stem domain and a luminal domain; in such embodiments, the HA2
might be soluble. In certain embodiments, an HA2 consists of a stem
domain; in such embodiments, the HA2 might be soluble.
[0028] As used herein, the term "heterologous" in the context of a
polypeptide, nucleic acid or virus refers to a polypeptide, nucleic
acid or virus, respectively, that is not normally found in nature
or not normally associated in nature with a polypeptide, nucleic
acid or virus of interest. For example, a "heterologous
polypeptide" may refer to a polypeptide derived from a different
virus, e.g., a different influenza strain or subtype, or an
unrelated virus or different species.
[0029] As used herein, the term "in combination," in the context of
the administration of two or more therapies to a subject, refers to
the use of more than one therapy (e.g., more than one prophylactic
agent and/or therapeutic agent). The use of the term "in
combination" does not restrict the order in which therapies are
administered to a subject. For example, a first therapy (e.g., a
first prophylactic or therapeutic agent) can be administered prior
to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2
hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72
hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6
weeks, 8 weeks, or 12 weeks before), concomitantly with, or
subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes,
1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48
hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5
weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a
second therapy to a subject.
[0030] As used herein, the term "infection" means the invasion by,
multiplication and/or presence of a virus in a cell or a subject.
In one embodiment, an infection is an "active" infection, i.e., one
in which the virus is replicating in a cell or a subject. Such an
infection is characterized by the spread of the virus to other
cells, tissues, and/or organs, from the cells, tissues, and/or
organs initially infected by the virus. An infection may also be a
latent infection, i.e., one in which the virus is not replicating.
In certain embodiments, an infection refers to the pathological
state resulting from the presence of the virus in a cell or a
subject, or by the invasion of a cell or subject by the virus.
[0031] As used herein, the term "influenza virus disease" refers to
the pathological state resulting from the presence of an influenza
(e.g., influenza A or B virus) virus in a cell or subject or the
invasion of a cell or subject by an influenza virus. In specific
embodiments, the term refers to a respiratory illness caused by an
influenza virus.
[0032] As used herein, the phrases "IFN deficient system" or
"IFN-deficient substrate" refer to systems, e.g., cells, cell lines
and animals, such as pigs, mice, chickens, turkeys, rabbits, rats,
etc., which do not produce one or more types of interferon
(IFN)(e.g., IFN-.gamma.) or produce low levels of IFN (i.e., a
reduction in IFN expression of 5-10%, 10-20%, 20-30%, 30-40%,
40-50%, 50-60%, 60-70%, 70-80%, 80-90% or more when compared to
IFN-competent systems under the same conditions), do not respond or
respond less efficiently to one or more types of IFN, and/or are
deficient in the activity of one or more antiviral genes induced by
one or more types of IFN.
[0033] As used herein, the numeric term "log" refers to
log.sub.10.
[0034] As used herein, the phrase "multiplicity of infection" or
"MOI" is the average number of infectious virus particles per
infected cell. The MOI is determined by dividing the number of
infectious virus particles added (ml added.times.PFU/ml) by the
number of cells added (ml added.times.cells/ml).
[0035] As used herein, the term "nucleic acid" is intended to
include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules
(e.g., mRNA) and analogs of the DNA or RNA generated using
nucleotide analogs. The nucleic acid can be single-stranded or
double-stranded. As used herein, a nucleic acid may include natural
(e.g., A, G, C, or T) or modified nucleotide bases (6-dimethylamino
purine, 5-fluoro cystine, 2-pyridone, 7-deazaguanosine, inosine,
etc.).
[0036] "Polypeptide" refers to a polymer of amino acids linked by
amide bonds as is known to those of skill in the art. The
polypeptide can be a polymer of 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200,
250, 300, 350, 400, 450, 500, 550 or more amino acids linked by
covalent amide bonds. In some embodiments, the polypeptide is a
polymer of 10 to 25, 10 to 30, 10 to 40, 10 to 50, or 25 to 50
amino acids linked by covalent amide bonds. In certain embodiments,
the polypeptide is a polymer of 100 to 150, 100 to 200, 100 to 250,
100 to 300, 100 to 350, 100 to 400, 100 to 450, 100 to 500, 100 to
550, 100 to 600, 100 to 650, 100 to 700, or 100 to 750 amino acids
linked by covalent amide bonds. In certain embodiments, the
polypeptide is a polymer of 50 to 55, 50 to 60, 50 to 65, 50 to 75,
50 to 80, 50 to 85, 50 to 90, 50 to 95, 50 to 100, 75 to 80, 75 to
85, 75 to 90, 75 to 95, or 75 to 100 amino acids linked by covalent
amide bonds. In some embodiments, the polypeptide is 55 to 60, 55
to 65, 55 to 70, 55 to 75, 55 to 80, 55 to 85, 55 to 90, 55 to 95,
55 to 100, or 60 to 75 amino acids linked by covalent amide bonds.
As used herein, the term can refer to a single polypeptide chain
linked by covalent amide bonds. The term can also refer to multiple
polypeptide chains associated by non-covalent interactions such as
ionic contacts, hydrogen bonds, Van der Waals contacts and
hydrophobic contacts. Those of skill in the art will recognize that
the term includes polypeptides that have been modified, for example
by post-translational processing such as signal peptide cleavage,
disulfide bond formation, glycosylation (e.g., N-linked
glycosylation), protease cleavage and lipid modification (e.g.,
S-palmitoylation).
[0037] As used herein, the terms "purified" and "isolated" when
used in the context of a polypeptide (including antibody) that is
obtained from a natural source, e.g., cells, refers to a
polypeptide which is substantially free of contaminating materials
from the natural source, e.g., soil particles, minerals, chemicals
from the environment, and/or cellular materials from the natural
source, such as but not limited to cell debris, cell wall
materials, membranes, organelles, the bulk of the nucleic acids,
carbohydrates, proteins, and/or lipids present in cells. Thus, a
polypeptide that is isolated includes preparations of a polypeptide
having less than about 30%, 20%, 10%, 5%, 2%, or 1% (by dry weight)
of cellular materials and/or contaminating materials. As used
herein, the terms "purified" and "isolated" when used in the
context of a polypeptide (including antibody) that is chemically
synthesized refers to a polypeptide which is substantially free of
chemical precursors or other chemicals which are involved in the
syntheses of the polypeptide. Accordingly, such preparations of the
polypeptide have less than about 30%, 20%, 10%, or 5% (by dry
weight) of chemical precursors or compounds other than the peptide
of interest. In a specific embodiment, a flu polypeptide is
chemically synthesized. In another specific embodiment, a flu
polypeptide is recombinantly expressed. In another specific
embodiment, a flu polypeptide is isolated.
[0038] As used herein, the terms "replication," "viral replication"
and "virus replication" in the context of a virus refer to one or
more, or all, of the stages of a viral life cycle which result in
the propagation of virus. The steps of a viral life cycle include,
but are not limited to, virus attachment to the host cell surface,
penetration or entry of the host cell (e.g., through receptor
mediated endocytosis or membrane fusion), uncoating (the process
whereby the viral capsid is removed and degraded by viral enzymes
or host enzymes thus releasing the viral genomic nucleic acid),
genome replication, synthesis of viral messenger RNA (mRNA), viral
protein synthesis, and assembly of viral ribonucleoprotein
complexes for genome replication, assembly of virus particles,
post-translational modification of the viral proteins, and release
from the host cell by lysis or budding and acquisition of a
phospholipid envelope which contains embedded viral glycoproteins.
In some embodiments, the terms "replication," "viral replication"
and "virus replication" refer to the replication of the viral
genome. In other embodiments, the terms "replication," "viral
replication" and "virus replication" refer to the synthesis of
viral proteins. In other embodiments, the terms "replication,"
"viral replication" and "virus replication" refer to the synthesis
of new viral particles.
[0039] As used herein, the terms "subject" or "patient" are used
interchangeably to refer to an animal (e.g., birds, reptiles, and
mammals). In a specific embodiment, a subject is a bird. In another
embodiment, a subject is a mammal including a non-primate (e.g., a
camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat,
and mouse) and a primate (e.g., a monkey, chimpanzee, and a human).
In certain embodiments, a subject is a non-human animal. In some
embodiments, a subject is a farm animal or pet (e.g., a dog, cat,
horse, goat, sheep, pig, donkey, or chicken). In another
embodiment, a subject is a human. In another embodiment, a subject
is a human infant. In another embodiment, a subject is a human
child. In another embodiment, a subject is a human adult. In
another embodiment, a subject is an elderly human. In another
embodiment, a subject is a premature human infant.
[0040] As used herein, the term "premature human infant" refers to
a human infant born at less than 37 weeks of gestational age.
[0041] As used herein, the term "human infant" refers to a newborn
to 1 year old human.
[0042] As used herein, the term "human toddler" refers to a human
that is 1 years to 3 years old.
[0043] As used herein, the term "human child" refers to a human
that is 1 year to 18 years old.
[0044] As used herein, the term "human adult" refers to a human
that is 18 years or older.
[0045] As used herein, the term "elderly human" refers to a human
65 years or older.
[0046] The terms "tertiary structure" and "quaternary structure"
have the meanings understood by those of skill in the art. Tertiary
structure refers to the three-dimensional structure of a single
polypeptide chain. Quaternary structure refers to the three
dimensional structure of a polypeptide having multiple polypeptide
chains.
[0047] As used herein, the terms "therapies" and "therapy" can
refer to any protocol(s), method(s), compound(s), composition(s),
formulation(s), and/or agent(s) that can be used in the prevention
or treatment of a viral infection or a disease or symptom
associated therewith. In certain embodiments, the terms "therapies"
and "therapy" refer to biological therapy, supportive therapy,
and/or other therapies useful in the treatment or prevention of a
viral infection or a disease or symptom associated therewith known
to one of skill in the art. In some embodiments, the term "therapy"
refers to a nucleic acid encoding a flu polypeptide, or a vector,
or composition comprising said nucleic acid encoding a flu
polypeptide. In some embodiments, the term "therapy" refers to an
antibody that specifically binds to a flu polypeptide.
[0048] As used herein, the terms "prevent," "preventing" and
"prevention" in the context of the administration of a therapy(ies)
to a subject to prevent an influenza virus disease refer to one or
more of the following effects resulting from the administration of
a therapy or a combination of therapies: (i) the inhibition of the
development or onset of an influenza virus disease or a symptom
thereof; (ii) the inhibition of the recurrence of an influenza
virus disease or a symptom associated therewith; and (iii) the
reduction or inhibition in influenza virus infection and/or
replication.
[0049] As used herein, the terms "prevent", "preventing" and
"prevention" in the context of administering a therapy to a subject
to prevent an influenza virus infection refers to the inhibition or
reduction of onset or development of one or more symptoms
associated with influenza virus infection.
[0050] As used herein, the terms "treat," "treatment," and
"treating" refer in the context of administration of a therapy(ies)
to a subject to treating an influenza virus disease to obtain a
beneficial or therapeutic effect of a therapy or a combination of
therapies. In specific embodiments, such terms refer to one, two,
three, four, five or more of the following effects resulting from
the administration of a therapy or a combination of therapies: (i)
the reduction or amelioration of the severity of an influenza virus
infection or a disease or a symptom associated therewith; (ii) the
reduction in the duration of an influenza virus infection or a
disease or a symptom associated therewith; (iii) the regression of
an influenza virus infection or a disease or a symptom associated
therewith; (iv) the reduction of the titer of an influenza virus;
(v) the reduction in organ failure associated with an influenza
virus, infection or a disease associated therewith; (vi) the
reduction in hospitalization of a subject; (vii) the reduction in
hospitalization length; (viii) the increase in the survival of a
subject; (ix) the elimination of an influenza virus infection or a
disease or symptom associated therewith; (x) the inhibition of the
progression of an influenza virus infection or a disease or a
symptom associated therewith; (xi) the prevention of the spread of
an influenza virus from a cell, tissue, organ or subject to another
cell, tissue, organ or subject; (xii) the inhibition or reduction
in the entry of an influenza virus into a host cell(s); (xiii) the
inhibition or reduction in the replication of an influenza virus
genome; (xiv) the inhibition or reduction in the synthesis of
influenza virus proteins; (xv) the inhibition or reduction in the
release of influenza virus particles from a host cell(s); and/or
(xvi) the enhancement or improvement the therapeutic effect of
another therapy.
[0051] As used herein, the terms "treat", "treatment" and
"treating" in the context of administering a therapy to a subject
to treat an influenza virus infection refers to: (i) the reduction
in influenza virus replication; (ii) the reduction in influenza
virus titers; (iii) the reduction in the spread of influenza virus
from one cell, organ or tissue to another cell, organ or tissue;
(iv) the reduction in the severity and/or number of symptoms
associated with an influenza virus infection; (v) the reduction in
the duration of a symptom(s) associated with an influenza virus
infection; and/or (vi) the inhibition or reduction in the
progression of an influenza virus infection.
[0052] As used herein, in some embodiments, the phrase "wild-type"
in the context of a virus refers to the types of a virus that are
prevalent, circulating naturally and producing typical outbreaks of
disease. In other embodiments, the term "wild-type" in the context
of a virus refers to a parental virus.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0053] FIG. 1: MAb 12D1 reacts by Western blot with truncated
hemagglutinin constructs. 12D1 makes dominant contacts with the HA2
subunit in the region of amino acids 30 to 106 (H3 numbering (see,
e.g., Wilson et al., Nature 1981; 289 (5796): 366-73)). Diminished
12D1 binding without diminished GFP expression in the HA2 76-184
and HA2 91-184 truncations along with loss of binding with the HA2
106-184 truncation suggests that the binding epitope lies in the
region from amino acids HA2 76-106. These 30 amino acids fall
within the membrane distal half of the long alpha-helix of HA2.
[0054] FIG. 2: Monoclonal antibody (mAb) 12D1 reacts with the long
alpha-helix of HA2. Lysates from 293T cells transfected with GFP or
GFP HA76-130 were incubated with mAb 12D1 and pulled-down with
protein G beads. Pulled-down fractions were blotted with mAb 12D1
or rabbit anti-GFP. Anti-mouse HRP used to detect 12D1 binding
reacts with the mouse Ig heavy and light chains within the
pulled-down fraction (A). Structural integrity of the 12D1 epitope
within the HApep-KLH conjugate was confirmed by direct binding
ELISA. MAb 36A7 binds outside of the 76-130 region of HA2 (B).
[0055] FIG. 3: Flu polypeptide (76-130)-KLH ("HApep-KLH") acts as a
robust immunogen and serum antibody elicited by HApep-KLH reacts
with multiple hemagglutinin subtypes. Sera from individual mice
were taken 10-days post primary and secondary immunization and were
tested for binding to H3 or H1 HA by ELISA (A). Pooled sera taken
post-secondary immunization with HApep-KLH was tested for
reactivity by Western blot with purified H3 or H1 subtype influenza
viruses or with Newcastle disease virus (NDV) (B). Pooled sera were
tested for binding activity against H2, H5 or H7 HA by ELISA (C).
Percent identity/similarity of core polypeptide (76-130) between
influenza subtypes (D).
[0056] FIG. 4: Immunization with flu polypeptide (76-130)-KLH
("HApep-KLH") protects mice against lethal challenge. Average
weight change of mice infected with lethal dose of H3N2 virus (A).
Vaccination with HApep-KLH protects H3N2-infected mice against
death (B). HApep-KLH vaccine protects 80% of mice against lethal
dose of H1N1 virus (C). Anti-H1 titer correlates with weight loss
after PR8 challenge (D).
[0057] FIG. 5: Sequence alignment between various influenza A virus
subtypes using selected strains from subtypes H1, H2, H3, H5 and H7
(A). Sequence alignment shows conserved amino acids between the
different subtypes (B).
[0058] FIG. 6: Sequence alignment between strains of various
influenza A virus subtypes including HINT (A) (SEQ ID NOS: 8-11),
H2N2 (B) (SEQ ID NOS: 13 and 14), H3N2 (C) (SEQ ID NOS: 16 and 18),
H5N1 (D) (SEQ ID NO: 22), H7 (E) (SEQ ID NOS: 31-35), H4 (F) (SEQ
ID NOS: 20 and 21), H6 (G) (SEQ ID NOS: 24-29), H8 (H) SEQ ID NOS:
37 and 38), H9 (I) (SEQ ID NOS: 40 and 41), H10 (J) (SEQ ID NOS: 43
and 44), H11 (K) (SEQ ID NOS: 46 and 47), H12 (L) (SEQ ID NOS: 49
and 50), H13 (M) (SEQ IDS NO: 52-54), H14 (N) (SEQ ID NO: 55), H15
(O) (SEQ ID NO: 56), and H16 (P) (SEQ ID NO: 57).
[0059] FIG. 7: Monoclonal antibody 12D1 reacts with the long
alpha-helix of HA2. (A) Hemagglutinin monomer. Amino acids 76-130
of HA2 are depicted. (B) Lysates from 293T cells were transfected
with pCAGGs-GFP or pCAGGs-GFP HA2 76-130, incubated with mAb 12D1
and pulled-down with protein G beads. Pulled-down fractions were
blotted with mAb 12D1 or rabbit anti-GFP. Arrows indicate location
of 76-130 GFP fusion protein. Anti-mouse HRP was used to detect
12D1 binding with the mouse Ig heavy and light chains within the
pulled-down fraction. (C) Structural integrity of the 12D1 epitope
within the LAH-KLH conjugate was confirmed by direct binding ELISA.
Hemagglutinin specific mAb 36A7 does not bind within the 76-130
region of HA2.
[0060] FIG. 8: LAH-KLH vaccine acts as a robust immunogen and serum
antibody elicited reacts with multiple hemagglutinin subtypes. (A,
B) Sera from individual mice were taken 10-days post primary and
secondary immunization and were tested for binding to H3 or H1
hemagglutinin by ELISA. (C) Pooled sera from 20 mice taken
post-secondary immunization with LAH-KLH was tested for reactivity
by Western blot with purified A/Hong Kong/1/1968 (H3) or
A/USSR/90/1977 (H1) subtype influenza viruses, purified
A/California/04/09 hemagglutinin (transmembrane domain absent), or
with Newcastle disease virus. (D) Pooled sera were tested for
binding activity against H2, H5 or H7 HA by ELISA. LAH-KLH
anti-serum has considerable binding activity against all 3 HA
subtypes. Positive sera used were from mice infected with either a
Group 1 influenza virus (for H2 and H5 ELISAs) or a Group 2 virus
(for H7 ELISA) (see Steel (2010) mBio 1(1):1-9). (E) LAH amino
acids 76-130 from the HA2 of different hemagglutinin subtypes.
Black highlight/white letters: residue conserved in all 5 HAs.
Conserved residues fall into one of four groups: 1)D/N/E/Q
2)I/L/V/M 3)K/R 4)S/T. Gray highlight/white letters: residue
conserved in 4/5 HAs OR less stringent conservation (R vs H near
middle conserves charge but change in size). Bold text, white
background: partial conservation (3/5) OR less stringent (L vs A at
middle, or F vs M towards left) (F) Isotype profile of
hemagglutinin specific antibody in serum pools from normal mice,
mice infected with A/Hong Kong/1/1968 (H3), or mice immunized with
LAH-KLH. Recombinant hemagglutinin from A/Hong Kong/1/1968 was used
to coat plates for ELISA.
[0061] FIG. 9: Immunization with LAH-KLH protects mice in vivo. (A,
B) Two weeks following secondary immunization, mice were challenged
with 4.times.10.sup.5 pfu of X31, a mouse adapted H3 influenza
virus, (C, D) 500 pfu of the mouse adapted H1 virus PR8, or (E, F)
with 500 pfu of an H5 highly-pathogenic avian influenza virus
modified to remove the poly-basic cleavage site in the viral
hemagglutinin (HAlo virus) (see Steel J, et al. (2009) J Virol
83(4):1742-1753). Each experimental group comprises 5 BALB/c mice.
Because of differences in pathogenicity, survival was defined as
20% weight loss for X31 (H3) and PR8 (H1) viruses, 30% weight loss
for VN/2004 virus (H5).
[0062] FIG. 10: Antibody mediates protection afforded by
immunization with LAH-KLH. (A) Analysis of pre-challenge serum from
mice infected with PR8 reveals a positive correlation between
hemagglutinin-specific antibody titer and increase in body weight
on days 1-3 following infection. (B, C) Pooled sera from mice
immunized with LAH-KLH, mice infected with H1 or H3 virus, or from
mice immunized with KLH alone were transferred to mice two hours
prior to infection with A/Georgia/81, a seasonal human H3 virus, or
with the H1 virus PR8. Lung titers were evaluated on day 2 post
infection. (D, E) Human sera taken pre or post-immunization with
the TIV were evaluated for binding activity with the LAH
polypeptide. Data shown are from serum samples diluted 1:3000. (D)
Subjects responded variably to seasonal vaccination and (E) serum
demonstrates minimal binding activity against the LAH peptide.
[0063] FIG. 11: LAH-KLH antiserum reacts with both Group 1 and
Group 2 hemagglutinin proteins while HA2 antiserum reacts with
Group 2 hemagglutinin proteins only. (A, B, and C) Activity of
antisera against Group 2 hemagglutinin proteins. HA2 and LAH-KLH
serum pools demonstrate comparable binding activity against the
HK/68 hemagglutinin but have different binding activities against
other Group 2 hemagglutinins. (D, E, F, G, and H) Activity of
antisera against Group 1 hemagglutinin proteins. The LAH-KLH serum
pool reacts with all Group 1 hemagglutinin proteins tested while
the HA2 antiserum does not. Positive control serum was from mice
infected with either the Group 2 H3 subtype X31 virus or the Group
1 H1 subtype PR8 virus.
5. DETAILED DESCRIPTION
[0064] 5.1 Flu Polypeptides
[0065] Provided herein are flu polypeptides. While not intending to
be bound by any particular theory of operation, it is believed that
the flu polypeptides are useful for presenting one or more
relatively conserved antigenic regions of the HA2 hemagglutinin
subunit (e.g., the HA2 hemagglutinin subunit long alpha-helix) to a
subject's immune system in order to generate an immune response
that is capable of cross reacting with, and preferably protecting
against, a plurality of influenza virus strains from a single
subtype or 2, 3, 4 or more different subtypes.
[0066] In certain embodiments, a flu polypeptide comprises a core
polypeptide or modified core polypeptide.
[0067] In certain embodiments, a flu polypeptide is acetylated at
its N- and/or C-terminus. In certain embodiments, a flu polypeptide
is pegylated.
[0068] In certain embodiments, a flu polypeptide comprises one,
two, three or more core polypeptides and/or modified
polypeptides.
[0069] In certain embodiments, a flu polypeptide comprises one,
two, three or more core polypeptides or modified polypeptides and
one, two, three or more T cell epitopes.
[0070] In some embodiments, a flu polypeptide comprises one, two,
three or more core polypeptides or modified polypeptides and one,
two, three or more immunogenic polypeptides.
[0071] In certain embodiments, a flu polypeptide comprises one,
two, three or more core polypeptides or modified core polypeptides
and a polypeptide that facilitates multimerization (e.g.,
trimerization of the flu polypeptide).
[0072] In certain embodiments, a flu polypeptide comprises one,
two, three or more core polypeptides or modified core polypeptides
and one, two, three or more, or all of the following: 1) one, two,
three or more carriers; 2) one, two, three or more T cell epitopes
(e.g., CD8 T cell epitopes); 3) one, two, three or more immunogenic
polypeptides (e.g., Salmonella flagellin, see, Section 5.1.6); 4)
one, two, three or more protein tags (e.g., His- or FLAG-tag, see,
Section 5.1.3); 5) one or more polypeptides that facilitate
multimerization of the flu polypeptide (e.g., T4 foldon domain,
see, Section 5.1.8)
[0073] In certain embodiments, a flu polypeptide comprises a core
polypeptide or a modified core polypeptide linked to a linker
polypeptide. In certain embodiments, a flu polypeptide comprises a
core polypeptide or modified core polypeptide linked to a carrier
protein.
[0074] 5.1.1 Core Polypeptides
[0075] In certain embodiments, the flu polypeptide comprises a core
polypeptide. In certain embodiments, the core polypeptide comprises
one or more relatively conserved antigenic regions of the HA2
hemagglutinin subunit long alpha-helix. In a specific embodiment,
the core polypeptide is capable of generating an immune response in
a subject that is capable of cross reacting with, and preferably
protecting against, a plurality of influenza virus strains from a
single subtype, or strains from 2, 3, 4 or more subtypes. The
ability of a core polypeptide to generate an immune response in a
subject that is capable of cross reacting with, and preferably
protecting against, a plurality of influenza virus strains from a
single subtype, or strains from 2, 3, 4 or more subtypes can be
assessed using methods known to those of skill in the art and
described herein (see Sections 5.13 and 6, infra). In another
specific embodiment, the core polypeptide is capable of generating
an immune response in a subject that is capable of neutralizing a
plurality of influenza virus strains from a single subtype, or
strains from 2, 3, 4 or more subtypes. The ability of a core
polypeptide to generate an immune response that is capable of
neutralizing a plurality of influenza virus strains from a single
subtype, or strains from 2, 3, 4 or more subtypes can be assessed
using methods known to those of skill in the art and described
herein (see Sections 5.13 and 6, infra). In another specific
embodiment, the core polypeptide is capable of generating an immune
response in a subject that is capable of inhibiting or reducing the
replication of a plurality of influenza virus strains from a single
subtype, or strains from 2, 3, 4 or more subtypes. The ability of a
core polypeptide to generate an immune response that is capable of
inhibiting or reducing the replication of a plurality of influenza
virus strains from a single subtype, or strains from 2, 3, 4 or
more subtypes can be assessed using methods known to those of skill
in the art and described herein (see Sections 5.13 and 6,
infra).
[0076] In a specific embodiment, a core polypeptide comprises the
long alpha-helix of the HA2 hemagglutinin subunit of an influenza
virus. In a specific embodiment, a core polypeptide comprises a
portion of the long alpha-helix of the HA2 hemagglutinin subunit of
an influenza virus. In a specific embodiment, a core polypeptide
comprises a portion of the long alpha-helix of the HA2, wherein the
native conformation of the portion is maintained. In a specific
embodiment, a core polypeptide comprises a portion of the long
alpha-helix of the HA2, wherein the portion maintains a native
alpha-helix conformation. One of skill in the art can determine
whether or not the alpha-helix conformation is maintained using any
method known in the art such as, e.g., NMR, X-ray crystallographic
methods, or secondary structure prediction methods, e.g., circular
dichroism.
[0077] In specific embodiments, a core polypeptide does not include
the amino acid sequence of a full length influenza virus
hemagglutinin. In certain embodiments, a core polypeptide comprises
or consists of between 25 to 50, 50 to 55, 50 to 60, 50 to 65, 50
to 70, 50 to 75, 50 to 80, 50 to 85, 50 to 90, 50 to 95, 50 to 100,
100 to 150, 100 to 200, or 100 to 250 amino acids. In other
embodiments, a core polypeptide comprises or consists of between 50
to 55, 50 to 60, 50 to 65, 50 to 75, 50 to 80, 50 to 85, 50 to 90,
50 to 95, 50 to 100, 75 to 80, 75 to 85, 75 to 90, 75 to 95, or 75
to 100 amino acids
[0078] In a specific embodiment, a core polypeptide comprises or
consists of amino acids 1(.+-.5) to 184(.+-.5), 16(.+-.5) to
184(.+-.5), 30(.+-.5) to 184(.+-.5), 31(.+-.5) to 184(.+-.5),
46(.+-.5) to 184(.+-.5), 61(.+-.5) to 184(.+-.5), 70(.+-.5) to
110(.+-.5), 76(.+-.5) to 106(.+-.5), 76(.+-.5) to 130(.+-.5) or
76(.+-.5) to 184(.+-.5) of a hemagglutinin polypeptide numbered
according to the classic H3 subtype numbering system. In some
embodiments, a core polypeptide comprises or consists of amino
acids 1(.+-.5) to 184(.+-.5), 16(.+-.5) to 184(.+-.5), 30(.+-.5) to
184(.+-.5), 31(.+-.5) to 184(.+-.5), 46(.+-.5) to 184(.+-.5),
61(.+-.5) to 184(.+-.5), 70(.+-.5) to 184(.+-.5), (70(.+-.5) to
110(.+-.5), 76(.+-.5) to 106(.+-.5), 76(.+-.5) to 130(.+-.5) or
76(.+-.5) to 184(.+-.5) of a hemagglutinin polypeptide numbered
according to the classic H3 subtype numbering system, wherein the
core polypeptide is less than 300, 275, 250, 200, 190, 185, or 180
amino acids in length. In a specific embodiment, a core polypeptide
comprises or consists of amino acids 76 to 106 of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system.
[0079] In another specific embodiment, a core polypeptide comprises
amino acids 76 to 130 of a hemagglutinin polypeptide numbered
according to the classic H3 subtype numbering system. In certain
embodiments, a core polypeptide comprises or consists of amino
acids 76 to 130 of a hemagglutinin polypeptide numbered according
to the classic H3 subtype numbering system, wherein the core
polypeptide is less than 300, 275, 250, 200, 190, 185, 180, 175,
150, 145, 130, 130, 125, 100, or 75 amino acids in length. In
another specific embodiment, a core polypeptide consists of amino
acids 76 to 130 of a hemagglutinin polypeptide numbered according
to the classic H3 subtype numbering system.
[0080] In a specific embodiment, a core polypeptide comprises or
consists of amino acids 70(.+-.5) to 125(.+-.5), 80(.+-.5) to
115(.+-.5), 90(.+-.5) to 105(.+-.5), or 76(.+-.5) to 95(.+-.5) of a
hemagglutinin polypeptide numbered according to the classic H3
subtype numbering system. In certain embodiments, a core
polypeptide comprises or consists of amino acids 70(+5) to
125(.+-.5), 80(.+-.5) to 115(.+-.5), 90(.+-.5) to 105(.+-.5), or
76(.+-.5) to 95(.+-.5) of a hemagglutinin polypeptide numbered
according to the classic H3 subtype numbering system, wherein the
core polypeptide is less than 300, 275, 250, 200, 190, 185, 180,
175, 150, 145, 130, 130, 125, 100, or 75 amino acids in length.
[0081] In a specific embodiment, a core polypeptide comprises or
consists of amino acids 70(.+-.5) to 130(.+-.5), 70(.+-.5) to
120(.+-.5), 70(.+-.5) to 110(.+-.5), 70(.+-.5) to 100(.+-.5), or
70(.+-.5) to 95(.+-.5) of a hemagglutinin polypeptide numbered
according to the classic H3 subtype numbering system. In certain
embodiments, a core polypeptide comprises or consists of amino
acids 70(.+-.5) to 130(.+-.5), 70(.+-.5) to 120(.+-.5), 70(.+-.5)
to 110(.+-.5), 70(.+-.5) to 100(.+-.5), or 70(.+-.5) to 95(.+-.5)
of a hemagglutinin polypeptide numbered according to the classic H3
subtype numbering system, wherein the core polypeptide is less than
300, 275, 250, 200, 190, 185, 180, 175, 150, 145, 130, 130, 125,
100, or 75 amino acids in length.
[0082] In a specific embodiment, a core polypeptide comprises or
consists of amino acids 70(.+-.5) to 130(.+-.5), 80(.+-.5) to
130(.+-.5), 90(.+-.5) to 130(.+-.5), 100(.+-.5) to 130(.+-.5), or
110(.+-.5) to 130(.+-.5) of a hemagglutinin polypeptide numbered
according to the classic H3 subtype numbering system. In certain
embodiments, a core polypeptide comprises or consists of amino
acids 70(.+-.5) to 130(.+-.5), 80(15) to 130(.+-.5), 90(.+-.5) to
130(.+-.5), 100(.+-.5) to 130(.+-.5), or 110(.+-.5) to 130(.+-.5)
of a hemagglutinin polypeptide numbered according to the classic H3
subtype numbering system, wherein the core polypeptide is less than
300, 275, 250, 200, 190, 185, 180, 175, 150, 145, 130, 130, 125,
100, or 75 amino acids in length.
[0083] In a specific embodiment, a core polypeptide comprises or
consists of amino acids 1-184, 10(.+-.5) to 184, 20(.+-.5) to 184,
30(.+-.5) to 184, 40(.+-.5) to 184, 50(.+-.5) to 184, 60(.+-.5) to
184, 70(.+-.5) to 184 or 80(.+-.5) to 184 of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system. In certain embodiments, a core polypeptide comprises or
consists of amino acids 1-184, 10(.+-.5) to 184, 20(.+-.5) to 184,
30(.+-.5) to 184, 40(.+-.5) to 184, 50(.+-.5) to 184, 60(.+-.5) to
184, 70(.+-.5) to 184 or 80(.+-.5) to 184 of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system, wherein the core polypeptide is less than 300, 275, 250,
200, 190, 185, 180, 175, 150, 145, 130, 130, 125, 100, or 75 amino
acids in length.
[0084] In a specific embodiment, a core polypeptide comprises or
consists of the long alpha-helix of the HA2 hemagglutinin subunit
of the influenza virus strain A/Hong Kong/1/1968 (H3) or a fragment
thereof (i.e., amino acids 76-130, numbered according to the
classic H3 subtype numbering system or a fragment thereof), i.e.
the core polypeptide comprises or consists of the following amino
acid sequence:
TABLE-US-00001 (SEQ ID NO: 2)
RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKT RRQLRENA or a
fragment thereof).
In some embodiments, the core polypeptide comprising the amino acid
sequence SEQ ID NO: 2 comprises at least 56 amino acids or more.
The core polypeptide corresponding to SEQ ID NO:2 can be modified
at the N-terminus, at the C-terminus, or both. In some embodiments,
the core polypeptide is modified at the N-terminus. In some
embodiments, the core polypeptide is modified at the C-terminus. In
a specific embodiment, the core polypeptide is acetylated at the
N-terminus. In another specific embodiment, the core polypeptide is
linked to a linker, such as a FLAG-tag, at the C-terminus. In
another specific embodiment, the C-terminus of the core polypeptide
is linked to a linker, e.g., a FLAG-tag, and a cysteine residue
which can be used, e.g., to couple/link the core polypeptide to a
carrier (e.g., KLH).
[0085] In a specific embodiment, a core polypeptide comprises or
consists of a region of hemagglutinin subunit of the influenza
virus strain A/Hong Kong/1/1968 (H3) or a fragment thereof that
corresponds to amino acids 79-134, numbered according to the
classic H3 subtype numbering system or a fragment thereof (i.e.,
the core polypeptide comprises or consists of the following amino
acid sequence:
TABLE-US-00002 LEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKT
RRQLRENAEDMG or a fragment thereof).
[0086] In a specific embodiment, a core polypeptide is linked to a
FLAG-tag and a C-terminal cysteine residue, and such a polypeptide
with the FLAG-tag and cysteine residue comprises or consists of the
following amino acid sequence:
TABLE-US-00003 (SEQ ID NO: 1)
RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKT
RRQLRENADYKDDDDKC.
In some embodiments, such a polypeptide is acetylated at the
N-terminus.
[0087] In certain embodiments, the core polypeptide shares at least
50% amino acid sequence identity with the amino acid sequence of
SEQ ID NO: 1 or SEQ ID NO: 2 and maintains the native conformation
of amino acids 76-130 of the influenza virus strain A/Hong
Kong/1/1968 (H3), numbered according to the classic H3 subtype
numbering system. In certain embodiments, the core polypeptide
shares at least 60% amino acid sequence identity with the amino
acid sequence of SEQ ID NO:1 or SEQ ID NO:2 and maintains the
native conformation of amino acids 76-130 of the influenza virus
strain A/Hong Kong/1/1968 (H3), numbered according to the classic
H3 subtype numbering system. In certain embodiments, the core
polypeptide shares at least 65% amino acid sequence identity with
the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 and maintains
the native conformation of amino acids 76-130 of the influenza
virus strain A/Hong Kong/1/1968 (1-13), numbered according to the
classic H3 subtype numbering system. In certain embodiments, the
core polypeptide shares at least 70% amino acid sequence identity
with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 and
maintains the native conformation of amino acids 76-130 of the
influenza virus strain A/Hong Kong/1/1968 (H3), numbered according
to the classic H3 subtype numbering system. In certain embodiments,
the core polypeptide shares at least 75% amino acid sequence
identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2
and maintains the native conformation of amino acids 76-130 of the
influenza virus strain A/Hong Kong/1/1968 (H3), numbered according
to the classic H3 subtype numbering system. In certain embodiments,
the core polypeptide shares at least 80% amino acid sequence
identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2
and maintains the native conformation of amino acids 76-130 of the
influenza virus strain A/Hong Kong/1/1968 (H3), numbered according
to the classic H3 subtype numbering system. In certain embodiments,
the core polypeptide shares at least 85% amino acid sequence
identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2
and maintains the native conformation of amino acids 76-130 of the
influenza virus strain A/Hong Kong/1/1968 (H3), numbered according
to the classic H3 subtype numbering system. In certain embodiments,
the core polypeptide shares at least 90% amino acid sequence
identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2
and maintains the native conformation of amino acids 76-130 of the
influenza virus strain A/Hong Kong/1/1968 (H3), numbered according
to the classic H3 subtype numbering system. In certain embodiments,
the core polypeptide shares at least 95% amino acid sequence
identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2
and maintains the native conformation of amino acids 76-130 of the
influenza virus strain A/Hong Kong/1/1968 (H3), numbered according
to the classic H3 subtype numbering system. In certain embodiments,
the core polypeptide shares at least 98% amino acid sequence
identity with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2
and maintains the native conformation of amino acids 76-130 of the
influenza virus strain A/Hong Kong/1/1968 (1-13), numbered
according to the classic H3 subtype numbering system. In certain
embodiments, the core polypeptide shares at least 99% amino acid
sequence identity with the amino acid sequence of SEQ ID NO:1 or
SEQ ID NO:2 and maintains the native conformation of amino acids
76-130 of the influenza virus strain A/Hong Kong/1/1968 (H3),
numbered according to the classic H3 subtype numbering system.
[0088] In certain embodiments, the core polypeptide shares at least
50% amino acid sequence similarity with the amino acid sequence of
SEQ ID NO:1 or SEQ ID NO:2 and maintains the native conformation of
amino acids 76-130 of the influenza virus strain A/Hong Kong/1/1968
(1-13), numbered according to the classic H3 subtype numbering
system. In certain embodiments, the core polypeptide shares at
least 60% amino acid sequence similarity with the amino acid
sequence of SEQ ID NO:1 or SEQ ID NO:2 and maintains the native
conformation of amino acids 76-130 of the influenza virus strain
A/Hong Kong/1/1968 (H3), numbered according to the classic H3
subtype numbering system. In certain embodiments, the core
polypeptide shares at least 65% amino acid sequence similarity with
the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 and maintains
the native conformation of amino acids 76-130 of the influenza
virus strain A/Hong Kong/1/1968 (H3), numbered according to the
classic H3 subtype numbering system. In certain embodiments, the
core polypeptide shares at least 70% amino acid sequence similarity
with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 and
maintains the native conformation of amino acids 76-130 of the
influenza virus strain A/Hong Kong/1/1968 (H3), numbered according
to the classic H3 subtype numbering system. In certain embodiments,
the core polypeptide shares at least 75% amino acid sequence
similarity with the amino acid sequence of SEQ ID NO:1 or SEQ ID
NO:2 and maintains the native conformation of amino acids 76-130 of
the influenza virus strain A/Hong Kong/1/1968 (H3), numbered
according to the classic H3 subtype numbering system. In certain
embodiments, the core polypeptide shares at least 80% amino acid
sequence similarity with the amino acid sequence of SEQ ID NO:1 or
SEQ ID NO:2 and maintains the native conformation of amino acids
76-130 of the influenza virus strain A/Hong Kong/1/1968 (H3),
numbered according to the classic H3 subtype numbering system. In
certain embodiments, the core polypeptide shares at least 85% amino
acid sequence similarity with the amino acid sequence of SEQ ID
NO:1 or SEQ ID NO:2 and maintains the native conformation of amino
acids 76-130 of the influenza virus strain A/Hong Kong/1/1968 (H3),
numbered according to the classic H3 subtype numbering system. In
certain embodiments, the core polypeptide shares at least 90% amino
acid sequence similarity with the amino acid sequence of SEQ ID
NO:1 or SEQ ID NO:2 and maintains the native conformation of amino
acids 76-130 of the influenza virus strain A/Hong Kong/1/1968 (H3),
numbered according to the classic H3 subtype numbering system. In
certain embodiments, the core polypeptide shares at least 95% amino
acid sequence similarity with the amino acid sequence of SEQ ID
NO:1 or SEQ ID NO:2 and maintains the native conformation of amino
acids 76-130 of the influenza virus strain A/Hong Kong/1/1968 (H3),
numbered according to the classic H3 subtype numbering system. In
certain embodiments, the core polypeptide shares at least 98% amino
acid sequence similarity with the amino acid sequence of SEQ ID
NO:1 or SEQ ID NO:2 and maintains the native conformation of amino
acids 76-130 of the influenza virus strain A/Hong Kong/1/1968 (H3),
numbered according to the classic H3 subtype numbering system. In
certain embodiments, the core polypeptide shares at least 99% amino
acid sequence similarity with the amino acid sequence of SEQ ID
NO:1 or SEQ ID NO:2 and maintains the native conformation of amino
acids 76-130 of the influenza virus strain A/Hong Kong/1/1968 (H3),
numbered according to the classic H3 subtype numbering system.
[0089] In a specific embodiment, a core polypeptide does not
comprise the long alpha-helix of the HA2 hemagglutinin subunit of
the influenza virus strain A/Hong Kong/1/1968 (H3) (i.e., amino
acids 76-130, numbered according to the classic H3 subtype
numbering system), i.e., the core polypeptide does not comprise the
following amino acid sequence:
TABLE-US-00004 (SEQ ID NO: 2)
RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKT RRQLRENA.
[0090] In certain embodiments, a core polypeptide is not linked to
a FLAG-tag and a C-terminal cysteine residue which can be used,
e.g., to couple/link the core polypeptide to a carrier (e.g., KLH).
In some specific embodiments, a core polypeptide described herein
does not comprise the following amino acid sequence:
TABLE-US-00005 (SEQ ID NO: 1)
RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKT
RRQLRENADYKDDDDKC,
wherein the FLAG-tag is represented by the amino acid sequence
DYKDDDDK. In some embodiments, a core polypeptide is not acetylated
at the N-terminus.
[0091] In a specific embodiment, a core polypeptide does not
comprise amino acids 70(.+-.5) to 125(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 80(.+-.5) to 115(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 90(.+-.5) to 105(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 76(.+-.5) to 95(.+-.5), of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system.
[0092] In a specific embodiment, a core polypeptide does not
comprise amino acids 70(.+-.5) to 130(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 70(.+-.5) to 120(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 70(.+-.5) to 110(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 70(.+-.5) to 100(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 70(.+-.5) to 95(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system.
[0093] In a specific embodiment, a core polypeptide does not
comprise amino acids 70(.+-.5) to 130(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 80(.+-.5) to 130(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 90(.+-.5) to 130(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 100(.+-.5) to 130(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 110(.+-.5) to 130(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype numbering
system.
[0094] In a specific embodiment, a core polypeptide does not
comprise amino acids 1-184 of a hemagglutinin polypeptide numbered
according to the classic H3 subtype numbering system. In a specific
embodiment, a core polypeptide does not comprise amino acids
10(.+-.5) to 184 of a hemagglutinin polypeptide numbered according
to the classic H3 subtype numbering system. In a specific
embodiment, a core polypeptide does not comprise amino acids
20(.+-.5) to 184 of a hemagglutinin polypeptide numbered according
to the classic H3 subtype numbering system. In a specific
embodiment, a core polypeptide does not comprise amino acids
30(.+-.5) to 184 of a hemagglutinin polypeptide numbered according
to the classic H3 subtype numbering system. In a specific
embodiment, a core polypeptide does not comprise amino acids
40(.+-.5) to 184 of a hemagglutinin polypeptide numbered according
to the classic H3 subtype numbering system. In a specific
embodiment, a core polypeptide does not comprise amino acids
50(.+-.5) to 184 of a hemagglutinin polypeptide numbered according
to the classic H3 subtype numbering system. In a specific
embodiment, a core polypeptide does not comprise amino acids
60(.+-.5) to 184 of a hemagglutinin polypeptide numbered according
to the classic H3 subtype numbering system. In a specific
embodiment, a core polypeptide does not comprise amino acids
70(.+-.5) to 184 of a hemagglutinin polypeptide numbered according
to the classic H3 subtype numbering system. In a specific
embodiment, a core polypeptide does not comprise amino acids
80(.+-.5) to 184 of a hemagglutinin polypeptide numbered according
to the classic H3 subtype numbering system.
[0095] In a specific embodiment, a core polypeptide does not
comprise amino acids 1(.+-.5) to 184(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype number
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 16(.+-.5) to 184(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype number
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 30(.+-.5) to 184(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype number
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 31(.+-.5) to 184(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype number
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 46(.+-.5) to 184(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype number
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 61(.+-.5) to 184(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype number
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 70(.+-.5) to 184(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype number
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 76(.+-.5) to 106(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype number
system. In a specific embodiment, a core polypeptide does not
comprise amino acids 76(.+-.5) to 184(.+-.5) of a hemagglutinin
polypeptide numbered according to the classic H3 subtype number
system.
[0096] In a specific embodiment, the core polypeptide is a generic
core polypeptide comprising the amino acid sequence:
TABLE-US-00006 (SEQ ID NO: 3)
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10DX.-
sub.11X.sub.12X.sub.13X.sub.14X.sub.15WX.sub.16YX.sub.17AELLVX.sub.18X.sub-
.19EN
X.sub.20X.sub.21TX.sub.22DX.sub.23X.sub.24DSX.sub.25X.sub.26X.sub.27X.sub.-
28LX.sub.29X.sub.30X.sub.31X.sub.32X.sub.33X.sub.34QLX.sub.35X.sub.36NX.su-
b.37,
[0097] wherein X.sub.1 is a hydrophilic amino acid; X.sub.2 is a
hydrophobic amino acid; X.sub.3 is a hydrophilic amino acid;
X.sub.4 is a hydrophilic amino acid; X.sub.5 is a hydrophobic amino
acid; X.sub.6 is N, E or I; X.sub.7 is a hydrophilic amino acid;
X.sub.8 is K, R, Y or W; X.sub.9 is M, V or T; X.sub.10 is a
hydrophilic residue; X.sub.11 is A, G, T or S; X.sub.12 is F, I, K,
L or M; X.sub.13 is L, I or T; X.sub.14 is a hydrophilic, acidic
amino acid; X.sub.15 is a hydrophobic amino acid; X.sub.16 is a
hydrophilic amino acid; amino acid X.sub.17 is a hydrophilic amino
acid; X.sub.18 is a hydrophobic amino acid; X.sub.19 is a
hydrophobic amino acid; X.sub.20 is a hydrophilic amino acid;
X.sub.21 is a hydrophilic, basic amino acid; X.sub.22 is a
hydrophobic amino acid; X.sub.23 is a hydrophobic amino acid;
X.sub.24 is H, T or A; X.sub.25 is a hydrophilic amino acid;
X.sub.26 is a hydrophobic amino acid; X.sub.27 is a hydrophilic
amino acid; X.sub.28 is a hydrophilic amino acid; X.sub.29 is a
hydrophobic amino acid; X.sub.30 is a hydrophilic, acidic amino
acid; X.sub.31 is a hydrophilic, basic amino acid; X.sub.32 is T or
V; X.sub.33 is a hydrophilic, basic amino acid; X.sub.34 is K, L,
M, S or R; X.sub.35 is a hydrophilic, basic amino acid; X.sub.36 is
a hydrophilic amino acid and X.sub.37 is a hydrophobic amino
acid.
[0098] In specific embodiments X.sub.1 is R or Q; X.sub.2 is L, M
or I; X.sub.3 is E, D, Q or G; X.sub.4 is D or N; X.sub.5 is L, M
or V; X.sub.6 is N, E or I; X.sub.7 is K or N; X.sub.8 is K, R, Y
or W; X.sub.9 is M, V or T; X.sub.10 is E, D, K or R; X.sub.11 is
A, G, T or S; X.sub.I, is F, I, K, L or M; X.sub.13 is L, I or T;
X.sub.14 is D or E; X.sub.15 is V, I or L; X.sub.16 is S or T;
X.sub.17 is N or Q; X.sub.18 is A or L; X.sub.19 is L or M;
X.sub.20 is E or Q; X.sub.21 is R or H; X.sub.22 is L or I;
X.sub.73 is F, V, M, Y or L; X.sub.24 is H, T or A; X.sub.25 is N
or E; X.sub.26 is V or M; X.sub.27 is K, N, R or S; X.sub.28 is K
or N; X.sub.29 is Y or F; X.sub.30 is D or E; X.sub.31 is K or R;
X.sub.37 is T or V; X.sub.33 is K or R; X.sub.34 is K, L, M, S or
R; X.sub.35 is K or R; X.sub.36 is D, N, Q or E and X.sub.37 is A
or V. In certain embodiments, the core polypeptide is acetylated at
the N-terminus.
[0099] In certain embodiments, the core polypeptide is a fragment
of a generic core polypeptide. In specific embodiments, the core
polypeptide is a fragment of a generic core polypeptide, wherein
the fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of a generic core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of a generic core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of a generic core polypeptide, wherein the fragment lacks
1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5), 20(.+-.5) or
25(.+-.5) amino acids from a generic core polypeptide's N-terminus
and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5), 20(.+-.5) or
25(.+-.5) amino acids from a generic core polypeptide's C-terminus.
In specific embodiments, the core polypeptide has an alpha-helical
conformation.
[0100] In some embodiments, the core polypeptide is a generic core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is a generic core polypeptide
that is between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to
200, 51 to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to
50, 20 to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15
to 30, 20 to 30 or 20 to 25 amino acids in length. In some
embodiments, the core polypeptide is a generic core polypeptide
that is less than 500, 450, 400, 350, 300, 275, 250, 225, 200, 175,
150, 125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35,
30 or 25 amino acids in length. In certain embodiments, the core
polypeptide is a generic core polypeptide less than 150, 125, 95,
90, 85, 80, 75, 65, 60, 55, 50, 45 or 40 amino acids in length but
at least 15, 20, 25, 30 or 35 amino acids in length.
[0101] In specific embodiments, the core polypeptide is a
derivative of the generic core polypeptide, wherein the derivative
comprises a generic core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10(.+-.5) amino acids attached to either of a generic core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0102] In specific embodiments, the core polypeptide is a
derivative of the generic core polypeptide, wherein the derivative
comprises a generic core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10(.+-.5) amino acids attached to the generic core
polypeptide's N-terminus and 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to the generic core polypeptide's
C-terminus and wherein the core polypeptide maintains an
alpha-helical conformation.
[0103] In a specific embodiment, the core polypeptide is a
consensus core polypeptide comprising or consisting of the amino
acid sequence:
TABLE-US-00007 (SEQ ID NO: 4)
RIENLNKKX.sub.1EDGFLDVWTYNAELLVLMENERTLDX.sub.2HDSNVKNLYE
KVRX.sub.3QLRX.sub.4NA,
[0104] wherein X.sub.1 is M, V, T; X.sub.2 is a hydrophobic amino
acid; X.sub.3 is L, M, S, K, R; and X.sub.4 is a hydrophilic amino
acid. In a specific embodiment, X.sub.1 is M, V, T; X.sub.2 is F, Y
or L; X.sub.3 is L, M, S, K, R; and X.sub.4 is D, N or E. In
certain embodiments, the core polypeptide is acetylated at the
N-terminus.
[0105] In certain embodiments, the core polypeptide is a fragment
of the consensus core polypeptide. In specific embodiments, the
core polypeptide is a fragment of a consensus core polypeptide,
wherein the fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5),
15(.+-.5), 20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino
acids from either of a consensus core polypeptide's N- or
C-terminus. In some embodiments, the core polypeptide is a fragment
of a consensus core polypeptide, wherein the fragment lacks
24(.+-.5) amino acids from its C-terminus. In specific embodiments,
the core polypeptide is a fragment of a consensus core polypeptide,
wherein the fragment lacks 1, 2, 3, 4, 5, or more amino acids from
both of a consensus core polypeptide's N- and C-termini. In
specific embodiments, the core polypeptide has an alpha-helical
conformation.
[0106] In some embodiments, the core polypeptide is a consensus
core polypeptide that is not full length influenza virus HA. In
some embodiments, the core polypeptide is a consensus core
polypeptide that is between 51 to 300, 51 to 275, 51 to 250, 51 to
225, 51 to 200, 51 to 175, 51 to 150, 51 to 125, 51 to 100, or 51
to 75, 15 to 50, 20 to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37,
20 to 35, 15 to 30, 20 to 30 or 20 to 25 amino acids in length. In
some embodiments, the core polypeptide is a consensus core
polypeptide that is less than 500, 450, 400, 350, 300, 275, 250,
225, 200, 175, 150, 125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55,
50, 45, 40, 35, 30 or 25 amino acids in length. In certain
embodiments, the core polypeptide is a consensus core polypeptide
less than 150, 125, 95, 90, 85, 80, 75, 65, 60, 55, 50, 45 or 40
amino acids in length but at least 15, 20, 25, 30 or 35 amino acids
in length.
[0107] In specific embodiments, the core polypeptide is a
derivative of the consensus core polypeptide, wherein the
derivative comprises a consensus core polypeptide with 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10(.+-.5) amino acids attached to either of a
consensus core polypeptide's N- or C-terminus and wherein the core
polypeptide maintains an alpha-helical conformation.
[0108] In specific embodiments, the core polypeptide is a
derivative of the consensus core polypeptide, wherein the
derivative comprises a consensus core polypeptide with 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10(.+-.5) amino acids attached to both of the
consensus core polypeptide's N- and C-termini and wherein the core
polypeptide maintains an alpha-helical conformation.
[0109] In a specific embodiment, the core polypeptide is a group 1
core polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00008 (SEQ ID NO: 5)
RX.sub.1ENLNKKX.sub.2X.sub.3DGFLDX.sub.4WTYNAELLVLX.sub.5ENERTLDX.sub.6HDS-
NVKNL YX.sub.7KVR X.sub.8QLX.sub.9X.sub.10NX.sub.11,
[0110] wherein X.sub.1 is a hydrophobic amino acid; X.sub.2 is a
hydrophobic amino acid; X.sub.3 is a hydrophilic amino acid;
X.sub.4 is a hydrophobic amino acid; X.sub.5 is a hydrophobic amino
acid; X.sub.6 is a hydrophobic acidic amino acid; X.sub.7 is a
hydrophilic, acidic amino acid; X.sub.8 is L, M, or S; X.sub.9 is a
hydrophilic, basic amino acid; X.sub.10 is a hydrophilic amino acid
and X.sub.11 is a hydrophobic amino acid. In specific embodiments,
X.sub.1 is L or I; X.sub.2 M or V; X.sub.3 is E or D; X.sub.4 V or
I; X.sub.5 is M or L; X.sub.6 is F or Y; X.sub.7 is D or E; X.sub.8
L, M or S; X.sub.9 R or K; X.sub.10 is D or N and X.sub.11 is A or
V. In a specific embodiment, this core polypeptide can be used to
induce an immune response against group 1 hemagglutinin subtypes.
In certain embodiments, the immune response induced neutralizes 2
or more influenza virus group 1 hemagglutinin subtypes. In certain
embodiments, the core polypeptide is acetylated at the
N-terminus.
[0111] In certain embodiments, the core polypeptide is a fragment
of a group 1 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of a group 1 core polypeptide, wherein
the fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of a group 1 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of a group 1 core
polypeptide, wherein the fragment lacks 24(+5) amino acids from its
C-terminus. In specific embodiments, the core polypeptide is a
fragment of the group 1 core polypeptide, wherein the fragment
lacks 1, 2, 3, 4, 5, or more amino acids from both of a group 1
core polypeptide's N- and C-termini. In specific embodiments, the
core polypeptide has an alpha-helical conformation.
[0112] In some embodiments, the core polypeptide is a group 1 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is a group 1 core polypeptide
that is between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to
200, 51 to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to
50, 20 to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15
to 30, 20 to 30 or 20 to 25 amino acids in length. In some
embodiments, the core polypeptide is a group 1 core polypeptide
that is less than 500, 450, 400, 350, 300, 275, 250, 225, 200, 175,
150, 125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35,
30 or 25 amino acids in length. In certain embodiments, the core
polypeptide is a group 1 core polypeptide less than 150, 125, 95,
90, 85, 80, 75, 65, 60, 55, 50, 45 or 40 amino acids in length but
at least 15, 20, 25, 30 or 35 amino acids in length.
[0113] In specific embodiments, the core polypeptide is a
derivative of the group 1 core polypeptide, wherein the derivative
comprises a group 1 core polypeptide with either 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10(.+-.5) amino acids attached to either of a group 1
core polypeptide's N- or C-terminus and wherein the core
polypeptide maintains an alpha-helical conformation.
[0114] In specific embodiments, the core polypeptide is a
derivative of the group 1 core polypeptide, wherein the derivative
comprises a group 1 core polypeptide with either 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10(.+-.5) amino acids attached to both of a group 1
core polypeptide's N- and C-termini and wherein the core
polypeptide maintains an alpha-helical conformation.
[0115] In a specific embodiment, the core polypeptide is a group 2
core polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00009 (SEQ ID NO: 6)
X.sub.1IX.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9DX.sub.10-
X.sub.11X.sub.12X.sub.13X.sub.14WSYNAELLVAX.sub.15E
NQHTIDLX.sub.16DSEMNKLX.sub.17E
X.sub.18X.sub.19X.sub.20RQLRENA,
[0116] wherein X.sub.1 is a hydrophilic amino acid; X.sub.2 is a
hydrophilic amino acid; X.sub.3 is a hydrophilic amino acid;
X.sub.4 is a hydrophobic amino acid; X.sub.5 is E or I; X.sub.6 is
a hydrophilic amino acid; X.sub.7 is a hydrophobic amino acid;
X.sub.8 is V or T; X.sub.9 is a hydrophilic amino acid; X.sub.10 is
a hydrophilic amino acid; X.sub.11 is K or M; X.sub.12 is I or T;
X.sub.13 is a hydrophilic, acidic amino acid; X.sub.14 is a
hydrophobic amino acid; X.sub.15 is a hydrophobic amino acid;
X.sub.16 is T or A; X.sub.17 is a hydrophobic amino acid; X.sub.18
is a hydrophilic basic amino acid; X.sub.19 is T or V, and X.sub.20
is a hydrophilic, basic amino acid. In specific embodiments,
X.sub.1 is R or Q; X.sub.2 is Q or G; X.sub.3 is D or N; X.sub.4 is
L or V; X.sub.5 is E or I; X.sub.6 is K or N; X.sub.7 Y or W;
X.sub.8 V or T; X.sub.9 is E or R; X.sub.10 is T or S; X.sub.11 is
K or M; X.sub.1, is I or T; X.sub.13 is D or E; X.sub.14 is L or V;
X.sub.15 is L or M; X.sub.16 is T or A; X.sub.17 F or Y; X.sub.18
is K or R; X.sub.19 is T or V and X.sub.20 is K or R. In a specific
embodiment, this core polypeptide can be used to induce an immune
response against group 2 hemagglutinin subtypes. In certain
embodiments, the immune response induced neutralizes 2 or more
influenza virus group 2 hemagglutinin subtypes. In certain
embodiments, the core polypeptide is acetylated at the
N-terminus.
[0117] In certain embodiments, the core polypeptide is a fragment
of a group 2 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of a group 2 core polypeptide, wherein
the fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of a group 2 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of a group 2 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of a group 2 core polypeptide, wherein the fragment lacks
1, 2, 3, 4, 5, or more amino acids from both of a group 2 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0118] In some embodiments, the core polypeptide is a group 2 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is a group 2 core polypeptide
that is between 51 to 511, 51 to 500, 51 to 450, 51 to 400, 51 to
350, 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200, 51 to
175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50, 20 to
50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, to 30, 20 to
30 or 20 to 25 amino acids in length. In some embodiments, the core
polypeptide is a group 2 core polypeptide that is less than 500,
450, 400, 350, 300, 275, 250, 225, 200, 175, 150, 125, 100, 95, 90,
85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25 amino acids in
length. In certain embodiments, the core polypeptide is a group 2
core polypeptide less than 150, 125, 95, 90, 85, 80, 75, 65, 60,
55, 50, 45 or 40 amino acids in length but at least 15, 20, 25, 30
or 35 amino acids in length.
[0119] In specific embodiments, the core polypeptide is a
derivative of a group 2 core polypeptide, wherein the derivative
comprises a group 2 core polypeptide with either 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10(.+-.5) amino acids attached to either of a group 2
core polypeptide's N- or C-terminus and wherein the core
polypeptide maintains an alpha-helical conformation.
[0120] In specific embodiments, the core polypeptide is a
derivative of a group 2 core polypeptide, wherein the derivative
comprises a group 2 core polypeptide with either 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10(.+-.5) amino acids attached to both of a group 2
core polypeptide's N- and C-termini and wherein the core
polypeptide maintains an alpha-helical conformation.
[0121] In a specific embodiment, the core polypeptide is an H1 core
polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00010 (SEQ ID NO: 7)
RX.sub.1ENLNKKVDDGFX.sub.2DIWTYNAELLVLLENERTLDX.sub.3HDSNVX.sub.4NLY
EKVX.sub.5SQLKNNA,
[0122] wherein X.sub.1 is a hydrophobic amino acid; X.sub.2 is a
hydrophobic amino acid; X.sub.3 is a hydrophobic amino acid;
X.sub.4 is a hydrophilic, basic amino acid, and X.sub.5 is a
hydrophilic, basic amino acid. In specific embodiments, X.sub.1 is
M or I; X.sub.2 is L or I; X.sub.3 is F or Y; X.sub.4 is K or R;
and X.sub.5 is K or R. This sequence corresponds with amino acids
76-130 of an H1 subtype hemagglutinin numbered according to the
classic H3 subtype numbering system. In certain embodiments, the
core polypeptide is acetylated at the N-terminus. In a specific
embodiment, a core polypeptide comprises any one of the amino acid
sequences shown in FIG. 6A (SEQ ID NOS: 8-11) or a fragment
thereof. In certain embodiments, the core polypeptide is acetylated
at the N-terminus. In a specific embodiment, this core polypeptide
can be used to induce an immune response against influenza virus
strains of subtype H1. In certain embodiments, the immune response
induced neutralizes strains of influenza virus subtype H1.
[0123] In certain embodiments, the core polypeptide is a fragment
of an H1 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of a H1 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H1 core polypeptide's N- or C-term inus. In some
embodiments, the core polypeptide is a fragment of an H1 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H1 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H1 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0124] In some embodiments, the core polypeptide is an H1 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H1 core polypeptide that is
between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200, 51
to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50, 20
to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to 30,
20 to 30 or 20 to 25 amino acids in length. In some embodiments,
the core polypeptide is an H1 core polypeptide that is less than
500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150, 125, 100,
95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25 amino
acids in length. In certain embodiments, the core polypeptide is an
H1 polypeptide less than 150, 125, 95, 90, 85, 80, 75, 65, 60, 55,
50, 45 or 40 amino acids in length but at least 15, 20, 25, 30 or
35 amino acids in length.
[0125] In specific embodiments, the core polypeptide is a
derivative of an H1 core polypeptide, wherein the derivative
comprises an H1 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to either of the H1 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0126] In specific embodiments, the core polypeptide is a
derivative of an H1 core polypeptide, wherein the derivative
comprises an H1 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to both of the H1 core polypeptide's
N- or C-termini and wherein the core polypeptide maintains an
alpha-helical conformation.
[0127] In a specific embodiment, the core polypeptide is an H2 core
polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00011 (SEQ ID NO: 12)
RLENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYX KVRMQLRDNV,
[0128] wherein X is a hydrophilic, acidic amino acid. In specific
embodiments, X is D or E. In certain embodiments, the core
polypeptide is acetylated at the N-terminus. In a specific
embodiment, a core polypeptide comprises any one of the amino acid
sequences shown in FIG. 6B (SEQ ID NO: 13 or 14) or a fragment
thereof. In certain embodiments, the core polypeptide is acetylated
at the N-terminus. In a specific embodiment, this core polypeptide
can be used to induce an immune response against influenza virus
strains of subtype H2. In certain embodiments, the immune response
induced neutralizes strains of influenza virus subtype H2.
[0129] In certain embodiments, the core polypeptide is a fragment
of an H2 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H2 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H2 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H2 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H2 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H2 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0130] In some embodiments, the core polypeptide is an H2 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H2 core polypeptide that is
between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200, 51
to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50, 20
to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to 30,
20 to 30 or 20 to 25 amino acids in length. In some embodiments,
the core polypeptide is an H2 core polypeptide that is less than
500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150, 125, 100,
95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25 amino
acids in length. In certain embodiments, the core polypeptide is an
H2 polypeptide less than 150, 125, 95, 90, 85, 80, 75, 65, 60, 55,
50, 45 or 40 amino acids in length but at least 15, 20, 25, 30 or
35 amino acids in length.
[0131] In specific embodiments, the core polypeptide is a
derivative of an H2 core polypeptide, wherein the derivative
comprises an H2 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to either of the H2 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0132] In specific embodiments, the core polypeptide is a
derivative of an H2 core polypeptide, wherein the derivative
comprises an H2 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to both of the H2 core polypeptide's
N- and C-termini and wherein the core polypeptide maintains an
alpha-helical conformation.
[0133] In a specific embodiment, the core polypeptide is an H3 core
polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00012 (SEQ ID NO: 15)
RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEX.sub.1T
X.sub.2X.sub.3QLRENA,
[0134] wherein X.sub.1 is a hydrophilic, basic amino acid; X.sub.2
is a hydrophilic, basic amino acid, and X.sub.3 is a hydrophilic,
basic amino acid. In specific embodiments, X.sub.1 is K or R;
X.sub.2 is K or R, and X.sub.3 is K or R. In certain embodiments,
the core polypeptide is acetylated at the N-terminus. In a specific
embodiment, a core polypeptide comprises any one of the amino acid
sequences shown in FIG. 6C (SEQ ID NOS: 16-18) or a fragment
thereof. In certain embodiments, the core polypeptide is acetylated
at the N-terminus. In a specific embodiment, this core polypeptide
can be used to induce an immune response against influenza virus
strains of subtype H3. In certain embodiments, the immune response
induced neutralizes strains of influenza virus subtype H3.
[0135] In certain embodiments, the core polypeptide is a fragment
of an H3 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H3 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(+5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H3 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H3 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H3 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H3 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0136] In some embodiments, the core polypeptide is an H3 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H3 core polypeptide that is
between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200, 51
to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50, 20
to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to 30,
20 to 30 or 20 to 25 amino acids in length. In some embodiments,
the core polypeptide is an H3 core polypeptide that is less than
500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150, 125, 100,
95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25 amino
acids in length. In certain embodiments, the core polypeptide is an
H3 polypeptide less than 150, 125, 95, 90, 85, 80, 75, 65, 60, 55,
50, 45 or 40 amino acids in length but at least 15, 20, 25, 30 or
35 amino acids in length.
[0137] In specific embodiments, the core polypeptide is a
derivative of an H3 core polypeptide, wherein the derivative
comprises an H3 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to either of the H3 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0138] In specific embodiments, the core polypeptide is a
derivative of an H3 core polypeptide, wherein the derivative
comprises an H3 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to both of the H3 core polypeptide's
N- and C-termini and wherein the core polypeptide maintains an
alpha-helical conformation.
[0139] In a specific embodiment, the core polypeptide is an H4 core
polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00013 (SEQ ID NO: 19)
RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDVTDSEMNKLFERV RX.sub.1QLRENA,
[0140] wherein X.sub.1 is a hydrophilic, basic amino acid. In
specific embodiments, X.sub.1 is R or H. In certain embodiments,
the core polypeptide is acetylated at the N-terminus. In a specific
embodiment, a core polypeptide comprises any one of the amino acid
sequences shown in FIG. 6F (SEQ ID NOS: 20 and 21) or a fragment
thereof. In certain embodiments, the core polypeptide is acetylated
at the N-terminus. In a specific embodiment, this core polypeptide
can be used to induce an immune response against influenza virus
strains of subtype H4. In certain embodiments, the immune response
induced neutralizes strains of influenza virus subtype H4.
[0141] In certain embodiments, the core polypeptide is a fragment
of an H4 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H4 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H4 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H4 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H4 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H4 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0142] In some embodiments, the core polypeptide is an H4 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H4 core polypeptide that is
between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200, 51
to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50, 20
to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to 30,
20 to 30 or 20 to 25 amino acids in length. In some embodiments,
the core polypeptide is an H4 core polypeptide that is less than
500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150, 125, 100,
95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25 amino
acids in length. In certain embodiments, the core polypeptide is an
H4 polypeptide less than 150, 125, 95, 90, 85, 80, 75, 65, 60, 55,
50, 45 or 40 amino acids in length but at least 15, 20, 25, 30 or
35 amino acids in length.
[0143] In specific embodiments, the core polypeptide is a
derivative of an H4 core polypeptide, wherein the derivative
comprises an H4 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to either of the H4 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0144] In specific embodiments, the core polypeptide is a
derivative of an 1-14 core polypeptide, wherein the derivative
comprises an H4 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to both of the H4 core polypeptide's
N- and C-termini and wherein the core polypeptide maintains an
alpha-helical conformation.
[0145] In a specific embodiment, the core polypeptide is an H5 core
polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00014 (SEQ ID NO: 22)
RIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDK VRLQLRDNA.
In certain embodiments, the core polypeptide is acetylated at the
N-terminus. In a specific embodiment, a core polypeptide comprises
any one of the amino acid sequences shown in FIG. 6D (SEQ ID NO:
22) or a fragment thereof. In certain embodiments, the core
polypeptide is acetylated at the N-terminus. In a specific
embodiment, this core polypeptide can be used to induce an immune
response against influenza virus strains of subtype H5. In certain
embodiments, the immune response induced neutralizes strains of
influenza virus subtype H5.
[0146] In certain embodiments, the core polypeptide is a fragment
of an H5 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H5 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H5 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H5 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H5 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of the H5 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0147] In some embodiments, the core polypeptide is an H5 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H5 core polypeptide that is
between 51 to 511, 51 to 500, 51 to 450, 51 to 400, 51 to 350, 51
to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200, 51 to 175, 51
to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50, 20 to 50, 25
to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to 30, 20 to 30
or 20 to 25 amino acids in length. In some embodiments, the core
polypeptide is an H5 core polypeptide that is less than 500, 450,
400, 350, 300, 275, 250, 225, 200, 175, 150, 125, 100, 95, 90, 85,
80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25 amino acids in
length. In certain embodiments, the core polypeptide is an H5
polypeptide less than 150, 125, 95, 90, 85, 80, 75, 65, 60, 55, 50,
45 or 40 amino acids in length but at least 15, 20, 25, 30 or 35
amino acids in length.
[0148] In specific embodiments, the core polypeptide is a
derivative of an H5 core polypeptide, wherein the derivative
comprises an H5 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to either of the H5 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0149] In specific embodiments, the core polypeptide is a
derivative of an H5 core polypeptide, wherein the derivative
comprises an H5 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to both of the H5 core polypeptide's
N- and C-termini and wherein the core polypeptide maintains an
alpha-helical conformation.
[0150] In a specific embodiment, the core polypeptide is an H6 core
polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00015 (SEQ ID NO: 23)
RIX.sub.1NX.sub.2NKRMEDGFLDVWTYNAELLVLLENX.sub.3RTLDX.sub.4HDANVKX.sub.5L
X.sub.6EKVKSX.sub.7LX.sub.8DNA,
[0151] wherein X.sub.I is G or D; X.sub.2 is a hydrophobic amino
acid; X.sub.3 is a hydrophilic amino acid; X.sub.4 is a hydrophobic
amino acid; X.sub.5 is a hydrophilic amino acid; X.sub.6 is H or Y;
X.sub.7 is Q or L and X.sub.8 is a hydrophilic, basic amino acid.
In specific embodiments, X.sub.1 is G or D; X.sub.2 is L or M;
X.sub.3 is E or G; X.sub.4 is L or M; X.sub.5 N or S; X.sub.6 is H
or Y; X.sub.7 is Q or L and X.sub.8 is R or K. In certain
embodiments, the core polypeptide is acetylated at the N-terminus.
In a specific embodiment, a core polypeptide comprises any one of
the amino acid sequences shown in FIG. 6G (SEQ ID NOS: 24-29) or a
fragment thereof. In certain embodiments, the core polypeptide is
acetylated at the N-terminus. In a specific embodiment, this core
polypeptide can be used to induce an immune response against
influenza virus strains of subtype H6. In certain embodiments, the
immune response induced neutralizes strains of influenza virus
subtype H6.
[0152] In certain embodiments, the core polypeptide is a fragment
of an H6 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H6 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H6 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H6 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H6 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H6 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0153] In some embodiments, the core polypeptide is an H6 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H6 core polypeptide that is
between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200, 51
to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50, 20
to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to 30,
20 to 30 or 20 to 25 amino acids in length. In some embodiments,
the core polypeptide is an H6 core polypeptide that is less than
500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150, 125, 100,
95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25 amino
acids in length. In certain embodiments, the core polypeptide is an
H6 polypeptide less than 150, 125, 95, 90, 85, 80, 75, 65, 60, 55,
50, 45 or 40 amino acids in length but at least 15, 20, 25, 30 or
35 amino acids in length.
[0154] In specific embodiments, the core polypeptide is a
derivative of an H6 core polypeptide, wherein the derivative
comprises an H6 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to either of the H6 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0155] In specific embodiments, the core polypeptide is a
derivative of an H6 core polypeptide, wherein the derivative
comprises an H6 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(+5) amino acids attached to both of the H6 core polypeptide's N-
and C-termini and wherein the core polypeptide maintains an
alpha-helical conformation.
[0156] In a specific embodiment, the core polypeptide is an H7 core
polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00016 (SEQ ID NO: 30)
QIGNVINWTRDX.sub.1MTEX.sub.2WSYNAELLVAMENQHTIDLADSEMX.sub.3KLY
ERVX.sub.4KQLRENA,
[0157] wherein X.sub.1 is a hydrophobic amino acid or a hydrophilic
amino acid; X.sub.2 is a hydrophobic amino acid; X.sub.3 is a
hydrophilic amino acid, and X.sub.4 is a hydrophilic, basic amino
acid. In specific embodiments, X.sub.1 is A or S; X.sub.2 is V or
I; X.sub.3 is N or S; and X.sub.4 is K or R. In certain
embodiments, the core polypeptide is acetylated at the N-terminus.
In a specific embodiment, a core polypeptide comprises any one of
the amino acid sequences shown in FIG. 6E (SEQ ID NOS: 31-35) or a
fragment thereof. In certain embodiments, the core polypeptide is
acetylated at the N-terminus. In a specific embodiment, this core
polypeptide can be used to induce an immune response against
influenza virus strains of subtype H7. In certain embodiments, the
immune response induced neutralizes strains of influenza virus
subtype H7.
[0158] In certain embodiments, the core polypeptide is a fragment
of an H7 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H7 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H7 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H7 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H7 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H7 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0159] In some embodiments, the core polypeptide is an H7 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H7 core polypeptide that is
between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200, 51
to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50, 20
to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to 30,
20 to 30 or 20 to 25 amino acids in length. In some embodiments,
the core polypeptide is an 1-17 core polypeptide that is less than
500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150, 125, 100,
95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25 amino
acids in length. In certain embodiments, the core polypeptide is an
H7 polypeptide less than 150, 125, 95, 90, 85, 80, 75, 65, 60, 55,
50, 45 or 40 amino acids in length but at least 15, 20, 25, 30 or
35 amino acids in length.
[0160] In specific embodiments, the core polypeptide is a
derivative of an H7 core polypeptide, wherein the derivative
comprises an H7 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to either of the H7 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0161] In specific embodiments, the core polypeptide is a
derivative of an H7 core polypeptide, wherein the derivative
comprises an H7 core polypeptide with either 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10(.+-.5) amino acids attached to both of the H7 core
polypeptide's N- and C-termini and wherein the core polypeptide
maintains an alpha-helical conformation.
[0162] In a specific embodiment, the core polypeptide is an 1-18
core polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00017 (SEQ ID NO: 36)
RINMINDKIDDQIEX.sub.1LWAYNAELLVLLENQKTLDEHDSNVKNLFDEVKR RLSANA,
wherein X.sub.1 is a hydrophilic amino acid. In certain
embodiments, X.sub.1 is D or N. In a specific embodiment, a core
polypeptide comprises any one of the amino acid sequences shown in
FIG. 6H (SEQ ID NOS: 37 and 38) or a fragment thereof. In a
specific embodiment, this core polypeptide can be used to induce an
immune response against influenza virus strains of subtype H8. In
certain embodiments, the immune response induced neutralizes
strains of influenza virus subtype H8.
[0163] In certain embodiments, the core polypeptide is a fragment
of an H8 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H8 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H8 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H8 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H8 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H8 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0164] In some embodiments, the core polypeptide is an H8 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H8 core polypeptide that is
between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200, 51
to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50, 20
to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to 30,
20 to 30 or 20 to 25 amino acids in length. In some embodiments,
the core polypeptide is an H8 core polypeptide that is less than
500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150, 125, 100,
95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25 amino
acids in length. In certain embodiments, the core polypeptide is an
H8 polypeptide less than 150, 125, 95, 90, 85, 80, 75, 65, 60, 55,
50, 45 or 40 amino acids in length but at least 15, 20, 25, 30 or
35 amino acids in length.
[0165] In specific embodiments, the core polypeptide is a
derivative of an H8 core polypeptide, wherein the derivative
comprises an 1-18 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to either of the 1-18 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0166] In specific embodiments, the core polypeptide is a
derivative of an H8 core polypeptide, wherein the derivative
comprises an H8 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to both of the H8 core polypeptide's
N- and C-termini and wherein the core polypeptide maintains an
alpha-helical conformation.
[0167] In a specific embodiment, the core polypeptide is an H9 core
polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00018 (SEQ ID NO: 39)
RLNMINNKIDDQIQDX.sub.1WAYNAELLVLLENQKTLDEHDANVNNLYN KVKRALGSNA,
[0168] wherein X.sub.1 is a hydrophobic amino acid. In specific
embodiments X.sub.1 is V or I. In certain embodiments, the core
polypeptide is acetylated at the N-terminus. In a specific
embodiment, a core polypeptide comprises any one of the amino acid
sequences shown in FIG. 6I (SEQ ID NOS: 40 and 41) or a fragment
thereof. In certain embodiments, the core polypeptide is acetylated
at the N-terminus. In a specific embodiment, this core polypeptide
can be used to induce an immune response against influenza virus
strains of subtype H9. In certain embodiments, the immune response
induced neutralizes strains of influenza virus subtype H9.
[0169] In certain embodiments, the core polypeptide is a fragment
of an H9 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H9 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H9 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H9 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H9 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H9 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0170] In some embodiments, the core polypeptide is an H9 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H9 core polypeptide that is
between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200, 51
to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50, 20
to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to 30,
20 to 30 or 20 to 25 amino acids in length. In some embodiments,
the core polypeptide is an H9 core polypeptide that is less than
500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150, 125, 100,
95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25 amino
acids in length. In certain embodiments, the core polypeptide is an
H9 polypeptide less than 150, 125, 95, 90, 85, 80, 75, 65, 60, 55,
50, 45 or 40 amino acids in length but at least 15, 20, 25, 30 or
35 amino acids in length.
[0171] In specific embodiments, the core polypeptide is a
derivative of an H9 core polypeptide, wherein the derivative
comprises an H9 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to either of the H9 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0172] In specific embodiments, the core polypeptide is a
derivative of an H9 core polypeptide, wherein the derivative
comprises an H9 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10(.+-.5) amino acids attached to both of the H9 core polypeptide's
N- and C-termini and wherein the core polypeptide maintains an
alpha-helical conformation.
[0173] In a specific embodiment, the core polypeptide is an H10
core polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00019 (SEQ ID NO: 42)
QIGNVINWTKDSITDIWTYX.sub.1AELLVAMENQHTIDMADSEMLNLYER VRKQLRQNA,
wherein X.sub.1 is a hydrophilic amino acid. In specific
embodiments, X.sub.1 is Q or N. In certain embodiments, the core
polypeptide is acetylated at the N-terminus. In a specific
embodiment, a core polypeptide comprises any one of the amino acid
sequences shown in FIG. 6J (SEQ ID NOS: 43 and 44) or a fragment
thereof. In certain embodiments, the core polypeptide is acetylated
at the N-terminus. In a specific embodiment, this core polypeptide
can be used to induce an immune response against influenza virus
strains of subtype H10. In certain embodiments, the immune response
induced neutralizes strains of influenza virus subtype H10.
[0174] In certain embodiments, the core polypeptide is a fragment
of an H10 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H10 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H10 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H10 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H10 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H10 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0175] In some embodiments, the core polypeptide is an H10 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H10 core polypeptide that
is between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200,
51 to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50,
20 to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to
30, 20 to 30 or 20 to 25 amino acids in length. In some
embodiments, the core polypeptide is an H10 core polypeptide that
is less than 500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150,
125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or
25 amino acids in length. In certain embodiments, the core
polypeptide is an H10 polypeptide less than 150, 125, 95, 90, 85,
80, 75, 65, 60, 55, 50, 45 or 40 amino acids in length but at least
15, 20, 25, 30 or 35 amino acids in length.
[0176] In specific embodiments, the core polypeptide is a
derivative of an H10 core polypeptide, wherein the derivative
comprises an H10 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(+5) amino acids attached to either of the H10 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0177] In specific embodiments, the core polypeptide is a
derivative of an H10 core polypeptide, wherein the derivative
comprises an H10 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to both of the H10 core
polypeptide's N- and C-termini and wherein the core polypeptide
maintains an alpha-helical conformation.
[0178] In a specific embodiment, the core polypeptide is an H11
core polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00020 (SEQ ID NO: 45)
RINQLSKHVDDSVX.sub.1DIWSYNAQLLVLLENEKTLDLHDSNVRNLHEK VRRMLKDNA,
[0179] wherein X.sub.1 is a hydrophobic amino acid. In specific
embodiments, X.sub.1 is V or I. In certain embodiments, the core
polypeptide is acetylated at the N-terminus. In a specific
embodiment, a core polypeptide comprises any one of the amino acid
sequences shown in FIG. 6K (SEQ ID NOS: 46 and 47) or a fragment
thereof. In certain embodiments, the core polypeptide is acetylated
at the N-terminus. In a specific embodiment, this core polypeptide
can be used to induce an immune response against influenza virus
strains of subtype H11. In certain embodiments, the immune response
induced neutralizes strains of influenza virus subtype H11.
[0180] In certain embodiments, the core polypeptide is a fragment
of an H11 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H11 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(+5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H11 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H11 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C=terminus. In specific embodiments, the core polypeptide is a
fragment of an H11 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H11 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0181] In some embodiments, the core polypeptide is an H11 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H11 core polypeptide that
is between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200,
51 to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50,
20 to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to
30, 20 to 30 or 20 to 25 amino acids in length. In some
embodiments, the core polypeptide is an H11 core polypeptide that
is less than 500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150,
125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or
25 amino acids in length. In certain embodiments, the core
polypeptide is an H11 polypeptide less than 150, 125, 95, 90, 85,
80, 75, 65, 60, 55, 50, 45 or 40 amino acids in length but at least
15, 20, 25, 30 or 35 amino acids in length.
[0182] In specific embodiments, the core polypeptide is a
derivative of an H11 core polypeptide, wherein the derivative
comprises an H11 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to either of the H11 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0183] In specific embodiments, the core polypeptide is a
derivative of an H11 core polypeptide, wherein the derivative
comprises an H11 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to both of the H11 core
polypeptide's N- and C-termini and wherein the core polypeptide
maintains an alpha-helical conformation.
[0184] In a specific embodiment, the core polypeptide is an H12
core polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00021 (SEQ ID NO: 48)
RINMINSKIDDQITDIWAYNAELLVLLENQKTLDEHDANVRNLHDRV
RRX.sub.1LX.sub.2ENA,
[0185] wherein X.sub.1 is a hydrophobic amino acid and X.sub.2 is a
hydrophilic, basic amino acid. In specific embodiments, X.sub.1 is
V or I and X.sub.2 is R or K. In certain embodiments, the core
polypeptide is acetylated at the N-terminus. In a specific
embodiment, a core polypeptide comprises any one of the amino acid
sequences shown in FIG. 6L (SEQ ID NOS: 49 and 50) or a fragment
thereof. In certain embodiments, the core polypeptide is acetylated
at the N-terminus. In a specific embodiment, this core polypeptide
can be used to induce an immune response against influenza virus
strains of subtype H12. In certain embodiments, the immune response
induced neutralizes strains of influenza virus subtype H12.
[0186] In certain embodiments, the core polypeptide is a fragment
of an H12 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H12 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H12 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H12 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H12 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H12 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0187] In some embodiments, the core polypeptide is an H12 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H12 core polypeptide that
is between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200,
51 to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50,
20 to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to
30, 20 to 30 or 20 to 25 amino acids in length. In some
embodiments, the core polypeptide is an H12 core polypeptide that
is less than 500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150,
125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or
25 amino acids in length. In certain embodiments, the core
polypeptide is an H12 polypeptide less than 150, 125, 95, 90, 85,
80, 75, 65, 60, 55, 50, 45 or 40 amino acids in length but at least
15, 20, 25, 30 or 35 amino acids in length.
[0188] In specific embodiments, the core polypeptide is a
derivative of an H12 core polypeptide, wherein the derivative
comprises an H12 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to either of the H12 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0189] In specific embodiments, the core polypeptide is a
derivative of an H12 core polypeptide, wherein the derivative
comprises an H12 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to both of the H12 core
polypeptide's N- and C-termini and wherein the core polypeptide
maintains an alpha-helical conformation.
[0190] In a specific embodiment, the core polypeptide is an H13
core polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00022 (SEQ ID NO: 51)
RINMLADRIDDAVTDX.sub.1WSYNAKLLVLLENDKTLDMHDANVRNLHX.sub.2 QVRR
X.sub.3LKX.sub.4NA,
[0191] wherein X.sub.1 is a hydrophobic amino acid; X.sub.2 is a
hydrophilic, acidic amino acid; X.sub.3 is A, S or E and X.sub.4 is
a hydrophilic amino acid. In specific embodiments, X.sub.I is V or
I; X.sub.2 is D or E; X.sub.3 is A, S or E and X.sub.4 is T or D.
In certain embodiments, the core polypeptide is acetylated at the
N-terminus. In a specific embodiment, a core polypeptide comprises
any one of the amino acid sequences shown in FIG. 6M (SEQ ID NOS:
52-54) or a fragment thereof. In certain embodiments, the core
polypeptide is acetylated at the N-terminus. In a specific
embodiment, this core polypeptide can be used to induce an immune
response against influenza virus strains of subtype H13. In certain
embodiments, the immune response induced neutralizes strains of
influenza virus subtype H13.
[0192] In certain embodiments, the core polypeptide is a fragment
of an H13 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H13 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H13 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H13 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H13 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H13 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0193] In some embodiments, the core polypeptide is an H13 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H13 core polypeptide that
is between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200,
51 to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50,
20 to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to
30, 20 to 30 or 20 to 25 amino acids in length. In some
embodiments, the core polypeptide is an H13 core polypeptide that
is less than 500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150,
125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or
25 amino acids in length. In certain embodiments, the core
polypeptide is an H13 polypeptide less than 150, 125, 95, 90, 85,
80, 75, 65, 60, 55, 50, 45 or 40 amino acids in length but at least
15, 20, 25, 30 or 35 amino acids in length.
[0194] In specific embodiments, the core polypeptide is a
derivative of an H13 core polypeptide, wherein the derivative
comprises an H13 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to either of the H13 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0195] In specific embodiments, the core polypeptide is a
derivative of an H13 core polypeptide, wherein the derivative
comprises an H13 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to both of the H13 core
polypeptide's N- and C-termini and wherein the core polypeptide
maintains an alpha-helical conformation.
[0196] In a specific embodiment, the core polypeptide is an H14
core polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00023 (SEQ ID NO: 55)
RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDVTDSEMNKLFERV RRQLRENA.
In certain embodiments, the core polypeptide is acetylated at the
N-terminus. In a specific embodiment, a core polypeptide comprises
any one of the amino acid sequences shown in FIG. 6N (SEQ ID NO:
55) or a fragment thereof. In certain embodiments, the core
polypeptide is acetylated at the N-terminus. In a specific
embodiment, this core polypeptide can be used to induce an immune
response against influenza virus strains of subtype H14. In certain
embodiments, the immune response induced neutralizes strains of
influenza virus subtype H14.
[0197] In certain embodiments, the core polypeptide is a fragment
of an H14 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H14 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H14 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H14 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H14 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H14 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0198] In some embodiments, the core polypeptide is an H14 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H14 core polypeptide that
is between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200,
51 to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50,
20 to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to
30, 20 to 30 or 20 to 25 amino acids in length. In some
embodiments, the core polypeptide is an H14 core polypeptide that
is less than 500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150,
125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or
25 amino acids in length. In certain embodiments, the core
polypeptide is an H14 polypeptide less than 150, 125, 95, 90, 85,
80, 75, 65, 60, 55, 50, 45 or 40 amino acids in length but at least
15, 20, 25, 30 or 35 amino acids in length.
[0199] In specific embodiments, the core polypeptide is a
derivative of an H14 core polypeptide, wherein the derivative
comprises an H14 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to either of the H14 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0200] In specific embodiments, the core polypeptide is a
derivative of an 1-114 core polypeptide, wherein the derivative
comprises an H14 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to both of the H14 core
polypeptide's N- and C-termini and wherein the core polypeptide
maintains an alpha-helical conformation.
[0201] In a specific embodiment, the core polypeptide is an H15
core polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00024 (SEQ ID NO: 56)
QIGNVINWTRDSLTEIWSYNAELLVAMENQHTIDLADSEMNKLYERV RRQLRENA.
In certain embodiments, the core polypeptide is acetylated at the
N-terminus. In a specific embodiment, a core polypeptide comprises
any one of the amino acid sequences shown in FIG. 6O or a fragment
thereof. In certain embodiments, the core polypeptide is acetylated
at the N-terminus. In a specific embodiment, this core polypeptide
can be used to induce an immune response against influenza virus
strains of subtype H15. In certain embodiments, the immune response
induced neutralizes strains of influenza virus subtype H15
[0202] In certain embodiments, the core polypeptide is a fragment
of an H15 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H15 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(.+-.5), 30(.+-.5), or 35(.+-.5) amino acids from
either of an H15 core polypeptide's N- or C-terminus. In some
embodiments, the core polypeptide is a fragment of an H15 core
polypeptide, wherein the fragment lacks 24(.+-.5) amino acids from
its C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H15 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H15 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0203] In some embodiments, the core polypeptide is an H15 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H15 core polypeptide that
is between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200,
51 to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50,
20 to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to
30, 20 to 30 or 20 to 25 amino acids in length. In some
embodiments, the core polypeptide is an H15 core polypeptide that
is less than 500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150,
125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or
25 amino acids in length. In certain embodiments, the core
polypeptide is an H15 polypeptide less than 150, 125, 95, 90, 85,
80, 75, 65, 60, 55, 50, 45 or 40 amino acids in length but at least
15, 20, 25, 30 or 35 amino acids in length.
[0204] In specific embodiments, the core polypeptide is a
derivative of an H15 core polypeptide, wherein the derivative
comprises an H15 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to either of the H15 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0205] In specific embodiments, the core polypeptide is a
derivative of an H15 core polypeptide, wherein the derivative
comprises an H15 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to both of the H15 core
polypeptide's N- and C-termini and wherein the core polypeptide
maintains an alpha-helical conformation.
[0206] In a specific embodiment, the core polypeptide is an H16
core polypeptide comprising or consisting of the amino acid
sequence:
TABLE-US-00025 (SEQ ID NO: 57)
RINMLADRVDDAVTDIWSYNAKLLVLX.sub.1ENDRTLDLHDANVX.sub.2NLH
X.sub.3QVKRALKX.sub.4NA,
[0207] wherein X.sub.1 is a hydrophobic amino acid; X.sub.2 is a
hydrophilic, basic amino acid; X.sub.3 is a hydrophilic, acidic
amino acid and X.sub.4 is a hydrophilic amino acid. In specific
embodiments, X.sub.1 is L or I; X.sub.2 is K or R; X.sub.3 is D or
E and X.sub.4 is S or N. In certain embodiments, the core
polypeptide is acetylated at the N-terminus. In a specific
embodiment, a core polypeptide comprises any one of the amino acid
sequences shown in FIG. 6P (SEQ ID NOS: 58-60) or a fragment
thereof. In certain embodiments, the core polypeptide is acetylated
at the N-terminus. In a specific embodiment, this core polypeptide
can be used to induce an immune response against influenza virus
strains of subtype H16. In certain embodiments, the immune response
induced neutralizes strains of influenza virus subtype H16.
[0208] In certain embodiments, the core polypeptide is a fragment
of an H16 core polypeptide. In specific embodiments, the core
polypeptide is a fragment of an H16 core polypeptide, wherein the
fragment lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10(.+-.5), 15(.+-.5),
20(.+-.5), 25(+5), 30(.+-.5), or 35(.+-.5) amino acids from either
of an H16 core polypeptide's N- or C-terminus. In some embodiments,
the core polypeptide is a fragment of an H16 core polypeptide,
wherein the fragment lacks 24(.+-.5) amino acids from its
C-terminus. In specific embodiments, the core polypeptide is a
fragment of an H16 core polypeptide, wherein the fragment lacks 1,
2, 3, 4, 5, or more amino acids from both of an H16 core
polypeptide's N- and C-termini. In specific embodiments, the core
polypeptide has an alpha-helical conformation.
[0209] In some embodiments, the core polypeptide is an H16 core
polypeptide that is not full length influenza virus HA. In some
embodiments, the core polypeptide is an H16 core polypeptide that
is between 51 to 300, 51 to 275, 51 to 250, 51 to 225, 51 to 200,
51 to 175, 51 to 150, 51 to 125, 51 to 100, or 51 to 75, 15 to 50,
20 to 50, 25 to 50, 15 to 37, 15 to 35, 20 to 37, 20 to 35, 15 to
30, 20 to 30 or 20 to 25 amino acids in length. In some
embodiments, the core polypeptide is an H16 core polypeptide that
is less than 500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150,
125, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or
25 amino acids in length. In certain embodiments, the core
polypeptide is an H16 polypeptide less than 150, 125, 95, 90, 85,
80, 75, 65, 60, 55, 50, 45 or 40 amino acids in length but at least
15, 20, 25, 30 or 35 amino acids in length.
[0210] In specific embodiments, the core polypeptide is a
derivative of an H16 core polypeptide, wherein the derivative
comprises an H16 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to either of an H16 core
polypeptide's N- or C-terminus and wherein the core polypeptide
maintains an alpha-helical conformation.
[0211] In specific embodiments, the core polypeptide is a
derivative of an H16 core polypeptide, wherein the derivative
comprises an H16 core polypeptide with 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10(.+-.5) amino acids attached to both of an H16 core
polypeptide's N- and C-termini and wherein the core polypeptide
maintains an alpha-helical conformation.
[0212] In specific embodiments, the core polypeptide comprises or
consists of the sequence:
TABLE-US-00026 EILELDEKVDDLRADTISSQIELAVLLSNEGIINSEDEHLLALERKLKK
MLGPSA.
In certain embodiments, the core polypeptide is acetylated at the
N-terminus.
[0213] In a specific embodiment, a core polypeptide comprises or
consists of any one of the amino acid sequences shown in FIG. 5A or
a fragment thereof. In certain embodiments, the core polypeptide is
acetylated at the N-terminus. In a specific embodiment, a core
polypeptide comprises or consists of any one of the amino acid
sequences shown in FIG. 5B or fragment thereof. In some
embodiments, the core polypeptide is between 51 to 300, 51 to 275,
51 to 250, 51 to 225, 51 to 200, 51 to 175, 51 to 150, 51 to 125,
51 to 100, or 51 to 75, 15 to 50, 20 to 50, 25 to 50, 15 to 37, 15
to 35, 20 to 37, 20 to 35, 15 to 30, 20 to 30 or 20 to 25 amino
acids in length. In some embodiments, the core polypeptide is less
than 500, 450, 400, 350, 300, 275, 250, 225, 200, 175, 150, 125,
100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30 or 25
amino acids in length. In certain embodiments, the core polypeptide
is less than 150, 125, 95, 90, 85, 80, 75, 65, 60, 55, 50, 45 or 40
amino acids in length but at least 15, 20, 25, 30 or 35 amino acids
in length.
[0214] 5.1.2 Flu Polypeptides and Core Polypeptides with Increased
Half-Life
[0215] In some embodiments, the flu polypeptides and core
polypeptides described herein are modified to have an extended (or
increased) half-life in vivo (i.e., modified core polypeptides). In
particular, provided herein are modified flu and core polypeptides
which have a half-life in a subject of from about 3 days to about
180 days (or more), and in some embodiments greater than 3 days,
greater than 7 days, greater than 10 days, greater than 15 days,
greater than 20 days, greater than 25 days, greater than 30 days,
greater than 35 days, greater than 40 days, greater than 45 days,
greater than 50 days, at least about 60 days, greater than 75 days,
greater than 90 days, greater than 105 days, greater than 120 days,
greater than 135 days, greater than 150 days, greater than 165
days, or greater than 180 days.
[0216] In some embodiments, flu or core polypeptides having an
increased half-life in vivo are generated by acetylation of the
N-terminus of the flu or core polypeptides. Acetylation of
polypeptides is a technique well-known to those of skill in the art
and comprises the addition of an acetyl group to the N-terminus of
the polypeptide. Acetylation of the core polypeptide can render the
core polypeptide less vulnerable to degradation by
exopeptidases.
[0217] In some embodiments, flu or core polypeptides having an
increased half-life in vivo are generated by amidation of the
C-terminus of the flu or core polypeptides.
[0218] In some embodiments, flu or core polypeptides having an
increased half-life in vivo are generated by pegylation, i.e.,
attaching inert polymer molecules such as high molecular weight
polyethyleneglycol (PEG) to the flu or core polypeptides with or
without a multifunctional linker either through site-specific
conjugation of the PEG to the N- or C-terminus of the flu or core
polypeptides or via epsilon-amino groups present on lysine
residues. PEG of various average molecular weights can be used such
as 1000 Da, 4000 Da, 5000 Da, 8000 Da, 10000 Da, 120000 Da or even
higher. In a specific embodiment, the N-terminus of the flu or core
polypeptides is pegylated. Linear or branched polymer
derivatization that results in minimal loss of biological activity
can be used. The degree of conjugation can be closely monitored by
SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG
molecules to the flu or core polypeptides. Unreacted PEG can be
separated from flu or core polypeptide-PEG conjugates by
size-exclusion or by ion-exchange chromatography. PEG-derivatized
flu or core polypeptides can be tested for in vivo efficacy using
methods well-known to those of skill in the art, for example, by
using animal model systems described herein.
[0219] In another embodiment, flu or core polypeptides can be
conjugated to albumin in order to make the core polypeptides more
stable in vivo or have a longer half-life in vivo. Such techniques
are well-known in the art, see, e.g., International Publication
Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent
No. EP 413,622, all of which are incorporated herein by
reference.
[0220] In some embodiments, flu or core polypeptides having an
increased half-life in vivo are generated by substitution of
terminal L-amino acids of the core polypeptides with D-amino
acids.
[0221] 5.1.3 Flu Polypeptides Comprising a Core Polypeptide and a
Linker
[0222] In some embodiments, the flu polypeptides described herein
comprises a core polypeptide or modified core polypeptide linked to
a linker. The linkers encompassed herein can be any linker known to
those of skill in the art that does not interfere with the native
structure of the core polypeptide with which the linker is
associated. In specific embodiments, the linkers encompassed herein
are not hydrophobic.
[0223] The length of the linker may be varied to provide optimal
linkage between a core polypeptide or a modified core polypeptide
described herein and a substrate (e.g., carrier protein, T cell
epitope, immunogenic polypeptide) to which the core polypeptide or
modified core polypeptide is to be linked. Further, the length of
the linker may be optimized to prevent immunogenic responses due to
linker. Linker molecules are commonly known in the art and
described in Denardo et al., 1998, Clin. Cancer Res. 4:2483-90;
Peterson et al., 1999, Bioconjug. Chem. 10:553; and Zimmerman et
al., 1999, Nucl. Med. Biol. 26:943-50 each of which is incorporated
by reference herein in its entirety.
[0224] Linkers may be one, two, three, four, five, six, seven,
eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,
twenty, or more than twenty amino acids in length. In some
embodiments, the linker is less than 20 amino acids in length. In
some embodiments, the linker is less than 15 amino acids in length.
In some embodiments, the linker is less than 10 amino acids in
length. In some embodiments, the linker is less than 9 amino acids
in length. In some embodiments, the linker is less than 8 amino
acids in length. In some embodiments, the linker is less than 7
amino acids in length. In some embodiments, the linker is less than
6 amino acids in length. In some embodiments, the linker is less
than 5 amino acids in length. In some embodiments, the linker is
less than 4 amino acids in length. In some embodiments, the linker
is less than 3 amino acids in length. In some embodiments, the
linker is less than 2 amino acids in length.
[0225] In some embodiments, a linker is between 1 and 50 amino
acids in length. In some embodiments, a linker is between 1 to 40
amino acids, 1 to 30 amino acids, 1 to 20 amino acids, 1 to 10
amino acids, 1 to 5 amino acids, 1 to 4 amino acids, 1 to 3 amino
acids, 1 to 2 amino acids or 1 amino acid in length.
[0226] In some embodiments, the linker is covalently attached to
the core polypeptide or modified core polypeptide. In specific
embodiments, the linker is attached to the core polypeptide or
modified core polypeptide through a peptide bond. In some
embodiments, the linker is attached to the N-terminus of the core
polypeptide or modified core polypeptide. In some embodiments, the
linker is attached to the C-terminus of the core polypeptide or
modified core polypeptide.
[0227] In some embodiments, the linker comprises one or more
glycine residues. In some embodiments, the linker comprises two or
more glycine residues. In some embodiments, the linker comprises
three or more glycine residues. In some embodiments, the linker
comprises four or more glycine residues. In some embodiments, the
linker comprises five or more glycine residues. In some
embodiments, the linker comprises ten or more glycine residues. In
some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 or more glycine residues. In some embodiments, the linker
comprises 2 to 4, 2 to 6, 2 to 10, 3 to 6, 3 to 8, 3 to 10, 5 to
10, 8 to 10, 10 to 15 or 10 to 20 glycine residues. In a specific
embodiment, the linker comprises three glycine residues.
[0228] In some embodiments, the linker comprises one or more
cysteine amino acid residues. In some embodiments, the linker
comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more cysteine
residues. In some embodiments, the linker comprises 2 to 4, 2 to 6,
2 to 10, 3 to 6, 3 to 8, 3 to 10, 5 to 10, 8 to 10, 10 to 15 or 10
to 20 cysteine residues.
[0229] In certain embodiments, the linker comprises a protein tag.
Protein tags can be useful for the isolation of protein complexes,
isolation of a flu polypeptide, affinity chromatography and/or
localization studies. In addition, protein tags may increase the
solubility of a flu polypeptide. Examples of protein tags include,
but are not limited to, His tag, Strep II tag, T7-tag, FLAG-tag,
S-tag, HA tag, c-Myc tag, DHFR tag, and green fluorescent protein
(GFP). Protein tags may be covalently attached to the N- or
C-terminus of the flu polypeptide. In some embodiments, the linker
comprises a FLAG-Tag protein tag, i.e., the linker comprises the
amino acid DYKDDDDK. In specific embodiments, the linker comprises
a FLAG-Tag covalently linked to a cysteine residue (i.e.,
DYKDDDDKC). In some embodiments, a flu polypeptide comprises 1, 2,
3, 4 or more protein tags. In certain embodiments, the protein tag
is not used as a linker in a flu polypeptide.
[0230] 5.1.4 Flu Polypeptides Comprising Multiple Core
Polypeptides
[0231] In certain embodiments, provided herein are flu polypeptides
comprising two or more core polypeptides or modified core
polypeptides. The two or more core polypeptides or modified core
polypeptides can be directly or indirectly linked/coupled to each
other. Without being bound to any particular theory of operation,
it is believed that administration of flu polypeptides comprising
two, three or more core polypeptides or modified core polypeptides
can elicit serum antibodies with broad reactivity within a subject
without co-administration of a carrier protein. In certain
embodiments, the core polypeptides or modified core polypeptides
are linked together via a linker such as described in Section 5.1.3
supra. In other words, in certain embodiments, a flu polypeptide
can comprise, for example, a core polypeptide or modified core
polypeptide, linked to a linker, which is in turned linked to a
core polypeptide or modified core polypeptide.
[0232] In a specific embodiment, a flu polypeptide has a sequence
X-(L-X).sub.n, wherein X is any core polypeptide or modified core
polypeptide described herein, L is any linker described herein,
n=1-20 and wherein X is covalently linked to L. In a specific
embodiment, the flu polypeptide comprises the sequence X-L-X. In a
specific embodiment, the flu polypeptide comprises the sequence
X-L-X-L-X. In a specific embodiment, the flu polypeptide comprises
the sequence X-L-X-L-X-L-X. In a specific embodiment, the flu
polypeptide comprises the sequence X-L-X-L-X-L-X-L-X. In a specific
embodiment, the flu polypeptide comprises the sequence
X-L-X-L-X-L-X-L-X-L-X. In a specific embodiment, the flu
polypeptide comprises the sequence X-L-X-L-X-L-X-L-X-L-X-L-X. In a
specific embodiment, the flu polypeptide comprises the sequence
X-L-X-L-X-L-X-L-X-L-X-L-X-L-X. In a specific embodiment, the flu
polypeptide comprises the sequence
X-L-X-L-X-L-X-L-X-L-X-L-X-L-X-L-X. In a specific embodiment, the
flu polypeptide comprises the sequence
X-L-X-L-X-L-X-L-X-L-X-L-X-L-X-L-X-L-X. In a specific embodiment,
the flu polypeptide comprises the sequence
X-L-X-L-X-L-X-L-X-L-X-L-X-L-X-L-X-L-X-L-X.
[0233] In some embodiments, L is absent. In other words, each of
the core polypeptide or modified core polypeptide of the flu
polypeptide is directly linked to one or more other core
polypeptide (e.g., X-X, X-X-X, X-X-X-X, X-X-X-X-X, etc.)
[0234] In certain embodiments, a flu polypeptide comprises two or
more of the same core polypeptides or modified core polypeptides.
In other embodiments, the flu polypeptide comprises or two or more
different core polypeptides or core polypeptides. In a specific
embodiment, the flu polypeptide comprises two or more of the same
core polypeptides or modified core polypeptides. In a specific
embodiment, the flu polypeptide comprises three or more of the same
core polypeptides or modified core polypeptides. In a specific
embodiment, the flu polypeptide comprises four or more of the same
core polypeptides or modified core polypeptides. In a specific
embodiment, the flu polypeptide comprises five or more of the same
core polypeptides or modified core polypeptides. In a specific
embodiment, each of the core polypeptide or modified core
polypeptide of the flu polypeptide is the same.
[0235] In a specific embodiment, the flu polypeptide comprises two
or more different core polypeptides or modified core polypeptides.
In a specific embodiment, the flu polypeptide comprises three or
more different core polypeptides or modified core polypeptides. In
a specific embodiment, the flu polypeptide comprises four or more
different core polypeptides or modified core polypeptides. In a
specific embodiment, the flu polypeptide comprises five or more
different core polypeptides or modified core polypeptides.
[0236] In some embodiments, a flu polypeptide comprises the
sequence X-L-X-L-X wherein X is a core polypeptide comprising the
amino acid sequence RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKT
RRQLRENA (SEQ ID NO:2) and L is a linker consisting of three
glycine residues. In other embodiments, X is not
RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKT RRQLRENA (SEQ ID
NO: 2) and is the same or varies.
[0237] In some embodiments, a flu polypeptide, in addition to
comprising two or more modified core polypeptides, comprises one,
two, three or more or all of the following: a T cell epitope, a
polypeptide that facilitates multimerization (e.g., a T4 foldon
domain), a protein tag, or an immunogenic polypeptide as described
herein. In a specific embodiment, the flu polypeptide comprises a
His-tag at its N-terminus. In a specific embodiment, a flu
polypeptide comprises a FLAG-tag at its C-terminus. In a specific
embodiment, a flu polypeptide comprises a His-tag at its N-terminus
and a FLAG-tag at its C-terminus. In a specific embodiment, a flu
polypeptide comprises an influenza A nuclear protein (NP) at its
C-terminus. In certain embodiments, a flu polypeptide comprises an
FIjB flagellin at its C-terminus.
[0238] In some embodiments, a flu polypeptide comprises the amino
acid sequence H-X-L-X-L-X-F, wherein H is a His tag or another
protein tag that facilitates purification and/or solubility of the
flu polypeptide, X is a core polypeptide or a modified core
polypeptide, L is a linker and F is a FLAG-tag or another protein
tag different from H that facilitates the purification and/or
solubility of the flu polypeptide. In some embodiments, L is 3
glycine residues. In some embodiments, L is the same and in other
embodiments L is different.
[0239] 5.1.5 Flu Polypeptides Comprising a T Cell Epitope
[0240] In certain embodiments, a flu polypeptide comprises a core
polypeptide or modified core polypeptide described herein and a T
cell epitope (e.g., CD4 or CD8 T cell epitope). In a specific
embodiment, a flu polypeptide comprises a core polypeptide or
modified core polypeptide and a CD4 T cell epitope. In a specific
embodiment, a flu polypeptide comprises a core polypeptide or
modified core polypeptide and a CD8 T cell epitope. In another
specific embodiment, the T-cell epitope is an influenza virus CD8 T
cell epitope (e.g., an influenza virus protein that contains a
highly conserved T cell epitope). The T cell epitope can be
directly or indirectly linked/coupled to a core polypeptide by a
modified core polypeptide.
[0241] Without being bound to any particular theory of operation,
it is believed that flu polypeptides comprising a core polypeptide
or modified core polypeptide and a CD8 T cell epitope can elicit
broadly neutralizing antibodies and prime for a broad spectrum CD8
T cell response. Highly conserved influenza virus regions that
contain CD8 T cell epitopes are found, for example, in influenza
nuclear protein (NP), matrix 1 (M1), neuraminidase (NA) and
polymerase basic-1 (PB1). See Alexander et al., 2010, Hum Immunol
71: 468-74, incorporated by reference herein in its entirety. In
some embodiments, the CD8 T cell epitope is a nuclear protein (NP)
or fragment thereof. In other embodiments, the CD8 T cell epitope
polypeptide is a matrix 1 (M1) protein or fragment the or fragment
thereof
[0242] In a specific embodiment, a flu polypeptide comprises a core
polypeptide or a modified core polypeptide linked to a T cell
epitope. The T cell epitope (e.g., CD8 T cell epitope) can be
attached to the core polypeptide or modified core polypeptide
described herein using any technique known to one of skill in the
art, including, but not limited to, single point attachment to a
primary amino group or sulfhydryl group using amine to amine
crosslinker BS (Bis[sulfosuccinimidyl]suberate), an
amine-to-sulfhydryl NHS-PEG-Maleimide cross linker, or a
sulfhydryl-to-sulfhydryl BM(PEG).sub.n PEG crosslinker. In other
embodiments, the T cell epitope (e.g., CD8 T cell epitope) is
covalently linked to the N- or C-terminus of a flu polypeptide. In
other embodiments, the T cell epitope (e.g., CD8 T cell epitope) is
linked to a core polypeptide or modified core polypeptide via a
linker as described herein in Section 5.1.3 supra.
[0243] In certain embodiments, a flu polypeptide in addition to
comprising one or more core polypeptides or modified core
polypeptides and a T cell epitope, comprises one, two, three or
more or all of the following: a protein tag, an immunogenic
polypeptide, a carrier and/or a polypeptide that facilitates the
multimerization of the flu polypeptide (e.g., a T4 foldon
domain).
[0244] In some embodiments, a flu polypeptide comprises one, two,
three, four or more core polypeptides or modified core polypeptides
and a T cell epitope(s). In specific embodiments, a flu polypeptide
comprises the formula H-X-L-X-L-X-F-T, wherein H (which is
optional) is a His tag or another protein tag that facilitates
purification and/or solubility, X is a core polypeptide or a
modified core polypeptide, L is a linker (such as described in
Section 5.1.3), F (which is optional) is a FLAG-tag or another
protein tag different that H which facilitates purification and/or
solubility, and T is a T cell epitope. In certain embodiments, the
polypeptide comprises the formula H-X-L-X-L-X-T, wherein H (which
is optional) is a His tag or another protein tag that facilitates
purification and/or solubility, X is a core polypeptide or a
modified core polypeptide, L is a linker (such as described in
Section 5.1.3), and T is a T cell epitope.
[0245] In certain embodiments, L is three glycine residues. In some
embodiments, L is the same each time it occurs in the formula and
in other embodiments, L is different or varies each time it occurs
in the formula. In certain embodiments, there is linker between F
and T. In other embodiments, F is directly linked to T. In certain
embodiments, the core polypeptide or modified core polypeptide is
the same each time it occurs in the formula. In other embodiments,
the core polypeptide or modified core polypeptide is different or
varies each time it occurs in the formula.
[0246] In some embodiments, there is a linker between X and T. In
other embodiments, X is directly linked to T.
[0247] 5.1.6 Flu Polypeptide Comprising an Immunogenic
Polypeptide
[0248] In certain embodiments provided herein, a flu polypeptide
comprises one or more core polypeptides or modified core
polypeptides and an immunogenic polypeptide. In a specific
embodiment, a flu polypeptide comprises a core polypeptide or a
modified core polypeptide directly or indirectly linked/coupled to
an immunogenic polypeptide. The immunogenic polypeptide can be
linked/coupled to the N- and/or C-terminus of the core polypeptide
or modified core polypeptide. In certain embodiments, the
immunogenic polypeptide is linked to the core polypeptide or
modified core polypeptide via a linker such as described in Section
5.1.3 supra.
[0249] Examples of immunogenic polypeptides include, but are not
limited to, Toll Like Receptor (TLR) ligands, such as the
Salmonella flagellin (a Toll like receptor 5 ligand). See, e.g.,
Huleatt et al., 2008, Vaccine 26: 201-14; Song et al., 2009,
Vaccine 27: 5875-84; and Wang et al., 2010, PLos One 5: e13972. In
a specific embodiment, a flu polypeptide comprises a core
polypeptide linked to FljB flagellin from Salmonella enterica.
[0250] In certain embodiments, a flu polypeptide comprises one or
more core polypeptides or modified core polypeptides and an
immunogenic polypeptide. In certain embodiments, a flu polypeptide,
in addition to comprising one or more core polypeptides or modified
polypeptides and an immunogenic polypeptide, comprises one, two
three, or more or all of the following: a protein tag that
facilitates purification and/or increases solubility of the flu
polypeptide, a T cell epitope and/or a polypeptide (e.g., a T4
foldon domain) that facilitates multimerization of the flu
polypeptide.
[0251] In certain embodiments, a flu polypeptide comprises two,
three, four or more core polypeptides or modified core polypeptides
and an immunogenic polypeptide(s). In specific embodiments, a flu
polypeptide comprises H-X-L-X-L-X-F-I, wherein H is an optional His
tag or another protein tag that facilitates purification and/or
solubility, X is a core polypeptide or modified core polypeptide, L
is an optional linker, such as described in Section 5.1.3, F is an
optional FLAG-tag or another protein tag different than H that
facilitates purification and/or solubility of the flu polypeptide,
and I is an immunogenic polypeptide. In certain embodiments, L is
three glycine residues. In some embodiments, L is the same
throughout the flu polypeptide and in other embodiments, L is
different throughout the polypeptide. In certain embodiments there
is a linker between F and I. In other embodiments, F is directly
linked to I.
[0252] In specific embodiments, a flu polypeptide comprises
H-X-L-X-L-X-I, wherein H is an optional His tag or another protein
tag that facilitates purification and/or solubility, X is a core
polypeptide or modified core polypeptide, L is an optional linker,
such as described in Section 5.1.3, and I is an immunogenic
polypeptide. In certain embodiments, L is three glycine residues.
In some embodiments, L is the same throughout the flu polypeptide
and in other embodiments, L is different throughout the
polypeptide. In certain embodiments there is a linker between X and
I. In other embodiments, X is directly linked to I.
[0253] 5.1.7 Flu Polypeptides Comprising a Carrier
[0254] In some embodiments, a flu polypeptides comprise a core
polypeptide or modified core polypeptide described herein and a
carrier. In a specific embodiment, a flu polypeptide comprises a
core polypeptide or a modified core polypeptide coupled/linked to a
carrier. The core polypeptide or modified core polypeptide can be
directly or indirectly linked/coupled to a carrier. A core
polypeptide or modified core polypeptide described herein can be
coupled/linked (e.g., directly linked by a linker) to a carrier,
including but not limited to, tetanus toxoid (e.g.,
chemically-inactivated tetanus toxin), diphtheria toxin (e.g.,
chemically-inactivated diphtheria toxoid or CRM 197--a non-toxic
diphtheria toxin point mutant), keyhole limpet hemocyanin (KLH),
bovine serum albumin, ovalbumin, thyroglobulin or meningococcal
outer membrane protein, using methods known to those of skill in
the art. In specific embodiments, a core polypeptide(s) or modified
core polypeptide(s) described herein are linked to KLH.
[0255] In certain embodiments, a core polypeptide(s) or modified
core polypeptide(s) described herein are directly linked to a
carrier protein, i.e., the core polypeptide or modified core
polypeptide and carrier protein are linked to one another without
an intervening linker molecule. In certain embodiments, the core
polypeptide(s) or modified core polypeptide(s) described herein are
linked to a carrier protein by a linker. In specific embodiments, a
core polypeptide(s) or modified core polypeptide(s) described
herein is linked to a carrier protein by a linker described in
section 5.1.3, supra.
[0256] In certain embodiments, a flu polypeptide comprises a core
polypeptide or modified core polypeptide coupled/linked to more
than one carrier. In specific embodiments, a flu polypeptide
comprises a core polypeptide or modified core polypeptide
coupled/linked to 2, 3, 4, 5 or more carriers.
[0257] In certain embodiments, 2, 3, 4, 5, 6 or more of the same
core polypeptide or modified core polypeptide described herein are
linked to a carrier. In some embodiments, 2, 3, 4, 5, 6 or more
different core polypeptides or modified core polypeptides described
herein are linked to a carrier.
[0258] In certain embodiments, the core polypeptides or modified
core polypeptides described herein are couple/linked to a carrier
by chemical cross-linking. For example, the cross-linker
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide ("EDC") or the
cross-linker Sulfosuccinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate ("Sulfo-SMCC") can
be used to cross-link a core polypeptide to a carrier. Other
cross-linkers include Glutaraldehyde and Bis-Diazotized Benzidine.
Methods of cross-linking are well known to those of skill in the
art and common cross-linking chemistries can be found at the
website:
www.piercenet.com/browse.cfm?fldID=CE4D6C5C-5946-4814-9904-C46E01232683.
[0259] In a particular embodiment, a flu polypeptide comprises (i)
the long alpha-helix of the HA2 hemagglutinin subunit of the
influenza virus strain A/Hong Kong/1/1968 (H3) (i.e., amino acids
76-130, numbered according to the classic H3 subtype numbering
system); (ii) a FLAG-tag; and (iii) a C-terminal cysteine residue
which can be used, e.g., to couple/link the core polypeptide to a
carrier (e.g., KLH). In a specific embodiment, such a flu
polypeptide comprises the following amino acid sequence:
TABLE-US-00027 (SEQ ID NO: 1)
RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKT
RRQLRENADYKDDDDKC,
wherein the FLAG-tag is represented by the amino acid sequence
DYKDDDDK. In some embodiments, the N-terminus of the modified core
polypeptide is acetylated.
[0260] 5.1.8 Multimerization Polypeptides
[0261] In certain embodiments, a flu polypeptide comprises a core
polypeptide or a modified core polypeptide described herein and a
polypeptide that facilitates the formation of multimers (e.g.,
trimers). In some embodiments, the core polypeptide or modified
polypeptide is coupled/linked to a polypeptide, such as a T4 foldon
domain, to allow/facilitate the formation of a trimer.
[0262] In specific embodiments the core'polypeptide or modified
core polypeptide is indirectly or directly linke/coupled to a
polypeptide that facilitates multimerization (e.g., trimerization,
such as by a T4 foldon domain) at its C-terminus. Meier et al.,
2004, J Mol Biol 344: 1051-69, incorporated by reference herein in
its entirety. Without being bound by any particular theory of
operation, a T4 foldon domain may allow for the formation of the
trimeric configuration of the influenza A long alpha helix seen in
the native hemagglutinin molecule.
[0263] In certain embodiments, a flu polypeptide comprises two or
more core polypeptides or modified polypeptides and a polypeptide
that facilitates the formation of a trimer. In a specific
embodiment, the polypeptide that facilitates formation of a trimer
is a T4 foldon domain.
[0264] In certain embodiments, the polypeptide that facilitates the
formation of a multimer is linked/coupled to a core polypeptide or
a modified pore polypeptide by a linker, such as described in
section 5.1.3 supra. In other words, in certain embodiments, the
flu polypeptide comprises a core polypeptide or a modified core
polypeptide, a linker and a polypeptide, such as a T4 foldon
domain, that facilitates the formation of multimers.
[0265] In certain embodiments a flu polypeptide in addition to
comprising 2, 3, 4 or more core polypeptides or modified core
polypeptides and a polypeptide that facilitates multimerization,
such as a T4 foldon domain, comprises one, two, three or more, or
all of the following: a protein tag facilitates purification and/or
solubility of the flu polypeptide, an immunogenic polypeptide,
and/or carrier such as described herein. In specific embodiments, a
flu polypeptide comprises a protein tag (e.g., a His tag) that
facilitates purification and/or solubility, a core polypeptide or a
modified core polypeptide and a polypeptide that facilitates
trimerization, such as a T4 foldon domain.
[0266] 5.2 Nucleic Acids Encoding Flu Polypeptides
[0267] Provided herein are nucleic acids that encode flu
polypeptides described herein. Due to the degeneracy of the genetic
code, any nucleic acid that encodes a flu polypeptide described
herein is encompassed herein. In certain embodiments, nucleic acids
corresponding to naturally occurring influenza virus nucleic acids
encoding a region of the HA2 domain (e.g., the long alpha helix
region) of the hemagglutinin protein are used to produce a flu
polypeptide.
[0268] Also provided herein are nucleic acids capable of
hybridizing to a nucleic acid encoding a flu polypeptide. In
certain embodiments, provided herein are nucleic acids capable of
hybridizing to a fragment of a nucleic acid encoding a flu
polypeptide. In other embodiments, provided herein are nucleic
acids capable of hybridizing to the full length of a nucleic acid
encoding a flu polypeptide. General parameters for hybridization
conditions for nucleic acids are described in Sambrook et al.,
Molecular Cloning--A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989), and in
Ausubel et al., Current Protocols in Molecular Biology, vol. 2,
Current Protocols Publishing, New York (1994). Hybridization may be
performed under high stringency conditions, medium stringency
conditions, or low stringency conditions. Those of skill in the art
will understand that low, medium and high stringency conditions are
contingent upon multiple factors, all of which interact and are
also dependent upon the nucleic acids in question. For example,
high stringency conditions may include temperatures within
5.degree. C. melting temperature of the nucleic acid(s), a low salt
concentration (e.g., less than 250 mM), and a high co-solvent
concentration (e.g., 1-20% of co-solvent, e.g., DMSO). Low
stringency conditions, on the other hand, may include temperatures
greater than 10.degree. C. below the melting temperature of the
nucleic acid(s), a high salt concentration (e.g., greater than 1000
mM) and the absence of co-solvents.
[0269] In some embodiments, a nucleic acid encoding an influenza
virus flu polypeptide is isolated, i.e., a flu polypeptide
described herein is isolated. In some embodiments, a nucleic acid
encoding an influenza virus core polypeptide or modified core
polypeptide as described herein is isolated. In certain
embodiments, an "isolated" nucleic acid refers to a nucleic acid
molecule which is separated from other nucleic acid molecules which
are present in the natural source of the nucleic acid. In other
words, the isolated nucleic acid can comprise heterologous nucleic
acids that are not associated with it in nature. In other
embodiments, an "isolated" nucleic acid, such as a cDNA molecule,
can be substantially free of other cellular material, or culture
medium when produced by recombinant techniques, or substantially
free of chemical precursors or other chemicals when chemically
synthesized. The term "substantially free of cellular material"
includes preparations of nucleic acid in which the nucleic acid is
separated from cellular components of the cells from which it is
isolated or recombinantly produced. Thus, nucleic acid that is
substantially free of cellular material includes preparations of
nucleic acid having less than about 30%, 20%, 10%, or 5% (by dry
weight) of other nucleic acids. The term "substantially free of
culture medium" includes preparations of nucleic acid in which the
culture medium represents less than about 50%, 20%, 10%, or 5% of
the volume of the preparation. The term "substantially free of
chemical precursors or other chemicals" includes preparations in
which the nucleic acid is separated from chemical precursors or
other chemicals which are involved in the synthesis of the nucleic
acid. In specific embodiments, such preparations of the nucleic
acid have less than about 50%, 30%, 20%, 10%, 5% (by dry weight) of
chemical precursors or compounds other than the nucleic acid of
interest.
[0270] In certain embodiments, provided herein are nucleic acids
that encode a flu polypeptide comprising a core polypeptide and one
or more additional components, e.g., a linker, a carrier, a protein
tag, and/or a protein that is/are associated with a core
polypeptide.
[0271] 5.3 Production and Purification of Flu Polypeptides
[0272] The flu polypeptides described herein can be produced by any
method known in the art for the synthesis of polypeptides, in
particular, by chemical synthesis or by recombinant expression
techniques. The methods provided herein encompass, unless otherwise
indicated, conventional techniques in molecular biology,
microbiology, genetic analysis, recombinant DNA, organic chemistry,
biochemistry, PCR, oligonucleotide synthesis and modification,
nucleic acid hybridization, and related fields within the skill of
the art. These techniques are described in the references cited
herein and are fully explained in the literature. See, e.g.,
Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press; Sambrook et al. (1989), Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory Press; Sambrook et al. (2001) Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y.; Ausubel et al., Current Protocols in Molecular
Biology, John Wiley & Sons (1987 and annual updates); Current
Protocols in Immunology, John Wiley & Sons (1987 and annual
updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical
Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and
Analogues: A Practical Approach, IRL Press; Birren et al. (eds.)
(1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor
Laboratory Press.
[0273] 5.3.1 Synthetic Production of Flu Polypeptides
[0274] The flu polypeptides described herein may be prepared using
conventional step-wise solution or solid phase synthesis (see,
e.g., Chemical Approaches to the Synthesis of Peptides and
Proteins, Williams et al., Eds., 1997, CRC Press, Boca Raton Fla.,
and references cited therein; Solid Phase Peptide Synthesis: A
Practical Approach, Atherton & Sheppard, Eds., 1989, IRL Press,
Oxford, England, and references cited therein).
[0275] Alternatively, the flu polypeptides described herein may be
prepared by way of segment condensation, as described, for example,
in Liu et al., 1996, Tetrahedron Lett. 37(7):933-936; Baca, et al.,
1995, J. Am. Chem. Soc. 117:1881-1887; Tam et al., 1995, Int. J.
Peptide Protein Res. 45:209-216; Schnolzer and Kent, 1992, Science
256:221-225; Liu and Tam, 1994, J. Am. Chem. Soc.
116(10):4149-4153; Liu and Tam, 1994, Proc. Natl. Acad. Sci. USA
91:6584-6588; Yamashiro and Li, 1988, Int. J. Peptide Protein Res.
31:322-334. Other methods useful for synthesizing the flu
polypeptides described herein are described in Nakagawa et al.,
1985, J. Am. Chem. Soc. 107:7087-7092.
[0276] Flu polypeptides comprising core polypeptides and linkers
can be synthesized by adding the linker(s) to the core polypeptide
chain at the appropriate step in the synthesis. Suitable protecting
schemes and chemistries are well known, and will be apparent to
those of skill in the art.
[0277] Formation of disulfide linkages, if desired, is generally
conducted in the presence of mild oxidizing agents. Chemical
oxidizing agents may be used, or the compounds may simply be
exposed to atmospheric oxygen to effect these linkages. Various
methods are known in the art, including those described, for
example, by Tam et al., 1979, Synthesis 955-957; Stewart et al.,
1984, Solid Phase Peptide Synthesis, 2d Ed., Pierce Chemical
Company Rockford, Ill.; Ahmed et al., 1975, J. Biol. Chem.
250:8477-8482; and Pennington et al., 1991 Peptides 1990 164-166,
Giralt and Andreu, Eds., ESCOM Leiden, The Netherlands. An
additional alternative is described by Kamber et al., 1980, Helv.
Chim. Acta 63:899-915. A method conducted on solid supports is
described by Albericio, 1985, Int. J. Peptide Protein Res.
26:92-97, each of which is incorporated by reference herein in its
entirety.
[0278] 5.3.2 Recombinant Expression of Flu Polypeptides
[0279] Recombinant expression of a flu polypeptide requires
construction of an expression vector containing a polynucleotide
that encodes the flu polypeptide. Once a polynucleotide encoding a
flu polypeptide has been obtained, the vector for the production of
the flu polypeptide may be produced by recombinant DNA technology
using techniques well-known in the art. Thus, methods for preparing
a flu polypeptide by expressing a polynucleotide containing a flu
polypeptide-encoding nucleotide sequence are described herein.
Methods which are well known to those skilled in the art can be
used to construct expression vectors containing flu polypeptide
coding sequences and appropriate transcriptional and translational
control signals. These methods include, for example, in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. Thus, provided herein are replicable
expression vectors comprising a nucleotide sequence encoding a flu
polypeptide operably linked to a promoter.
[0280] An expression vector comprises a nucleic acid encoding a flu
polypeptide in a form suitable for expression of the nucleic acid
in a host cell. In specific embodiments, the host cell is an
isolated host cell. In a specific embodiment, an expression vector
includes one or more regulatory sequences, selected on the basis of
the host cells to be used for expression, which is operably linked
to the nucleic acid to be expressed. Within an expression vector,
"operably linked" is intended to mean that a nucleic acid of
interest is linked to the regulatory sequence(s) in a manner which
allows for expression of the nucleic acid (e.g., in an in vitro
transcription/translation system or in a host cell when the vector
is introduced into the host cell). Regulatory sequences include
promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Regulatory sequences include those which
direct constitutive expression of a nucleic acid in many types of
host cells, those which direct expression of the nucleic acid only
in certain host cells (e.g., tissue-specific regulatory sequences),
and those which direct the expression of the nucleic acid upon
stimulation with a particular agent (e.g., inducible regulatory
sequences). It will be appreciated by those skilled in the art that
the design of the expression vector can depend on such factors as
the choice of the host cell to be transformed, the level of
expression of protein desired, etc. The term "host cell" is
intended to include a particular subject cell transformed or
transfected with a nucleic acid and the progeny or potential
progeny of such a cell. Progeny of such a cell may not be identical
to the parent cell transformed or transfected with the nucleic acid
due to mutations or environmental influences that may occur in
succeeding generations or integration of the nucleic acid into the
host cell genome. In specific embodiments, the host cell is
isolated.
[0281] An expression vector can be introduced into host cells via
conventional transformation or transfection techniques. Such
techniques include, but are not limited to, calcium phosphate or
calcium chloride co-precipitation, DEAE-dextran-mediated
transfection, lipofection, and electroporation. Suitable methods
for transforming or transfecting host cells can be found in
Sambrook et al., 1989, Molecular Cloning--A Laboratory Manual, 2nd
Edition, Cold Spring Harbor Press, New York, and other laboratory
manuals. In certain embodiments, a host cell is transiently
transfected with an expression vector containing a nucleic acid
encoding a flu polypeptide. In other embodiments, a host cell is
stably transfected with an expression vector containing a nucleic
acid encoding a flu polypeptide. Thus, provided herein are host
cells containing a polynucleotide encoding a flu polypeptide
described herein or generated in accordance with the methods
provided herein.
[0282] A variety of host-expression vector systems may be utilized
to express a flu polypeptide. Such host-expression systems
represent vehicles by which the coding sequences of interest may be
produced and subsequently purified, but also represent cells which
may, when transformed or transfected with the appropriate
nucleotide coding sequences, express a flu polypeptide in situ.
These include but are not limited to microorganisms such as
bacteria (e.g., E. coli and B. subtilis) transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing flu polypeptide coding sequences; yeast (e.g.,
Saccharomyces Pichia) transformed with recombinant yeast expression
vectors containing flu polypeptide coding sequences; insect cell
systems infected with recombinant virus expression vectors (e.g.,
baculovirus) containing flu polypeptide coding sequences; plant
cell systems infected with recombinant virus expression vectors
(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV)
or transformed with recombinant plasmid expression vectors (e.g.,
Ti plasmid) containing flu polypeptide coding sequences; or
mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3
cells) harboring recombinant expression constructs containing
promoters derived from the genome of mammalian cells (e.g.,
metallothionein promoter) or from mammalian viruses (e.g., the
adenovirus late promoter; the vaccinia virus 7.5K promoter).
Preferably, bacterial cells such as Escherichia coli, and more
preferably, eukaryotic cells are used for the expression of a flu
polypeptide. For example, mammalian cells such as Chinese hamster
ovary cells (CHO), in conjunction with a vector such as the major
intermediate early gene promoter element from human cytomegalovirus
is an effective expression system for flu polypeptides (Foecking et
al., 1986, Gene 45:101; and Cockett et al., 1990, Bio/Technology
8:2). In a specific embodiment, the expression of nucleotide
sequences encoding the flu polypeptides described herein or
generated in accordance with the methods provided herein is
regulated by a constitutive promoter, inducible promoter or tissue
specific promoter.
[0283] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the flu
polypeptide being expressed. For example, when a large quantity of
flu polypeptide is to be produced, for the generation of
pharmaceutical compositions of a flu polypeptide, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited to, the E. coli expression vector pUR278
(Ruther et al., 1983, EMBO 12:1791), in which the flu polypeptide
coding sequence may be ligated individually into the vector in
frame with the lac Z coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids
Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.
24:5503-5509); and the like. pGEX vectors may also be used to
express foreign polypeptides as fusion proteins with glutathione
5-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to matrix glutathione agarose beads followed by elution in
the presence of free glutathione. The pGEX vectors are designed to
include thrombin or factor Xa protease cleavage sites so that the
cloned target gene product can be released from the GST moiety.
[0284] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. The flu
polypeptide coding sequence may be cloned individually into
non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example
the polyhedrin promoter).
[0285] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, the flu polypeptide coding sequence of interest
may be ligated to an adenovirus transcription/translation control
complex, e.g., the late promoter and tripartite leader sequence.
This chimeric gene may then be inserted in the adenovirus genome by
in vitro or in vivo recombination. Insertion in a non-essential
region of the viral genome (e.g., region E1 or E3) will result in a
recombinant virus that is viable and capable of expressing the flu
polypeptide in infected hosts (e.g., see Logan & Shenk, 1984,
Proc. Natl. Acad. Sci. USA 8 1:355-359). Specific initiation
signals may also be required for efficient translation of inserted
flu polypeptide coding sequences. These signals include the ATG
initiation codon and adjacent sequences. Furthermore, the
initiation codon must be in phase with the reading frame of the
desired coding sequence to ensure translation of the entire insert.
These exogenous translational control signals and initiation codons
can be of a variety of origins, both natural and synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, etc. (see, e.g., Bittner et al., 1987, Methods in
Enzymol. 153:51-544).
[0286] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the flu
polypeptide. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used. Such mammalian
host cells include but are not limited to CHO, VERY, BHK, Hela,
COS, Vero, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and
T47D, NS0 (a murine myeloma cell line that does not endogenously
produce any immunoglobulin chains), CRL7O3O and HsS78Bst cells.
[0287] For long-term, high-yield production of recombinant flu
polypeptide, stable expression is preferred. For example, cell
lines which stably express the flu polypeptide molecule may be
engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the flu polypeptide. Such engineered cell lines
may be particularly useful in screening and evaluation of
compositions that interact directly or indirectly with the flu
polypeptide. Methods commonly known in the art of recombinant DNA
technology may be routinely applied to select the desired
recombinant clone, and such methods are described, for example, in
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley & Sons, NY (1993); Kriegler, Gene Transfer and
Expression, A Laboratory Manual, Stockton Press, NY (1990); and in
Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in
Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin
et al., 1981, J. Mol. Biol. 150:1, which are incorporated by
reference herein in their entireties.
[0288] The expression levels of a flu polypeptide can be increased
by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning, Vol.
3 (Academic Press, New York, 1987)). When a marker in the vector
system expressing the flu polypeptide is amplifiable, increase in
the level of inhibitor present in culture of host cell will
increase the number of copies of the marker gene. Since the
amplified region is associated with the flu polypeptide, production
of the flu polypeptide will also increase (Crouse et al., 1983,
Mol. Cell. Biol. 3:257).
[0289] As an alternative to recombinant expression of a flu
polypeptide using a host cell, an expression vector containing a
nucleic acid encoding a flu polypeptide can be transcribed and
translated in vitro using, e.g., T7 promoter regulatory sequences
and T7 polymerase. In a specific embodiment, a coupled
transcription/translation system, such as Promega TNT.RTM., or a
cell lysate or cell extract comprising the components necessary for
transcription and translation may be used to produce a flu
polypeptide.
[0290] Accordingly, provided herein are methods for producing a flu
polypeptide. In one embodiment, the method comprises culturing a
host cell containing a nucleic acid encoding the polypeptide in a
suitable medium such that the polypeptide is produced. In some
embodiments, the method further comprises isolating the polypeptide
from the medium or the host cell.
[0291] In certain embodiments, plants (e.g., plants of the genus
Nicotiana) may be engineered to express a flu polypeptide described
herein. In specific embodiments, plants are engineered to express a
flu polypeptide described herein via an agroinfiltration procedure
using methods known in the art. For example, nucleic acids encoding
a gene of interest, e.g., a gene encoding a flu polypeptide
described herein, are introduced into a strain of Agrobacterium.
Subsequently the strain is grown in a liquid culture and the
resulting bacteria are washed and suspended into a buffer solution.
The plants are then exposed (e.g., via injection or submersion) to
the Agrobacterium that comprises the nucleic acids encoding a flu
polypeptide described herein such that the Agrobacterium transforms
the gene of interest to a portion of the plant cells. The flu
polypeptide is then transiently expressed by the plant and can be
isolated using methods known in the art and described herein. (For
specific examples see Shoji et al., 2008, Vaccine,
26(23):2930-2934; and D'Aoust et al., 2008, J. Plant Biotechnology,
6(9):930-940). In a specific embodiment, the plant is, a tobacco
plant (i.e., Nicotiana tabacum). In another specific embodiment,
the plant is a relative of the tobacco plant (e.g., Nicotiana
benthamiana).
[0292] In some embodiments, a plant cell culture system is used for
expression of a flu polypeptide. See, e.g., U.S. Pat. Nos.
5,929,304; 7,504,560; 6,770,799; 6,551,820; 6,136,320; 6,034,298;
5,914,935; 5,612,487; and 5,484,719, U.S. patent application
publication Nos. 2009/0208477, 2009/0082548, 2009/0053762,
2008/0038232, 2007/0275014 and 2006/0204487, and Shoji et al.,
2008, Vaccine, 26(23):2930-2934, and D'Aoust et al., 2008, J. Plant
Biotechnology, 6(9):930-940 (which are incorporated herein by
reference in their entirety) for plant cells and methods for the
production of proteins utilizing plant cell culture systems. In a
specific embodiment, carrot cells are engineered to express a flu
polypeptide. In certain embodiments, algae (e.g., Chlamydomonas
reinhardtii) may be engineered to express a flu polypeptide (see,
e.g., Rasala et al., 2010, Plant Biotechnology Journal (Published
online Mar. 7, 2010, which is incorporated herein by reference in
its entirety).
[0293] 5.3.3 Purification of Flu Polypeptides
[0294] The flu polypeptides described herein and generated using
the approaches described in Sections 5.3.1 and 5.3.1, supra, may be
purified by any method known in the art for purification of a
polypeptide, for example, by chromatography (e.g., ion exchange,
affinity, particularly by affinity for the specific antigen after
Protein A, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard technique for the
purification of proteins. Further, the flu polypeptides may be
fused to heterologous polypeptide sequences described herein or
otherwise known in the art to facilitate purification. The actual
conditions used to purify a particular flu polypeptide will depend,
in part, on the synthesis strategy (e.g., synthetic production vs.
recombinant production) and on factors such as net charge,
hydrophobicity, and/or hydrophilicity of the flu polypeptide, and
will be apparent to those having skill in the art.
[0295] 5.4 Influenza Virus Vectors
[0296] In one aspect, provided herein are influenza viruses
containing a flu polypeptide. In a specific embodiment, the flu
polypeptide is incorporated into the virions of the influenza
virus. The influenza viruses may be conjugated to moieties that
target the viruses to particular cell types, such as immune cells.
In some embodiments, the virions of the influenza virus have
incorporated into them or express a heterologous polypeptide in
addition to a flu polypeptide. The heterologous polypeptide may be
a polypeptide that has immunopotentiating activity, or that targets
the influenza virus to a particular cell type, such as an antibody
that binds to an antigen on a specific cell type or a ligand that
binds a specific receptor on a specific cell type.
[0297] Influenza viruses containing a flu polypeptide may be
produced by supplying in trans the flu polypeptide during
production of virions using techniques known to one skilled in the
art, such as reverse genetics and helper-free plasmid rescue.
Alternatively, a parental influenza virus comprises a genome
engineered to express a flu polypeptide in cells susceptible to
infection with the virus wherein hemagglutinin function is provided
in trans to produce progeny influenza viruses containing the
influenza flu polypeptide.
[0298] In another aspect, provided herein are influenza viruses
comprising a genome engineered to express a flu polypeptide. In a
specific embodiment, the genome of a parental influenza virus is
engineered to encode a flu polypeptide, which is expressed by
progeny influenza virus. In another specific embodiment, the genome
of a parental influenza virus is engineered to encode a flu
polypeptide, which is expressed and incorporated into the virions
of progeny influenza virus. Thus, the progeny influenza virus
resulting from the replication of the parental influenza virus
contain a flu polypeptide.
[0299] In some embodiments, the virions of the parental influenza
virus have incorporated into them a heterologous polypeptide. In
certain embodiments, the genome of a parental influenza virus is
engineered to encode a heterologous polypeptide and an influenza
virus flu polypeptide, which are expressed by progeny influenza
virus. In specific embodiments, the influenza flu polypeptide, the
heterologous polypeptide or both are incorporated into virions of
the progeny influenza virus.
[0300] The heterologous polypeptide may be a polypeptide that
targets the influenza virus to a particular cell type, such as an
antibody that recognizes an antigen on a specific cell type or a
ligand that binds a specific receptor on a specific cell type. In
some embodiments, the targeting polypeptide replaces the target
cell recognition function of the virus. In a specific embodiment,
the heterologous polypeptide targets the influenza virus to the
same cell types that influenza virus infects in nature. In other
specific embodiments, the heterologous polypeptide targets the
progeny influenza virus to immune cells, such as B cells, T cells,
macrophages or dendritic cells. In some embodiments, the
heterologous polypeptide recognizes and binds to cell-specific
markers of antigen presenting cells, such as dendritic cells (e.g.,
such as CD44): In one embodiment, the heterologous polypeptide is
DC-SIGN which targets the virus to dendritic cells. In another
embodiment, the heterologous polypeptide is an antibody (e.g., a
single-chain antibody) that targets the virus to an immune cell,
which may be fused with a transmembrane domain from another
polypeptide so that it is incorporated into the influenza virus
virion. In some embodiments, the antibody is a CD20 antibody, a
CD34 antibody, or an antibody against DEC-205. Techniques for
engineering viruses to express polypeptides with targeting
functions are known in the art. See, e.g., Yang et al., 2006, PNAS
103: 11479-11484 and United States patent application Publication
No. 20080019998, published Jan. 24, 2008, and No. 20070020238,
published Jan. 25, 2007, the contents of each of which are
incorporated herein in their entirety.
[0301] In another embodiment, the heterologous polypeptide is a
viral attachment protein. Non-limiting examples of viruses whose
attachment protein(s) can be used in this aspect are viruses
selected from the group of: Lassa fever virus, Hepatitis B virus,
Rabies virus, Newcastle disease virus (NDV), a retrovirus such as
human immunodeficiency virus, tick-borne encephalitis virus,
vaccinia virus, herpesvirus, poliovirus, alphaviruses such as
Semliki Forest virus, Ross River virus, and Aura virus (which
comprise surface glycoproteins such as E1, E2, and E3), Borna
disease virus, Hantaan virus, foamyvirus, and SARS-CoV virus.
[0302] In a specific embodiment, an influenza A virus is engineered
to encode a flu polypeptide and an influenza C HEF protein, wherein
the influenza C HEF protein is substituted for the influenza A
neuraminidase (NA) protein.
[0303] In one embodiment, a flavivirus surface glycoprotein may be
used, such as Dengue virus (DV) E protein. In some embodiments, a
Sindbis virus glycoprotein from the alphavirus family is used (K.
S. Wang, R. J. Kuhn, E. G. Strauss, S. Ou, J. H. Strauss, J. Virol.
66, 4992 (1992)). In certain embodiments, the heterologous
polypeptide is derived from an NDV HN or F protein; a human
immunodeficiency virus (HIV) gp160 (or a product thereof, such as
gp41 or gp120); a hepatitis B virus surface antigen (HBsAg); a
glycoprotein of herpesvirus (e.g., gD, gE); or VPI of
poliovirus.
[0304] In another embodiment, the heterologous polypeptide is
derived from any non-viral targeting system known in the art. In
certain embodiments, a protein of a nonviral pathogen such as an
intracellular bacteria or protozoa is used. In some embodiments,
the bacterial polypeptide is provided by, e.g., Chlamydia,
Rikettsia, Coxelia, Listeria, Brucella, or Legionella. In some
embodiments, protozoan polypeptide is provided by, e.g., Plasmodia
species, Leishmania spp., Toxoplasma gondii, or Trypanosoma cruzi.
Other exemplary targeting systems are described in Waehler et al.,
2007, "Engineering targeted viral vectors for gene therapy," Nature
Reviews Genetics 8: 573-587, which is incorporated herein in its
entirety.
[0305] In certain embodiments, the heterologous polypeptide
expressed by an influenza virus has immunopotentiating (immune
stimulating) activity. Non-limiting examples of immunopotentiating
polypeptides include, but are not limited to, stimulation
molecules, cytokines, chemokines, antibodies and other agents such
as Flt-3 ligands. Specific examples of polypeptides with
immunopotentiating activity include: interferon type 1, alpha,
beta, or gamma interferon, colony stimulating factors such as
granulocyte-macrophage colony-stimulating factor (GM-CSF),
interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15,
IL-18, IL-21, IL-23, tumor necrosis factor (TNF)-.beta.,
TNF.alpha.., B7.1, B7.2, 4-1BB, CD40 ligand (CD40L), and
drug-inducible CD40 (CD40) (see, e.g., Hanks, B. A., et al. 2005.
Nat Med 11:130-137, which is incorporated herein by reference in
its entirety.)
[0306] Since the genome of influenza A and B viruses consist of
eight (8) single-stranded, negative sense segments (influenza C
viruses consist of seven (7) single-stranded, negative sense
segments), the genome of a parental influenza virus may be
engineered to express a flu polypeptide (and any other polypeptide,
such as a heterologous polypeptide) using a recombinant segment and
techniques known to one skilled in the art, such a reverse genetics
and helper-free plasmid rescue. In one embodiment, the recombinant
segment comprises a nucleic acid encoding the flu polypeptide as
well as the 3' and 5' incorporation signals which are required for
proper replication, transcription and packaging of the vRNAs (Gao
et al., 2010, J. of Virology 84:8062-8074, Fujii et al., 2003,
Proc. Natl. Acad. Sci. USA 100:2002-2007; Zheng, et al., 1996,
Virology 217:242-251, and PCT/US2010/043697, all of which are
incorporated by reference herein in their entireties). In certain
embodiments, the recombinant segment encoding the flu polypeptide
may replace the HA segment of a parental influenza virus. In some
embodiments, the recombinant segment encoding the flu polypeptide
may replace the NS1 gene of the parental influenza virus. In some
embodiments, the recombinant segment encoding the flu polypeptide
may replace the NA gene of the parental influenza virus. Exemplary
influenza virus strains that can be used to express the flu
polypeptides include Ann Arbor/1/50, A/Puerto Rico/8/34, A/South
Dakota/6/2007, A/Uruguay/716/2007, and B/Brisbane/60/2008.
[0307] In some embodiments, the genome of a parental influenza
virus may be engineered to express a flu polypeptide using a
recombinant segment that is bicistronic. Bicistronic techniques
allow the engineering of coding sequences of multiple proteins into
a single mRNA through the use of internal ribosome entry site
(IRES) sequences. IRES sequences direct the internal recruitment of
ribosomes to the RNA molecule and allow downstream translation in a
cap independent manner. Briefly, a coding region of one protein is
inserted into the open reading frame (ORF) of a second protein. The
insertion is flanked by an IRES and any untranslated signal
sequences necessary for proper expression and/or function. The
insertion must not disrupt the ORF, polyadenylation or
transcriptional promoters of the second protein (see, e.g.,
Garcia-Sastre et al., 1994, J. Virol. 68:6254-6261 and
Garcia-Sastre et al., 1994 Dev. Biol. Stand. 82:237-246, each of
which is hereby incorporated by reference in its entirety). See
also, e.g., U.S. Pat. No. 6,887,699, U.S. Pat. No. 6,001,634, U.S.
Pat. No. 5,854,037 and U.S. Pat. No. 5,820,871, each of which is
incorporated herein by reference in its entirety. Any IRES known in
the art or described herein may be used in accordance with the
invention (e.g., the IRES of BiP gene, nucleotides 372 to 592 of
GenBank database entry HUMGRP78; or the IRES of
encephalomyocarditis virus (EMCV), nucleotides 1430-2115 of GenBank
database entry CQ867238.). Thus, in certain embodiments, a parental
influenza virus is engineered to contain a bicistronic RNA segment
that expresses a flu polypeptide and another polypeptide, such as
gene expressed by the parental influenza virus.
[0308] Techniques known to one skilled in the art may be used to
produce an influenza virus containing a flu polypeptide and an
influenza virus comprising a genome engineered to express a flu
polypeptide. For example, reverse genetics techniques may be used
to generate such an influenza virus. Briefly, reverse genetics
techniques generally involve the preparation of synthetic
recombinant viral RNAs that contain the non-coding regions of the
negative-strand, viral RNA which are essential for the recognition
by viral polymerases and for packaging signals necessary to
generate a mature virion. The recombinant RNAs are synthesized from
a recombinant DNA template and reconstituted in vitro with purified
viral polymerase complex to form recombinant ribonucleoproteins
(RNPs) which can be used to transfect cells. A more efficient
transfection is achieved if the viral polymerase proteins are
present during transcription of the synthetic RNAs either in vitro
or in vivo. The synthetic recombinant RNPs can be rescued into
infectious virus particles. The foregoing techniques are described
in U.S. Pat. No. 5,166,057 issued Nov. 24, 1992; in U.S. Pat. No.
5,854,037 issued Dec. 29, 1998; in European Patent Publication EP
0702085A 1, published Feb. 20, 1996; in U.S. patent application
Ser. No. 09/152,845; in International Patent Publications PCT WO
97/12032 published Apr. 3, 1997; WO 96/34625 published Nov. 7,
1996; in European Patent Publication EP A780475; WO 99/02657
published Jan. 21, 1999; WO 98/53078 published Nov. 26, 1998; WO
98/02530 published Jan. 22, 1998; WO 99/15672 published Apr. 1,
1999; WO 98/13501 published Apr. 2, 1998; WO 97/06270 published
Feb. 20, 1997; and EPO 780 475A1 published Jun. 25, 1997, each of
which is incorporated by reference herein in its entirety.
[0309] Alternatively, helper-free plasmid technology may be used to
produce an influenza virus containing a flu polypeptide and/or an
influenza virus comprising a genome engineered to express a flu
polypeptide. Briefly, full length cDNAs of viral segments are
amplified using PCR with primers that include unique restriction
sites, which allow the insertion of the PCR product into the
plasmid vector (Flandorfer et al., 2003, J. Virol. 77:9116-9123;
Nakaya et al., 2001, J. Virol. 75:11868-11873; both of which are
incorporated herein by reference in their entireties). The plasmid
vector is designed so that an exact negative (vRNA sense)
transcript is expressed. For example, the plasmid vector may be
designed to position the PCR product between a truncated human RNA
polymerase I promoter and a hepatitis delta virus ribozyme sequence
such that an exact negative (vRNA sense) transcript is produced
from the polymerase I promoter. Separate plasmid vectors comprising
each viral segment as well as expression vectors comprising
necessary viral proteins may be transfected into cells leading to
production of recombinant viral particles. In another example,
plasmid vectors from which both the viral genomic RNA and mRNA
encoding the necessary viral proteins are expressed may be used.
For a detailed description of helper-free plasmid technology see,
e.g., International Publication No. WO 01/04333; U.S. Pat. Nos.
6,951,754, 7,384,774, 6,649,372, and 7,312,064; Fodor et al., 1999,
J. Virol. 73:9679-9682; Quinlivan et al., 2005, J. Virol.
79:8431-8439; Hoffmann et al., 2000, Proc. Natl. Acad. Sci. USA
97:6108-6113; and Neumann et al., 1999, Proc. Natl. Acad. Sci. USA
96:9345-9350, which are incorporated herein by reference in their
entireties.
[0310] The influenza viruses described herein may be propagated in
any substrate that allows the virus to grow to titers that permit
their use in accordance with the methods described herein. In one
embodiment, the substrate allows the viruses to grow to titers
comparable to those determined for the corresponding wild-type
viruses. In certain embodiments, the substrate is one which is
biologically relevant to the influenza virus. In a specific
embodiment, an attenuated influenza virus by virtue of, e.g., a
mutation in the NS1 gene, may be propagated in an IFN-deficient
substrate. For example, a suitable IFN-deficient substrate may be
one that is defective in its ability to produce or respond to
interferon, or is one which an IFN-deficient substrate may be used
for the growth of any number of viruses which may require
interferon-deficient growth environment. See, for example, U.S.
Pat. Nos. 6,573,079, issued Jun. 3, 2003, 6,852,522, issued Feb. 8,
2005, and 7,494,808, issued Feb. 24, 2009, the entire contents of
each of which is incorporated herein by reference in its
entirety.
[0311] The influenza viruses described herein may be isolated and
purified by any method known to those of skill in the art. In one
embodiment, the virus is removed from cell culture and separated
from cellular components, typically by well known clarification
procedures, e.g., such as gradient centrifugation and column
chromatography, and may be further purified as desired using
procedures well known to those skilled in the art, e.g., plaque
assays.
[0312] In certain embodiments, the influenza viruses, or influenza
virus polypeptides, genes or genome segments for use as described
herein are obtained or derived from an influenza A virus. In
certain embodiments, the influenza viruses, or influenza virus
polypeptides, genes or genome segments for use as described herein
are obtained or derived from a single influenza A virus subtype or
strain. In other embodiments, the influenza viruses, or influenza
virus polypeptides, genes or genome segments for use as described
herein are obtained or derived from two or more influenza A virus
subtypes or strains.
[0313] In some embodiments, the influenza viruses, or influenza
virus polypeptides, genes or genome segments for use as described
herein are obtained or derived from an influenza B virus. In
certain embodiments, the influenza viruses, or influenza virus
polypeptides, genes or genome segments for use as described herein
are obtained or derived from a single influenza B virus subtype or
strain. In other embodiments, the influenza viruses, or influenza
virus polypeptides, genes or genome segments for use as described
herein are obtained or derived from two or more influenza B virus
subtypes or strains. In other embodiments, the influenza viruses,
or influenza virus polypeptides, genes or genome segments for use
as described herein are obtained or derived from a combination of
influenza A and influenza B virus subtypes or strains.
[0314] In some embodiments, the influenza viruses, or influenza
virus polypeptides, genes or genome segments for use as described
herein are obtained or derived from an influenza C virus. In
certain embodiments, the influenza viruses, or influenza virus
polypeptides, genes or genome segments for use as described herein
are obtained or derived from a single influenza C virus subtype or
strain. In other embodiments, the influenza viruses, or influenza
virus polypeptides, genes or genome segments for use as described
herein are obtained or derived from two or more influenza C virus
subtypes or strains. In other embodiments, the influenza viruses,
or influenza virus polypeptides, genes or genome segments for use
as described herein are obtained or derived from a combination of
influenza C virus and influenza A virus and/or influenza B virus
subtypes or strains.
[0315] Non-limiting examples of influenza A viruses include subtype
H10N4, subtype H10N5, subtype H10N7, subtype H10N8, subtype H10N9,
subtype H11N1, subtype H11N13, subtype H11N2, subtype H11N4,
subtype H11N6, subtype H11N8, subtype H11N9, subtype H12N1, subtype
H12N4, subtype H12N5, subtype H12N8, subtype H13N2, subtype H13N3,
subtype H13N6, subtype H13N7, subtype H14N5, subtype H14N6, subtype
H15N8, subtype H15N9, subtype H16N3, subtype HINT, subtype H1N2,
subtype H1N3, subtype H1N6, subtype H1N9, subtype H2N1, subtype
H2N2, subtype H2N3, subtype H2N5, subtype H2N7, subtype H2N8,
subtype H2N9, subtype H3N1, subtype H3N2, subtype H3N3, subtype
H3N4, subtype H3N5, subtype H3N6, subtype H3N8, subtype H3N9,
subtype H4N1, subtype H4N2, subtype H4N3, subtype H4N4, subtype
H4N5, subtype H4N6, subtype H4N8, subtype H4N9, subtype H.sub.5N1,
subtype H5N2, subtype H5N3, subtype H5N4, subtype H5N6, subtype
H5N7, subtype H5N8, subtype H5N9, subtype H.sub.6N1, subtype H6N2,
subtype H6N3, subtype H6N4, subtype H6N5, subtype H6N6, subtype
H6N7, subtype H6N8, subtype H6N9, subtype H7N1, subtype H7N2,
subtype H7N3, subtype H7N4, subtype H7N5, subtype H7N7, subtype
H7N8, subtype H7N9, subtype H8N4, subtype H8N5, subtype H.sub.9N1,
subtype H9N2, subtype H9N3, subtype H9N5, subtype H9N6, subtype
H9N7, subtype H9N8, and subtype H9N9.
[0316] Specific examples of strains of influenza A virus include,
but are not limited to: A/sw/Iowa/15/30 (H1N1); A/WSN/33 (H1N1);
A/eq/Prague/1/56 (H7N7); A/PR/8/34; A/mallard/Potsdam/178-4/83
(H2N2); A/herring gull/DE/712/88 (H16N3); A/sw/Hong Kong/168/1993
(HIND; A/mallard/Alberta/211/98 (H1N1); A/shorebird/Delaware/168/06
(H16N3); A/sw/Netherlands/25/80 (H1N1); A/sw/Germany/2/81 (H1N1);
A/sw/Hannover/1/81 (H1N1); A/sw/Potsdam/1/81 (H1N1);
A/sw/Potsdam/15/81 (H1N1); A/sw/Potsdam/268/81 (H1N1);
A/sw/Finistere/2899/82 (H1N1); A/sw/Potsdam/35/82 (H3N2); A/sw/Cote
d'Armor/3633/84 (H3N2); A/sw/Gent/1/84 (H3N2);
A/sw/Netherlands/12/85 (H1N1); A/sw/Karrenzien/2/87 (H3N2);
A/sw/Schwerin/103/89 (H1N1); A/turkey/Germany/3/91 (H1N1);
A/sw/Germany/8533/91 (H1N1); A/sw/Belgium/220/92 (H3N2);
A/sw/GentN230/92 (H1N1); A/sw/Leipzig/145/92 (H3N2);
A/sw/Re220/92hp (H3N2); A/sw/Bakum/909/93 (H3N2);
A/sw/Schleswig-Holstein/1/93 (H1N1); A/sw/Scotland/419440/94
(H1N2); A/sw/Bakum/5/95 (H1N1); A/sw/Best/5C/96 (H1N1);
A/sw/England/17394/96 (H1N2); A/sw/Jena/5/96 (H3N2);
A/sw/Oedenrode/7C/96 (H3N2); A/sw/Lohne/1/97 (H3N2); A/sw/Cote
d'Armor/790/97 (H1N2); A/sw/Bakum/1362/98 (H3N2);
A/sw/Italy/1521/98 (HIN2); A/sw/Italy/1553-2/98 (H3N2);
A/sw/Italy/1566/98 (H1N1); A/sw/Italy/1589/98 (H1N1);
A/sw/Bakum/8602/99 (H3N2); A/sw/Cotes d'Armor/604/99 (HIN2);
A/sw/Cote d'Armor/1482/99 (H1N1); A/sw/Gent/7625/99 (HIN2); A/Hong
Kong/1774/99 (H3N2); A/sw/Hong Kong/5190/99 (H3N2); A/sw/Hong
Kong/5200/99 (H3N2); A/sw/Hong Kong/5212/99 (H3N2); A/sw/Ille et
Villaine/1455/99 (H1N1); A/sw/Italy/1654-1/99 (H1N2);
A/sw/Italy/2034/99 (H1N1); A/sw/Italy/2064/99 (H1N2);
A/sw/Berlin/1578/00 (H3N2); A/sw/Bakum/1832/00 (HIN2);
A/sw/Bakum/1833/00 (HIN2); A/sw/Cote d'Armor/800/00 (HIN2);
A/sw/Hong Kong/7982/00 (H3N2); A/sw/Italy/1081/00 (H1N2);
A/sw/Belzig/2/01 (H1N1); A/sw/Belzig/54/01 (H3N2); A/sw/Hong
Kong/9296/01 (H3N2); A/sw/Hong Kong/9745/01 (H3N2);
A/sw/Spain/33601/01 (H3N2); A/sw/Hong Kong/1144/02 (H3N2);
A/sw/Hong Kong/1197/02 (H3N2); A/sw/Spain/39139/02 (H3N2);
A/sw/Spain/42386/02 (H3N2); A/Switzerland/8808/2002 (H1N1);
A/sw/Bakum/1769/03 (H3N2); A/sw/Bissendorf/IDT1864/03 (H3N2);
A/sw/Ehren/IDT2570/03 (HIN2); A/sw/Gescher/IDT2702/03 (HIN2);
A/sw/Haselunne/2617/03hp (H1N1); A/sw/Loningen/IDT2530/03 (H1N2);
A/sw/IVD/IDT2674/03 (HIN2); A/sw/Nordkirchen/IDT1993/03 (H3N2);
A/sw/Nordwalde/IDT2197/03 (HIN2); A/sw/Norden/IDT2308/03 (HIN2);
A/sw/Spain/50047/03 (H1N1); A/sw/Spain/51915/03 (H1N1);
A/sw/Vechta/2623/03 (H1N1); A/sw/Visbek/IDT2869/03 (HIN2);
A/sw/Waltersdorf/IDT2527/03 (HIN2); A/sw/Damme/IDT2890/04 (H3N2);
A/sw/Geldern/IDT2888/04 (H1N1); A/sw/Granstedt/IDT3475/04 (H1N2);
A/sw/Greven/IDT2889/04 (H1N1); A/sw/Gudensberg/IDT2930/04 (HIN2);
A/sw/Gudensberg/IDT2931/04 (HIN2); A/sw/Lohne/IDT3357/04 (H3N2);
A/sw/Nortrup/IDT3685/04 (HIN2); A/sw/Seesen/IDT3055/04 (H3N2);
A/sw/Spain/53207/04 (H1N1); A/sw/Spain/54008/04 (H3N2);
A/sw/Stolzenau/IDT3296/04 (H1N2); A/sw/Wedel/IDT2965/04 (H1N1);
A/sw/Bad Griesbach/IDT4191/05 (H3N2); A/sw/Cloppenburg/IDT4777/05
(HIN2); A/sw/Dotlingen/IDT3780/05 (HIN2); A/sw/Dotlingen/IDT4735/05
(H1N2); A/sw/Egglham/IDT5250/05 (H3N2); A/sw/Harkenblek/IDT4097/05
(H3N2); A/sw/Hertzen/IDT4317/05 (H3N2); A/sw/Krogel/IDT4192/05 (H1N
1); A/sw/Laer/IDT3893/05 (H1N1); A/sw/Laer/IDT4126/05 (H3N2);
A/sw/Merzen/IDT4114/05 (H3N2); A/sw/Muesleringen-S./IDT4263/05
(H3N2); A/sw/Osterhofen/IDT4004/05 (H3N2); A/sw/Sprenge/IDT3805/05
(HIN2); A/sw/Stadtlohn/IDT3853/05 (HIN2); A/swNoglarn/IDT4096/05
(H1N1); A/sw/Wohlerst/IDT4093/05 (H1N1); A/sw/Bad Griesbach/I
DT5604/06 (H1N1); A/sw/Herzlake/IDT5335/06 (H3N2);
A/sw/Herzlake/IDT5336/06 (H3N2); A/sw/Herzlake/IDT5337/06 (H3N2);
and A/wild boar/Germany/R169/2006 (H3N2).
[0317] Other specific examples of strains of influenza A virus
include, but are not limited to: A/Toronto/3141/2009 (H1N1);
A/Regensburg/D6/2009 (H1N1); A/Bayern/62/2009 (H1N1);
A/Bayern/62/2009 (H1N1); A/Bradenburg/19/2009 (H1N1);
A/Bradenburg/20/2009 (H1N1); A/Distrito Federal/2611/2009 (H1N1);
A/Mato Grosso/2329/2009 (H1N1); A/Sao Paulo/1454/2009 (H1N1); A/Sao
Paulo/2233/2009 (H1N1); A/Stockholm/37/2009 (H1N1);
A/Stockholm/41/2009 (H1N1); A/Stockholm/45/2009 (H1N1);
A/swine/Alberta/OTH-33-1/2009 (H1N1);
A/swine/Alberta/OTH-33-14/2009 (H1N1);
A/swine/Alberta/OTH-33-2/2009 (H1N1);
A/swine/Alberta/OTH-33-21/2009 (H1N1);
A/swine/Alberta/OTH-33-22/2009 (H1N1);
A/swine/Alberta/OTH-33-23/2009 (H1N1);
A/swine/Alberta/OTH-33-24/2009 (H1N1);
A/swine/Alberta/OTH-33-25/2009 (H1N1);
A/swine/Alberta/OTH-33-3/2009 (H1N1); A/swine/Alberta/OTH-33-7/2009
(H1N1); A/Beijing/502/2009 (H1N1); A/Firenze/10/2009 (H1N1); A/Hong
Kong/2369/2009 (H1N1); A/Italy/85/2009 (H1N1); A/Santo
Domingo/572N/2009 (H1N1); A/Catalonia/385/2009 (H1N1);
A/Catalonia/386/2009 (H1N1); A/Catalonia/387/2009 (H1N1);
A/Catalonia/390/2009 (H1N1); A/Catalonia/394/2009 (H1N1);
A/Catalonia/397/2009 (H1N1); A/Catalonia/398/2009 (H1N1);
A/Catalonia/399/2009 (H1N1); A/Sao Paulo/2303/2009 (H1N1);
A/Akita/1/2009 (H1N1); A/Castro/JXP/2009 (H1N1); A/Fukushima/1/2009
(H1N1); A/Israel/276/2009 (H1N1); A/Israel/277/2009 (H1N1);
A/Israel/70/2009 (H1N1); A/Iwate/1/2009 (H1N1); A/Iwate/2/2009
(H1N1); A/Kagoshima/1/2009 (H1N1); A/Osaka/180/2009 (H1N1);
A/Puerto Montt/Bio87/2009 (H1N1); A/Sao Paulo/2303/2009 (H1N1);
A/Sapporo/1/2009 (H1N1); A/Stockholm/30/2009 (H1N1);
A/Stockholm/31/2009 (H1N1); A/Stockholm/32/2009 (H1N1);
A/Stockholm/33/2009 (H1N1); A/Stockholm/34/2009 (H1N1);
A/Stockholm/35/2009 (H1N1); A/Stockholm/36/2009 (H1N1);
A/Stockholm/38/2009 (H1N1); A/Stockholm/39/2009 (H1N1);
A/Stockholm/40/2009 (H1N1;) A/Stockholm/42/2009 (H1N1);
A/Stockholm/43/2009 (H1N1); A/Stockholm/44/2009 (H1N1);
A/Utsunomiya/2/2009 (H1N1); A/WRAIR/0573N/2009 (H1N1); and
A/Zhejiang/DTID-ZJU01/2009 (HIND.
[0318] Non-limiting examples of influenza B viruses include strain
Aichi/5/88, strain Akita/27/2001, strain Akita/5/2001, strain
Alaska/16/2000, strain Alaska/1777/2005, strain Argentina/69/2001,
strain Arizona/146/2005, strain Arizona/148/2005, strain
Bangkok/163/90, strain Bangkok/34/99, strain Bangkok/460/03, strain
Bangkok/54/99, strain Barcelona/215/03, strain Beijing/15/84,
strain Beijing/184/93, strain Beijing/243/97, strain Beijing/43/75,
strain Beijing/5/76, strain Beijing/76/98, strain
Belgium/WV106/2002, strain Belgium/WV107/2002, strain
Belgium/WV109/2002, strain Belgium/WV114/2002, strain
Belgium/WV122/2002, strain Bonn/43, strain Brazil/952/2001, strain
Bucharest/795/03, strain Buenos Aires/161/00), strain Buenos
Aires/9/95, strain Buenos Aires/SW16/97, strain Buenos
AiresNL518/99, strain Canada/464/2001, strain Canada/464/2002,
strain Chaco/366/00, strain Chaco/R113/00, strain Cheju/303/03,
strain Chiba/447/98, strain Chongqing/3/2000, strain clinical
isolate SA1 Thailand/2002, strain clinical isolate SA10
Thailand/2002, strain clinical isolate SA100 Philippines/2002,
strain clinical isolate SA101 Philippines/2002, strain clinical
isolate SA110 Philippines/2002), strain clinical isolate SA112
Philippines/2002, strain clinical isolate SA113 Philippines/2002,
strain clinical isolate SA114 Philippines/2002, strain clinical
isolate SA2 Thailand/2002, strain clinical isolate SA20
Thailand/2002, strain clinical isolate SA38 Philippines/2002,
strain clinical isolate SA39 Thailand/2002, strain clinical isolate
SA99 Philippines/2002, strain CNIC/27/2001, strain
Colorado/2597/2004, strain CordobaNA418/99, strain
Czechoslovakia/16/89, strain Czechoslovakia/69/90, strain
Daeku/10/97, strain Daeku/45/97, strain Daeku/47/97, strain
Daeku/9/97, strain B/Du/4/78, strain B/Durban/39/98, strain
Durban/43/98, strain Durban/44/98, strain B/Durban/52/98, strain
Durban/55/98, strain Durban/56/98, strain England/1716/2005, strain
England/2054/2005), strain England/23/04, strain Finland/154/2002,
strain Finland/159/2002, strain Finland/160/2002, strain
Finland/161/2002, strain Finland/162/03, strain Finland/162/2002,
strain Finland/162/91, strain Finland/164/2003, strain
Finland/172/91, strain Finland/173/2003, strain Finland/176/2003,
strain Finland/184/91, strain Finland/188/2003, strain
Finland/190/2003, strain Finland/220/2003, strain
Finland/1115/2002, strain Fujian/36/82, strain Geneva/5079/03,
strain Genoa/11/02, strain Genoa/2/02, strain Genoa/21/02, strain
Genova/54/02, strain Genova/55/02, strain Guangdong/05/94, strain
Guangdong/08/93, strain Guangdong/5/94, strain Guangdong/55/89,
strain Guangdong/8/93, strain Guangzhou/7/97, strain
Guangzhou/86/92, strain Guangzhou/87/92, strain Gyeonggi/592/2005,
strain Hannover/2/90, strain Harbin/07/94, strain Hawaii/10/2001,
strain Hawaii/1990/2004, strain Hawaii/38/2001, strain
Hawaii/9/2001, strain Hebei/19/94, strain Hebei/3/94), strain
Henan/22/97, strain Hiroshima/23/2001, strain Hong Kong/110/99,
strain Hong Kong/1115/2002, strain Hong Kong/112/2001, strain Hong
Kong/123/2001, strain Hong Kong/1351/2002, strain Hong
Kong/1434/2002, strain Hong Kong/147/99, strain Hong Kong/156/99,
strain Hong Kong/157/99, strain Hong Kong/22/2001, strain Hong
Kong/22/89, strain Hong Kong/336/2001, strain Hong Kong/666/2001,
strain Hong Kong/9/89, strain Houston/1/91, strain Houston/1/96,
strain Houston/2/96, strain Hunan/4/72, strain Ibaraki/2/85, strain
ncheon/297/2005, strain India/3/89, strain India/77276/2001, strain
Israel/95/03, strain Israel/WV187/2002, strain Japan/1224/2005,
strain Jiangsu/10/03, strain Johannesburg/1/99, strain
Johannesburg/96/01, strain Kadoma/1076/99, strain Kadoma/122/99,
strain Kagoshima/15/94, strain Kansas/22992/99, strain
Khazkov/224/91, strain Kobe/1/2002, strain, strain Kouchi/193/99,
strain Lazio/1/02, strain Lee/40, strain Leningrad/129/91, strain
Lissabon/2/90), strain Los Angeles/1/02, strain Lusaka/270/99,
strain Lyon/1271/96, strain Malaysia/83077/2001, strain
Maputo/1/99, strain Mar del Plata/595/99, strain Maryland/1/01,
strain Memphis/1/01, strain Memphis/12/97-MA, strain
Michigan/22572/99, strain Mie/1/93, strain Milano/1/01, strain
Minsk/318/90, strain Moscow/3/03, strain Nagoya/20/99, strain
Nanchang/1/00, strain Nashville/107/93, strain Nashville/45/91,
strain Nebraska/2/01, strain Netherland/801/90, strain
Netherlands/429/98, strain New York/1/2002, strain NIB/48/90,
strain Ningxia/45/83, strain Norway/1/84, strain Oman/16299/2001,
strain Osaka/1059/97, strain Osaka/983/97-V2, strain
Oslo/1329/2002, strain Oslo/1846/2002, strain Panama/45/90, strain
Paris/329/90, strain Parma/23/02, strain Perth/211/2001, strain
Peru/1364/2004, strain Philippines/5072/2001, strain Pusan/270/99,
strain Quebec/173/98, strain Quebec/465/98, strain Quebec/7/01,
strain Roma/1/03, strain Saga/S172/99, strain Seoul/13/95, strain
Seoul/37/91, strain Shangdong/7/97, strain Shanghai/361/2002),
strain Shiga/T30/98, strain Sichuan/379/99, strain
Singapore/222/79, strain Spain/WV27/2002, strain Stockholm/10/90,
strain Switzerland/5441/90, strain Taiwan/0409/00, strain
Taiwan/0722/02, strain Taiwan/97271/2001, strain Tehran/80/02,
strain Tokyo/6/98, strain Trieste/28/02, strain Ulan Ude/4/02,
strain United Kingdom/34304/99, strain USSR/100/83, strain
Victoria/103/89, strain Vienna/1/99, strain Wuhan/356/2000, strain
WV194/2002, strain Xuanwu/23/82, strain Yamagata/1311/2003, strain
Yamagata/K500/2001, strain Alaska/12/96, strain GA/86, strain
NAGASAKI/1/87, strain Tokyo/942/96, and strain
Rochester/02/2001.
[0319] Non-limiting examples of influenza C viruses include strain
Aichi/1/81, strain Ann Arbor/1/50, strain Aomori/74, strain
California/78, strain England/83, strain Greece/79, strain
Hiroshima/246/2000, strain Hiroshima/252/2000, strain Hyogo/1/83,
strain Johannesburg/66, strain Kanagawa/1/76, strain Kyoto/1/79,
strain Mississippi/80, strain Miyagi/1/97, strain Miyagi/5/2000,
strain Miyagi/9/96, strain Nara/2/85, strain NewJersey/76, strain
pig/Beijing/115/81, strain Saitama/3/2000), strain Shizuoka/79,
strain Yamagata/2/98, strain Yamagata/6/2000, strain Yamagata/9/96,
strain BERLIN/1/85, strain ENGLAND/892/8, strain GREAT
LAKES/1167/54, strain JJ/50, strain PIG/BEIJING/10/81, strain
PIG/BEIJING/439/82), strain TAYLOR/1233/47, and strain
C/YAMAGATA/10/81.
[0320] In certain embodiments, the influenza viruses provided
herein have an attenuated phenotype. In specific embodiments, the
attenuated influenza virus is based on influenza A virus. In other
embodiments, the attenuated influenza virus is based on influenza B
virus. In yet other embodiments, the attenuated influenza virus is
based on influenza C virus. In other embodiments, the attenuated
influenza virus may comprise genes or genome segments from one or
more strains or subtypes of influenza A, influenza B, and/or
influenza C virus. In some embodiments, the attenuated backbone
virus comprises genes from an influenza A virus and an influenza B
virus.
[0321] In specific embodiments, attenuation of influenza virus is
desired such that the virus remains, at least partially, infectious
and can replicate in vivo, but only generate low titers resulting
in subclinical levels of infection that are non-pathogenic. Such
attenuated viruses are especially suited for embodiments described
herein wherein the virus or an immunogenic composition thereof is
administered to a subject to induce an immune response. Attenuation
of the influenza virus can be accomplished according to any method
known in the art, such as, e.g., selecting viral mutants generated
by chemical mutagenesis, mutation of the genome by genetic
engineering, selecting reassortant viruses that contain segments
with attenuated function, or selecting for conditional virus
mutants (e.g., cold-adapted viruses). Alternatively, naturally
occurring attenuated influenza viruses may be used as influenza
virus backbones for the influenza virus vectors.
[0322] In some embodiments, an influenza virus may be attenuated,
at least in part, by engineering the influenza virus to express a
mutated NS1 gene that impairs the ability of the virus to
antagonize the cellular interferon (IFN) response. Examples of the
types of mutations that can be introduced into the influenza virus
NS1 gene include deletions, substitutions, insertions and
combinations thereof. One or more mutations can be
introduced-anywhere throughout the NS1 gene (e.g., the N-terminus,
the C-terminus or somewhere in between) and/or the regulatory
element of the NS1 gene. In one embodiment, an attenuated influenza
virus comprises a genome having a mutation in an influenza virus
NS1 gene resulting in a deletion consisting of 5, preferably 10,
15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 99,
100, 105, 110, 115, 120, 125, 126, 130, 135, 140, 145, 150, 155,
160, 165, 170 or 175 amino acid residues from the C-terminus of
NS1, or a deletion of between 5-170, 25-170, 50-170, 100-170,
100-160, or 105-160 amino acid residues from the C-terminus. In
another embodiment, an attenuated influenza virus comprises a
genome having a mutation in an influenza virus NS1 gene such that
it encodes an NS1 protein of amino acid residues 1-130, amino acid
residues 1-126, amino acid residues 1-120, amino acid residues
1-115, amino acid residues 1-110, amino acid residues 1-100, amino
acid residues 1-99, amino acid residues 1-95, amino acid residues
1-85, amino acid residues 1-83, amino acid residues 1-80, amino
acid residues 1-75, amino acid residues 1-73, amino acid residues
1-70, amino acid residues 1-65, or amino acid residues 1-60,
wherein the N-terminus amino acid is number 1. In another
embodiment, the amino acid residues of NS1 are counted based on the
PR8 virus. For examples of NS1 mutations and influenza viruses
comprising a mutated NS1, see, e.g., U.S. Pat. Nos. 6,468,544 and
6,669,943; and Li et al., 1999, J. Infect. Dis. 179:1132-1138, each
of which is incorporated by reference herein in its entirety.
[0323] 5.5 Non-Influenza Virus Vectors
[0324] In one aspect, provided herein are non-influenza viruses
containing a flu polypeptide. In a specific embodiment, the flu
polypeptide is incorporated into the virions of the non-influenza
virus. The non-influenza viruses may be conjugated to moieties that
target the viruses to particular cell types, such as immune cells.
In some embodiments, the virions of the non-influenza virus have
incorporated into them or express a heterologous polypeptide in
addition to a flu polypeptide. The heterologous polypeptide may be
a polypeptide that has immunopotentiating activity, or that targets
the non-influenza virus to a particular cell type, such as an
antibody that recognizes an antigen on a specific cell type or a
ligand that binds a specific receptor on a specific cell type. See
Section 5.4, supra, for examples of such heterologous
polypeptides.
[0325] Non-influenza viruses containing a flu polypeptide may be
produced by supplying in trans the flu polypeptide during
production of virions using techniques known to one skilled in the
art. Alternatively, a parental non-influenza virus comprises a
genome engineered to express a flu polypeptide in cells susceptible
to infection with the virus wherein hemagglutinin function is
provided in trans to produce progeny viruses containing the
influenza flu polypeptide.
[0326] Any virus type, subtype or strain including, but not limited
to, naturally occurring strains, variants or mutants, mutagenized
viruses, reassortants and/or genetically modified viruses may be
used as a non-influenza virus vector. In a specific embodiment, the
parental non-influenza virus is not a naturally occurring virus. In
another specific embodiment, the parental non-influenza virus is a
genetically engineered virus. In certain embodiments, an enveloped
virus is preferred for the expression of a membrane bound flu
polypeptide described herein.
[0327] In an exemplary embodiment, the non-influenza virus vector
is a Newcastle disease virus (NDV). In another embodiment, the
non-influenza virus vector is a vaccinia virus. In other exemplary,
non-limiting, embodiments, the non-influenza virus vector is
adenovirus, adeno-associated virus (AAV), hepatitis B virus,
retrovirus (such as, e.g., a gammaretrovirus such as Mouse Stem
Cell Virus (MSCV) genome or Murine Leukemia Virus (MLV), e.g.,
Moloney murine leukemia virus, oncoretrovirus, or lentivirus), an
alphavirus (e.g., Venezuelan equine encephalitis virus), a
rhabdovirus, such as vesicular stomatitis virus or
papillomaviruses, poxvirus (such as, e.g., vaccinia virus, a MVA-T7
vector, or fowlpox), metapneumovirus, measles virus, herpesvirus,
such as herpes simplex virus, or foamyvirus. See, e.g., Lawrie and
Tumin, 1993, Cur. Opin. Genet. Develop. 3, 102-109 (retroviral
vectors); Bett et al., 1993, J. Virol. 67, 5911 (adenoviral
vectors); Zhou et al., 1994, J. Exp. Med. 179, 1867
(adeno-associated virus vectors); Dubensky et al., 1996, J. Virol.
70, 508-519 (viral vectors from the pox family including vaccinia
virus and the avian pox viruses and viral vectors from the alpha
virus genus such as those derived from Sindbis and Semliki Forest
Viruses); U.S. Pat. No. 5,643,576 (Venezuelan equine encephalitis
virus); WO 96/34625 (VSV); Ohe et al., 1995, Human Gene Therapy 6,
325-333; Woo et al., WO 94/12629; Xiao & Brandsma, 1996,
Nucleic Acids. Res. 24, 2630-2622 (papillomaviruses); and Bukreyev
and Collins, 2008, Curr Opin Mol Ther. 10:46-55 (NDV), each of
which is incorporated by reference herein in its entirety.
[0328] In a specific embodiment, the non-influenza virus vector is
NDV. Any NDV type, subtype or strain may serve as the backbone that
is engineered to express a flu polypeptide, including, but not
limited to, naturally-occurring strains, variants or mutants,
mutagenized viruses, reassortants and/or genetically engineered
viruses. In a specific embodiment, the NDV that serves as the
backbone for genetic engineering is a naturally-occurring strain.
In certain embodiments, the NDV that serves as the backbone for
genetic engineering is a lytic strain. In other embodiments, the
NDV that serves as the backbone for genetic engineering is a
non-lytic strain. In certain embodiments, the NDV that serves as
the backbone for genetic engineering is lentogenic strain. In some
embodiments, the NDV that serves as the backbone for genetic
engineering is a mesogenic strain. In other embodiments, the NDV
that serves as the backbone for genetic engineering is a velogenic
strain. Specific examples of NDV strains include, but are not
limited to, the 73-T strain, Ulster strain, MTH-68 strain, Italien
strain, Hickman strain, PV701 strain, Hitchner B1 strain, La Sota
strain, YG97 strain, MET95 strain, and F48E9 strain. In a specific
embodiment, the NDV that serves as the backbone for genetic
engineering is the Hitchner B1 strain. In another specific
embodiment, the NDV that serves as the backbone for genetic
engineering is the La Sota strain.
[0329] In one embodiment, the NDV used as the backbone for a
non-influenza virus vector is engineered to express a modified F
protein in which the cleavage site of the F protein is replaced
with one containing one or two extra arginine residues, allowing
the mutant cleavage site to be activated by ubiquitously expressed
proteases of the furin family. Specific examples of NDVs that
express such a modified F protein include, but are not limited to,
rNDV/F2aa and rNDV/F3aa. For a description of mutations introduced
into a NDV F protein to produce a modified F protein with a mutated
cleavage site, see, e.g., Park et al. (2006) "Engineered viral
vaccine constructs with dual specificity: Avian influenza and
Newcastle disease." PNAS USA 103: 8203-2808, which is incorporated
herein by reference in its entirety.
[0330] In one embodiment, the non-influenza virus vector is a
poxvirus. A poxvirus vector may be based on any member of the
poxyiridae, in particular, a vaccinia virus or an avipox virus
(e.g., such as canarypox, fowlpox, etc.) that provides suitable
sequences for vaccine vectors. In a specific embodiment, the
poxyiral vector is a vaccinia virus vector. Suitable vaccinia
viruses include, but are not limited to, the Copenhagen (VC-2)
strain (Goebel, et al., Virol 179: 247-266, 1990; Johnson, et al.,
Virol. 196: 381-401, 1993), modified Copenhagen strain (NYVAC)
(U.S. Pat. No. 6,265,189), the WYETH strain and the modified Ankara
(MVA) strain (Antoine, et al., Virol. 244: 365-396, 1998). Other
suitable poxviruses include fowlpox strains such as ALVAC and
TROVAC vectors that provide desirable properties and are highly
attenuated (see, e.g., U.S. Pat. No. 6,265,189; Tartaglia et al.,
In AIDS Research Reviews, Koff, et al., eds., Vol. 3, Marcel
Dekker, N.Y., 1993; and Tartaglia et al., 1990, Reviews in
Immunology 10: 13-30, 1990).
[0331] Methods of engineering non-influenza viruses to express a
flu polypeptide are well known in the art, as are methods for
attenuating, propagating, and isolating and purifying such viruses.
For such techniques with respect to NDV vectors, see, e.g.,
International Publication No. WO 01/04333; U.S. Pat. Nos.
7,442,379, 6,146,642, 6,649,372, 6,544,785 and 7,384,774; Swayne et
al. (2003). Avian Dis. 47:1047-1050; and Swayne et al. (2001). J.
Virol. 11868-11873, each of which is incorporated by reference in
its entirety. For such techniques with respect to poxviruses, see,
e.g., Piccini, et al., Methods of Enzymology 153: 545-563, 1987;
International Publication No. WO 96/11279; U.S. Pat. No. 4,769,330;
U.S. Pat. No. 4,722,848; U.S. Pat. No. 4,769,330; U.S. Pat. No.
4,603,112; U.S. Pat. No. 5,110,587; U.S. Pat. No. 5,174,993; EP 83
286; EP 206 920; Mayr et al., Infection 3: 6-14, 1975; and Sutter
and Moss, Proc. Natl. Acad. Sci. USA 89: 10847-10851, 1992. In
certain embodiments, the non-influenza virus is attenuated.
[0332] Exemplary considerations for the selection of a
non-influenza virus vector, particularly for use in compositions
for administration to a subject, are safety, low toxicity,
stability, cell type specificity, and immunogenicity, particularly,
antigenicity of the flu polypeptide expressed by the non-influenza
virus vector.
[0333] 5.6 Viral-Like Particles and Virosomes
[0334] Flu polypeptides can be incorporated into viral-like
particle (VLP) vectors. VLPs generally comprise a viral
polypeptide(s) typically derived from a structural protein(s) of a
virus. In some embodiments, the VLPs are not capable of
replicating. In certain embodiments, the VLPs may lack the complete
genome of a virus or comprise a portion of the genome of a virus.
In some embodiments, the VLPs are not capable of infecting a cell.
In some embodiments, the VLPs express on their surface one or more
of viral (e.g., virus surface glycoprotein) or non-viral (e.g.,
antibody or protein) targeting moieties known to one skilled in the
art or described herein. In some embodiments, the VLP comprises a
flu polypeptide and a viral structural protein such as HIV gag.
[0335] Methods for producing and characterizing recombinantly
produced VLPs have been described based on several viruses,
including influenza virus (Bright et al. (2007) Vaccine. 25:3871),
human papilloma virus type 1 (Hagnesee et al. (1991) J. Virol.
67:315), human papilloma virus type 16 (Kirnbauer et al. Proc.
Natl. Acad. Sci. (1992)89:12180), HIV-1 (Haffer et al., (1990) J.
Virol. 64:2653), and hepatitis A (Winokur (1991) 65:5029), each of
which is incorporated herein in its entirety. Methods for
expressing VLPs that contain NDV proteins are provided by Pantua et
al. (2006) J. Virol. 80:11062-11073, and in United States patent
application Publication No. 20090068221, published Mar. 12, 2009,
each of which is incorporated in its entirety herein.
[0336] In a specific embodiment, a flu polypeptide may be
incorporated into a virosome. A virosome containing a flu
polypeptide may be produced using techniques known to those skilled
in the art. For example, a virosome may be produced by disrupting a
purified virus, extracting the genome, and reassembling particles
with the viral proteins (e.g., a flu polypeptide) and lipids to
form lipid particles containing viral proteins.
[0337] 5.7 Bacterial Vectors
[0338] In a specific embodiment, bacteria may be engineered to
express a flu polypeptide described herein. Suitable bacteria for
expression of a flu polypeptide include, but are not limited to,
Listeria, Salmonella, Shigella sp., Mycobacterium tuberculosis, E.
coli, Neisseria meningitides, Brucella abortus, Brucella
melitensis, Borrelia burgdorferi, and Francisella tularensis. In a
specific embodiment, the bacteria engineered to express a flu
polypeptide are attenuated. Techniques for the production of
bacteria engineered to express a heterologous polypeptide are known
in the art and can be applied to the expression of a flu
polypeptide. See, e.g., United States Patent Application
Publication No. 20080248066, published Oct. 9, 2008, and United
States Patent Application Publication No. 20070207171, published
Sep. 6, 2007, each of which are incorporated by reference herein in
their entirety.
[0339] 5.8 Generation of Antibodies Against Flu Polypeptides
[0340] The flu polypeptides, nucleic acids encoding such
polypeptides, or vectors comprising such nucleic acids or
polypeptides described herein may be used to elicit neutralizing
antibodies against influenza, for example, against the long
alpha-helix region of the HA2 domain of the flu polypeptide. In a
specific embodiment, the flu polypeptides, nucleic acids encoding
such polypeptides, or vectors comprising such nucleic acids or
polypeptides described herein may be administered to a non-human
subject (e.g., a mouse, rabbit, rat, guinea pig, etc.) to induce an
immune response that includes the production of antibodies which
may be isolated using techniques known to one of skill in the art
(e.g., immunoaffinity chromatography, centrifugation,
precipitation, etc.).
[0341] Alternatively, the flu polypeptides described herein may be
used to screen for antibodies from antibody libraries. For example,
an isolated flu polypeptide comprising a flu polypeptide may be
immobilized to a solid support (e.g., a silica gel, a resin, a
derivatized plastic film, a glass bead, cotton, a plastic bead, a
polystyrene bead, an alumina gel, or a polysaccharide, a magnetic
bead), and screened for binding to antibodies. As an alternative,
the antibodies may be immobilized to a solid support and screened
for binding to the isolated flu polypeptide. Any screening assay,
such as a panning assay, ELISA, surface plasmon resonance, or other
antibody screening assay known in the art may be used to screen for
antibodies that bind to the flu polypeptide. The antibody library
screened may be a commercially available antibody library, an in
vitro generated library, or a library obtained by identifying and
cloning or isolating antibodies from an individual infected with
influenza. In particular embodiments, the antibody library is
generated from a survivor of an influenza virus outbreak. Antibody
libraries may be generated in accordance with methods known in the
art. In a particular embodiment, the antibody library is generated
by cloning the antibodies and using them in phage display libraries
or a phagemid display library.
[0342] Antibodies identified in the methods described herein may be
tested for neutralizing activity and lack of autoreactivity using
the biological assays known in the art or described herein. In one
embodiment, an antibody isolated from a non-human animal or an
antibody library neutralizes a hemagglutinin polypeptide from more
than one influenza subtype. In some embodiments, an antibody
elicited or identified using a flu polypeptide, a nucleic acid
encoding such a polypeptide, or a vector encoding such a nucleic
acid or polypeptide neutralizes an influenza H3 virus. In some
embodiments, an antibody elicited or identified using a flu
polypeptide, a nucleic acid encoding such a polypeptide, or a
vector comprising such a nucleic acid or polypeptide neutralizes 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or more
subtypes or strains of influenza virus. In one embodiment, the
neutralizing antibody neutralizes one or more influenza A viruses
and one or more influenza B viruses. In particular embodiments, the
neutralizing antibody is not an antibody described in Wang et al.
(2010) "Broadly Protective Monoclonal Antibodies against H3
Influenza Viruses following Sequential Immunization with Different
Hemagglutinins," PLOS Pathogens 6(2):1-9.
[0343] Antibodies identified or elicited using a flu polypeptide, a
nucleic acid encoding such a polypeptide, or a vector comprising
such a nucleic acid or polypeptide include immunoglobulin molecules
and immunologically active portions of immunoglobulin molecules,
i.e., molecules that contain an antigen binding site that
specifically binds to a flu polypeptide. The immunoglobulin
molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and
IgY), class (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA,
and IgA.sub.2) or subclass of immunoglobulin molecule. Antibodies
include, but are not limited to, monoclonal antibodies,
multispecific antibodies, human antibodies, humanized antibodies,
chimeric antibodies, single-chain Fvs (scFv), single chain
antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs
(sdFv), and anti-idiotypic (anti-Id) antibodies (including, e.g.,
anti-Id antibodies to antibodies elicited or identified using a
method described herein), and epitope-binding fragments of any of
the above. In a specific embodiment, an antibody elicited or
identified using a flu polypeptide, a nucleic acid encoding such a
polypeptide, or a vector comprising such a nucleic acid or
polypeptide is a human or humanized monoclonal antibody.
[0344] Antibodies elicited or identified using a flu polypeptide, a
nucleic acid encoding such a polypeptide, or a vector comprising
such a nucleic acid or polypeptide may be used to monitor the
efficacy of a therapy and/or disease progression. Any immunoassay
system known in the art may be used for this purpose including, but
not limited to, competitive and noncompetitive assay systems using
techniques such as radioimmunoassays, ELISA (enzyme linked
immunosorbent assays), "sandwich" immunoassays, precipitin
reactions, gel diffusion precipitin reactions, immunodiffusion
assays, agglutination assays, complement fixation assays,
immunoradiometric assays, fluorescent immunoassays, protein A
immunoassays and immunoelectrophoresis assays, to name but a
few.
[0345] Antibodies elicited or identified using a flu polypeptide, a
nucleic acid encoding such a polypeptide, or a vector comprising
such a nucleic acid or polypeptide may be used to detect influenza
virus, for example, from a plurality of influenza virus strains
from a single subtype or 2, 3, 4 or more different subtypes and/or
to diagnosis an influenza virus infection by, for example, a
plurality of influenza virus strains from a single subtype or 2, 3,
4 or more different subtypes.
[0346] Antibodies elicited or identified using a flu polypeptide, a
nucleic acid encoding such a polypeptide, or a vector comprising
such a nucleic acid or polypeptide may be used in the production of
antiidiotypic antibody. The antiidiotypic antibody can then in turn
be used for immunization, in order to produce a subpopulation of
antibodies that bind a particular antigen of influenza, e.g., a flu
polypeptide (Jerne, 1974, Ann. Immunol. (Paris) 125c:373; Jerne et
al., 1982, EMBO J. 1:234, incorporated herein by reference in its
entirety).
[0347] In certain embodiments, the non-human subjects administered
flu polypeptides, nucleic acids encoding such polypeptides, or
vectors comprising such nucleic acids or polypeptides to generate
antibodies in accordance with the methods described herein are
transgenic animals (e.g., transgenic mice) that are capable of
producing human antibodies. Human antibodies can be produced using
transgenic mice which are incapable of expressing functional
endogenous immunoglobulins, but which can express human
immunoglobulin genes. For example, the human heavy and light chain
immunoglobulin gene complexes may be introduced randomly or by
homologous recombination into mouse embryonic stem cells.
Alternatively, the human variable region, constant region, and
diversity region may be introduced into mouse embryonic stem cells
in addition to the human heavy and light chain genes. The mouse
heavy and light chain immunoglobulin genes may be rendered
non-functional separately or simultaneously with the introduction
of human immunoglobulin loci by homologous recombination. In
particular, homozygous deletion of the JH region prevents
endogenous antibody production. The modified embryonic stem cells
are expanded and microinjected into blastocysts to produce chimeric
mice. The chimeric mice are then bred to produce homozygous
offspring which express human antibodies. The human immunoglobulin
transgenes harbored by the transgenic mice rearrange during B cell
differentiation, and subsequently undergo class switching and
somatic mutation. Thus, using such a technique, it is possible to
produce therapeutically useful IgG, IgA, IgM and IgE antibodies.
For an overview of this technology for producing human antibodies,
see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g., PCT publications WO 98/24893;
WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598
877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;
5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and
5,939,598, which are incorporated by reference herein in their
entirety. Companies such as Abgenix, Inc. (Freemont, Calif.),
Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, N.J.)
can be engaged to provide human antibodies directed against a
selected antigen.
[0348] 5.9 Stimulation of Cells with Flu Polypeptides
[0349] In another aspect, provided herein are methods for
stimulating cells ex vivo with a flu polypeptide described herein.
Such cells, e.g., dendritic cells, may be used in vitro to generate
antibodies against the flu polypeptide or may themselves be
administered to a subject by, e.g., an adoptive transfer technique
known in the art. See, e.g., United States patent application
Publication No. 20080019998, published Jan. 24, 2008, which is
incorporated herein by reference in its entirety, for a description
of adoptive transfer techniques. In certain embodiments, when cells
that have been stimulated ex vivo with a flu polypeptide described
herein are administered to a subject, the cells are not mammalian
cells (e.g., CB-1 cells). In certain embodiments, when cells that
have been stimulated ex vivo with a flu polypeptide described
herein are administered to a subject, the cells are mammalian cells
(e.g., CB-1 cells).
[0350] In one non-limiting example, a vector, e.g., an influenza
virus vector, engineered to express a flu polypeptide described
herein can be used to generate dendritic cells (DCs) that express
the flu polypeptide and display immunostimulatory properties
directed against a flu polypeptide. Such DCs may be used to expand
memory T cells and are potent stimulators of T cells, including
influenza flu polypeptide-specific cytotoxic T lymphocyte clones.
See Strobel et al., 2000, Human Gene Therapy 11:2207-2218, which is
incorporated herein by reference in its entirety.
[0351] A flu polypeptide described herein may be delivered to a
target cell in any way that allows the polypeptide to contact the
target cell, e.g., a DC, and deliver the polypeptide to the target
cell. In certain embodiments, the flu polypeptide is delivered to a
subject, as described herein. In some such embodiments, cells
contacted with the polypeptide may be isolated and propagated.
[0352] In certain embodiments, a flu polypeptide is delivered to a
target cell in vitro. Techniques known to one of skill in the art
may be used to deliver the polypeptide to target cells. For
example, target cells may be contacted with the polypeptide in a
tissue culture plate, tube or other container. The polypeptide may
be suspended in media and added to the wells of a culture plate,
tube or other container. The media containing the polypeptide may
be added prior to plating of the cells or after the cells have been
plated. The target cells are preferably incubated with the
polypeptide for a sufficient amount of time to allow the
polypeptide to contact the cells. In certain embodiments, the cells
are incubated with the polypeptide for about 1 hour or more, about
5 hours or more, about 10 hours or more, about 12 hours or more,
about 16 hours or more, about 24, hours or more, about 48 hours or
more, about 1 hour to about 12 hours, about 3 hours to about 6
hours, about 6 hours to about 12 hours, about 12 hours to about 24
hours, or about 24 hours to about 48 hours. In certain embodiments,
wherein the flu polypeptide is in a virus, the contacting of the
target cells comprises infecting the cells with the virus.
[0353] The target cells may be from any species, including, e.g.,
humans, mice, rats, rabbits and guinea pigs. In some embodiments,
target cells are DCs obtained from a healthy subject or a subject
in need of treatment. In certain embodiments, target cells are DCs
obtained from a subject in whom it is desired to stimulate an
immune response to the polypeptide. Methods of obtaining cells from
a subject are well known in the art.
[0354] 5.10 Compositions
[0355] The flu polypeptides, nucleic acids, vectors, bacteria,
antibodies, or cells described herein (sometimes referred to herein
as "active compounds") may be incorporated into compositions. In a
specific embodiment, the compositions are pharmaceutical
compositions, such as immunogenic compositions (e.g., vaccine
formulations). The pharmaceutical compositions provided herein can
be in any form that allows for the composition to be administered
to a subject. In a specific embodiment, the pharmaceutical
compositions are suitable for veterinary and/or human
administration. The compositions may be used in methods of
preventing or treating an influenza virus disease.
[0356] In one embodiment, a pharmaceutical composition comprises a
flu polypeptide, in an admixture with a pharmaceutically acceptable
carrier. In another embodiment, a pharmaceutical composition
comprises a nucleic acid encoding a flu polypeptide described
herein, in an admixture with a pharmaceutically acceptable carrier.
In another embodiment, a pharmaceutical composition comprises an
expression vector comprising a nucleic acid encoding a flu
polypeptide, in an admixture with a pharmaceutically acceptable
carrier. In another embodiment, a pharmaceutical composition
comprises an influenza virus or non-influenza virus containing a
flu polypeptide, in an admixture with a pharmaceutically acceptable
carrier. In another embodiment, a pharmaceutical composition
comprises an influenza virus or non-influenza virus having a genome
engineered to express a flu polypeptide, in admixture with a
pharmaceutically acceptable carrier. In another embodiment, a
pharmaceutical composition comprises a viral-like particle or
virosome containing a flu polypeptide, in an admixture with a
pharmaceutically acceptable carrier. In another embodiment, a
pharmaceutical composition comprises a bacteria expressing or
engineered to express a flu polypeptide, in an admixture with a
pharmaceutically acceptable carrier. In another embodiment, a
pharmaceutical composition comprises cells stimulated with a flu
polypeptide, in an admixture with a pharmaceutically acceptable
carrier.
[0357] In some embodiments, a pharmaceutical composition may
comprise one or more other therapies in addition to an active
compound.
[0358] As used herein, the term "pharmaceutically acceptable" means
approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeiae for use in animals, and more particularly
in humans. The term "carrier" refers to a diluent, adjuvant,
excipient, or vehicle with which the pharmaceutical composition is
administered. Saline solutions and aqueous dextrose and glycerol
solutions can also be employed as liquid carriers, particularly for
injectable solutions. Suitable excipients include starch, glucose,
lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride,
dried skim milk, glycerol, propylene, glycol, water, ethanol and
the like. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
The formulation should suit the mode of administration.
[0359] In a specific embodiment, pharmaceutical compositions are
formulated to be suitable for the intended route of administration
to a subject. For example, the pharmaceutical composition may be
formulated to be suitable for subcutaneous, parenteral, oral,
intradermal, transdermal, colorectal, intraperitoneal, and rectal
administration. In a specific embodiment, the pharmaceutical
composition may be formulated for intravenous, oral,
intraperitoneal, intranasal, intratracheal, subcutaneous,
intramuscular, topical, intradermal, transdermal or pulmonary
administration.
[0360] In certain embodiments, biodegradable polymers, such as
ethylene vinyl acetate, polyanhydrides, polyethylene glycol
(PEGylation), polymethyl methacrylate polymers, polylactides,
poly(lactide-co-glycolides), polyglycolic acid, collagen,
polyorthoesters, and polylactic acid, may be used as carriers. In
some embodiments, the active compounds are prepared with carriers
that increase the protection of the compound against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Methods for preparation of such formulations will be
apparent to those skilled in the art. Liposomes or micelles can
also be used as pharmaceutically acceptable carriers. These can be
prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811. In certain
embodiments, the pharmaceutical compositions comprise one or more
adjuvants.
[0361] In specific embodiments, immunogenic compositions described
herein are monovalent formulations. In other embodiments,
immunogenic compositions described herein are multivalent
formulations. In one example, a multivalent formulation comprises
one or more vectors expressing a flu polypeptide derived from an
influenza A virus and one or more vectors expressing a flu
polypeptide derived from an influenza B virus. In another example,
a multivalent formulation comprises a vector expressing a flu
polypeptide derived from an H3 influenza A virus and a vector
expressing a flu polypeptide derived from an H1 influenza A virus.
In another example, a multivalent formulation comprises a vector
expressing a flu polypeptide derived from an H3 influenza A virus,
a vector expressing a flu polypeptide derived from an H1 influenza
A virus, and a vector expressing a flu polypeptide derived from an
influenza B virus. In certain embodiments, a multivalent
formulation may comprise one or more different flu polypeptides
expressed using a single vector.
[0362] In certain embodiments, the pharmaceutical compositions
described herein additionally comprise a preservative, e.g., the
mercury derivative thimerosal. In a specific embodiment, the
pharmaceutical compositions described herein comprises 0.001% to
0.01% thimerosal. In other embodiments, the pharmaceutical
compositions described herein do not comprise a preservative. In a
specific embodiment, thimerosal is used during the manufacture of a
pharmaceutical composition described herein and the thimerosal is
removed via purification steps following production of the
pharmaceutical composition, i.e., the pharmaceutical composition
contains trace amounts of thimerosal (<0.3 .mu.g of mercury per
dose after purification; such pharmaceutical compositions are
considered thimerosal-free products).
[0363] In certain embodiments, the pharmaceutical compositions
described herein additionally comprise egg protein (e.g., ovalbumin
or other egg proteins). The amount of egg protein in the
pharmaceutical compositions described herein may range from about
0.0005 to about 1.2 .mu.g of egg protein to 1 ml of pharmaceutical
composition. In other embodiments, the pharmaceutical compositions
described herein do not comprise egg protein.
[0364] In certain embodiments, the pharmaceutical compositions
described herein additionally comprise one or more antimicrobial
agents (e.g., antibiotics) including, but not limited to
gentamicin, neomycin, polymyxin (e.g., polymyxin B), and kanamycin,
streptomycin. In other embodiments, the pharmaceutical compositions
described herein do not comprise any antibiotics.
[0365] In certain embodiments, the pharmaceutical compositions
described herein additionally comprise one or more components used
to inactivate a virus, e.g., formalin or formaldehyde or a
detergent such as sodium deoxycholate, octoxynol 9 (Triton X-100),
and octoxynol 10. In other embodiments, the pharmaceutical
compositions described herein do not comprise any components used
to inactivate a virus.
[0366] In certain embodiments, the pharmaceutical compositions
described herein additionally comprise gelatin. In other
embodiments, the pharmaceutical compositions described herein do
not comprise gelatin.
[0367] In certain embodiments, the pharmaceutical compositions
described herein additionally comprise one or more buffers, e.g.,
phosphate buffer and sucrose phosphate glutamate buffer. In other
embodiments, the pharmaceutical compositions described herein do
not comprise buffers.
[0368] In certain embodiments, the pharmaceutical compositions
described herein additionally comprise one or more salts, e.g.,
sodium chloride, calcium chloride, sodium phosphate, monosodium
glutamate, and aluminum salts (e.g., aluminum hydroxide, aluminum
phosphate, alum (potassium aluminum sulfate), or a mixture of such
aluminum salts). In other embodiments, the pharmaceutical
compositions described herein do not comprise salts.
[0369] In specific embodiments, the pharmaceutical compositions
described herein are low-additive influenza virus vaccines, i.e.,
the pharmaceutical compositions do not comprise one or more
additives commonly found in influenza virus vaccines. Low-additive
influenza vaccines have been described (see, e.g., International
Application No. PCT/IB2008/002238 published as International
Publication No. WO 09/001,217 which is herein incorporated by
reference in its entirety).
[0370] The pharmaceutical compositions described herein can be
included in a container, pack, or dispenser together with
instructions for administration.
[0371] The pharmaceutical compositions described herein can be
stored before use, e.g., the pharmaceutical compositions can be
stored frozen (e.g., at about -20.degree. C. or at about
-70.degree. C.); stored in refrigerated conditions (e.g., at about
4.degree. C.); or stored at room temperature (see International
Application No. PCT/IB2007/001149 published as International
Publication No. WO 07/110,776, which is herein incorporated by
reference in its entirety, for methods of storing compositions
comprising influenza vaccines without refrigeration).
[0372] In certain embodiments, when the active compound in a
pharmaceutical composition described herein is a cell engineered to
express a flu polypeptide, the cells in the pharmaceutical
composition are not mammalian cells (e.g., CB-1 cells). In certain
embodiments, when the active compound in a pharmaceutical
composition described herein is a cell engineered to express a flu
polypeptide, the cells in the pharmaceutical composition are
mammalian cells.
[0373] 5.10.1 Subunit Vaccines
[0374] In a specific embodiment, provided herein are subunit
vaccines comprising a core polypeptide described herein. In some
embodiments, a subunit vaccine comprises a flu polypeptide and one
or more surface glycoproteins (e.g., influenza virus
neuraminidase), other targeting moieties or adjuvants. In specific
embodiments, a subunit vaccine comprises a single influenza flu
polypeptide. In other embodiments, a subunit vaccine comprises two,
three, four or more influenza flu polypeptides. In specific
embodiments, the influenza flu polypeptide(s) used in a subunit
vaccine is not membrane-bound, i.e., it is soluble.
[0375] In certain embodiments, provided herein are subunit vaccines
comprising about 10 .mu.g to about 60 .mu.g of one or more flu
polypeptides described herein, about 0.001% to 0.01% thimerosal,
about 0.1 .mu.g to about 1.0 .mu.g chicken egg protein, about 1.0
.mu.g to about 5.0 .mu.g polymyxin, about 1.0 .mu.g to about 5.0
.mu.g neomycin, about 0.1 .mu.g to about 0.5 .mu.g
betapropiolactone, and about 0.001 to about 0.05% w/v of
nonylphenol ethoxylate per dose.
[0376] In a specific embodiment, a subunit vaccine provided herein
comprises or consists of a 0.5 ml dose that comprises 45 .mu.g of a
flu polypeptide(s) provided herein, .ltoreq.1.0 .mu.g of mercury
(from thimerosal), .ltoreq.1.0 .mu.g chicken egg protein (i.e.,
ovalbumin), .ltoreq.3.75 .mu.g polymyxin, and .ltoreq.2.5 .mu.g
neomycin. In some embodiments, a subunit vaccine provided herein
additionally comprises or consists of not more than 0.5 .mu.g
betapropiolactone, and not more than 0.015% w/v of nonylphenol
ethoxylate per dose. In some embodiments, the 0.5 ml dose subunit
vaccine is packaged in a pre-filled syringe.
[0377] In a specific embodiment, a subunit vaccine provided herein
consists of a 5.0 ml multidose vial (0.5 ml per dose) that
comprises 45 .mu.g of a flu polypeptide(s) provided herein, 25.0
.mu.g of mercury (from thimerosal), .ltoreq.1.0 .mu.g chicken egg
protein (i.e., ovalbumin), .ltoreq.3.75 .mu.g polymyxin, and
.ltoreq.2.5 .mu.g neomycin. In some embodiments, a subunit vaccine
provided herein additionally comprises or consists of not more than
0.5 .mu.g betapropiolactone, and not more than 0.015% w/v of
nonylphenol ethoxylate per dose.
[0378] In a specific embodiment, the subunit vaccine is prepared
using influenza virus that was propagated in embryonated chicken
eggs (i.e., the components of the subunit vaccine (e.g., a flu
polypeptide) are isolated from virus that was propagated in
embryonated chicken eggs). In another specific embodiment, the
subunit vaccine is prepared using influenza virus that was not
propagated in embryonated chicken eggs (i.e., the components of the
subunit vaccine (e.g., a flu polypeptide) are isolated from virus
that was not propagated in embryonated chicken eggs). In another
specific embodiment, the subunit vaccine is prepared using
influenza virus that was propagated in mammalian cells, e.g.,
immortalized human cells (see, e.g., International Application No.
PCT/EP2006/067566 published as International Publication No. WO
07/045,674 which is herein incorporated by reference in its
entirety) or canine kidney cells such as MDCK cells (see, e.g.,
International Application No. PCT/IB2007/003536 published as
International Publication No. WO 08/032,219 which is herein
incorporated by reference in its entirety) (i.e., the components of
the subunit vaccine (e.g., a flu polypeptide) are isolated from
virus that was propagated in mammalian cells). In another specific
embodiment, the flu polypeptide(s) in a subunit vaccine are
prepared using an expression vector, e.g., a viral vector, plant
vector or a bacterial vector (i.e., the flu polypeptide(s) in the
subunit vaccine are obtained/isolated from an expression
vector).
[0379] 5.10.2 Live Virus Vaccines
[0380] In one embodiment, provided herein are immunogenic
compositions (e.g., vaccines) comprising live virus containing a
flu polypeptide. In another embodiment, provided herein are
immunogenic compositions (e.g., vaccines) comprising live virus
that is engineered to encode a flu polypeptide, which is expressed
by progeny virus produced in the subjects administered the
compositions. In specific embodiments, the flu polypeptide is
membrane-bound. In other specific embodiments, the influenza virus
flu polypeptide is not membrane-bound, i.e., soluble. In particular
embodiments, the live virus is an influenza virus, such as
described in Section 5.4, supra. In other embodiments, the live
virus is a non-influenza virus, such as described in Section 5.5,
supra. In some embodiments, the live virus is attenuated. In some
embodiments, an immunogenic composition comprises two, three, four
or more live viruses containing or engineered to express two,
three, four or more different flu polypeptides.
[0381] In certain embodiments, provided herein are immunogenic
compositions (e.g., vaccines) comprising about 10.sup.5 to about
10.sup.10 fluorescent focus units (FFU) of live attenuated
influenza virus containing one or more flu polypeptides described
herein, about 0.1 to about 0.5 mg monosodium glutamate, about 1.0
to about 5.0 mg hydrolyzed procine gelatin, about 1.0 to about 5.0
mg arginine, about 10 to about 15 mg sucrose, about 1.0 to about
5.0 mg dibasic potassium phosphate, about 0.5 to about 2.0 mg
monobasic potassium phosphate, and about 0.001 to about 0.05
.mu.g/mlgentamicin sulfate per dose. In some embodiments, the
immunogenic compositions (e.g., vaccines) are packaged as
pre-filled sprayers containing single 0.2 ml doses.
[0382] In a specific embodiment, provided herein are immunogenic
compositions (e.g., vaccines) comprising 10.sup.6.5 to 10.sup.7.5
FFU of live attenuated influenza virus containing one or more flu
polypeptides described herein, 0.188 mg monosodium glutamate, 2.0
mg hydrolyzed procine gelatin, 2.42 mg arginine, 13.68 mg sucrose,
2.26 mg dibasic potassium phosphate, 0.96 mg monobasic potassium
phosphate, and <0.015 .mu.g/ml gentamicin sulfate per dose. In
some embodiments, the immunogenic compositions (e.g., vaccines) are
packaged as pre-filled sprayers containing single 0.2 ml doses.
[0383] In a specific embodiment, the live virus that contains a flu
polypeptide is propagated in embryonated chicken eggs before its
use in an immunogenic composition described herein. In another
specific embodiment, the live virus that contains a flu polypeptide
is not propagated in embryonated chicken eggs before its use in an
immunogenic composition described herein. In another specific
embodiment, the live virus that contains a flu polypeptide is
propagated in mammalian cells, e.g., immortalized human cells (see,
e.g., International Application No. PCT/EP2006/067566 published as
International Publication No. WO 07/045,674 which is herein
incorporated by reference in its entirety) or canine kidney cells
such as MDCK cells (see, e.g., International Application No.
PCT/IB2007/003536 published as International Publication No. WO
08/032,219 which is herein incorporated by reference in its
entirety) before its use in an immunogenic composition described
herein.
[0384] An immunogenic composition comprising a live virus for
administration to a subject may be preferred because multiplication
of the virus in the subject may lead to a prolonged stimulus of
similar kind and magnitude to that occurring in natural infections,
and therefore, confer substantial, long lasting immunity.
[0385] 5.10.3 Inactivated Virus Vaccines
[0386] In one embodiment, provided herein are immunogenic
compositions (e.g., vaccines) comprising an inactivated virus
containing a flu polypeptide. In specific embodiments, the flu
polypeptide is membrane-bound. In particular embodiments, the
inactivated virus is an influenza virus, such as described in
Section 5.4, supra. In other embodiments, the inactivated virus is
a non-influenza virus, such as described in Section 5.5, supra. In
some embodiments, an immunogenic composition comprises two, three,
four or more inactivated viruses containing two, three, four or
more different flu polypeptides. In certain embodiments, the
inactivated virus immunogenic compositions comprise one or more
adjuvants.
[0387] Techniques known to one of skill in the art may be used to
inactivate viruses containing a flu polypeptide. Common methods use
formalin, heat, or detergent for inactivation. See, e.g., U.S. Pat.
No. 6,635,246, which is herein incorporated by reference in its
entirety. Other methods include those described in U.S. Pat. Nos.
5,891,705; 5,106,619 and 4,693,981, which are incorporated herein
by reference in their entireties.
[0388] In certain embodiments, provided herein are immunogenic
compositions (e.g., vaccines) comprising inactivated influenza
virus such that each dose of the immunogenic composition comprises
about 15 to about 60 .mu.g of flu polypeptide described herein,
about 1.0 to about 5.0 mg sodium chloride, about 20 to about 100
.mu.g monobasic sodium phosphate, about 100 to about 500 .mu.g
dibasic sodium phosphate, about 5 to about 30 .mu.g monobasic
potassium phosphate, about 5 to about 30 .mu.g potassium chloride,
and about 0.5 to about 3.0 .mu.g calcium chloride. In some
embodiments, the immunogenic compositions (e.g., vaccines) are
packaged as single 0.25 ml or single 0.5 ml doses. In other
embodiments, the immunogenic compositions (e.g., vaccines) are
packaged as multi-dose formulations.
[0389] In certain embodiments, provided herein are immunogenic
compositions (e.g., vaccines) comprising inactivated influenza
virus such that each dose of the immunogenic composition comprises
about 15 to about 60 .mu.g of flu polypeptide described herein,
about 0.001% to 0.01% thimerosal, about 1.0 to about 5.0 mg sodium
chloride, about 20 to about 100 .mu.g monobasic sodium phosphate,
about 100 to about 500 .mu.g dibasic sodium phosphate, about 5 to
about 30 .mu.g monobasic potassium phosphate, about 5 to about 30
mg potassium chloride, and about 0.5 to about 3.0 .mu.g calcium
chloride per dose. In some embodiments, the immunogenic
compositions (e.g., vaccines) are packaged as single 0.25 ml or
single 0.5 ml doses. In other embodiments, the immunogenic
compositions (e.g., vaccines) are packaged as multi-dose
formulations.
[0390] In a specific embodiment, immunogenic compositions (e.g.,
vaccines) provided herein are packaged as single 0.25 ml doses and
comprise 22.5 .mu.g of flu polypeptide described herein, 2.05 mg
sodium chloride, 40 .mu.g monobasic sodium phosphate, 150 .mu.g
dibasic sodium phosphate, 10 .mu.g monobasic potassium phosphate,
10 .mu.g potassium chloride, and 0.75 .mu.g calcium chloride per
dose.
[0391] In a specific embodiment, immunogenic compositions (e.g.,
vaccines) provided herein are packaged as single 0.5 ml doses and
comprise 45 .mu.g of flu polypeptide described herein, 4.1 mg
sodium chloride, 80 .mu.g monobasic sodium phosphate, 300 .mu.g
dibasic sodium phosphate, 20 .mu.g monobasic potassium phosphate,
20 .mu.g potassium chloride, and 1.5 .mu.g calcium chloride per
dose.
[0392] In a specific embodiment, immunogenic compositions (e.g.,
vaccines) are packaged as multi-dose formulations comprising or
consisting of 5.0 ml of vaccine (0.5 ml per dose) and comprise 24.5
.mu.g of mercury (from thimerosal), 45 .mu.g of flu polypeptide
described herein, 4.1 mg sodium chloride, 80 .mu.g monobasic sodium
phosphate, 300 .mu.g dibasic sodium phosphate, 20 .mu.g monobasic
potassium phosphate, 20 .mu.g potassium chloride, and 1.5 .mu.g
calcium chloride per dose.
[0393] In a specific embodiment, the inactivated virus that
contains a flu polypeptide was propagated in embryonated chicken
eggs before its inactivation and subsequent use in an immunogenic
composition described herein. In another specific embodiment, the
inactivated virus that contains a flu polypeptide was not
propagated in embryonated chicken eggs before its inactivation and
subsequent use in an immunogenic composition described herein. In
another specific embodiment, the inactivated virus that contains a
flu polypeptide was propagated in mammalian cells, e.g.,
immortalized human cells (see, e.g., International Application No.
PCT/EP2006/067566 published as International Publication No. WO
07/045,674 which is herein incorporated by reference in its
entirety) or canine kidney cells such as MDCK cells (see, e.g.,
International Application No. PCT/IB2007/003536 published as
International Publication No. WO 08/032,219 which is herein
incorporated by reference in its entirety) before its inactivation
and subsequent use in an immunogenic composition described
herein.
[0394] 5.10.4 Split Virus Vaccines
[0395] In one embodiment, an immunogenic composition comprising a
flu polypeptide is a split virus vaccine. In some embodiments,
split virus vaccine contains two, three, four or more different flu
polypeptides. In certain embodiments, the flu polypeptide is/was
membrane-bound. In certain embodiments, the split virus vaccines
comprise one or more adjuvants.
[0396] Techniques for producing split virus vaccines are known to
those skilled in the art. By way of non-limiting example, an
influenza virus split vaccine may be prepared using inactivated
particles disrupted with detergents. One example of a split virus
vaccine that can be adapted for use in accordance with the methods
described herein is the Fluzone.RTM., Influenza Virus Vaccine
(Zonal Purified, Subvirion) for intramuscular use, which is
formulated as a sterile suspension prepared from influenza viruses
propagated in embryonated chicken eggs. The virus-containing fluids
are harvested and inactivated with formaldehyde. Influenza virus is
concentrated and purified in a linear sucrose density gradient
solution using a continuous flow centrifuge. The virus is then
chemically disrupted using a nonionic surfactant, octoxinol-9,
(Triton.RTM. X-100--A registered trademark of Union Carbide, Co.)
producing a "split virus." The split virus is then further purified
by chemical means and suspended in sodium phosphate-buffered
isotonic sodium chloride solution.
[0397] In certain embodiments, provided herein are split virus
vaccines comprising about 10 .mu.g to about 60 .mu.g of one or more
flu polypeptides described herein, about 0.01 to about 1.0 mg
octoxynol-10 (TRITON X-1000, about 0.5 to 0.5 mg .alpha.-tocopheryl
hydrogen succinate, about 0.1 to 1.0 mg polysorbate 80 (Tween 80),
about 0.001 to about 0.003 .mu.g hydrocortisone, about 0.05 to
about 0.3 .mu.g gentamcin sulfate, about 0.5 to about 2.0
.mu.gchicken egg protein (ovalbumin), about 25 to 75 .mu.g
formaldehyde, and about 25 to 75 .mu.g sodium deoxycholate.
[0398] In a specific embodiment, a split virus vaccine provided
herein comprises or consists of a 0.5 ml dose that comprises 45
.mu.g of influenza flu polypeptide(s) provided herein,
.ltoreq.0.085 mg octoxynol-10 (TRITON X-100.RTM., .ltoreq.0.1 mg
.alpha.-tocopheryl hydrogen succinate, .ltoreq.0.415 mg polysorbate
80 (Tween 80), .ltoreq.0.0016 .mu.g hydrocortisone, .ltoreq.0.15
.mu.g gentamcin sulfate, .ltoreq.1.0 chicken egg protein
(ovalbumin), .ltoreq.50 mg formaldehyde, and .ltoreq.50 .mu.g
sodium deoxycholate. In some embodiments, the 0.5 ml dose subunit
vaccine is packaged in a pre-filled syringe.
[0399] In a specific embodiment, the split virus vaccine is
prepared using influenza virus that was propagated in embryonated
chicken eggs. In another specific embodiment, the split virus
vaccine is prepared using influenza virus that was not propagated
in embryonated chicken eggs. In another specific embodiment, the
split virus vaccine is prepared using influenza virus that was
propagated in mammalian cells, e.g., immortalized human cells (see,
e.g., PCT/EP2006/067566 published as WO 07/045,674 which is herein
incorporated by reference in its entirety) or canine kidney cells
such as MDCK cells (see, e.g., PCT/IB2007/003536 published as WO
08/032,219 which is herein incorporated by reference in its
entirety).
[0400] 5.10.5 Adjuvants
[0401] In certain embodiments, the compositions described herein
comprise, or are administered in combination with, an adjuvant. The
adjuvant for administration in combination with a composition
described herein may be administered before, concomitantly with, or
after administration of said composition. In some embodiments, the
term "adjuvant" refers to a compound that when administered in
conjunction with or as part of a composition described herein
augments, enhances and/or boosts the immune response to a flu
polypeptide, but when the compound is administered alone does not
generate an immune response to the polypeptide. In some
embodiments, the adjuvant generates an immune response to the
polypeptide and does not produce an allergy or other adverse
reaction. Adjuvants can enhance an immune response by several
mechanisms including, e.g., lymphocyte recruitment, stimulation of
B and/or T cells, and stimulation of macrophages.
[0402] In certain embodiments, an adjuvant augments the intrinsic
response to the flu polypeptide without causing conformational
changes in the polypeptide that affect the qualitative form of the
response. Specific examples of adjuvants include, but are not
limited to, aluminum salts (alum) (such as aluminum hydroxide,
aluminum phosphate, and aluminum sulfate), 3 De-O-acylated
monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis),
AS03 (GlaxoSmithKline), AS04 (GlaxoSmithKline), polysorbate 80
(Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see
International Application No. PCT/US2007/064857, published as
International Publication No. WO2007/109812), imidazoquinoxaline
compounds (see International Application No. PCT/US2007/064858,
published as International Publication No. WO2007/109813) and
saponins, such as QS21 (see Kensil et al., in Vaccine Design: The
Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum
Press, NY, 1995); U.S. Pat. No. 5,057,540). In some embodiments,
the adjuvant is Freund's adjuvant (complete or incomplete). Other
adjuvants are oil in water emulsions (such as squalene or peanut
oil), optionally in combination with immune stimulants, such as
monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336,
86-91 (1997)). Another adjuvant is CpG (Bioworld Today, Nov. 15,
1998). Such adjuvants can be used with or without other specific
immunostimulating agents such as MPL or 3-DMP, QS21, polymeric or
monomeric amino acids such as polyglutamic acid or polylysine, or
other immunopotentiating agents described in Section 5.4, supra. It
should be understood that different formulations of flu polypeptide
may comprise different adjuvants or may comprise the same
adjuvant.
[0403] 5.11 Prophylactic and Therapeutic Uses
[0404] In one aspect, provided herein are methods for inducing an
immune response in a subject utilizing an active compound, i.e., a
flu polypeptide described herein, a nucleic acid encoding such a
polypeptide, a vector (e.g., a viral vector, or a bacteria)
containing or expressing such a polypeptide, or cells stimulated
with such a polypeptide. In a specific embodiment, a method for
inducing an immune response to an influenza virus in a subject
comprises administering to a subject in need thereof an effective
amount of a flu polypeptide or an immunogenic composition thereof.
In another embodiment, a method for inducing an immune response to
an influenza virus in a subject comprises administering to a
subject in'need thereof an effective amount of a nucleic acid
encoding a flu polypeptide or an immunogenic composition thereof.
In another embodiment, a method for inducing an immune response to
an influenza virus in a subject comprises administering to a
subject in need thereof an effective amount of a viral vector
containing or expressing a flu polypeptide or an immunogenic
composition thereof. In yet another embodiment, a method for
inducing an immune response to an influenza virus in a subject
comprises administering to a subject in need thereof an effective
amount of cells stimulated with a flu polypeptide or a
pharmaceutical composition thereof. In certain embodiments, a flu
polypeptide used in the method is a purified flu polypeptide
derived from a mammalian cell, a plant cell, or an insect cell.
[0405] In a specific embodiment, a method for inducing an immune
response to an influenza virus in a subject comprises administering
to a subject in need thereof a subunit vaccine described herein. In
another embodiment, a method for inducing an immune response to an
influenza virus in a subject comprises administering to a subject
in need thereof a live virus vaccine described herein. In
particular embodiments, the live virus vaccine comprises an
attenuated virus. In another embodiment, a method for inducing an
immune response to an influenza virus in a subject comprises
administering to a subject in need thereof an inactivated virus
vaccine described herein. In another embodiment, a method for
inducing an immune response to an influenza virus in a subject
comprises administering to a subject in need thereof a split virus
vaccine described herein. In another embodiment, a method for
inducing an immune response to an influenza virus in a subject
comprises administering to a subject in need thereof a viral-like
particle vaccine described herein. In another embodiment, a method
for inducing an immune response to an influenza virus in a subject
comprises administering to a subject in need thereof a virosome
described herein. In another embodiment, a method for inducing an
immune response to an influenza virus in a subject comprises
administering to a subject in need thereof a bacteria expressing or
engineered to express a flu polypeptide or a composition thereof.
In certain embodiments, a flu polypeptide used in the method is a
purified flu polypeptide derived from a mammalian cell, a plant
cell, or an insect cell.
[0406] In some embodiments, the immune response induced by an
active compound or a composition described herein is effective to
prevent and/or treat an influenza virus infection caused by any
subtype or strain of influenza virus. In certain embodiments, the
immune response induced by an active compound or a composition
described herein is effective to prevent and/or treat an influenza
virus infection caused by a subtype of influenza virus that belongs
to one HA group (e.g., Group 1, which comprises H1, H2, H5, F16,
H8, H9, H11, H12, H13, and H16) and not the other HA group (e.g.,
Group 2, which comprises H3, H4, H7, H10, H14, and H15). For
example, the immune response induced may be effective to prevent
and/or treat an influenza virus infection caused by an influenza
virus that belongs to the HA group consisting of H11, H13, H16, H9,
H8, H12, H6, H1, H5 and H2. Alternatively, the immune response
induced may be effective to prevent and/or treat an influenza virus
infection caused by an influenza virus that belongs to the HA group
consisting of H3, H4, H14, H10, H15 and H7. In some embodiments,
the immune response induced by an active compound or a composition
described herein is effective to prevent and/or treat an influenza
virus infection caused by one, two, three, four or five subtypes of
influenza virus. In certain embodiments, the immune response
induced by an active compound or a composition described herein is
effective to prevent and/or treat an influenza virus infection
caused by six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen or fifteen subtypes of influenza virus. In some
embodiments, the immune response induced by an active compound or a
composition described herein is effective to prevent and/or treat
an influenza virus infection caused by one or more variants within
the same subtype of influenza virus.
[0407] In some embodiments, the immune response induced by an
active compound or a composition described herein is effective to
prevent and/or treat an influenza virus infection caused by both
H1N1 and H2N2 subtypes. In other embodiments, the immune response
induced by an active compound or a composition described herein is
not effective to prevent and/or treat an influenza virus infection
caused by both H1N1 and H2N2 subtypes. In some embodiments, the
immune response induced by an active compound or a composition
described herein is effective to prevent and/or treat an influenza
virus infection caused by H1N1, H2N2, and H3N2 subtypes. In some
embodiments, the immune response induced by an active compound or a
composition described herein is effective to prevent and/or treat
an influenza virus infection caused by H3N2 subtypes. In other
embodiments, the immune response induced by an active compound or a
composition described herein is not effective to prevent and/or
treat an influenza virus infection caused by H3N2 subtypes.
[0408] In some embodiments, the immune response induced by an
active compound or a composition described herein is effective to
prevent and/or treat an influenza virus disease caused by any
subtype or strain of influenza virus. In certain embodiments, the
immune response induced by an active compound or a composition
described herein is effective to prevent and/or treat an influenza
virus disease caused by a subtype of influenza virus that belongs
to one HA group and not the other HA group. For example, the immune
response induced may be effective to prevent and/or treat an
influenza virus disease caused by an influenza virus that belongs
to the HA group consisting of H11, H13, H16, H9, H8, H12, H6, H1,
H5 and H2. Alternatively, the immune response induced may be
effective to prevent and/or treat an influenza virus disease caused
by an influenza virus that belongs to the HA group consisting of
H3, H4, H14, H10, H15 and H7. In some embodiments, the immune
response induced by an active compound or a composition described
herein is effective to prevent and/or treat an influenza virus
disease caused by any of one, two, three, four or five subtypes of
influenza virus. In certain embodiments, the immune response
induced by an active compound or a composition described herein is
effective to prevent and/or treat an influenza virus disease caused
by any of six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen or fifteen subtypes of influenza virus. In some
embodiments, the immune response induced by an active compound or a
composition described herein is effective to prevent and/or treat
an influenza virus disease caused by one or more variants within
the same subtype of influenza virus.
[0409] In some embodiments, the immune response induced by an
active compound or a composition described herein is effective to
reduce symptoms resulting from an influenza virus
disease/infection. Symptoms of influenza virus disease/infection
include, but are not limited to, body aches (especially joints and
throat), fever, nausea, headaches, irritated eyes, fatigue, sore
throat, reddened eyes or skin, and abdominal pain.
[0410] In some embodiments, the immune response induced by an
active compound or a composition described herein is effective to
reduce the hospitalization of a subject suffering from an influenza
virus disease/infection. In some embodiments, the immune response
induced by an active compound or a composition described herein is
effective to reduce the duration of hospitalization of a subject
suffering from an influenza virus disease/infection.
[0411] In another aspect, provided herein are methods for
preventing and/or treating an influenza virus infection in a
subject utilizing an active compound (e.g., a flu polypeptide
described herein, a nucleic acid encoding such a polypeptide, a
vector containing or expressing such a polypeptide, or cells
stimulated with such a polypeptide) or a composition described
herein. In one embodiment, a method for preventing or treating an
influenza virus infection in a subject comprises administering to a
subject in need thereof a flu polypeptide, a nucleic acid encoding
such a polypeptide, a vector containing or expressing such a
polypeptide, or a composition of any one of the foregoing. In a
specific embodiment, a method for preventing or treating an
influenza virus infection in a subject comprises administering to a
subject in need thereof a subunit vaccine, a live virus vaccine, an
inactivated virus vaccine, a split virus vaccine or a viral-like
particle vaccine. In specific embodiments, the influenza virus
infection is caused by an influenza A virus. In other embodiments,
the influenza virus infection is caused by an influenza B or C
virus.
[0412] In another aspect, provided herein are methods for
preventing and/or treating an influenza virus disease in a subject
utilizing a flu polypeptide described herein, a nucleic acid
encoding such a polypeptide, a vector containing or expressing such
a polypeptide, or cells stimulated with such a polypeptide. In a
specific embodiment, a method for preventing or treating an
influenza virus disease in a subject comprises administering to a
subject in need thereof an effective amount of a flu polypeptide or
an immunogenic composition thereof. In another embodiment, a method
for preventing or treating an influenza virus disease in a subject
comprises administering to a subject in need thereof an effective
amount of a nucleic acid encoding a flu polypeptide or an
immunogenic composition thereof. In another embodiment, a method
for preventing or treating an influenza virus disease in a subject
comprises administering to a subject in need thereof an effective
amount of a viral vector containing or expressing a flu polypeptide
or an immunogenic composition thereof. In yet another embodiment, a
method for preventing or treating an influenza virus disease in a
subject comprises administering to a subject in need thereof an
effective amount of cells stimulated with a flu polypeptide or a
pharmaceutical composition thereof.
[0413] In a specific embodiment, a method for preventing or
treating an influenza virus disease in a subject comprises
administering to a subject in need thereof a subunit vaccine
described herein. In another embodiment, a method for preventing or
treating an influenza virus disease in a subject comprises
administering to a subject in need thereof a live virus vaccine
described herein. In particular embodiments, the live virus vaccine
comprises an attenuated virus. In another embodiment, a method for
preventing or treating an influenza virus disease in a subject
comprises administering to a subject in need thereof an inactivated
virus vaccine described herein. In another embodiment, a method for
preventing or treating an influenza virus disease in a subject
comprises administering to a subject in need thereof a split virus
vaccine described herein. In another embodiment, a method for
preventing or treating an influenza virus disease comprises
administering to a subject in need thereof a viral-like particle
vaccine described herein. In another embodiment, a method for
preventing or treating an influenza virus disease in a subject,
comprising administering to a subject in need thereof a virosome
described herein. In another embodiment, a method for preventing or
treating an influenza virus disease in a subject comprising
administering to a subject in need thereof a bacteria expressing or
engineered to express a flu polypeptide or a composition thereof.
In specific embodiments, the influenza virus disease is caused by
or associated with the presence of an influenza A virus. In other
embodiments, the influenza virus disease is caused by or associated
with the presence of an influenza B virus.
[0414] In another aspect, provided herein are methods of preventing
and/or treating an influenza virus disease in a subject by
administering neutralizing antibodies described herein. In a
specific embodiment, a method for preventing or treating an
influenza virus disease in a subject comprises administering to a
subject in need thereof an effective amount of a neutralizing
antibody described herein, or a pharmaceutical composition thereof.
In particular embodiments, the neutralizing antibody is a
monoclonal antibody. In certain embodiments, the neutralizing
antibody is not an antibody described in Wang et al. (2010)
"Broadly Protective Monoclonal Antibodies against H3 Influenza
Viruses following Sequential Immunization with Different
Hemagglutinins," PLOS Pathogens 6(2):1-9. In certain embodiments,
the neutralizing antibody is not an antibody described in
PCT/US2010/036170.
[0415] In certain embodiments, the methods for preventing or
treating an influenza virus disease or infection in a subject
(e.g., a human or non-human animal) provided herein result in a
reduction in the replication of the influenza virus in the subject
as measured by in vivo and in vitro assays known to those of skill
in the art and described herein. In some embodiments, the
replication of the influenza virus is reduced by approximately 1
log or more, approximately 2 logs or more, approximately 3 logs or
more, approximately 4 logs or more, approximately 5 logs or more,
approximately 6 logs or more, approximately 7 logs or more,
approximately 8 logs or more, approximately 9 logs or more,
approximately 10 logs or more, 1 to 3 logs, 1 to 5 logs, 1 to 8
logs, 1 to 9 logs, 2 to 10 logs, 2 to 5 logs, 2 to 7 logs, 2 logs
to 8 logs, 2 to 9 logs, 2 to 10 logs 3 to 5 logs, 3 to 7 logs, 3 to
8 logs, 3 to 9 logs, 4 to 6 logs, 4 to 8 logs, 4 to 9 logs, 5 to 6
logs, 5 to 7 logs, 5 to 8 logs, 5 to 9 logs, 6 to 7 logs, 6 to 8
logs, 6 to 9 logs, 7 to 8 logs, 7 to 9 logs, or 8 to 9 logs.
[0416] 5.11.1 Combination Therapies
[0417] In various embodiments, a flu polypeptide described herein,
a nucleic acid encoding such a polypeptide, a vector (e.g., a viral
vector or a bacteria) containing or expressing such a polypeptide,
cells stimulated with such a polypeptide, or a neutralizing
antibody may be administered to a subject in combination with one
or more other therapies (e.g., antiviral, antibacterial, or
immunomodulatory therapies). In some embodiments, a pharmaceutical
composition (e.g., an immunogenic composition) described herein may
be administered to a subject in combination with one or more
therapies. The one or more other therapies may be beneficial in the
treatment or prevention of an influenza virus disease or may
ameliorate a symptom or condition associated with an influenza
virus disease. In some embodiments, the one or more other therapies
are pain relievers, anti-fever medications, or therapies that
alleviate or assist with breathing. In certain embodiments, the
therapies are, administered less than 5 minutes apart, less than 30
minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to
about 2 hours apart, at about 2 hours to about 3 hours apart, at
about 3 hours to about 4 hours apart, at about 4 hours to about 5
hours apart, at about 5 hours to about 6 hours apart, at about 6
hours to about 7 hours apart, at about 7 hours to about 8 hours
apart, at about 8 hours to about 9 hours apart, at about 9 hours to
about 10 hours apart, at about 10 hours to about 11 hours apart, at
about 11 hours to about 12 hours apart, at about 12 hours to 18
hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours
apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52
hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84
hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours
part. In specific embodiments, two or more therapies are
administered within the same patent visit.
[0418] Any anti-viral agents well-known to one of skill in the art
may used in combination with an active compound or pharmaceutical
composition described herein. Non-limiting examples of anti-viral
agents include proteins, polypeptides, peptides, fusion proteins
antibodies, nucleic acid molecules, organic molecules, inorganic
molecules, and small molecules that inhibit and/or reduce the
attachment of a virus to its receptor, the internalization of a
virus into a cell, the replication of a virus, or release of virus
from a cell. In particular, anti-viral agents include, but are not
limited to, nucleoside analogs (e.g., zidovudine, acyclovir,
gangcyclovir, vidarabine, idoxuridine, trifluridine, and
ribavirin), foscarnet, amantadine, peramivir, rimantadine,
saquinavir, indinavir, ritonavir, alpha-interferons and other
interferons, AZT, zanamivir (Relenza.RTM.), and oseltamivir
(Tamiflue). Other anti-viral agents include influenza virus
vaccines, e.g., Fluarix.RTM. (GlaxoSmithKline), FluMist.RTM.
(Medlmmune Vaccines), Fluvirin.RTM. (Chiron Corporation),
Flulaval.RTM. (GlaxoSmithKline), Afluria.RTM. (CSL Biotherapies
Inc.), Agriflu.RTM. (Novartis) or Fluzone.RTM. (Aventis
Pasteur).
[0419] In specific embodiments, the anti-viral agent is an
immunomodulatory agent that is specific for a viral antigen. In
particular embodiments, the viral antigen is an influenza virus
polypeptide other than a hemagglutinin polypeptide. In other
embodiments, the viral antigen is an flu polypeptide.
[0420] Any anti-bacterial agents known to one of skill in the art
may used in combination with an active compound or pharmaceutical
composition described herein. Non-limiting examples of
anti-bacterial agents include Amikacin, Amoxicillin,
Amoxicillin-clavulanic acid, Amphothericin-B, Ampicillin,
Ampicllin-sulbactam, Apramycin, Azithromycin, Aztreonam,
Bacitracin, Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil,
Cefalexin, Cefalothin, Cefazolin, Cefdinir, Cefepime, Cefixime,
Cefinenoxime, Cefoperazone, Cefoperazone-sulbactam, Cefotaxime,
Cefoxitin, Cefpirorrie, Cefpodoxime, Cefpodoxime-clavulanic acid,
Cefpodoxime-sulbactam, Cefprozil, Cefquinome, Ceftazidime,
Ceftibutin, Ceftiofur, Ceftobiprole, Ceftriaxon, Cefuroxime,
Chloramphenicole, Florfenicole, Ciprofloxacin, Clarithromycin,
Clinafloxacin, Clindamycin, Cloxacillin, Colistin, Cotrimoxazol
(Trimthoprim/sulphamethoxazole), Dalbavancin,
Dalfopristin/Quinopristin, Daptomycin, Dibekacin, Dicloxacillin,
Doripenem, Doxycycline, Enrofloxacin, Ertapenem, Erythromycin,
Flucloxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidic acid,
Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem,
Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin,
Linezolid, Loracarbef, Mecillnam (amdinocillin), Meropenem,
Metronidazole, Meziocillin, Mezlocillin-sulbactam, Minocycline,
Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin, Netilmicin,
Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Pefloxacin,
Penicillin V, Piperacillin, Piperacillin-sulbactam,
Piperacillin-tazobactam, Rifampicin, Roxythromycin, Sparfloxacin,
Spectinomycin, Spiramycin, Streptomycin, Sulbactam,
Sulfamethoxazole, Teicoplanin, Telavancin, Telithromycin,
Temocillin, Tetracyklin, Ticarcillin, Ticarcillin-clavulanic acid,
Tigecycline, Tobramycin, Trimethoprim, Trovafloxacin, Tylosin,
Vancomycin, Virginiamycin, and Voriconazole.
[0421] In some embodiments, a combination therapy comprises
administration of two or more different vectors described in
Sections 5.4-5.7. In one example, one or more vectors expressing a
flu polypeptide derived from an influenza A virus and one or more
vectors expressing a flu polypeptide derived from an influenza B
virus are administered in combination. In some embodiments, a
combination therapy comprises administration of a vector expressing
a flu polypeptide derived from an H3 influenza A virus and a vector
expressing a flu polypeptide derived from an H1 influenza A virus.
In some embodiments, the combination therapy comprises
administration of a vector expressing a flu polypeptide derived
from an H3 influenza A virus, a vector expressing a flu polypeptide
derived from an H1 influenza A virus, and a vector expressing a flu
polypeptide derived from an influenza B virus.
[0422] In some embodiments, a combination therapy comprises active
immunization with an active compound that induces an immune
response to one, two, three, or more HA subtypes in one HA group
(e.g., Group 1) in combination with an active compound that induces
an immune response to one, two, three, or more HA subtypes in the
other HA group (e.g., Group 2).
[0423] In some embodiments, a combination therapy comprises active
immunization with two or more flu polypeptides described in Section
5.1.
[0424] In certain embodiments, a combination therapy comprises
active immunization with one, two, or more flu polypeptides derived
from an influenza A virus and one or more flu polypeptides derived
from an influenza B virus.
[0425] 5.11.2 Patient Populations
[0426] In certain embodiments, an active compound or composition
described herein may be administered to a naive subject, i.e., a
subject that does not have a disease caused by influenza virus
infection or has not been and is not currently infected with an
influenza virus infection. In one embodiment, an active compound or
composition described herein is administered to a naive subject
that is at risk of acquiring an influenza virus infection. In one
embodiment, an active compound or composition described herein is
administered to a subject that does not have a disease caused by
the specific influenza virus, or has not been and is not infected
with the specific influenza virus to which the flu polypeptide
induces an immune response. An active compound or composition
described herein may also be administered to a subject that is
and/or has been infected with the influenza virus or another type,
subtype or strain of the influenza virus to which the flu
polypeptide induces an immune response.
[0427] In certain embodiments, an active compound or composition
described herein is administered to a patient who has been
diagnosed with an influenza virus infection. In some embodiments,
an active compound or composition described herein is administered
to a patient infected with an influenza virus before symptoms
manifest or symptoms become severe (e.g., before the patient
requires hospitalization). In some embodiments, an active compound
or composition described herein is administered to a patient that
is infected with or has been diagnosed with a different type of
influenza virus than that of the influenza virus from which the flu
polypeptide of the active compound or composition was derived.
[0428] In certain embodiments, an active compound or composition
described herein is administered to a patient that may be or is
infected with an influenza virus that belongs to the same HA group
as that of the influenza flu polypeptide. In certain embodiments,
an active compound or composition described herein is administered
to a patient that may be or is infected with an influenza virus of
the same subtype as that of the influenza flu polypeptide.
[0429] In some embodiments, a subject to be administered an active
compound or composition described herein is an animal. In certain
embodiments, the animal is a bird. In certain embodiments, the
animal is a canine. In certain embodiments, the animal is a feline.
In certain embodiments, the animal is a horse. In certain
embodiments, the animal is a cow. In certain embodiments, the
animal is a mammal, e.g., a horse, swine, mouse, or primate,
preferably a human.
[0430] In certain embodiments, a subject to be administered an
active compound or composition described herein is a human adult.
In certain embodiments, a subject to be administered an active
compound or composition described herein is a human adult more than
50 years old. In certain embodiments, a subject to be administered
an active compound or composition described herein is an elderly
human subject.
[0431] In certain embodiments, a subject to be administered an
active compound or composition described herein is a human child.
In certain embodiments, a subject to be administered an active
compound or composition described herein is a human infant. In
certain embodiments, a subject to be administered an active
compound or composition described herein is a premature human
infant. In some embodiments, a patient treated or prevented in
accordance with the methods provided herein is a human toddler. In
certain embodiments, a subject to whom an active compound or
composition described herein is administered is not an infant of
less than 6 months old. In a specific embodiment, a subject to be
administered an active compound or composition described herein is
2 years old or younger.
[0432] In specific embodiments, a subject to be administered an
active compound or composition described herein is any infant or
child more than 6 months of age and any adult over 50 years of age.
In other embodiments, the subject is an individual who is pregnant.
In another embodiment, the subject is an individual who may or will
be pregnant during the influenza season (e.g., November to April).
In specific embodiments, a subject to be administered an active
compound or composition described herein is a woman who has given
birth 1, 2, 3, 4, 5, 6, 7, or 8 weeks earlier.
[0433] In some embodiments, the human subject to be administered an
active compound or composition described herein is any individual
at increased risk of influenza virus infection or disease resulting
from influenza virus infection (e.g., an immunocompromised or
immunodeficient individual). In some embodiments, the human subject
to be administered an active compound or composition described
herein is any individual in close contact with an individual with
increased risk of influenza virus infection or disease resulting
from influenza virus infection (e.g., immunocompromised or
immunosuppressed individuals).
[0434] In some embodiments, the human subject to be administered an
active compound or composition described herein is an individual
affected by any condition that increases susceptibility to
influenza virus infection or complications or disease resulting
from influenza virus infection. In other embodiments, an active
compound or composition described herein is administered to a
subject in which an influenza virus infection has the potential to
increase complications of another condition that the individual is
affected by, or for which they are at risk. In particular
embodiments, such conditions that increase susceptibility to
influenza virus complications or for which influenza virus
increases complications associated with the condition are, e.g.,
conditions that affect the lung, such as cystic fibrosis,
emphysema, asthma, or bacterial infections (e.g., infections caused
by Haemophilus influenzae, Streptococcus pneumoniae, Legionella
pneumophila, and Chlamydia trachomatus); cardiovascular disease
(e.g., congenital heart disease, congestive heart failure, and
coronary artery disease); endocrine disorders (e.g., diabetes),
neurological and neuron-developmental conditions (e.g., disorders
of the brain, the spinal cord, the peripheral nerve, and muscle
(such as cerebral palsy, epilepsy (seizure disorders), stroke,
intellectual disability (e.g., mental retardation), muscular
dystrophy, and spinal cord injury)).
[0435] In some embodiments, the human subject to be administered an
active compound or composition described herein is an individual
that resides in a group home, such as a nursing home. In some
embodiments, the human subject to be administered an active
compound or composition described herein works in, or spends a
significant amount of time in, a group home, e.g., a nursing home.
In some embodiments, the human subject to be administered an active
compound or composition described herein is a health care worker
(e.g., a doctor or nurse). In some embodiments, the human subject
to be administered an active compound or composition described
herein is a smoker. In a specific embodiment, the human subject to
be administered an active compound or composition described herein
is immunocompromised or immunosuppressed.
[0436] In addition, subjects at increased risk of developing
complications from influenza who may be administered an active
compound or composition described herein include: any individual
who can transmit influenza viruses to those at high risk for
complications, such as, e.g., members of households with high-risk
individuals, including households that will include infants younger
than 6 months, individuals coming into contact with infants less
than 6 months of age, or individuals who will come into contact
with individuals who live in nursing homes or other long-term care
facilities; individuals with long-term disorders of the lungs,
heart, or circulation; individuals with metabolic diseases (e.g.,
diabetes); individuals with kidney disorders; individuals with
blood disorders (including anemia or sickle cell disease);
individuals with weakened immune systems (including
immunosuppression caused by medications, malignancies such as
cancer, organ transplant, or HIV infection); children who receive
long-term aspirin therapy (and therefore have a higher chance of
developing Reye syndrome if infected with influenza).
[0437] In other embodiments, subjects for administration of an
active compound or composition described herein include healthy
individuals six months of age or older, who: plan to travel to
foreign countries and areas where flu outbreaks may be occurring,
such, e.g., as the tropics and the Southern Hemisphere from April
through September; travel as a part of large organized tourist
groups that may include persons from areas of the world where
influenza viruses are circulating; attend school or college and
reside in dormitories, or reside in institutional settings; or wish
to reduce their risk of becoming ill with influenza.
[0438] In some embodiments, a subject for whom administration of an
active compound or composition described herein is contraindicated
include any individual for whom influenza vaccination is
contraindicated, such as: infants younger than six months of age;
and individuals who have had an anaphylactic reaction (allergic
reactions that cause difficulty breathing, which is often followed
by shock) to eggs, egg products, or other components used in the
production of the immunogenic formulation. In certain embodiments,
when administration of an active compound or composition described
herein is contraindicated due to one or more components used in the
production of the immunogenic formulation (e.g., due to the
presence of egg or egg products), the active compound or
composition may be produced in a manner that does not include the
component that causes the administration of an active compound or
composition to be contraindicated (e.g., the active compound or
composition may be produced without the use of eggs or egg
products).
[0439] In some embodiments, it may be advisable not to administer a
live virus vaccine to one or more of the following patient
populations: elderly humans; infants younger than 6 months old;
pregnant individuals; infants under the age of 1 years old;
children under the age of 2 years old; children under the age of 3
years old; children under the age of 4 years old; children under
the age of 5 years old; adults under the age of 20 years old;
adults under the age of 25 years old; adults under the age of 30
years old; adults under the age of 35 years old; adults under the
age of 40 years old; adults under the age of 45 years old; adults
under the age of 50 years old; elderly humans over the age of 70
years old; elderly humans over the age of 75 years old; elderly
humans over the age of 80 years old; elderly humans over the age of
85 years old; elderly humans over the age of 90 years old; elderly
humans over the age of 95 years old; children and adolescents (2-17
years of age) receiving aspirin or aspirin-containing medications,
because of the complications associated with aspirin and wild-type
influenza virus infections in this age group; individuals with a
history of asthma or other reactive airway diseases; individuals
with chronic underlying medical conditions that may predispose them
to severe influenza infections; individuals with a history of
Guillain-Barre syndrome; individuals with acute serious illness
with fever; or individuals who are moderately or severely ill. For
such individuals, administration of inactivated virus vaccines,
split virus vaccines, subunit vaccines, virosomes, viral-like
particles or the non-viral vectors described herein may be
preferred. In certain embodiments, subjects preferably administered
a live virus vaccine may include healthy children and adolescents,
ages 2-17 years, and healthy adults, ages 18-49.
[0440] In certain embodiments, an immunogenic formulation
comprising a live virus vector is not given concurrently with other
live-virus vaccines.
[0441] 5.12 Modes of Administration
[0442] 5.12.1 Routes of Delivery
[0443] An active compound or composition described herein may be
delivered to a subject by a variety of routes. These include, but
are not limited to, intranasal, intratracheal, oral, intradermal,
intramuscular, intraperitoneal, transdermal, intravenous,
conjunctival and subcutaneous routes. In a specific embodiment, an
active compound or composition described herein is delivered to a
subject by the subcutaneous route. In some embodiments, a
composition is formulated for topical administration, for example,
for application to the skin. In specific embodiments, the route of
administration is nasal, e.g., as part of a nasal spray. In certain
embodiments, a composition is formulated for intramuscular
administration. In some embodiments, a composition is formulated
for subcutaneous administration. In certain embodiments, a
composition is not formulated for administration by injection. In
specific embodiments for live virus vaccines, the vaccine is
formulated for administration by a route other than injection.
[0444] In cases where the antigen is a viral vector, a virus-like
particle vector, or a bacterial vector, for example, it may be
preferable to introduce an immunogenic composition via the natural
route of infection of the backbone virus or bacteria from which the
vector was derived. Alternatively, it may be preferable to
introduce a flu polypeptide via the natural route of infection of
the influenza virus from which polypeptide is derived. The ability
of an antigen, particularly a viral vector, to induce a vigorous
secretory and cellular immune response can be used advantageously.
For example, infection of the respiratory tract by a viral vector
may induce a strong secretory immune response, for example in the
urogenital system, with concomitant protection against an influenza
virus. In addition, in a preferred embodiment it may be desirable
to introduce the pharmaceutical compositions into the lungs by any
suitable route. Pulmonary administration can also be employed,
e.g., by use of an inhaler or nebulizer, and formulation with an
aerosolizing agent for use as a spray.
[0445] In a specific embodiment, a subunit vaccine is administered
intranasally. In a specific embodiment, a subunit vaccine is
administered intramuscularly. In another specific embodiment, a
subunit vaccine is administered subcutaneously. In another specific
embodiment, a subunit vaccine is administered intradermally.
[0446] In a specific embodiment, a live virus vaccine is
administered intranasally. In a specific embodiment, a live virus
vaccine is administered intramuscularly. In another specific
embodiment, a live virus vaccine is administered subcutaneously. In
another specific embodiment, a live virus vaccine is administered
intradermally.
[0447] In a specific embodiment, an inactivated virus vaccine is
administered intranasally. In a specific embodiment, an inactivated
virus vaccine is administered intramuscularly. In another specific
embodiment, an inactivated virus vaccine is administered
subcutaneously. In another specific embodiment, an inactivated
virus vaccine is administered intradermally.
[0448] In a specific embodiment, a split virus vaccine is
administered intranasally. In a specific embodiment, a split virus
vaccine is administered intramuscularly. In another specific
embodiment, a split virus vaccine is administered subcutaneously.
In another specific embodiment, a split virus vaccine is
administered intradermally.
[0449] In a specific embodiment, a viral-like particle or
composition thereof is administered intranasally. In a specific
embodiment, a viral-like particle or composition thereof is
administered intramuscularly. In another specific embodiment, a sp
viral-like particle or composition thereof is administered
subcutaneously. In another specific embodiment, a viral-like
particle or composition thereof is administered intradermally.
[0450] In some embodiments, cells stimulated with a flu polypeptide
in vitro may be introduced (or re-introduced) into a subject using
techniques known to one of skill in the art. In some embodiments,
the cells can be introduced into the dermis, under the dermis, or
into the peripheral blood stream. In some embodiments, the cells
introduced into a subject are preferably cells derived from that
subject, to avoid an adverse immune response. In other embodiments,
cells also can be used that are derived from a donor host having a
similar immune background. Other cells also can be used, including
those designed to avoid an adverse immunogenic response.
[0451] 5.12.2 Dosage and Frequency of Administration
[0452] The amount of an active compound or composition which will
be effective in the treatment and/or prevention of an influenza
virus infection or an influenza virus disease will depend on the
nature of the disease, and can be determined by standard clinical
techniques.
[0453] The precise dose to be employed in the formulation will also
depend on the route of administration, and the seriousness of the
infection or disease caused by it, and should be decided according
to the judgment of the practitioner and each subject's
circumstances. For example, effective doses may also vary depending
upon means of administration, target site, physiological state of
the patient (including age, body weight, health), whether the
patient is human or an animal, other medications administered, and
whether treatment is prophylactic or therapeutic. Usually, the
patient is a human but nonhuman mammals including transgenic
mammals can also be treated. Treatment dosages are optimally
titrated to optimize safety and efficacy.
[0454] In certain embodiments, an in vitro assay is employed to
help identify optimal dosage ranges. Effective doses may be
extrapolated from dose response curves derived from in vitro or
animal model test systems.
[0455] Exemplary doses for nucleic acids encoding flu polypeptides
range from about 10 ng to 1 g, 100 ng to 100 mg, 1 .mu.g to 10 mg,
or 30-300 .mu.g nucleic acid, e.g., DNA, per patient.
[0456] In certain embodiments, exemplary doses for flu
polypeptide(s) (e.g., as provided in split virus vaccines and
subunit vaccines) range from about 5 .mu.g to 100 mg, 15 .mu.g to
50 mg, 15 .mu.g to 25 mg, 15 .mu.g to 10 mg, 15 .mu.g to 5 mg, 15
.mu.g to 1 mg, 15 .mu.g to 100 .mu.g, 15 .mu.g to 75 .mu.g, 5 .mu.g
to 50 .mu.g, 10 .mu.g to 50 .mu.g, 15 .mu.g to 45 .mu.g, 20 .mu.g
to 40 .mu.g, or to 35 .mu.g per kilogram of the patient. In other
embodiments, exemplary doses for flu polypeptide(s) range from
about 1 .mu.g to 50 mg, 5 .mu.g to 50 mg, 1 .mu.g to 100 mg, 5
.mu.g to 100 mg, 15 .mu.g to 50 mg, 15 .mu.g to 25 mg, 15 .mu.g to
10 mg, 15 .mu.g to 5 mg, 15 .mu.g to 1 mg, 15 .mu.g to 100 .mu.g,
15 .mu.g to 75 .mu.g, 5 .mu.g to 50 .mu.g, 10 .mu.g to 50 .mu.g, 15
.mu.g to 45 .mu.g, 20 .mu.g to 40 .mu.g, or 25 to 35 .mu.g of flu
polypeptide(s) per dose. In certain embodiments, an exemplary dose
for a flu polypeptide(s) comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, or 50 .mu.g of flu polypeptide(s) per dose. In
certain embodiments, an exemplary dose for a flu polypeptide(s)
comprises 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 .mu.g of
flu polypeptide(s) per dose.
[0457] Doses for infectious viral vectors may vary from 10-100, or
more, virions per dose. In some embodiments, suitable dosages of a
virus vector are 10.sup.2, 5.times.10.sup.2, 10.sup.3,
5.times.10.sup.3, 10.sup.4, 5.times.10.sup.4, 10.sup.5,
5.times.10.sup.5, 10.sup.6, 5.times.10.sup.6, 10.sup.7,
5.times.10.sup.7, 10.sup.8, 5.times.10.sup.8, 1.times.10.sup.9,
5.times.10.sup.9, 1.times.10.sup.10, 5.times.10.sup.10,
1.times.10.sup.11, 5.times.10.sup.11 or 10.sup.12 pfu, and can be
administered to a subject once, twice, three or more times with
intervals as often as needed.
[0458] In certain embodiments, exemplary doses for VLPs range from
about 0.01 .mu.g to about 100 mg, about 0.1 .mu.g to about 100 mg,
about 5 .mu.g to about 100 mg, about 15 .mu.g to about 50 mg, about
15 .mu.g to about 25 mg, about 15 .mu.g to about 10 mg, about 15
.mu.g to about 5 mg, about 15 .mu.g to about 1 mg, about 15 .mu.g
to about 100 .mu.g, about 15 .mu.g to about 75 .mu.g, about 5 .mu.g
to about 50 .mu.g, about 10 .mu.g to about 50 .mu.g, about 15 .mu.g
to about 45 .mu.g, about 20 .mu.g to about 40 .mu.g, or about 25 to
about 35 .mu.g per kilogram of the patient. In other embodiments,
exemplary doses for flu polypeptides range from about 1 .mu.g to
about 50 mg, about 5 .mu.g to about 50 mg, about 1 .mu.g to about
100 mg, about 5 .mu.g to about 100 mg, about 15 .mu.g to about 50
mg, about 15 .mu.g to about 25 mg, about 15 .mu.g to about 10 mg,
about 15 .mu.g to about 5 mg, about 15 .mu.g to about 1 mg, about
15 .mu.g to about 100 .mu.g, about 15 .mu.g to about 75 .mu.g,
about 5 .mu.g to about 50 .mu.g, about 10 .mu.g to about 50 .mu.g,
about 15 .mu.g to about 45 .mu.g, about 20 .mu.g to about 40 .mu.g,
or about 25 to about 35 .mu.g of flu polypeptide(s) per dose, and
can be administered to a subject once, twice, three or more times
with intervals as often as needed.
[0459] In one embodiment, an inactivated vaccine is formulated such
that it contains about 5 .mu.g to about 50 .mu.g, about 10 .mu.g to
about 50 .mu.g, about 15 .mu.g to about 100 .mu.g, about 15 .mu.g
to about 75 .mu.g, about 15 .mu.g to about 50 .mu.g, about 15 .mu.g
to about 30 .mu.g, about 20 .mu.g to about 50 .mu.g, about 25 .mu.g
to about 40 .mu.g, about 25 .mu.g to about 35 .mu.g of a flu
polypeptide(s). Such a vaccine may contain a combination of one or
more different flu polypeptides, for example, one or more flu
polypeptides from an influenza A virus and one or more flu
polypeptides from an influenza B virus. In one embodiment, a live
attenuated influenza-vaccine (LAIV) is formulated such that a
0.2-mL dose contains 10.sup.6.5-7.5 fluorescent focal units of live
attenuated influenza viruses from three strains expressing at least
one influenza flu polypeptide.
[0460] In certain embodiments, an active compound or composition is
administered to a subject once as a single dose. In certain
embodiments, an active compound or composition is administered to a
subject as a single dose followed by a second dose 3 to 6 weeks
later. In accordance with these embodiments, booster inoculations
may be administered to the subject at 6 to 12 month intervals
following the second inoculation. In certain embodiments, the
booster inoculations may utilize a different active compound or
composition. In some embodiments, the administration of the same
active compound or composition may be repeated and the
administrations may be separated by at least 1 day, 2 days, 3 days,
5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3
months, or at least 6 months. In certain embodiments, an active
compound or composition is administered to a subject as a single
dose once per year.
[0461] In specific embodiments for administration to children, two
doses of an active compound or composition, given at least one
month apart, are administered to a child. In specific embodiments
for administration to adults, a single dose is given. In another
embodiment, two doses of an active compound or composition, given
at least one month apart, are administered to an adult. In another
embodiment, a young child (six months to nine years old) may be
administered an active compound or composition for the first time
in two doses given one month apart. In a particular embodiment, a
child who received only one dose in their first year of vaccination
should receive two doses in the following year. In some
embodiments, two doses administered 4 weeks apart are preferred for
children 2-8 years of age who are administered an influenza
vaccine, e.g., an immunogenic formulation described herein, for the
first time. In certain embodiments, for children 6-35 months of
age, a half dose (0.25 ml) may be preferred, in contrast to 0.5 ml
which may be preferred for subjects over three years of age.
[0462] In particular embodiments, an active compound or composition
is administered to a subject in the fall or winter, i.e., prior to
or during the influenza season in each hemisphere. In one
embodiment, children are administered their first dose early in the
season, e.g., late September or early October, so that the second
dose can be given prior to the peak of the influenza season.
[0463] In certain embodiments, an active compound or composition
thereof is administered to a subject as 2, 3, 4, 5 or more doses 2
weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks apart. In some
embodiments, 2, 3, 4, 5 or more doses of an active compound or
compositions thereof are administered to a subject 2, 3, 4, 5 or 6
weeks apart at a dosage of 1 .mu.g to 20 mg, 10 .mu.g to 20 mg, 500
.mu.g to 20 mg, 1 mg to 20 mg or 5 mg to 20 mg. In certain
embodiments, the active compounds or compositions thereof
administered is the same each time. In certain embodiments, the flu
polypeptides or compositions thereof administered are different
each time.
[0464] For passive immunization with an antibody that binds to a
flu polypeptide, the dosage ranges from about 0.0001 to 100 mg/kg,
and more usually 0.01 to 5 mg/kg, of the patient body weight. For
example, dosages can be 1 mg/kg body weight or 10 mg/kg body weight
or within the range of 1-10 mg/kg or in other words, 70 mg or 700
mg or within the range of 70-700 mg, respectively, for a 70 kg
patient. An exemplary treatment regime entails administration once
per every two weeks or once a month or once every 3 to 6 months for
a period of one year or over several years, or over several
year-intervals. In some methods, two or more monoclonal antibodies
with different binding specificities are administered
simultaneously, in which case the dosage of each antibody
administered falls within the ranges indicated. Antibody is usually
administered on multiple occasions. Intervals between single
dosages can be weekly, monthly or yearly. Intervals can also be
irregular as indicated by measuring blood levels of antibody to the
flu polypeptide in the patient.
[0465] 5.13 Biological Assays
[0466] 5.13.1 Assays for Testing Activity of Influenza Flu
polypeptide
[0467] Assays for testing the expression of a flu polypeptide in a
vector disclosed herein may be conducted using any assay known in
the art. For example, an assay for incorporation into a viral
vector comprises growing the virus as described in this section or
Sections 5.4 or 5.5, purifying the viral particles by
centrifugation through a sucrose cushion, and subsequent analysis
for flu polypeptide expression by an immunoassay, such as Western
blotting, using methods well known in the art.
[0468] In one embodiment, a flu polypeptide disclosed herein is
assayed for proper folding and functionality by testing its ability
to bind specifically to an antibody directed to a flu polypeptide
using any assay for antibody-antigen interaction known in the art.
Antibodies for use in such assays include, for example the
neutralizing antibodies described in Wang et al. (2010) "Broadly
Protective Monoclonal Antibodies against H3 Influenza Viruses
following Sequential Immunization with Different Hemagglutinins,"
PLOS Pathogens 6(2):1-9, International Publication No.
PCT/US2010/036170 and U.S. Ser. No. 12/778,103.
[0469] In another embodiment, a flu polypeptide disclosed herein is
assayed for proper folding by determination of the structure or
conformation of the flu polypeptide using any method known in the
art such as, e.g., NMR, X-ray crystallographic methods, or
secondary structure prediction methods, e.g., circular
dichroism.
[0470] 5.13.2 Assays for Testing Activity of Antibodies Generated
Using Influenza Flu Polypeptides
[0471] Antibodies described herein may be characterized in a
variety of ways known to one of skill in the art (e.g. ELISA,
Surface Plasmon resonance display (BIAcore), Western blot,
immunofluorescence, immunostaining and/or microneutralization
assays). In some embodiments, antibodies are assayed for the
ability to specifically bind to a flu polypeptide, or a vector
comprising said polypeptide. Such an assay may be performed in
solution (e.g., Houghten, 1992, Bio/Techniques 13:412 421), on
beads (Lam, 1991, Nature 354:82 84), on chips (Fodor, 1993, Nature
364:555 556), on bacteria (U.S. Pat. No. 5,223,409), on spores
(U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), on plasmids
(Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865 1869) or on
phage (Scott and Smith, 1990, Science 249:386 390; Cwirla et al.,
1990, Proc. Natl. Acad. Sci. USA 87:6378 6382; and Felici, 1991, J.
Mol. Biol. 222:301 310) (each of these references is incorporated
herein in its entirety by reference).
[0472] Specific binding of an antibody to the flu polypeptide and
cross-reactivity with other antigens can be assessed by any method
known in the art. Immunoassays which can be used to analyze
specific binding and cross-reactivity include, but are not limited
to, competitive and non-competitive assay systems using techniques
such as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al., eds., 1994, Current
Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,
Inc., New York, which is incorporated by reference herein in its
entirety).
[0473] The binding affinity of an antibody to a flu polypeptide and
the off-rate of an antibody-antigen interaction can be determined
by competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., .sup.3H or .sup.125I) with the antibody of interest
in the presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody for a flu polypeptide and the binding
off-rates can be determined from the data by Scatchard plot
analysis. Competition with a second antibody can also be determined
using radioimmunoassays. In this case, a flu polypeptide is
incubated with the test antibody conjugated to a labeled compound
(e.g., .sup.3H or .sup.125I) in the presence of increasing amounts
of an unlabeled second antibody.
[0474] In certain embodiments, antibody binding affinity and rate
constants are measured using the KinExA 3000 System (Sapidyne
Instruments, Boise, Id.). In some embodiments, surface plasmon
resonance (e.g., BIAcore kinetic) analysis is used to determine the
binding on and off rates of the antibodies to a flu polypeptide.
BIAcore kinetic analysis comprises analyzing the binding and
dissociation of flu polypeptide from chips with immobilized
antibodies to a flu polypeptide on their surface. A typical BIAcore
kinetic study involves the injection of 250 .mu.L of an antibody
reagent (mAb, Fab) at varying concentration in HBS buffer
containing 0.005% Tween-20 over a sensor chip surface, onto which
has been immobilized the flu polypeptide. The flow rate is
maintained constant at 75 .mu.L/min. Dissociation data is collected
for 15 min or longer as necessary. Following each
injection/dissociation cycle, the bound antibody is removed from
the flu polypeptide surface using brief, 1 min pulses of dilute
acid, typically 10-100 mM HCl, though other regenerants are
employed as the circumstances warrant. More specifically, for
measurement of the rates of association, k.sub.on, and
dissociation, k.sub.off, the polypeptide is directly immobilized
onto the sensor chip surface through the use of standard amine
coupling chemistries, namely the EDC/NHS method
(EDC=N-diethylaminopropyl)-carbodiimide). Briefly, a 5-100 nM
solution of the polypeptide in 10 mM NaOAc, pH 4 or pH 5 is
prepared and passed over the EDC/NHS-activated surface until
approximately 30-50 RU's worth of polypeptide are immobilized.
Following this, the unreacted active esters are "capped" off with
an injection of 1 M Et-NH.sub.2. A blank surface, containing no
polypeptide, is prepared under identical immobilization conditions
for reference purposes. Once an appropriate surface has been
prepared, a suitable dilution series of each one of the antibody
reagents is prepared in HBS/Tween-20, and passed over both the
polypeptide and reference cell surfaces, which are connected in
series. The range of antibody concentrations that are prepared
varies, depending on what the equilibrium binding constant,
K.sub.D, is estimated to be. As described above, the bound antibody
is removed after each injection/dissociation cycle using an
appropriate regenerant.
[0475] The neutralizing activity of an antibody can be determined
utilizing any assay known to one skilled in the art. Antibodies
described herein can be assayed for their ability to inhibit the
binding of an influenza virus to its host cell receptor (i.e.,
sialic acid) using techniques known to those of skill in the art.
For example, cells expressing influenza virus receptors can be
contacted with a composition comprising an influenza virus in the
presence or absence of the antibody and the ability of the antibody
to inhibit the influenza virus' binding can be measured.
Alternatively, the ability of antibodies to inhibit an influenza
virus from binding to its receptor can be determined in cell-free
assays.
[0476] In other embodiments, an antibody suitable for use in the
methods described herein does not inhibit influenza virus receptor
binding, yet is still found to be neutralizing in an assay
described herein. In some embodiments, an antibody suitable for use
in accordance with the methods described herein reduces or inhibits
virus-host membrane fusion in an assay known in the art or
described herein.
[0477] In one embodiment, virus-host membrane fusion is assayed in
an in vitro assay using an influenza virus containing a reporter
and a host cell capable of being infected with the virus. An
antibody inhibits fusion if reporter activity is inhibited or
reduced compared to a negative control (e.g., reporter activity in
the presence of a control antibody or in the absence of
antibody).
[0478] 5.13.3 Assays for Testing Activity of Stimulated Cells
[0479] Cells stimulated in accordance with the methods described
herein may be analyzed, for example, for integration, transcription
and/or expression of the polynucleotide or gene(s) of interest, the
number of copies of the gene integrated, and the location of the
integration. Such analysis may be carried out at any time and may
be carried out by any methods known in the art. In other
embodiments, successful stimulation of the target cell with a flu
polypeptide described herein is determined by detecting production
of neutralizing antibodies against the flu polypeptide using
methods known in the art or described herein.
[0480] In certain embodiments, subjects in which the stimulated
cells, e.g., DCs, are administered can be analyzed for location of
the cells, expression of a vector-delivered polynucleotide or gene
encoding the flu polypeptide, stimulation of an immune response
(e.g., production of neutralizing antibodies against the flu
polypeptide), and/or monitored for symptoms associated with
influenza virus infection or a disease associated therewith by any
methods known in the art or described herein.
[0481] Reporter assays can be used to determine the specificity of
the targeting of the flu polypeptide. For example, a mixed
population of bone marrow cells can be obtained from a subject and
cultured in vitro. The flu polypeptide can be administered to the
mixed population of bone marrow cells, and expression of a reporter
gene associated with the flu polypeptide can be assayed in the
cultured cells. In some embodiments, at least about 50%, more
preferably at least about 60%, 70%, 80% or 90%, still more
preferably at least about 95% of stimulated cells in the mixed cell
population are dendritic cells.
[0482] 5.13.4 Viral Activity Assays
[0483] Antibodies described herein or compositions thereof can be
assessed in vitro for antiviral activity. In one embodiment, the
antibodies, or compositions thereof are tested in vitro for their
effect on growth of an influenza virus. Growth of influenza virus
can be assessed by any method known in the art or described herein
(e.g. in cell culture). In a specific embodiment, cells are
infected at a MOI of 0.0005 and 0.001, 0.001 and 0.01, 0.01 and
0.1, 0.1 and 1, or 1 and 10, or a MOI of 0.0005, 0.001, 0.005,
0.01, 0.05, 0.1, 0.5, 1, 5 or 10 and incubated with serum free
media supplemented. Viral titers are determined in the supernatant
by hemagglutinin plaques or any other viral assay described herein.
Cells in which viral titers can be assessed include, but are not
limited to, EFK-2 cells, Vero cells, MDCK cells, primary human
umbilical vein endothelial cells (HUVEC), H292 human epithelial
cell line and HeLa cells. In vitro assays include those that
measure altered viral replication (as determined, e.g., by plaque
formation) or the production of viral proteins (as determined,
e.g., by Western blot analysis) or viral RNAs (as determined, e.g.,
by RT-PCR or northern blot analysis) in cultured cells in vitro
using methods which are well known in the art or described
herein.
[0484] In one non-limiting example, a monolayer of the target
mammalian cell line is infected with different amounts (e.g.,
multiplicity of 3 plaque forming units (pfu) or 5 pfu) of virus
(e.g., influenza) and subsequently cultured in the presence or
absence of various dilutions of antibodies (e.g., 0.1 .mu.g/ml, 1
.mu.g/ml, 5 .mu.g/ml, or 10 .mu.g/ml). Infected cultures are
harvested 48 hours or 72 hours post infection and titered by
standard plaque assays known in the art on the appropriate target
cell line (e.g., Vero cells).
[0485] In a non-limiting example of a hemagglutination assay, cells
are contacted with an antibody and are concurrently or subsequently
infected with the virus (e.g., at an MOI of 1) and the virus is
incubated under conditions to permit virus replication (e.g., 20-24
hours). The antibodies are preferably present throughout the course
of infection. Viral replication and release of viral particles is
then determined by hemagglutination assays using 0.5% chicken red
blood cells. See, e.g., Kashyap et al., PNAS USA 105: 5986-5991. In
some embodiments, a compound is considered an inhibitor of viral
replication if it reduces viral replication by at least 2 wells of
HA, which equals approximately a 75% reduction in viral titer. In
specific embodiments, an inhibitor reduces viral titer in this
assay by 50% or more, by 55% or more, by 60% or more, by 65% or
more, by 70% or more, by 75% or more, by 80% or more, by 85% or
more, by 90% or more, or by 95% or more. In other specific
embodiments an inhibitor results in a reduction of approximately 1
log or more, approximately 2 logs or more, approximately 3 logs or
more, approximately 4 logs or more, approximately 5 logs or more,
approximately 6 logs or more, approximately 7 logs or more,
approximately 8 logs or more, approximately 9 logs or more,
approximately 10 logs or more, 1 to 3 logs, 1 to 5 logs, 1 to 8
logs, 1 to 9 logs, 2 to 10 logs, 2 to 5 logs, 2 to 7 logs, 2 logs
to 8 logs, 2 to 9 logs, 2 to 10 logs 3 to 5 logs, 3 to 7 logs, 3 to
8 logs, 3 to 9 logs, 4 to 6 logs, 4 to 8 logs, 4 to 9 logs, 5 to 6
logs, 5 to 7 logs, 5 to 8 logs, 5 to 9 logs, 6 to 7 logs, 6 to 8
logs, 6 to 9 logs, 7 to 8 logs, 7 to 9 logs, or 8 to 9 logs in
influenza virus titer in the subject. The log-reduction in
influenza virus titer may be as compared to a negative control, as
compared to another treatment, or as compared to the titer in the
patient prior to antibody administration.
[0486] 5.13.5 Cytotoxicity Assays
[0487] Many assays well-known in the art can be used to assess
viability of cells (infected or uninfected) or cell lines following
exposure to an active compound or a composition thereof and, thus,
determine the cytotoxicity of the compound or composition. For
example, cell proliferation can be assayed by measuring
Bromodeoxyuridine (BrdU) incorporation (See, e.g., Hoshino et al.,
1986, Int. J. Cancer 38, 369; Campana et al., 1988, J. Immunol.
Meth. 107:79), (3H) thymidine incorporation (See, e.g., Chen, J.,
1996, Oncogene 13:1395-403; Jeoung, J., 1995, J. Biol. Chem.
270:18367 73), by direct cell count, or by detecting changes in
transcription, translation or activity of known genes such as
proto-oncogenes (e.g., fos, myc) or cell cycle markers (Rb, cdc2,
cyclin A, D1, D2, D3, E, etc). The levels of such protein and mRNA
and activity can be determined by any method well known in the art.
For example, protein can be quantitated by known immunodiagnostic
methods such as ELISA, Western blotting or immunoprecipitation
using antibodies, including commercially available antibodies. mRNA
can be quantitated using methods that are well known and routine in
the art, for example, using northern analysis, RNase protection, or
polymerase chain reaction in connection with reverse transcription.
Cell viability can be assessed by using trypan-blue staining or
other cell death or viability markers known in the art. In a
specific embodiment, the level of cellular ATP is measured to
determined cell viability.
[0488] In specific embodiments, cell viability is measured in
three-day and seven-day periods using an assay standard in the art,
such as the CellTiter-Glo Assay Kit (Promega) which measures levels
of intracellular ATP. A reduction in cellular ATP is indicative of
a cytotoxic effect. In another specific embodiment, cell viability
can be measured in the neutral red uptake assay. In other
embodiments, visual observation for morphological changes may
include enlargement, granularity, cells with ragged edges, a filmy
appearance, rounding, detachment from the surface of the well, or
other changes. These changes are given a designation of T (100%
toxic), PVH (partially toxic--very heavy--80%), PH (partially
toxic--heavy--60%), P (partially toxic--40%), Ps (partially
toxic--slight--20%), or 0 (no toxicity--0%), conforming to the
degree of cytotoxicity seen. A 50% cell inhibitory (cytotoxic)
concentration (IC.sub.50) is determined by regression analysis of
these data.
[0489] In a specific embodiment, the cells used in the cytotoxicity
assay are animal cells, including primary cells and cell lines. In
some embodiments, the cells are human cells. In certain
embodiments, cytotoxicity is assessed in one or more of the
following cell lines: 0937, a human monocyte cell line; primary
peripheral blood mononuclear cells (PBMC); Huh7, a human
hepatoblastoma cell line; 293T, a human embryonic kidney cell line;
and THP-1, monocytic cells. In certain embodiments, cytotoxicity is
assessed in one or more of the following cell lines: MDCK, MEF, Huh
7.5, Detroit, or human tracheobronchial epithelial (HTBE)
cells.
[0490] Active compounds or compositions thereof can be tested for
in vivo toxicity in animal models. For example, animal models,
described herein and/or others known in the art, used to test the
activities of active compounds can also be used to determine the in
vivo toxicity of these compounds. For example, animals are
administered a range of concentrations of active compounds.
Subsequently, the animals are monitored over time for lethality,
weight loss or failure to gain weight, and/or levels of serum
markers that may be indicative of tissue damage (e.g., creatine
phosphokinase level as an indicator of general tissue damage, level
of glutamic oxalic acid transaminase or pyruvic acid transaminase
as indicators for possible liver damage). These in vivo assays may
also be adapted to test the toxicity of various administration mode
and/or regimen in addition to dosages.
[0491] The toxicity and/or efficacy of an active compound can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD.sub.50/ED.sub.50. An active
compound that exhibits large therapeutic indices is preferred.
While an active compound that exhibits toxic side effects may be
used, care should be taken to design a delivery system that targets
such agents to the site of affected tissue in order to minimize
potential damage to uninfected cells and, thereby, reduce side
effects.
[0492] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage of an active
compound for use in humans. The dosage of such agents lies
preferably within a range of circulating concentrations that
include the ED.sub.50 with little or no toxicity. The dosage may
vary within this range depending upon the dosage form employed and
the route of administration utilized. For any active compound used
in a method described herein, the effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma may
be measured, for example, by high-performance liquid
chromatography. Additional information concerning dosage
determination is provided herein.
[0493] Further, any assays known to those skilled in the art can be
used to evaluate the prophylactic and/or therapeutic utility of the
active compounds and compositions described herein, for example, by
measuring viral infection or a condition or symptoms associated
therewith.
[0494] 5.13.6 Assay for Assessing Ability of Flu Polypeptides to
Induce an Immune Response
[0495] The ability of a flu polypeptide to generate an immune
response in a subject that is capable of cross-reacting with, and
preferably protecting against, a plurality of influenza virus
strains can be assessed using any approach known to those of skill
in the art or described herein. In some embodiments, the ability of
a flu polypeptide to generate an immune response in a subject that
is capable of cross-reacting with, and preferably protecting
against, a plurality of influenza virus strains can be assessed by
immunizing a subject (e.g., a mouse) or set of subjects with a flu
polypeptide described herein and immunizing an additional subject
(e.g., a mouse) or set of subjects with a control (PBS). The
subjects or set of subjects can subsequently be challenged with a
plurality of virulent influenza virus strains and the ability of
the virulent influenza virus strains to cause influenza virus
disease in the subjects or set of subjects can be determined. Those
skilled in the art will recognize that if the subject or set of
subjects immunized with the control suffer from an influenza virus
disease subsequent to challenge with the virulent influenza virus
strains but the subject or set of subjects immunized with a flu
polypeptide described herein do not suffer from influenza virus
disease, then the flu polypeptide is able to generate an immune
response in a subject that is capable of cross-reacting with a
plurality of influenza virus strains. Further, in certain
embodiments, a flu polypeptide described herein is able to generate
an immune response that is capable of cross-reacting with a
plurality of influenza virus strains if the subject or set of
subjects immunized with the flu polypeptide suffer from influenza
virus disease for shorter periods of time, receive less
hospitalization time, exhibit a reduction in/absence of one or more
symptoms associated with influenza virus disease or have symptoms
that manifest themselves for shorter periods of time compared to
subjects immunized with control. Methods for determining whether a
subject suffers from influenza virus disease are known in the art
and described herein. See, e.g., Sections 5.13.7 and 6.3, infra.
The ability of a flu polypeptide to induce antiserum that simply
cross-reacts with a plurality of influenza virus strains, or with
multiple hemagglutinin subtypes can be tested by an immunoassay,
such as an ELISA.
[0496] 5.13.7 Methods of Assaying Influenza Activity in Animals
[0497] Active compounds and compositions thereof are preferably
assayed in vivo for the desired therapeutic or prophylactic
activity prior to use in humans. For example, in vivo assays can be
used to determine whether it is preferable to administer an active
compound or composition thereof and/or another therapy. For
example, to assess the use of an active compound or composition
thereof to prevent an influenza virus disease, the composition can
be administered before the animal is infected with influenza virus.
Alternatively, or in addition, an active compound or composition
thereof can be administered to the animal at the same time that the
animal is infected with influenza virus. To assess the use of an
active compound or composition thereof to treat an influenza virus
infection or disease associated therewith, the compound or
composition may be administered after infecting the animal with
influenza virus. In a specific embodiment, an active compound or
composition thereof is administered to the animal more than one
time.
[0498] Active compounds and compositions thereof can be tested for
antiviral activity in animal model systems including, but are not
limited to, rats, mice, chicken, cows, monkeys, pigs, goats, sheep,
dogs, rabbits, guinea pigs, etc. In a specific embodiment, active
compounds and compositions thereof are tested in a mouse model
system. Such model systems are widely used and well-known to the
skilled artisan. In a specific embodiment, active compounds and
compositions thereof are tested in a mouse model system.
Non-limiting examples of animal models for influenza virus are
provided in this section.
[0499] In general, animals are infected with influenza virus and
concurrently or subsequently treated with an active compound or
composition thereof, or placebo. Alternatively, animals are treated
with an active compound or composition thereof or placebo and
subsequently infected with influenza virus. Samples obtained from
these animals (e.g., serum, urine, sputum, semen, saliva, plasma,
or tissue sample) can be tested for viral replication via well
known methods in the art, e.g., those that measure altered viral
titers (as determined, e.g., by plaque formation), the production
of viral proteins (as determined, e.g., by Western blot, ELISA, or
flow cytometry analysis) or the production of viral nucleic acids
(as determined, e.g., by RT-PCR or northern blot analysis). For
quantitation of virus in tissue samples, tissue samples are
homogenized in phosphate-buffered saline (PBS), and dilutions of
clarified homogenates are adsorbed for 1 hour at 37.degree. C. onto
monolayers of cells (e.g., Vero, CEF or MDCK cells). In other
assays, histopathologic evaluations are performed after infection,
preferably evaluations of the organ(s) the virus is known to target
for infection. Virus immunohistochemistry can be performed using a
viral-specific monoclonal antibody.
[0500] The effect of an active compound or composition thereof on
the virulence of a virus can also be determined using in vivo
assays in which the titer of the virus in an infected subject
administered an active compound or composition thereof, the length
of survival of an infected subject administered an active compound
or composition thereof, the immune response in an infected subject
administered an active compound or composition thereof, the number,
duration and/or severity of the symptoms in an infected subject
administered an active compound or composition thereof, and/or the
time period before onset of one or more symptoms in an infected
subject administered an active compound or composition thereof, is
assessed. Techniques known to one of skill in the art can be used
to measure such effects. In certain embodiments, an active compound
or composition thereof results in a 0.5 fold, 1 fold, 2 fold, 4
fold, 6 fold, 8 fold, 10 fold, 15 fold, 20 fold, 25 fold, 50 fold,
75 fold, 100 fold, 125 fold, 150 fold, 175 fold, 200 fold, 300
fold, 400 fold, 500 fold, 750 fold, or 1,000 fold or greater
reduction in titer of influenza virus relative to an untreated
subject. In some embodiments, an active compound or composition
thereof results in a reduction in titer of influenza virus relative
to an untreated subject of approximately 1 log or more,
approximately 2 logs or more, approximately 3 logs or more,
approximately 4 logs or more, approximately 5 logs or more,
approximately 6 logs or more, approximately 7 logs or more,
approximately 8 logs or more, approximately 9 logs or more,
approximately 10 logs or more, 1 to 3 logs, 1 to 5 logs, 1 to 8
logs, 1 to 9 logs, 2 to 10 logs, 2 to 5 logs, 2 to 7 logs, 2 logs
to 8 logs, 2 to 9 logs, 2 to 10 logs 3 to 5 logs, 3 to 7 logs, 3 to
8 logs, 3 to 9 logs, 4 to 6 logs, 4 to 8 logs, 4 to 9 logs, 5 to 6
logs, 5 to 7 logs, 5 to 8 logs, 5 to 9 logs, 6 to 7 logs, 6 to 8
logs, 6 to 9 logs, 7 to 8 logs, 7 to 9 logs, or 8 to 9 logs.
[0501] Influenza virus animal models, such as ferret, mouse, guinea
pig, squirrel monkey, macaque, and chicken, developed for use to
test antiviral agents against influenza virus have been described.
See, e.g., Sidwell et al., Antiviral Res., 2000, 48:1-16; Lowen A.
C. et al. PNAS., 2006, 103: 9988-92; and McCauley et al., Antiviral
Res., 1995, 27:179-186 and Rimmelzwann et al., Avian Diseases,
2003, 47:931-933. For mouse models of influenza, non-limiting
examples of parameters that can be used to assay antiviral activity
of active compounds administered to the influenza-infected mice
include pneumonia-associated death, serum .alpha.1-acid
glycoprotein increase, animal weight, lung virus assayed by
hemagglutinin, lung virus assayed by plaque assays, and
histopathological change in the lung. Statistical analysis is
carried out to calculate significance (e.g., a P value of 0.05 or
less).
[0502] In other assays, histopathologic evaluations are performed
after infection of an animal model subject. Nasal turbinates and
trachea may be examined for epithelial changes and subepithelial
inflammation. The lungs may be examined for bronchiolar epithelial
changes and peribronchiolar inflammation in large, medium, and
small or terminal bronchioles. The alveoli are also evaluated for
inflammatory changes. The medium bronchioles are graded on a scale
of 0 to 3+ as follows: 0 (normal: lined by medium to tall columnar
epithelial cells with ciliated apical borders and basal
pseudostratified nuclei; minimal inflammation); 1+ (epithelial
layer columnar and even in outline with only slightly increased
proliferation; cilia still visible on many cells); 2+ (prominent
changes in the epithelial layer ranging from attenuation to marked
proliferation; cells disorganized and layer outline irregular at
the luminal border); 3+ (epithelial layer markedly disrupted and
disorganized with necrotic cells visible in the lumen; some
bronchioles attenuated and others in marked reactive
proliferation).
[0503] The trachea is graded on a scale of 0 to 2.5+ as follows: 0
(normal: Lined by medium to tall columnar epithelial cells with
ciliated apical border, nuclei basal and pseudostratified.
Cytoplasm evident between apical border and nucleus. Occasional
small focus with squamous cells); 1+ (focal squamous metaplasia of
the epithelial layer); 2+ (diffuse squamous metaplasia of much of
the epithelial layer, cilia may be evident focally); 2.5+ (diffuse
squamous metaplasia with very few cilia evident).
[0504] Virus immunohistochemistry is performed using a
viral-specific monoclonal antibody (e.g. NP-, N- or HN-specific
monoclonal antibodies). Staining is graded 0 to 3+ as follows: 0
(no infected cells); 0.5+ (few infected cells); 1+ (few infected
cells, as widely separated individual cells); 1.5+ (few infected
cells, as widely separated singles and in small clusters); 2+
(moderate numbers of infected cells, usually affecting clusters of
adjacent cells in portions of the epithelial layer lining
bronchioles, or in small sublobular foci in alveoli); 3+ (numerous
infected cells, affecting most of the epithelial layer in
bronchioles, or widespread in large sublobular foci in
alveoli).
[0505] In one example, the ability to induce lung lesions and cause
infection in an animal model of virus infection is compared using
wild-type virus and mock virus. Lung lesions can be assessed as a
percentage of lung lobes that are healthy by visual inspection.
Animals are euthanized 5 days p.i. by intravenous administration of
pentobarbital, and their lungs are removed in toto. The percentage
of the surface of each pulmonary lobe that is affected by
macroscopic lesions is estimated visually. The percentages are
averaged to obtain a mean value for the 7 pulmonary lobes of each
animal. In other assays, nasal swabs can be tested to determine
virus burden or titer. Nasal swabs can be taken during necropsy to
determine viral burden post-infection.
[0506] In one embodiment, virus is quantified in tissue samples.
For example, tissue samples are homogenized in phosphate-buffered
saline (PBS), and dilutions of clarified homogenates adsorbed for 1
h at 37.degree. C. onto monolayers of cells (e.g., MDCK cells).
Infected monolayers are then overlaid with a solution of minimal
essential medium containing 0.1% bovine serum albumin (BSA), 0.01%
DEAE-dextran, 0.1% NaHCO.sub.3, and 1% agar. Plates are incubated 2
to 3 days until plaques could be visualized. Tissue culture
infectious dose (TCID) assays to titrate virus from PR8-infected
samples are carried out as follows. Confluent monolayers of cells
(e.g., MDCK cells) in 96-well plates are incubated with log
dilutions of clarified tissue homogenates in media. Two to three
days after inoculation, 0.05-ml aliquots from each well are
assessed for viral growth by hemagglutination assay (HA assay).
[0507] 5.13.8 Methods of Assaying Influenza Activity in Humans
[0508] In one embodiment, an active compound or composition thereof
is assessed in infected human subjects. In accordance with this
embodiment, an active compound or composition thereof is
administered to the human subject, and the effect of the active
compound or composition on viral replication and/or survival is
determined by, e.g., analyzing the level of the virus or viral
nucleic acids in a biological sample (e.g., serum or plasma). An
active compound or composition thereof that alters virus
replication and/or survival can be identified by comparing the
level of virus replication and/or survival in a subject or group of
subjects treated with a control to that in a subject or group of
subjects treated with an active compound or composition thereof.
Alternatively, alterations in viral replication and/or survival can
be identified by comparing the level of the virus replication
and/or survival in a subject or group of subjects before and after
the administration of an active compound or composition thereof.
Techniques known to those of skill in the art can be used to obtain
the biological sample and analyze the mRNA or protein
expression.
[0509] In another embodiment, the effect of an active compound or
composition thereof on the severity of one or more symptoms
associated with an influenza virus infection/disease are assessed
in an infected subject. In accordance with this embodiment, an
active compound or composition thereof or a control is administered
to a human subject suffering from influenza virus infection and the
effect of the active compound or composition on one or more
symptoms of the virus infection is determined. An active compound
or composition thereof that reduces one or more symptoms can be
identified by comparing the subjects treated with a control to the
subjects treated with the active compound or composition. In
another embodiment, an active compound or composition thereof is
administered to a healthy human subject and monitored for efficacy
as a vaccine (e.g., the subject is monitored for the onset of
symptoms of influenza virus infection; reduction in
hospitalization, the ability of influenza virus to infect the
subject; and/or a reduction in/absence of one or more symptoms
and/or duration of symptoms associated with influenza virus
infection). Techniques known to physicians familiar with infectious
diseases can be used to determine whether an active compound or
composition thereof reduces one or more symptoms associated with
the influenza virus disease.
[0510] 5.14 Kits
[0511] Provided herein is a pharmaceutical pack or kit comprising
one or more containers filled with one or more of the ingredients
of the pharmaceutical compositions described herein, such as one or
more active compounds provided herein. Optionally associated with
such container(s) can be a notice in the form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects
approval by the agency of manufacture, use or sale for human
administration.
[0512] The kits encompassed herein can be used in the above
methods. In one embodiment, a kit comprises an active compound
described herein, preferably one or more influenza flu
polypeptides, in one or more containers. In certain embodiments, a
kit comprises a vaccine described herein, e.g., a split virus
vaccine, a subunit vaccine, an inactivated influenza virus vaccine,
or a live influenza virus vaccine.
6. EXAMPLES
[0513] 6.1 Monoclonal Antibody 12D1
[0514] This example demonstrates that an anti-influenza virus
antibody, monoclonal antibody 12D1, reacts with the long
alpha-helix of HA2.
[0515] 6.1.1 Materials and Methods
[0516] 6.1.1.1 Truncated Hemagglutinin Subunit 2 (HA2)
[0517] The whole coding region of A/HK/1/68 HA was
reversed-transcribed and amplified from viral RNA and subsequently
sub-clone into a pCAGGs expression vector. Truncated versions of
the HA2 portion were generated by PCR amplification from
pCAGGs-HK68 HA and sub-cloned further into a pCAGGs-green
fluorescent protein (GFP) expression plasmid. The resulting plasmid
thus consists of a expression vector encoding a GFP fused to a
portion of a truncated HA2. All constructs were sequenced and
confirmed.
[0518] 6.1.1.2 Western Blot
[0519] Blots were produced by methods previously described (Towbin
et al., Proc Natl Acad Sci U S A, 1979. 76(9):4350-4). Samples were
boiled for 5 minutes at 100.degree. C. in loading buffer containing
SDS and 0.6M DTT. Immuno-precipitated complexes, cell lysates or
purified virus were resolved in a 4-20% Tris-HCl SDS-PAGE gel
(Bio-Rad, Inc.) and samples were blotted onto a Protran
nitrocellulose membrane (Whatman). GFP and fusion GFP-HA truncated
peptides were detected using rabbit anti-GFP (Santa Cruz
Biotechnology, Inc) and/or mAb 12D1. Secondary antibodies were
anti-rabbit IgG HRP (Dako) and anti-mouse Ig (GE Healtchare,
Inc.).
[0520] 6.1.1.3 Immunoprecipitation
[0521] 293T cells were transfected with various pCAGGs encoding the
GFP-truncated HA2 fusion proteins using Lipofectamine 2000
(Invitrogen, Inc). At 24 hours post transfection cells were lysed
with radioimmuno-precipitation assay (RIPA) buffer and the
truncated fusion peptides were immuno-precipitated with 1 to 5
.mu.g of mAb 12D1 bound to protein G-Agarose (Roche, Inc) overnight
at 4.degree. C. Immuno-precipitation was analyzed by Western
blotting under reducing and denaturing conditions.
[0522] 6.1.1.4 ELISA
[0523] 96 well plates (Nunc Immulon 2) were coated with 2 ug/ml
HApep-KLH conjugate (FIG. 2B) or purified HA (FIG. 2A,C) in PBS
overnight, 4.degree. C. Plates were blocked for 30 minutes at room
temperature with 1% BSA/PBS and washed twice with PBS/0.025% Tween.
Antibodies or anti-serum were serially diluted in 1% BSA/PBS, added
to the plate followed by 3 hour incubation at 37.degree. C. Plates
were washed three times, anti-mouse-AP (Southern Biotech) diluted
1:2000 was added to wells followed by 3 hour incubation, 37.degree.
C. P-nitrophenyl phosphate (PNPP) substrate was then added to wells
and allowed to develop for 20-30 minutes at room temperature.
Optical density measurements were taken at 405 nm.
[0524] 6.1.2 Results
[0525] As demonstrated in FIG. 1, mAb 12D1 reacts within the region
of amino acids 76-130 of the HA2 molecule; this region comprises
the "long alpha-helix" of HA2. As mAb 12D1 is known to have
protective activity in vivo against H3 virus infection
(demonstrated by passive transfer of mAb 12D1 prior to virus
challenge),
[0526] 6.2 Design and Production of Flu Polypeptide
[0527] It was hypothesized that immunization with the 76-130 region
of HA2 might elicit a similarly protective immune response against
influenza viruses of the H3 subtype or of multiple subtypes.
[0528] In order to increase the immunogenicity of the 76-130
peptide of HA2 a construct with a C-terminal spacer domain was
designed consisting of eight amino acids followed by a cysteine
residue which facilitated primary amine-mediated coupling to the
carrier protein Keyhole limpet hemacyanin (KLH). In order to
increase serum half-life the peptide was acetylated at the
N-terminus.
[0529] To verify the structural integrity of the long alpha-helix
within the KLH conjugate, binding of mAb 12D1 to the conjugate was
tested by direct-binding ELISA and found that the 12D1 binding
region was intact (FIG. 1B).
[0530] 6.2.1 The HA2 Binding Region of mAb 12D1
[0531] The identity of the region of the H3 hemagglutinin that
might elicit antibodies similar to the 12D1 monoclonal antibody
(mAb) was examined. Sixteen passages of A/HK/1968 virus in the
presence of the anti-H3 mAb did not yield escape variants which
might have assisted in identification of the binding epitopes. The
hemagglutinin of six plaques present after incubation of A/HK/1968
virus with 50 ug/ml mAb 12D1 in a plaque assay was sequenced and no
changes from the wild-type hemagglutinin were found. Because mAb
12D1 mediates protection against influenza disease in vivo and
reacts with a continuous epitope of the viral hemagglutinin (no
trimeric structure required), as evidenced by reactivity with the
denatured hemagglutinin monomer by Western blot, the 12D1 binding
epitope was focused on. Truncated hemagglutinin constructs
consisting of hemagglutinin segments of varying length fused to GFP
were generated. GFP expression was utilized to assess expression of
the constructs in transfected 293T cells. By analysis of the
truncated hemagglutinin constructs, it was determined that the 12D1
paratope makes dominant interactions with the HA2 subunit in the
region of amino acids 30-106. Diminished 12D1 binding without
diminished GFP expression in the 76-184 and 91-184 truncations
along with loss of binding with the 106-184 truncation suggested
that 12D1 binding is dependent on contacts with amino acids in the
HA2 76-106 region (FIG. 1). Additional truncated HAs were designed
and constructed to further narrow down the minimal binding site of
12D1. Amongs those, the region spanning from aa 76 to aa
130--representing the long alpha-helix of HA2 not only was detected
by 12D1 in a Western blot, but also positive by
immuno-precipitation.
[0532] These 30 amino acids fall within the membrane distal half of
the long alpha-helix of HA2. The 12D1 paratope may have additional
contacts with amino acids outside of this region (in HA1 or HA2)
that are not required for binding by Western blot.
[0533] 6.3. Serum Antibodies Induced by Flu Polypeptide React with
Multiple HA Subtypes
[0534] 6.3.1 Materials and Methods
[0535] Western blots and ELISA were performed as described in
Section 6.1.1, supra.
[0536] 6.3.2 Results
[0537] As demonstrated in FIG. 3, flu polypeptide (76-130)-KLH
("HApep-KLH") acts as a robust immunogen and serum antibody
elicited by HApep-KLH reacts with multiple hemagglutinin
subtypes.
[0538] To evaluate the efficacy of the HApep-KLH construct as an
immunogen, sera were taken from mice 10 days post primary and
secondary immunizations. These sera were tested for reactivity with
recombinantly expressed, purified hemagglutinins of different
subtypes. First, it was noted that the HApep-KLH vaccine construct
did elicit serum antibody reactive with purified hemagglutinin
protein. Second, a marked increase in anti-HA titer following
secondary immunization was evident, indicating that the construct
did act to elicit a robust humoral immune response in mice.
Finally, the heterosubtypic reactivity of the HApep-KLH anti-sera
was intriguing. Sera from immunized mice demonstrated binding
activity with hemagglutinins of H3, H1, H2, H9 and H7 subtypes.
[0539] 6.4 Immunization with Flu Polypeptides Protects Mice from
Lethal Virus Challenge
[0540] 6.4.1. Materials and Methods
[0541] 6-8 week old BALB/C mice (Jackson Laboratories) were
immunized with 25 ug HApep-KLH or KLH alone in Complete Freund's
adjuvant (Sigma) by subcutaneous administration. Three weeks
following primary immunization, mice were boosted with 25 ug
HApep-KLH or KLH alone in Incomplete Freund's adjuvant. Two to
three weeks following boost, mice were challenged with virus.
Before virus infection, mice were anesthetized by intraperitoneal
administration of a ketamine (75 mg/kg of body weight)/xylazine (15
mg/kg of body weight) mixture. Virus was administered intranasally
in 50 ug total PBS; challenge doses consisted of 40,000 pfu X31 or
500 pfu PR8. Body weights were monitored daily.
[0542] 6.4.2. Results
[0543] As demonstrated in FIG. 4, immunization with flu polypeptide
(76-130)-KLH ("HApep-KLH") protects mice against lethal
challenge.
[0544] Mice were immunized with 25 ug HApep-KLH by subcutaneous
administration in a prime-boost immunization schedule.
Immunizations were spaced 3 weeks apart and mice were challenged
with virus 2-3 weeks following secondary immunization. Following
virus challenge, mice weights were taken daily as a read-out of
disease severity. Immunization with HApep-KLH was found to protect
100% of mice from lethal challenge with X31, a mouse-adapted virus
expressing the HA and NA from Hong Kong/1/1968 (H3) (FIG. 3B).
Average weights from mice immunized with the HApep-KLH construct or
mice receiving PBS were significantly different on all days (FIG.
3A).
[0545] In a similar challenge experiment, mice received a lethal
dose of mouse adapted PR/8 virus (H1). By seven days post
challenge, all mice receiving PBS had succumbed to disease while
80% of mice receiving the HApep-KLH vaccine were protected (FIG.
4A). Serum antibody titer against H1 subtype hemagglutinin was
found to correlate with weight change in the days following virus
challenge (4B).
[0546] 6.5 Vaccination with Flu Polypeptides Provides Protection
Against Distinct Viral Subtypes
[0547] This example demonstrates that a flu polypeptide can provide
protection in mice against influenza viruses of the structurally
divergent subtypes H3N2, H1N1 and H5N1.
[0548] 6.5.1 Materials and Methods
[0549] 6.5.1.1 Viruses and Purified Hemagglutinins
[0550] Viruses used were: X31 virus (A/Hong Kong/1/1968
hemagglutinin and neuraminidase with remaining 6 segments from
PR8), A/Puerto Rico/8/34 (PR8) virus, A/USSR/90/1977 virus,
A/Georgia/81 virus, HAlo virus (A/Viet Nam/4/2005 virus with
hemagglutinin modified to remove the poly-basic cleavage site).
Purified hemagglutinin used were from: A/Hong Kong/I/1968;
A/Brisbane/10/2007; ANiet Nam/1203/2004 (H5); A/Singapore/1/57
(H2); A/Teal/HK/312/97 (H6); A/Netherlands/219/2003 (H7);
A/HK/1073/99 (H9); and A/California/04/2009 (H1).
[0551] 6.5.1.2 Western Blot
[0552] Blots were produced by methods previously described (see
Towbin et al., (1979) Proc Natl Acad Sci USA 76(9):4350-4354).
Samples were boiled for 5 minutes at 100.degree. C. in loading
buffer containing SDS and 0.6M DTT. Immuno-precipitated complexes,
cell lysates or purified virus were resolved in a 4-20% Tris-HCl
SDS-PAGE gel (Bio-Rad, Inc.) and samples were blotted onto a
Protran nitrocellulose membrane (Whatman). GFP and fusion GFP-HA
truncated peptides were detected using rabbit anti-GFP (Santa Cruz
Biotechnology, Inc) and/or mAb 12D1. Secondary antibodies were
anti-rabbit IgG HRP (Dako) and anti-mouse Ig-HRP(GE Healtchare,
Inc.).
[0553] 6.5.1.3 Immunoprecipitation
[0554] The 76-130 (LAH) region of the A/HK/1/68 HA2 was generated
by PCR amplification of viral RNA and sub-cloned into a pCAGG-green
fluorescent protein (GFP) plasmid (see Basler et al., (2001) Proc
Natl Acad Sci USA 98(5):2746-2751). GFP was present at the
N-terminus of the HA2 truncation. 293T cells were transfected with
the GFP-LAH construct using Lipofectamine 2000 (Invitrogen, Inc).
At 24 hours post transfection cells were lysed with
radioimmuno-precipitation assay (RIPA) buffer and the GFP-LAH
fusion protein was immuno-precipitated with 1 to 5 .mu.g of mAb
12D1 bound to protein G-Agarose (Roche, Inc) overnight at 4.degree.
C.
[0555] 6.5.1.4 Long-Alpha Helix-KLH Vaccine
[0556] The A/HK/1/68 HA2 LAH polypeptide (amino acids 76-130)
sequence used was:
TABLE-US-00028 Ac-RIQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEK
TRRQLRENADYKDDDDKC.
The construct was acetylated at the N-terminus and consisted of
amino acids 76-130 of the H3 A/Hong Kong/I/1968 HA2 molecule
followed by a FLAG-tag (DYKDDDDK), followed by a cysteine. The
polypeptide was coupled to the carrier protein keyhole limpet
hemocyanin (KLH) by thiol to primary amine coupling. This conjugate
was produced by CHI Scientific, Inc., Maynard, Mass. USA.
[0557] 6.5.1.6 ELISA
[0558] 96-well plates (Nunc Immulon 2) were coated with 2 .mu.g/ml
LAH-KLH conjugate (FIG. 7B), purified hemagglutinin (FIG. 7A, C),
or influenza virus vaccine (FLUVIRON(R), obtained from BEI
Resources) purified surface antigen (Novartis Vaccines) in PBS
overnight, 4.degree. C. Plates were blocked for 30 minutes at room
temperature with 1% BSA/PBS and washed twice with PBS/0.025% Tween.
Antibodies, anti-serum or serum from individuals vaccinated with
the 2008-2009 trivalent inactivated influenza virus vaccine (TIV)
were serially diluted in 1% BSA/PBS and added to the plate followed
by a 3 hour incubation at 37.degree. C. An anti-flag antibody
(Sigma) was used as a positive control in wells coated with the
LAH-KLH conjugate. Plates were washed three times, and
anti-mouse-alkaline phosphatase (AP) (Southern Biotech) diluted
1:2000 was added to wells followed by a 3 hour incubation at
37.degree. C. For human sera, anti-human IgG (Fc specific)-AP
(Sigma) antibody was used at 1:500 dilution. Anti-rabbit Ig-AP
(Southern Biotech) at 1:500 dilution was used as the secondary for
the anti-flag antibody. P-nitrophenyl phosphate (PNPP) substrate
was then added to wells and allowed to develop for 20-30 minutes at
room temperature. Optical density measurements were taken at 405
nm.
[0559] 6.5.1.6 Mouse Immunizations and Challenge Experiments
[0560] 6-8 week old BALB/C mice (Jackson Laboratories) were
immunized with 25 .mu.g LAH-KLH, HA2, KLH alone or PBS in Complete
Freund's adjuvant (Sigma) by subcutaneous administration. Three
weeks following primary immunization, mice were boosted with 25
.mu.g of the same immunogen or PBS in Incomplete Freund's adjuvant.
Two to three weeks following boost, mice were challenged with
virus. Before virus infection, mice were anesthetized by
intraperitoneal administration of a ketamine (75 mg/kg of body
weight)/xylazine (15 mg/kg of body weight) mixture. Virus was
administered intranasally in 50 .mu.l total PBS; challenge doses
consisted of 4.times.10.sup.5 pfu X31 or 500 pfu PR8 or HAlo virus.
Body weights were monitored daily. For passive transfer
experiments, mice were bled two weeks following the last
immunization with KLH or LAH-KLH or three weeks following infection
with PR8 virus or A/Hong Kong/1/1968 virus. Sera from mice were
pooled according to vaccination antigen or virus infection and 200
.mu.l serum was transferred to each recipient mouse by
intraperitoneal administration 2 hours prior to infection with
either 50 pfu PR8 virus or 3700 pfu Georgia/81 virus. Lung titers
were assessed by plaque assay 2 days post infection.
[0561] 6.5.2 Results
[0562] Mouse monoclonal antibody 12D1 binds a continuous portion of
the HA2 molecule and has broad-neutralizing activity against
influenza viruses of the H3 subtype. By generating constructs
designed to express short regions of the HA2 molecule, it was
determined that mAb 12D1 binds amino acids within the highly
conserved `long alpha-helix` (LAH) region of the protein. The
portion of the hemagglutinin that interacts with mAb 12D1 was
originally identified by interpretation of binding data using
multiple HA2 truncations of varying lengths. Based on the
cumulative truncation data, it was determined that mAb 12D1 binds
within the 76-106 region of HA2 (see Wang et al., (2010) PLoS
Pathog 6(2):e 1000796). Subsequent work, however, revealed that a
peptide representing the entire LAH (amino acids 76-130) of the H3
virus A/Hong Kong/1/1968 provided the necessary structural elements
for maximal binding by mAb 12D1 (FIG. 7A). This region, amino acids
76-130 of HA2, was expressed in 293T cells and was pulled-down by
mAb 12D1 (FIG. 7B). Whether immunization with the 76-130 region of
HA2 might elicit an antibody repertoire with functional similarity
to that of mAb 12D1 and provide protection against influenza
viruses of the H3 subtype or of multiple subtypes was thus
determined.
[0563] The antigenicity of the 76-130 polypeptide (LAH) was
enhanced by designing a conjugate vaccine consisting of the LAH
plus a C-terminal spacer domain of eight amino acids (FLAG-tag)
followed by a cysteine residue which facilitated thiol to primary
amine-mediated coupling to the carrier protein keyhole limpet
hemocyanin (KLH). To extend serum half-life, the LAH peptide was
acetylated at the N-terminus (see Werle and Bernkop-Schnurch,
(2006) Amino Acids 30(4):351-367). The structural integrity of the
mAb 12D1 binding region within the conjugate was confirmed by
direct-binding ELISA (FIG. 7C).
[0564] To test the construct in vivo, mice were immunized with the
LAH-KLH conjugate in a prime-boost schedule with three weeks
lapsing between immunizations. Sera were taken from mice 10 days
post primary and secondary immunizations. To evaluate the conjugate
for its ability to elicit the production of antibodies of relevant
specificity, anti-sera were tested for reactivity with purified
hemagglutinin protein of different subtypes. It was first noted
that the LAH anti-serum reacted with hemagglutinin protein by both
ELISA and by Western blot (FIG. 8A-C). Second, a marked increase in
anti-HA titer following secondary immunization demonstrated that
the construct acted as a productive immunogen in mice (FIGS. 8A and
8B). Finally, sera from immunized mice had substantial
heterosubtypic binding activity. Anti-LAH sera demonstrated
activity by ELISA with hemagglutinins from the 1968 pandemic H3
virus A/Hong Kong/1/1968, the 2009 pandemic H1 virus
A/California/04/09, as well as hemagglutinins of H2, H5 and H7
subtypes (FIG. 8D). Alignment of the 76-130 region of
hemagglutinins from these subtypes demonstrates a high degree of
conservation in amino-acid sequence and amino-acid type (FIG. 8E).
Further serologic analysis demonstrated that antibody generated in
LAH-KLH vaccination boosts serum IgM and IgG subtypes specific for
the viral hemagglutinin. The significant boost in IgG subtypes
indicates T-cell dependent antibody production and suggests an
affinity matured anti-hemagglutinin response (FIG. 8F) (see Jumper
et al., (1994) J Immunol 152(2):438-445).
[0565] Two to three weeks following secondary immunization, mice
were challenged by intranasal administration with 4.times.10.sup.5
pfu of X31, a mouse adapted virus expressing the hemagglutinin and
neuraminidase of the 1968 pandemic H3 influenza virus. Mice
immunized with the LAH-KLH construct lost significantly less weight
at all time points than did mice that received PBS with adjuvant.
In addition, all immunized mice survived challenge, while control
mice succumbed to infection by day 4 (FIGS. 9A and 9B).
[0566] Next, immunized mice were challenged with other virus
subtypes that cause human influenza disease, but that belong to a
distinct phylogenetic class from H3 subtype viruses (see Fields B
N, Knipe D M, & Howley P M (2007) Fields' virology (Lippincott
Williams & Wilkins, Philadelphia) 5th Ed pp 2 v. (xix, 3091,
1-3086 p.)). Mice were infected with 500 pfu (10-15 mLD50) of PR8,
a mouse-adapted H1 virus or with 500 pfu of an H5 highly-pathogenic
avian influenza virus modified to remove the poly-basic cleavage
site in the viral hemagglutinin (see Steel et al., (2009) J Virol
83(4):1742-1753). Vaccination with the LAH-KLH conjugate was
protective against weight loss caused by H1 and H5 influenza
disease to a highly significant degree on virtually every day
during infection. Vaccinated mice infected with PR8 showed a
significant delay in kinetics of weight loss, while 60% of
vaccinated mice infected with the H5 avian virus survived lethal
challenge to ten days post infection (FIG. 9C-F).
[0567] Analysis of pre-challenge sera from mice that were
subsequently infected with PR8 revealed a positive correlation
between hemagglutinin-specific antibody titer and increase in body
weight in days following infection (FIG. 10A). Animals productively
immunized (with anti-H1 serum antibody) gained weight during days
1-3 post infection, whereas animals without H1-specific antibody
lost weight during this critical period. These data suggested that
antibody induced by LAH-KLH vaccination was a requisite component
in protection of mice against disease.
[0568] To further investigate the role of anti-LAH antibody in
protection, in vivo passive transfer experiments were performed.
Two hours prior to infection, recipient mice were given 200 .mu.l
of serum by intraperitoneal administration from donor mice that had
been infected with H1 or H3 virus, vaccinated with KLH alone or
vaccinated with the LAH-KLH vaccine. Recipient mice were then
infected with a human seasonal H3 virus, A/Georgia/81, or with the
H1 virus PR8. Two days following infection, lung titers were
evaluated. The transfer of LAH-KLH antiserum was found to
significantly reduce lung titers in animals infected with either
the human seasonal H3 virus (p=0.0009) or the H1 (p=0.0008) virus
(FIGS. 10B and 10C). This transfer experiment suggests that the LAH
construct induces neutralizing antibodies in the vaccinated
mouse.
[0569] Next, whether seasonal influenza vaccination in humans
induces antibody specific for the LAH region of the hemagglutinin
was investigated. To explore this possibility, binding activity in
human sera taken pre and post-immunization with the 2008-2009
trivalent inactivated influenza virus vaccine (TIV) was evaluated.
This seasonal vaccine composition contained an A/Brisbane/59/2007
(H1N1)-like virus, an A/Brisbane/10/2007 (H3N2)-like virus and a
B/Florida/4/2006-like virus (see Administration UFaD (2010)
Influenza Virus Vaccine for the 2008-2009 Season). Serum samples
from human patients were evaluated for a post-vaccination boost in
IgG antibody titer against the seasonal TIV composition as a
measure of vaccine response. Minimal serum antibody specific for
the LAH peptide was detected even in subjects demonstrating the
highest response to seasonal vaccination (FIGS. 10D and 10E).
[0570] As demonstrated in FIG. 8D, the breadth of reactivity seen
in the LAH-KLH antiserum is greater than what has been previously
described in studies of hemagglutinin stalk vaccine constructs (see
Bommakanti et al., (2010) Proc Natl Acad Sci USA; and Steel, (2010)
mBio 1(1):1-9). In order to probe the importance of the design of
the conjugate complex in eliciting this broad response, the serum
activity elicited by vaccination with the LAH-KLH construct was
compared with that elicited by vaccination with the intact HA2
molecule. The ectodomain of the A/Hong Kong/1/1968 HA2 protein was
recombinantly expressed as previously described (see Chen et al.,
(1999) Proc Natl Acad Sci USA 96(16):8967-8972). Mice were
vaccinated with pure, uncoupled, HA2 protein by the same methods
used to vaccinate mice with LAH-KLH. Pooled antisera from 20 mice
taken ten days post secondary vaccination with either LAH-KLH or
HA2 protein was evaluated for binding activity against a panel of
recombinantly expressed hemagglutinins. While the LAH-KLH antiserum
reacted with all hemagglutinin subtypes tested, the HA2 antiserum
contained antibody reactive with Group 2 hemagglutinin proteins
only (FIG. 11A-H and Table 1). Since the LAH structure is present
in the HA2 protein, the broad reactivity seen in the LAH-KLH
antiserum must be a consequence of the manner in which the LAH is
presented as an antigen within the conjugate complex. Elimination
of immunodominant regions of the HA2 may cause the LAH-KLH vaccine
to induce a more focused anti-LAH immune response that mediates
broad reactivity between hemagglutinin subtypes. Alternately, the
induction of broadly-reactive antibody may be a consequence of
anchoring the LAH at the C-terminus to a carrier protein, thus
rendering regions of the LAH immunogenic that are otherwise
antigenically silent in the context of the intact HA2 protein.
[0571] 6.5.3 Conclusion
[0572] This LAH core polypeptide linked to KLH has protective
activity against antigenically divergent influenza virus subtypes
that currently cause seasonal and pandemic disease in humans.
Additionally, the LAH core polypeptide linked to KLH has protective
activity against an avian H5N1 virus, a subtype with potential to
cause pandemic influenza disease in humans. Thus, the LAH core
polypeptide linked to KLH represents a peptide-based influenza
virus vaccine that would be inexpensive and uncomplicated to
manufacture.
TABLE-US-00029 TABLE 1 Summary of ELISA data Anti-LAH- KLH Anti-HA2
Group II HK/68 H3 + + Bris/07 H3 + + Neth/03 H7 + + Cal/09 H1 + -
Group I HK/99 H9 + - Sing/57 H2 + - Viet/04 H5 + - HK/97 H6 + -
[0573] All publications, patents and patent applications cited in
this specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Although
the foregoing invention has been described in some detail by way of
illustration and example for purposes of clarity of understanding,
it will be readily apparent to those of ordinary skill in the art
in light of the teachings of this invention that certain changes
and modifications may be made thereto without departing from the
spirit or scope of the appended claims.
Sequence CWU 1
1
60164PRTArtificial SequenceThe long alpha helix domain of the
hemagglutinin polypeptide of Influenza strain A/Hong Kong/1/1968
(H3) + flag tag 1Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr
Lys Ile Asp Leu1 5 10 15 Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala
Leu Glu Asn Gln His Thr 20 25 30 Ile Asp Leu Thr Asp Ser Glu Met
Asn Lys Leu Phe Glu Lys Thr Arg 35 40 45 Arg Gln Leu Arg Glu Asn
Ala Asp Tyr Lys Asp Asp Asp Asp Lys Cys 50 55 60 255PRTArtificial
SequenceThe long alpha helix domain of the hemagglutinin
polypeptide of Influenza strain A/Hong Kong/1/1968 (H3) generic
core peptide 2Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys
Ile Asp Leu1 5 10 15 Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu
Glu Asn Gln His Thr 20 25 30 Ile Asp Leu Thr Asp Ser Glu Met Asn
Lys Leu Phe Glu Lys Thr Arg 35 40 45 Arg Gln Leu Arg Glu Asn Ala 50
55 355PRTUnknowngeneric core polypeptide 3Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa1 5 10 15 Trp Xaa Tyr Xaa
Ala Glu Leu Leu Val Xaa Xaa Glu Asn Xaa Xaa Thr 20 25 30 Xaa Asp
Xaa Xaa Asp Ser Xaa Xaa Xaa Xaa Leu Xaa Xaa Xaa Xaa Xaa 35 40 45
Xaa Gln Leu Xaa Xaa Asn Xaa 50 55 455PRTArtificial
SequenceConsensus core polypeptide 4Arg Ile Glu Asn Leu Asn Lys Lys
Xaa Glu Asp Gly Phe Leu Asp Val1 5 10 15 Trp Thr Tyr Asn Ala Glu
Leu Leu Val Leu Met Glu Asn Glu Arg Thr 20 25 30 Leu Asp Xaa His
Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val Arg 35 40 45 Xaa Gln
Leu Arg Xaa Asn Ala 50 55 555PRTArtificial SequenceGroup 1 core
polypeptide 5Arg Xaa Glu Asn Leu Asn Lys Lys Xaa Xaa Asp Gly Phe
Leu Asp Xaa1 5 10 15 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Xaa
Glu Asn Glu Arg Thr 20 25 30 Leu Asp Xaa His Asp Ser Asn Val Lys
Asn Leu Tyr Xaa Lys Val Arg 35 40 45 Xaa Gln Leu Xaa Xaa Asn Xaa 50
55 655PRTArtificial SequenceGroup 2 core polypeptide 6Xaa Ile Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa1 5 10 15 Trp
Ser Tyr Asn Ala Glu Leu Leu Val Ala Xaa Glu Asn Gln His Thr 20 25
30 Ile Asp Leu Xaa Asp Ser Glu Met Asn Lys Leu Xaa Glu Xaa Xaa Xaa
35 40 45 Arg Gln Leu Arg Glu Asn Ala 50 55 755PRTArtificial
SequenceH1 core polypeptides 7Arg Xaa Glu Asn Leu Asn Lys Lys Val
Asp Asp Gly Phe Xaa Asp Ile1 5 10 15 Trp Thr Tyr Asn Ala Glu Leu
Leu Val Leu Leu Glu Asn Glu Arg Thr 20 25 30 Leu Asp Xaa His Asp
Ser Asn Val Xaa Asn Leu Tyr Glu Lys Val Xaa 35 40 45 Ser Gln Leu
Lys Asn Asn Ala 50 55 855PRTArtificial SequenceThe long alpha helix
domain of the hemagglutinin polypeptide of Influenza strain A/New
Caledonia/20/1999 8Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly
Phe Leu Asp Ile1 5 10 15 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
Leu Glu Asn Glu Arg Thr 20 25 30 Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu Tyr Glu Lys Val Lys 35 40 45 Ser Gln Leu Lys Asn Asn
Ala 50 55 955PRTArtificial SequenceThe long alpha helix domain of
the hemagglutinin polypeptide of Influenza strain
A/Brisbane/59/2007-2 9Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp
Gly Phe Ile Asp Ile1 5 10 15 Trp Thr Tyr Asn Ala Glu Leu Leu Val
Leu Leu Glu Asn Glu Arg Thr 20 25 30 Leu Asp Phe His Asp Ser Asn
Val Lys Asn Leu Tyr Glu Lys Val Lys 35 40 45 Ser Gln Leu Lys Asn
Asn Ala 50 55 1055PRTArtificial SequenceThe long alpha helix domain
of the hemagglutinin polypeptide of Influenza strain A/South
Carolina/1/1918 10Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp Gly
Phe Leu Asp Ile1 5 10 15 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
Leu Glu Asn Glu Arg Thr 20 25 30 Leu Asp Phe His Asp Ser Asn Val
Arg Asn Leu Tyr Glu Lys Val Lys 35 40 45 Ser Gln Leu Lys Asn Asn
Ala 50 55 1155PRTArtificial SequenceThe long alpha helix domain of
the hemagglutinin polypeptide of Influenza strain
A/California/07/2009 11Arg Ile Glu Asn Leu Asn Lys Lys Val Asp Asp
Gly Phe Leu Asp Ile1 5 10 15 Trp Thr Tyr Asn Ala Glu Leu Leu Val
Leu Leu Glu Asn Glu Arg Thr 20 25 30 Leu Asp Tyr His Asp Ser Asn
Val Lys Asn Leu Tyr Glu Lys Val Arg 35 40 45 Ser Gln Leu Lys Asn
Asn Ala 50 55 1255PRTArtificial SequenceH2 core polypeptide 12Arg
Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val1 5 10
15 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr
20 25 30 Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Xaa Lys
Val Arg 35 40 45 Met Gln Leu Arg Asp Asn Val 50 55
1355PRTArtificial SequenceThe long alpha helix domain of the
hemagglutinin polypeptide of Influenza strain A/Singapore/1/1957
13Arg Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val1
5 10 15 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg
Thr 20 25 30 Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp
Lys Val Arg 35 40 45 Met Gln Leu Arg Asp Asn Val 50 55
1455PRTArtificial SequenceThe long alpha helix domain of the
hemagglutinin polypeptide of Influenza strain A/Czech
Republic/1/1966 14Arg Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly
Phe Leu Asp Val1 5 10 15 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
Met Glu Asn Glu Arg Thr 20 25 30 Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu Tyr Glu Lys Val Arg 35 40 45 Met Gln Leu Arg Asp Asn
Val 50 55 1555PRTArtificial SequenceH3 core polypeptide 15Arg Ile
Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu1 5 10 15
Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr 20
25 30 Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Xaa Thr
Xaa 35 40 45 Xaa Gln Leu Arg Glu Asn Ala 50 55 1655PRTArtificial
SequenceThe long alpha helix domain of the hemagglutinin
polypeptide of Infuenza strain A/Denmark/191/2005 16Arg Ile Gln Asp
Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu1 5 10 15 Trp Ser
Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr 20 25 30
Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Arg Thr Lys 35
40 45 Lys Gln Leu Arg Glu Asn Ala 50 55 1755PRTArtificial
SequenceThe long alpha helix domain of the hemagglutinin
polypeptide of Infuenza strain A/Arkansas/05/2008 17Arg Ile Gln Asp
Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu1 5 10 15 Trp Ser
Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr 20 25 30
Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Lys 35
40 45 Lys Gln Leu Arg Glu Asn Ala 50 55 1855PRTArtificial
SequenceThe long alpha helix domain of the hemagglutinin
polypeptide of Infuenza strain A/Hong Kong/1/68 18Arg Ile Gln Asp
Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu1 5 10 15 Trp Ser
Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His Thr 20 25 30
Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe Glu Lys Thr Arg 35
40 45 Arg Gln Leu Arg Glu Asn Ala 50 55 1955PRTArtificial
SequenceH4 core polypeptide 19Arg Ile Gln Asp Leu Glu Lys Tyr Val
Glu Asp Thr Lys Ile Asp Leu1 5 10 15 Trp Ser Tyr Asn Ala Glu Leu
Leu Val Ala Leu Glu Asn Gln His Thr 20 25 30 Ile Asp Val Thr Asp
Ser Glu Met Asn Lys Leu Phe Glu Arg Val Arg 35 40 45 Xaa Gln Leu
Arg Glu Asn Ala 50 55 2055PRTArtificial SequenceThe long alpha
helix domain of the hemagglutinin polypeptide of Influenza strain
A/mallard/Ohia/324/1988 20Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu
Asp Thr Lys Ile Asp Leu1 5 10 15 Trp Ser Tyr Asn Ala Glu Leu Leu
Val Ala Leu Glu Asn Gln His Thr 20 25 30 Ile Asp Val Thr Asp Ser
Glu Met Asn Lys Leu Phe Glu Arg Val Arg 35 40 45 Arg Gln Leu Arg
Glu Asn Ala 50 55 2155PRTArtificial SequenceThe long alpha helix
domain of the hemagglutinin polypeptide of Influenza strain
A/mallard/Minnesota/271/1999 21Arg Ile Gln Asp Leu Glu Lys Tyr Val
Glu Asp Thr Lys Ile Asp Leu1 5 10 15 Trp Ser Tyr Asn Ala Glu Leu
Leu Val Ala Leu Glu Asn Gln His Thr 20 25 30 Ile Asp Val Thr Asp
Ser Glu Met Asn Lys Leu Phe Glu Arg Val Arg 35 40 45 His Gln Leu
Arg Glu Asn Ala 50 55 2255PRTArtificial SequenceH5 core polypeptide
22Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val1
5 10 15 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg
Thr 20 25 30 Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp
Lys Val Arg 35 40 45 Leu Gln Leu Arg Asp Asn Ala 50 55
2355PRTArtificial SequenceH6 core polypeptide 23Arg Ile Xaa Asn Xaa
Asn Lys Arg Met Glu Asp Gly Phe Leu Asp Val1 5 10 15 Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Xaa Arg Thr 20 25 30 Leu
Asp Xaa His Asp Ala Asn Val Lys Xaa Leu Xaa Glu Lys Val Lys 35 40
45 Ser Xaa Leu Xaa Asp Asn Ala 50 55 2455PRTArtificial SequenceThe
long alpha helix domain of the hemagglutinin polypeptide of
Influenza strain A/blue winged teal/ALB/69/1985 24Arg Ile Gly Asn
Leu Asn Lys Arg Met Glu Asp Gly Phe Leu Asp Val1 5 10 15 Trp Thr
Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr 20 25 30
Leu Asp Met His Asp Ala Asn Val Lys Asn Leu His Glu Lys Val Lys 35
40 45 Ser Gln Leu Arg Asp Asn Ala 50 55 2555PRTArtificial
SequenceThe long alpha helix domain of the hemagglutinin
polypeptide of Influenza strain A/Canada goose/Ohio/127/1989 25Arg
Ile Asp Asn Leu Asn Lys Arg Met Glu Asp Gly Phe Leu Asp Val1 5 10
15 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr
20 25 30 Leu Asp Met His Asp Ala Asn Val Lys Asn Leu His Glu Lys
Val Lys 35 40 45 Ser Gln Leu Lys Asp Asn Ala 50 55
2655PRTArtificial SequenceThe long alpha helix domain of the
hemagglutinin polypeptide of Influenza strain A/blu-winged
teal/ALB/368/1978 26Arg Ile Asp Asn Leu Asn Lys Arg Met Glu Asp Gly
Phe Leu Asp Val1 5 10 15 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
Leu Glu Asn Gly Arg Thr 20 25 30 Leu Asp Met His Asp Ala Asn Val
Lys Asn Leu His Glu Lys Val Lys 35 40 45 Ser Leu Leu Arg Asp Asn
Ala 50 55 2755PRTArtificial SequenceThe long alpha helix domain of
the hemagglutinin polypeptide of Influenza strain A/northern
pintail/California/HKWF151/2007 27Arg Ile Asp Asn Leu Asn Lys Arg
Met Glu Asp Gly Phe Leu Asp Val1 5 10 15 Trp Thr Tyr Asn Ala Glu
Leu Leu Val Leu Leu Glu Asn Glu Arg Thr 20 25 30 Leu Asp Leu His
Asp Ala Asn Val Lys Asn Leu Tyr Glu Lys Val Lys 35 40 45 Ser Gln
Leu Arg Asp Asn Ala 50 55 2855PRTArtificial SequenceThe long alpha
helix domain of the hemagglutinin polypeptide of Influenza strain
A/chicken/CA/S0403106/2004 28Arg Ile Asp Asn Met Asn Lys Arg Met
Glu Asp Gly Phe Leu Asp Val1 5 10 15 Trp Thr Tyr Asn Ala Glu Leu
Leu Val Leu Leu Glu Asn Glu Arg Thr 20 25 30 Leu Asp Leu His Asp
Ala Asn Val Lys Ser Leu His Glu Lys Val Lys 35 40 45 Ser Gln Leu
Arg Asp Asn Ala 50 55 2955PRTArtificial SequenceH6 Consensus 29Arg
Ile Asp Asn Leu Asn Lys Arg Met Glu Asp Gly Phe Leu Asp Val1 5 10
15 Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg Thr
20 25 30 Leu Asp Met His Asp Ala Asn Val Lys Asn Leu His Glu Lys
Val Lys 35 40 45 Ser Gln Leu Arg Asp Asn Ala 50 55
3055PRTArtificial SequenceH7 core polypeptide 30Gln Ile Gly Asn Val
Ile Asn Trp Thr Arg Asp Xaa Met Thr Glu Xaa1 5 10 15 Trp Ser Tyr
Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr 20 25 30 Ile
Asp Leu Ala Asp Ser Glu Met Xaa Lys Leu Tyr Glu Arg Val Xaa 35 40
45 Lys Gln Leu Arg Glu Asn Ala 50 55 3155PRTArtificial SequenceThe
long alpha helix domain of the hemagglutinin polypeptide of
Influenza strain A/green winged teal/California/AKS1370/2008 31Gln
Ile Gly Asn Val Ile Asn Trp Thr Arg Asp Ser Met Thr Glu Val1 5 10
15 Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr
20 25 30 Ile Asp Leu Ala Asp Ser Glu Met Asn Lys Leu Tyr Glu Arg
Val Arg 35 40 45 Lys Gln Leu Arg Glu Asn Ala 50 55
3255PRTArtificial SequenceThe long alpha helix domain of the
hemagglutinin polypeptide of Influenza strain A/duck/New
Jersey/Sg-00286/1996 32Gln Ile Gly Asn Val Ile Asn Trp Thr Arg Asp
Ala Met Thr Glu Val1 5 10 15 Trp Ser Tyr Asn Ala Glu Leu Leu Val
Ala Met Glu Asn Gln His Thr 20 25 30 Ile Asp Leu Ala Asp Ser Glu
Met Asn Lys Leu Tyr Glu Arg Val Arg 35 40 45 Lys Gln Leu Arg Glu
Asn Ala 50 55 3355PRTArtificial SequenceThe long alpha helix domain
of the hemagglutinin polypeptide of Influenza strain A/avian/New
York/Sg-00338/1999 33Gln Ile Gly Asn Val Ile Asn Trp Thr Arg Asp
Ala Met Thr Glu Ile1 5 10 15 Trp Ser Tyr Asn Ala Glu Leu Leu Val
Ala Met Glu Asn Gln His Thr 20 25 30 Ile Asp Leu Ala Asp Ser Glu
Met Ser Lys Leu Tyr Glu Arg Val Arg 35 40 45 Lys Gln Leu Arg Glu
Asn Ala 50 55 3455PRTArtificial SequenceThe long alpha helix domain
of the hemagglutinin polypeptide of Influenza strain
A/chicken/New
Jersey/Sg-00431/2004 34Gln Ile Gly Asn Val Ile Asn Trp Thr Arg Asp
Ala Met Thr Glu Ile1 5 10 15 Trp Ser Tyr Asn Ala Glu Leu Leu Val
Ala Met Glu Asn Gln His Thr 20 25 30 Ile Asp Leu Ala Asp Ser Glu
Met Ser Lys Leu Tyr Glu Arg Val Lys 35 40 45 Lys Gln Leu Arg Glu
Asn Ala 50 55 3555PRTArtificial SequenceH7 Consensus 35Gln Ile Gly
Asn Val Ile Asn Trp Thr Arg Asp Ala Met Thr Glu Val1 5 10 15 Trp
Ser Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr 20 25
30 Ile Asp Leu Ala Asp Ser Glu Met Asn Lys Leu Tyr Glu Arg Val Arg
35 40 45 Lys Gln Leu Arg Glu Asn Ala 50 55 3655PRTArtificial
SequenceH8 core polypeptide 36Arg Ile Asn Met Ile Asn Asp Lys Ile
Asp Asp Gln Ile Glu Xaa Leu1 5 10 15 Trp Ala Tyr Asn Ala Glu Leu
Leu Val Leu Leu Glu Asn Gln Lys Thr 20 25 30 Leu Asp Glu His Asp
Ser Asn Val Lys Asn Leu Phe Asp Glu Val Lys 35 40 45 Arg Arg Leu
Ser Ala Asn Ala 50 55 3755PRTArtificial SequenceThe long alpha
helix domain of the hemagglutinin polypeptide of Influenza strain
A/pintail duck/Alberta/114/1979 37Arg Ile Asn Met Ile Asn Asp Lys
Ile Asp Asp Gln Ile Glu Asp Leu1 5 10 15 Trp Ala Tyr Asn Ala Glu
Leu Leu Val Leu Leu Glu Asn Gln Lys Thr 20 25 30 Leu Asp Glu His
Asp Ser Asn Val Lys Asn Leu Phe Asp Glu Val Lys 35 40 45 Arg Arg
Leu Ser Ala Asn Ala 50 55 3855PRTArtificial SequenceThe long alpha
helix domain of the hemagglutinin polypeptide of Influenza strain
A/pintail/Barrow/38/2005 38Arg Ile Asn Met Ile Asn Asp Lys Ile Asp
Asp Gln Ile Glu Asn Leu1 5 10 15 Trp Ala Tyr Asn Ala Glu Leu Leu
Val Leu Leu Glu Asn Gln Lys Thr 20 25 30 Leu Asp Glu His Asp Ser
Asn Val Lys Asn Leu Phe Asp Glu Val Lys 35 40 45 Arg Arg Leu Ser
Ala Asn Ala 50 55 3955PRTArtificial SequenceH9 core polypeptide
39Arg Leu Asn Met Ile Asn Asn Lys Ile Asp Asp Gln Ile Gln Asp Xaa1
5 10 15 Trp Ala Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gln Lys
Thr 20 25 30 Leu Asp Glu His Asp Ala Asn Val Asn Asn Leu Tyr Asn
Lys Val Lys 35 40 45 Arg Ala Leu Gly Ser Asn Ala 50 55
4055PRTArtificial SequenceThe long alpha helix domain of the
hemagglutinin polypeptide of Influenza strain A/chicken/Middle
East/ED-1/1999 40Arg Leu Asn Met Ile Asn Asn Lys Ile Asp Asp Gln
Ile Gln Asp Val1 5 10 15 Trp Ala Tyr Asn Ala Glu Leu Leu Val Leu
Leu Glu Asn Gln Lys Thr 20 25 30 Leu Asp Glu His Asp Ala Asn Val
Asn Asn Leu Tyr Asn Lys Val Lys 35 40 45 Arg Ala Leu Gly Ser Asn
Ala 50 55 4155PRTArtificial SequenceThe long alpha helix domain of
the hemagglutinin polypeptide of Influenza strain A/duck/Hong
Kong/702/1979 41Arg Leu Asn Met Ile Asn Asn Lys Ile Asp Asp Gln Ile
Gln Asp Ile1 5 10 15 Trp Ala Tyr Asn Ala Glu Leu Leu Val Leu Leu
Glu Asn Gln Lys Thr 20 25 30 Leu Asp Glu His Asp Ala Asn Val Asn
Asn Leu Tyr Asn Lys Val Lys 35 40 45 Arg Ala Leu Gly Ser Asn Ala 50
55 4255PRTArtificial SequenceH10 core polypeptide 42Gln Ile Gly Asn
Val Ile Asn Trp Thr Lys Asp Ser Ile Thr Asp Ile1 5 10 15 Trp Thr
Tyr Xaa Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr 20 25 30
Ile Asp Met Ala Asp Ser Glu Met Leu Asn Leu Tyr Glu Arg Val Arg 35
40 45 Lys Gln Leu Arg Gln Asn Ala 50 55 4355PRTArtificial
SequenceThe long alpha helix domain of the hemagglutinin
polypeptide of Infuenza strain A/mallard/Maryland/161/2001 43Gln
Ile Gly Asn Val Ile Asn Trp Thr Lys Asp Ser Ile Thr Asp Ile1 5 10
15 Trp Thr Tyr Gln Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr
20 25 30 Ile Asp Met Ala Asp Ser Glu Met Leu Asn Leu Tyr Glu Arg
Val Arg 35 40 45 Lys Gln Leu Arg Gln Asn Ala 50 55
4455PRTArtificial SequenceThe long alpha helix domain of the
hemagglutinin polypeptide of Infuenza strain
A/chicken/Germany/n/1949 44Gln Ile Gly Asn Val Ile Asn Trp Thr Lys
Asp Ser Ile Thr Asp Ile1 5 10 15 Trp Thr Tyr Asn Ala Glu Leu Leu
Val Ala Met Glu Asn Gln His Thr 20 25 30 Ile Asp Met Ala Asp Ser
Glu Met Leu Asn Leu Tyr Glu Arg Val Arg 35 40 45 Lys Gln Leu Arg
Gln Asn Ala 50 55 4555PRTArtificial SequenceH11 core polypeptide
45Arg Ile Asn Gln Leu Ser Lys His Val Asp Asp Ser Val Xaa Asp Ile1
5 10 15 Trp Ser Tyr Asn Ala Gln Leu Leu Val Leu Leu Glu Asn Glu Lys
Thr 20 25 30 Leu Asp Leu His Asp Ser Asn Val Arg Asn Leu His Glu
Lys Val Arg 35 40 45 Arg Met Leu Lys Asp Asn Ala 50 55
4655PRTArtificial SequenceThe long alpha helix domain of the
hemagglutinin polypeptide of Influenza strain A/GF/NY/26410-17/1995
46Arg Ile Asn Gln Leu Ser Lys His Val Asp Asp Ser Val Ile Asp Ile1
5 10 15 Trp Ser Tyr Asn Ala Gln Leu Leu Val Leu Leu Glu Asn Glu Lys
Thr 20 25 30 Leu Asp Leu His Asp Ser Asn Val Arg Asn Leu His Glu
Lys Val Arg 35 40 45 Arg Met Leu Lys Asp Asn Ala 50 55
4755PRTArtificial SequenceThe long alpha helix domain of the
hemagglutinin polypeptide of Influenza strain A/duck/England/1/1956
47Arg Ile Asn Gln Leu Ser Lys His Val Asp Asp Ser Val Val Asp Ile1
5 10 15 Trp Ser Tyr Asn Ala Gln Leu Leu Val Leu Leu Glu Asn Glu Lys
Thr 20 25 30 Leu Asp Leu His Asp Ser Asn Val Arg Asn Leu His Glu
Lys Val Arg 35 40 45 Arg Met Leu Lys Asp Asn Ala 50 55
4855PRTArtificial SequenceH12 core polypeptide 48Arg Ile Asn Met
Ile Asn Ser Lys Ile Asp Asp Gln Ile Thr Asp Ile1 5 10 15 Trp Ala
Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr 20 25 30
Leu Asp Glu His Asp Ala Asn Val Arg Asn Leu His Asp Arg Val Arg 35
40 45 Arg Xaa Leu Xaa Glu Asn Ala 50 55 4955PRTArtificial
SequenceThe long alpha helix domain of the hemagglutinin
polypeptide of Influenza strain A/duck/Alberta/60/1976 49Arg Ile
Asn Met Ile Asn Ser Lys Ile Asp Asp Gln Ile Thr Asp Ile1 5 10 15
Trp Ala Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr 20
25 30 Leu Asp Glu His Asp Ala Asn Val Arg Asn Leu His Asp Arg Val
Arg 35 40 45 Arg Val Leu Arg Glu Asn Ala 50 55 5055PRTArtificial
SequenceThe long alpha helix domain of the hemagglutinin
polypeptide of Influenza strain A/duck/Hokkaido/66/01 50Arg Ile Asn
Met Ile Asn Ser Lys Ile Asp Asp Gln Ile Thr Asp Ile1 5 10 15 Trp
Ala Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Gln Lys Thr 20 25
30 Leu Asp Glu His Asp Ala Asn Val Arg Asn Leu His Asp Arg Val Arg
35 40 45 Arg Ile Leu Lys Glu Asn Ala 50 55 5155PRTArtificial
SequenceH13 core polypeptide 51Arg Ile Asn Met Leu Ala Asp Arg Ile
Asp Asp Ala Val Thr Asp Xaa1 5 10 15 Trp Ser Tyr Asn Ala Lys Leu
Leu Val Leu Leu Glu Asn Asp Lys Thr 20 25 30 Leu Asp Met His Asp
Ala Asn Val Arg Asn Leu His Xaa Gln Val Arg 35 40 45 Arg Xaa Leu
Lys Xaa Asn Ala 50 55 5255PRTArtificial SequenceThe long alpha
helix domain of the hemagglutinin polypeptide of Influenza strain
A/black-headed gull/Asktrakhan/227/1984 52Arg Ile Asn Met Leu Ala
Asp Arg Ile Asp Asp Ala Val Thr Asp Val1 5 10 15 Trp Ser Tyr Asn
Ala Lys Leu Leu Val Leu Leu Glu Asn Asp Lys Thr 20 25 30 Leu Asp
Met His Asp Ala Asn Val Arg Asn Leu His Asp Gln Val Arg 35 40 45
Arg Ala Leu Lys Thr Asn Ala 50 55 5355PRTArtificial SequenceThe
long alpha helix domain of the hemagglutinin polypeptide of
Influenza strain A/American while pelican/Minnesota/Sg-0611/2008
53Arg Ile Asn Met Leu Ala Asp Arg Ile Asp Asp Ala Val Thr Asp Val1
5 10 15 Trp Ser Tyr Asn Ala Lys Leu Leu Val Leu Leu Glu Asn Asp Lys
Thr 20 25 30 Leu Asp Met His Asp Ala Asn Val Arg Asn Leu His Asp
Gln Val Arg 35 40 45 Arg Ser Leu Lys Thr Asn Ala 50 55
5455PRTArtificial SequenceThe long alpha helix domain of the
hemagglutinin polypeptide of Influenza strain
A/duck/Siberia/272PF/1998 54Arg Ile Asn Met Leu Ala Asp Arg Ile Asp
Asp Ala Val Thr Asp Ile1 5 10 15 Trp Ser Tyr Asn Ala Lys Leu Leu
Val Leu Leu Glu Asn Asp Lys Thr 20 25 30 Leu Asp Met His Asp Ala
Asn Val Arg Asn Leu His Glu Gln Val Arg 35 40 45 Arg Glu Leu Lys
Asp Asn Ala 50 55 5555PRTArtificial SequenceH14 core polypeptide
55Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr Lys Ile Asp Leu1
5 10 15 Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu Asn Gln His
Thr 20 25 30 Ile Asp Val Thr Asp Ser Glu Met Asn Lys Leu Phe Glu
Arg Val Arg 35 40 45 Arg Gln Leu Arg Glu Asn Ala 50 55
5655PRTArtificial SequenceH15 core polypeptide 56Gln Ile Gly Asn
Val Ile Asn Trp Thr Arg Asp Ser Leu Thr Glu Ile1 5 10 15 Trp Ser
Tyr Asn Ala Glu Leu Leu Val Ala Met Glu Asn Gln His Thr 20 25 30
Ile Asp Leu Ala Asp Ser Glu Met Asn Lys Leu Tyr Glu Arg Val Arg 35
40 45 Arg Gln Leu Arg Glu Asn Ala 50 55 5755PRTArtificial
SequenceH16 core polypeptide 57Arg Ile Asn Met Leu Ala Asp Arg Val
Asp Asp Ala Val Thr Asp Ile1 5 10 15 Trp Ser Tyr Asn Ala Lys Leu
Leu Val Leu Xaa Glu Asn Asp Arg Thr 20 25 30 Leu Asp Leu His Asp
Ala Asn Val Xaa Asn Leu His Xaa Gln Val Lys 35 40 45 Arg Ala Leu
Lys Xaa Asn Ala 50 55 5855PRTArtificial SequenceThe long alpha
helix domain of the hemagglutinin polypeptide of Influenza strain
A/black-headed gull/Turkenistan/13/76 58Arg Ile Asn Met Leu Ala Asp
Arg Val Asp Asp Ala Val Thr Asp Ile1 5 10 15 Trp Ser Tyr Asn Ala
Lys Leu Leu Val Leu Ile Glu Asn Asp Arg Thr 20 25 30 Leu Asp Leu
His Asp Ala Asn Val Arg Asn Leu His Asp Gln Val Lys 35 40 45 Arg
Ala Leu Lys Ser Asn Ala 50 55 5955PRTArtificial SequenceThe long
alpha helix domain of the hemagglutinin polypeptide of Influenza
strain A/slender-billed gull/Astrakhan/28/76 59Arg Ile Asn Met Leu
Ala Asp Arg Val Asp Asp Ala Val Thr Asp Ile1 5 10 15 Trp Ser Tyr
Asn Ala Lys Leu Leu Val Leu Leu Glu Asn Asp Arg Thr 20 25 30 Leu
Asp Leu His Asp Ala Asn Val Arg Asn Leu His Asp Gln Val Lys 35 40
45 Arg Ala Leu Lys Ser Asn Ala 50 55 6055PRTArtificial SequenceThe
long alpha helix domain of the hemagglutinin polypeptide of
Influenza strain A/herring gull/DE/712/1988 60Arg Ile Asn Met Leu
Ala Asp Arg Val Asp Asp Ala Val Thr Asp Ile1 5 10 15 Trp Ser Tyr
Asn Ala Lys Leu Leu Val Leu Ile Glu Asn Asp Arg Thr 20 25 30 Leu
Asp Leu His Asp Ala Asn Val Lys Asn Leu His Glu Gln Val Lys 35 40
45 Arg Ala Leu Lys Asn Asn Ala 50 55
* * * * *
References