U.S. patent application number 13/782381 was filed with the patent office on 2014-03-13 for novel influenza hemagglutinin protein-based vaccines.
The applicant listed for this patent is The United States of America, as represented by the Secretary, Dept. of Health and Human Services, The United States of America, as represented by the Secretary, Dept. of Health and Human Services. Invention is credited to Jeffrey C. BOYINGTON, Masaru KANEKIYO, Patrick M. MCTAMNEY, Gary J. NABEL, Chih-Jen WEI, Hadi M. YASSINE.
Application Number | 20140072958 13/782381 |
Document ID | / |
Family ID | 47915112 |
Filed Date | 2014-03-13 |
United States Patent
Application |
20140072958 |
Kind Code |
A1 |
NABEL; Gary J. ; et
al. |
March 13, 2014 |
NOVEL INFLUENZA HEMAGGLUTININ PROTEIN-BASED VACCINES
Abstract
Novel vaccines are provided that elicit broadly neutralizing
anti-influenza antibodies. Some vaccines comprise nanoparticles
that display hemagglutinin trimers from influenza virus on their
surface. The nanoparticles comprise fusion proteins comprising a
monomeric subunit of ferritin joined to at least a portion of an
influenza hemagglutinin protein. Some portions comprise the
ectodomain while some portions are limited to the stem region. The
fusion proteins self-assemble to form the hemagglutinin-displaying
nanoparticles. Some vaccines comprise only the stem region of an
influenza hemagglutinin protein joined to a trimerization domain.
Such vaccines can be used to vaccinate an individual against
infection by heterologous influenza viruses and influenza virus
that are antigenically divergent from the virus from which the
nanoparticle hemagglutinin protein was obtained. Also provided are
fusion proteins and nucleic acid molecules encoding such proteins.
Finally, also provided are assays using nanoparticles of the
invention to detect anti-influenza antibodies.
Inventors: |
NABEL; Gary J.; (Washington,
DC) ; KANEKIYO; Masaru; (Silver Spring, MD) ;
WEI; Chih-Jen; (Potomac, MD) ; MCTAMNEY; Patrick
M.; (Bethesda, MD) ; YASSINE; Hadi M.;
(Rockville, MD) ; BOYINGTON; Jeffrey C.;
(Clarksburg, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Dept. of Health and Human Services |
Bethesda |
MD |
US |
|
|
Family ID: |
47915112 |
Appl. No.: |
13/782381 |
Filed: |
March 1, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US12/56822 |
Sep 24, 2012 |
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13782381 |
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61661209 |
Jun 18, 2012 |
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61538663 |
Sep 23, 2011 |
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Current U.S.
Class: |
435/5 ;
530/400 |
Current CPC
Class: |
C12N 2760/16134
20130101; C12N 2760/16034 20130101; A61P 37/04 20180101; C07K
2319/40 20130101; A61K 2039/55566 20130101; C12N 7/00 20130101;
A61K 39/12 20130101; C12N 2760/16234 20130101; C07K 14/205
20130101; C12N 2760/16071 20130101; A61K 39/105 20130101; C07K
2319/35 20130101; A61K 39/145 20130101; C07K 2319/00 20130101; C07K
14/005 20130101; C12N 2760/16022 20130101; A61K 2039/55555
20130101; A61K 2039/575 20130101; C07K 2319/21 20130101; A61K
2039/70 20130101; C07K 2319/735 20130101; G01N 33/56983 20130101;
A61K 2039/6031 20130101; A61P 31/16 20180101 |
Class at
Publication: |
435/5 ;
530/400 |
International
Class: |
G01N 33/569 20060101
G01N033/569 |
Claims
1. A method for detecting anti-influenza virus antibodies in a
sample, the method comprising: a. contacting at least a portion of
the sample with a nanoparticle comprising a fusion protein, wherein
the fusion protein comprises at least 25 contiguous amino acids
from a monomeric ferritin subunit protein joined to at least one
epitope from an influenza hemagglutinin (HA) protein such that the
nanoparticle comprises trimers of the influenza virus HA protein
epitope on its surface, and wherein the at least a portion of the
sample and the nanoparticle are contacted under conditions suitable
for forming a complex between the nanoparticle and the
anti-influenza virus antibodies, if present; and, b. analyzing the
contacted sample for the presence of a nanoparticle/antibody
complex, wherein the presence of such a complex indicates the
sample contains anti-influenza antibodies.
2. The method of claim 1, wherein the monomeric ferritin subunit
protein is selected from the group consisting of a bacterial
ferritin, a plant ferritin, an algal ferritin and a mammalian
ferritin.
3. The nanoparticle of claim 1, wherein the monomeric ferritin
subunit protein comprises at least 25 contiguous amino acids an
amino acid sequence selected from the group consisting of SEQ ID
NO:2 and SEQ ID NO:5, where in the fusion protein is capable of
self-assembling into nanoparticles.
4. The nanoparticle of claim 1, wherein the hemagglutinin protein
is from an influenza virus selected from the group consisting of
A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009
CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968
(1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3),
A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo,
B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris,
H1), B/Brisbane/60/2008 (2008 Bris, B).
5. The nanoparticle of claim 1, wherein the fusion protein
comprises an amino acid sequence at least 80% identical to a
sequence selected from the group consisting of SEQ ID NO:41, SEQ ID
NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ
ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, wherein the
nanoparticle elicits an immune response against an influenza
virus.
6. The nanoparticle of claim 1, wherein the nanoparticle comprises
a second fusion protein comprising a second influenza hemagglutinin
protein, wherein the first and second influenza hemagglutinin
proteins are from different types, sub-types or strains of
influenza viruses.
7. The method of claim 1, wherein the step of analyzing comprises
an assay selected from the group consisting of a hemagglutinin
inhibition assay, an immunodiffusion assay, an enzyme-linked
immunoassay, a radioimmunoassay, a fluorescence immunoassay, a
chemiluminescent assay, a lateral flow assay, a flow-through assay,
a precipitation assay, an immunoprecipitation assay, a BioCoreJ
assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s
Immunodot System, Fribourg, Switzerland), an immunoblot assay
(e.g., a western blot), an phosphorescence assay, a chromatography
assay, a PAGe-based assay, a surface plasmon resonance assay, a
spectrophotometric assay, and an electronic sensory assay.
8. A method for identifying an individual having anti-influenza
antibodies, comprising: a. contacting at least a portion of a
sample from an individual with a nanoparticle comprising a fusion
protein, wherein the fusion protein comprises at least 25
contiguous amino acids from a monomeric ferritin subunit protein
joined to at least one epitope from an influenza hemagglutinin (HA)
protein such that the nanoparticle comprises trimers of the
influenza virus HA protein epitope on its surface, and wherein the
at least a portion of the sample and the nanoparticle are contacted
under conditions suitable for forming a complex between the
nanoparticle and the anti-influenza virus antibodies, if present;
b. analyzing the contacted sample for the presence of a
nanoparticle/antibody complex, wherein the presence of such a
complex indicates the individual has anti-influenza antibodies.
9. The method of claim 8, wherein the monomeric ferritin subunit
protein is selected from the group consisting of a bacterial
ferritin, a plant ferritin, an algal ferritin and a mammalian
ferritin.
10. The nanoparticle of claim 8, wherein the monomeric ferritin
subunit protein comprises at least 25 contiguous amino acids an
amino acid sequence selected from the group consisting of SEQ ID
NO:2 and SEQ ID NO:5, where in the fusion protein is capable of
self-assembling into nanoparticles.
11. The nanoparticle of claim 8, wherein the hemagglutinin protein
is from an influenza virus selected from the group consisting of
A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009
CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968
(1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3),
A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo,
B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris,
H1), B/Brisbane/60/2008 (2008 Bris, B).
12. The nanoparticle of claim 8, wherein the fusion protein
comprises an amino acid sequence at least 80% identical to a
sequence selected from the group consisting of SEQ ID NO:41, SEQ ID
NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ
ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, wherein the
nanoparticle elicits an immune response against an influenza
virus.
13. The nanoparticle of claim 8, wherein the nanoparticle comprises
a second fusion protein comprising a second influenza hemagglutinin
protein, wherein the first and second influenza hemagglutinin
proteins are from different types, sub-types or strains of
influenza viruses.
14. The method of claim 8, wherein the step of analyzing comprises
an assay selected from the group consisting of a hemagglutinin
inhibition assay, an immunodiffusion assay, an enzyme-linked
immunoassay, a radioimmunoassay, a fluorescence immunoassay, a
chemiluminescent assay, a lateral flow assay, a flow-through assay,
a precipitation assay, an immunoprecipitation assay, a BioCoreJ
assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s
Immunodot System, Fribourg, Switzerland), an immunoblot assay
(e.g., a western blot), an phosphorescence assay, a chromatography
assay, a PAGe-based assay, a surface plasmon resonance assay, a
spectrophotometric assay, and an electronic sensory assay.
15. A method for measuring the response of an individual to a
vaccine, comprising: a. administering to the individual a vaccine
for influenza virus; b. contacting at least a portion of a sample
from the individual with a nanoparticle comprising a fusion
protein, wherein the fusion protein comprises at least 25
contiguous amino acids from a monomeric ferritin subunit protein
joined to at least one epitope from an influenza hemagglutinin (HA)
protein such that the nanoparticle comprises trimers of the
influenza virus HA protein epitope on its surface, and wherein the
at least a portion of the sample and the nanoparticle are contacted
under conditions suitable for forming a complex between the
nanoparticle and the anti-influenza virus antibodies, if present;
and, c. determining the level of antibody present in the sample by
determining the level of nanoparticle/antibody complex; wherein an
increase in the level of antibody in the sample over the
pre-vaccination level of antibody indicates the vaccine induced an
immune response in the individual.
16. The method of claim 15, wherein the monomeric ferritin subunit
protein is selected from the group consisting of a bacterial
ferritin, a plant ferritin, an algal ferritin and a mammalian
ferritin.
17. The nanoparticle of claim 15, wherein the monomeric ferritin
subunit protein comprises at least 25 contiguous amino acids an
amino acid sequence selected from the group consisting of SEQ ID
NO:2 and SEQ ID NO:5, where in the fusion protein is capable of
self-assembling into nanoparticles.
18. The nanoparticle of claim 15, wherein the hemagglutinin protein
is from an influenza virus selected from the group consisting of
A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009
CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968
(1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3),
A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo,
B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris,
H1), B/Brisbane/60/2008 (2008 Bris, B).
19. The nanoparticle of claim 15, wherein the fusion protein
comprises an amino acid sequence at least 80% identical to a
sequence selected from the group consisting of SEQ ID NO:41, SEQ ID
NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ
ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, wherein the
nanoparticle elicits an immune response against an influenza
virus.
20. The method of claim 15, wherein the step of analyzing comprises
an assay selected from the group consisting of a hemagglutinin
inhibition assay, an immunodiffusion assay, an enzyme-linked
immunoassay, a radioimmunoassay, a fluorescence immunoassay, a
chemiluminescent assay, a lateral flow assay, a flow-through assay,
a precipitation assay, an immunoprecipitation assay, a BioCoreJ
assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s
Immunodot System, Fribourg, Switzerland), an immunoblot assay
(e.g., a western blot), an phosphorescence assay, a chromatography
assay, a PAGe-based assay, a surface plasmon resonance assay, a
spectrophotometric assay, and an electronic sensory assay.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C.
.sctn.119(e) to P.C.T. Patent Application Serial No. PCT/US12/56822
entitled "NOVEL INFLUENZA HEMAGGLUTININ PROTEIN-BASED VACCINES"
filed Sep. 24, 2012; which claims priority to U.S. Provisional
Patent Application Ser. No. 61/661,209 entitled "SELF-ASSEMBLED
FERRITIN NANOPARTICLES EXPRESSING HEMAGGLUTININ AS AN INFLUENZA
VACCINE" filed Jun. 18, 2012; and U.S. Provisional Patent
Application Ser. No. 61/538,663 entitled "SELF-ASSEMBLED FERRITIN
NANOPARTICLES EXPRESSING HEMAGGLUTININ AS AN INFLUENZA VACCINE"
filed Sep. 23, 2011, all of which are incorporated herein in their
entirety by this reference.
SUMMARY OF THE INVENTION
[0002] The present invention provides novel hemagglutinin
protein-based influenza vaccines that are easily manufactured,
potent, and which elicit broadly neutralizing influenza antibodies.
In particular, the present invention provides influenza
hemagglutinin proteins, and portions thereof, that are useful in
inducing the production of neutralizing antibodies. It also
provides novel HA-ferritin nanoparticle (np) vaccines. Such
nanoparticles comprise fusion proteins, each of which comprises a
monomeric subunit of ferritin joined to an immunogenic portion of
an influenza hemagglutinin protein. Because such nanoparticles
display influenza hemagglutinin protein on their surface, they can
be used to vaccinate an individual against influenza virus.
[0003] In one embodiment, the invention is a nanoparticle that
comprises a fusion protein, and in this embodiment the fusion
protein comprises at least 25 contiguous amino acids from a
monomeric ferritin subunit protein joined to a first influenza
hemagglutinin (HA) protein, such that the nanoparticle comprises
influenza virus HA protein trimers on its surface. The nanoparticle
can form an octahedron, which can consist of 24 subunits having 432
symmetry. Further, the monomeric ferritin subunit protein can be
selected from a bacterial ferritin, a plant ferritin, an algal
ferritin, an insect ferritin, a fungal ferritin and a mammalian
ferritin, and in a preferred embodiment, is a Helicobacter pylori
ferritin protein.
[0004] In this embodiment, the monomeric ferritin subunit protein
can comprise at least 25 contiguous amino acids of an amino acid
sequence selected from SEQ ID NO:2 and SEQ ID NO:5 or can comprise
an amino acid at least about 80% identical, at least about 85%
identical, at least about 90% identical, at least about 95%
identical, at least about 97% identical, at least about 99%
identical to those sequences or can comprise those sequences. In
another embodiment, the monomeric subunit comprises a region
corresponding to amino acids 5-167 of SEQ ID NO:2.
[0005] In this embodiment, the hemagglutinin protein can comprise
at least 25 contiguous amino acids from the hemagglutinin protein
of an influenza virus selected from A/New Calcdonia/20/1999 (1999
NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957
(1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3),
A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo,
H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per,
H3), A/Brisbane/59/2007 (2007 Bris, H1), B/Brisbane/60/2008 (2008
Bris, B). Also, the hemagglutinin protein can comprise an amino
acid sequence that is selected from the amino acid sequences of SEQ
ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20,
SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID
NO:35, and SEQ ID NO:38 or one that is at least 80% identical, at
least about 85% identical, at least about 90% identical, at least
about 95% identical, at least about 97% identical, at least about
99% identical thereto. Alternatively, the hemagglutinin protein can
comprise an amino acid sequence that is selected from the amino
acid sequences of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID
NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ
ID NO:95 and SEQ ID NO:98 or one that is at least 80% identical, at
least about 85% identical, at least about 90% identical, at least
about 95% identical, at least about 97% identical, at least about
99% identical thereto.
[0006] In this embodiment, the hemagglutinin protein can be capable
of eliciting an immune response to a protein comprising an amino
acid sequence selected from SEQ ID NO:8, SEQ ID NO:11, SEQ ID
NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ
ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38 or it can
comprise a region selected from a region capable of allowing
formation of a hemagglutinin trimer, a stem region, an ectodomain,
and a region comprising the amino acid sequence from the amino acid
residue immediately distal to the last amino acid of the second
helical coiled coil to the amino acid residue proximal to the first
amino acid of the transmembrane domain.
[0007] The hemagglutinin protein can also comprise a hemagglutinin
spike domain, a region corresponding to amino acids 1-519 of SEQ ID
NO:8 or an amino acid sequence selected from the group consisting
of amino acids 1-519 of SEQ ID NO:8 and SEQ ID NO:11.
[0008] In this embodiment, the fusion protein can comprise a linker
sequence.
[0009] In this embodiment, the nanoparticle can elicit an immune
response against a stem region of influenza hemagglutinin, a spike
of influenza hemagglutinin, an influenza virus strain that is
heterologous to the strain influenza virus from which the
hemagglutinin protein was obtained or an influenza virus that is
antigenically divergent from the influenza virus from which the
hemagglutinin protein was obtained.
[0010] In this embodiment, the fusion protein can comprise an amino
acid sequence at least about 80% identical, at least about 85%
identical, at least about 90% identical, at least about 95%
identical, at least about 97% identical, at least about 99%
identical to a sequence selected from the group consisting of SEQ
ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53,
SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID
NO:68, wherein the nanoparticle elicits an immune response against
an influenza virus or can comprise an amino acid sequence selected
from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID
NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ
ID NO:62, SEQ ID NO:65, and SEQ ID NO:68. The fusion protein can
also comprise an amino acid sequence at least 80% identical, at
least about 85% identical, at least about 90% identical, at least
about 95% identical, at least about 97% identical, at least about
99% identical to a sequence selected from the group consisting of
SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID
NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and
SEQ ID NO:128, wherein the nanoparticle elicits an immune response
against an influenza virus.
[0011] In this embodiment, the nanoparticle can comprise a second
fusion protein comprising a second influenza hemagglutinin protein,
wherein the first and second influenza hemagglutinin proteins are
from different Types, from different sub-types or different strains
of influenza viruses.
[0012] Another embodiment of the present invention is a vaccine
composition comprising any of the foregoing nanoparticle. The
vaccine composition can further comprise at least one additional
nanoparticle that comprises at least one hemagglutinin protein from
a different strain of influenza than the first hemagglutinin
protein and the second hemagglutinin protein.
[0013] A further embodiment of the invention is a method to produce
a vaccine against influenza virus. The method includes expressing a
fusion protein comprising a monomeric ferritin protein joined to an
influenza hemagglutinin protein under conditions such that the
fusion proteins form a nanoparticle displaying hemagglutinin
trimers on its surface and recovering the nanoparticle.
[0014] The invention also includes a method to vaccinate an
individual against influenza that includes administering a
nanoparticle to an individual such that the nanoparticle elicits an
immune response against influenza virus. In this embodiment, the
nanoparticle comprises a monomeric subunit of ferritin joined to an
influenza hemagglutinin protein and the nanoparticle displays
influenza hemagglutinin trimers on its surface. In this embodiment,
the nanoparticle can elicit an immune response to an influenza
virus strain that is heterologous to the sub-type or strain of or
that is antigenically divergent from the influenza virus from which
the hemagglutinin protein was obtained.
[0015] This method can further include administering to the
individual a first vaccine composition and then at a later time,
administering a second vaccine composition comprising a
nanoparticle that comprises an HA-SS-ferritin fusion protein. The
HA SS-ferritin fusion protein can comprise an amino acid sequence
selected from SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID
NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ
ID NO:95 and SEQ ID NO:98 or one that is at least 80% identical, at
least 85% identical, at least 90% identical, at least 95%
identical, at least 97% identical or at least 99% identical
thereto, wherein the HA 55-ferritin fusion protein elicits an
immune response to an influenza virus. The HA 55-ferritin fusion
protein can comprise an amino acid sequence selected from the group
consisting of SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID
NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ
ID NO:125 and SEQ ID NO:128, or one at least 80% identical, at
least 85% identical, at least 90% identical, at least 95%
identical, at least 97% identical or at least 99% identical
thereto, wherein the HA 55-ferritin fusion protein elicits an
immune response to an influenza virus.
[0016] In this method, the first vaccine composition can comprise a
nanoparticle comprising an ectodomain from the hemagglutinin
protein of an influenza virus selected from the group consisting of
A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009
CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968
(1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3),
A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo,
B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris,
H1), B/Brisbane/60/2008 (2008 Bris, B). Alternatively, the
hemagglutinin of the first vaccine composition protein can comprise
an amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:11, SEQ
ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26,
SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38 or one
that is at least about 80% identical, at least about 85% identical,
at least about 90% identical, at least about 95% identical, at
least about 97% identical, at least about 99% identical thereto.
Further, the first vaccine composition can comprise an HA-ferritin
fusion protein comprising an amino acid sequence selected from SEQ
ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53,
SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID
NO:68 or an amino acid sequence that is at least 80% identical, at
least about 85% identical, at least about 90% identical, at least
about 95% identical, at least about 97% identical, at least about
99% identical thereto, wherein the nanoparticle elicits an immune
response against an influenza virus.
[0017] Administration of the boosting composition is generally
weeks or months after administration of the priming
composition.
[0018] A further embodiment of the present invention is a fusion
protein comprising a monomeric ferritin subunit protein joined to
an influenza hemagglutinin protein. The monomeric ferritin subunit
protein can be selected from a bacterial ferritin, a plant
ferritin, an algal ferritin, an insect ferritin, a fungal ferritin
and a mammalian ferritin or can be a monomeric subunit of a
Helicobacter pylori ferritin protein. The monomeric ferritin
subunit protein can comprise a domain that allows the fusion
protein to self-assemble into nanoparticles. In this embodiment,
the monomeric ferritin subunit protein can comprise SEQ ID NO:2 or
SEQ ID NO:5 or comprise at least 25 contiguous amino acids from or
be at least about 80% identical, at least about 85% identical, at
least about 90% identical, at least about 95% identical, at least
about 97% identical, at least about 99% to a sequence selected from
SEQ ID NO:2 and SEQ ID NO:5 and the fusion protein can be capable
of self-assembling into nanoparticles. Additionally, the monomeric
subunit can comprise a region corresponding to amino acids 5-167 of
SEQ ID NO:2.
[0019] In this embodiment, the hemagglutinin protein can comprise
at least 25 amino acids from an influenza virus selected from A/New
Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA,
H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968
HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005
(2005 Indo, H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009
(2009 Per, H3), A/Brisbane/59/2007 (2007 Bris, H1), and
B/Brisbane/60/2008 (2008 Bris, B). Alternatively, the hemagglutinin
protein can be capable of eliciting an immune response to a protein
comprising an amino acid sequence selected from SEQ ID NO:8, SEQ ID
NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ
ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID
NO:38 or one that is at least about 80% identical, at least about
85% identical, at least about 90% identical, at least about 95%
identical, at least about 97% identical, at least about 99%
thereto.
[0020] In this embodiment, the fusion protein can comprise an amino
acid sequence selected from SEQ ID NO:41, SEQ ID NO:44, SEQ ID
NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ
ID NO:62, SEQ ID NO:65, and SEQ ID NO:68 or one that is at least
about 80% identical, at least about 85% identical, at least about
90% identical, at least about 95% identical, at least about 97%
identical, at least about 99% thereto.
[0021] Further in this embodiment, the hemagglutinin protein can
comprise a region selected from a region capable of allowing
trimerization of the hemagglutinin protein, a stem region, an
ectodomain, and a region comprising the amino acid sequence from
the amino acid residue immediately distal to the last amino acid of
the second helical coiled coil to the amino acid residue proximal
to the first amino acid of the transmembrane domain. The
hemagglutinin protein alternatively can comprise a region
corresponding to amino acids 1-519 of SEQ ID NO:8, an amino acid
sequence selected from the group consisting of amino acids 1-519 of
SEQ ID NO:8 and SEQ ID NO:11, or a hemagglutinin spike domain.
Further, the hemagglutinin protein can comprise the stem region
from an influenza virus selected from A/New Calcdonia/20/1999 (1999
NC, H1), A/California/04/2009 (2009 CA, H1), A/Singapore/1/1957
(1957 Sing, H2), A/Hong Kong/1/1968 (1968 HK, H3),
A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005 (2005 Indo,
H5), B/Florida/4/2006 (2006 Flo, B), A/Perth/16/2009 (2009 Per,
H3), A/Brisbane/59/2007 (2007 Bris, H1), or B/Brisbane/60/2008
(2008 Bris, B). The hemagglutinin protein can also comprise an
amino acid sequence at least about 80% identical, at least about
85% identical, at least about 90% identical, at least about 95%
identical, at least about 97% identical, at least about 99% to SEQ
ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83,
SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID
NO:98.
[0022] In this embodiment, the fusion protein can comprise one or
more linker sequences or an amino acid sequence of selected from
the group consisting of SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107
SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID
NO:122 SEQ ID NO:125 and SEQ ID NO:128 or a sequence that is at
least about 80% identical, at least about 85% identical, at least
about 90% identical, at least about 95% identical, at least about
97% identical, at least about 99% thereto.
[0023] A further embodiment of the present invention is a nucleic
acid molecule encoding any of the fusion proteins described above.
In this embodiment, the nucleic acid molecule can be functionally
linked to a promoter. Other embodiments of the invention include
recombinant cells and viruses that comprise such nucleic acid
molecules.
[0024] Another embodiment of the invention is a protein comprising
an amino acid sequence at least 80% identical, at least about 85%
identical, at least about 90% identical, at least about 95%
identical, at least about 97% identical, at least about 99% to an
amino acid selected from SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77,
SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID
NO:92, SEQ ID NO:95, and SEQ ID NO:98, wherein the protein is
joined to one or more trimerization domains. In this embodiment,
the protein can be joined to at least a portion of the head region
of an influenza hemagglutinin protein, comprise one or more linker
regions or elicit an immune response against an influenza virus. A
further embodiment is a nucleic acid molecule encoding such a
protein.
BACKGROUND
[0025] Protective immune responses induced by vaccination against
influenza virus are primarily directed to the viral hemagglutinin
(HA) protein, which is a glycoprotein on the surface of the virus
responsible for interaction of the virus with host cell receptors.
HA proteins on the virus surface are trimers of hemagglutinin
protein monomers that are enzymatically cleaved to yield
amino-terminal HA1 and carboxy-terminal HA2 polypeptides. The
globular head consists exclusively of the major portion of the HA1
polypeptide, whereas the stem that anchors the hemagglutinin
protein into the viral lipid envelope is comprised of HA2 and part
of HA1. The globular head of a hemagglutinin protein includes two
domains: the receptor binding domain (RBD), an .about.148-amino
acid residue domain that includes the sialic acid-binding site, and
the vestigial esterase domain, a smaller .about.75-amino acid
residue region just below the RBD. The top part of the RBD adjacent
to the 2,6-sialic acid recognition sites includes a large region
(amino acids 131-143, 170-182, 205-215 and 257-262, 1918 numbering)
(referred to herein as the RBD-A region) of over 6000 A.sup.2 per
trimer that is 95% conserved between A/South Carolina/1/1918 (1918
SC) and A/California/04/2009 (2009 CA) pandemic strains. The
globular head includes several antigenic sites that include
immunodominant epitopes. Examples include the Sa, Sb, Ca.sub.1,
Ca.sub.2 and Cb antigenic sites (see, for example, Caton A J et al,
1982, Cell 31, 417-427). The RBD-A region includes the Sa antigenic
site and part of the Sb antigenic site.
[0026] Antibodies against influenza often target variable antigenic
sites in the globular head of HA, which surround a conserved sialic
acid binding site, and thus, neutralize only antigenically closely
related viruses. The variability of the HA head is due to the
constant antigenic drift of influenza viruses and is responsible
for seasonal endemics of influenza. In contrast, gene segments of
the viral genome can undergo reassortment (antigenic shift) in host
species, creating new viruses with altered antigenicity that are
capable of becoming pandemics [Salomon, R. et al. Cell 136, 402-410
(2009)]. Until now, each year, influenza vaccine is updated to
reflect the predicted HA and neuraminidase (NA) for upcoming
circulating viruses.
[0027] Current vaccine strategies for influenza use either a
chemically inactivated or a live attenuated influenza virus. Both
vaccines are generally produced in embryonated eggs which present
major manufacturing limitations due to the time consuming process
and limited production capacity. Another more critical limitation
of current vaccines is its highly strain-specific efficacy. These
challenges became glaring obvious during emergence of the 2009 H1N1
pandemic, thus validating the necessity for new vaccine platforms
capable of overcoming these limitations. Virus-like particles
represent one of such alternative approaches and are currently
being evaluated in clinical trials [Roldao, A. et al. Expert Rev
Vaccines 9, 1149-1176 (2010); Sheridan, C. Nat Biotechnol 27,
489-491 (2009)]. Instead of embryonated eggs, VLPs that often
comprise HA, NA and matrix protein 1 (M1) can be mass-produced in
mammalian or insect cell expression systems [Haynes, J. R. Expert
Rev Vaccines 8, 435-445 (2009)]. The advantages of this approach
are its particulate, multivalent nature and the authentic display
of properly folded, trimeric HA spikes that faithfully mimic the
infectious virion. In contrast, by the nature of its assembly, the
enveloped VLPs contain a small but finite host cell component that
may present potential safety, immunogenicity challenges following
repeated use of this platform [Wu, C. Y. et al. PLoS One 5, e9784
(2010)]. Moreover, the immunity induced by the VLPs is essentially
the same as current vaccines do, and thus, does not likely improve
both potency and breadth of vaccine-induced protective immunity. In
addition to VLPs, a recombinant HA protein has also been evaluated
in humans [Treanor, J. J. et al. Vaccine 19, 1732-1737 (2001);
Treanor, J. J. JAMA 297, 1577-1582 (2007)], though the ability to
induce protective neutralizing antibody titers are limited. The
recombinant HA proteins used in those trials were produced in
insect cells and might not form native trimer preferentially
[Stevens, J. Science 303, 1866-1870 (2004)].
[0028] Recently, entirely new classes of broadly neutralizing
antibodies against influenza viruses were isolated. One class of
antibodies recognizes the highly conserved HA stem [Corti, D. et
al. J Clin Invest 120, 1663-1673 (2010); Ekiert, D. C. et al.
Science 324, 246-251 (2009); Kashyap, A. K. et al. Proc Natl Acad
Sci USA 105, 5986-5991 (2008); Okuno, Y. et al. J Virol 67,
2552-2558 (1993); Sui, J. et al. Nat Struct Mol Biol 16, 265-273
(2009); Ekiert, D. C. et al. Science 333, 843-850 (2011); Corti, D.
et al. Science 333, 850-856 (2011)], and another class of
antibodies precisely recognizes the sialic acid binding site of the
RBD on the variable HA head [Whittle, J. R. et al. Proc Natl Acad
Sci USA 108, 14216-14221 (2011); Krause, J. C. et al. J Virol 85,
10905-10908 (2011)]. Unlike strain-specific antibodies, those
antibodies are capable of neutralizing multiple antigenically
distinct viruses, and hence inducing such antibodies has been a
focus of next generation universal vaccine [Nabel, G. J. et al. Nat
Med 16, 1389-1391 (2010)]. However, robustly eliciting these
antibodies with such heterologous neutralizing profile by
vaccination has been difficult [Steel, J. et al. MBio 1, e0018
(2010); Wang, T. T. et al. PLoS Pathog 6, e1000796 (2010); Wei, C.
J. et al. Science 329, 1060-1064 (2010)].
[0029] Despite several alternatives to conventional influenza
vaccines, advances in biotechnology in past decades have allowed
engineering of biological materials to be exploited for the
generation of novel vaccine platforms. Ferritin, an iron storage
protein found in almost all living organisms, is an example which
has been extensively studied and engineered for a number of
potential biochemical/biomedical purposes [Iwahori, K. U.S. Patent
2009/0233377 (2009); Meldrum, F. C. et al. Science 257, 522-523
(1992); Naitou, M. et al. U.S. Patent 2011/0038025 (2011);
Yamashita, I. Biochim Biophys Acta 1800, 846-857 (2010)], including
a potential vaccine platform for displaying exogenous epitope
peptides [Carter, D. C. et al. U.S. Patent 2006/0251679 (2006); Li,
C. Q. et al. Industrial Biotechnol 2, 143-147 (2006)]. Its use as a
vaccine platform is particularly interesting because of its
self-assembly and multivalent presentation of antigen which induces
stronger B cell responses than monovalent form as well as induce
T-cell independent antibody responses [Bachmann, M. F. et al. Annu
Rev Immunol 15, 235-270 (1997); Dintzis, H. M. et al. Proc Natl
Acad Sci USA 73, 3671-3675 (1976)]. Further, the molecular
architecture of ferritin, which consists of 24 subunits assembling
into an octahedral cage with 432 symmetry has the potential to
display multimeric antigens on its surface.
[0030] There remains a need for an efficacious influenza vaccine
that provides robust protection against influenza virus. There
particularly remains a need for an influenza vaccine that protects
individuals from heterologous strains of influenza virus, including
evolving seasonal and pandemic influenza virus strains of the
future. The present invention meets this need by providing a novel
HA-ferritin nanoparticle (HA-ferritin np) influenza vaccine that is
easily manufactured, potent, and elicits broadly neutralizing
influenza antibodies.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1. Molecular design and construction of ferritin
particles displaying influenza virus hemagglutinin. (a) Ribbon
diagram of a subunit of H. pylori nonheme ferritin (PDB: 3bve)
(left). Amino- and carboxyl-termini are labeled as N and C,
respectively. Three ferritin subunits surrounding an octahedral
three-fold axis are shown as a ribbon diagram (middle). Residue
Asp5 is indicated. The octahedral assembly of the ferritin particle
(viewed at 8 .ANG. resolution along an octahedral three-fold axis)
and A/Solomon Islands/3/2006 (H1N1) HA trimer (PDB: 3sm5) (viewed
down from membrane proximal side) (right). The measured average
distance between the Asp5 residues in each ferritin subunit
surrounding an octahedral three-fold axis is shown as a triangle.
The same equilateral triangle (a=b=c=28 .ANG.) is also drawn on the
HA trimer (right). (b) Schematic representation of the HA-ferritin
expression vector used for protein production. (c) Chromatogram of
the size exclusion chromatography of ferritin nanoparticles (np)
and HA-np (left). Molecular weights (kDa) of calibration standards
are indicated above the curves with vertical lines. Calculated
molecular weights for ferritin nanoparticles and HA-np were 419 and
2,165 kDa, respectively, and were within 10% of the predicted
molecular weights (408 and 2,040 kDa, respectively). Particle size
distribution (radius) of purified ferritin nanoparticles and HA-np
was determined by dynamic light scattering (middle). Measured mean
diameters (d) are indicated. The polydispersity indices of purified
ferritin np and HA-np were 0.035 and 0.011, respectively. Purified
HA trimer (thrombin uncleaved), HA-np and ferritin nanoparticles
were analyzed by SDS-PAGE (right). (d) Negatively stained
transmission electron microscopy images of ferritin nanoparticles
(left) and HA-np (right). Images were originally recorded at
67,000.times. magnification. (e) Models representing octahedral
four-, three- and two-fold symmetries of HA-ferritin np (top
panels) and actual TEM image (bottom panels) are shown. Visible HA
spikes are numbered in the images.
[0032] FIG. 2. Genetic and structural comparison of ferritins. (a)
Phylogenetic tree analysis of ferritins found in RSCB PDB.
Twenty-two sequences contain 16 ferritins including Vc (Vibrio
cholerae), Ec (E. coli), Hp (H. pylori), Af (Archaeoglobus
fulgidus), Pf (Pyrococcus furiosus), Tm (Thermatoga maritime), Pm
(Pseudo-nitzschia multiseries), Tn (L) (Trichoplusia ni light
chain), Soybean (chloroplastic), Horse (L) (light chain), Human
(L), (H) and (M) (light, heavy chains and mitochondrial,
respectively), Mouse (L) (light chain), and Frog (M) and (L)
(middle and lower subunits, respectively), and 6 bacterioferritins
(B) including Mt (B) (Mycobacterium tuberculosis), Pa (B)
(Pseudomonas aeruginosa), Rs (B) (Rhodabacter sphaeroides), Bm (B)
(Brucella melitensis), Av (B) (Azobacter vinelandii), and Ec (B).
Protein sequences were aligned using Clustal W2
(www.ebi.ac.uk/Tools/msa/clustalw2) with Gonnet matrix and a
phylogenetic tree was generated with the Phylodendron program
(http://iubio.bio.indiana.edu/treeapp/treeprint-form.html) using
the neighbor joining method. (b) Comparison of surface exposed
residues between H. pylori and mouse (light chain) (left) or human
(light chain) (middle), and mouse and human (light chains) (right).
Conservation of surface exposed residues was rendered by UCSF
Chimera using a protein sequence alignment generated by Clustal W2.
Conserved and varied residues between the two ferritins are shown
as light and dark residues, respectively. PDB files 3bve (H.
pylori) (left and middle) and 1h96 (mouse light chain) (right) were
used for surface rendering.
[0033] FIG. 3. Antigenic characterization of HA-ferritin np. (a)
Binding of mAbs directed to globular head and stem of HA was
measured by ELISA. Equal amount (200 ng of HA per well) of HA
trimer (), TIV (.box-solid.), HA-ferritin ( ) or Ferr (equimolar
amount as HA-Ferr) (.largecircle.) were coated on the plates and
wells were probed with anti-head mAb (3u-u) and anti-stem mAb
CR6261. The half maximal effective concentrations (EC.sub.50) of
binding were calculated for each antibody and showed as ng
ml.sup.-1 (b) Inhibition of antibody-mediated neutralization of
1999 NC pseudotyped virus by using HA trimer, HA-Ferr or Ferr as a
competitor. Inhibition of neutralization was plotted as percent
inhibition respect to no competitor control. The anti-stem
neutralizing mAbs, F10 (left) and CR6261 (right) were used at 3.125
and 25 .mu.g ml.sup.-1, respectively. Competitor proteins were
added to the reactions at a final concentration of 20 .mu.g
ml.sup.-1.
[0034] FIG. 4. Immune responses in HA-np-immunized mice. (a) HAI
(left), IC.sub.90 neutralization (middle), and anti-HA ab endpoint
titers (right) against 1999 NC HA after two immunizations with 0.17
.mu.g (amount of H1 HA) of TIV or HA-np with or without Ribi
adjuvant and a 3-week interval. The immune sera were collected 2
weeks after the second immunization. The data are presented as
box-and-whiskers plots (boxed from lower to upper quartile with
whiskers from minimum to maximum) with lines at the mean (n=5). (b)
Neutralization breadth of the immune sera elicited by HA-trimer,
TIV, or HA-np. An additional group of mice (n=4) was immunized
twice with 20 .mu.g of trimeric HA protein using Ribi adjuvant and
a 4-week interval. The immune sera were collected 2 weeks after the
second immunization. IC.sub.50 neutralization titers against a
panel of H1N1 pseudotyped viruses were determined. (c) Cellular and
humoral immune responses against H. pylori (top) and mouse (bottom)
ferritins. Mice were immunized twice with 1.67 .mu.g (amount of H1
HA) of TIV or HA-np, or 0.57 .mu.g of ferritin nanoparticles
(equimolar to HA-np) using Ribi adjuvant and a 3-week interval. The
splenocytes and immune sera were harvested 11 days after the second
immunization. Cytokine-producing CD4.sup.+ and CD8.sup.+ T cells
were measured by ICS (left), and ab response was detected by ELISA
(right). All cells expressing IFN-.gamma., TNF.alpha., or IL-2 were
identified as cytokine.sup.+ cells. The percentage of
cytokine.sup.+ cells in CD4.sup.+ and CD8.sup.+ T cells that were
activated in response to stimulation with specific peptides
covering the entire H. pylori or mouse ferritins (heavy and light
chains combined) were plotted. Recombinant H. pylori and purified
mouse (liver) ferritins were used for detecting anti-ferritin ab
responses. The data are presented as box-and-whiskers plots with
lines at the mean (n=5).
[0035] FIG. 5. Successive immunization of HA-nanoparticles in mice.
Mice were pre-immunized with 1.67 .mu.g (amount of HA) of 2009
Perth (H3) HA-nanoparticles or 0.57 .mu.g (equimolar to
HA-nanoparticle) of empty ferritin nanoparticles at week 0 and then
immunized with 1.67 .mu.g (amount of HA) of 1999 NC (H1)
HA-nanoparticles at week 3. Ribi was used as an adjuvant. Another
group of mice was immunized with 1999 NC (H1) HA-nanoparticles
without pre-immunization of empty ferritin nanoparticles or H3
HA-nanoparticles. (a) Ab responses to H. pylori ferritin (left) and
2009 Perth H3 HA (right). Immune sera collected 2 weeks after the
immunization with H3 HA-nanoparticles or empty ferritin
nanoparticles were analyzed by ELISA. (b) Immune responses to 1999
NC (H1) after 1999 NC (H1) HA-nanoparticle immunization. Naive mice
or mice with pre-immunity to ferritin or H3 HA were immunized with
H1 HA-nanoparticles at week 3 and HAI (left), IC.sub.90
neutralization (middle) and ELISA (right) Ab titers were measured 2
weeks after the immunization. The data are presented as
box-and-whiskers plots with lines at the mean (n=5).
[0036] FIG. 6. Development of trivalent HA-np. (a) HA-np consisting
of HAs from 2009 CA (H1), 2009 Perth (H3) or 2006 FL (type B) were
purified and visualized by TEM. (b) HAI titers against 2009 CA
(H1N1) and 2009 Perth (H3N2) viruses in immunized mice. Mice were
immunized twice with 1.67 .mu.g (amount of HA) of monovalent H1,
monovalent H3, monovalent type B, or 5.0 .mu.g (total amount of HA)
of trivalent HA-np or TIV (2011-2012 season) using Ribi adjuvant
with a 3-week interval. Immune sera were collected 2 weeks after
the second immunization. The data are presented as box-and-whiskers
plots with lines at the mean (n=5).
[0037] FIG. 7. Protective immunity induced in ferrets immunized
with the HA-np. Ferrets were immunized twice with PBS (control),
7.5 ug (2.5 ug of H1 HA) of TIV or 2.5 ug (amount of HA) 1999 NC
HA-np using Ribi adjuvant and a 4-week interval. Control animals
received PBS. (a) HA1 (left), IC90 neutralization (middle), and
anti-HA ab endpoint titers (right) in immunized ferrets against
homologous 1999 NC HA were determined. Immune sera were collected 3
and 2 weeks after the first (R. Salomon, R. G. Webster, The
influenza virus enigma. Cell 136, 402-410 (2009) and second (L. C.
Lambert, A. S. Fauci, Influenza vaccines for the future. N Engl J
Med 363, 2036-2044 (2010)) immunizations, respectively. The data
are presented as box and whisker plots with lines at the mean
(n=6). (b) Protection of immunized ferrets from an unmatched 2007
Bris virus challenge. Ferrets were challenged with 10.sup.6.5 50%
egg infectious dose (EID50) of 2007 Bris virus 5 weeks after the
second immunization. Virus titers in the nasal washes from 1, 3 and
5 days post challenge were determined by a 50% tissue culture
infectious dose (TCID.sub.50) assay (left). One of six ferrets in
the TIV-immunized group showed measurable virus on day 5. Virus
titers in 4 out of 6 ferrets on day 3 and 6 out of 6 ferrets on day
5 in the HA-np-immunized group were under the detection limit
(<102). The mean viral loads with standard deviation (s.d.) at
each time point were plotted (n=6). Change in the body weight after
the virus challenge was also monitored (right). Each data point
represents the mean percent change in body weight from the
pre-challenge (day 0). The mean body weight changes with standard
error (s.e.) at each time point were plotted (n=6).
[0038] FIG. 8. Improved neutralization breadth and detection of
stem- and RBS-directed abs. (a) Neutralization breadth of immune
sera in ferrets. IC.sub.50 neutralization titers against a panel of
H1N1 pseudotyped viruses (left) and HAI titers against 1934 PR8 and
2007 Bris H1N1 viruses (right) were determined. The HAI titers are
presented as box-and-whiskers plots with lines at the mean (n=6).
(b) Stem- and RBS-directed abs elicited by HA-np immunization.
Ferret immune sera (diluted 1:100) were pre-absorbed with
.DELTA.Stem HA-expressing cells and their binding to WT or
.DELTA.Stem HA were analyzed by ELISA (left). The immune sera
(diluted 1:1,000) were pre-absorbed with .DELTA.RBS HA-expressing
cells and their binding to WT or .DELTA.RBS HA were analyzed by
ELISA (middle). The mean endpoint dilution titers were plotted with
s.d. (n=6). Competition ELISA with stem-directed mAb CR6261
(right). The immune sera pre-absorbed with .DELTA.Stem were tested
for binding to HA in the presence of an isotype control IgG or
CR6261. Each symbol represents the titer of an individual ferret
(n=6). (c) Neutralization competition with WT, .DELTA.Stem or
.DELTA.RBS HA protein (left). The neutralization of HA-np immune
sera against 1986 Sing, 1995 Beijing, 1999 NC and 2007 Bris was
measured in the presence of irrelevant protein (control), WT,
.DELTA.Stem or .DELTA.RBS HA as a competitor. Percent
neutralizations at serum dilution 1:200 (1986 Sing, 2007 Bris),
1:800 (1995 Beijing) or 1:3,200 (1999 NC) were plotted. Each symbol
represents the individual ferret serum and mean is indicated as a
red line with s.d. (n=6 for 1986 Sing, 1995 Beijing and 1999 NC;
n=3 for 2007 Bris). The relative contribution of the stem- and
RBS-directed neutralization was determined by the inhibition of
neutralization for each competitor protein (right). Mean percent
contributions in neutralizing each virus were plotted as pile-up
bars (n=6).
[0039] FIG. 9. Characterization of .DELTA.RBS HA probe. (a) Crystal
structure of HA (A/Solomon Islands/3/2006) complex with an anti-RBS
mAb CH65 Fab (PDB: 3sm5) (J. R. Whittle et al., Broadly
neutralizing human antibody that recognizes the receptor-binding
pocket of influenza virus hemagglutinin. Proc Natl Acad Sci USA
108, 14216-14221 (2011)) (left). Close up view of CH65 contact area
(right). The residue HA1 190 which has been mutated to be
glycosylated in .DELTA.RBS mutant is highlighted. The CH65
Fab-bound HA1 protomer is darkened. (b) Characterization of the
soluble trimer of WT and .DELTA.RBS HAs from 1999 NC and 2007 Bris.
The WT and .DELTA.RBS HA proteins were immunoprecipitated with
anti-RBS (CH65), stem (CR6261) and control (anti-HIV, VRC01) mAbs.
Immune complexes were then dissolved in Lamini buffer and analyzed
by SDS-PAGE. Antibody heavy and light chains are labeled as HC and
LC, respectively.
[0040] FIG. 10. Purification of HA-np. HA-np were purified by
routine iodixanol gradient ultracentrifugation routinely. Fractions
containing HA-np were confirmed by SDS-PAGE and Western blotting
using a mAb against 1999 NC HA. The HA-np were enriched in the
fraction with density of .about.1.15 g/ml.
[0041] FIG. 11. Protocol for immunization of mice and ferrets using
pan-group 1 HA-ferritin np. Mice were injected intramuscularly
twice (Week 0 and week 4) with PBS (control) or 6.8 ug (1.7 ug of
each HA-ferritin np) pan-group 1 vaccine in Ribi. Ferrets were
injected intramuscularly twice (Week 0 and week 4) with PBS
(control) or 10 ug (2.5 ug of each HA-ferritin np) pan-group 1
vaccine in Ribi.
[0042] FIG. 12. Neutralization activity of mouse antisera against
Group1 HA pseudotyped viruses. Neutralization activity of murine
antisera from control or pan Group1 HA-np immunized mice against
the indicated HA pseudotyped viruses. IC50 titers are shown for all
panels.
[0043] FIG. 13. Neutralization activity of ferret antisera against
Group1 HA pseudotyped viruses. Neutralization activity of ferret
antisera from control or pan Group1 HA np immunized ferrets against
the indicated HA pseudotyped viruses. IC50 titers are shown for all
panels.
[0044] FIG. 14. H1 hemaggulutination inhbitions (HAI) assays were
performed using the sera obtained from the ferritin immunization
studies. These studies were performed using actual H1 virus, and H2
and H5 HAI were performed using HA-ferritin np.
[0045] FIG. 15. Protection of ferrets from viral challenge with
Influenza A/Brisbane/59/2007 Brisbane (H1N1) (2007 Bris). Two
groups of ferrets (n=6 for control and n=5 for pan-group1 immune)
were immunized with pan Group1 HA np vaccine or PBS (control) and
challenged with heterologous 2007 Bris virus (10.sup.6.5
EID.sub.50). Virus titers were measured in nasal swabs collected on
day 3 and day 5 post challenge. Titers were determined using
end-point titration in MDCK cells.
[0046] FIG. 16. Protection of ferrets from viral challenge with
Influenza A/Mexico/2009 (H1N1) (2009 Mex). Two groups of ferrets
(n=6) were immunized with pan Group1 HA np vaccine or PBS (control)
and challenged with heterologous 2009 Mex virus (10.sup.6.5
EID.sub.50). Virus titers were measured in nasal swabs collected on
day 3 and day 5 post challenge. Titers were determined using
end-point titration in MDCK cells.
[0047] FIG. 17. Conservation of the influenza HA stem region.
(left, right) Neutralizing antibodies that react with both Group 1
and Group 2 viruses act at the sites of vulnerability shown in the
Figure. (Right) Space filling model of influenza HA protein
illustrating amino acid sequence conservation in over 800 human
H1N1 strains. Light residues indicate residues that are 100%
conserved. Dark residues as indicate sites of variation.
[0048] FIG. 18. Design of HA Stabilized Stem protein. (A) Schematic
of the HA SS (bottom) in comparison to HA (top). HA SS was
constructed by inserting a GWG linker between residues 42 and 314
of HA1 RBD head, a gp41 post-fusion trimerization motif inserted in
place of residues 59 through 93 of HA2, a GG linker between HA2 and
the gp41 HR2 helix and an NGTGGGSG linker between the two gp41
helices. The gene sequence of H1 NC 99 SS is provided in the
supplemental materials. (B) Trimeric and monomeric representation
of HA (PDB entry 1RU7) in comparison to the HA SS model. Coloring
is respective to above panel, with the monomeric representation
also illustrating the CR6261 epitope as yellow and HA residues
which are omitted in the stabilized HA stem as grey. (C) CR6261,
FI6v3, and the germline of the VH1-69 Ab 70-5B03 have similar
affinity to HA and SS by ELISA. HA SS competes with CR6261 (D)
binding to HA and (E) neutralization of H1N1 pseudovirus similar to
soluble HA trimer.
[0049] FIG. 19. Size exclusion chromatogram of HA and HA SS probes.
Calibration standards are shown above the curves as vertical
lines.
[0050] FIG. 20. Electron microscopic analysis of nanoparticles.
Purified SS-np were subjected to transmission electron microscopic
analysis. The samples were negatively stained with ammonium
molybdate and images were recorded on a Tecnai T12 microscope (FEI)
at 80 kV with a CCD camera (AMT Corp.). Images of lower (left) and
higher (right) magnifications are shown. The SS spikes were
protruding perpendicularly from the particle core and clearly
visible.
[0051] FIG. 21. Antigenic characterization of HA SS-ferritin np.
The ability of purified HA SS and HA SS-np to bind to monoclonal
Abs CR6261 (left) and FI6v3 (right) was characterized by ELISA. HA
and HIV gp120 proteins served as controls.
[0052] FIG. 22. Immune sera of mice immunized heterologously with
HA-np prime and HA SS-np boost are reactive to the conserved HA
stem epitope. Antibodies elicited by vaccination target the
conserved HA stem epitope as individual mice possess differential
binding (a minimum of 2-fold difference in endpoint dilution)
between wt and .DELTA.stem HA variants. The percentage of mice
displaying differential binding is given above matched wt and
.DELTA.stem constructs. Error bars represent standard error.
[0053] FIG. 23. Immune sera of mice immunized with HA SS
neutralizes diverse pseudovirus stains. IC.sub.50 values are shown
for individual mice against H1 homosubtypic strains and H2, H5 and
H9 group-1 heterosubtypic strains. Dashed lines represents the
lowest dilution assayed (50). Error bars represent standard
error.
[0054] FIG. 24. Boosting with HA SS-np increases neutralizing
titers in ferrets against H1N1 New Calendonia. Pseudovirus
neutralizing titers were calculated for preimmune, HA FL-np primed,
and HA SS-np boosted sera from individual mice. Error bars
represent standard deviation of values.
[0055] FIG. 25. Map and sequence of CMV8x/R-H1NC HA(517)_SGG-egm
and the related HA-ferritin fusion protein.
[0056] FIG. 26. Map and sequence of CMV8x/R-H1CA HA(518)_SGG-egm
and the related HA-ferritin fusion protein.
[0057] FIG. 27. Map and sequence of CMV8x/R-H2Sing HA(514)_SGG-egm
and the related HA-ferritin fusion protein.
[0058] FIG. 28. Map and sequence of CMV8x/R-H3HK HA(519)_SGG-egm
and the related HA-ferritin fusion protein.
[0059] FIG. 29. Map and sequence of CMV8x/R-H3Bris HA(519)_SGG-egm
and the related HA-ferritin fusion protein.
[0060] FIG. 30. Map and sequence of CMV8x/R-H5Indo HA(520)_SGG-egm
and the related HA-ferritin fusion protein.
[0061] FIG. 31. Map and sequence of CMV8x/R-B.Florida
HA(534)_SGG-egm and the related HA-ferritin fusion protein.
[0062] FIG. 32. Map and sequence of CMV8x/R-H3Perth HA(519)_SGG-egm
and the related HA-ferritin fusion protein.
[0063] FIG. 33. Map and sequence of CMV8x/R-H1Bris HA(517)_SGG-egm
and the related HA-ferritin fusion protein.
[0064] FIG. 34. Map and sequence of CMV8x/R-B.Bris HA(535)_SGG-egm
and the related HA-ferritin fusion protein.
[0065] FIG. 35. Map and sequence of CMV8x/R--H1NC SS
Gen4.55_SGG-egm and the related HA SS-ferritin fusion protein.
[0066] FIG. 36. Map and sequence of CMV/R H1 CA
SS/Gen4.55/ferritin.
[0067] FIG. 37. Map and sequence of CMV/R H1 Bris
SS/Gen4.55/ferritin.
[0068] FIG. 38. Map and sequence of CMV/R H2 Sing
SS/Gen4.55/ferritin.
[0069] FIG. 39. Map and sequence of CMV/R H3 Bris
SS/Gen4.55/ferritin.
[0070] FIG. 40. Map and sequence of CMV/R H3 Perth
SS/Gen4.55/ferritin.
[0071] FIG. 41. Map and sequence of CMV/R H3 HK68
SS/Gen4.55/ferritin.
[0072] FIG. 42. Map and sequence of CMV/R H5 Indo
SS/Gen4.55/ferritin.
[0073] FIG. 43. Map and sequence of CMV/R B Bris
SS/Gen4.55/ferritin.
[0074] FIG. 44. Map and sequence of CMV/R B FL
SS/Gen4.55/ferritin.
[0075] FIG. 45. Illustration showing hemagglutination reaction.
[0076] FIG. 46. Comparison of the hemagglutination activities of
HA-ferritin nanoparticle and H5N1 vaccine. Inactivated vaccine (A)
and HA-ferritin nanoparticles (B) were serially diluted in PBS and
incubated with 0.5% chicken red blood cells (RBCs) for 30 minutes
at room temperature. A series of dilutions were also performed
using PBS as a control (C). Hemagglutination mediated through
sialic acid moiety on RBC surface and the sialic acid-binding site
on HA was determined by formation of lattice-structure of multiple
RBCs (lack of red dot in the well).
[0077] FIG. 47. Mutation in sialic acid-binding site (.DELTA.SA;
Y98F) diminishes hemagglutination of HA-ferritin nanoparticles.
Hemagglutination activity of HA-ferritin nanoparticles was
diminished by a mutation in the sialic acid-binding site on HA.
[0078] FIG. 48. Titration of antibodies inhibiting hemagglutination
using two assay systems (traditional HAI assay and
nanoparticle-based HAI assay). Sera from twenty-four ferrets were
pretreated with receptor-destroying enzyme (RDE) and heat
inactivated were titrated by using A/New Calcdonia/20/99 (H1N1)
virus or HA-np having the same HA. The samples include 6 negative
(pre-immune) and 24 test (post-immune) sera. The titers are shown
as scattered dots (left) and comparative (middle) plots.
Correlation between the two assays was assessed (right).
DETAILED DESCRIPTION OF THE INVENTION
[0079] The present invention relates to a novel vaccine for
influenza virus. More specifically, the present invention relates
to novel, influenza hemagglutinin protein-based vaccines that
elicit an immune response against a broad range of influenza
viruses. It also relates to self-assembling ferritin-based,
nanoparticles that display immunogenic portions of influenza
hemagglutinin protein on their surface. Such nanoparticles are
useful for vaccinating individuals against influenza virus.
Accordingly, the present invention also relates to fusion proteins
for producing such nanoparticles and nucleic acid molecules
encoding such proteins. Additionally, the present invention relates
to, methods of producing nanoparticles of the present invention,
and methods of using such nanoparticles to vaccinate
individuals.
[0080] Before the present invention is further described, it is to
be understood that this invention is not limited to particular
embodiments described, as such may, of course, vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the claims.
[0081] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise. For
example, a nucleic acid molecule refers to one or more nucleic acid
molecules. As such, the terms "a", "an", "one or more" and "at
least one" can be used interchangeably. Similarly the terms
"comprising", "including" and "having" can be used interchangeably.
It is further noted that the claims may be drafted to exclude any
optional element. As such, this statement is intended to serve as
antecedent basis for use of such exclusive terminology as "solely,"
"only" and the like in connection with the recitation of claim
elements, or use of a "negative" limitation.
[0082] In addition to the above, unless specifically defined
otherwise, the following terms and phrases, which are common to the
various embodiments disclosed herein, are defined as follows:
[0083] As used herein, the term immunogenic refers to the ability
of a specific protein, or a specific region thereof, to elicit an
immune response to the specific protein, or to proteins comprising
an amino acid sequence having a high degree of identity with the
specific protein. According to the present invention, two proteins
having a high degree of identity have amino acid sequences at least
80% identical, at least 85% identical, at least 87% identical, at
least 90% identical, at least 92% identical, at least 94%
identical, at least 96% identical, at least 98% identical or at
least 99% identical.
[0084] As used herein, an immune response to a vaccine, or
nanoparticle, of the present invention is the development in a
subject of a humoral and/or a cellular immune response to a
hemagglutinin protein present in the vaccine. For purposes of the
present invention, a "humoral immune response" refers to an immune
response mediated by antibody molecules, including secretory (IgA)
or IgG molecules, while a "cellular immune response" is one
mediated by T-lymphocytes and/or other white blood cells. One
important aspect of cellular immunity involves an antigen-specific
response by cytolytic T-cells ("CTL"s). CTLs have specificity for
peptide antigens that are presented in association with proteins
encoded by the major histocompatibility complex (MHC) and expressed
on the surfaces of cells. CTLs help induce and promote the
destruction of intracellular microbes, or the lysis of cells
infected with such microbes. Another aspect of cellular immunity
involves an antigen-specific response by helper T-cells. Helper
T-cells act to help stimulate the function, and focus the activity
of, nonspecific effector cells against cells displaying peptide
antigens in association with MHC molecules on their surface. A
cellular immune response also refers to the production of
cytokines, chemokines and other such molecules produced by
activated T-cells and/or other white blood cells, including those
derived from CD4+ and CD8+ T-cells.
[0085] Thus, an immunological response may be one that stimulates
CTLs, and/or the production or activation of helper T-cells. The
production of chemokines and/or cytokines may also be stimulated.
The vaccine may also elicit an antibody-mediated immune response.
Hence, an immunological response may include one or more of the
following effects: the production of antibodies (e.g., IgA or IgG)
by B-cells; and/or the activation of suppressor, cytotoxic, or
helper T-cells and/or T-cells directed specifically to a
hemagglutinin protein present in the vaccine. These responses may
serve to neutralize infectivity, and/or mediate
antibody-complement, or antibody dependent cell cytotoxicity (ADCC)
to provide protection to an immunized individual. Such responses
can be determined using standard immunoassays and neutralization
assays, well known in the art.
[0086] According to the present invention all nomenclature used to
classify influenza virus is that commonly used by those skilled in
the art. Thus, a Type, or Group, of influenza virus refers to
influenza Type A, influenza Type B or influenza type C. It is
understood by those skilled in the art that the designation of a
virus as a specific Type relates to sequence difference in the
respective M1 (matrix) protein or NP (nucleoprotein). Type A
influenza viruses are further divided into Group1 and Group 2.
These Groups are further divided into subtypes, which refers to
classification of a virus based on the sequence of its HA protein.
Examples of current commonly recognized subtypes are H1, H2, H3,
H4, H5, H6, H7, H8, H8, H10, H11, H12, H13, H14, H15 or H16. Group
1 influenza subtypes are H1, H2, H5, H7 and H9. Group 2 influenza
subtypes are H4, H4, H6, H8, H10, H11, H12, H13, H14, H15 and H16.
Finally, the term strain refers to viruses within a subtype that
differ from one another in that they have small, genetic variations
in their genome.
[0087] As used herein, neutralizing antibodies are antibodies that
prevent influenza virus from completing one round of replication.
As defined herein, one round of replication refers the life cycle
of the virus, starting with attachment of the virus to a host cell
and ending with budding of newly formed virus from the host cell.
This life cycle includes, but is not limited to, the steps of
attaching to a cell, entering a cell, cleavage and rearrangement of
the HA protein, fusion of the viral membrane with the endosomal
membrane, release of viral ribonucleoproteins into the cytoplasm,
formation of new viral particles and budding of viral particles
from the host cell membrane.
[0088] As used herein, broadly neutralizing antibodies are
antibodies that neutralize more than one type, subtype and/or
strain of influenza virus. For example, broadly neutralizing
antibodies elicited against an HA protein from a Type A influenza
virus may neutralize a Type B or Type C virus. As a further
example, broadly neutralizing antibodies elicited against an HA
protein from Group I influenza virus may neutralize a Group 2
.mu.m. AS an additional example, broadly neutralizing antibodies
elicited against an HA protein from one sub-type or strain of
virus, may neutralize another sub-type or strain of virus. For
example, broadly neutralizing antibodies elicited against an HA
protein from an H1 influenza virus may neutralize viruses from one
or more sub-types selected from the group consisting of H2, H3, H4,
H5, H6, H7, H8, H8, H10, H11, H12, H13, H14, H15 or H16.
[0089] As used herein, an influenza hemagglutinin protein, or HA
protein, refers to a full-length influenza hemagglutinin protein or
any portion thereof, that is capable of eliciting an immune
response. Preferred HA proteins are those that are capable of
forming a trimer. An epitope of a full-length influenza
hemagglutinin protein refers to a portion of such protein that can
elicit a neutralizing antibody response against the homologous
influenza strain, i.e., a strain from which the HA is derived. In
some embodiments, such an epitope can also elicit a neutralizing
antibody response against a heterologous influenza strain, i.e., a
strain having an HA that is not identical to that of the HA of the
immunogen.
[0090] With regard to hemagglutinin proteins, it is understood by
those skilled in the art that hemagglutinin proteins from different
influenza viruses may have different lengths due to mutations
(insertions, deletions) in the protein. Thus, reference to a
corresponding region refers to a region of another proteins that is
identical, or nearly so (e.g., at least 95%, identical, at least
98% identical or at least 99% identical), in sequence, structure
and/or function to the region being compared. For example, with
regard to the stem region of a hemagglutinin protein, the
corresponding region in another hemagglutinin protein may not have
the same residue numbers, but will have a nearly identical sequence
and will perform the same function. To better clarify sequences
comparisons between viruses, numbering systems are used by those in
the field, which relate amino acid positions to a reference
sequence. Thus, corresponding amino acid residues in hemagglutinin
proteins from different strains of influenza may not have the same
residue number with respect to their distance from the n-terminal
amino acid of the protein. For example, using the H3 numbering
system, reference to residue 100 in A/New Calcdonia/20/1999 (1999
NC, H1) does not mean it is the 100.sup.th residue from the
N-terminal amino acid. Instead, residue 100 of A/New
Calcdonia/20/1999 (1999 NC, H1) aligns with residue 100 of
influenza H3N2 strain. The use of such numbering systems is
understood by those skilled in the art. Unless otherwise noted,
reference to amino acids in hemagglutinin proteins herein is made
using the H3 numbering system.
[0091] According to the present invention, a trimerization domain
is a series of amino acids that when joined (also referred to as
fused) to a protein or peptide, allow the fusion protein to
interact with other fusion proteins containing the trimerization
domain, such that a trimeric structure is formed. Any known
trimerization domain can be used in the present invention. Examples
of trimerization domains include, but are not limited to, the HIV-1
gp41 trimerization domain, the SIV gp41 trimerization domain, the
Ebola virus gp-2 trimerization domain, the HTLV-1 gp-21
trimerization domain, the T4 fibritin trimerization domain (i.e.,
foldon), the yeast heat shock transcription factor trimerization
domain, and the human collagen trimerization domain.
[0092] As used herein, a variant refers to a protein, or nucleic
acid molecule, the sequence of which is similar, but not identical
to, a reference sequence, wherein the activity of the variant
protein (or the protein encoded by the variant nucleic acid
molecule) is not significantly altered. These variations in
sequence can be naturally occurring variations or they can be
engineered through the use of genetic engineering technique know to
those skilled in the art. Examples of such techniques are found in
Sambrook J, Fritsch E F, Maniatis T et al., in Molecular Cloning--A
Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory
Press, 1989, pp. 9.31-9.57), or in Current Protocols in Molecular
Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, both of
which are incorporated herein by reference in their entirety.
[0093] With regard to variants, any type of alteration in the amino
acid, or nucleic acid, sequence is permissible so long as the
resulting variant protein retains the ability to elicit
neutralizing antibodies against an influenza virus. Examples of
such variations include, but are not limited to, deletions,
insertions, substitutions and combinations thereof. For example,
with regard to proteins, it is well understood by those skilled in
the art that one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10),
amino acids can often be removed from the amino and/or carboxy
terminal ends of a protein without significantly affecting the
activity of that protein. Similarly, one or more (e.g., 2, 3, 4, 5,
6, 7, 8, 9 or 10) amino acids can often be inserted into a protein
without significantly affecting the activity of the protein.
[0094] As noted, variant proteins of the present invention can
contain amino acid substitutions relative to the influenza HA
proteins disclosed herein. Any amino acid substitution is
permissible so long as the activity of the protein is not
significantly affected. In this regard, it is appreciated in the
art that amino acids can be classified into groups based on their
physical properties. Examples of such groups include, but are not
limited to, charged amino acids, uncharged amino acids, polar
uncharged amino acids, and hydrophobic amino acids. Preferred
variants that contain substitutions are those in which an amino
acid is substituted with an amino acid from the same group. Such
substitutions are referred to as conservative substitutions.
[0095] Naturally occurring residues may be divided into classes
based on common side chain properties:
[0096] 1) hydrophobic: Met, Ala, Val, Leu, Ile;
[0097] 2) neutral hydrophilic: Cys, Ser, Thr;
[0098] 3) acidic: Asp, Glu;
[0099] 4) basic: Asn, Gln, His, Lys, Arg;
[0100] 5) residues that influence chain orientation: Gly, Pro;
and
[0101] 6) aromatic: Trp, Tyr, Phe.
[0102] For example, non-conservative substitutions may involve the
exchange of a member of one of these classes for a member from
another class.
[0103] In making amino acid changes, the hydropathic index of amino
acids may be considered. Each amino acid has been assigned a
hydropathic index on the basis of its hydrophobicity and charge
characteristics. The hydropathic indices are: isoleucine (+4.5);
valine (+4.2); leucine (+3.8); phenylalanine (+2.8);
cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine
(-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9);
tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate
(-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5);
lysine (-3.9); and arginine (-4.5). The importance of the
hydropathic amino acid index in conferring interactive biological
function on a protein is generally understood in the art (Kyte et
al., 1982, J. Mol. Biol. 157:105-31). It is known that certain
amino acids may be substituted for other amino acids having a
similar hydropathic index or score and still retain a similar
biological activity. In making changes based upon the hydropathic
index, the substitution of amino acids whose hydropathic indices
are within .+-.2 is preferred, those within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly
preferred.
[0104] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity, particularly where the biologically functionally
equivalent protein or peptide thereby created is intended for use
in immunological invention, as in the present case. The greatest
local average hydrophilicity of a protein, as governed by the
hydrophilicity of its adjacent amino acids, correlates with its
immunogenicity and antigenicity, i.e., with a biological property
of the protein. The following hydrophilicity values have been
assigned to these amino acid residues: arginine (+3.0); lysine
(+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine
(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine
(-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5);
cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and
tryptophan (-3.4). In making changes based upon similar
hydrophilicity values, the substitution of amino acids whose
hydrophilicity values are within .+-.2 is preferred, those within
.+-.1 are particularly preferred, and those within .+-.0.5 are even
more particularly preferred. One may also identify epitopes from
primary amino acid sequences on the basis of hydrophilicity.
[0105] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. For example, amino acid
substitutions can be used to identify important residues of the HA
protein, or to increase or decrease the immunogenicity, solubility
or stability of the HA proteins described herein. Exemplary amino
acid substitutions are shown below in Table 1.
TABLE-US-00001 TABLE 1 Amino Acid Substitutions Original Amino Acid
Exemplary Substitutions Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln
Asp Glu Cys Ser, Ala Gln Asn Glu Asp Gly Pro, Ala His Asn, Gln,
Lys, Arg Ile Leu, Val, Met, Ala Leu Ile, Val, Met, Ala Lys Arg,
Gln, Asn Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro Ala Ser
Thr, Ala, Cys Thr Ser Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser Val Ile,
Met, Leu, Phe, Ala
[0106] As used herein, the phrase significantly affect a proteins
activity refers to a decrease in the activity of a protein by at
least 10%, at least 20%, at least 30%, at least 40% or at least
50%. With regard to the present invention, such an activity may be
measured, for example, as the ability of a protein to elicit
neutralizing antibodies against an influenza virus. Such activity
may be measured by measuring the titer of such antibodies against
influenza virus, or by measuring the number of types, subtypes or
strains neutralized by the elicited antibodies. Methods of
determining antibody titers and methods of performing virus
neutralization assays are known to those skilled in the art. In
addition to the activities described above, other activities that
may be measured include the ability to agglutinate red blood cells
and the binding affinity of the protein for a cell. Methods of
measuring such activities are known to those skilled in the
art.
[0107] As used herein, a fusion protein is a recombinant protein
containing amino acid sequence from at least two unrelated proteins
that have been joined together, via a peptide bond, to make a
single protein. The unrelated amino acid sequences can be joined
directly to each other or they can be joined using a linker
sequence. As used herein, proteins are unrelated, if their amino
acid sequences are not normally found joined together via a peptide
bond in their natural environment(s) (e.g., inside a cell). For
example, the amino acid sequences of monomeric subunits that make
up ferritin, and the amino acid sequences of influenza
hemagglutinin proteins are not normally found joined together via a
peptide bond.
[0108] The terms individual, subject, and patient are
well-recognized in the art, and are herein used interchangeably to
refer to any human or other animal susceptible to influenza
infection. Examples include, but are not limited to, humans and
other primates, including non-human primates such as chimpanzees
and other apes and monkey species; farm animals such as cattle,
sheep, pigs, seals, goats and horses; domestic mammals such as dogs
and cats; laboratory animals including rodents such as mice, rats
and guinea pigs; birds, including domestic, wild and game birds
such as chickens, turkeys and other gallinaceous birds, ducks,
geese, and the like. The terms individual, subject, and patient by
themselves, do not denote a particular age, sex, race, and the
like. Thus, individuals of any age, whether male or female, are
intended to be covered by the present disclosure and include, but
are not limited to the elderly, adults, children, babies, infants,
and toddlers. Likewise, the methods of the present invention can be
applied to any race, including, for example, Caucasian (white),
African-American (black), Native American, Native Hawaiian,
Hispanic, Latino, Asian, and European. An infected subject is a
subject that is known to have influenza virus in their body.
[0109] As used herein, a vaccinated subject is a subject that has
been administered a vaccine that is intended to provide a
protective effect against an influenza virus.
[0110] As used herein, the terms exposed, exposure, and the like,
indicate the subject has come in contact with a person of animal
that is known to be infected with an influenza virus.
[0111] The publications discussed herein are provided solely for
their disclosure prior to the filing date of the present
application. Nothing herein is to be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention. Further, the dates of publication
provided may be different from the actual publication dates, which
may need to be independently confirmed.
[0112] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications mentioned herein are incorporated herein by
reference to disclose and describe the methods and/or materials in
connection with which the publications are cited.
[0113] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable sub-combination.
All combinations of the embodiments are specifically embraced by
the present invention and are disclosed herein just as if each and
every combination was individually and explicitly disclosed. In
addition, all sub-combinations are also specifically embraced by
the present invention and are disclosed herein just as if each and
every such sub-combination was individually and explicitly
disclosed herein.
[0114] According to the present invention, vaccines are provided
that elicit a broad immune response against influenza viruses. Some
vaccines disclosed herein may elicit an immune response against the
entire HA protein, while others may elicit an immune response
against a specific region or portion of an influenza HA protein.
Moreover, the inventors have discovered that specific fusion
proteins comprising portions of hemagglutinin protein are useful
for eliciting a broad immune response against influenza viruses.
Each of these embodiments will now be disclosed in detail
below.
Vaccines Against the Stem Region of Influenza HA Protein
[0115] As stated previously, the amino acid sequence of the stem
region of the hemagglutinin protein is highly conserved across
types, sub-types and strains of influenza viruses and contains a
site of vulnerability for group 1 viruses. Thus, an immune response
directed this region of the HA protein may protect individuals
against influenza viruses from several types, sub-types and/or
strains.
[0116] Consequently, one embodiment of the present invention is a
protein that elicits an immune response against the stem region of
an influenza HA protein. In one embodiment, the immune response can
be directed against the stem region of an HA protein from a virus
selected from the group consisting of influenza A viruses,
influenza B viruses and influenza C viruses. In one embodiment, the
immune response can be directed against the stem region of an HA
protein from a virus selected from the group consisting of an H1
influenza virus, an H2 influenza virus, an influenza H3 virus, an
influenza H4 virus, an influenza H5 virus, an influenza H6 virus,
an H7 influenza virus, an H8 influenza virus, an H9 influenza
virus, an H10 influenza virus, an H11 influenza virus, an H12
influenza virus, an H13 influenza virus, an H14 influenza virus, an
H15 influenza virus and an H16 influenza virus. In one embodiment,
the immune response can be directed against the stem region of an
HA protein from a strain of virus selected from the group of
viruses listed in Table 2.
TABLE-US-00002 TABLE 2 SEQ ID NO Comments FERRITIN 1 Coding
sequence for ferritin monomeric subunit protein from H. pylori 2
Amino acid sequence encoded by SEQ ID NO: 1 3 Complement of SEQ ID
NO1 4 Nucleic acid sequence encoding amino acids 5-167 from SEQ ID
NO: 2; Asn19 has been replaced with Gln 5 Amino acid sequence
encoded by SEQ ID NO: 3 6 Complement of SEQ ID NO3 FULL LENGTH HA 7
Nucleic acid sequence encoding full length hemagglutinin protein
from A/New Caledonia/20/1999 (1999 NC, H1)(GenBank: AY289929) 8
Amino acid sequence encoded by SEQ ID NO: 7 (full length
hemagglutinin protein from A/New Caledonia/20/1999 (1999 NC,
H1)(GenBank: AY289929)) 9 Complement of SEQ ID NO: 7 ECTODOMAINS 10
Nucleic acid sequence encoding ectodomain from hemagglutinin
protein from A/New Caledonia/20/1999 (1999 NC, H1). 11 Amino acid
sequence encoded by SEQ ID NO: 10 (ectodomain from hemagglutinin
protein from A/New Caledonia/20/1999 (1999 NC, H1). Amino acids
1-517 from SEQ ID NO: 8. 12 Complement of SEQ ID NO: 10 13 Nucleic
acid sequence encoding ectodomain from hemagglutinin protein from
A/California/04/2009 (2009 CA, H1). 14 Amino acid sequence encoded
by SEQ ID NO: 13 (ectodomain from hemagglutinin protein from
A/California/04/2009 (2009 CA, H1)) 15 Complement of SEQ ID NO: 13
16 Nucleic acid sequence encoding ectodomain from hemagglutinin
protein from A/Singapore/1/1957 (1957 Sing, H2). 17 Amino acid
sequence encoded by SEQ ID NO: 16 (ectodomain from hemagglutinin
protein from A/Singapore/1/1957 (1957 Sing, H2)) 18 Complement of
SEQ ID NO: 16 19 Nucleic acid sequence encoding ectodomain from
hemagglutinin protein from A/Hong Kong/1/1968 (1968 HK, H3). 20
Amino acid sequence encoded by SEQ ID NO: 19) ectodomain from
hemagglutinin protein from A/Hong Kong/1/1968 (1968 HK, H3)) 21
Complement of SEQ ID NO: 19 22 Nucleic acid sequence encoding
ectodomain from hemagglutinin protein from A/Brisbane/10/2007 (2007
Bris, H3). 23 Amino acid sequence encoded by SEQ ID NO: 22
(ectodomain from hemagglutinin protein from A/Brisbane/10/2007
(2007 Bris, H3)) 24 Complement of SEQ ID NO: 22. 25 Nucleic acid
sequence encoding ectodomain from hemagglutinin protein from
A/Indonesia/05/2005 (2005 Indo, H5) 26 Amino acid sequence encoded
by SEQ ID NO: 25 (ectodomain from hemagglutinin protein from
A/Indonesia/05/2005 (2005 Indo, H5)) 27 Complement of SEQ ID NO: 25
28 Nucleic acid sequence encoding ectodomain from hemagglutinin
protein from B/Florida/4/2006 (2006 Flo, B) 29 Amino acid sequence
encoded by SEQ ID NO: 28 (ectodomain from hemagglutinin protein
from B/Florida/4/2006 (2006 Flo, B)) 30 Complement of SEQ ID NO: 28
31 Nucleic acid sequence encoding ectodomain from hemagglutinin
protein from A/Perth/16/2009 (2009 Per, H3) 32 Amino acid sequence
encoded by SEQ ID NO: 31 (ectodomain from hemagglutinin protein
from A/Perth/16/2009 (2009 Per, H3)) 33 Complement of SEQ ID NO: 31
34 Nucleic acid sequence encoding ectodomain from hemagglutinin
protein from A/Brisbane/59/2007 (2007 Bris, H1) 35 Amino acid
sequence encoded by SEQ ID NO: 34 (ectodomain from hemagglutinin
protein from A/Brisbane/59/2007 (2007 Bris, H1)) 36 Complement of
SEQ ID NO: 34 37 Nucleic acid sequence encoding ectodomain from
hemagglutinin protein from B/Brisbane/60/2008 (2008 Bris, B) 38
Amino acid sequence encoded by SEQ ID NO: 37 (ectodomain from
hemagglutinin protein from B/Brisbane/60/2008 (2008 Bris, B)) 39
Complement of SEQ ID NO: 37 FERRITIN-HA ECTODOMAIN FUSION 40
Nucleic acid sequence encoding SEQ ID NO: 41 41 Amino acid sequence
of ferritin-HA fusion (ectodomain from hemagglutinin protein from
A/New Caledonia/20/1999 (1999 NC, H1)) 42 Complement of SEQ ID NO:
40 43 Nucleic acid sequence encoding SEQ ID NO: 44 44 Amino acid
sequence of ferritin-HA fusion (ectodomain from hemagglutinin
protein from A/California/04/2009 (2009 CA, H1)) 45 Complement of
SEQ ID NO: 43 46 Nucleic acid sequence encoding SEQ ID NO: 47 47
Amino acid sequence of ferritin-HA fusion (ectodomain from
hemagglutinin protein from A/Singapore/1/1957 (1957 Sing, H2)) 48
Complement of SEQ ID NO: 46 49 Nucleic acid sequence encoding SEQ
ID NO: 50 50 Amino acid sequence of ferritin-HA fusion (ectodomain
from hemagglutinin protein from A/Hong Kong/1/1968 (1968 HK, H3))
51 Complement of SEQ ID NO: 49 52 Nucleic acid sequence encoding
SEQ ID NO: 53 53 Amino acid sequence of ferritin-HA fusion
(ectodomain from hemagglutinin protein from A/Brisbane/10/2007
(2007 Bris, H3)) 54 Complement of SEQ ID NO: 52 55 Nucleic acid
sequence encoding SEQ ID NO: 56 56 Amino acid sequence of
ferritin-HA fusion (ectodomain from hemagglutinin protein from
A/Indonesia/05/2005 (2005 Indo, H5)) 57 Compliment of SEQ ID NO: 55
58 Nucleic acid sequence encoding SEQ ID NO: 59 59 Amino acid
sequence of ferritin-HA fusion protein (ectodomain from
hemagglutinin protein from B/Florida/4/2006 (2006 Flo, B)) 60
Complement of SEQ ID NO: 58 61 Nucleic acid sequence encoding SEQ
ID NO: 62 62 Amino acid sequence of ferritin-HA fusion protein
(ectodomain from hemagglutinin protein from A/Perth/16/2009 (2009
Per, H3)) 63 Complement of SEQ ID NO: 61 64 Nucleic acid sequence
encoding SEQ ID NO: 65 65 Amino acid sequence of ferritin-HA fusion
protein (ectodomain from hemagglutinin protein from
A/Brisbane/59/2007 (2007 Bris, H1)) 66 Complement of SEQ ID NO: 64
67 Nucleic acid sequence encoding SEQ ID NO: 68 68 Amino acid
sequence of ferritin-HA fusion protein (ectodomain from
hemagglutinin protein from B/Brisbane/60/2008 (2008 Bris, B)) 69
Complement of SEQ ID NO: 67 STEM REGION 70 Nucleic acid molecule
encoding SEQ ID NO: 71 71 Amino acid sequence of stem region from
A/New Caledonia/20/1999 (1999 NC, H1)(GenBank: AY289929) 72
Complement of SEQ ID NO: 70 73 Nucleic acid sequence encoding SEQ
ID NO: 74 74 Amino acid sequence of stem region from
A/California/04/2009 (2009 CA, H1) 75 Complement of SEQ ID NO: 73
76 Nucleic acid sequence encoding SEQ ID NO: 77 77 Amino acid
sequence of stem region from A/Singapore/1/1957 (1957 Sing, H2) 78
Complement of SEQ ID NO: 76 79 Nucleic acid sequence encoding SEQ
ID NO: 80 80 Amino acid sequence of stem region from A/Hong
Kong/1/1968 (1968 HK, H3) 81 Complement of SEQ ID NO: 79 82 Nucleic
acid sequence encoding SEQ ID NO: 83 83 Amino acid sequence of stem
region from A/Brisbane/10/2007 (2007 Bris, H3) 84 Complement of SEQ
ID NO: 82 85 Nucleic acid sequence encoding SEQ ID NO: 86 86 Amino
acid sequence of stem region from A/Indonesia/05/2005 (2005 Indo,
H5) 87 Complement of SEQ ID NO: 85 88 Nucleic acid sequence
encoding SEQ ID NO: 89 89 Amino acid sequence of stem region from
B/Florida/4/2006 (2006 Flo, B) 90 Complement of SEQ ID NO: 88 91
Nucleic acid sequence encoding SEQ ID NO: 92 92 Amino acid sequence
of stem region from A/Perth/16/2009 (2009 Per, H3) 93 Complement of
SEQ ID NO: 91 94 Nucleic acid sequence encoding SEQ ID NO: 95 95
Amino acid sequence of stem region from A/Brisbane/59/2007 (2007
Bris, H1) 96 Complement of SEQ ID NO: 94 97 Nucleic acid sequence
encoding SEQ ID NO: 98 98 Amino acid sequence of stem region from
B/Brisbane/60/2008 (2008 Bris, B) 99 Complement of SEQ ID NO: 97
FERRITIN-HA STEM REGION FUSION 100 Nucleic acid sequence encoding
SEQ ID NO: 101 101 Amino acid sequence of ferritin-HA stem region
fusion protein A/New Caledonia/20/1999 (1999 NC, H1) 102 Complement
of SEQ ID NO: 100 103 Nucleic acid sequence encoding SEQ ID NO: 104
104 Amino acid sequence of ferritin-HA stem region fusion protein
(H1 CA) 105 Complement of SEQ ID NO: 103 106 Nucleic acid sequence
encoding SEQ ID NO: 107 107 Amino acid sequence of ferritin-HA stem
region fusion protein (H2 Sing) 108 Complement of SEQ ID NO: 106
109 Nucleic acid sequence encoding SEQ ID NO: 110 110 Amino acid
sequence of ferritin-HA stem region fusion protein (H3 Hong Kong)
111 Complement of SEQ ID NO: 109 112 Nucleic acid sequence encoding
SEQ ID NO: 113 113 Amino acid sequence of ferritin-HA stem region
fusion protein (H5 Indonesia) 114 Complement of SEQ ID NO: 112 115
Nucleic acid sequence encoding SEQ ID NO: 116 116 Amino acid
sequence of ferritin-HA stem region fusion protein
(A/Brisbane/59/2007 (2007 Bris, H1)) 117 Complement of SEQ ID NO:
115 118 Nucleic acid sequence encoding SEQ ID NO: 119 119 Amino
acid sequence of ferritin-HA stem region fusion protein
(A/Brisbane/10/2007 (2007 Bris, H3)) 120 Complement of SEQ ID NO:
118 121 Nucleic acid sequence encoding SEQ ID NO: 122 122 Amino
acid sequence of ferritin-HA stem region fusion protein
(A/Perth/16/2009 (2009 Per, H3)) 123 Complement of SEQ ID NO: 121
124 Nucleic acid sequence encoding SEQ ID NO: 125 125 Amino acid
sequence of ferritin-HA stem region fusion protein
(B/Brisbane/60/2008 (2008 Bris, B) 126 Complement of SEQ ID NO: 124
127 Nucleic acid sequence encoding SEQ ID NO: 128 128 Amino acid
sequence of ferritin-HA stem region fusion protein
(B/Florida/4/2006 (2006 Flo, B)) 129 Complement of SEQ ID NO:
127
[0117] One type of immune response is a B-cell response, which
results in the production of antibodies against the antigen that
elicited the immune response. Thus, one embodiment of the present
invention is a protein that elicits antibodies that bind to the
stem region of influenza HA protein from a virus selected from the
group consisting of influenza A viruses, influenza B viruses and
influenza C viruses. One embodiment of the present invention is a
protein that elicits antibodies that bind to the stem region of
influenza HA protein selected from the group consisting of an H1
influenza virus HA protein, an H2 influenza virus HA protein, an
influenza H3 virus HA protein, an influenza H4 virus HA protein, an
influenza H5 virus HA protein, an influenza H6 virus HA protein, an
H7 influenza virus HA protein, an H8 influenza virus HA protein, an
H9 influenza virus HA protein, an H10 influenza virus HA protein HA
protein, an H11 influenza virus HA protein, an H12 influenza virus
HA protein, an H13 influenza virus HA protein, an H14 influenza
virus HA protein, an H15 influenza virus HA protein and an H16
influenza virus HA protein. One embodiment of the present invention
is a protein that elicits antibodies that bind to the stem region
of influenza HA protein from a strain of virus selected from the
viruses listed in Table 2.
[0118] While all antibodies are capable of binding to the antigen
which elicited the immune response that resulted in antibody
production, preferred antibodies are those that neutralize an
influenza virus. Thus, one embodiment of the present invention is a
protein that elicits neutralizing antibodies that bind to the stem
region of influenza HA protein from a virus selected from the group
consisting of influenza A viruses, influenza B viruses and
influenza C viruses. One embodiment of the present invention is a
protein that elicits neutralizing antibodies that bind to the stem
region of influenza HA protein selected from the group consisting
of an H1 influenza virus HA protein, an H2 influenza virus HA
protein, an influenza H3 virus HA protein, an influenza H4 virus HA
protein, an influenza H5 virus HA protein, an influenza H6 virus HA
protein, an H7 influenza virus HA protein, an H8 influenza virus HA
protein, an H9 influenza virus HA protein, an H10 influenza virus
HA protein HA protein, an H11 influenza virus HA protein, an H12
influenza virus HA protein, an H13 influenza virus HA protein, an
H14 influenza virus HA protein, an H15 influenza virus HA protein
and an H16 influenza virus HA protein. One embodiment of the
present invention is a protein that elicits neutralizing antibodies
that bind to the stem region of influenza HA protein from a strain
of virus selected from the viruses listed in Table 2. One
embodiment of the present invention is a protein that elicits
neutralizing antibodies that bind to a protein comprising an amino
acid sequence at least 80% identical to a sequence selected from
the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77,
SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID
NO:92, SEQ ID NO:95 and SEQ ID NO:98. One embodiment of the present
invention is a protein that elicits neutralizing antibodies that
bind to a protein comprising an amino acid sequence selected from
the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77,
SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID
NO:92, SEQ ID NO:95 and SEQ ID NO:98.
[0119] Neutralizing antibodies elicited by proteins of the present
invention can neutralize viral infections by affecting any step in
the life cycle of the virus. For example, neutralizing antibodies
may prevent an influenza virus from attaching to a cell, entering a
cell, releasing viral ribonucleoproteins into the cytoplasm,
forming new viral particles in the infected cell and budding new
viral particles from the infected host cell membrane. In one
embodiment, neutralizing antibodies elicited by proteins of the
present invention prevent influenza virus from attaching to the
host cell. In one embodiment, neutralizing antibodies elicited by
proteins of the present invention prevent influenza virus from
entering the host cell. In one embodiment, neutralizing antibodies
elicited by proteins of the present invention prevent fusion of
viral membranes with endosomal membranes. In one embodiment,
neutralizing antibodies elicited by proteins of the present
invention prevent release of ribonucleoproteins into the cytoplasm
of the host cell. In one embodiment, neutralizing antibodies
elicited by proteins of the present invention prevent assembly of
new virus in the infected host cell. In one embodiment,
neutralizing antibodies elicited by proteins of the present
invention prevent release of newly formed virus from the infected
host cell.
[0120] Because the amino acid sequence of the stem region of
influenza virus is highly conserved, neutralizing antibodies
elicited by proteins of the present invention may be broadly
neutralizing. That is, neutralizing antibodies elicited by proteins
of the present invention may neutralize influenza viruses of more
than one type, subtype and/or strain. Thus, one embodiment of the
present invention is a protein that elicits broadly neutralizing
antibodies that bind the stem region of influenza HA protein. One
embodiment is a protein that elicits antibodies that bind the stem
region of an HA protein from more than one type of influenza virus
selected from the group consisting of influenza type A viruses,
influenza type B viruses and influenza type C viruses. One
embodiment is a protein that elicits antibodies that bind the stem
region of an HA protein from more than one sub-type of influenza
virus selected from the group consisting of an H1 influenza virus,
an H2 influenza virus, an influenza H3 virus, an influenza H4
virus, an influenza H5 virus, an influenza H6 virus, an H7
influenza virus, an H8 influenza virus, an H9 influenza virus, an
H10 influenza virus, an H11 influenza virus, an H12 influenza
virus, an H13 influenza virus, an H14 influenza virus, an H15
influenza virus and an H16 influenza virus. One embodiment is a
protein that elicits antibodies that bind the stem region of an HA
protein from more than strain of influenza virus. One embodiment is
a protein that elicits antibodies that bind the stem region of an
HA protein from more than one strain of influenza virus selected
from the viruses listed in Table 2. One embodiment of the present
invention is a protein that elicits antibodies that bind more than
one protein comprising an amino acid sequence at least 80%
identical to a sequence selected from the group consisting of SEQ
ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83,
SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID
NO:98. One embodiment of the present invention is a protein that
elicits neutralizing antibodies that bind to more than one protein
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID
NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ
ID NO:95 and SEQ ID NO:98.
[0121] Particularly useful proteins of the present invention are
those comprising an immunogenic portion of an influenza HA protein.
Thus, one embodiment of the present invention is a protein
comprising at least one immunogenic portion from the stem region of
influenza HA protein, wherein the protein elicits neutralizing
antibodies against an influenza virus. Such a protein is referred
to as a stem-region protein (or a stem-region immunogen). One
embodiment of the present invention is a protein comprising at
least one immunogenic portion from the stem region of an HA protein
from a virus selected from the group consisting of influenza type A
viruses, influenza type B viruses and influenza type C viruses,
wherein the protein elicits neutralizing antibodies against an
influenza virus. One embodiment of the present invention is a
protein comprising at least one immunogenic portion from the stem
region of an HA protein selected from the group consisting of an H1
influenza virus HA protein, an H2 influenza virus HA protein, an
influenza H3 virus HA protein, an influenza H4 virus HA protein, an
influenza H5 virus HA protein, an influenza H6 virus HA protein, an
H7 influenza virus HA protein, an H8 influenza virus HA protein, an
H9 influenza virus HA protein, an H10 influenza virus HA protein HA
protein, an H11 influenza virus HA protein, an H12 influenza virus
HA protein, an H13 influenza virus HA protein, an H14 influenza
virus HA protein, an H15 influenza virus HA protein and an H16
influenza virus HA protein. One embodiment of the present invention
is a protein comprising at least one immunogenic portion from the
stem region of an HA protein from the viruses listed in Table 2.
One embodiment of the present invention is a protein comprising at
least one immunogenic portion from a protein comprising an amino
acid sequence at least 80% identical to a sequence selected from
the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77,
SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID
NO:92, SEQ ID NO:95 and SEQ ID NO:98. One embodiment of the present
invention is a protein comprising at least one immunogenic portion
from a protein comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ
ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92,
SEQ ID NO:95 and SEQ ID NO:98. In one embodiment, such proteins
comprising immunogenic portions of the HA protein elicit the
production of broadly neutralizing antibodies against influenza
virus.
[0122] Immunogenic portions of proteins comprise epitopes, which
are clusters of amino acid residues that are recognized by the
immune system, thereby eliciting an immune response. Such epitopes
may consist of contiguous amino acids residues (i.e., amino acid
residues that are adjacent to one another in the protein), or they
may consist of non-contiguous amino acid residues (i.e., amino acid
residues that are not adjacent one another in the protein) but
which are in close special proximity in the finally folded protein.
It is well understood by those skilled in the art that epitopes
require a minimum of six amino acid residues in order to be
recognized by the immune system. Thus, in one embodiment the
immunogenic portion from the influenza HA protein comprises at
least one epitope. One embodiment of the present invention is a
protein comprising at least 6 amino acids, at least 10 amino acids,
at least 25 amino acids, at least 50 amino acids, at least 75 amino
acids or at least 100 amino acids from the stem region of influenza
HA protein. One embodiment of the present invention is a protein
comprising at least 6 amino acids, at least 10 amino acids, at
least 25 amino acids, at least 50 amino acids, at least 75 amino
acids or at least 100 amino acids from the stem region of an HA
protein from a virus selected from the group consisting of
influenza type A viruses, influenza type B viruses and influenza
type C viruses. One embodiment of the present invention is a
protein comprising at least 6 amino acids, at least 10 amino acids,
at least 25 amino acids, at least 50 amino acids, at least 75 amino
acids or at least 100 amino acids from the stem region of an HA
protein selected from the group consisting an H1 influenza virus HA
protein, an H2 influenza virus HA protein, an influenza H3 virus HA
protein, an influenza H4 virus HA protein, an influenza H5 virus HA
protein, an influenza H6 virus HA protein, an H7 influenza virus HA
protein, an H8 influenza virus HA protein, an H9 influenza virus HA
protein, an H10 influenza virus HA protein HA protein, an H11
influenza virus HA protein, an H12 influenza virus HA protein, an
H13 influenza virus HA protein, an H14 influenza virus HA protein,
an H15 influenza virus HA protein and an H16 influenza virus HA
protein. One embodiment of the present invention is a protein
comprising at least 6 amino acids, at least 10 amino acids, at
least 25 amino acids, at least 50 amino acids, at least 75 amino
acids or at least 100 amino acids from the stem region of an HA
protein from a strain of virus selected from the viruses listed in
Table 2. In one embodiment, the amino acids are contiguous amino
acids from the stem region of the HA protein. In one embodiment,
such proteins comprising at least 6 amino acids, at least 10 amino
acids, at least 25 amino acids, at least 50 amino acids, at least
75 amino acids or at least 100 amino acids from the stem region of
an HA protein elicit the production of broadly neutralizing
antibodies against influenza virus. One embodiment of the present
invention is a protein comprising at least 6 amino acids, at least
10 amino acids, at least 25 amino acids, at least 50 amino acids,
at least 75 amino acids or at least 100 amino acids from the stem
region of an HA protein comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID
NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ
ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one
embodiment, the amino acids are contiguous amino acids from the
stem region of the HA protein. In one embodiment, the amino acids
are non-contiguous, but are in close spatial proximity in the final
protein.
[0123] While the present application discloses the use of stem
regions from several exemplary HA proteins having specific
sequences, the invention may also be practiced using stem regions
from proteins comprising variations of the disclosed HA sequences.
Thus, one embodiment of the present invention is a stem-region
protein comprising an amino acid sequence at least 80%, at least
85%, at least 90%, at least 92%, at least 94%, at least 96%, at
least 98% or at least 99% identical the stem region of an HA
protein comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID
NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. One embodiment of the
present invention is a stem-region protein comprising an amino acid
sequence at least 80%, at least 85%, at least 90%, at least 92%, at
least 94%, at least 96%, at least 98% or at least 99% identical to
a sequence selected from the group consisting of SEQ ID NO:71, SEQ
ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86,
SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. One
embodiment of the present invention is a stem-region protein
comprising the stem region of an HA protein comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:8,
SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID
NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and
SEQ ID NO:38. One embodiment of the present invention is a
stem-region protein comprising an amino acid sequence selected from
the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77,
SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID
NO:92, SEQ ID NO:95, and SEQ ID NO:98.
[0124] While the proteins disclosed thus far may elicit broadly
neutralizing antibodies against an influenza virus, the inventors
have discovered that such proteins are more stable and easier to
purify when they exist in a trimeric form. Thus, one embodiment is
a protein comprising the stem-region protein of the present
invention joined to a trimerization domain. In one embodiment, the
stem region is from an HA protein comprising an amino acid sequence
at least 80% identical, at least 85% identical, at least 90%
identical, at least 92% identical, at least 94% identical, at least
96% identical, at least 98% identical or at least 99% identical to
a sequence selected from the group consisting of SEQ ID NO:8, SEQ
ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23,
SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID
NO:38. In one embodiment, the stem region is from an HA protein
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID
NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the
stem region protein comprises an amino acid sequence at least 80%
identical, at least 85% identical, at least 90% identical, at least
92% identical, at least 94% identical, at least 96% identical, at
least 98% identical or at least 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NO:71, SEQ ID
NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ
ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one
embodiment, the stem region protein comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:71, SEQ ID
NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ
ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one
embodiment, the trimerization domain is selected from the group
consisting of the HIV-1 gp41 trimerization domain, the SIV gp41
trimerization domain, the Ebola virus gp-2 trimerization domain,
the HTLV-1 gp-21 trimerization domain, the T4 fibritin
trimerization domain (i.e., foldon), the yeast heat shock
transcription factor trimerization domain, and the human collagen
trimerization domain. In one embodiment, the trimerization domain
is an HIV gp41 trimerization domain.
[0125] The inventors have also found that, in some instances, stem
region proteins of the present invention may be more stable when
joined to at least part of the head region of the HA protein. Thus,
one embodiment of the present invention is a protein comprising a
stem region protein joined to the head region of an HA protein and
a trimerization domain. In one embodiment, the stem region protein
is from an HA protein comprising an amino acid sequence at least
80% identical, at least 85% identical, at least 90% identical, at
least 92% identical, at least 94% identical, at least 96%
identical, at least 98% identical or at least 99% identical to a
sequence selected from the group consisting of SEQ ID NO:8, SEQ ID
NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ
ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID
NO:38. In one embodiment, the stem region protein is from an HA
protein comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID
NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the
stem region protein comprises an amino acid sequence at least 80%
identical, at least 85% identical, at least 90% identical, at least
92% identical, at least 94% identical, at least 96% identical, at
least 98% identical or at least 99% identical to an amino acid
sequence selected from the group consisting of SEQ ID NO:71, SEQ ID
NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ
ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one
embodiment, the stem region protein comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:71, SEQ ID
NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ
ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98.
[0126] In some embodiments of the present invention, the various
protein domains (e.g., stem region protein, trimerization domain,
head region, etc.) may be joined directly to one another. In other
embodiments, it may be necessary to employ linkers (also referred
to as a spacer sequences) so that the various domains are in the
proper special orientation. The linker sequence is designed to
position the hemagglutinin protein in such a way to that it
maintains the ability to elicit an immune response to the influenza
virus. Linker sequences of the present invention comprise amino
acids. Preferable amino acids to use are those having small side
chains and/or those which are not charged. Such amino acids are
less likely to interfere with proper folding and activity of the
fusion protein. Accordingly, preferred amino acids to use in linker
sequences, either alone or in combination are serine, glycine and
alanine Examples of such linker sequences include, but are not
limited to, SGG, GSG, GG and NGTGGSG. Amino acids can be added or
subtracted as needed. Those skilled in the art are capable of
determining appropriate linker sequences for proteins of the
present invention.
[0127] One embodiment of the present invention is a fusion protein
comprising a stem region protein joined to at least a portion of
the head region of an HA protein and a trimerization domain,
wherein the fusion protein comprises one or more linker sequences.
In one embodiment, the stem region protein is from an HA protein
comprising an amino acid sequence at least 80% identical, at least
85% identical, at least 90% identical, at least 92% identical, at
least 94% identical, at least 96% identical, at least 98% identical
or at least 99% identical to a sequence selected from the group
consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID
NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the
stem region protein is from an HA protein comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:8, SEQ ID
NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ
ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID
NO:38. In one embodiment, the stem region protein comprises an
amino acid sequence at least 80% identical, at least 85% identical,
at least 90% identical, at least 92% identical, at least 94%
identical, at least 96% identical, at least 98% identical or at
least 99% identical to an amino acid sequence selected from the
group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ
ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92,
SEQ ID NO:95, and SEQ ID NO:98. In one embodiment, the stem region
protein comprises an amino acid sequence selected from the group
consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID
NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ
ID NO:95, and SEQ ID NO:98. In one embodiment, the linker is
selected from the group consisting of GG, GSG and NGTGGSG. In one
embodiment, the protein elicits antibodies that neutralize at least
one virus that is a different Type, sub-type or strain than the
Type, sub-type or strain of the virus from which the HA protein was
obtained.
Vaccines Comprising HA-Ferritin Fusion Proteins
[0128] The inventors have also discovered that fusion of influenza
HA protein with ferritin protein (HA-ferritin fusion protein)
results in a vaccine that elicits a robust immune response to
influenza virus. Such HA-ferritin fusion proteins self-assemble
into nanoparticles that display immunogenic portions of influenza
hemagglutinin protein on their surface. These nanoparticles are
useful for vaccinating individuals against a broad range of
influenza viruses. Thus, one embodiment of the present invention is
an HA-ferritin fusion protein comprising a monomeric ferritin
subunit disclosed herein joined to an influenza hemagglutinin
protein disclosed herein, wherein the HA-ferritin fusion protein is
capable of self-assembling into nanoparticles.
[0129] Ferritin is a globular protein found in all animals,
bacteria, and plants, that acts primarily to control the rate and
location of polynuclear Fe(III).sub.2O.sub.3 formation through the
transportation of hydrated iron ions and protons to and from a
mineralized core. The globular form of ferritin is made up of
monomeric subunit proteins (also referred to as monomeric ferritin
subunits), which are polypeptides having a molecule weight of
approximately 17-20 kDa. An example of the sequence of one such
monomeric ferritin subunit is represented by SEQ ID NO:2. Each
monomeric ferritin subunit has the topology of a helix bundle which
includes a four antiparallel helix motif, with a fifth shorter
helix (the c-terminal helix) lying roughly perpendicular to the
long axis of the 4 helix bundle. According to convention, the
helices are labeled `A, B, C, and D & E` from the N-terminus
respectively. The N-terminal sequence lies adjacent to the capsid
three-fold axis and extends to the surface, while the E helices
pack together at the four-fold axis with the C-terminus extending
into the particle core. The consequence of this packing creates two
pores on the capsid surface. It is expected that one or both of
these pores represent the point by which the hydrated iron diffuses
into and out of the capsid. Following production, these monomeric
ferritin subunit proteins self-assemble into the globular ferritin
protein. Thus, the globular form of ferritin comprises 24
monomeric, ferritin subunit proteins, and has a capsid-like
structure having 432 symmetry.
[0130] According to the present invention, a monomeric ferritin
subunit of the present invention is a full length, single
polypeptide of a ferritin protein, or any portion thereof, which is
capable of directing self-assembly of monomeric ferritin subunits
into the globular form of the protein. Amino acid sequences from
monomeric ferritin subunits of any known ferritin protein can be
used to produce fusion proteins of the present invention, so long
as the monomeric ferritin subunit is capable of self-assembling
into a nanoparticle displaying hemagglutinin on its surface. In one
embodiment, the monomeric subunit is from a ferritin protein
selected from the group consisting of a bacterial ferritin protein,
a plant ferritin protein, an algal ferritin protein, an insect
ferritin protein, a fungal ferritin protein and a mammalian
ferritin protein. In one embodiment, the ferritin protein is from
Helicobacter pylori.
[0131] HA-ferritin fusion proteins of the present invention need
not comprise the full-length sequence of a monomeric subunit
polypeptide of a ferritin protein. Portions, or regions, of the
monomeric ferritin subunit protein can be utilized so long as the
portion comprises an amino acid sequence that directs self-assembly
of monomeric ferritin subunits into the globular form of the
protein. One example of such a region is located between amino
acids 5 and 167 of the Helicobacter pylori ferritin protein. More
specific regions are described in Zhang, Y. Self-Assembly in the
Ferritin Nano-Cage Protein Super Family. 2011, Int. J. Mol. Sci.,
12, 5406-5421, which is incorporated herein by reference in its
entirety.
[0132] One embodiment of the present invention is an HA-ferritin
fusion protein comprising an HA protein of the present invention
joined to at least 25 contiguous amino acids, at least 50
contiguous amino acids, at least 75 contiguous amino acids, at
least 100 contiguous amino acids, or at least 150 contiguous amino
acids from a monomeric ferritin subunit, wherein the HA-ferritin
fusion protein is capable of self-assembling into nanoparticles.
One embodiment of the present invention is an HA-ferritin fusion
protein comprising an HA protein of the present invention joined to
at least 25 contiguous amino acids, at least 50 contiguous amino
acids, at least 75 contiguous amino acids, at least 100 contiguous
amino acids, or at least 150 contiguous amino acids from the region
of a ferritin protein corresponding to the amino acid sequences of
the Helicobacter pylori ferritin monomeric subunit that direct
self-assembly of the monomeric subunits into the globular form of
the ferritin protein, wherein the HA-ferritin fusion protein is
capable of self-assembling into nanoparticles. One embodiment of
the present invention is an HA-ferritin fusion protein comprising
an HA protein of the present invention joined to at least 25
contiguous amino acids, at least 50 contiguous amino acids, at
least 75 contiguous amino acids, at least 100 contiguous amino
acids, or at least 150 contiguous amino acids from SEQ ID NO:2 that
are capable of directing self-assembly of the monomeric subunits
into the globular ferritin protein, wherein the HA-ferritin fusion
protein is capable of self-assembling into nanoparticles. One
embodiment of the present invention is an HA-ferritin fusion
protein comprising an HA-protein of the present invention joined to
at least 25 contiguous amino acids, at least 50 contiguous amino
acids, at least 75 contiguous amino acids, at least 100 contiguous
amino acids, or at least 150 contiguous amino acids from amino acid
residues 5-167 of SEQ ID NO:2, wherein the HA-ferritin fusion
protein is capable of self-assembling into nanoparticles. One
embodiment of the present invention is an HA-ferritin fusion
protein comprising an HA protein of the present invention joined to
at least 25 contiguous amino acids, at least 50 contiguous amino
acids, at least 75 contiguous amino acids, at least 100 contiguous
amino acids, or at least 150 contiguous amino acids from SEQ ID
NO:5, wherein the HA-ferritin fusion protein is capable of
self-assembling into nanoparticles. One embodiment of the present
invention is an HA-ferritin fusion protein comprising an HA protein
of the present invention joined to amino acid residues 5-167 from
SEQ ID NO:2, or SEQ ID NO:5, wherein the HA-ferritin fusion protein
is capable of self-assembling into nanoparticles. As has been
previously discussed, it is well-known in the art that some
variations can be made in the amino acid sequence of a protein
without affecting the activity of the protein. Such variations
include insertion of amino acid residues, deletions of amino acid
residues, and substitutions of amino acid residues. Thus, in one
embodiment, the sequence of the monomeric ferritin subunit is
divergent enough from the sequence of a ferritin subunit naturally
found in a mammal, such that when the variant monomeric ferritin
subunit is introduced into the mammal, it does not result in the
production of antibodies that react with the mammal's natural
ferritin protein. According to the present invention, such a
monomeric subunit is referred to as immunogenically neutral. One
embodiment of the present invention is an HA-ferritin fusion
protein comprising an HA protein of the present invention joined to
an amino acid sequence at least 80%, at least 85%, at least 90%, at
least 95%, and at least 97% identical to the amino acid sequence of
a monomeric ferritin subunit that is responsible for directing
self-assembly of the monomeric ferritin subunits into the globular
form of the protein, wherein the HA-ferritin fusion protein is
capable of self-assembling into nanoparticles. In one embodiment,
the HA-ferritin fusion protein comprises a polypeptide sequence
identical in sequence to a monomeric ferritin subunit. One
embodiment of the present invention is an HA-ferritin fusion
protein comprising an HA protein of the present invention joined to
an amino acid sequence at least 80%, at least 85%, at least 90%, at
least 95%, and at least 97% identical to the amino acid sequence of
a monomeric ferritin subunit from Helicobacter pylori, wherein the
HA-ferritin fusion protein is capable of self-assembling into
nanoparticles. One embodiment of the present invention is an
HA-ferritin fusion protein comprising an HA protein of the present
invention joined to an amino acid sequence at least 80%, at least
85%, at least 90%, at least 95%, and at least 97% identical to a
sequence selected from amino acid residues 5-167 from SEQ ID NO:2
and SEQ ID NO:5, wherein the HA-ferritin fusion protein is capable
of self-assembling into nanoparticles. One embodiment of the
present invention is an HA-ferritin fusion protein comprising an HA
protein of the present invention joined to a sequence selected from
amino acid residues 5-167 from SEQ ID NO:2 and SEQ ID NO:5.
[0133] In some embodiments, it may be useful to engineer mutations
into the amino acid sequences of proteins of the present invention.
For example, it may be useful to alter sites such as enzyme
recognition sites or glycosylation sites in the monomeric ferritin
subunit, the trimerization domain, or linker sequences, in order to
give the fusion protein beneficial properties (e.g., solubility,
half-life, mask portions of the protein from immune surveillance).
In this regard, it is known that the monomeric subunit of ferritin
is not glycosylated naturally. However, it can be glycosylated if
it is expressed as a secreted protein in mammalian or yeast cells.
Thus, in one embodiment, potential N-linked glycosylation sites in
the amino acid sequences from the monomeric ferritin subunit are
mutated so that the mutated ferritin subunit sequences are no
longer glycosylated at the mutated site. One such sequence of a
mutated monomeric ferritin subunit is represented by SEQ ID
NO:5.
[0134] According to the present invention, the hemagglutinin
protein portion of HA-ferritin fusion proteins of the present
invention can be from any influenza virus, so long as the
HA-ferritin fusion protein elicits an immune response against an
influenza virus. Thus, one embodiment of the preset invention is an
HA-ferritin fusion protein comprising a ferritin protein of the
present invention joined to an amino acid sequence from an HA
protein from an influenza A virus, an influenza B virus or an
influenza C virus. One embodiment of the preset invention is an
HA-ferritin fusion protein comprising a ferritin protein of the
present invention joined to an amino acid sequence from an
influenza A Group 1 virus HA protein. One embodiment of the preset
invention is an HA-ferritin fusion protein comprising a ferritin
protein of the present invention joined to an amino acid sequence
from an influenza A Group 2 virus HA protein. One embodiment of the
preset invention is an HA-ferritin fusion protein comprising a
ferritin protein of the present invention joined to an amino acid
sequence from an HA protein selected from the group consisting of
an H1 influenza virus HA protein, an H2 influenza virus HA protein,
an H5 influenza virus HA protein, an H7 virus influenza HA protein
and an H9 influenza virus HA protein. One embodiment of the preset
invention is an HA-ferritin fusion protein comprising a ferritin
protein of the present invention joined to an amino acid sequence
from an HA protein selected from the group consisting of an H3
influenza virus HA protein, an H4 influenza virus HA protein, an H6
influenza virus HA protein, an H8 influenza virus HA protein, an
H10 influenza virus HA protein, an H11 influenza virus HA protein,
an H12 influenza virus HA protein, an H13 influenza virus HA
protein, an H14 influenza virus HA protein, an H15 influenza virus
HA protein, and an H15 influenza virus HA protein. One embodiment
of the preset invention is an HA-ferritin fusion protein comprising
a ferritin protein of the present invention joined to an amino acid
sequence of an HA protein from a virus listed in Table 2.
[0135] Preferred hemagglutinin proteins to use in constructing
HA-ferritin fusion proteins of the present invention are those that
elicit an immune response against an influenza virus. Even more
preferred hemagglutinin proteins are those that are capable of
eliciting antibodies to an influenza virus. One embodiment of the
present invention is an HA-ferritin fusion protein that elicits
antibodies to a virus selected from the group consisting of
influenza A viruses, influenza B viruses and influenza C viruses.
One embodiment of the present invention is a HA-ferritin fusion
protein that elicits antibodies to a virus selected from the group
consisting of an H1 influenza virus, an H2 influenza virus, an
influenza H3 virus, an influenza H4 virus, an influenza H5 virus,
an influenza H6 virus, an H7 influenza virus, an H8 influenza
virus, an H9 influenza virus, an H10 influenza virus, an H11
influenza virus, an H12 influenza virus, an H13 influenza virus, an
H14 influenza virus, an H15 influenza virus and an H16 influenza
virus. One embodiment of the present invention is an HA-ferritin
fusion protein that elicits antibodies to a virus listed in Table
2. Preferred antibodies elicited by HA-ferritin fusion proteins of
the present invention are those that neutralize an influenza virus.
Thus, one embodiment of the present invention is an HA-ferritin
fusion protein that elicits neutralizing antibodies to a virus
selected from the group consisting of influenza A viruses,
influenza B viruses and influenza C viruses. One embodiment of the
present invention is an HA-ferritin fusion protein that elicits
neutralizing antibodies to a virus having a subtype selected from
the group consisting of an H1 influenza virus, an H2 influenza
virus, an influenza H3 virus, an influenza H4 virus, an influenza
H5 virus, an influenza H6 virus, an H7 influenza virus, an H8
influenza virus, an H9 influenza virus, an H10 influenza virus, an
H11 influenza virus, an H12 influenza virus, an H13 influenza
virus, an H14 influenza virus, an H15 influenza virus and an H16
influenza virus. One embodiment of the present invention is an
HA-ferritin fusion protein that elicits neutralizing antibodies to
a virus listed in Table 2.
[0136] As has been discussed, neutralizing antibodies elicited by a
HA-ferritin fusion protein of the present invention can neutralize
viral infections by affecting any step in the life cycle of the
virus. Thus, in one embodiment of the present invention, an
HA-ferritin fusion protein elicits neutralizing antibodies that
prevent influenza virus from attaching to the host cell. In one
embodiment of the present invention, an HA-ferritin fusion protein
may elicit neutralizing antibodies that prevent influenza virus
from entering the host cell. In one embodiment of the present
invention, an HA-ferritin fusion protein may elicit neutralizing
antibodies that prevent fusion of viral membranes with endosomal
membranes. In one embodiment of the present invention, an
HA-ferritin fusion protein may elicit neutralizing antibodies that
prevent influenza virus from releasing ribonucleoproteins into the
cytoplasm of the host cell. In one embodiment of the present
invention, an HA-ferritin fusion protein may elicit neutralizing
antibodies that prevent assembly of new virus in the infected host
cell. In one embodiment of the present invention, an HA-ferritin
fusion protein may elicit neutralizing antibodies that prevent
release of newly formed virus from the infected host cell.
[0137] Preferred HA-ferritin fusion proteins of the present
invention are those that elicit broadly neutralizing antibodies.
Thus, one embodiment is an HA-ferritin fusion protein that elicits
antibodies that neutralizes more than one type of influenza virus
selected from the group consisting of influenza type A viruses,
influenza type B viruses and influenza type C viruses. One
embodiment is an HA-ferritin fusion protein that elicits antibodies
that neutralize more than one sub-type of influenza virus selected
from the group consisting of an H1 influenza virus, an H2 influenza
virus, an influenza H3 virus, an influenza H4 virus, an influenza
H5 virus, an influenza H6 virus, an H7 influenza virus, an H8
influenza virus, an H9 influenza virus, an H10 influenza virus, an
H11 influenza virus, an H12 influenza virus, an H13 influenza
virus, an H14 influenza virus, an H15 influenza virus and an H16
influenza virus. One embodiment is an HA-ferritin protein that
elicits antibodies that neutralize from more than one strain of
influenza virus selected from the viruses listed in Table 2.
[0138] It will be understood by those skilled in the art that
particularly useful HA-ferritin useful proteins of the present
invention are those comprising an immunogenic portion of influenza
HA protein. Thus, one embodiment of the present invention is an
HA-ferritin fusion protein comprising a ferritin protein of the
present invention joined to at least one immunogenic portion of an
influenza HA protein. One embodiment of the present invention is an
HA-ferritin protein comprising a ferritin protein of the present
invention joined to at least one immunogenic portion of an HA
protein from a virus selected from the group consisting of
influenza type A viruses, influenza type B viruses and influenza
type C viruses. One embodiment of the present invention is an
HA-ferritin protein comprising a ferritin protein of the present
invention joined to at least one immunogenic portion of an HA
protein selected from the group consisting of an H1 influenza virus
HA protein, an H2 influenza virus HA protein, an H5 influenza virus
HA protein, an H7 virus influenza HA protein and an H9 influenza
virus HA protein. One embodiment of the preset invention is an
HA-ferritin fusion protein comprising a ferritin protein of the
present invention joined to at least one immunogenic portion of an
HA protein selected from the group consisting of an H3 influenza
virus HA protein, an H4 influenza virus HA protein, an H6 influenza
virus HA protein, an H8 influenza virus HA protein, an H10
influenza virus HA protein, an H11 influenza virus HA protein, an
H12 influenza virus HA protein, an H13 influenza virus HA protein,
an H14 influenza virus HA protein, an H15 influenza virus HA
protein, and an H16 influenza virus HA protein, joined to a
ferritin protein of the present invention. One embodiment of the
present invention is an HA-ferritin protein comprising a ferritin
protein of the present invention joined to at least one immunogenic
portion of an HA protein from virus listed in Table 2. In one
embodiment, an HA-ferritin fusion protein comprising an immunogenic
portion of an HA protein elicits the production of broadly
neutralizing antibodies against influenza virus.
[0139] Immunogenic portions of proteins comprise epitopes, which
are clusters of amino acid residues that are recognized by the
immune system, thus eliciting an immune response. Such epitopes may
consist of contiguous amino acids residues (i.e., amino acid
residues that are adjacent to one another in the protein), or they
may consist of non-contiguous amino acid residues (i.e., amino acid
residues that are not adjacent one another in the protein) but
which are in close special proximity in the finally folded protein.
It is well understood by those skilled in the art that such
epitopes require a minimum of six amino acid residues in order to
be recognized by the immune system. Thus, one embodiment of the
present invention is an HA-ferritin fusion comprising an
immunogenic portion from the influenza HA protein, wherein the
immunogenic portion comprises at least one epitope.
[0140] It is known in the art that some variation in a protein
sequence can be tolerated without significantly affecting the
activity of the protein. Thus, one embodiment of the present
invention is an HA-ferritin fusion protein comprising a ferritin
protein of the present invention joined to an amino acid sequence
that is a variant of an HA protein from a virus selected from the
group consisting of influenza Type A viruses influenza Type B
viruses and influenza type C viruses. One embodiment of the present
invention is an HA-ferritin fusion protein comprising a ferritin
protein of the present invention joined to an amino acid sequence
at least about 80%, at least about 85%, at least about 90%, at
least about 95%, at least about 97% or at least about 99% identical
to the sequence of a HA protein from a virus selected from the
group consisting of influenza Type A viruses influenza Type B
viruses and influenza type C viruses, wherein the HA-ferritin
fusion protein elicits the production of neutralizing antibodies
against an influenza protein. One embodiment of the present
invention is an HA-ferritin fusion protein comprising a ferritin
protein of the present invention joined to an amino acid sequence
at least about 80%, at least about 85%, at least about 90%, at
least about 95%, at least about 97% or at least about 99% identical
to the sequence of a HA protein selected from the group consisting
an H1 influenza virus HA protein, an H2 influenza virus HA protein,
an H5 influenza virus HA protein, an H7 virus influenza HA protein
and an H9 influenza virus HA protein, wherein the HA-ferritin
fusion protein elicits the production of neutralizing antibodies
against an influenza protein. One embodiment of the present
invention is an HA-ferritin fusion protein comprising a ferritin
protein of the present invention joined to an amino acid sequence
at least about 80%, at least about 85%, at least about 90%, at
least about 95%, at least about 97% or at least about 99% identical
to the sequence of a HA protein selected from the group consisting
of an H3 influenza virus HA protein, an H4 influenza virus HA
protein, an H6 influenza virus HA protein, an H8 influenza virus HA
protein, an H10 influenza virus HA protein, an H11 influenza virus
HA protein, an H12 influenza virus HA protein, an H13 influenza
virus HA protein, an H14 influenza virus HA protein, an H15
influenza virus HA protein, and an H16 influenza virus HA protein,
joined to a ferritin protein of the present invention, wherein the
HA-ferritin fusion protein elicits the production of neutralizing
antibodies against an influenza protein. One embodiment of the
present invention is an HA-ferritin fusion protein comprising a
ferritin protein of the present invention joined to an amino acid
sequence at least about 80%, at least about 85%, at least about
90%, at least about 95%, at least about 97% or at least about 99%
identical to the sequence of a HA protein from a virus listed in
Table 2, wherein the HA-ferritin fusion protein elicits the
production of neutralizing antibodies against an influenza protein.
One embodiment of the present invention is an HA-ferritin fusion
protein comprising a ferritin protein of the present invention
joined to amino acid sequence at least about 80%, at least about
85%, at least about 90%, at least about 95%, at least about 97% or
at least about 99% identical to a sequence selected from the group
consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID
NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:32, SEQ ID NO:35, and SEQ ID NO:38, wherein the HA-ferritin
fusion protein elicits the production of neutralizing antibodies
against an influenza protein. One embodiment of the present
invention is an HA-ferritin fusion protein comprising a ferritin
protein of the present invention joined to amino acid sequence
selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11,
SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID
NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID
NO:38.
[0141] One embodiment of the present invention is an HA-ferritin
fusion protein comprising an amino acid sequence at least 80%, at
least about 85%, at least about 90%, at least about 95%, at least
about 97% or at least about 99% identical to a sequence selected
from the group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID
NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ
ID NO:62, SEQ ID NO:65, and SEQ ID NO:68. One embodiment of the
present invention is an HA-ferritin fusion protein comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ
ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID
NO:68.
[0142] It is known in the art that influenza hemagglutinin proteins
have various regions, or domains, each possessing specific
activities. For example, the globular head extends out from the
lipid membrane and comprises two domains: the receptor binding
domain (RBD) and the vestigial esterase domain. The RB domain is
involved in binding of the HA protein to receptors. The globular
head also includes several antigenic sites that include
immunodominant epitopes. The stem region is responsible for
anchoring the HA protein into the viral lipid envelope. Thus, it
will be understood by those skilled in the art that HA-ferritin
fusion proteins of the present invention need not comprise the
entire sequence of the HA protein. Instead, an HA-ferritin fusion
protein can comprise only those portions, regions, domains, and the
like, that contain the necessary activities for practicing the
present invention. For example, an HA-ferritin fusion protein may
contain only those amino acid sequences from the HA protein that
contain antigenic sites, epitopes, immunodominant epitopes, and the
like.
[0143] One embodiment of the present invention is an HA-ferritin
fusion protein comprising a ferritin protein of the present
invention joined to at least 25 amino acids, at least 50 amino
acids, at least 75 amino acids, at least 100 amino acids, at least
150 amino acids, at least 200 amino acids, at least 300 amino
acids, at least 400 amino acids, or at least 500 amino acids from
an HA protein from a virus selected from the group consisting of
influenza Type A viruses influenza Type B viruses and influenza
type C viruses, wherein the HA-ferritin fusion protein elicits the
production of neutralizing antibodies against an influenza protein.
One embodiment of the present invention is an HA-ferritin fusion
protein comprising a ferritin protein of the present invention
joined to at least 25 amino acids, at least 50 amino acids, at
least 75 amino acids, at least 100 amino acids, at least 150 amino
acids, at least 200 amino acids, at least 300 amino acids, at least
400 amino acids, or at least 500 amino acids from an HA protein
selected from the group consisting an H1 influenza virus HA
protein, an H2 influenza virus HA protein, an H5 influenza virus HA
protein, an H7 virus influenza HA protein and an H9 influenza virus
HA protein, wherein the HA-ferritin fusion protein elicits the
production of neutralizing antibodies against an influenza protein.
One embodiment of the present invention is an HA-ferritin fusion
protein comprising a ferritin protein of the present invention
joined to at least 25 amino acids, at least 50 amino acids, at
least 75 amino acids, at least 100 amino acids, at least 150 amino
acids, at least 200 amino acids, at least 300 amino acids, at least
400 amino acids, or at least 500 amino acids from an HA protein
selected from the group consisting of an H3 influenza virus HA
protein, an H4 influenza virus HA protein, an H6 influenza virus HA
protein, an H8 influenza virus HA protein, an H10 influenza virus
HA protein, an H11 influenza virus HA protein, an H12 influenza
virus HA protein, an H13 influenza virus HA protein, an H14
influenza virus HA protein, an H15 influenza virus HA protein, and
an H16 influenza virus HA protein, wherein the HA-ferritin fusion
protein elicits the production of neutralizing antibodies against
an influenza protein. One embodiment of the present invention is an
HA-ferritin fusion protein comprising a ferritin protein of the
present invention joined to at least 25 amino acids, at least 50
amino acids, at least 75 amino acids, at least 100 amino acids, at
least 150 amino acids, at least 200 amino acids, at least 300 amino
acids, at least 400 amino acids, or at least 500 amino acids from
and HA protein from a virus listed in Table 2, wherein the
HA-ferritin fusion protein elicits the production of neutralizing
antibodies against in influenza virus. One embodiment of the
present invention is an HA-ferritin fusion protein comprising a
ferritin protein of the present invention joined to at least 25
amino acids, at least 50 amino acids, at least 75 amino acids, at
least 100 amino acids, at least 150 amino acids, at least 200 amino
acids, at least 300 amino acids, at least 400 amino acids, or at
least 500 amino acids from a protein consisting of a sequence
selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11,
SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID
NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID
NO:38.
[0144] One embodiment of the present invention is an HA-ferritin
fusion protein comprising a ferritin protein of the present
invention joined to at least one domain from a HA protein from a
virus listed in Table 2, wherein the domain is selected from the
group consisting of an ectodomain, an RDB domain, a stem domain,
and a domain comprising the region stretching from the amino acid
residue immediately distal to the last amino acid of second helical
coil to the amino acid residue proximal to the first amino acid of
the transmembrane domain. According to the present invention, an
ectodomain of an influenza hemagglutinin protein refers to the
portion of the hemagglutinin protein that lies outside its
transmembrane domain. In one embodiment, the HA-ferritin fusion
protein comprises a ferritin protein of the present invention
joined to a region of a HA protein from a virus listed in Table 2,
wherein the region consists of the amino acid immediately distal to
the last amino acid of the second helical coiled coil and proximal
to the first amino acid of the transmembrane domain. In one
embodiment, the HA-ferritin fusion protein comprises a ferritin
protein of the present invention joined to a region of a HA protein
from a virus listed in Table 2, wherein the region comprises an
amino acid sequence distal to the second helical coiled coil and
proximal to the transmembrane domain. In one embodiment, the
HA-ferritin fusion protein comprises a ferritin protein of the
present invention joined to the ectodomain of a HA protein from a
virus listed in Table 2. One embodiment of the present invention is
an HA-ferritin fusion protein comprising a ferritin protein of the
present invention joined a sequence selected from the group
consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID
NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:32, SEQ ID NO:35, and SEQ ID NO:38.
[0145] The stem region of an influenza HA protein is a particularly
useful domain for constructing fusion proteins of the present
invention. Thus, one embodiment of the present invention is a
ferritin protein of the present invention joined to at least one
immunogenic portion from the stem region of influenza HA protein.
According to the preset invention, such a protein is referred to an
HA SS-ferritin fusion protein. As used herein, the HA stem region
of the hemagglutinin protein consists of the amino acids from the
membrane up to the head region of the protein. More specifically,
the stem region consists of the amino terminal amino acid up to the
cysteine at position 52, and all residues after the cysteine
residue at position 277 (using standard H3 numbering). Sequences of
exemplary stem regions are represented by SEQ ID NO:71, SEQ ID
NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ
ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98.
[0146] One embodiment of the present invention is an HA-ferritin
fusion protein comprising a ferritin protein of the present
invention joined to at least 25 amino acids, at least 50 amino
acids, at least 75 amino acids, at least 100 amino acids, at least
150 amino acids, or at least 200 amino acids from the stem region
of an HA protein from a virus selected from the group consisting of
influenza Type A viruses influenza Type B viruses and influenza
type C viruses, wherein the HA-ferritin fusion protein elicits the
production of neutralizing antibodies against an influenza protein.
One embodiment of the present invention is an HA-ferritin fusion
protein comprising a ferritin protein of the present invention
joined to at least 25 amino acids, at least 50 amino acids, at
least 75 amino acids, at least 100 amino acids, at least 150 amino
acids, or at least 200 amino acids from the stem region of an HA
protein selected from the group consisting an H1 influenza virus HA
protein, an H2 influenza virus HA protein, an H5 influenza virus HA
protein, an H7 virus influenza HA protein and an H9 influenza virus
HA protein, wherein the HA-ferritin fusion protein elicits the
production of neutralizing antibodies against an influenza protein.
One embodiment of the present invention is an HA-ferritin fusion
protein comprising a ferritin protein of the present invention
joined to at least 25 amino acids, at least 50 amino acids, at
least 75 amino acids, at least 100 amino acids, at least 150 amino
acids, or at least 200 amino acids from the stem region of an HA
protein selected from the group consisting of an H3 influenza virus
HA protein, an H4 influenza virus HA protein, an H6 influenza virus
HA protein, an H8 influenza virus HA protein, an H10 influenza
virus HA protein, an H11 influenza virus HA protein, an H12
influenza virus HA protein, an H13 influenza virus HA protein, an
H14 influenza virus HA protein, an H15 influenza virus HA protein,
and an H16 influenza virus HA protein, wherein the HA-ferritin
fusion protein elicits the production of neutralizing antibodies
against an influenza protein. One embodiment of the present
invention is an HA-ferritin fusion protein comprising a ferritin
protein of the present invention joined to at least 25 amino acids,
at least 50 amino acids, at least 75 amino acids, at least 100
amino acids, at least 150 amino acids, or at least 200 amino acids
from the stem region of an HA protein from a virus listed in Table
2, wherein the HA-ferritin fusion protein elicits the production of
neutralizing antibodies against in influenza virus. One embodiment
of the present invention is an HA-ferritin fusion protein
comprising a ferritin protein of the present invention joined to at
least 25 amino acids, at least 50 amino acids, at least 75 amino
acids, at least 100 amino acids, at least 150 amino acids, or at
least 200 amino acids from the stem region of an HA protein
comprising a sequence selected from the group consisting of SEQ ID
NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ
ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID
NO:38. One embodiment of the present invention is an HA-ferritin
fusion protein comprising a ferritin protein of the present
invention joined to at least 25 amino acids, at least 50 amino
acids, at least 75 amino acids, at least 100 amino acids, at least
150 amino acids, or at least 200 amino acids from the stem region
comprising a sequence selected from the group consisting of SEQ ID
NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ
ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID
NO:98.
[0147] One embodiment of the present invention is an HA-ferritin
fusion protein comprising a ferritin protein of the present
invention joined to an amino acid sequence at least about 80%, at
least about 85%, at least about 90%, at least about 95%, at least
about 97% or at least about 99% identical to the sequence of the
stem region of an HA protein from a virus selected from the group
consisting of influenza Type A viruses influenza Type B viruses and
influenza type C viruses, wherein the Ha-ferritin fusion protein
elicits the production of neutralizing antibodies against an
influenza protein. One embodiment of the present invention is an
HA-ferritin fusion protein comprising a ferritin protein of the
present invention joined to an amino acid sequence at least about
80%, at least about 85%, at least about 90%, at least about 95%, at
least about 97% or at least about 99% identical to the sequence of
the stem region of an HA protein selected from the group consisting
an H1 influenza virus HA protein, an H2 influenza virus HA protein,
an H5 influenza virus HA protein, an H7 virus influenza HA protein
and an H9 influenza virus HA protein, wherein the Ha-ferritin
fusion protein elicits the production of neutralizing antibodies
against an influenza protein. One embodiment of the present
invention is an HA-ferritin fusion protein comprising a ferritin
protein of the present invention joined to an amino acid sequence
at least about 80%, at least about 85%, at least about 90%, at
least about 95%, at least about 97% or at least about 99% identical
to the sequence of the stem region of an HA protein selected from
the group consisting of an H3 influenza virus HA protein, an H4
influenza virus HA protein, an H6 influenza virus HA protein, an H8
influenza virus HA protein, an H10 influenza virus HA protein, an
H11 influenza virus HA protein, an H12 influenza virus HA protein,
an H13 influenza virus HA protein, an H14 influenza virus HA
protein, an H15 influenza virus HA protein, and an H16 influenza
virus HA protein, joined to a ferritin protein of the present
invention, wherein the HA-ferritin fusion protein elicits the
production of neutralizing antibodies against an influenza protein.
One embodiment of the present invention is an HA-ferritin fusion
protein comprising a ferritin protein of the present invention
joined to an amino acid sequence at least about 80%, at least about
85%, at least about 90%, at least about 95%, at least about 97% or
at least about 99% identical to the sequence of the stem region of
an HA protein from a virus listed in Table 2, wherein the
HA-ferritin fusion protein elicits the production of neutralizing
antibodies against an influenza protein. One embodiment of the
present invention is an HA-ferritin fusion protein comprising a
ferritin protein of the present invention joined to an amino acid
sequence at least about 80%, at least about 85%, at least about
90%, at least about 95%, at least about 97% or at least about 99%
identical to the stem region of an HA protein comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:11,
SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID
NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38.,
wherein the HA-ferritin fusion protein elicits the production of
neutralizing antibodies against an influenza protein. One
embodiment of the present invention is an HA-ferritin fusion
protein comprising a ferritin protein of the present invention
joined to an amino acid sequence at least about 80%, at least about
85%, at least about 90%, at least about 95%, at least about 97% or
at least about 99% identical to an amino acid sequence selected
from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID
NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ
ID NO:92, SEQ ID NO:95 and SEQ ID NO:98, wherein the HA-ferritin
fusion protein elicits the production of neutralizing antibodies
against an influenza protein. One embodiment of the present
invention is an HA-ferritin fusion protein comprising a ferritin
protein of the present invention joined to an amino acid sequence
selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74,
SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID
NO:89, SEQ ID NO:92, SEQ ID NO:95 and SEQ ID NO:98.
[0148] As has been described for stem region proteins of the
present invention, the inventors have found that HA-ferritin fusion
proteins are more stable and easier to purify when they exist in a
trimeric form. Thus, in one embodiment of the present invention the
HA portion of the HA-ferritin fusion protein is joined to one or
more trimerization domains. In one embodiment, the HA protein
comprises an amino acid sequence at least 80% identical, at least
85% identical, at least 90% identical, at least 92% identical, at
least 94% identical, at least 96% identical, at least 98% identical
or at least 99% identical to a sequence selected from the group
consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID
NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:32, SEQ ID NO:35, and SEQ ID NO:38, joined to one or more
trimerization domains. In one embodiment, the HA protein comprises
an amino acid sequence selected from the group consisting of SEQ ID
NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ
ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35,
and SEQ ID NO:38 joined to one or more trimerization domains. In
one embodiment, the HA protein comprises an amino acid sequence at
least 80% identical, at least 85% identical, at least 90%
identical, at least 92% identical, at least 94% identical, at least
96% identical, at least 98% identical or at least 99% identical to
an amino acid sequence selected from the group consisting of SEQ ID
NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ
ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID
NO:98 joined to one or more trimerization domains. In one
embodiment, the HA protein comprises an amino acid sequence
selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74,
SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID
NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98 joined to one
or more trimerization domains. In one embodiment, the trimerization
domain is selected from the group consisting of the HIV-1 gp41
trimerization domain, the SIV gp41 trimerization domain, the Ebola
virus gp-2 trimerization domain, the HTLV-1 gp-21 trimerization
domain, the T4 fibritin trimerization domain (i.e., foldon), the
yeast heat shock transcription factor trimerization domain, and the
human collagen trimerization domain. In one embodiment, the
trimerization domain is an HIV gp41 trimerization domain.
[0149] Additionally, the inventors have found that, in some
instances, HA-ferritin fusion proteins in which the HA portion is
limited to HA stem region sequences may be more stable when joined
to at least part of the head region of the HA protein. Thus, one
embodiment of the present invention is an HA 55-ferritin fusion
protein, wherein, the HA portion of the fusion protein is joined to
an amino acid sequence from at least a portion of an HA protein
head region.
[0150] HA-ferritin proteins of the present invention are
constructed by joining ferritin proteins of the present invention
with HA proteins of the present invention. In addition, HA-ferritin
fusion proteins may contain other domains (e.g., stem region
protein, trimerization domain, head region, etc.) that improve the
functionality of the final HA-ferritin fusion protein. In some
embodiments, joining of the various proteins and/or domains can be
done such that the sequences are directly linked. In other
embodiments, it may be necessary to employ linkers (also referred
to as a spacer sequences) between the various proteins and/or
domains so that the so that they are in the proper special
orientation. More specifically, linker sequence can be inserted so
that the hemagglutinin protein is positioned in such a way to
maintain the ability to elicit an immune response to the influenza
virus. Linker sequences of the present invention comprise amino
acids. Preferable amino acids to use are those having small side
chains and/or those which are not charged. Such amino acids are
less likely to interfere with proper folding and activity of the
fusion protein. Accordingly, preferred amino acids to use in linker
sequences, either alone or in combination are serine, glycine and
alanine Examples of such linker sequences include, but are not
limited to, SGG, GSG, GG and NGTGGSG. Amino acids can be added or
subtracted as needed. Those skilled in the art are capable of
determining appropriate linker sequences for proteins of the
present invention.
[0151] In accordance with the invention, suitable portions of the
hemagglutinin protein can be joined to the ferritin protein either
as an exocapsid product by fusion with the N-terminal sequence
lying adjacent to the capsid three-fold axis, as an endocapsid
product by fusion with the C-terminus extending inside the capsid
core, or a combination thereof. In one embodiment, the
hemagglutinin portion of the fusion protein is joined to the
N-terminal sequence of the ferritin portion of the fusion
protein.
[0152] One embodiment of the present invention is an HA-ferritin
fusion protein comprising an amino acid sequence at least about
80%, at least about 85%, at least about 90%, at least about 95%, at
least about 97% or at least about 99% identical to SEQ ID NO:101,
SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID
NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128,
wherein the HA-ferritin fusion protein elicits the production of
neutralizing antibodies against an influenza protein. One
embodiment of the present invention is an HA-ferritin fusion
protein comprising SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ
ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122
SEQ ID NO:125 and SEQ ID NO:128.
[0153] Proteins of the present invention are encoded by nucleic
acid molecules of the present invention. In addition, they are
expressed by nucleic acid constructs of the present invention. As
used herein a nucleic acid construct is a recombinant expression
vector, i.e., a vector linked to a nucleic acid molecule encoding a
protein such that the nucleic acid molecule can effect expression
of the protein when the nucleic acid construct is administered to,
for example, a subject or an organ, tissue or cell. The vector also
enables transport of the nucleic acid molecule to a cell within an
environment, such as, but not limited to, an organism, tissue, or
cell culture. A nucleic acid construct of the present disclosure is
produced by human intervention. The nucleic acid construct can be
DNA, RNA or variants thereof. The vector can be a DNA plasmid, a
viral vector, or other vector. In one embodiment, a vector can be a
cytomegalovirus (CMV), retrovirus, adenovirus, adeno-associated
virus, herpes virus, vaccinia virus, poliovirus, sindbis virus, or
any other DNA or RNA virus vector. In one embodiment, a vector can
be a pseudotyped lentiviral or retroviral vector. In one
embodiment, a vector can be a DNA plasmid. In one embodiment, a
vector can be a DNA plasmid comprising viral components and plasmid
components to enable nucleic acid molecule delivery and expression.
Methods for the construction of nucleic acid constructs of the
present disclosure are well known. See, for example, Molecular
Cloning: a Laboratory Manual, 3.sup.rd edition, Sambrook et al.
2001 Cold Spring Harbor Laboratory Press, and Current Protocols in
Molecular Biology, Ausubel et al. eds., John Wiley & Sons,
1994. In one embodiment, the vector is a DNA plasmid, such as a
CMV/R plasmid such as CMV/R or CMV/R 8 KB (also referred to herein
as CMV/R 8 kb). Examples of CMV/R and CMV/R 8 kb are provided
herein. CMV/R is also described in U.S. Pat. No. 7,094,598 B2,
issued Aug. 22, 2006.
[0154] As used herein, a nucleic acid molecule comprises a nucleic
acid sequence that encodes a stem region immunogen, a ferritin
monomeric subunit, a hemagglutinin protein, and/or an HA-ferritin
fusion protein of the present invention. A nucleic acid molecule
can be produced recombinantly, synthetically, or by a combination
of recombinant and synthetic procedures. A nucleic acid molecule of
the disclosure can have a wild-type nucleic acid sequence or a
codon-modified nucleic acid sequence to, for example, incorporate
codons better recognized by the human translation system. In one
embodiment, a nucleic acid molecule can be genetically-engineered
to introduce, or eliminate, codons encoding different amino acids,
such as to introduce codons that encode an N-linked glycosylation
site. Methods to produce nucleic acid molecules of the disclosure
are known in the art, particularly once the nucleic acid sequence
is know. It is to be appreciated that a nucleic acid construct can
comprise one nucleic acid molecule or more than one nucleic acid
molecule. It is also to be appreciated that a nucleic acid molecule
can encode one protein or more than one protein.
[0155] Preferred nucleic acid molecules are those that encode a
stem-region protein, a ferritin monomeric subunit, a hemagglutinin
protein, and/or an HA-ferritin fusion protein comprising a
monomeric subunit of a ferritin protein joined to an influenza
hemagglutinin protein. Thus, one embodiment of the present
invention is a nucleic acid molecule comprising a nucleic acid
sequence encoding a protein that comprises a monomeric subunit of a
ferritin protein joined to an influenza hemagglutinin protein. In
one embodiment, the monomeric subunit of ferritin is from the
ferritin protein of Helicobacter pylori. In one embodiment, the
monomeric subunit comprises an amino acid sequence at least 80%, at
least 85%, at least 90%, at least 92%, at least 94%, at least 96%,
at least 98% or at least 99% identical to a sequence selected from
the group consisting of SEQ ID NO:2 and SEQ ID NO:5. In one
embodiment, the monomeric subunit comprises an amino acid sequence
selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5.
In one embodiment the influenza hemagglutinin protein comprises an
amino acid sequence at least 80%, at least 85%, at least 90%, at
least 92%, at least 94%, at least 96%, at least 98% or at least 99%
identical to a sequence selected from the group consisting of SEQ
ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20,
SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID
NO:35, and SEQ ID NO:38. In one embodiment the influenza
hemagglutinin protein comprises a sequence selected from the group
consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID
NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment the
influenza hemagglutinin protein comprises an amino acid sequence at
least 80%, at least 85%, at least 90%, at least 92%, at least 94%,
at least 96%, at least 98% or at least 99% identical to a sequence
selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74,
SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID
NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one
embodiment the influenza hemagglutinin protein comprises a sequence
selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74,
SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID
NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one
embodiment the influenza hemagglutinin protein comprises at least
25 amino acids, at least 50 amino acids, at least 75 amino acids,
at least 100 amino acids, at least 150 amino acids, or at least 200
amino acids from a sequence selected from the group consisting of
SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID
NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ
ID NO:35, and SEQ ID NO:38. In one embodiment the influenza
hemagglutinin protein comprises at least 25 amino acids, at least
50 amino acids, at least 75 amino acids, at least 100 amino acids,
at least 150 amino acids, or at least 200 amino acids from a
sequence selected from the group consisting of SEQ ID NO:71, SEQ ID
NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ
ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98.
[0156] One embodiment of the present invention is a nucleic acid
molecule comprising a nucleic sequence encoding a protein
comprising an amino acid sequence at least 80%, at least 85%, at
least 90%, at least 92%, at least 94%, at least 96%, at least 98%
or at least 99% identical to a sequence selected from the group
consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID
NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ
ID NO:65, and SEQ ID NO:68. One embodiment of the present invention
is a nucleic acid molecule comprising a nucleic sequence encoding
an amino acid sequence selected from the group consisting of SEQ ID
NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ
ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID
NO:68.
[0157] One embodiment of the present invention is a nucleic acid
molecule comprising a nucleic sequence encoding a protein
comprising an amino acid sequence at least 80%, at least 85%, at
least 90%, at least 92%, at least 94%, at least 96%, at least 98%
or at least 99% identical to SEQ ID NO:101, SEQ ID NO:104 SEQ ID
NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ
ID NO:122 SEQ ID NO:125 and SEQ ID NO:128. One embodiment of the
present invention is a nucleic acid molecule comprising a nucleic
sequence encoding an amino acid sequence selected from the group
consisting of SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID
NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ
ID NO:125 and SEQ ID NO:128.
[0158] Also embodied in the present invention are nucleic acid
sequences that are variants of nucleic acid sequence encoding
protein of the present invention. Such variants include nucleotide
insertions, deletions, and substitutions, so long as they do not
affect the ability of fusion proteins of the present invention to
self-assemble into nanoparticles, or significantly affect the
ability of the hemagglutinin portion of fusion proteins to elicit
an immune response to an influenza virus. Thus, one embodiment of
the present invention is a nucleic acid molecule encoding a fusion
protein of the present invention, wherein the monomeric subunit is
encoded by a nucleotide sequence at least 85%, at least 90%, at
least 95%, or at least 97% identical to a nucleotide sequence
selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4.
One embodiment of the present invention is a nucleic acid molecule
encoding an HA-ferritin fusion protein of the present invention,
wherein the HA protein is encoded by a nucleotide sequence at least
85%, at least 90%, at least 95%, at least 97% identical or at least
99% identical to a nucleic acid sequence encoding an amino acid
sequence selected from the group consisting of SEQ ID NO:8, SEQ ID
NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ
ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID
NO:38. One embodiment of the present invention is a nucleic acid
molecule encoding an HA-ferritin fusion protein of the present
invention, wherein the HA protein is encoded by a nucleotide
sequence at least 85%, at least 90%, at least 95%, at least 97%
identical or at least 99% identical to a nucleic acid sequence
encoding an amino acid sequence selected from the group consisting
of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID
NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and
SEQ ID NO:98.
[0159] One embodiment of the present invention is a nucleic acid
molecule comprising a nucleic acid sequence at least about 80%, at
least about 85%, at least about 90%, at least about 95%, at least
about 97% or at least about 99% identical to a nucleic acid
sequence selected from the group consisting of SEQ ID NO:7, SEQ ID
NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ
ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, and SEQ ID
NO:37. One embodiment of the present invention is a nucleic acid
molecule comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID
NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ
ID NO:31, SEQ ID NO:34, and SEQ ID NO:37.
[0160] One embodiment of the present invention is a nucleic acid
molecule comprising a nucleic acid sequence at least about 80%, at
least about 85%, at least about 90%, at least about 95%, at least
about 97% or at least about 99% identical to a nucleic acid
sequence selected from the group consisting of SEQ ID NO:40, SEQ ID
NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ
ID NO:58, SEQ ID NO:61, SEQ ID NO:64, and SEQ ID NO:67. One
embodiment of the present invention is a nucleic acid molecule
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID
NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ
ID NO:64, and SEQ ID NO:67.
[0161] One embodiment of the present invention is a nucleic acid
molecule comprising a nucleic acid sequence at least about 80%, at
least about 85%, at least about 90%, at least about 95%, at least
about 97% or at least about 99% identical to a nucleic acid
sequence selected from the group consisting of SEQ ID NO:70, SEQ ID
NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ
ID NO:88, SEQ ID NO:91, SEQ ID NO:94, and SEQ ID NO:97. One
embodiment of the present invention is a nucleic acid molecule
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NO:70, SEQ ID NO:73, SEQ ID NO:76, SEQ ID
NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ
ID NO:94, and SEQ ID NO:97.
[0162] One embodiment of the present invention is a nucleic acid
molecule comprising a nucleic acid sequence at least about 80%, at
least about 85%, at least about 90%, at least about 95%, at least
about 97% or at least about 99% identical to a nucleic acid
sequence selected from the group consisting of SEQ ID NO:100, SEQ
ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID
NO:115, SEQ ID NO:121, SEQ ID NO:124, and SEQ ID NO:127. One
embodiment of the present invention is a nucleic acid molecule
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:106, SEQ ID
NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:121, SEQ ID NO:124,
and SEQ ID NO:127.
[0163] Also encompassed by the present invention are expression
systems for producing fusion proteins of the present invention. In
one embodiment, nucleic acid molecules of the present invention are
operationally linked to a promoter. As used herein, operationally
linked means that proteins encoded by the linked nucleic acid
molecules can be expressed when the linked promoter is activated.
Promoters useful for practicing the present invention are known to
those skilled in the art. One embodiment of the present invention
is a recombinant cell comprising a nucleic acid molecule of the
present invention. One embodiment of the present invention is a
recombinant virus comprising a nucleic acid molecule of the present
invention.
[0164] As indicated above, the recombinant production of the
ferritin fusion proteins of the present invention can take place
using any suitable conventional recombinant technology currently
known in the field. For example, molecular cloning a fusion
protein, such as ferritin with a suitable protein such as the
recombinant influenza hemagglutinin protein, can be carried out via
expression in E. coli with the suitable monomeric subunit protein,
such as the helicobacter pylori ferritin monomeric subunit. The
construct may then be transformed into protein expression cells,
grown to suitable size, and induced to produce the fusion
protein.
[0165] As has been described, because HA-ferritin fusion proteins
of the present invention comprise a monomeric subunit of ferritin,
they can self-assemble. According to the present invention, the
supramolecule resulting from such self-assembly is referred to as a
hemagglutinin expressing ferritin based nanoparticle. For ease of
discussion, the hemagglutinin expressing ferritin based
nanoparticle will simply be referred to as a, or the, nanoparticle
(np). Nanoparticles of the present invention have the same
structural characteristics as the ferritin proteins described
earlier. That is, they contain 24 subunits and have 432 symmetry.
In the case of nanoparticles of the present invention, the subunits
are the fusion proteins comprising a ferritin monomeric subunit
joined to an influenza hemagglutinin protein. Such nanoparticles
display at least a portion of the hemagglutinin protein on their
surface as hemagglutinin trimers. In such a construction, the
hemagglutinin trimer is accessible to the immune system and thus
can elicit an immune response. Thus, one embodiment of the present
invention is a nanoparticle comprising an HA-ferritin fusion
protein, wherein the fusion protein comprises a monomeric ferritin
subunit joined to an influenza hemagglutinin protein. In one
embodiment, the nanoparticle is an octahedron. In one embodiment,
the nanoparticle displays the hemagglutinin protein on its surface
as a hemagglutinin trimer. In one embodiment, the influenza
hemagglutinin protein is capable of eliciting neutralizing
antibodies to an influenza virus. In one embodiment, the monomeric
ferritin subunit comprises at least 50 amino acids, at least 100
amino acids, or at least 150 amino acids from an amino acid
sequence selected from the group consisting of SEQ ID NO:2 and SEQ
ID NO:5, and/or comprises an amino acid sequence at least 85%, at
least 90%, at least 95%, at least 97% at least 99% identical to an
amino acid sequence selected from the group consisting of SEQ ID
NO:2 and SEQ ID NO:5. In one embodiment, the monomeric ferritin
subunit comprises SEQ ID NO:2 or SEQ ID NO:5.
[0166] In one embodiment, the influenza hemagglutinin protein
comprises at least one epitope from an influenza hemagglutinin
protein listed in Table 2. In one embodiment, the influenza
hemagglutinin protein comprises at least 25 amino acids, at least
50 amino acids, at least 75 amino acids, at least 100 amino acids,
at least 150 amino acids, at least 200 amino acids, at least 300
amino acids, at least 400 amino acids, or at least 500 amino acids
from a hemagglutinin protein of a virus listed in Table 2. In one
embodiment, the hemagglutinin protein comprises at least 25 amino
acids, at least 50 amino acids, at least 75 amino acids, at least
100 amino acids, at least 150 amino acids, at least 200 amino
acids, at least 300 amino acids, at least 400 amino acids, or at
least 500 amino acids from a protein consisting of a sequence
selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11,
SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID
NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID NO:38.
In one embodiment, the hemagglutinin protein comprises a sequence
selected from the group consisting of SEQ ID NO:8, SEQ ID NO:11,
SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID
NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID
NO:38.
[0167] In one embodiment, the influenza hemagglutinin protein
comprises an amino acid sequence at least 80%, at least 85%, at
least 90%, at least 95%, at least 97%, at least 99% identical to
the sequence of an hemagglutinin protein from a virus listed in
Table 2. In one embodiment, the influenza hemagglutinin protein
comprises an amino acid sequence at least 80%, at least 85%, at
least 90%, at least 95%, at least 97%, at least 99% identical to a
protein sequence selected from the group consisting of SEQ ID NO:8,
SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID
NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and
SEQ ID NO:38.
[0168] In one embodiment, the hemagglutinin protein comprises at
least 25 amino acids, at least 50 amino acids, at least 75 amino
acids, at least 100 amino acids, at least 150 amino acids, at least
200 amino acids, at least 300 amino acids, at least 400 amino
acids, or at least 500 amino acids from a protein consisting of a
sequence selected from the group consisting of SEQ ID NO:71, SEQ ID
NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ
ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one
embodiment, the influenza hemagglutinin protein comprises an amino
acid sequence at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at least 99% identical to a protein sequence
selected from the group consisting of SEQ ID NO:71, SEQ ID NO:74,
SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID
NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one
embodiment, the hemagglutinin protein comprises a sequence selected
from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID
NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ
ID NO:92, SEQ ID NO:95, and SEQ ID NO:98.
[0169] In one embodiment, the HA-ferritin fusion protein comprises
an amino acid sequence at least 80%, at least 85%, at least 90%, at
least 95%, at least 97%, at least 99% identical to a protein
sequence selected from the group consisting of SEQ ID NO:41, SEQ ID
NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ
ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68. In one
embodiment, the HA-ferritin fusion protein comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:41, SEQ ID
NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ
ID NO:59, SEQ ID NO:62, SEQ ID NO:65, and SEQ ID NO:68. In one
embodiment, the HA-ferritin fusion protein comprises an amino acid
sequence at least 80%, at least 85%, at least 90%, at least 95%, at
least 97%, at least 99% identical to SEQ ID NO:101, SEQ ID NO:104
SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID
NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID NO:128. In one
embodiment, the HA-ferritin fusion protein comprises SEQ ID NO:101,
SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID
NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID
NO:128.
[0170] Because stem region immunogens, HA-ferritin fusion proteins
and nanoparticles of the present invention can elicit an immune
response to an influenza virus, they can be used as vaccines to
protect individuals against infection by influenza virus. According
to the present invention a vaccine can be a stem region immunogen,
an HA-ferritin fusion protein, or a nanoparticle of the present
invention. Thus, one embodiment of the present invention is a
vaccine comprising a stem region immunogen, an HA-ferritin fusion
protein, or a nanoparticle of the present invention. Vaccines of
the present invention can also contain other components such as
adjuvants, buffers and the like. Although any adjuvant can be used,
preferred embodiments can contain: chemical adjuvants such as
aluminum phosphate, benzyalkonium chloride, ubenimex, and QS21;
genetic adjuvants such as the IL-2 gene or fragments thereof, the
granulocyte macrophage colony-stimulating factor (GM-CSF) gene or
fragments thereof, the IL-18 gene or fragments thereof, the
chemokine (C--C motif) ligand 21 (CCL21) gene or fragments thereof,
the IL-6 gene or fragments thereof, CpG, LPS, TLR agonists, and
other immune stimulatory genes; protein adjuvants such IL-2 or
fragments thereof, the granulocyte macrophage colony-stimulating
factor (GM-CSF) or fragments thereof, IL-18 or fragments thereof,
the chemokine (C--C motif) ligand 21 (CCL21) or fragments thereof,
IL-6 or fragments thereof, CpG, LPS, TLR agonists and other immune
stimulatory cytokines or fragments thereof; lipid adjuvants such as
cationic liposomes, N3 (cationic lipid), monophosphoryl lipid A
(MPL1); other adjuvants including cholera toxin, enterotoxin,
Fms-like tyrosine kinase-3 ligand (Flt-3L), bupivacaine, marcaine,
and levamisole.
[0171] One embodiment of the disclosure is a ferritin-based
nanoparticle vaccine that includes more than one influenza
hemagglutinin protein. Such a vaccine can include a combination of
different influenza hemagglutinin proteins, either on a single
nanoparticle or as a mixture of nanoparticles, at least two of
which have a unique influenza hemagglutinin proteins. A multivalent
vaccine can comprise as many influenza hemagglutinin proteins as
necessary in order to result in production of the immune response
necessary to protect against a desired breadth of virus strains. In
one embodiment, the vaccine comprises a hemagglutinin protein from
at least two different influenza strains (bi-valent). In one
embodiment, the vaccine comprises a hemagglutinin protein from at
least three different influenza strains (tri-valent). In one
embodiment, the vaccine comprises a hemagglutinin protein from at
least four different influenza strains (tetra-valent). In one
embodiment, the vaccine comprises a hemagglutinin protein from at
least five different influenza strains (penta-valent). In one
embodiment, the vaccine comprises a hemagglutinin protein from at
least six different influenza strains (hexa-valent). In various
embodiments, a vaccine comprises a hemagglutinin protein from each
of 7, 8, 9, or 10 different strains of influenza virus. An example
of such a combination is a ferritin-based nanoparticle vaccine that
comprises influenza A group 1 hemagglutinin protein, an influenza A
group 2 hemagglutinin protein, and an influenza B hemagglutinin
protein. In one embodiment, the influenza hemagglutinin proteins
are H1 HA, H3 HA, and B HA. In one embodiment, the influenza
hemagglutinin proteins are those included in the 2011-2012
influenza vaccine. Another example of a multivalent vaccine is a
ferritin based nanoparticle vaccine that comprises hemagglutinin
proteins from four different influenza viruses. In one embodiment,
the multivalent vaccine comprises hemagglutinin proteins from H1
A/NC/20/1999, H1 A/CA/04/2009, H2 A/Singapore/1/1957 and H5
A/Indonesia/05/2005. Such a vaccine is described in Example 2.
[0172] One embodiment of the present invention is a method to
vaccinate an individual against influenza virus, the method
comprising administering a nanoparticle to an individual such that
an immune response against influenza virus is produced in the
individual, wherein the nanoparticle comprises a monomeric subunit
from ferritin joined to an influenza hemagglutinin protein, and
wherein the nanoparticle displays the influenza hemagglutinin on
its surface. In one embodiment, the nanoparticle is a monovalent
nanoparticle. In one embodiment, the nanoparticle is multivalent
nanoparticle. Another embodiment of the present invention is a
method to vaccinate an individual against infection with influenza
virus, the method comprising:
[0173] a) obtaining a nanoparticle comprising monomeric subunits,
wherein the monomeric subunits comprise a ferritin protein joined
to an influenza hemagglutinin protein, and wherein the nanoparticle
displays the influenza hemagglutinin on its surface; and,
[0174] b) administering the nanoparticle to an individual such that
an immune response against an influenza virus is produced.
[0175] One embodiment of the present invention is a method to
vaccinate an individual against influenza virus, the method
comprising administering a vaccine of the embodiments to an
individual such that an immune response against influenza virus is
produced in the individual, wherein the vaccine comprises at least
one nanoparticle comprising a monomeric subunit from ferritin
joined to an influenza hemagglutinin protein, and wherein the
nanoparticle displays the influenza hemagglutinin on its surface.
In one embodiment, the vaccine is a stem region immunogen. In one
embodiment, the vaccine is a nanoparticle. In one embodiment, the
vaccine is a monovalent vaccine. In one embodiment, the vaccine is
multivalent vaccine. Another embodiment of the present invention is
a method to vaccinate an individual against infection with
influenza virus, the method comprising:
[0176] a) obtaining a vaccine comprising at least one nanoparticle
comprising an HA-ferritin fusion protein, wherein the fusion
protein comprises a ferritin protein joined to an influenza HA
protein, and wherein the nanoparticle displays the influenza HA on
its surface; and,
[0177] b) administering the vaccine to an individual such that an
immune response against an influenza virus is produced.
[0178] In one embodiment, the nanoparticle is a monovalent
nanoparticle. In one embodiment, the nanoparticle is multivalent
nanoparticle.
[0179] In one embodiment, the nanoparticle is an octahedron. In one
embodiment, the influenza hemagglutinin protein is capable of
eliciting neutralizing antibodies to an influenza virus. In one
embodiment, the influenza HA protein is capable of eliciting
broadly neutralizing antibodies to an influenza virus. In one
embodiment, the ferritin portion of the fusion protein comprise at
least 50 amino acids, at least 100 amino acids, or at least 150
amino acids from an amino acid sequence selected from the group
consisting of SEQ ID NO:2 and SEQ ID NO:5, and/or comprises an
amino acid sequence at least 85%, at least 90%, at least 95%, at
least 97% at least 99% identical to an amino acid sequence selected
from the group consisting of SEQ ID NO:2 and SEQ ID NO:5. In one
embodiment, the HA portion of the fusion protein comprises at least
one epitope from an influenza hemagglutinin protein listed in Table
2. In one embodiment, the HA portion of the fusion protein
comprises at least 25 amino acids, at least 50 amino acids, at
least 75 amino acids, at least 100 amino acids, at least 150 amino
acids, at least 200 amino acids, at least 300 amino acids, at least
400 amino acids, or at least 500 amino acids from a hemagglutinin
protein of a virus listed in Table 2. In one embodiment, the HA
portion of the fusion protein comprises at least 25 amino acids, at
least 50 amino acids, at least 75 amino acids, at least 100 amino
acids, at least 150 amino acids, at least 200 amino acids, at least
300 amino acids, at least 400 amino acids, or at least 500 amino
acids from a protein consisting of a sequence selected from the
group consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID
NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:32, SEQ ID NO:35, and SEQ ID NO:38. In one embodiment, the HA
portion of the fusion protein comprises an amino acid sequence at
least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 99% identical to the sequence of an HA protein from a
virus listed in Table 2. In one embodiment, the HA portion of the
fusion protein comprises an amino acid sequence at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 99%
identical to a sequence selected from the group consisting of SEQ
ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20,
SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID
NO:35, and SEQ ID NO:38. In one embodiment, the HA portion of the
fusion protein comprises at least 25 amino acids, at least 50 amino
acids, at least 75 amino acids, at least 100 amino acids, at least
150 amino acids, at least 200 amino acids, at least 300 amino
acids, at least 400 amino acids, or at least 500 amino acids from a
sequence selected from the group consisting of SEQ ID NO:71, SEQ ID
NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ
ID NO:89, SEQ ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one
embodiment, the HA portion of the fusion protein comprises an amino
acid sequence at least 80%, at least 85%, at least 90%, at least
95%, at least 97%, at least 99% identical to a sequence selected
from the group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID
NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ
ID NO:92, SEQ ID NO:95, and SEQ ID NO:98. In one embodiment, the
HA-ferritin fusion protein comprises an amino acid sequence at
least 80%, at least 85%, at least 90%, at least 95%, at least 97%,
at least 99% identical to a protein sequence selected from the
group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ
ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62,
SEQ ID NO:65, and SEQ ID NO:68. In one embodiment, the HA-ferritin
fusion protein comprises an amino acid sequence selected from the
group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ
ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62,
SEQ ID NO:65, and SEQ ID NO:68. In one embodiment, the HA-ferritin
fusion protein comprises an amino acid sequence at least 80%, at
least 85%, at least 90%, at least 95%, at least 97%, at least 99%
identical to SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID
NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ
ID NO:125 and SEQ ID NO:128. In one embodiment, the HA-ferritin
fusion protein comprises SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107
SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID
NO:122 SEQ ID NO:125 and SEQ ID NO:128.
[0180] Vaccines of the present invention can be used to vaccinate
individuals using a prime/boost protocol. Such a protocol is
described in U.S. Patent Publication No. 20110177122, which is
incorporated herein by reference in its entirety. In such a
protocol, a first vaccine composition may be administered to the
individual (prime) and then after a period of time, a second
vaccine composition may be administered to the individual (boost).
Administration of the boosting composition is generally weeks or
months after administration of the priming composition, preferably
about 2-3 weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks,
or 24 weeks, or 28 weeks, or 32 weeks. In one embodiment, the
boosting composition is formulated for administration about 1 week,
or 2 weeks, or 3 weeks, or 4 weeks, or 5 weeks, or 6 weeks, or 7
weeks, or 8 weeks, or 9 weeks, or 16 weeks, or 20 weeks, or 24
weeks, or 28 weeks, or 32 weeks after administration of the priming
composition.
[0181] The first and second vaccine compositions can be, but need
not be, the same composition. Thus, in one embodiment of the
present invention, the step of administering the vaccine comprises
administering a first vaccine composition, and then at a later
time, administering a second vaccine composition. In one
embodiment, the first vaccine composition comprises a nanoparticle
comprising an HA-ferritin fusion protein of the present invention.
In one embodiment, the first vaccine composition comprises a
nanoparticle comprising an ectodomain from the hemagglutinin
protein of an influenza virus selected from the group consisting of
A/New Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009
CA, H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968
(1968 HK, H3), A/Brisbane/10/2007 (2007 Bris, H3),
A/Indonesia/05/2005 (2005 Indo, H5), B/Florida/4/2006 (2006 Flo,
B), A/Perth/16/2009 (2009 Per, H3), A/Brisbane/59/2007 (2007 Bris,
H1), B/Brisbane/60/2008 (2008 Bris, B). In one embodiment, the
hemagglutinin of the first vaccine composition comprises an amino
acid sequence at least about 80% identical to an amino acid
sequence selected from the group consisting of SEQ ID NO:8, SEQ ID
NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ
ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, and SEQ ID
NO:38. In one embodiment, the first vaccine composition comprises
an HA-ferritin fusion protein comprising an amino acid sequence at
least 80% identical, at least 85% identical, at least 90%
identical, at least 95% identical, at least 97% identical or at
least 99% identical to a sequence selected from the group
consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID
NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ
ID NO:65, and SEQ ID NO:68, wherein the nanoparticle elicits an
immune response against an influenza virus. In one embodiment, the
first vaccine composition comprises an HA-ferritin fusion protein
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID
NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ
ID NO:65, and SEQ ID NO:68. In one embodiment, the second vaccine
composition comprises a nanoparticle comprising an HA SS-ferritin
fusion protein of the present invention. In one embodiment, the HA
SS-ferritin fusion protein comprises an amino acid sequence at
least 80% identical, at least 85% identical, at least 90%
identical, at least 95% identical, at least 97% identical or at
least 99% identical to a sequence selected from the group
consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ ID
NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ
ID NO:95 and SEQ ID NO:98. In one embodiment, the HA SS-ferritin
fusion protein comprises an amino acid sequence selected from the
group consisting of SEQ ID NO:71, SEQ ID NO:74, SEQ ID NO:77, SEQ
ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92,
SEQ ID NO:95 and SEQ ID NO:98. In one embodiment, the HA
55-ferritin fusion protein comprises an amino acid sequence at
least 80% identical, at least 85% identical, at least 90%
identical, at least 95% identical, at least 97% identical or at
least 99% identical to SEQ ID NO:101, SEQ ID NO:104 SEQ ID NO:107
SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ ID
NO:122 SEQ ID NO:125 and SEQ ID NO:128, wherein the HA 55-ferritin
fusion protein elicits an immune response to an influenza virus. In
one embodiment, the HA SS-ferritin fusion protein comprises SEQ ID
NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ
ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID
NO:128. In one embodiment, the individual is at risk for infection
with influenza virus. In one embodiment, the individual has been
exposed to influenza virus. As used herein, the terms exposed,
exposure, and the like, indicate the subject has come in contact
with a person of animal that is known to be infected with an
influenza virus. Vaccines of the present invention may be
administered using techniques well known to those in the art.
Techniques for formulation and administration may be found, for
example, in "Remington's Pharmaceutical Sciences", 18.sup.th ed.,
1990, Mack Publishing Co., Easton, Pa. Vaccines may be administered
by means including, but not limited to, traditional syringes,
needleless injection devices, or microprojectile bombardment gene
guns. Suitable routes of administration include, but are not
limited to, parenteral delivery, such as intramuscular,
intradermal, subcutaneous, intramedullary injections, as well as,
intrathecal, direct intraventricular, intravenous, intraperitoneal,
intranasal, or intraocular injections, just to name a few. For
injection, the compounds of one embodiment of the invention may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hanks' solution, Ringer's solution, or
physiological saline buffer.
[0182] In one embodiment, vaccines, or nanoparticles, of the
present invention can be used to protect an individual against
infection by heterologous influenza virus. That is, a vaccine made
using hemagglutinin protein from one strain of influenza virus is
capable of protecting an individual against infection by different
strains of influenza. For example, a vaccine made using
hemagglutinin protein from influenza A/New Calcdonia/20/1999 (1999
NC, H1), can be used to protect an individual against infection by
an influenza virus including, but not limited to A/New
Calcdonia/20/1999 (1999 NC, H1), A/California/04/2009 (2009 CA,
H1), A/Singapore/1/1957 (1957 Sing, H2), A/Hong Kong/1/1968 (1968
HK, H3), A/Brisbane/10/2007 (2007 Bris, H3), A/Indonesia/05/2005
(2005 indo, H5), A/Perth/16/2009 (2009 Per, H3), and/or
A/Brisbane/59/2007 (2007 Bris, H1).
[0183] In one embodiment, vaccines, or nanoparticles, of the
present invention can be used to protect an individual against
infection by an antigenically divergent influenza virus.
Antigenically divergent refers to the tendency of a strain of
influenza virus to mutate over time, thereby changing the amino
acids that are displayed to the immune system. Such mutation over
time is also referred to as antigenic drift. Thus, for example, a
vaccine made using hemagglutinin protein from a A/New
Calcdonia/20/1999 (1999 NC, H1) strain of influenza virus is
capable of protecting an individual against infection by earlier,
antigenically divergent New Calcdonia strains of influenza, and by
evolving (or diverging) influenza strains of the future.
[0184] Because nanoparticles of the present invention display
hemagglutinin proteins that are antigenically similar to an intact
hemagglutinin, they can be used in assays for detecting antibodies
against influenza virus (anti-influenza antibodies).
[0185] Thus, one embodiment of the present invention is a method
for detecting anti-influenza virus antibodies using nanoparticles
of the present invention. A detection method of the present
invention can generally be accomplished by:
[0186] a. contacting at least a portion of a sample being tested
for the presence of anti-influenza antibodies with a nanoparticle
of the present invention; and,
[0187] b. detecting the presence of a nanoparticle/antibody
complex;
[0188] wherein the presence of a nanoparticle/antibody complex
indicates that the sample contains anti-influenza antibodies.
[0189] In one embodiment of the present invention, a sample is
obtained, or collected, from an individual to be tested for the
presence of anti-influenza virus antibodies. The individual may or
may not be suspected of having anti-influenza antibodies or of
having been exposed to influenza virus. A sample is any specimen
obtained from the individual that can be used to test for the
presence of anti-influenza virus antibodies. A preferred sample is
a body fluid that can be used to detect the presence of
anti-influenza virus antibodies. Examples of body fluids that may
be used to practice the present method include, but are not limited
to, blood, plasma, serum, lacrimal fluid and saliva. Those skilled
in the art can readily identify samples appropriate for practicing
the disclosed methods.
[0190] Blood, or blood-derived fluids such as plasma, serum, and
the like, are particularly suitable as the sample. Such samples can
be collected and prepared from individuals using methods known in
the art. The sample may be refrigerated or frozen before assay.
[0191] Any nanoparticle of the present invention can be used to
practice the disclosed method as long as the nanoparticle binds to
anti-influenza virus antibodies. Useful nanoparticles, and methods
of their production, have been described in detail herein. In a
preferred embodiment, the nanoparticle comprises a fusion protein,
wherein the fusion protein comprises at least 25, at least 50, at
least 75, at least 100, or at least 150 contiguous amino acids from
a monomeric ferritin subunit protein joined to (fused to) at least
one epitope from an influenza hemagglutinin protein (i.e., an
HA-ferritin fusion protein) such that the nanoparticle comprises
trimers of the influenza virus HA protein epitope on its surface,
and wherein the fusion protein is capable of self-assembling into
nanoparticles. In one embodiment the at least 25, at least 50, at
least 75, at least 100, or at least 150 contiguous amino acids are
from the region of a monomeric ferritin protein corresponding to
the amino acid sequences of the Helicobacter pylori ferritin
monomeric subunit that direct self-assembly of the monomeric
subunits into the globular form of the ferritin protein. In one
embodiment the at least 25, at least 50, at least 75, at least 100,
or at least 150 contiguous amino acids are from SEQ ID NO:2, and
are capable of directing self-assembly of the monomeric subunits
into the globular ferritin protein. In one embodiment the at least
25, at least 50, at least 75, at least 100, or at least 150
contiguous amino acids are from amino acid residues 5-167 of SEQ ID
NO:2, or from SEQ ID NO:5, wherein the HA-ferritin fusion protein
is capable of self-assembling into nanoparticles.
[0192] In one embodiment the nanoparticle comprises a fusion
protein comprising an amino acid sequence at least 80%, at least
85%, at least 90%, at least 95%, or at least 97% identical to the
amino acid sequence of a monomeric ferritin subunit that is
responsible for directing self-assembly of the monomeric ferritin
subunits into the globular form of the protein, wherein the fusion
protein is capable of self-assembling into nanoparticles. In one
embodiment, the fusion protein comprises a polypeptide sequence
identical in sequence to a monomeric ferritin subunit. In one
embodiment the fusion protein comprises an amino acid sequence at
least 80%, at least 85%, at least 90%, at least 95%, or at least
97% identical to the amino acid sequence of a monomeric ferritin
subunit from Helicobacter pylori, wherein the HA-ferritin fusion
protein is capable of self-assembling into nanoparticles. In one
embodiment the fusion protein comprises an amino acid sequence at
least 80%, at least 85%, at least 90%, at least 95%, or at least
97% identical to the amino acid sequence of to a sequence selected
from the group consisting of (1) amino acid residues 5-167 from SEQ
ID NO:2 and (2) SEQ ID NO:5, wherein the HA-ferritin fusion protein
is capable of self-assembling into nanoparticles. In one embodiment
the fusion protein comprises a sequence selected from the group
consisting of (1) amino acid residues 5-167 from SEQ ID NO:2 and
(2) SEQ ID NO:5.
[0193] In one embodiment, the nanoparticle comprises a fusion
protein comprising a ferritin protein of the present invention
joined to at least one immunogenic portion of an HA protein from a
virus selected from the group consisting of influenza type A
viruses, influenza type B viruses and influenza type C viruses. In
one embodiment the fusion protein comprises a ferritin protein of
the present invention joined to at least one immunogenic portion of
an HA protein selected from the group consisting of an H1 influenza
virus HA protein, an H2 influenza virus HA protein, H3 influenza
virus HA protein, an H4 influenza virus HA protein, an H5 influenza
virus HA protein, an H6 influenza virus HA protein, an H7 virus
influenza HA protein, an H8 influenza virus HA protein, an H9
influenza virus HA protein, an H10 influenza virus HA protein, an
H11 influenza virus HA protein, an H12 influenza virus HA protein,
an H13 influenza virus HA protein, an H14 influenza virus HA
protein, an H15 influenza virus HA protein, and an H16 influenza
virus HA protein. In one embodiment, the fusion protein comprises a
ferritin protein of the present invention joined to at least one
immunogenic portion of an HA protein from virus listed in Table 2.
In, one embodiment the immunogenic portion comprises at least one
epitope.
[0194] In one embodiment, the nanoparticle comprises a fusion
protein comprising a ferritin protein of the present invention
joined to an amino acid sequence that is a variant of an HA protein
from a virus selected from the group consisting of influenza Type A
viruses influenza type B viruses and influenza type C viruses,
wherein the fusion protein is capable of selectively binding
anti-influenza antibodies. In one embodiment, the fusion protein
comprises a ferritin protein of the present invention joined to an
amino acid sequence at least about 80%, at least about 85%, at
least about 90%, at least about 95%, at least about 97% or at least
about 99% identical to the sequence of a HA protein from a virus
selected from the group consisting of influenza Type A viruses
influenza Type B viruses and influenza type C viruses, wherein the
fusion protein is capable of selectively binding anti-influenza
antibodies. In one embodiment, the fusion protein comprises a
ferritin protein of the present invention joined to an amino acid
sequence at least about 80%, at least about 85%, at least about
90%, at least about 95%, at least about 97% or at least about 99%
identical to the sequence of a HA protein selected from the group
consisting an H1 influenza virus HA protein, an H2 influenza virus
HA protein, H3 influenza virus HA protein, an H4 influenza virus HA
protein, an H5 influenza virus HA protein, an H6 influenza virus HA
protein, an H7 virus influenza HA protein, an H8 influenza virus HA
protein, an H9 influenza virus HA protein, an H10 influenza virus
HA protein, an H11 influenza virus HA protein, an H12 influenza
virus HA protein, an H13 influenza virus HA protein, an H14
influenza virus HA protein, an H15 influenza virus HA protein, and
an H16 influenza virus HA protein, wherein the fusion protein is
capable of selectively binding anti-influenza antibodies. In one
embodiment, the fusion protein comprises a ferritin protein of the
present invention joined to an amino acid sequence at least about
80%, at least about 85%, at least about 90%, at least about 95%, at
least about 97% or at least about 99% identical to the sequence of
a HA protein from a virus listed in Table 2, wherein the fusion
protein is capable of selectively binding anti-influenza
antibodies. In one embodiment, the fusion protein comprises a
ferritin protein of the present invention joined to amino acid
sequence at least about 80%, at least about 85%, at least about
90%, at least about 95%, at least about 97% or at least about 99%
identical to a sequence selected from the group consisting of SEQ
ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20,
SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID
NO:35, and SEQ ID NO:38, wherein the fusion protein is capable of
selectively binding anti-influenza antibodies. In one embodiment,
the fusion protein comprises a ferritin protein of the present
invention joined to amino acid sequence selected from the group
consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID
NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:32, SEQ ID NO:35, and SEQ ID NO:38, wherein the fusion
protein is capable of selectively binding anti-influenza
antibodies.
[0195] In one embodiment the nanoparticle comprises a fusion
protein comprising an amino acid sequence at least 80%, at least
about 85%, at least about 90%, at least about 95%, at least about
97% or at least about 99% identical to a sequence selected from the
group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ
ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62,
SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:101, SEQ ID NO:104 SEQ ID
NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ
ID NO:122 SEQ ID NO:125 and SEQ ID NO:128, wherein the fusion
protein is capable of selectively binding anti-influenza
antibodies. In one embodiment the fusion protein comprises an amino
acid sequence selected from the group consisting of SEQ ID NO:41,
SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID
NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, SEQ
ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113
SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID
NO:128.
[0196] As used herein, the term contacting refers to the
introduction of a sample being tested for the presence of
anti-influenza antibodies to a nanoparticle of the present
invention, for example, by combining or mixing the sample and the
nanoparticle of the present invention, such that the nanoparticle
is able to come into physical contact with antibodies in the
sample, if present. When anti-influenza virus antibodies are
present in the sample, an antibody/nanoparticle complex is then
formed. Such complex formation refers to the ability of an
anti-influenza virus antibodies to selectively bind to the HA
portion of the fusion protein in the nanoparticle in order to form
a stable complex that can be detected. Binding of anti-influenza
virus antibodies in the sample to the nanoparticle is accomplished
under conditions suitable to form a complex. Such conditions (e.g.,
appropriate concentrations, buffers, temperatures, reaction times)
as well as methods to optimize such conditions are known to those
skilled in the art. Binding can be measured using a variety of
methods standard in the art including, but not limited to,
agglutination assays, precipitation assays, enzyme immunoassays
(e.g., ELISA), immunoprecipitation assays, immunoblot assays and
other immunoassays as described, for example, in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Labs
Press, 1989), and Harlow et al., Antibodies, a Laboratory Manual
(Cold Spring Harbor Labs Press, 1988), both of which are
incorporated by reference herein in their entirety. These
references also provide examples of complex formation
conditions.
[0197] As used herein, the phrases selectively binds hemagglutinin,
selective binding to hemagglutinin, and the like, refer to the
ability of an antibody to preferentially bind a HA protein as
opposed to binding proteins unrelated to HA, or non-protein
components in the sample or assay. An antibody that selectively
binds HA is one that binds albumin but does not significantly bind
other molecules or components that may be present in the sample or
assay. Significant binding, is considered, for example, binding of
an anti-HA antibody to a non-HA molecule with an affinity or
avidity great enough to interfere with the ability of the assay to
detect and/or determine the level of, anti-influenza antibodies in
the sample. Examples of other molecules and compounds that may be
present in the sample, or the assay, include, but are not limited
to, non-HA proteins, such as albumin, lipids and carbohydrates.
[0198] In one embodiment, an anti-influenza virus
antibody/nanoparticle complex, also referred to herein as an
antibody/nanoparticle complex, can be formed in solution. In one
embodiment an antibody/nanoparticle complex can be formed in which
the nanoparticle is immobilized on (e.g., coated onto) a substrate.
Immobilization techniques are known to those skilled in the art.
Suitable substrate materials include, but are not limited to,
plastic, glass, gel, celluloid, fabric, paper, and particulate
materials. Examples of substrate materials include, but are not
limited to, latex, polystyrene, nylon, nitrocellulose, agarose,
cotton, PVDF (poly-vinylidene-fluoride), and magnetic resin.
Suitable shapes for substrate material include, but are not limited
to, a well (e.g., microtiter dish well), a microtiter plate, a
dipstick, a strip, a bead, a lateral flow apparatus, a membrane, a
filter, a tube, a dish, a celluloid-type matrix, a magnetic
particle, and other particulates. Particularly preferred substrates
include, for example, an ELISA plate, a dipstick, an immunodot
strip, a radioimmunoassay plate, an agarose bead, a plastic bead, a
latex bead, a cotton thread, a plastic chip, an immunoblot
membrane, an immunoblot paper and a flow-through membrane. In one
embodiment, a substrate, such as a particulate, can include a
detectable marker. For descriptions of examples of substrate
materials, see, for example, Kemeny, D. M. (1991) A Practical Guide
to ELISA, Pergamon Press, Elmsford, N.Y. pp 33-44, and Price, C.
and Newman, D. eds. Principles and Practice of Immunoassay, 2nd
edition (1997) Stockton Press, NY, N.Y., both of which are
incorporated herein by reference in their entirety.
[0199] In accordance with the present invention, once formed, an
anti-influenza virus antibody/nanoparticle complex is detected.
Detection can be qualitative, quantitative, or semi-quantitative.
As used herein, the phrases detecting complex formation, detecting
the complex, and the like, refer to identifying the presence of
anti-influenza virus antibody complexed with the nanoparticle. If
complexes are formed, the amount of complexes formed can, but need
not be, quantified. Complex formation, or selective binding,
between a putative anti-influenza virus antibody and a nanoparticle
can be measured (i.e., detected, determined) using a variety of
methods standard in the art (see, for example, Sambrook et al.
supra.), examples of which are disclosed herein. A complex can be
detected in a variety of ways including, but not limited to use of
one or more of the following assays: a hemagglutination inhibition
assay, a radial diffusion assay, an enzyme-linked immunoassay, a
competitive enzyme-linked immunoassay, a radioimmunoassay, a
fluorescence immunoassay, a chemiluminescent assay, a lateral flow
assay, a flow-through assay, a particulate-based assay (e.g., using
particulates such as, but not limited to, magnetic particles or
plastic polymers, such as latex or polystyrene beads), an
immunoprecipitation assay, a BioCoreJ assay (e.g., using colloidal
gold), an immunodot assay (e.g., CMG=s Immunodot System, Fribourg,
Switzerland), and an immunoblot assay (e.g., a western blot), an
phosphorescence assay, a flow-through assay, a chromatography
assay, a PAGe-based assay, a surface plasmon resonance assay, a
spectrophotometric assay, and an electronic sensory assay. Such
assays are well known to those skilled in the art.
[0200] Assays can be used to give qualitative or quantitative
results depending on how they are used. Some assays, such as
agglutination, particulate separation, and precipitation assays,
can be observed visually (e.g., either by eye or by a machines,
such as a densitometer or spectrophotometer) without the need for a
detectable marker.
[0201] In other assays, conjugation (i.e., attachment) of a
detectable marker to the nanoparticle, or to a reagent that
selectively binds to the nanoparticle, aids in detecting complex
formation. A detectable marker can be conjugated to the
nanoparticle, or nanoparticle-binding reagent, at a site that does
not interfere with ability of the nanoparticle to bind to an
anti-influenza virus antibody. Methods of conjugation are known to
those of skill in the art. Examples of detectable markers include,
but are not limited to, a radioactive label, a fluorescent label, a
chemiluminescent label, a chromophoric label, an enzyme label, a
phosphorescent label, an electronic label; a metal sol label, a
colored bead, a physical label, or a ligand. A ligand refers to a
molecule that binds selectively to another molecule. Preferred
detectable markers include, but are not limited to, fluorescein, a
radioisotope, a phosphatase (e.g., alkaline phosphatase), biotin,
avidin, a peroxidase (e.g., horseradish peroxidase),
beta-galactosidase, and biotin-related compounds or avidin-related
compounds (e.g., streptavidin or ImmunoPure7 NeutrAvidin).
[0202] In one embodiment, an antibody/nanoparticle complex can be
detected by contacting a sample with a specific compound, such as
an antibody, that binds to an anti-influenza antibody, ferritin, or
to the antibody/nanoparticle complex, conjugated to a detectable
marker. A detectable marker can be conjugated to the specific
compound in such a manner as not to block the ability of the
compound to bind to the complex being detected. Preferred
detectable markers include, but are not limited to, fluorescein, a
radioisotope, a phosphatase (e.g., alkaline phosphatase), biotin,
avidin, a peroxidase (e.g., horseradish peroxidase),
beta-galactosidase, and biotin-related compounds or avidin-related
compounds (e.g., streptavidin or ImmunoPure7 NeutrAvidin).
[0203] In another embodiment, a complex is detected by contacting
the complex with an indicator molecule. Suitable indicator
molecules include molecules that can bind to the anti-influenza
virus antibody/nanoparticle complex, the anti-influenza virus
antibody, or the nanoparticle. As such, an indicator molecule can
comprise, for example, a reagent that binds the anti-influenza
virus antibody, such as an antibody that recognizes
immunoglobulins. Preferred indicator molecules that are antibodies
include, for example, antibodies reactive with the antibodies from
species of individual in which the anti-influenza virus antibodies
are produced. An indicator molecule itself can be attached to a
detectable marker of the present invention. For example, an
antibody can be conjugated to biotin, horseradish peroxidase,
alkaline phosphatase or fluorescein.
[0204] The present invention can further comprise one or more
layers and/or types of secondary molecules or other binding
molecules capable of detecting the presence of an indicator
molecule. For example, an untagged (i.e., not conjugated to a
detectable marker) secondary antibody that selectively binds to an
indicator molecule can be bound to a tagged (i.e., conjugated to a
detectable marker) tertiary antibody that selectively binds to the
secondary antibody. Suitable secondary antibodies, tertiary
antibodies and other secondary or tertiary molecules can be readily
selected by those skilled in the art. Preferred tertiary molecules
can also be selected by those skilled in the art based upon the
characteristics of the secondary molecule. The same strategy can be
applied for subsequent layers.
[0205] Preferably, the indicator molecule is conjugated to a
detectable marker. A developing agent is added, if required, and
the substrate is submitted to a detection device for analysis. In
some protocols, washing steps are added after one or both complex
formation steps in order to remove excess reagents. If such steps
are used, they involve conditions known to those skilled in the art
such that excess reagents are removed but the complex is
retained.
[0206] While the assays described thus far directly detect the
antibody/nanoparticle complex, it is also possible to detect the
complex using indirect methods. One example of an indirect assay is
the hemagglutination inhibition (HAI) assay, which is a standard
assay used to identify the levels of influenza virus in the serum
of people thought to be infected with the virus. General methods
for performing hemagglutinin inhibition assays are taught herein
and are also disclosed in, for example, (see, for example, the WHO
Reference on Animal Influenza Diagnosis and Surveillance, 2002,
Department of Communicable Disease Surveillance and Response, World
Health Organization).
[0207] The HAI assay is based on the fact that hemagglutinin
protein on influenza virus is able to bind sialic acid molecules on
red blood cells. Thus, when influenza virus is mixed with red blood
cells, the virus and the red blood cells bind together forming a
lattice (see FIG. 45), with the result that the lattice settles to
the bottom of the reaction vessel (e.g., the well of an 96-well
plate) forming a diffuse, red color over the entire surface. In
contrast, if the reaction contains anything that interferes with
binding of the HA protein to the sialic acid molecules on the red
blood cells (such as anti-HA antibodies), formation of the lattice
is prevented causing the red blood cells to fall out of solution
and pool in the bottom of the container. In the latter case, the
red blood cells form a characteristic red dot or "button" in the
bottom of the well (see, for example, FIG. 46). In the HAI assay, a
sample to be tested for the presence of anti-influenza antibodies
is added to the influenza virus/red blood cell mixture (the assay
mix) and the effect on button formation is observed. If the test
sample contains anti-influenza antibodies, they will bind to the
virus in the assay mixture, preventing, or disrupting, formation of
the lattice causing the red blood cells to fall out of solution,
pool in the well and form a button (or dot). The titer of the
antibody present in the test sample is determined by counting how
far the sample can be diluted before its hemagglutination
inhibiting activity is lost. For example, if a sample diluted
1:1000 stil produces a dot nd the next dilution (1:2000) results in
lattice formation (no dot) then the titer of antibody in the sample
is 1:1000. Such methods of determining antibody titers are known to
those skilled in the art.
[0208] A problem with the HAI assay is that it requires the use of
live influenza virus, which due to the inherent dangers of growing
such virus, requires the use of BSL2 and BSL3 facilities. However,
such problems can be alleviated by using nanoparticles of the
present invention in the HAI assay since they are fully recombinant
and are made without the need to produce potentially dangerous live
virus in eggs or in cell cultures. Moreover, because the fusion
proteins making up the nanoparticles are recombinant, they offer
the opportunity to test the effect of mutations in the HA protein
that would otherwise inactivate viral replication. Thus, a HAI
assay using nanoparticles of the present invention offers
improvements and benefits not found in the currently available HAI
assay.
[0209] Accordingly, in one embodiment, detection of an
anti-influenza virus antibody/nanoparticle complex is conducted
using a hemagglutination inhibition assay, wherein the
hemagglutination inhibition assay is performed using nanoparticles
of the present invention instead of influenza virus. More
specifically, one embodiment of the present invention is a method
for detecting anti-influenza virus antibodies in a sample, the
method comprising: [0210] a. contacting at least a portion of the
sample with a nanoparticle of the present invention and with red
blood cells comprising sialic acid molecules, to form a test
mixture; [0211] b. analyzing the test mixture for the presence of
pooled red blood cells (i.e., a button), wherein the presence of
pooled red blood cells (i.e., a button) in indicative of the
presence anti-influenza antibodies in the sample.
[0212] As has been described, the presence of anti-influenza
antibodies in the test sample (at least at sufficient levels)
causes the red blood cells to fall out of solution and pool,
thereby forming a "button" (or dot) at the bottom of the vessel
(e.g., ELISA well) housing the test mixture. Thus, formation of a
button in the vessel containing the test mixture is indicative of
the presence of anti-influenza antibodies in the test sample.
[0213] The level of red blood cell pooling (i.e., button formation)
can be determined using any of the techniques known in the art for
conducting hemagglutinin inhibition assays. In some embodiments,
the level of button formation may be determined by simple visual
inspection using the naked eye. In some embodiments, the level of
button formation may be determined using a magnifying device such
as, for example, a dissecting scope) or a microscope. In some
embodiments, the level of button formation may be determined using
a device such as, for example, a spectrophotometer or a
refractometer. Methods of detecting or measuring button formation
are known to those skilled in the art.
[0214] Any nanoparticle of the present invention can be used to
practice hemagglutination inhibition assays of the present
invention as long as it is capable of binding to sialic acid
residues and to anti-influenza virus antibodies. Useful
nanoparticles and methods of their production have been disclosed
in detail herein. In a preferred embodiment, the nanoparticle
comprises a fusion protein, wherein the fusion protein comprises at
least 25, at least 50, at least 75, at least 100, or at least 150
contiguous amino acids from a monomeric ferritin subunit protein
joined to at least one epitope from an influenza hemagglutinin
protein (i.e., an HA-ferritin fusion protein) such that the
nanoparticle comprises trimers of the influenza virus HA protein
epitope on its surface, and wherein the fusion protein is capable
of self-assembling into nanoparticles. In one embodiment the at
least 25, at least 50, at least 75, at least 100, or at least 150
contiguous amino acids are from the region of a monomeric ferritin
protein corresponding to the amino acid sequences of the
Helicobacter pylori ferritin monomeric subunit that direct
self-assembly of the monomeric subunits into the globular form of
the ferritin protein. In one embodiment the at least 25, at least
50, at least 75, at least 100, or at least 150 contiguous amino
acids are from SEQ ID NO:2, and are capable of directing
self-assembly of the monomeric subunits into the globular ferritin
protein. In one embodiment the at least 25, at least 50, at least
75, at least 100, or at least 150 contiguous amino acids are from
amino acid residues 5-167 of SEQ ID NO:2, or from SEQ ID NO:5,
wherein the HA-ferritin fusion protein is capable of
self-assembling into nanoparticles.
[0215] In one embodiment the nanoparticle comprises a fusion
protein comprising an amino acid sequence at least 80%, at least
85%, at least 90%, at least 95%, or at least 97% identical to the
amino acid sequence of a monomeric ferritin subunit that is
responsible for directing self-assembly of the monomeric ferritin
subunits into the globular form of the protein, wherein the fusion
protein is capable of self-assembling into nanoparticles. In one
embodiment, the fusion protein comprises a polypeptide sequence
identical in sequence to a monomeric ferritin subunit. In one
embodiment the fusion protein comprises an amino acid sequence at
least 80%, at least 85%, at least 90%, at least 95%, or at least
97% identical to the amino acid sequence of a monomeric ferritin
subunit from Helicobacter pylori, wherein the HA-ferritin fusion
protein is capable of self-assembling into nanoparticles. In one
embodiment the fusion protein comprises an amino acid sequence at
least 80%, at least 85%, at least 90%, at least 95%, or at least
97% identical to the amino acid sequence of to a sequence selected
from the group consisting of (1) amino acid residues 5-167 from SEQ
ID NO:2 and (2) SEQ ID NO:5, wherein the HA-ferritin fusion protein
is capable of self-assembling into nanoparticles. In one embodiment
the fusion protein comprises a sequence selected from the group
consisting of (1) amino acid residues 5-167 from SEQ ID NO:2 and
(2) SEQ ID NO:5.
[0216] In one embodiment, the nanoparticle comprises a fusion
protein comprising a ferritin protein of the present invention
joined to at least one immunogenic portion of an HA protein from a
virus selected from the group consisting of influenza type A
viruses, influenza type B viruses and influenza type C viruses. In
one embodiment the fusion protein comprises a ferritin protein of
the present invention joined to at least one immunogenic portion of
an HA protein selected from the group consisting of an H1 influenza
virus HA protein, an H2 influenza virus HA protein, H3 influenza
virus HA protein, an H4 influenza virus HA protein, an H5 influenza
virus HA protein, an H6 influenza virus HA protein, an H7 virus
influenza HA protein, an H8 influenza virus HA protein, an H9
influenza virus HA protein, an H10 influenza virus HA protein, an
H11 influenza virus HA protein, an H12 influenza virus HA protein,
an H13 influenza virus HA protein, an H14 influenza virus HA
protein, an H15 influenza virus HA protein, and an H16 influenza
virus HA protein. In one embodiment, the fusion protein comprises a
ferritin protein of the present invention joined to at least one
immunogenic portion of an HA protein from an influenza virus of the
Victoria of Yamagata lineage. In one embodiment, the fusion protein
comprises a ferritin protein of the present invention joined to at
least one immunogenic portion of an HA protein from virus listed in
Table 2. In, one embodiment the immunogenic portion comprises at
least one epitope.
[0217] In one embodiment, the nanoparticle comprises a fusion
protein comprising a ferritin protein of the present invention
joined to an amino acid sequence that is a variant of an HA protein
from a virus selected from the group consisting of influenza Type A
viruses influenza Type B viruses and influenza type C viruses,
wherein the fusion protein is capable of selectively binding
anti-influenza antibodies. In one embodiment, the fusion protein
comprises a ferritin protein of the present invention joined to an
amino acid sequence at least about 80%, at least about 85%, at
least about 90%, at least about 95%, at least about 97% or at least
about 99% identical to the sequence of a HA protein from a virus
selected from the group consisting of influenza Type A viruses
influenza Type B viruses and influenza type C viruses, wherein the
fusion protein is capable of selectively binding anti-influenza
antibodies. In one embodiment, the fusion protein comprises a
ferritin protein of the present invention joined to an amino acid
sequence at least about 80%, at least about 85%, at least about
90%, at least about 95%, at least about 97% or at least about 99%
identical to the sequence of a HA protein selected from the group
consisting an H1 influenza virus HA protein, an H2 influenza virus
HA protein, H3 influenza virus HA protein, an H4 influenza virus HA
protein, an H5 influenza virus HA protein, an H6 influenza virus HA
protein, an H7 virus influenza HA protein, an H8 influenza virus HA
protein, an H9 influenza virus HA protein, an H10 influenza virus
HA protein, an H11 influenza virus HA protein, an H12 influenza
virus HA protein, an H13 influenza virus HA protein, an H14
influenza virus HA protein, an H15 influenza virus HA protein, and
an H16 influenza virus HA protein, wherein the fusion protein is
capable of selectively binding anti-influenza antibodies. In one
embodiment, the fusion protein comprises a ferritin protein of the
present invention joined to an amino acid sequence at least about
80%, at least about 85%, at least about 90%, at least about 95%, at
least about 97% or at least about 99% identical to the sequence of
a HA protein from a virus listed in Table 2, wherein the fusion
protein is capable of selectively binding anti-influenza
antibodies. In one embodiment, the fusion protein comprises a
ferritin protein of the present invention joined to amino acid
sequence at least about 80%, at least about 85%, at least about
90%, at least about 95%, at least about 97% or at least about 99%
identical to a sequence selected from the group consisting of SEQ
ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20,
SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID
NO:35, and SEQ ID NO:38, wherein the fusion protein is capable of
selectively binding anti-influenza antibodies. In one embodiment,
the fusion protein comprises a ferritin protein of the present
invention joined to amino acid sequence selected from the group
consisting of SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID
NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ
ID NO:32, SEQ ID NO:35, and SEQ ID NO:38, wherein the fusion
protein is capable of selectively binding anti-influenza
antibodies.
[0218] In one embodiment the nanoparticle comprises a fusion
protein comprising an amino acid sequence at least 80%, at least
about 85%, at least about 90%, at least about 95%, at least about
97% or at least about 99% identical to a sequence selected from the
group consisting of SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ
ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62,
SEQ ID NO:65, SEQ ID NO:68, SEQ ID NO:101, SEQ ID NO:104 SEQ ID
NO:107 SEQ ID NO:110 SEQ ID NO:113 SEQ ID NO:116 SEQ ID NO:119 SEQ
ID NO:122 SEQ ID NO:125 and SEQ ID NO:128, wherein the fusion
protein is capable of selectively binding anti-influenza
antibodies. In one embodiment the fusion protein comprises an amino
acid sequence selected from the group consisting of SEQ ID NO:41,
SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID
NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68, SEQ
ID NO:101, SEQ ID NO:104 SEQ ID NO:107 SEQ ID NO:110 SEQ ID NO:113
SEQ ID NO:116 SEQ ID NO:119 SEQ ID NO:122 SEQ ID NO:125 and SEQ ID
NO:128.
[0219] Any red blood cell can be used to perform a HAI assay of the
present invention as long as the red blood cells comprise sialic
acid residues that are able to bind to influenza hemagglutinin
protein. Examples of suitable red bloods cells include chicken red
bloods cells, and turkey red bloods cells? Those skilled in the art
are capable of identifying red blood cells useful for practicing
the disclosed invention. Likewise, conditions suitable for
formation of a complex between a nanoparticle and an anti-influenza
antibody, if present, are known to those skilled in the art.
[0220] Because assays of the present invention can detect
anti-influenza virus antibodies in a sample, including a blood
sample, such assays can be used to identify individuals having
anti-influenza antibodies. Thus, one embodiment of the present
invention is a method to identify an individual having
anti-influenza virus antibodies, the method comprising: [0221] a.
contacting a sample from an individual being tested for
anti-influenza antibodies with a nanoparticle of the present
invention; and, [0222] b. analyzing the contacted sample for the
presence of a nanoparticle/antibody complex [0223] wherein the
presence of a nanoparticle/antibody complex indicates the
individual has anti-influenza antibodies.
[0224] Any of the disclosed assay formats can be used to conduct
the disclosed method. Examples of useful assay formats include, but
are not limited to, a hemagglutination inhibition assay, a radial
diffusion assay, an enzyme-linked immunoassay, a competitive
enzyme-linked immunoassay, a radioimmunoassay, a fluorescence
immunoassay, a chemiluminescent assay, a lateral flow assay, a
flow-through assay, a particulate-based assay (e.g., using
particulates such as, but not limited to, magnetic particles or
plastic polymers, such as latex or polystyrene beads), an
immunoprecipitation assay, a BioCoreJ assay (e.g., using colloidal
gold), an immunodot assay (e.g., CMG=s Immunodot System, Fribourg,
Switzerland), and an immunoblot assay (e.g., a western blot), an
phosphorescence assay, a flow-through assay, a chromatography
assay, a PAGe-based assay, a surface plasmon resonance assay, a
spectrophotometric assay, and an electronic sensory assay.
[0225] If no anti-influenza antibodies are detected in the sample,
such a result indicates the individual does not have anti-influenza
virus antibodies. The individual being tested may or may not be
suspected of having antibodies to influenza virus. The disclosed
methods may also be used to determine if an individual has been
exposed to one or more specific type, group, sub-group or strain of
influenza virus. To make such a determination, a sample is obtained
from an individual that has tested negative for antibodies (i.e.,
lacked antibodies) to one or more specific type, group, sub-group
or strain of influenza virus sometime in their past (e.g., greater
than about 1 year, greater than about 2 years, greater than about 3
years, greater than about 4 years, greater than about 5 years,
etc.). The sample is then tested for the presence of anti-influenza
virus antibodies to one or more type, group, sub-group or strain,
of influenza virus using a nanoparticle-based assay of the present
invention. If the assay indicates the presence of such antibodies,
the individual is then identified as having been exposed to one or
more type, group sub-group or strain, of influenza virus sometime
after the test identifying them as negative for anti-influenza
antibodies. Thus, one embodiment of the present invention is method
to identify an individual that has been exposed to influenza virus,
the method comprising: [0226] a. contacting at least a portion of a
sample from an individual being tested for anti-influenza
antibodies with a nanoparticle of the present invention; and,
[0227] b. analyzing the contacted sample for the presence or level
of a antibody/nanoparticle complex, wherein the presence or level
of antibody/nanoparticle complex indicates the presence or level of
recent anti-influenza antibodies; [0228] c. comparing the recent
anti-influenza antibody level with a past anti-influenza antibody
level; [0229] wherein an increase in the recent anti-influenza
antibody level over the past anti-influenza antibody level
indicates the individual has been exposed to influenza virus
subsequent to determination of the past anti-influenza antibody
level.
[0230] Methods of the present invention are also useful for
determining the response of an individual to a vaccine. Thus, one
embodiment is a method for measuring the response of an individual
to an influenza vaccine, the method comprising: [0231] a.
administering to the individual a vaccine for influenza virus;
[0232] b. contacting at least a portion of a sample from the
individual with a nanoparticle of the present invention; [0233] c.
analyzing the contacted sample for the presence or level of a
antibody/nanoparticle complex, wherein the presence or level of
antibody/nanoparticle complex indicates the presence or level of
recent anti-influenza antibodies [0234] wherein an increase in the
level of antibody in the sample over the pre-vaccination level of
antibody in the individual indicates the vaccine induced an immune
response in the individual.
[0235] The influenza vaccine administered to the individual may,
but need not, comprise a vaccine of the present invention, as long
as the nanoparticle comprises an HA protein that can bind an
anti-influenza antibody induced by the administered vaccine.
Methods of administering influenza vaccines are known to those of
skill in the art.
[0236] Analysis of the sample obtained from the individual may be
performed using any of the disclosed assay formats. In one
embodiment, analysis of the sample is performed using an assay
format selected from the group consisting of, a hemagglutination
inhibition assay, a radial diffusion assay, an enzyme-linked
immunoassay, a competitive enzyme-linked immunoassay, a
radioimmunoassay, a fluorescence immunoassay, a chemiluminescent
assay, a lateral flow assay, a flow-through assay, a
particulate-based assay (e.g., using particulates such as, but not
limited to, magnetic particles or plastic polymers, such as latex
or polystyrene beads), an immunoprecipitation assay, a BioCoreJ
assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG=s
Immunodot System, Fribourg, Switzerland), and an immunoblot assay
(e.g., a western blot), an phosphorescence assay, a flow-through
assay, a chromatography assay, a PAGe-based assay, a surface
plasmon resonance assay, a spectrophotometric assay, and an
electronic sensory assay.
[0237] In one embodiment, the method includes a step of determining
the level of anti-influenza antibody present in the individual
prior to administering the vaccine. However, it is also possible to
determine the level of anti-influenza antibody present in the
individual from prior medical records, if such information is
available.
[0238] While not necessary to perform the disclosed method, it may
be preferable to wait some period of time between the step of
administering the vaccine and the step of determining the level of
anti-influenza antibody in the individual. In one embodiment,
determination of the level of anti-influenza antibodies present in
the individual is performed at least 1 day, at least 2 days, at
least 3 days, at least 4 days, at least 5 days, at least 6 days, at
least one week, at least two weeks, at least three weeks, at least
four weeks, at least two months, at least three months or at least
six months, following administration of the vaccine.
[0239] The present invention also includes kits suitable for
detecting anti-influenza antibodies. Suitable means of detection
include the techniques disclosed herein, utilizing nanoparticles of
the present invention. Kits may also comprise a detectable marker,
such as an antibody that selectively binds to the nanoparticle, or
other indicator molecules. The kit can also contain associated
components, such as, but not limited to, buffers, labels,
containers, inserts, tubings, vials, syringes and the like.
EXAMPLES
[0240] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the embodiments, and are not
intended to limit the scope of what the inventors regard as their
invention nor are they intended to represent that the experiments
below are all or the only experiments performed. Efforts have been
made to ensure accuracy with respect to numbers used (e.g. amounts,
temperature, etc.) but some experimental errors and deviations
should be accounted for. Unless indicated otherwise, parts are
parts by weight, molecular weight is weight average molecular
weight, and temperature is in degrees Celsius. Standard
abbreviations are used.
Example 1
Design and Production of Ferritin-Based Nanoparticles Expressing
Influenza Virus HA
[0241] This Example demonstrates the ability of HA-ferritin fusion
proteins to form nanoparticles. Analysis of ferritin structure
suggested that it was possible to insert a heterologous protein,
specifically influenza virus HA, so that it mimics a
physiologically relevant trimeric viral spike (FIG. 1A). Ferritin
forms a nearly spherical particle consisting of 24 subunits
arranged with octahedral symmetry around a hollow interior. The
symmetry of the ferritin nanoparticles includes eight three-fold
axes on the surface. The aspartic acid (Asp) at residue 5 near the
NH.sub.2 terminus is readily accessible, and the distance (28
.ANG.) between each Asp5 on the three-fold axis is almost identical
to the distance between the central axes of each HA2 subunit of
trimeric HA (FIG. 1A, right).
Vector Construction.
[0242] The HA-ferritin fusion proteins were constructed by joining
the ectodomain of A/New Calcdonia/20/1999 (1999 NC) HA to ferritin
(FIG. 1B). Specifically, the gene encoding H. pylori nonheme
iron-containing ferritin (GenBank NP.sub.--223316) with a point
mutation (N19Q) to abolish a potential N-linked glycosylation site
was synthesized by PCR-based accurate synthesis (M. F. Bachmann, R.
M. Zinkernagel, Neutralizing antiviral B cell responses. Annu Rev
Immunol 15, 235-270 (1997)) using human-preferred codons. The human
CD5 leader sequence and a serine-glycine-glycine (SGG) spacer were
joined to the gene fragment encoding ferritin (residues 5-167) to
generate a secreted protein. The plasmids encoding various
influenza virus HAs, including A/South Carolina/1/1918 (1918 SC),
GenBank AF117241; A/Puerto Rico/8/1934 (1934 PR8), UniProt P03452;
A/Singapore/6/1986 (1986 Sing), GenBank AB038395;
A/Beijing/262/1995 (1995 Beijing), GenBank AAP34323; A/New
Calcdonia/20/1999 (1999 NC), GenBank AY289929; A/Solomon
Islands/3/2006 (2006 SI), GenBank ABU99109; A/Brisbane/59/2007
(2007 Bris), GenBank ACA28844; A/California/04/2009 (2009 CA),
GenBank ACP41105; A/Perth/16/2009 (H3 2009 Perth), GenBank
ACS71642; B/Florida/04/2006 (B 2006 Florida), GenBank ACA33493 and
their corresponding NAs with human preferred codons were
synthesized as previously reported (C. J. Wei et al., Induction of
broadly neutralizing H1N1 influenza antibodies by vaccination.
Science 329, 1060-1064 (2010)). The gene fragments encoding HAs
(residues HA1 1-HA2 174, H3 numbering) from 1999 NC HA, 2009 CA HA,
2009 Perth H3 and 2006 Florida B were amplified and joined to the
ferritin gene fragment (residues 5-167) with an SGG linker to give
rise to the HA-ferritin fusion gene. To produce soluble trimeric
HA, the 1999 NC HA gene fragment (residues HA1 1-HA2 174, H3
numbering) was joined to a thrombin cleavage site followed by a
foldon trimerization motif and a poly-histidine tag as described
previously (A. S. Xiong et al., PCR-based accurate synthesis of
long DNA sequences. Nat Protoc 1, 791-797 (2006)). Both full length
and soluble forms of 1999 NC .DELTA.Stem (C. J. Wei et al.,
Induction of broadly neutralizing H1N1 influenza antibodies by
vaccination. Science 329, 1060-1064 (2010)) and .DELTA.RBS HA
mutants were generated by introducing an N-linked glycosylation
site at residues HA2 45 (145N/G47T) and HA1 190 (Q192T),
respectively. The soluble form of 2007 Bris .DELTA.RBS HA mutant
was generated by introducing an N-linked glycosylation site at the
same site. All genes were then cloned into mammalian expression
vectors for efficient expression (C. J. Wei et al., Comparative
efficacy of neutralizing antibodies elicited by recombinant
hemagglutinin proteins from avian H5N1 influenza virus. J Virol 82,
6200-6208 (2008)). Plasmids encoding the mAbs, CR6261 (D. C. Ekiert
et al., Antibody recognition of a highly conserved influenza virus
epitope. Science 324, 246-251 (2009)), CH65 (J. R. Whittle et al.,
Broadly neutralizing human antibody that recognizes the
receptor-binding pocket of influenza virus hemagglutinin. Proc Natl
Acad Sci USA 108, 14216-14221 (2011)) and a single-chain variable
fragment F10 (J. Sui et al., Structural and functional bases for
broad-spectrum neutralization of avian and human influenza A
viruses. Nat Struct Mol Biol 16, 265-273 (2009)) were also
synthesized as described by C. J. Wei et al., (Science 329,
1060-1064 (2010).
Protein Biosyntheses and Purifications.
[0243] To produce ferritin nanoparticles, HA-np and trimeric HA,
the expression vectors were transfected into 293F cells
(Invitrogen), a human embryonic kidney cell line using 293fectin
(Invitrogen) according to the manufacturer's instructions. Matched
NAs were co-transfected at 20:1 HA:NA (wt:wt). The cells were grown
in Freestyle 293 expression medium (Invitrogen) and the culture
supernatants were collected 4 days post-transfection by
centrifugation and filtered through a 0.22 .mu.m pore filter unit
(Nalgene) to remove cell debris. The supernatants were concentrated
with a 30 kDa molecular weight cut-off filter unit (Pall Corp.) and
then buffer exchanged to a Tris buffer (20 mM Tris, 50 mM NaCl, pH
7.5 for ferritin nanoparticles; 20 mM Tris, 500 mM NaCl, pH 7.5 for
HA-np). The ferritin nanoparticles were purified by ion-exchange
chromatography using a HiLoad 16/10 Q Sepharose HP column (GE
Healthcare). The HA-np were purified by affinity column
chromatography using Erythrina cristagalli agglutinin (ECA, coral
tree lectin; EY Laboratories, Inc.) specific for galactose
.beta.(1,4) N-acetylglucosamine. The ferritin nanoparticles and
HA-np were further purified by size exclusion chromatography with a
Superose 6 PG XK 16/70 column (GE Healthcare) in PBS. The peak
fraction was collected and used for further studies. The molecular
weights of the ferritin nanoparticle and HA-np were calculated
based on two equations generated by least squares linear regression
on a semi-log plot using gel filtration low and high molecular
weight standards (Bio-Rad), respectively. The yield of 1999 NC
HA-np is .about.4 mg liter.sup.-1 and appears stable at 4.degree.
C. or frozen at -80.degree. C. The trimeric HA proteins were
purified as described by A. S. Xiong et at (Nat Protoc 1, 791-797
(2006)) with slight modifications. Briefly, HA proteins were first
purified by affinity chromatography using Ni Sepharose HP resin (GE
Healthcare), and then were separated by size exclusion
chromatography with a HiLoad 16/60 Superdex 200 PG column (GE
Healthcare). To remove the foldon trimerization motif and
poly-histidine tag, HA proteins were digested with thrombin (EMD
Chemicals, Inc.) (3 U mg ml.sup.-1) overnight at 4.degree. C.
Undigested proteins were removed by passing over Ni Sepharose HP
resin and the digested HAs were purified on a HiLoad 16/60 Superdex
200 PG column. All purified proteins were verified by SDS-PAGE.
Protein purity and size distribution were examined by dynamic light
scattering using a DynaPro system (Wyatt Technology). All human
mAbs and a single-chain variable fragment were also produced in
293F cells and purified as described previously (C. J. Wei et al.,
Induction of broadly neutralizing H1N1 influenza antibodies by
vaccination. Science 329, 1060-1064 (2010); W. P. Kong et al.,
Protective immunity to lethal challenge of the 1918 pandemic
influenza virus by vaccination. Proc Natl Acad Sci USA 103,
15987-15991 (2006)). MAbs against 1999 NC HA were purified from
hybridoma supernatants as previously described (C. J. Wei et al.,
Induction of broadly neutralizing H1N1 influenza antibodies by
vaccination. Science 329, 1060-1064 (2010)).
Iodixanol-Based Gradient Centrifugation.
[0244] Alternatively, HA-np were purified by iodixanol gradient
ultracentrifugation (FIG. 10) routinely used for virus and VLP
purifications (C. J. Wei et al., Cross-neutralization of 1918 and
2009 influenza viruses: role of glycans in viral evolution and
vaccine design. Sci Transl Med 2, 24ra21 (2010)). Fractions
containing HA np were confirmed by SDS-PAGE and Western blotting
using a mAb against 1999 NC HA.
Electron Microscopic Analysis.
[0245] Purified ferritin nanoparticles and HA-np were subjected to
transmission electron microscopic analysis. The samples were
negatively stained with phosphotungstic acid (ferritin
nanoparticles) or ammonium molybdate (HA-np) and images were
recorded on a Tecnai T12 microscope (FEI) at 80 kV with a CCD
camera (AMT Corp.).
Analysis of HA-Ferritin np.
[0246] Among the various ferritins, Helicobacter (H.) pylori
nonheme ferritin (K. J. Cho et al., The crystal structure of
ferritin from Helicobacter pylori reveals unusual conformational
changes for iron uptake. J Mol Biol 390, 83-98 (2009)) was selected
as a prototype because of its highly divergent sequence compared to
mammalian ferritins (FIG. 2), thus minimizing the likelihood of
inducing autoimmunity after vaccination. The final purification
step for recombinant HA-ferritin was size exclusion chromatography
(FIG. 1C, left) and dynamic light scattering was used to confirm
that both ferritin and HA-ferritin self-assembled into
supramolecules with diameters of 14.61 and 37.23 nm, respectively
(FIG. 1C, middle). HA-ferritin and ferritin subunits from these
nanoparticles migrated at the expected respective molecular weights
of 85 and 17 kDa by SDS-PAGE compared to 68 kDa for purified HA
(FIG. 1C, right). While the morphology of the ferritin
nanoparticles was smooth, as visualized by transmission electron
microscopy (TEM), HA-ferritin formed np that exhibited clearly
visible spikes around the spherical core (FIG. 1D, Ferritin np vs.
HA-np). Remarkably, the placement of these spikes clearly
illustrated the octahedral symmetry of the HA-np design. Octahedral
two-, three- and four-fold axes were distinctly observed in the TEM
image (FIG. 1E, right). These data demonstrated the formation of HA
spikes on self-assembling HA-ferritin nanoparticles. More
importantly, this design enabled HAs from different subtypes or
influenza B viruses to be readily joined to a ferritin core without
substantial modification.
Example 2
Antigenicity and Immunogenicity of HA-np in Mice
[0247] To verify the antigenicity of the HA spikes on the np,
HA-ferritin np were analyzed for their ability to react with
anti-HA head ab and a conformation-dependent monoclonal ab (mAb),
CR6261, that recognizes a highly conserved structure in the
trimeric HA stem and neutralizes diverse influenza A group 1
viruses D. C. Ekiert et al., Antibody recognition of a highly
conserved influenza virus epitope. Science 324, 246-251 (2009)),
using ELISA and a virus neutralization assay.
Analysis by ELISA.
[0248] Purified trimeric HA, HA-np, and TIV (2 .mu.g of H1 HA
ml.sup.-1), ferritin nanoparticles (0.68 .mu.g ml.sup.-1 for FIG. 3
or 2 .mu.g ml.sup.-1 for the rest), mouse liver ferritin (2 .mu.g
ml.sup.-1, Alpha Diagnostic International, Inc.), .DELTA.Stem and
.DELTA.RBS HA trimer (2 .mu.g ml.sup.-1) were coated (100
.mu.l/well) onto MaxiSorp.TM. plates (Nunc) and the wells were
probed with the anti-HA mAbs, anti-mouse liver ferritin IgG (Alpha
Diagnostic International, Inc.) or immune sera followed by
peroxidase-conjugated secondary antibodies (anti-mouse IgG and
anti-human IgG, SouthernBiotech; anti-ferret IgG, Rockland
Immunochemicals, Inc.). The wells were developed using a SureBlue
chromogen (KPL) and the reaction was stopped by adding 0.5 M
sulfuric acid. For the ELISA-based competition assay, HA trimer (2
.mu.g ml.sup.-1) was coated onto the plates. Plates were incubated
with an anti-stem mAb, CR6261 (8 .mu.g ml.sup.-1) or an isotype
control Ab, VRC01 (8 .mu.g ml.sup.-1) (Z. Y. Yang et al.,
Immunization by avian H5 influenza hemagglutinin mutants with
altered receptor binding specificity. Science 317, 825-828 (2007);
X. Wu et al., Rational design of envelope identifies broadly
neutralizing human monoclonal antibodies to HIV-1. Science 329,
856-861 (2010)) before adding serially diluted pre-absorbed ferret
immune sera. The wells were probed with anti-ferret IgG and
developed as described above. Absorbance at 450 nm was measured by
SpectraMax M2e (Molecular Devices). The endpoint titers were
determined by calculating the intersection of the observed binding
curve and the absorbance threshold (four times background).
Neutralization Assays.
[0249] HA/NA-pseudotyped lentiviral vectors encoding luciferase
were used. Immune sera used for the assay were pretreated with RDE
as described above. Pre-titrated pseudotyped viruses (Gag
p24.apprxeq.6.25 ng ml.sup.-1) were incubated with serially diluted
sera for 20 minutes at room temperature and added to 293A cells
(10,000 cells/well in a 96-well plate; 50 .mu.l/well; in
triplicate). Plates were then washed and replaced with fresh media
2 hours later, and luciferase activity was measured after 24 hours.
For the protein competition assay, neutralizing activity of the
mAbs F10, CR6261 or immune sera was measured in the presence of
competitor proteins, trimeric HA (WT, .DELTA.Stem or .DELTA.RBS),
HA-np, ferritin nanoparticles or irrelevant protein (HIV-1 gp120)
at final concentration of 20 and 25 .mu.g ml.sup.-1 for mAbs and
immune sera, respectively. The HA-np was able to bind to anti-head
or anti-stem mAbs with affinities similar to trimeric HA or
trivalent inactivated vaccine (TIV) containing the same 1999 NC HA
at equimolar concentrations of HA, in contrast to a ferritin
nanoparticle control (FIG. 3A). Analogous to trimeric HA, the HA-np
also blocked neutralization by CR6261 and another stem-directed
mAb, F10 (4 J. Sui et al., Structural and functional bases for
broad-spectrum neutralization of avian and human influenza A
viruses. Nat Struct Mol Biol 16, 265-273 (2009)) (FIG. 3B). These
results indicated that HA molecules on the HA-np antigenically
resembled the physiological trimeric viral spike.
Example 3
Immunogenicity of HA-Ferritin np In Vivo
[0250] This Example demonstrates the ability of HA-ferritin np of
the present invention to elicit neutralizing antibodies.
[0251] To assess the immunogenicity of the HA-ferritin np in vivo,
mice were immunized twice with HA-np or TIV's from the 2006-2007
season, with HAs from A/New Calcdonia/20/1999 (H1N1),
A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2504/04 (type B), or from
the 2011-2012 season, with HAs from A/California/07/09-like (H1N1),
A/Perth/16/09 (H3N2) and B/Brisbane/60/08 (type B). Briefly, female
BALB/c mice (6-8 weeks old; Charles River Laboratories) were
immunized (5 mice/group) intramuscularly with 5 or 0.5 .mu.g (1.67
or 0.17 .mu.g of H1 HA) of TIV, 2.24 or 0.22 .mu.g (1.67 or 0.17
.mu.g of HA) of HA-np or 0.57 .mu.g of ferritin nanoparticles
(equimolar to 2.24 .mu.g of HA-np) in 100 .mu.l of PBS or in 100
.mu.l of 50% (v/v) mixture of Ribi adjuvant (Sigma) in PBS at weeks
0 and 3. A group of BALB/c mice (n=4) was immunized with 20 .mu.g
of trimeric HA (thrombin cleaved) in 100 .mu.l of 50% (v/v) mixture
of Ribi adjuvant in PBS at weeks 0 and 4. For the experiment using
trivalent HA-np, mice were immunized (n=5) with 6.72 .mu.g (1.67
.mu.g of each HA component) of trivalent HA-np in 100 .mu.l of 50%
(v/v) mixture of Ribi adjuvant in PBS at weeks 0 and 3. Blood
samples were collected prior to the first dose, and at 2 weeks
after each immunization.
[0252] The resulting antibody titers were determined as described
in Example 2. The HA-np induced significantly higher HAI titers
than TIV (FIG. 4A, left; p<0.0001), and a similar effect was
observed in the neutralization assay and ELISA (FIG. 4A, middle and
right; p<0.0001). For example, neutralization titers elicited by
HA-np as assessed by the concentration of ab needed to inhibit
viral entry by 90% (IC.sub.90) were .about.34 times higher than TIV
(FIG. 4A, middle). Because higher titers were observed in groups
with the adjuvant Ribi, further comparisons were performed with
this adjuvant. Neutralization against a panel of H1N1 strains
revealed not only increased potency but also enhanced breadth
stimulated by HA-np compared with TIV or trimeric HA (FIG. 4B).
Neutralization against two highly divergent H1N1 viruses, A/Puerto
Rico/8/1934 (1934 PR8) and A/Singapore/6/1986 (1986 Sing) were only
observed in mice immunized with the HA-np, and the titer against
the contemporary virus A/Brisbane/59/2007 (2007 Bris) was more than
one log higher in mice immunized with HA-np than with TIV (FIG.
4B).
[0253] To assess whether the preexisting immune responses to
ferritin nanoparticles or to other HA subtypes would attenuate the
immunogenicity of the subsequent immunization of HA-np, mice were
pre-immunized with either H3 (A/Perth/16/09, 2009 Perth) HA-np or
empty ferritin nanoparticles to elicit anti-H3 HA and/or anti-H.
pylori ferritin immune responses (FIG. 5A). These animals were then
immunized with H1 (1999 NC) HA-np. Comparable HAI, IC.sub.90
neutralization and ELISA titers against 1999 NC HA were observed in
naive animals as well as in groups pre-immunized with H3 HA-np or
empty ferritin nanoparticles (FIG. 5B). These results indicated
that preexisting anti-H. pylori ferritin immunity did not diminish
the HA-specific ab response.
Example 4
Lack of Autoreactivity of H. pylori Ferritin Nanoparticles
[0254] This Example demonstrates analyzes the ability of
HA-ferritin np of the present invention to elicit an auto-immune
response against autologous ferritin in mice.
[0255] Although the overall structural architecture and
physiological functions of ferritin are conserved across organisms,
murine ferritin has only 27% amino acid sequence identity to H.
pylori ferritin. This homology nonetheless raised the possibility
that immunization with H. pylori ferritin in mice might abrogate
immune tolerance and induce autoimmunity. To address this concern,
CD4, CD8 T-cell and ab responses against both murine and H. pylori
ferritins were analyzed by intracellular cytokine staining (ICS)
and ELISA in mice immunized with HA-np. ELISAs were performed
according to the procedure in Example 2. For intracellular cytokine
analysis, CD4.sup.+ and CD8.sup.+ T-cell responses were evaluated
for interferon-.gamma. (IFN-.gamma.), tumor necrosis factor .alpha.
(TNF.alpha.), and interleukin-2 (IL-2) as described by T. Zhou et
al. (Science 329, 811-817 (2010)). Individual peptide pools (15-mer
overlapping by 11 residues, 2.5 .mu.g ml.sup.-1 for each peptide)
covering H. pylori ferritin or mouse ferritin light and heavy
chains were used to stimulate cells. After stimulation, cells were
fixed, permeabilized and stained using anti-mouse CD3, CD4, CD8,
IFN-.gamma., TNF.alpha. and IL-2 mAbs (BD Pharmingen) together with
aqua blue dye for live/dead stain (Invitrogen). The data were
collected by LSR II Flow Cytometer (BD Biosciences) and
IFN-.gamma.-, TNF.alpha.- and IL-2-positive cells in the CD4.sup.+
and CD8.sup.+ cell populations were analyzed with FlowJo software
(Tree Star).
[0256] Although an increase in the ICS staining of CD4.sup.+ T
cells stimulated with H. pylori ferritin peptides (FIG. 4C, top
left) was observed, no increases in the CD4.sup.+ and CD8.sup.+ ICS
responses were seen with murine ferritin peptide stimulation (FIG.
4C, bottom left and middle). In addition, while high titers
(>10.sup.6) of anti-H. pylori ferritin abs were detected in
ferritin nanoparticle- and HA-np-immune sera, abs to mouse ferritin
were undetectable (FIG. 4C, right). These results demonstrate that
HA-ferritin np of the present invention do not elicit
autoreactivity to autologous ferritin in mice.
Example 5
Generation of Trivalent HA-np and Immunogenicity in Mice
[0257] The Example analyzes whether multivalent HA-np were similar
in immunogenicity to monovalent np.
[0258] HA-np expressing HAs from H1 (A/California/04/09, 2009 CA),
H3 (2009 Perth) or influenza B (B/Florida/04/06, 2006 FL) were
generated. The 2009 CA (H1)-, 2009 Perth (H3)- and 2006 FL (type
B)-HA-np self-assembled and displayed the same morphology observed
for 1999 NC HA-np (FIG. 6A). Trivalent HA-np were generated by
combining three monovalent HA-np, and their immunogenicity was
compared to a seasonal TIV containing the same H1 and H3 strains
and a mismatched type B (B/Brisbane/60/08). HAI titers against
homologous H1N1 and H3N2 viruses were significantly increased in
animals immunized with trivalent HA-np relative to TIV-immunized
animals (FIG. 6B; p=0.0125 and 0.0036, respectively). When compared
to animals immunized with the corresponding monovalent HA-np, HAI
titers against 2009 CA (H1) and 2009 Perth (H3) induced by
trivalent HA-np were comparable (FIG. 6B). These results
demonstrate that no substantial antigenic competition between H1
and H3 HA-np was observed with a trivalent HA-np vaccine.
Example 6
Cross-Protective Immunity Elicited by HA-np in Ferrets
[0259] This Example demonstrates that vaccination of ferrets with
1999 NC HA-np elicits a protective immunity similar to that
observed in human disease.
[0260] Male Fitch ferrets (6 months old; Triple F Farms),
seronegative for exposure to H1N1, H3N2 and type B influenza
viruses, were housed and cared for at BIOQUAL, Inc. (Rockville,
Md.). Prior to study start, a temperature transponder (Biomedic
Data Systems, Inc.) was implanted into the neck of each ferret.
Ferrets were immunized (6 ferrets/group) intramuscularly with 500
.mu.l of PBS, 7.5 .mu.g (2.5 .mu.g of H1 HA) of TIV or 3.35 .mu.g
(2.5 .mu.g of HA) of HA-np in 500 .mu.l of 50% (v/v) mixture of
Ribi adjuvant in PBS at weeks 0 and 4. Blood was collected 3 and 2
weeks after the first and the second immunization,
respectively.
[0261] Three weeks after the first immunization, all ferrets
receiving HA-np generated protective HAI titers against homologous
H1 1999 NC virus (>1:40), while only 50% (3/6) of TIV-immunized
ferrets induced HAI titers greater than 1:40 (FIG. 7A, left;
p=0.0056). The same trend was also observed for both neutralization
and anti-HA ab titers (FIG. 7A, middle and right; p=0.0047 and
p=0.0045, respectively), documenting the superior potency of HA-np
in a second species. After boosting, the HAI and IC.sub.90
neutralization titers of the HA-np-immune sera were .about.10-fold
higher than those of TIV-immunized ferrets (FIG. 7A, left and
middle; 457.+-.185 vs. 5760.+-.1541, p=0.0066, and 598.+-.229 vs.
5515.+-.1074, p=0.0012, respectively). A similar enhancement in
HA-np vs. TIV immunization was also observed by ELISA titers (FIG.
7A, right; p=0.0038). Remarkably, a single immunization with HA-np
induced immune responses comparable to two immunizations with TIV
(FIG. 7A).
[0262] To determine whether HA-np could confer protection against
an unmatched H1N1 virus, five weeks after the last immunization
ferrets immunized with 1999 NC HA-np or TIV containing the same H1
HA were challenged with 10.sup.6.5 EID.sub.50 of 2007 Bris virus.
(1999 NC and 2007 Bris viruses are 8 years apart and their
antigenic characteristics are sufficiently different to require the
production of two different vaccines to confer protection in
humans.) The virus was expanded in embryonated chicken eggs from a
seed stock obtained from CDC (Atlanta, Ga.) and has a titer of
10.sup.6.5 EID.sub.50 ml.sup.-1. The virus stock was inoculated
intranasally into ferrets, which had been anesthetized with
ketamine/xylazine, in a volume of 500 .mu.l per nostril. The
ferrets were observed for clinical signs twice daily and weight and
temperature measurements recorded daily by technicians blind to the
treatment groups. Nasal washes were obtained on days 1, 3 and 5 and
infectious viral titers were determined by TCID.sub.50 assay using
MDCK cells as described previously (C. J. Wei et al., Induction of
broadly neutralizing H1N1 influenza antibodies by vaccination.
Science 329, 1060-1064 (2010)).
[0263] Ferrets immunized with HA-np showed a significant reduction
in viral shedding beginning 1 day after challenge compared to the
sham control group (FIG. 7B, left; p=0.0259). At the same time
point, no reduction in viral shedding was seen in the TIV-immunized
group. Four of six animals immunized with HA-np had no detectable
viral load after 3 days and by day 5, all animals in this group
cleared the virus, while all animals in the sham control group
still had detectable virus (FIG. 7B). In addition, HA-np-immunized
ferrets suffered less body weight loss compared to the
TIV-immunized and sham control groups (FIG. 7B, right). These
results demonstrate faster virus clearance in ferrets immunized
with HA-np than with TIV and further demonstrate that HA-np
effectively induced cross-protective immunity in vaccinated
ferrets.
Example 7
Induction of Two Types of Neutralizing abs (nAbs) in Ferrets
[0264] This Example demonstrates the breadth and specificity of
nAbs in ferret immune sera.
[0265] IC.sub.50 neutralization titers against 1986 Sing,
A/Beijing/262/1995 (1995 Beijing), A/Solomon Islands/3/2006 (2006
SI) and 2007 Bris were significantly higher in animals immunized
with HA-np compared to immunization with TIV (FIG. 8A, left). This
enhanced breadth was due not only to a quantitative increase in
overall ab titer (.about.9-fold against matched virus) but also
reflected a qualitative difference in the types of abs elicited
(>40-fold enhancement against an unmatched strain). To determine
whether the cross-reactivity induced by HA-np was due to nAbs to
the conserved HA stem epitope, ferret immune sera were pre-absorbed
with cells expressing a stem mutant (.DELTA.Stem) HA to remove
non-stem directed antibodies. Briefly, ferret immune sera taken 2
weeks after the second immunization were subjected to the assay.
The plasmids encoding for .DELTA.Stem and .DELTA.RBS HAs were
transfected into 293F cells. Three days after transfection, the
cells were analyzed by flow cytometry to confirm expression of HA
on the cell surface and used for serum absorption. One ml of the
immune sera diluted at 1:100 and 1:1,000 was incubated with 100
.mu.l of pre-washed .DELTA.Stem and .DELTA.RBS HA-expressing 293F
cell pellets, respectively. After incubating for 1 hour at
4.degree. C., supernatants were harvested by centrifugation and
binding to WT and mutant HAs was examined by ELISA previously
described (C. J. Wei et al., Induction of broadly neutralizing H1N1
influenza antibodies by vaccination. Science 329, 1060-1064
(2010)). The .DELTA.Stem HA-pre-absorbed sera were also used for
competition ELISA.
[0266] Stem-specific abs were detected in HA-np-immunized ferrets
(6/6) in greater frequency and magnitude than TIV-immune ferrets
(2/6) (FIG. 8B, left; p=0.0056). Moreover, binding of these
pre-absorbed sera to HA was inhibited by CR6261 mAb (FIG. 8B,
right; p=0.0019), further documenting the specificity of HA-np
immune sera to the stem epitope. The HAI titers against
heterologous 2007 Bris virus were also significantly higher in
ferrets immunized with HA-np (6/6, 1:80-1:640) than with TIV (3/6,
1:40-1:80) (FIG. 8A, right; p=0.0054). Interestingly, in contrast
to a previous study in which DNA prime/TIV boost was used to elicit
anti-stem broadly neutralizing abs (bnAbs) (C. J. Wei et al.,
Induction of broadly neutralizing H1N1 influenza antibodies by
vaccination. Science 329, 1060-1064 (2010)), sera from animals
immunized with HA-np showed HAI ab titers against a highly
divergent 1934 PR8 strain, with titers .gtoreq.1:40 in all ferrets.
However, no HAI titers against 1934 PR8 were detected in
TIV-immunized ferrets (FIG. 8A, right). These data suggested that
the HA-np vaccine might elicit another class of nAb directed
towards the conserved RBS in the HA head.
[0267] To determine whether HA-np elicited abs against RBS, an RBS
mutant HA (.DELTA.RBS) was generated by introducing a glycosylation
site in the sialic acid binding pocket at residue 190 (FIG. 9) (D.
Lingwood et al., Structural and genetic basis for development of
broadly neutralizing influenza antibodies. Nature, in press).
Ferret immune sera were absorbed with .DELTA.RBS HA-expressing
cells to remove abs to HA outside of this region and tested for
binding against WT or .DELTA.RBS HA. RBS-directed abs were detected
with titers of >1:2,000 in all HA-np-immunized ferrets, but only
1 out of 6 ferrets that received TIV (FIG. 8B, middle).
[0268] To define the relative contributions of these stem and RBS
abs to the breadth of neutralization, neutralization assays were
performed in the presence of competitor proteins: WT, .DELTA.Stem
or .DELTA.RBS HA. In the presence of excess .DELTA.Stem HA, only
stem-directed abs can neutralize viruses; similarly, .DELTA.RBS HA
interferes with all antibodies in the serum except those proximal
to the RBS. The relative contribution of stem- and RBS-directed
neutralization was measured as activity remaining in the presence
of the respective competitor HA. For example, with 2007 Bris,
.DELTA.RBS HA only partially inhibited neutralization, while either
WT or .DELTA.Stem HA almost completely abolished the neutralization
activity of the sera; hence, the neutralization against 2007 Bris
was due almost entirely to RBS-directed abs (FIG. 8C). Four H1N1
strains were tested in this assay. The pattern of neutralization
inhibition varied by strain. Neutralization of 1999 NC or 2007 Bris
was mediated predominantly by RBS-directed abs. However,
neutralization of 1986 Sing was due mainly to stem-directed abs.
Interestingly, the neutralization of 1995 Beijing was more complex.
Both stem- and RBS-directed abs contributed to neutralization of
this virus (FIG. 8C).
[0269] These results demonstrate that HA-np induce both known types
of bnAbs-stem-directed and RBS-directed. Together, these abs
contribute to the breadth and potency of the immune sera elicited
by HA-np. The synergy between them explains mechanistically the
observed superior efficacy of the HA-np vaccine and decreases the
likelihood of viral escape mutations from either antibody
alone.
[0270] Taken together the above-disclosed Examples demonstrate that
a ferritin-based nanoparticle is able to present trimeric HA in its
native fold, rigidly and symmetrically, with sufficient spacing to
ensure optimal access to potential bnAbs directed to the stem. They
also demonstrate that the nanoparticles have enhanced
immunogenicity and an expanded neutralization breadth to both stem
and RBD antibodies.
Example 8
Immunization of Mice and Ferrets Using a Tetravalent Vaccine
[0271] This Example demonstrates the ability of a multivalent
vaccine to elicit an immune response against several strains and
sub-types of influenza virus.
[0272] The ability of a pan-group 1 vaccine to stimulate
neutralizing antibodies against a variety of influenza viruses was
tested in mice and ferrets using a protocol similar to that
described in Example 1, and outlined in FIG. 11. Briefly, a
pan-group 1 HA-ferritin np vaccine was produced by combining four
different monovalent HA-ferritin np vaccines. Specifically,
HA-ferritin np, each expressing either H1 A/NC/20/1999, H1
A/CA/04/2009, H2 A/Singapore/1/1957 or H5 A/Indonesia/05/2005, were
combined to produce a single vaccine containing all four HA
proteins. Mice were immunized twice in a four week interval using
6.8 ug total of the pan-group 1 vaccine (1.7 ug of each HA-ferritin
np) in Ribi. Ferrets were immunized twice in a four week interval
using 10 ug total of the pan-group 1 vaccine (2.5 ug of each
HA-ferritin np) in Ribi. Blood was obtained from the immunized
animals and the titer of neutralizing antibodies against various
influenza viruses measured. The results of this analysis are shown
in FIGS. 12-14. Immunized ferrets were also challenged with either
influenza A/Brisbane/59/2007 Brisbane (H1N1) (2207 Bris) (FIG. 15)
or influenza A/Mexico/2009 (H1N1) (2009 Mex) (FIG. 16) and the
resulting virus titers measured on day 3 and 5 post-challenge.
Example 9
Design and Construction HA-Ferritin Stem-Region Fusion Proteins
[0273] This Example demonstrates the construction of HA-ferritin
proteins and nanoparticles that present the stem region of the
influenza HA protein.
[0274] As illustrated in FIG. 17, the stem region of the influenza
HA protein is highly conserved among different influenza strains,
and possesses a site of vulnerability for Group 1 viruses. Thus, a
vaccine that elicits neutralizing antibodies against the stem
region of the influenza HA protein should be broadly neutralizing.
A nanoparticle displaying the stem region of the influenza stem
region was constructed as a vaccine.
Design of an HA-Stabilized Stem Fusion Protein
[0275] An HA-stabilized stem fusion protein (HA SS) was constructed
as follows: residues 43-313 of the head domain of HA1 were replace
with a Gly-Trp-Gly linker. The membrane distal end of HA2 (residues
59 to 93) was replaced by an HIV-1 Bal gp41 HR2 helix followed by a
six residue glycine-rich linker (Asn-Gly-Thr-Gly-Gly-Gly-Ser-Gly)
and the gp41 HR1 helix. The HR1 helix of gp41 was added in frame
with helix C of HA2 so as to generate a long central chimeric
helix. The resulting six helix bundle sitting atop the modified
hemagglutinin stem provides stability to the SS trimer in lieu of
the missing head residues. A schematic of the resulting protein is
shown in FIG. 18A, while a ribbon diagram is shown in FIG. 18B. A
second trimerization domain consisting of a 28 residue T4 foldon
domain was joined to the membrane proximal C-terminus of HA2. The
HA 55-ferritin nanoparticle (HA SS-np) protein was generated by
joining residue 174 (H3 numbering) of HA SS to H. pylori ferritin
(residues 5-167) with a Ser-Gly-Gly linker.
[0276] In constructing HA-SS fusion proteins, genes encoding
wild-type HA proteins (A/Puerto Rico/8/1934 (H1 1934 PR8),
A/Singapore/6/1986 (H1 1986 Sing), A/New Calcdonia/20/1999 (H1 1999
NC), A/Brisbane/59/2007 (H1 2007 Bris), A/Vietnam/1203/2004 (H5
2004 VN), A/Canada/720/05 (H2 2005 CAN), A/Hong Kong/1/1968 (H3
1968 HK), A/Hong Kong/1073/1999 (H9 1999 HK) and their
corresponding NAs, H1 NC 99 SS, RSC3 HIV gp120 control protein, and
all Abs (CR6261, F16v3, and VRC01) were synthesized with human
preferred codons as previously described (Wei et al. Science 2010,
329(5995):1060-4). Helicobacter pylori nonheme iron-containing
ferritin (GenBank NP.sub.--223316) with a point mutation (N19Q) to
abolish a potential N-linked glycosylation site was synthesized by
PCR-based accurate synthesis (Xiong et al. Nat Protoc 2006,
1(2):791-797) using human-preferred codons. Coding sequences for
the human CD5 leader sequence and a serine-glycine-glycine (SGG)
spacer were joined to the gene fragment encoding ferritin (residues
5-167) to generate a secreted protein. HA and HA SS-- np fusion
proteins were generated by overlap PCR by joining the HA
ectodomains at residue HA2 174 (H3 numbering) to H. pylori ferritin
(residues 5-167) with a Ser-Gly-Gly linker. Stem mutant probes
.DELTA.stem (glycosylation insertion into the CR6261 binding
epitope at position 45 in HA2; H3 numbering) which prevent binding
at the conserved H1 stem epitope were generated using site directed
mutagenesis. Genes encoding these proteins were cloned into a CMVR
plasmid backbone for efficient mammalian cell expression.
Protein Expression and Purification
[0277] Plasmids encoding soluble proteins were transfected (HA
ectodomain genes were cotransfected with the corresponding NA
encoding plasmids) into the human embryonic kidney cell line 293F
and isolated from expression supernatants 72-96 hrs
post-transfection. All HA and HA SS trimeric proteins were purified
first by metal chelation affinity chromatography and then by size
exclusion chromatography as previously described (Wei et al. J
Virol. 2008, 82(13):6200-8). IgG Abs were purified using a Protein
G affinity column (GE Healthcare). The HA- and HA SS-np were
purified by affinity column chromatography using Erythrina
cristagalli agglutinin (ECA, coral tree lectin; EY Laboratories,
Inc.) specific for galactose .beta.(1,4) N-acetylglucosamine and
Galanthus nivalis agglutinin (GNA, snowdrop lectin; EY
Laboratories, Inc.) specific for .alpha.(1,3) and .alpha.(1,6)
linked high mannose structures, respectively. HA- and HA SS-np were
further purified by size exclusion chromatography with a Superose 6
PG XK 16/70 column (GE Healthcare) in PBS (FIG. 19).
HA SS-Ferritin Characterization.
[0278] HA SS-ferritin np were visualized by electron microscopy.
Briefly, purified HA SS-np were negatively stained with
phosphotungstic acid and ammonium molybdate, respectively, and
images were recorded on a Tecnai T12 microscope (FEI) at 80 kV with
a CCD camera (AMT Corp.). The results of this analysis are shown in
FIG. 20. IN addition, the ability of purified HA SS and HA SS-np to
bind to monoclonal Abs CR6261 and FI6v3 (1.7.times.10.sup.-4 to 10
.mu.g/mL) was characterized by ELISA. HA and HIV gp120 proteins
served as controls. Ab binding was detected by
peroxidase-conjugated goat anti-human IgG. The results of this
analysis, which are shown in FIG. 21, demonstrate that
HASS-ferritin is antigenically similar to HA protein.
Example 10
Immune Response to HA SS-Ferritin Nanoparticles
[0279] This Example demonstrates the immune response generated in
animals following immunization with HA SS-ferritin np.
[0280] BALB/c mice were immunized twice intramuscularly with
protein (2 or 10 .mu.g each) formulated with Ribi adjuvant system
(Sigma) at a 3 week interval. Mice received either homologous (HA
SS-np prime and boost) or heterologous (HA-np prime and HA SS-np
boost) immunizations. Ferrets were immunized three times
intramuscularly with HA SS-np (10 .mu.g each) formulated with Ribi
adjuvant system (Sigma) at weeks 0, 4 and 14. Serum was collected
from animals 2 weeks after each immunization and 1 week prior to
the first immunization and heat inactivated (30 min at 56.degree.
C.).
[0281] Pre- and post-immune sera from immunized mice and ferrets
were assayed for binding to HA and HA SS by ELISA. Briefly, sera
were serially diluted (diluted 50 to 2.3.times.10.sup.6) and
assayed for reactivity to soluble trimeric HA and HA SS proteins,
as well as control proteins (200 ng/well with molar equivalents
plated according to HA SS). Binding was detected by peroxidase
conjugated anti-mouse or anti-ferret IgG, respectively. Endpoint
dilutions were determined from nonlinear fit dose-response curves
using a detection limit of 2.times. background absorbance. The
result from this analysis are shown in FIG. 22 and demonstrate that
stem specific cross-reactive antibodies which recognize the
conserved stem-epitope are elicited by HA SS-np vaccination.
[0282] Sera were also analyzed for neutralization of pseudotyped
recombinant lentiviruses expressing wild-type HA with the
corresponding NA with a luciferase reporter gene as previously
described (Wei et al. Science 2010, 329(5995):1060-4) following
pretreatment with receptor-destroying enzyme (RDE II; Denka Seiken
Co., Ltd.). Psuedotype neutralization competition of ferret serum
was performed by incubating serially diluted serum in the presence
of either H1 1999 NC SS, H1 1999 NC SS .DELTA.stem probe or gp120
control (10 .mu.g/mL) for 1 hr (RT) before addition to pseudotyped
recombinant lentiviruses and assaying for neutralization. The
results from this analysis are shown in FIG. 23 and demonstrate
that vaccination with HA SS-np elicits neutralizing antibodies
against various group-1 strains.
Example 11
Immune Response to HA SS-Ferritin Heterologous Immunization
Boost
[0283] This example demonstrates that HA SS-np can be utilized to
boost antibodies directed to the conserved stem epitope.
[0284] BALB/c mice were immunized twice intramuscularly with
heterologous ferritin proteins (HA-np prime and HA SS-np boost; 2
.mu.g each) formulated with Ribi adjuvant system (Sigma) at a 3
week interval. Serum was collected from animals 2 weeks after each
immunization and 1 week prior to the first immunization and heat
inactivated (30 min at 56.degree. C.).
[0285] Pre- and post-immune sera from immunized mice were assayed
for binding to HA and HA SS by ELISA. Briefly, sera were serially
diluted (diluted 50 to 2.3.times.10.sup.6) and assayed for
reactivity to soluble trimeric HA and HA SS proteins, as well as
control proteins (200 ng/well with molar equivalents plated
according to HA SS). Binding was detected by peroxidase conjugated
anti-mouse or anti-ferret IgG, respectively. Endpoint dilutions
were determined from nonlinear fit dose-response curves using a
detection limit of 2.times. background absorbance. The results from
this analysis are shown in FIG. 22 and demonstrate that
cross-reactive stem-epitope specific antibodies are being
elicited.
[0286] Sera were also analyzed for neutralization of pseudotyped
recombinant lentiviruses expressing wild-type HA with the
corresponding NA with a luciferase reporter gene as previously
described (Wei et al. Science 2010, 329(5995):1060-4) following
pretreatment with receptor-destroying enzyme (RDE II; Denka Seiken
Co., Ltd.). The results from this analysis are shown in FIG. 24 and
demonstrate that mice which have preexisting stem antibodies titers
can be boosted with HA SS-np.
[0287] While the present invention has been described with
reference to the specific embodiments thereof, it should be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objective, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the
claims.
Example 12
Use of HA-Ferritin Nanoparticles in a Hemagglutination Inhibition
Assay
[0288] This Example demonstrates the ability of HA-ferritin
nanoparticles of the invention to agglutinate red blood cells as
well as the utility of such nanoparticles in performing
hemagglutination inhibition assays.
[0289] To compare the ability of HA-ferritin nanoparticles to
agglutinate red blood cells, agglutination assays were performed
using either inactivated influenza virus of HA-ferritin
nanoparticles. Briefly, using 96-well plates, inactivated vaccine
and HA-ferritin nanoparticles were each serially diluted in PBS and
incubated with 0.5% chicken RBCs (vol/vol using PBS) for 30 minutes
at room temperature. Hemagglutination mediated through sialic acid
moiety on RBC surface and the sialic acid-binding site on HA was
determined by lack of a red dot in the well, which indicates
formation of a lattice-structure comprising RBCs and either virus
or HA-ferritin nanoparticles. The results of this analysis, which
are shown in FIG. 46, demonstrate that the ability of HA-ferritin
nanoparticles of the present invention to agglutinate red blood
cells is equivalent to that of inactivated influenza virus.
[0290] The importance of the HA protein sialic acid binding domain
on the ability of the nanoparticles to agglutinate RBCs was
examined by constructing nanoparticles using HA-ferritin fusion
proteins in which the sialic acid binding domain was disrupted by a
mutation. These nanoparticles were then used in an agglutination
assay, as described above. The results of this analysis, which are
shown in FIG. 46, demonstrate that the sialic acid binding domain
is essential to the ability of the nanoparticles to agglutinate
RBCs.
[0291] The ability of HA-ferritin nanoparticles to be used in a
hemagglutinin inhibition assay was compared to that of live virus
as follows:
HAI Assay Using Live Virus
[0292] Seed stocks of influenza viruses were obtained from the CDC
(Atlanta, Ga.) and the viruses expanded in embryonated chicken eggs
or in Madin-Darby canine kidney (MDCK) cells. Serum samples were
pretreated with receptor-destroying enzyme (RDE II; Denka Seiken
Co., Ltd.) overnight at 37.degree. C. followed by heat inactivation
at 56.degree. C. for 30 minutes. HAI assays were conducted in
V-bottom 96-well plates (Corning, Inc.) using four hemagglutinating
units of virus per well and 0.5% turkey or chicken red blood cells
(RBCs). Sera pretreated with RDE were serially diluted in PBS (25
.mu.l) and mixed with virus (25 .mu.l) for 30 min at room
temperature. RBCs (50 .mu.l) were then added to the reaction and
incubated for another 30 min at room temperature. Hemagglutination
was determined by visual inspection of the wells for the presence
(inhibition; anti-influenza antibodies present) or absence (lack of
inhibition; no anti-influenza antibodies present) of a red dot in
the bottom of the well.
HAI Assay Using HA-Ferritin Nanoparticles
[0293] HA-ferritin nanoparticles were produced using the methods
disclosed herein. Briefly, vectors expressing influenza HA were
transfected into 293F cells (Invitrogen) using 293fectin
(Invitrogen) according to the manufacturer's instructions. Matched
neuraminidase (NA) vectors were co-transfected at a ratio of 20:1
HA:NA (wt:wt) (NA cleaves the sialic acid from the HA to prevent
the HA proteins from binding to each other and forming aggregates).
The cells were grown in Freestyle 293 expression medium
(Invitrogen) and the culture supernatants were collected 4 days
post-transfection. The supernatants were concentrated and then
buffer exchanged to PBS. The HA ferritin-nanoparticles were
purified by affinity column chromatography using Erythrina
cristagalli agglutinin (ECA, coral tree lectin; EY Laboratories,
Inc.) specific for galactose .beta.(1,4) N-acetylglucosamine. The
HA ferritin-nanoparticles were further purified by size exclusion
chromatography with a Superose 6 PG XK 16/70 column (GE Healthcare)
in PBS. HAI assays were performed as described above, using
V-bottom 96-well plates (Corning, Inc.) and four hemagglutinating
units of HA ferritin-nanoparticles per well.
[0294] The HA-ferritin nanoparticles were then tested for their
ability to work in a HAI assay. Briefly, serum samples were
pretreated with receptor-destroying enzyme (RDE II; Denka Seiken
Co., Ltd.) overnight at 37.degree. C. followed by heat inactivation
at 56.degree. C. for 30 minutes. HAI assays were conducted in
V-bottom 96-well plates (Corning, Inc.) using four hemagglutinating
units of HA ferritin nanoparticles per well and 0.5% turkey or
chicken red blood cells (RBCs). Sera pretreated with RDE were
serially diluted in PBS (25 .mu.l) and mixed with HA ferritin
nanoparticles (25 .mu.l) for 30 min at room temperature. RBCs (50
.mu.l) were then added to the reaction and incubated for another 30
min at room temperature. Hemagglutination was determined by visual
inspection of the wells for the presence (inhibition;
anti-influenza antibodies present) or absence (lack of inhibition;
no anti-influenza antibodies present) of a red dot in the bottom of
the well.
[0295] The results of these studies, which are shown in FIG. 48,
demonstrate that the level of anti-influenza antibodies obtained
using the HA-ferritin nanoparticle HAI assay is highly correlated
with the level obtained using a conventional virus HAI assay.
Sequence CWU 1
1
1291504DNAHelicobacter pylori 1atgttatcaa aagacatcat taagttgcta
aacgaacaag tgaataagga aatgaactct 60tccaacttgt atatgagcat gagttcatgg
tgctataccc atagcttaga tggcgcgggg 120cttttcttgt ttgaccatgc
ggctgaagaa tacgagcatg ctaaaaagct tattatcttc 180ttgaatgaaa
acaatgtgcc tgtgcaattg accagcatca gcgcgcctga gcataagttt
240gaaggtttga ctcaaatttt ccaaaaagcc tatgaacatg agcaacacat
cagcgagtct 300attaacaata tcgtagatca cgccataaaa agcaaagatc
atgcgacttt caatttcttg 360caatggtatg tggctgaaca gcatgaagaa
gaagtgcttt tcaaggatat tttggataaa 420attgagttga ttggtaatga
aaaccatggc ttgtatttag ccgatcagta tgtcaaaggg 480atcgctaaaa
gcaggaaatc ttaa 5042167PRTHelicobacter pylori 2Met Leu Ser Lys Asp
Ile Ile Lys Leu Leu Asn Glu Gln Val Asn Lys 1 5 10 15 Glu Met Asn
Ser Ser Asn Leu Tyr Met Ser Met Ser Ser Trp Cys Tyr 20 25 30 Thr
His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp His Ala Ala 35 40
45 Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu Asn Glu Asn
50 55 60 Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu His
Lys Phe 65 70 75 80 Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu
His Glu Gln His 85 90 95 Ile Ser Glu Ser Ile Asn Asn Ile Val Asp
His Ala Ile Lys Ser Lys 100 105 110 Asp His Ala Thr Phe Asn Phe Leu
Gln Trp Tyr Val Ala Glu Gln His 115 120 125 Glu Glu Glu Val Leu Phe
Lys Asp Ile Leu Asp Lys Ile Glu Leu Ile 130 135 140 Gly Asn Glu Asn
His Gly Leu Tyr Leu Ala Asp Gln Tyr Val Lys Gly 145 150 155 160 Ile
Ala Lys Ser Arg Lys Ser 165 3504DNAHelicobacter pylori 3ttaagatttc
ctgcttttag cgatcccttt gacatactga tcggctaaat acaagccatg 60gttttcatta
ccaatcaact caattttatc caaaatatcc ttgaaaagca cttcttcttc
120atgctgttca gccacatacc attgcaagaa attgaaagtc gcatgatctt
tgctttttat 180ggcgtgatct acgatattgt taatagactc gctgatgtgt
tgctcatgtt cataggcttt 240ttggaaaatt tgagtcaaac cttcaaactt
atgctcaggc gcgctgatgc tggtcaattg 300cacaggcaca ttgttttcat
tcaagaagat aataagcttt ttagcatgct cgtattcttc 360agccgcatgg
tcaaacaaga aaagccccgc gccatctaag ctatgggtat agcaccatga
420actcatgctc atatacaagt tggaagagtt catttcctta ttcacttgtt
cgtttagcaa 480cttaatgatg tcttttgata acat 5044489DNAHelicobacter
pylori 4gacatcatca agctgctgaa cgagcaggtg aacaaggaga tgcagagcag
caacctgtac 60atgagcatga gcagctggtg ctacacccac agcctggacg gcgccggcct
gttcctgttc 120gaccacgccg ccgaggagta cgagcacgcc aagaagctga
tcatcttcct gaacgagaac 180aacgtgcccg tgcagctgac cagcatcagc
gcccccgagc acaagttcga gggcctgacc 240cagatcttcc agaaggccta
cgagcacgag cagcacatca gcgagagcat caacaacatc 300gtggaccacg
ccatcaagag caaggaccac gccaccttca acttcctgca gtggtacgtg
360gccgagcagc acgaggagga ggtgctgttc aaggacatcc tggacaagat
cgagctgatc 420ggcaacgaga accacggcct gtacctggcc gaccagtacg
tgaagggcat cgccaagagc 480aggaagagc 4895163PRTHelicobacter pylori
5Asp Ile Ile Lys Leu Leu Asn Glu Gln Val Asn Lys Glu Met Gln Ser 1
5 10 15 Ser Asn Leu Tyr Met Ser Met Ser Ser Trp Cys Tyr Thr His Ser
Leu 20 25 30 Asp Gly Ala Gly Leu Phe Leu Phe Asp His Ala Ala Glu
Glu Tyr Glu 35 40 45 His Ala Lys Lys Leu Ile Ile Phe Leu Asn Glu
Asn Asn Val Pro Val 50 55 60 Gln Leu Thr Ser Ile Ser Ala Pro Glu
His Lys Phe Glu Gly Leu Thr 65 70 75 80 Gln Ile Phe Gln Lys Ala Tyr
Glu His Glu Gln His Ile Ser Glu Ser 85 90 95 Ile Asn Asn Ile Val
Asp His Ala Ile Lys Ser Lys Asp His Ala Thr 100 105 110 Phe Asn Phe
Leu Gln Trp Tyr Val Ala Glu Gln His Glu Glu Glu Val 115 120 125 Leu
Phe Lys Asp Ile Leu Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn 130 135
140 His Gly Leu Tyr Leu Ala Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser
145 150 155 160 Arg Lys Ser 6489DNAHelicobacter pylori 6gctcttcctg
ctcttggcga tgcccttcac gtactggtcg gccaggtaca ggccgtggtt 60ctcgttgccg
atcagctcga tcttgtccag gatgtccttg aacagcacct cctcctcgtg
120ctgctcggcc acgtaccact gcaggaagtt gaaggtggcg tggtccttgc
tcttgatggc 180gtggtccacg atgttgttga tgctctcgct gatgtgctgc
tcgtgctcgt aggccttctg 240gaagatctgg gtcaggccct cgaacttgtg
ctcgggggcg ctgatgctgg tcagctgcac 300gggcacgttg ttctcgttca
ggaagatgat cagcttcttg gcgtgctcgt actcctcggc 360ggcgtggtcg
aacaggaaca ggccggcgcc gtccaggctg tgggtgtagc accagctgct
420catgctcatg tacaggttgc tgctctgcat ctccttgttc acctgctcgt
tcagcagctt 480gatgatgtc 48971695DNAInfluenza virus 7atgaaggcca
aactgctggt gctgctgtgt acctttaccg ccacctacgc cgacacaatc 60tgtatcggct
accacgccaa caatagcacc gacaccgtgg atacagtgct ggagaagaac
120gtgaccgtga cccactctgt gaacctgctg gaggacagcc acaatggcaa
gctgtgtctg 180ctgaaaggca ttgcccctct gcagctgggc aattgttctg
tggccggatg gattctgggc 240aaccccgagt gtgagctgct gatttctaag
gagagctgga gctacatcgt ggagaccccc 300aatcctgaga atggcacctg
ctaccctggc tacttcgccg attacgagga gctgcgcgag 360cagctgtcta
gcgtgtccag cttcgagaga ttcgagatct tccccaagga gtccagctgg
420cctaatcaca cagtgacagg cgtgtctgcc agctgtagcc acaacggcaa
aagcagcttc 480taccggaacc tgctgtggct gacaggcaag aatggcctgt
accccaacct gagcaagagc 540tacgtgaaca acaaggaaaa ggaagtgctg
gtgctgtggg gagtgcacca ccctcccaac 600atcggaaatc agcgggccct
gtaccacaca gagaacgcct atgtgagcgt ggtgtccagc 660cactacagca
gaagattcac ccccgagatc gccaagagac ccaaagtgag agaccaggag
720ggccggatca attactactg gaccctgctg gagcctggcg ataccatcat
cttcgaggcc 780aacggcaatc tgatcgcccc ttggtatgcc tttgccctga
gcagaggctt tggcagcggc 840atcatcacaa gcaacgcccc catggatgag
tgtgatgcca agtgccagac acctcagggc 900gccatcaata gcagcctgcc
cttccagaat gtgcaccctg tgaccatcgg cgagtgcccc 960aagtatgtga
gaagcgccaa gctgagaatg gtgaccggcc tgagaaacat ccctagcatc
1020cagagcagag gactgtttgg agccatcgcc ggattcatcg agggaggatg
gacaggcatg 1080gtggatggct ggtacggcta ccaccaccag aatgagcagg
gctctggata tgccgccgat 1140cagaagtcta cccagaacgc catcaacggc
atcaccaaca aggtgaacag cgtgatcgag 1200aagatgaaca cccagtttac
cgctgtgggc aaggagttca acaagctgga gcggaggatg 1260gagaacctga
acaagaaggt ggacgacggc tttctggaca tctggaccta caatgccgaa
1320ctcctggtcc tcctcgagaa tgagaggacc ctggacttcc acgacagcaa
cgtgaagaac 1380ctgtatgaga aggtgaagag ccagctgaag aacaacgcca
aggagatcgg caacggctgc 1440ttcgagttct accacaagtg taacaacgag
tgtatggaga gcgtgaagaa cggcacctac 1500gactacccta agtacagcga
ggagagcaag ctgaaccggg agaagatcga tggcgtgaag 1560ctggagagca
tgggcgtgta tcagatcctg gccatctaca gcacagtggc ctcttctctg
1620gtgctgctgg tgtctctggg cgccatctcc ttttggatgt gctccaacgg
cagcctgcag 1680tgcaggatct gtatc 16958565PRTInfluenza virus 8Met Lys
Ala Lys Leu Leu Val Leu Leu Cys Thr Phe Thr Ala Thr Tyr 1 5 10 15
Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20
25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val
Asn 35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys Leu Cys Leu Leu
Lys Gly Ile 50 55 60 Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala
Gly Trp Ile Leu Gly 65 70 75 80 Asn Pro Glu Cys Glu Leu Leu Ile Ser
Lys Glu Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Asn Pro Glu
Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 100 105 110 Ala Asp Tyr Glu Glu
Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe
Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr 130 135 140 Val
Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys Ser Ser Phe 145 150
155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly Leu Tyr Pro
Asn 165 170 175 Leu Ser Lys Ser Tyr Val Asn Asn Lys Glu Lys Glu Val
Leu Val Leu 180 185 190 Trp Gly Val His His Pro Pro Asn Ile Gly Asn
Gln Arg Ala Leu Tyr 195 200 205 His Thr Glu Asn Ala Tyr Val Ser Val
Val Ser Ser His Tyr Ser Arg 210 215 220 Arg Phe Thr Pro Glu Ile Ala
Lys Arg Pro Lys Val Arg Asp Gln Glu 225 230 235 240 Gly Arg Ile Asn
Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 245 250 255 Ile Phe
Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe Ala 260 265 270
Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Pro Met 275
280 285 Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly Ala Ile Asn
Ser 290 295 300 Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile Gly
Glu Cys Pro 305 310 315 320 Lys Tyr Val Arg Ser Ala Lys Leu Arg Met
Val Thr Gly Leu Arg Asn 325 330 335 Ile Pro Ser Ile Gln Ser Arg Gly
Leu Phe Gly Ala Ile Ala Gly Phe 340 345 350 Ile Glu Gly Gly Trp Thr
Gly Met Val Asp Gly Trp Tyr Gly Tyr His 355 360 365 His Gln Asn Glu
Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr 370 375 380 Gln Asn
Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu 385 390 395
400 Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu
405 410 415 Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly
Phe Leu 420 425 430 Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
Leu Glu Asn Glu 435 440 445 Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu Tyr Glu Lys 450 455 460 Val Lys Ser Gln Leu Lys Asn Asn
Ala Lys Glu Ile Gly Asn Gly Cys 465 470 475 480 Phe Glu Phe Tyr His
Lys Cys Asn Asn Glu Cys Met Glu Ser Val Lys 485 490 495 Asn Gly Thr
Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn 500 505 510 Arg
Glu Lys Ile Asp Gly Val Lys Leu Glu Ser Met Gly Val Tyr Gln 515 520
525 Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val
530 535 540 Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser
Leu Gln 545 550 555 560 Cys Arg Ile Cys Ile 565 91695DNAInfluenza
virus 9gatacagatc ctgcactgca ggctgccgtt ggagcacatc caaaaggaga
tggcgcccag 60agacaccagc agcaccagag aagaggccac tgtgctgtag atggccagga
tctgatacac 120gcccatgctc tccagcttca cgccatcgat cttctcccgg
ttcagcttgc tctcctcgct 180gtacttaggg tagtcgtagg tgccgttctt
cacgctctcc atacactcgt tgttacactt 240gtggtagaac tcgaagcagc
cgttgccgat ctccttggcg ttgttcttca gctggctctt 300caccttctca
tacaggttct tcacgttgct gtcgtggaag tccagggtcc tctcattctc
360gaggaggacc aggagttcgg cattgtaggt ccagatgtcc agaaagccgt
cgtccacctt 420cttgttcagg ttctccatcc tccgctccag cttgttgaac
tccttgccca cagcggtaaa 480ctgggtgttc atcttctcga tcacgctgtt
caccttgttg gtgatgccgt tgatggcgtt 540ctgggtagac ttctgatcgg
cggcatatcc agagccctgc tcattctggt ggtggtagcc 600gtaccagcca
tccaccatgc ctgtccatcc tccctcgatg aatccggcga tggctccaaa
660cagtcctctg ctctggatgc tagggatgtt tctcaggccg gtcaccattc
tcagcttggc 720gcttctcaca tacttggggc actcgccgat ggtcacaggg
tgcacattct ggaagggcag 780gctgctattg atggcgccct gaggtgtctg
gcacttggca tcacactcat ccatgggggc 840gttgcttgtg atgatgccgc
tgccaaagcc tctgctcagg gcaaaggcat accaaggggc 900gatcagattg
ccgttggcct cgaagatgat ggtatcgcca ggctccagca gggtccagta
960gtaattgatc cggccctcct ggtctctcac tttgggtctc ttggcgatct
cgggggtgaa 1020tcttctgctg tagtggctgg acaccacgct cacataggcg
ttctctgtgt ggtacagggc 1080ccgctgattt ccgatgttgg gagggtggtg
cactccccac agcaccagca cttccttttc 1140cttgttgttc acgtagctct
tgctcaggtt ggggtacagg ccattcttgc ctgtcagcca 1200cagcaggttc
cggtagaagc tgcttttgcc gttgtggcta cagctggcag acacgcctgt
1260cactgtgtga ttaggccagc tggactcctt ggggaagatc tcgaatctct
cgaagctgga 1320cacgctagac agctgctcgc gcagctcctc gtaatcggcg
aagtagccag ggtagcaggt 1380gccattctca ggattggggg tctccacgat
gtagctccag ctctccttag aaatcagcag 1440ctcacactcg gggttgccca
gaatccatcc ggccacagaa caattgccca gctgcagagg 1500ggcaatgcct
ttcagcagac acagcttgcc attgtggctg tcctccagca ggttcacaga
1560gtgggtcacg gtcacgttct tctccagcac tgtatccacg gtgtcggtgc
tattgttggc 1620gtggtagccg atacagattg tgtcggcgta ggtggcggta
aaggtacaca gcagcaccag 1680cagtttggcc ttcat 1695101551DNAInfluenza
virus 10atgaaggcca aactgctggt gctgctgtgt acctttaccg ccacctacgc
cgacacaatc 60tgtatcggct accacgccaa caatagcacc gacaccgtgg atacagtgct
ggagaagaac 120gtgaccgtga cccactctgt gaacctgctg gaggacagcc
acaatggcaa gctgtgtctg 180ctgaaaggca ttgcccctct gcagctgggc
aattgttctg tggccggatg gattctgggc 240aaccccgagt gtgagctgct
gatttctaag gagagctgga gctacatcgt ggagaccccc 300aatcctgaga
atggcacctg ctaccctggc tacttcgccg attacgagga gctgcgcgag
360cagctgtcta gcgtgtccag cttcgagaga ttcgagatct tccccaagga
gtccagctgg 420cctaatcaca cagtgacagg cgtgtctgcc agctgtagcc
acaacggcaa aagcagcttc 480taccggaacc tgctgtggct gacaggcaag
aatggcctgt accccaacct gagcaagagc 540tacgtgaaca acaaggaaaa
ggaagtgctg gtgctgtggg gagtgcacca ccctcccaac 600atcggaaatc
agcgggccct gtaccacaca gagaacgcct atgtgagcgt ggtgtccagc
660cactacagca gaagattcac ccccgagatc gccaagagac ccaaagtgag
agaccaggag 720ggccggatca attactactg gaccctgctg gagcctggcg
ataccatcat cttcgaggcc 780aacggcaatc tgatcgcccc ttggtatgcc
tttgccctga gcagaggctt tggcagcggc 840atcatcacaa gcaacgcccc
catggatgag tgtgatgcca agtgccagac acctcagggc 900gccatcaata
gcagcctgcc cttccagaat gtgcaccctg tgaccatcgg cgagtgcccc
960aagtatgtga gaagcgccaa gctgagaatg gtgaccggcc tgagaaacat
ccctagcatc 1020cagagcagag gactgtttgg agccatcgcc ggattcatcg
agggaggatg gacaggcatg 1080gtggatggct ggtacggcta ccaccaccag
aatgagcagg gctctggata tgccgccgat 1140cagaagtcta cccagaacgc
catcaacggc atcaccaaca aggtgaacag cgtgatcgag 1200aagatgaaca
cccagtttac cgctgtgggc aaggagttca acaagctgga gcggaggatg
1260gagaacctga acaagaaggt ggacgacggc tttctggaca tctggaccta
caatgccgaa 1320ctcctggtcc tcctcgagaa tgagaggacc ctggacttcc
acgacagcaa cgtgaagaac 1380ctgtatgaga aggtgaagag ccagctgaag
aacaacgcca aggagatcgg caacggctgc 1440ttcgagttct accacaagtg
taacaacgag tgtatggaga gcgtgaagaa cggcacctac 1500gactacccta
agtacagcga ggagagcaag ctgaaccggg agaagatcga t 155111517PRTInfluenza
virus 11Met Lys Ala Lys Leu Leu Val Leu Leu Cys Thr Phe Thr Ala Thr
Tyr 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser
Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val
Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser His Asn Gly Lys
Leu Cys Leu Leu Lys Gly Ile 50 55 60 Ala Pro Leu Gln Leu Gly Asn
Cys Ser Val Ala Gly Trp Ile Leu Gly 65 70 75 80 Asn Pro Glu Cys Glu
Leu Leu Ile Ser Lys Glu Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr
Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 100 105 110 Ala
Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120
125 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr
130 135 140 Val Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Lys Ser
Ser Phe 145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn
Gly Leu Tyr Pro Asn 165 170 175 Leu Ser Lys Ser Tyr Val Asn Asn Lys
Glu Lys Glu Val Leu Val Leu 180 185 190 Trp Gly Val His His Pro Pro
Asn Ile Gly Asn Gln Arg Ala Leu Tyr 195 200 205 His Thr Glu Asn Ala
Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg 210 215 220 Arg Phe Thr
Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asp Gln Glu 225 230 235 240
Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 245
250 255 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp Tyr Ala Phe
Ala 260
265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Thr Ser Asn Ala Pro
Met 275 280 285 Asp Glu Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly Ala
Ile Asn Ser 290 295 300 Ser Leu Pro Phe Gln Asn Val His Pro Val Thr
Ile Gly Glu Cys Pro 305 310 315 320 Lys Tyr Val Arg Ser Ala Lys Leu
Arg Met Val Thr Gly Leu Arg Asn 325 330 335 Ile Pro Ser Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340 345 350 Ile Glu Gly Gly
Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His 355 360 365 His Gln
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr 370 375 380
Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu 385
390 395 400 Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn
Lys Leu 405 410 415 Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp
Asp Gly Phe Leu 420 425 430 Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu
Val Leu Leu Glu Asn Glu 435 440 445 Arg Thr Leu Asp Phe His Asp Ser
Asn Val Lys Asn Leu Tyr Glu Lys 450 455 460 Val Lys Ser Gln Leu Lys
Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 465 470 475 480 Phe Glu Phe
Tyr His Lys Cys Asn Asn Glu Cys Met Glu Ser Val Lys 485 490 495 Asn
Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn 500 505
510 Arg Glu Lys Ile Asp 515 121551DNAInfluenza virus 12atcgatcttc
tcccggttca gcttgctctc ctcgctgtac ttagggtagt cgtaggtgcc 60gttcttcacg
ctctccatac actcgttgtt acacttgtgg tagaactcga agcagccgtt
120gccgatctcc ttggcgttgt tcttcagctg gctcttcacc ttctcataca
ggttcttcac 180gttgctgtcg tggaagtcca gggtcctctc attctcgagg
aggaccagga gttcggcatt 240gtaggtccag atgtccagaa agccgtcgtc
caccttcttg ttcaggttct ccatcctccg 300ctccagcttg ttgaactcct
tgcccacagc ggtaaactgg gtgttcatct tctcgatcac 360gctgttcacc
ttgttggtga tgccgttgat ggcgttctgg gtagacttct gatcggcggc
420atatccagag ccctgctcat tctggtggtg gtagccgtac cagccatcca
ccatgcctgt 480ccatcctccc tcgatgaatc cggcgatggc tccaaacagt
cctctgctct ggatgctagg 540gatgtttctc aggccggtca ccattctcag
cttggcgctt ctcacatact tggggcactc 600gccgatggtc acagggtgca
cattctggaa gggcaggctg ctattgatgg cgccctgagg 660tgtctggcac
ttggcatcac actcatccat gggggcgttg cttgtgatga tgccgctgcc
720aaagcctctg ctcagggcaa aggcatacca aggggcgatc agattgccgt
tggcctcgaa 780gatgatggta tcgccaggct ccagcagggt ccagtagtaa
ttgatccggc cctcctggtc 840tctcactttg ggtctcttgg cgatctcggg
ggtgaatctt ctgctgtagt ggctggacac 900cacgctcaca taggcgttct
ctgtgtggta cagggcccgc tgatttccga tgttgggagg 960gtggtgcact
ccccacagca ccagcacttc cttttccttg ttgttcacgt agctcttgct
1020caggttgggg tacaggccat tcttgcctgt cagccacagc aggttccggt
agaagctgct 1080tttgccgttg tggctacagc tggcagacac gcctgtcact
gtgtgattag gccagctgga 1140ctccttgggg aagatctcga atctctcgaa
gctggacacg ctagacagct gctcgcgcag 1200ctcctcgtaa tcggcgaagt
agccagggta gcaggtgcca ttctcaggat tgggggtctc 1260cacgatgtag
ctccagctct ccttagaaat cagcagctca cactcggggt tgcccagaat
1320ccatccggcc acagaacaat tgcccagctg cagaggggca atgcctttca
gcagacacag 1380cttgccattg tggctgtcct ccagcaggtt cacagagtgg
gtcacggtca cgttcttctc 1440cagcactgta tccacggtgt cggtgctatt
gttggcgtgg tagccgatac agattgtgtc 1500ggcgtaggtg gcggtaaagg
tacacagcag caccagcagt ttggccttca t 1551131554DNAInfluenza virus
13atgaaggcca tcctggtggt gctgctgtac accttcgcca ccgccaacgc cgacaccctg
60tgcatcggct accacgccaa caacagcacc gacaccgtgg acaccgtgct ggagaagaac
120gtgaccgtga cccacagcgt gaacctgctg gaggacaagc acaacggcaa
gctgtgcaag 180ctgcggggcg tggcccccct gcacctgggc aagtgcaaca
tcgccggctg gattctgggc 240aaccccgagt gcgagagcct gagcaccgcc
agcagctgga gctacatcgt ggagaccccc 300agcagcgaca acggcacctg
ctaccccggc gacttcatcg actacgagga gctgcgggag 360cagctgagca
gcgtgagcag cttcgagcgg ttcgagatct tccccaagac cagcagctgg
420cccaaccacg acagcaacaa gggcgtgacc gccgcctgcc cccacgccgg
cgccaagagc 480ttctacaaga acctgatctg gctggtgaag aagggcaaca
gctaccccaa gctgagcaag 540agctacatca acgacaaggg caaggaggtg
ctggtgctgt ggggcatcca ccaccccagc 600accagcgccg accagcagag
cctgtaccag aacgccgaca cctacgtgtt cgtgggcagc 660agccggtaca
gcaagaagtt caagcccgag atcgccatcc ggcccaaggt gcgggaccag
720gagggccgga tgaactacta ctggaccctg gtggagcccg gcgacaagat
caccttcgag 780gccaccggca acctggtggt gccccggtac gccttcgcca
tggagcggaa cgccggcagc 840ggcatcatca tcagcgacac ccccgtgcac
gactgcaaca ccacctgcca gacccccaag 900ggcgccatca acaccagcct
gcccttccag aacatccacc ccatcaccat cggcaagtgc 960cccaagtacg
tgaagagcac caagctgcgg ctggccaccg gcctgcggaa catccccagc
1020atccagagcc ggggcctgtt cggcgccatc gccggcttca tcgagggcgg
ctggaccggc 1080atggtggacg gctggtacgg ctaccaccac cagaacgagc
agggcagcgg ctacgccgcc 1140gacctgaaga gcacccagaa cgccatcgac
gagatcacca acaaggtgaa cagcgtgatc 1200gagaagatga acacccagtt
caccgccgtg ggcaaggagt tcaaccacct ggagaagcgg 1260atcgagaacc
tgaacaagaa ggtggacgac ggcttcctgg acatctggac ctacaacgcc
1320gagctgctgg tgctgctgga gaacgagcgg accctggact accacgacag
caacgtgaag 1380aacctgtacg agaaggtgcg gagccagctg aagaacaacg
ccaaggagat cggcaacggc 1440tgcttcgagt tctaccacaa gtgcgacaac
acctgcatgg agagcgtgaa gaacggcacc 1500tacgactacc ccaagtacag
cgaggaggcc aagctgaacc gggaggagat cgac 155414518PRTInfluenza virus
14Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn 1
5 10 15 Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp
Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
Ser Val Asn 35 40 45 Leu Leu Glu Asp Lys His Asn Gly Lys Leu Cys
Lys Leu Arg Gly Val 50 55 60 Ala Pro Leu His Leu Gly Lys Cys Asn
Ile Ala Gly Trp Ile Leu Gly 65 70 75 80 Asn Pro Glu Cys Glu Ser Leu
Ser Thr Ala Ser Ser Trp Ser Tyr Ile 85 90 95 Val Glu Thr Pro Ser
Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe 100 105 110 Ile Asp Tyr
Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu
Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro Asn His Asp 130 135
140 Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His Ala Gly Ala Lys Ser
145 150 155 160 Phe Tyr Lys Asn Leu Ile Trp Leu Val Lys Lys Gly Asn
Ser Tyr Pro 165 170 175 Lys Leu Ser Lys Ser Tyr Ile Asn Asp Lys Gly
Lys Glu Val Leu Val 180 185 190 Leu Trp Gly Ile His His Pro Ser Thr
Ser Ala Asp Gln Gln Ser Leu 195 200 205 Tyr Gln Asn Ala Asp Thr Tyr
Val Phe Val Gly Ser Ser Arg Tyr Ser 210 215 220 Lys Lys Phe Lys Pro
Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln 225 230 235 240 Glu Gly
Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp Lys 245 250 255
Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg Tyr Ala Phe 260
265 270 Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile Ile Ser Asp Thr
Pro 275 280 285 Val His Asp Cys Asn Thr Thr Cys Gln Thr Pro Lys Gly
Ala Ile Asn 290 295 300 Thr Ser Leu Pro Phe Gln Asn Ile His Pro Ile
Thr Ile Gly Lys Cys 305 310 315 320 Pro Lys Tyr Val Lys Ser Thr Lys
Leu Arg Leu Ala Thr Gly Leu Arg 325 330 335 Asn Ile Pro Ser Ile Gln
Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly 340 345 350 Phe Ile Glu Gly
Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr 355 360 365 His His
Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys Ser 370 375 380
Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile 385
390 395 400 Glu Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe
Asn His 405 410 415 Leu Glu Lys Arg Ile Glu Asn Leu Asn Lys Lys Val
Asp Asp Gly Phe 420 425 430 Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu
Leu Val Leu Leu Glu Asn 435 440 445 Glu Arg Thr Leu Asp Tyr His Asp
Ser Asn Val Lys Asn Leu Tyr Glu 450 455 460 Lys Val Arg Ser Gln Leu
Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly 465 470 475 480 Cys Phe Glu
Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val 485 490 495 Lys
Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu 500 505
510 Asn Arg Glu Glu Ile Asp 515 151554DNAInfluenza virus
15gtcgatctcc tcccggttca gcttggcctc ctcgctgtac ttggggtagt cgtaggtgcc
60gttcttcacg ctctccatgc aggtgttgtc gcacttgtgg tagaactcga agcagccgtt
120gccgatctcc ttggcgttgt tcttcagctg gctccgcacc ttctcgtaca
ggttcttcac 180gttgctgtcg tggtagtcca gggtccgctc gttctccagc
agcaccagca gctcggcgtt 240gtaggtccag atgtccagga agccgtcgtc
caccttcttg ttcaggttct cgatccgctt 300ctccaggtgg ttgaactcct
tgcccacggc ggtgaactgg gtgttcatct tctcgatcac 360gctgttcacc
ttgttggtga tctcgtcgat ggcgttctgg gtgctcttca ggtcggcggc
420gtagccgctg ccctgctcgt tctggtggtg gtagccgtac cagccgtcca
ccatgccggt 480ccagccgccc tcgatgaagc cggcgatggc gccgaacagg
ccccggctct ggatgctggg 540gatgttccgc aggccggtgg ccagccgcag
cttggtgctc ttcacgtact tggggcactt 600gccgatggtg atggggtgga
tgttctggaa gggcaggctg gtgttgatgg cgcccttggg 660ggtctggcag
gtggtgttgc agtcgtgcac gggggtgtcg ctgatgatga tgccgctgcc
720ggcgttccgc tccatggcga aggcgtaccg gggcaccacc aggttgccgg
tggcctcgaa 780ggtgatcttg tcgccgggct ccaccagggt ccagtagtag
ttcatccggc cctcctggtc 840ccgcaccttg ggccggatgg cgatctcggg
cttgaacttc ttgctgtacc ggctgctgcc 900cacgaacacg taggtgtcgg
cgttctggta caggctctgc tggtcggcgc tggtgctggg 960gtggtggatg
ccccacagca ccagcacctc cttgcccttg tcgttgatgt agctcttgct
1020cagcttgggg tagctgttgc ccttcttcac cagccagatc aggttcttgt
agaagctctt 1080ggcgccggcg tgggggcagg cggcggtcac gcccttgttg
ctgtcgtggt tgggccagct 1140gctggtcttg gggaagatct cgaaccgctc
gaagctgctc acgctgctca gctgctcccg 1200cagctcctcg tagtcgatga
agtcgccggg gtagcaggtg ccgttgtcgc tgctgggggt 1260ctccacgatg
tagctccagc tgctggcggt gctcaggctc tcgcactcgg ggttgcccag
1320aatccagccg gcgatgttgc acttgcccag gtgcaggggg gccacgcccc
gcagcttgca 1380cagcttgccg ttgtgcttgt cctccagcag gttcacgctg
tgggtcacgg tcacgttctt 1440ctccagcacg gtgtccacgg tgtcggtgct
gttgttggcg tggtagccga tgcacagggt 1500gtcggcgttg gcggtggcga
aggtgtacag cagcaccacc aggatggcct tcat 1554161542DNAInfluenza virus
16atggccatca tctacctgat cctgctgttt acagctgtgc ggggcgatca gatctgtatc
60ggctaccacg ccaacaatag caccgagaag gtggacacca tcctggaaag aaatgtgacc
120gtgacccacg ccaaggatat tctggaaaag acccacaacg gcaagctgtg
caagctgaat 180ggcattcctc ctctggaact gggcgattgt tctattgctg
gctggctgct gggaaatcct 240gagtgcgata gactgctgtc tgtgcctgag
tggagctaca tcatggaaaa agagaaccct 300agggacggac tgtgttaccc
cggcagcttc aacgattacg aggaactgaa gcacctgctg 360tccagcgtga
agcacttcga gaaagtgaag atcctgccca aggatagatg gacccagcat
420acaacaacag gcggaagcag agcttgtgct gtgtccggca accccagctt
cttcagaaat 480atggtctggc tgaccaagaa gggctctaat tatcctgtgg
ccaagggcag ctacaataat 540acaagcggcg agcagatgct gattatttgg
ggcgtgcacc accctaatga tgagacagag 600cagagaaccc tgtaccagaa
tgtgggcaca tacgtgtctg tgggcaccag cacactgaat 660aagagaagca
cccccgatat tgccaccaga cccaaagtga atggacaggg cggcagaatg
720gaattttcct ggaccctgct ggatatgtgg gacaccatca actttgagag
caccgggaat 780ctgattgccc ctgagtacgg cttcaagatc agcaagagag
gcagcagcgg catcatgaaa 840acagagggca ccctggaaaa ctgtgaaacc
aagtgtcaga cacctctggg cgccattaat 900accaccctgc ccttccataa
tgtgcaccct ctgacaatcg gcgagtgccc taagtacgtg 960aagtctgaga
aactggtgct ggccacagga ctgagaaatg tgccccagat cgagtcaaga
1020ggcctgtttg gagccattgc cggctttatt gaaggcggat ggcagggaat
ggtggatggg 1080tggtacggct atcaccacag caatgatcag ggatctggct
atgccgccga taaagagagc 1140acccagaagg cctttgacgg catcaccaac
aaagtgaaca gcgtgatcga gaagatgaac 1200acccagtttg aggccgtggg
caaagagttc agcaatctgg aaagacggct ggaaaacctg 1260aacaagaaaa
tggaagatgg cttcctggac gtgtggacat ataatgccga gctgctggtg
1320ctgatggaaa acgagaggac cctggacttt cacgacagca acgtgaagaa
cctgtacgac 1380aaagtgcgga tgcagctgag agacaatgtg aaagagctgg
gcaacggctg ctttgagttc 1440taccacaagt gcgacgacga gtgcatgaat
agcgtgaaga acggcaccta cgactaccct 1500aagtatgagg aagagagcaa
gctgaacaga aacgagatca ag 154217514PRTInfluenza virus 17Met Ala Ile
Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Asp 1 5 10 15 Gln
Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp 20 25
30 Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp Ile Leu
35 40 45 Glu Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile
Pro Pro 50 55 60 Leu Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu
Leu Gly Asn Pro 65 70 75 80 Glu Cys Asp Arg Leu Leu Ser Val Pro Glu
Trp Ser Tyr Ile Met Glu 85 90 95 Lys Glu Asn Pro Arg Asp Gly Leu
Cys Tyr Pro Gly Ser Phe Asn Asp 100 105 110 Tyr Glu Glu Leu Lys His
Leu Leu Ser Ser Val Lys His Phe Glu Lys 115 120 125 Val Lys Ile Leu
Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly 130 135 140 Gly Ser
Arg Ala Cys Ala Val Ser Gly Asn Pro Ser Phe Phe Arg Asn 145 150 155
160 Met Val Trp Leu Thr Lys Lys Gly Ser Asn Tyr Pro Val Ala Lys Gly
165 170 175 Ser Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp
Gly Val 180 185 190 His His Pro Asn Asp Glu Thr Glu Gln Arg Thr Leu
Tyr Gln Asn Val 195 200 205 Gly Thr Tyr Val Ser Val Gly Thr Ser Thr
Leu Asn Lys Arg Ser Thr 210 215 220 Pro Asp Ile Ala Thr Arg Pro Lys
Val Asn Gly Gln Gly Gly Arg Met 225 230 235 240 Glu Phe Ser Trp Thr
Leu Leu Asp Met Trp Asp Thr Ile Asn Phe Glu 245 250 255 Ser Thr Gly
Asn Leu Ile Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys 260 265 270 Arg
Gly Ser Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys 275 280
285 Glu Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro
290 295 300 Phe His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys
Tyr Val 305 310 315 320 Lys Ser Glu Lys Leu Val Leu Ala Thr Gly Leu
Arg Asn Val Pro Gln 325 330 335 Ile Glu Ser Arg Gly Leu Phe Gly Ala
Ile Ala Gly Phe Ile Glu Gly 340 345 350 Gly Trp Gln Gly Met Val Asp
Gly Trp Tyr Gly Tyr His His Ser Asn 355 360 365 Asp Gln Gly Ser Gly
Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala 370 375 380 Phe Asp Gly
Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn 385 390 395 400
Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Ser Asn Leu Glu Arg Arg 405
410 415 Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val
Trp 420 425 430 Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu
Arg Thr Leu 435 440 445 Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr
Asp Lys Val Arg Met 450 455 460 Gln Leu Arg Asp Asn Val Lys Glu Leu
Gly Asn Gly Cys Phe Glu Phe 465 470 475 480 Tyr His Lys Cys Asp Asp
Glu Cys Met Asn Ser Val Lys Asn Gly Thr 485 490 495 Tyr Asp Tyr Pro
Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu 500 505 510 Ile Lys
181542DNAInfluenza virus 18cttgatctcg tttctgttca gcttgctctc
ttcctcatac ttagggtagt cgtaggtgcc 60gttcttcacg ctattcatgc actcgtcgtc
gcacttgtgg tagaactcaa agcagccgtt 120gcccagctct ttcacattgt
ctctcagctg catccgcact ttgtcgtaca ggttcttcac 180gttgctgtcg
tgaaagtcca gggtcctctc gttttccatc agcaccagca gctcggcatt
240atatgtccac acgtccagga agccatcttc cattttcttg ttcaggtttt
ccagccgtct
300ttccagattg ctgaactctt tgcccacggc ctcaaactgg gtgttcatct
tctcgatcac 360gctgttcact ttgttggtga tgccgtcaaa ggccttctgg
gtgctctctt tatcggcggc 420atagccagat ccctgatcat tgctgtggtg
atagccgtac cacccatcca ccattccctg 480ccatccgcct tcaataaagc
cggcaatggc tccaaacagg cctcttgact cgatctgggg 540cacatttctc
agtcctgtgg ccagcaccag tttctcagac ttcacgtact tagggcactc
600gccgattgtc agagggtgca cattatggaa gggcagggtg gtattaatgg
cgcccagagg 660tgtctgacac ttggtttcac agttttccag ggtgccctct
gttttcatga tgccgctgct 720gcctctcttg ctgatcttga agccgtactc
aggggcaatc agattcccgg tgctctcaaa 780gttgatggtg tcccacatat
ccagcagggt ccaggaaaat tccattctgc cgccctgtcc 840attcactttg
ggtctggtgg caatatcggg ggtgcttctc ttattcagtg tgctggtgcc
900cacagacacg tatgtgccca cattctggta cagggttctc tgctctgtct
catcattagg 960gtggtgcacg ccccaaataa tcagcatctg ctcgccgctt
gtattattgt agctgccctt 1020ggccacagga taattagagc ccttcttggt
cagccagacc atatttctga agaagctggg 1080gttgccggac acagcacaag
ctctgcttcc gcctgttgtt gtatgctggg tccatctatc 1140cttgggcagg
atcttcactt tctcgaagtg cttcacgctg gacagcaggt gcttcagttc
1200ctcgtaatcg ttgaagctgc cggggtaaca cagtccgtcc ctagggttct
ctttttccat 1260gatgtagctc cactcaggca cagacagcag tctatcgcac
tcaggatttc ccagcagcca 1320gccagcaata gaacaatcgc ccagttccag
aggaggaatg ccattcagct tgcacagctt 1380gccgttgtgg gtcttttcca
gaatatcctt ggcgtgggtc acggtcacat ttctttccag 1440gatggtgtcc
accttctcgg tgctattgtt ggcgtggtag ccgatacaga tctgatcgcc
1500ccgcacagct gtaaacagca ggatcaggta gatgatggcc at
1542191557DNAInfluenza virus 19atgaaaacca tcattgccct gagctacatc
ttttgtctgg ctctgggcca ggatctgccc 60ggcaatgata atagcaccgc caccctgtgt
ctgggacacc acgccgtgcc taatggcacc 120ctggtgaaaa ccattaccga
cgaccagatc gaagtgacca atgccaccga gctggtgcag 180agcagcagca
ccggcaagat ctgcaacaac ccccacagaa tcctggatgg catcgactgt
240accctgatcg atgccctgct gggcgatcct cactgcgacg tgttccagaa
cgagacatgg 300gacctgttcg tggagagaag caaggccttc agcaactgct
acccctacga tgtgcccgat 360tacgcctctc tgagaagcct ggtggccagc
agcggcacac tggaattcat caccgagggc 420tttacctgga caggcgtgac
ccagaatggc ggcagcaatg cctgtaaaag aggccctggc 480agcggcttct
tcagcagact gaactggctg accaagtccg gcagcaccta ccctgtgctg
540aacgtgacca tgcccaacaa cgacaacttc gacaagctgt acatctgggg
cgtgcaccac 600cctagcacca atcaggaaca gaccagcctg tacgtgcagg
ccagcggcag agtgaccgtg 660tctaccagac ggtcccagca gaccatcatc
cccaacatcg agtcaagacc ttgggtgcgc 720ggcctgagca gcagaatcag
catctactgg accatcgtga aacctggcga cgtgctggtg 780atcaacagca
atggcaacct gatcgccccc agaggctact tcaagatgcg gaccggcaag
840agcagcatca tgagaagcga cgcccccatc gatacctgta tcagcgagtg
catcaccccc 900aacggcagca tccccaacga caagcccttc cagaacgtga
acaagatcac ctacggcgcc 960tgccctaagt acgtgaagca gaacaccctg
aagctggcca ccggcatgag aaatgtgccc 1020gagaagcaga caagaggcct
gtttggcgcc attgccggct ttatcgagaa cggctgggag 1080ggcatgatcg
atgggtggta cggcttcaga caccagaatt ctgagggcac aggacaggcc
1140gccgatctga agtctacaca ggccgccatc gaccagatca acggcaagct
gaacagagtg 1200atcgagaaaa ccaacgagaa gttccaccag atcgagaaag
aattcagcga ggtggagggc 1260agaatccagg acctggaaaa atacgtggag
gacaccaaga tcgacctgtg gagctacaat 1320gccgaactgc tggtcgccct
ggaaaaccag cacaccatcg acctgaccga cagcgagatg 1380aataagctgt
tcgaaaagac cagacggcag ctgagagaaa acgccgagga catgggcaac
1440ggctgcttca agatctacca caagtgcgac aacgcctgca tcgagagcat
cagaaacggc 1500acctacgacc acgatgtgta cagggacgag gccctgaaca
acagattcca gatcaag 155720519PRTInfluenza virus 20Met Lys Thr Ile
Ile Ala Leu Ser Tyr Ile Phe Cys Leu Ala Leu Gly 1 5 10 15 Gln Asp
Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly 20 25 30
His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asp Asp 35
40 45 Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser
Thr 50 55 60 Gly Lys Ile Cys Asn Asn Pro His Arg Ile Leu Asp Gly
Ile Asp Cys 65 70 75 80 Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro His
Cys Asp Val Phe Gln 85 90 95 Asn Glu Thr Trp Asp Leu Phe Val Glu
Arg Ser Lys Ala Phe Ser Asn 100 105 110 Cys Tyr Pro Tyr Asp Val Pro
Asp Tyr Ala Ser Leu Arg Ser Leu Val 115 120 125 Ala Ser Ser Gly Thr
Leu Glu Phe Ile Thr Glu Gly Phe Thr Trp Thr 130 135 140 Gly Val Thr
Gln Asn Gly Gly Ser Asn Ala Cys Lys Arg Gly Pro Gly 145 150 155 160
Ser Gly Phe Phe Ser Arg Leu Asn Trp Leu Thr Lys Ser Gly Ser Thr 165
170 175 Tyr Pro Val Leu Asn Val Thr Met Pro Asn Asn Asp Asn Phe Asp
Lys 180 185 190 Leu Tyr Ile Trp Gly Val His His Pro Ser Thr Asn Gln
Glu Gln Thr 195 200 205 Ser Leu Tyr Val Gln Ala Ser Gly Arg Val Thr
Val Ser Thr Arg Arg 210 215 220 Ser Gln Gln Thr Ile Ile Pro Asn Ile
Glu Ser Arg Pro Trp Val Arg 225 230 235 240 Gly Leu Ser Ser Arg Ile
Ser Ile Tyr Trp Thr Ile Val Lys Pro Gly 245 250 255 Asp Val Leu Val
Ile Asn Ser Asn Gly Asn Leu Ile Ala Pro Arg Gly 260 265 270 Tyr Phe
Lys Met Arg Thr Gly Lys Ser Ser Ile Met Arg Ser Asp Ala 275 280 285
Pro Ile Asp Thr Cys Ile Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile 290
295 300 Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Lys Ile Thr Tyr Gly
Ala 305 310 315 320 Cys Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu
Ala Thr Gly Met 325 330 335 Arg Asn Val Pro Glu Lys Gln Thr Arg Gly
Leu Phe Gly Ala Ile Ala 340 345 350 Gly Phe Ile Glu Asn Gly Trp Glu
Gly Met Ile Asp Gly Trp Tyr Gly 355 360 365 Phe Arg His Gln Asn Ser
Glu Gly Thr Gly Gln Ala Ala Asp Leu Lys 370 375 380 Ser Thr Gln Ala
Ala Ile Asp Gln Ile Asn Gly Lys Leu Asn Arg Val 385 390 395 400 Ile
Glu Lys Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser 405 410
415 Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr
420 425 430 Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala
Leu Glu 435 440 445 Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met
Asn Lys Leu Phe 450 455 460 Glu Lys Thr Arg Arg Gln Leu Arg Glu Asn
Ala Glu Asp Met Gly Asn 465 470 475 480 Gly Cys Phe Lys Ile Tyr His
Lys Cys Asp Asn Ala Cys Ile Glu Ser 485 490 495 Ile Arg Asn Gly Thr
Tyr Asp His Asp Val Tyr Arg Asp Glu Ala Leu 500 505 510 Asn Asn Arg
Phe Gln Ile Lys 515 211557DNAInfluenza virus 21cttgatctgg
aatctgttgt tcagggcctc gtccctgtac acatcgtggt cgtaggtgcc 60gtttctgatg
ctctcgatgc aggcgttgtc gcacttgtgg tagatcttga agcagccgtt
120gcccatgtcc tcggcgtttt ctctcagctg ccgtctggtc ttttcgaaca
gcttattcat 180ctcgctgtcg gtcaggtcga tggtgtgctg gttttccagg
gcgaccagca gttcggcatt 240gtagctccac aggtcgatct tggtgtcctc
cacgtatttt tccaggtcct ggattctgcc 300ctccacctcg ctgaattctt
tctcgatctg gtggaacttc tcgttggttt tctcgatcac 360tctgttcagc
ttgccgttga tctggtcgat ggcggcctgt gtagacttca gatcggcggc
420ctgtcctgtg ccctcagaat tctggtgtct gaagccgtac cacccatcga
tcatgccctc 480ccagccgttc tcgataaagc cggcaatggc gccaaacagg
cctcttgtct gcttctcggg 540cacatttctc atgccggtgg ccagcttcag
ggtgttctgc ttcacgtact tagggcaggc 600gccgtaggtg atcttgttca
cgttctggaa gggcttgtcg ttggggatgc tgccgttggg 660ggtgatgcac
tcgctgatac aggtatcgat gggggcgtcg cttctcatga tgctgctctt
720gccggtccgc atcttgaagt agcctctggg ggcgatcagg ttgccattgc
tgttgatcac 780cagcacgtcg ccaggtttca cgatggtcca gtagatgctg
attctgctgc tcaggccgcg 840cacccaaggt cttgactcga tgttggggat
gatggtctgc tgggaccgtc tggtagacac 900ggtcactctg ccgctggcct
gcacgtacag gctggtctgt tcctgattgg tgctagggtg 960gtgcacgccc
cagatgtaca gcttgtcgaa gttgtcgttg ttgggcatgg tcacgttcag
1020cacagggtag gtgctgccgg acttggtcag ccagttcagt ctgctgaaga
agccgctgcc 1080agggcctctt ttacaggcat tgctgccgcc attctgggtc
acgcctgtcc aggtaaagcc 1140ctcggtgatg aattccagtg tgccgctgct
ggccaccagg cttctcagag aggcgtaatc 1200gggcacatcg taggggtagc
agttgctgaa ggccttgctt ctctccacga acaggtccca 1260tgtctcgttc
tggaacacgt cgcagtgagg atcgcccagc agggcatcga tcagggtaca
1320gtcgatgcca tccaggattc tgtgggggtt gttgcagatc ttgccggtgc
tgctgctctg 1380caccagctcg gtggcattgg tcacttcgat ctggtcgtcg
gtaatggttt tcaccagggt 1440gccattaggc acggcgtggt gtcccagaca
cagggtggcg gtgctattat cattgccggg 1500cagatcctgg cccagagcca
gacaaaagat gtagctcagg gcaatgatgg ttttcat 1557221557DNAInfluenza
virus 22atgaaaacca tcattgccct gagctacatc ctgtgcctgg tgttcacaca
gaagctgccc 60ggcaacgata atagcaccgc cacactgtgt ctgggacacc acgccgtgcc
taatggcacc 120atcgtgaaaa caatcaccaa cgaccagatc gaagtgacca
atgccacaga gctggtgcag 180agcagcagca caggcgagat ctgtgacagc
ccccaccaga tcctggatgg cgagaactgt 240accctgatcg atgccctgct
gggcgatcct cagtgcgacg gcttccagaa caagaaatgg 300gacctgttcg
tggagagaag caaggcctac agcaactgct acccctacga cgtgcctgat
360tacgccagcc tgagaagcct ggtggcctct agcggcaccc tggaattcaa
caacgagagc 420ttcaactgga ccggcgtgac acagaatggc accagcagcg
cctgcatcag acggtccaac 480aacagcttct tcagtagact gaattggctg
acccacctga agttcaagta ccccgccctg 540aacgtgacca tgcccaacaa
tgagaagttc gacaagctgt acatctgggg agtgcaccac 600cctggcaccg
acaacgatca gatcttccct tacgcccagg ccagcggcag aatcaccgtg
660tccaccaaga gaagccagca gaccgtgatc cccaatatcg gcagcagacc
cagagtgcgg 720aacatcccca gcaggatcag catctactgg acaatcgtga
agcctggcga catcctgctg 780atcaacagca ccggcaacct gatcgcccct
cggggctact ttaagatcag aagcggcaag 840agcagcatca tgagatccga
cgcccccatc ggcaagtgca acagcgagtg catcacccca 900aacggcagca
tccccaacga caagcccttc cagaacgtga acaggatcac ctacggcgcc
960tgccctagat acgtgaagca gaacaccctg aagctggcca ccggcatgag
aaatgtgccc 1020gagaagcaga ccagaggcat ctttggcgcc attgccggct
ttatcgagaa tggctgggag 1080ggaatggtgg atgggtggta cggcttcaga
caccagaata gcgagggaat tggacaggcc 1140gccgatctga aatctaccca
ggccgccatc gaccagatca acggcaagct gaacaggctg 1200atcggcaaga
ccaacgagaa gttccaccag atcgagaaag aattcagcga ggtggagggc
1260agaatccagg acctggaaaa atacgtggag gacaccaaga tcgacctgtg
gagctacaat 1320gccgaactgc tggtcgccct ggaaaaccag cacacaattg
atctgacaga cagtgagatg 1380aataagctgt tcgagaaaac caagaagcag
ctgagagaaa acgccgagga catgggcaac 1440ggctgcttca agatctacca
caagtgcgac aacgcctgca tcggcagcat cagaaacggc 1500acctacgacc
acgacgtgta cagagatgag gccctgaaca accggtttca gatcaag
155723519PRTInfluenza virus 23Met Lys Thr Ile Ile Ala Leu Ser Tyr
Ile Leu Cys Leu Val Phe Thr 1 5 10 15 Gln Lys Leu Pro Gly Asn Asp
Asn Ser Thr Ala Thr Leu Cys Leu Gly 20 25 30 His His Ala Val Pro
Asn Gly Thr Ile Val Lys Thr Ile Thr Asn Asp 35 40 45 Gln Ile Glu
Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr 50 55 60 Gly
Glu Ile Cys Asp Ser Pro His Gln Ile Leu Asp Gly Glu Asn Cys 65 70
75 80 Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro Gln Cys Asp Gly Phe
Gln 85 90 95 Asn Lys Lys Trp Asp Leu Phe Val Glu Arg Ser Lys Ala
Tyr Ser Asn 100 105 110 Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser
Leu Arg Ser Leu Val 115 120 125 Ala Ser Ser Gly Thr Leu Glu Phe Asn
Asn Glu Ser Phe Asn Trp Thr 130 135 140 Gly Val Thr Gln Asn Gly Thr
Ser Ser Ala Cys Ile Arg Arg Ser Asn 145 150 155 160 Asn Ser Phe Phe
Ser Arg Leu Asn Trp Leu Thr His Leu Lys Phe Lys 165 170 175 Tyr Pro
Ala Leu Asn Val Thr Met Pro Asn Asn Glu Lys Phe Asp Lys 180 185 190
Leu Tyr Ile Trp Gly Val His His Pro Gly Thr Asp Asn Asp Gln Ile 195
200 205 Phe Pro Tyr Ala Gln Ala Ser Gly Arg Ile Thr Val Ser Thr Lys
Arg 210 215 220 Ser Gln Gln Thr Val Ile Pro Asn Ile Gly Ser Arg Pro
Arg Val Arg 225 230 235 240 Asn Ile Pro Ser Arg Ile Ser Ile Tyr Trp
Thr Ile Val Lys Pro Gly 245 250 255 Asp Ile Leu Leu Ile Asn Ser Thr
Gly Asn Leu Ile Ala Pro Arg Gly 260 265 270 Tyr Phe Lys Ile Arg Ser
Gly Lys Ser Ser Ile Met Arg Ser Asp Ala 275 280 285 Pro Ile Gly Lys
Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile 290 295 300 Pro Asn
Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala 305 310 315
320 Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met
325 330 335 Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala
Ile Ala 340 345 350 Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp
Gly Trp Tyr Gly 355 360 365 Phe Arg His Gln Asn Ser Glu Gly Ile Gly
Gln Ala Ala Asp Leu Lys 370 375 380 Ser Thr Gln Ala Ala Ile Asp Gln
Ile Asn Gly Lys Leu Asn Arg Leu 385 390 395 400 Ile Gly Lys Thr Asn
Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser 405 410 415 Glu Val Glu
Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr 420 425 430 Lys
Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu 435 440
445 Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe
450 455 460 Glu Lys Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu Asp Met
Gly Asn 465 470 475 480 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn
Ala Cys Ile Gly Ser 485 490 495 Ile Arg Asn Gly Thr Tyr Asp His Asp
Val Tyr Arg Asp Glu Ala Leu 500 505 510 Asn Asn Arg Phe Gln Ile Lys
515 241557DNAInfluenza virus 24cttgatctga aaccggttgt tcagggcctc
atctctgtac acgtcgtggt cgtaggtgcc 60gtttctgatg ctgccgatgc aggcgttgtc
gcacttgtgg tagatcttga agcagccgtt 120gcccatgtcc tcggcgtttt
ctctcagctg cttcttggtt ttctcgaaca gcttattcat 180ctcactgtct
gtcagatcaa ttgtgtgctg gttttccagg gcgaccagca gttcggcatt
240gtagctccac aggtcgatct tggtgtcctc cacgtatttt tccaggtcct
ggattctgcc 300ctccacctcg ctgaattctt tctcgatctg gtggaacttc
tcgttggtct tgccgatcag 360cctgttcagc ttgccgttga tctggtcgat
ggcggcctgg gtagatttca gatcggcggc 420ctgtccaatt ccctcgctat
tctggtgtct gaagccgtac cacccatcca ccattccctc 480ccagccattc
tcgataaagc cggcaatggc gccaaagatg cctctggtct gcttctcggg
540cacatttctc atgccggtgg ccagcttcag ggtgttctgc ttcacgtatc
tagggcaggc 600gccgtaggtg atcctgttca cgttctggaa gggcttgtcg
ttggggatgc tgccgtttgg 660ggtgatgcac tcgctgttgc acttgccgat
gggggcgtcg gatctcatga tgctgctctt 720gccgcttctg atcttaaagt
agccccgagg ggcgatcagg ttgccggtgc tgttgatcag 780caggatgtcg
ccaggcttca cgattgtcca gtagatgctg atcctgctgg ggatgttccg
840cactctgggt ctgctgccga tattggggat cacggtctgc tggcttctct
tggtggacac 900ggtgattctg ccgctggcct gggcgtaagg gaagatctga
tcgttgtcgg tgccagggtg 960gtgcactccc cagatgtaca gcttgtcgaa
cttctcattg ttgggcatgg tcacgttcag 1020ggcggggtac ttgaacttca
ggtgggtcag ccaattcagt ctactgaaga agctgttgtt 1080ggaccgtctg
atgcaggcgc tgctggtgcc attctgtgtc acgccggtcc agttgaagct
1140ctcgttgttg aattccaggg tgccgctaga ggccaccagg cttctcaggc
tggcgtaatc 1200aggcacgtcg taggggtagc agttgctgta ggccttgctt
ctctccacga acaggtccca 1260tttcttgttc tggaagccgt cgcactgagg
atcgcccagc agggcatcga tcagggtaca 1320gttctcgcca tccaggatct
ggtgggggct gtcacagatc tcgcctgtgc tgctgctctg 1380caccagctct
gtggcattgg tcacttcgat ctggtcgttg gtgattgttt tcacgatggt
1440gccattaggc acggcgtggt gtcccagaca cagtgtggcg gtgctattat
cgttgccggg 1500cagcttctgt gtgaacacca ggcacaggat gtagctcagg
gcaatgatgg ttttcat 1557251560DNAInfluenza virus 25atggaaaaga
tcgtgctgct gctggccatt gtgagcctgg tgaagagcga ccagatctgc 60attggctacc
acgccaacaa tagcacagag caggtggaca ccatcatgga aaaaaacgtg
120accgtgaccc acgctcagga catcctggaa aagacccaca acggcaagct
gtgtgatctg 180gacggcgtga agcctctgat cctgagagat tgtagcgtgg
ctggatggct gctgggcaac 240cctatgtgcg acgagttcat caacgtgccc
gagtggagct atatcgtgga gaaggccaac 300cccaccaacg atctgtgtta
ccccggcagc ttcaacgatt acgaggaact gaagcacctg 360ctgtcccgga
tcaaccactt cgagaagatc cagatcatcc ccaagtcctc ttggagcgat
420cacgaagcct ctagcggagt gtctagcgcc tgtccttacc tgggcagccc
cagcttcttc 480agaaacgtgg tgtggctgat caagaagaac agcacctacc
ccaccatcaa gaagagctac 540aacaacacca accaggaaga tctgctggtc
ctgtggggaa tccaccaccc taatgatgcc 600gccgagcaga ccagactgta
ccagaacccc accacctata tcagcatcgg caccagcacc 660ctgaatcaga
gactggtgcc caagatcgcc accagatcca aggtgaacgg ccagagcggc
720aggatggaat tcttctggac catcctgaag cccaacgacg ccatcaactt
cgagagcaac 780ggcaacttta tcgcccctga gtacgcctac aagatcgtga
agaagggcga cagcgccatc 840atgaagagcg agctggaata cggcaactgc
aacaccaagt gccagacacc tatgggcgcc 900atcaacagca gcatgccctt
ccacaacatc caccctctga ccatcggcga gtgccctaag 960tacgtgaaga
gcaacagact ggtgctggcc acaggcctga gaaatagccc ccagcgggag
1020agcagaagaa agaagagggg cctgtttgga gccatcgccg gctttattga
aggcggctgg 1080cagggaatgg tggatggctg gtacggctac caccacagca
atgagcaggg ctctggatat 1140gccgccgaca aagagtctac ccagaaggcc
atcgacggcg tcaccaacaa ggtgaacagc 1200atcatcgaca agatgaacac
ccagttcgag gctgtgggca gagagttcaa caacctggaa 1260cggcggatcg
agaacctgaa caagaaaatg gaagatggct tcctggatgt gtggacctac
1320aatgccgaac tgctggtgct gatggaaaac gagcggaccc tggacttcca
cgacagcaac 1380gtgaagaacc tgtacgacaa agtgcggctg cagctgagag
acaacgccaa agagctgggc 1440aacggctgct tcgagttcta ccacaagtgc
gacaacgagt gcatggaaag catcaggaac 1500ggcacctaca actaccctca
gtacagcgag gaagccaggc tgaagaggga agagatcagc 156026520PRTInfluenza
virus 26Met Glu Lys Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val Lys
Ser 1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr
Glu Gln Val 20 25 30 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr
His Ala Gln Asp Ile 35 40 45 Leu Glu Lys Thr His Asn Gly Lys Leu
Cys Asp Leu Asp Gly Val Lys 50 55 60 Pro Leu Ile Leu Arg Asp Cys
Ser Val Ala Gly Trp Leu Leu Gly Asn 65 70 75 80 Pro Met Cys Asp Glu
Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val 85 90 95 Glu Lys Ala
Asn Pro Thr Asn Asp Leu Cys Tyr Pro Gly Ser Phe Asn 100 105 110 Asp
Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 115 120
125 Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser
130 135 140 Ser Gly Val Ser Ser Ala Cys Pro Tyr Leu Gly Ser Pro Ser
Phe Phe 145 150 155 160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser
Thr Tyr Pro Thr Ile 165 170 175 Lys Lys Ser Tyr Asn Asn Thr Asn Gln
Glu Asp Leu Leu Val Leu Trp 180 185 190 Gly Ile His His Pro Asn Asp
Ala Ala Glu Gln Thr Arg Leu Tyr Gln 195 200 205 Asn Pro Thr Thr Tyr
Ile Ser Ile Gly Thr Ser Thr Leu Asn Gln Arg 210 215 220 Leu Val Pro
Lys Ile Ala Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225 230 235 240
Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 245
250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys
Ile 260 265 270 Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Leu
Glu Tyr Gly 275 280 285 Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly
Ala Ile Asn Ser Ser 290 295 300 Met Pro Phe His Asn Ile His Pro Leu
Thr Ile Gly Glu Cys Pro Lys 305 310 315 320 Tyr Val Lys Ser Asn Arg
Leu Val Leu Ala Thr Gly Leu Arg Asn Ser 325 330 335 Pro Gln Arg Glu
Ser Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile 340 345 350 Ala Gly
Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355 360 365
Gly Tyr His His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370
375 380 Glu Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn
Ser 385 390 395 400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val
Gly Arg Glu Phe 405 410 415 Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu
Asn Lys Lys Met Glu Asp 420 425 430 Gly Phe Leu Asp Val Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Met 435 440 445 Glu Asn Glu Arg Thr Leu
Asp Phe His Asp Ser Asn Val Lys Asn Leu 450 455 460 Tyr Asp Lys Val
Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly 465 470 475 480 Asn
Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu 485 490
495 Ser Ile Arg Asn Gly Thr Tyr Asn Tyr Pro Gln Tyr Ser Glu Glu Ala
500 505 510 Arg Leu Lys Arg Glu Glu Ile Ser 515 520
271560DNAInfluenza virus 27gctgatctct tccctcttca gcctggcttc
ctcgctgtac tgagggtagt tgtaggtgcc 60gttcctgatg ctttccatgc actcgttgtc
gcacttgtgg tagaactcga agcagccgtt 120gcccagctct ttggcgttgt
ctctcagctg cagccgcact ttgtcgtaca ggttcttcac 180gttgctgtcg
tggaagtcca gggtccgctc gttttccatc agcaccagca gttcggcatt
240gtaggtccac acatccagga agccatcttc cattttcttg ttcaggttct
cgatccgccg 300ttccaggttg ttgaactctc tgcccacagc ctcgaactgg
gtgttcatct tgtcgatgat 360gctgttcacc ttgttggtga cgccgtcgat
ggccttctgg gtagactctt tgtcggcggc 420atatccagag ccctgctcat
tgctgtggtg gtagccgtac cagccatcca ccattccctg 480ccagccgcct
tcaataaagc cggcgatggc tccaaacagg cccctcttct ttcttctgct
540ctcccgctgg gggctatttc tcaggcctgt ggccagcacc agtctgttgc
tcttcacgta 600cttagggcac tcgccgatgg tcagagggtg gatgttgtgg
aagggcatgc tgctgttgat 660ggcgcccata ggtgtctggc acttggtgtt
gcagttgccg tattccagct cgctcttcat 720gatggcgctg tcgcccttct
tcacgatctt gtaggcgtac tcaggggcga taaagttgcc 780gttgctctcg
aagttgatgg cgtcgttggg cttcaggatg gtccagaaga attccatcct
840gccgctctgg ccgttcacct tggatctggt ggcgatcttg ggcaccagtc
tctgattcag 900ggtgctggtg ccgatgctga tataggtggt ggggttctgg
tacagtctgg tctgctcggc 960ggcatcatta gggtggtgga ttccccacag
gaccagcaga tcttcctggt tggtgttgtt 1020gtagctcttc ttgatggtgg
ggtaggtgct gttcttcttg atcagccaca ccacgtttct 1080gaagaagctg
gggctgccca ggtaaggaca ggcgctagac actccgctag aggcttcgtg
1140atcgctccaa gaggacttgg ggatgatctg gatcttctcg aagtggttga
tccgggacag 1200caggtgcttc agttcctcgt aatcgttgaa gctgccgggg
taacacagat cgttggtggg 1260gttggccttc tccacgatat agctccactc
gggcacgttg atgaactcgt cgcacatagg 1320gttgcccagc agccatccag
ccacgctaca atctctcagg atcagaggct tcacgccgtc 1380cagatcacac
agcttgccgt tgtgggtctt ttccaggatg tcctgagcgt gggtcacggt
1440cacgtttttt tccatgatgg tgtccacctg ctctgtgcta ttgttggcgt
ggtagccaat 1500gcagatctgg tcgctcttca ccaggctcac aatggccagc
agcagcacga tcttttccat 1560281602DNAInfluenza virus 28atgaaggcca
tcatcgtgct gctgatggtg gtgaccagca acgccgatag aatctgcacc 60ggcatcacca
gcagcaatag cccccatgtg gtgaaaacag ccacccaggg cgaagtgaat
120gtgacaggcg tgatccctct gaccaccacc cccaccaaga gctacttcgc
caacctgaag 180ggcaccagaa ccagaggcaa gctgtgcccc gattgcctga
actgcaccga tctggatgtg 240gctctgggca gacctatgtg tgtgggcacc
acaccatctg ccaaggccag catcctgcac 300gaagtgaagc ctgtgaccag
cggctgcttc cccatcatgc acgaccggac caagatcaga 360cagctgccca
acctgctgag aggctacgag aacatccggc tgtccaccca gaatgtgatc
420gatgccgaga aagcccctgg cggaccttat agactgggca ccagcggctc
ttgtcccaat 480gccacctcca agagcggctt ttttgccaca atggcctggg
ccgtgcctaa ggacaacaac 540aagaacgcca ccaaccctct gaccgtggag
gtgccctaca tctgtacaga gggcgaggat 600cagatcacag tgtggggctt
ccacagcgac gacaagaccc agatgaagaa cctgtacggc 660gacagcaacc
cccagaagtt taccagcagc gccaatggcg tgaccaccca ctacgtgtcc
720cagatcggca gctttcccga tcagacagag gatggcggac tgcctcagtc
tggcaggatc 780gtggtggact acatgatgca gaagcctggc aagaccggca
ccatcgtgta tcagagaggc 840gtgctgctgc ctcagaaagt gtggtgtgcc
agcggcaggt ctaaagtgat caagggcagc 900ctgcctctga ttggcgaggc
cgactgtctg cacgaaaagt acggcggcct gaacaagagc 960aagccctact
acacaggcga gcacgccaag gccatcggca attgccccat ctgggtgaaa
1020acccccctga agctggccaa tggcaccaag tacagacctc ccgccaagct
gctgaaagag 1080agaggcttct ttggcgccat tgccggattt ctggaaggcg
gctgggaggg aatgattgcc 1140ggctggcacg gctatacatc tcatggggcc
catggcgtgg ctgtggccgc cgatctgaag 1200tctacccagg aagccatcaa
caagatcacc aagaacctga acagcctgag cgagctggaa 1260gtgaagaatc
tgcagagact gagcggcgcc atggatgagc tgcacaacga gatcctggaa
1320ctggacgaga aagtggatga tctccgcgcc gatacaattt cctcccagat
tgaactggcc 1380gtgctgctgt ccaacgaggg catcatcaac agcgaggatg
aacacctgct ggccctggaa 1440cggaagctga agaagatgct gggcccttct
gccgtggaga tcggcaacgg ctgcttcgag 1500acaaagcaca agtgcaacca
gacctgcctg gatagaatcg ccgctggcac cttcaatgcc 1560ggcgagttca
gcctgcctac cttcgacagc ctgaatatca cc 160229534PRTInfluenza virus
29Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp 1
5 10 15 Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val
Lys 20 25 30 Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile
Pro Leu Thr 35 40 45 Thr Thr Pro Thr Lys Ser Tyr Phe Ala Asn Leu
Lys Gly Thr Arg Thr 50 55 60 Arg Gly Lys Leu Cys Pro Asp Cys Leu
Asn Cys Thr Asp Leu Asp Val 65 70 75 80 Ala Leu Gly Arg Pro Met Cys
Val Gly Thr Thr Pro Ser Ala Lys Ala 85 90 95 Ser Ile Leu His Glu
Val Lys Pro Val Thr Ser Gly Cys Phe Pro Ile 100 105 110 Met His Asp
Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly 115 120 125 Tyr
Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asp Ala Glu Lys 130 135
140 Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser Gly Ser Cys Pro Asn
145 150 155 160 Ala Thr Ser Lys Ser Gly Phe Phe Ala Thr Met Ala Trp
Ala Val Pro 165 170 175 Lys Asp Asn Asn Lys Asn Ala Thr Asn Pro Leu
Thr Val Glu Val Pro 180 185 190 Tyr Ile Cys Thr Glu Gly Glu Asp Gln
Ile Thr Val Trp Gly Phe His 195 200 205 Ser Asp Asp Lys Thr Gln Met
Lys Asn Leu Tyr Gly Asp Ser Asn Pro 210 215 220 Gln Lys Phe Thr Ser
Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser 225 230 235 240 Gln Ile
Gly Ser Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu Pro Gln 245 250 255
Ser Gly Arg Ile Val Val Asp Tyr Met Met Gln Lys Pro Gly Lys Thr 260
265 270 Gly Thr Ile Val Tyr Gln Arg Gly Val Leu Leu Pro Gln Lys Val
Trp 275 280 285 Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu
Pro Leu Ile 290 295 300 Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly
Gly Leu Asn Lys Ser 305 310 315 320 Lys Pro Tyr Tyr Thr Gly Glu His
Ala Lys Ala Ile Gly Asn Cys Pro 325 330 335 Ile Trp Val Lys Thr Pro
Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg 340 345 350 Pro Pro Ala Lys
Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala 355 360 365 Gly Phe
Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly 370 375 380
Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys 385
390 395 400 Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn
Ser Leu 405 410 415 Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser
Gly Ala Met Asp 420 425 430 Glu Leu His Asn Glu Ile Leu Glu Leu Asp
Glu Lys Val Asp Asp Leu 435 440 445 Arg Ala Asp Thr Ile Ser Ser Gln
Ile Glu Leu Ala Val Leu Leu Ser 450 455 460 Asn Glu Gly Ile Ile Asn
Ser Glu Asp Glu His Leu Leu Ala Leu Glu 465 470 475 480 Arg Lys Leu
Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly Asn 485 490 495 Gly
Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg 500 505
510 Ile Ala Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe
515 520 525 Asp Ser Leu Asn Ile Thr 530 301602DNAInfluenza virus
30ggtgatattc aggctgtcga aggtaggcag gctgaactcg ccggcattga aggtgccagc
60ggcgattcta tccaggcagg tctggttgca cttgtgcttt gtctcgaagc agccgttgcc
120gatctccacg gcagaagggc ccagcatctt cttcagcttc cgttccaggg
ccagcaggtg 180ttcatcctcg ctgttgatga tgccctcgtt ggacagcagc
acggccagtt caatctggga 240ggaaattgta tcggcgcgga gatcatccac
tttctcgtcc agttccagga tctcgttgtg 300cagctcatcc atggcgccgc
tcagtctctg cagattcttc acttccagct cgctcaggct 360gttcaggttc
ttggtgatct tgttgatggc ttcctgggta gacttcagat cggcggccac
420agccacgcca tgggccccat gagatgtata gccgtgccag ccggcaatca
ttccctccca 480gccgccttcc agaaatccgg caatggcgcc aaagaagcct
ctctctttca gcagcttggc 540gggaggtctg tacttggtgc cattggccag
cttcaggggg gttttcaccc agatggggca 600attgccgatg gccttggcgt
gctcgcctgt gtagtagggc ttgctcttgt tcaggccgcc 660gtacttttcg
tgcagacagt cggcctcgcc aatcagaggc aggctgccct tgatcacttt
720agacctgccg ctggcacacc acactttctg aggcagcagc acgcctctct
gatacacgat 780ggtgccggtc ttgccaggct tctgcatcat gtagtccacc
acgatcctgc cagactgagg 840cagtccgcca tcctctgtct gatcgggaaa
gctgccgatc tgggacacgt agtgggtggt 900cacgccattg gcgctgctgg
taaacttctg ggggttgctg tcgccgtaca ggttcttcat 960ctgggtcttg
tcgtcgctgt ggaagcccca cactgtgatc tgatcctcgc cctctgtaca
1020gatgtagggc acctccacgg tcagagggtt ggtggcgttc ttgttgttgt
ccttaggcac 1080ggcccaggcc attgtggcaa aaaagccgct cttggaggtg
gcattgggac aagagccgct 1140ggtgcccagt ctataaggtc cgccaggggc
tttctcggca tcgatcacat tctgggtgga 1200cagccggatg ttctcgtagc
ctctcagcag gttgggcagc tgtctgatct tggtccggtc 1260gtgcatgatg
gggaagcagc cgctggtcac aggcttcact tcgtgcagga tgctggcctt
1320ggcagatggt gtggtgccca cacacatagg tctgcccaga gccacatcca
gatcggtgca 1380gttcaggcaa tcggggcaca gcttgcctct ggttctggtg
cccttcaggt tggcgaagta 1440gctcttggtg ggggtggtgg tcagagggat
cacgcctgtc acattcactt cgccctgggt 1500ggctgttttc accacatggg
ggctattgct gctggtgatg ccggtgcaga ttctatcggc 1560gttgctggtc
accaccatca gcagcacgat gatggccttc at 1602311493DNAInfluenza virus
31atgacaactc aacagccacg ctctgcttgg ggcaccatgc cgtccctaac gggaccattg
60tgaaaaccat tactaacgat cagatagagg tgactaatgc caccgagctg gtgcaaagta
120gctccacagg agagatctgc gatagtcccc accagattct ggacggaaag
aattgtacgc 180tgatcgacgc gctgttgggc gaccctcagt gtgacggatt
tcagaataag aagtgggatc 240tgtttgtgga aaggtcaaag gcttattcaa
attgctaccc ttacgatgtg cctgattatg 300ccagcctgcg gtccctcgtc
gcgtctagtg ggactctgga gttcaacaac gagtcattta 360actggactgg
cgttacacag aacgggacta gttccgcttg cataaggaga agcaaaaata
420gtttcttcag cagactgaat tggctgacac atctgaactt caagtaccct
gcactgaatg 480taaccatgcc caacaacgag cagttcgata agctttacat
ttggggagtt catcatcctg 540gcactgacaa ggatcagatc tttctgtatg
cccaggcttc cggcaggatt accgtgtcta 600caaagagaag ccagcaaact
gtgtctccca atatcggcag tagacccaga gtacggaaca 660tccctagtcg
catcagtatt tactggacca tcgtgaaacc aggcgatatt ctcctgatta
720acagtactgg caacctgatc gccccccggg gatactttaa aatccgctct
ggaaagtcct 780ccattatgag atcagatgca ccgatcggaa aatgcaactc
tgagtgtatc acacccaatg 840ggagcattcc caatgacaaa cctttccaga
acgttaatcg aataacttat ggggcctgtc 900cacggtacgt gaagcaaaat
accttgaaac tggcgaccgg tatgcgcaat gtccccgaaa 960aacagacccg
cgggatattt ggggctatcg caggctttat cgagaatggc tgggaaggga
1020tggtggatgg ttggtatggt tttagacatc aaaactccga aggcagaggc
caggctgccg 1080atctcaagag cacgcaggcc gctatagatc agatcaatgg
aaagctcaac agactgatcg 1140ggaaaaccaa cgaaaaattc catcagatcg
agaaagagtt ctccgaagtc gaggggcgca 1200tacaggacct ggagaagtat
gttgaggata caaagattga tctgtggtcc tacaatgccg 1260agctgctggt
ggctctggag aatcagcaca ctattgacct gaccgattca gagatgaaca
1320aactttttga gaagacgaag aagcagctta gagaaaatgc agaggacatg
gggaacggat 1380gctttaaaat atatcataag tgtgataatg cctgcatcgg
atcaattaga aatggtacct 1440atgatcacga tgtttacagg gacgaagcgc
tgaataacag gttccagata aaa 149332519PRTInfluenza virus 32Met Lys Thr
Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala 1 5 10 15 Gln
Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly 20 25
30 His His Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile Thr Asn Asp
35 40 45 Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser
Ser Thr 50 55 60 Gly Glu Ile Cys Asp Ser Pro His Gln Ile Leu Asp
Gly Lys Asn Cys 65 70 75 80 Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro
Gln Cys Asp Gly Phe Gln 85 90 95 Asn Lys Lys Trp Asp Leu Phe Val
Glu Arg Ser Lys Ala Tyr Ser Asn 100
105 110 Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu
Val 115 120 125 Ala Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu Ser Phe
Asn Trp Thr 130 135 140 Gly Val Thr Gln Asn Gly Thr Ser Ser Ala Cys
Ile Arg Arg Ser Lys 145 150 155 160 Asn Ser Phe Phe Ser Arg Leu Asn
Trp Leu Thr His Leu Asn Phe Lys 165 170 175 Tyr Pro Ala Leu Asn Val
Thr Met Pro Asn Asn Glu Gln Phe Asp Lys 180 185 190 Leu Tyr Ile Trp
Gly Val His His Pro Gly Thr Asp Lys Asp Gln Ile 195 200 205 Phe Leu
Tyr Ala Gln Ala Ser Gly Arg Ile Thr Val Ser Thr Lys Arg 210 215 220
Ser Gln Gln Thr Val Ser Pro Asn Ile Gly Ser Arg Pro Arg Val Arg 225
230 235 240 Asn Ile Pro Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys
Pro Gly 245 250 255 Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile
Ala Pro Arg Gly 260 265 270 Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser
Ile Met Arg Ser Asp Ala 275 280 285 Pro Ile Gly Lys Cys Asn Ser Glu
Cys Ile Thr Pro Asn Gly Ser Ile 290 295 300 Pro Asn Asp Lys Pro Phe
Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala 305 310 315 320 Cys Pro Arg
Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met 325 330 335 Arg
Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala 340 345
350 Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly
355 360 365 Phe Arg His Gln Asn Ser Glu Gly Arg Gly Gln Ala Ala Asp
Leu Lys 370 375 380 Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys
Leu Asn Arg Leu 385 390 395 400 Ile Gly Lys Thr Asn Glu Lys Phe His
Gln Ile Glu Lys Glu Phe Ser 405 410 415 Glu Val Glu Gly Arg Ile Gln
Asp Leu Glu Lys Tyr Val Glu Asp Thr 420 425 430 Lys Ile Asp Leu Trp
Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu 435 440 445 Asn Gln His
Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe 450 455 460 Glu
Lys Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 465 470
475 480 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly
Ser 485 490 495 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp
Glu Ala Leu 500 505 510 Asn Asn Arg Phe Gln Ile Lys 515
331493DNAInfluenza virus 33ttttatctgg aacctgttat tcagcgcttc
gtccctgtaa acatcgtgat cataggtacc 60atttctaatt gatccgatgc aggcattatc
acacttatga tatattttaa agcatccgtt 120ccccatgtcc tctgcatttt
ctctaagctg cttcttcgtc ttctcaaaaa gtttgttcat 180ctctgaatcg
gtcaggtcaa tagtgtgctg attctccaga gccaccagca gctcggcatt
240gtaggaccac agatcaatct ttgtatcctc aacatacttc tccaggtcct
gtatgcgccc 300ctcgacttcg gagaactctt tctcgatctg atggaatttt
tcgttggttt tcccgatcag 360tctgttgagc tttccattga tctgatctat
agcggcctgc gtgctcttga gatcggcagc 420ctggcctctg ccttcggagt
tttgatgtct aaaaccatac caaccatcca ccatcccttc 480ccagccattc
tcgataaagc ctgcgatagc cccaaatatc ccgcgggtct gtttttcggg
540gacattgcgc ataccggtcg ccagtttcaa ggtattttgc ttcacgtacc
gtggacaggc 600cccataagtt attcgattaa cgttctggaa aggtttgtca
ttgggaatgc tcccattggg 660tgtgatacac tcagagttgc attttccgat
cggtgcatct gatctcataa tggaggactt 720tccagagcgg attttaaagt
atccccgggg ggcgatcagg ttgccagtac tgttaatcag 780gagaatatcg
cctggtttca cgatggtcca gtaaatactg atgcgactag ggatgttccg
840tactctgggt ctactgccga tattgggaga cacagtttgc tggcttctct
ttgtagacac 900ggtaatcctg ccggaagcct gggcatacag aaagatctga
tccttgtcag tgccaggatg 960atgaactccc caaatgtaaa gcttatcgaa
ctgctcgttg ttgggcatgg ttacattcag 1020tgcagggtac ttgaagttca
gatgtgtcag ccaattcagt ctgctgaaga aactattttt 1080gcttctcctt
atgcaagcgg aactagtccc gttctgtgta acgccagtcc agttaaatga
1140ctcgttgttg aactccagag tcccactaga cgcgacgagg gaccgcaggc
tggcataatc 1200aggcacatcg taagggtagc aatttgaata agcctttgac
ctttccacaa acagatccca 1260cttcttattc tgaaatccgt cacactgagg
gtcgcccaac agcgcgtcga tcagcgtaca 1320attctttccg tccagaatct
ggtggggact atcgcagatc tctcctgtgg agctactttg 1380caccagctcg
gtggcattag tcacctctat ctgatcgtta gtaatggttt tcacaatggt
1440cccgttaggg acggcatggt gccccaagca gagcgtggct gttgagttgt cat
1493341551DNAInfluenza virus 34atgaaagtga agctgctggt gctgctgtgt
acctttaccg ccacctacgc cgataccatc 60tgtatcggct accacgccaa caatagcacc
gacaccgtgg ataccgtgct ggaaaagaac 120gtgaccgtga cccacagcgt
gaacctgctg gaaaacagcc acaacggcaa gctgtgtctg 180ctgaaaggca
ttgcccctct gcagctggga aattgtagcg tggccggctg gattctgggc
240aatcctgagt gcgagctgct gatttccaaa gagtcctggt cctacatcgt
ggagaagccc 300aaccctgaga atggcacctg ctaccctggc cacttcgccg
attacgagga actgagagaa 360cagctgtcca gcgtgtccag cttcgagaga
ttcgagatct tccccaaaga gagcagctgg 420cccaatcata cagtgaccgg
cgtgagcgcc tcttgtagcc acaatggcga gagcagcttc 480tacagaaacc
tgctgtggct gaccggcaag aacggcctgt accccaacct gagcaagagc
540tacgccaaca acaaagaaaa agaagtgctg gtcctctggg gagtgcacca
ccctcctaac 600atcggcatcc agaaggccct gtaccacacc gagaatgcct
acgtgtccgt ggtgtccagc 660cactacagca gaaagttcac ccccgagatc
gccaaaagac ccaaagtgcg ggaccaggaa 720ggcaggatca actactactg
gaccctgctg gaacctggcg acaccatcat cttcgaggcc 780aacggcaatc
tgatcgcccc tagatacgcc tttgccctga gcagaggctt tggcagcggc
840atcatcaaca gcaacgcccc catggacaag tgtgacgcca agtgtcagac
accacaggga 900gctatcaata gcagcctgcc cttccagaat gtgcaccctg
tgaccatcgg cgagtgtcct 960aaatacgtgc ggagcgccaa gctgagaatg
gtgaccggcc tgaggaatat ccccagcatc 1020cagagcagag gcctgtttgg
cgccattgcc ggctttatcg agggcggatg gacaggcatg 1080gtggatgggt
ggtacggcta ccaccaccag aatgagcagg gatctggcta tgccgccgat
1140cagaagagca cccagaacgc catcaacggc atcaccaaca aagtgaacag
cgtgatcgag 1200aagatgaaca cccagttcac cgccgtgggc aaagagttca
acaagctgga acggcggatg 1260gaaaacctga acaagaaggt ggacgacggc
ttcatcgaca tctggaccta caacgccgaa 1320ctcctggtcc tcctggaaaa
tgagaggacc ctggacttcc acgacagcaa cgtgaagaac 1380ctgtacgaga
aagtgaagag ccagctgaag aacaacgcca aagagatcgg caacggctgc
1440ttcgagttct accacaagtg caacgacgag tgcatggaaa gcgtgaagaa
cggcacctac 1500gactacccca agtacagcga ggaaagcaag ctgaaccggg
agaagatcga t 155135517PRTInfluenza virus 35Met Lys Val Lys Leu Leu
Val Leu Leu Cys Thr Phe Thr Ala Thr Tyr 1 5 10 15 Ala Asp Thr Ile
Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp
Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40 45
Leu Leu Glu Asn Ser His Asn Gly Lys Leu Cys Leu Leu Lys Gly Ile 50
55 60 Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu
Gly 65 70 75 80 Asn Pro Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser Trp
Ser Tyr Ile 85 90 95 Val Glu Lys Pro Asn Pro Glu Asn Gly Thr Cys
Tyr Pro Gly His Phe 100 105 110 Ala Asp Tyr Glu Glu Leu Arg Glu Gln
Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro
Lys Glu Ser Ser Trp Pro Asn His Thr 130 135 140 Val Thr Gly Val Ser
Ala Ser Cys Ser His Asn Gly Glu Ser Ser Phe 145 150 155 160 Tyr Arg
Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly Leu Tyr Pro Asn 165 170 175
Leu Ser Lys Ser Tyr Ala Asn Asn Lys Glu Lys Glu Val Leu Val Leu 180
185 190 Trp Gly Val His His Pro Pro Asn Ile Gly Ile Gln Lys Ala Leu
Tyr 195 200 205 His Thr Glu Asn Ala Tyr Val Ser Val Val Ser Ser His
Tyr Ser Arg 210 215 220 Lys Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys
Val Arg Asp Gln Glu 225 230 235 240 Gly Arg Ile Asn Tyr Tyr Trp Thr
Leu Leu Glu Pro Gly Asp Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly
Asn Leu Ile Ala Pro Arg Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly
Phe Gly Ser Gly Ile Ile Asn Ser Asn Ala Pro Met 275 280 285 Asp Lys
Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly Ala Ile Asn Ser 290 295 300
Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile Gly Glu Cys Pro 305
310 315 320 Lys Tyr Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu
Arg Asn 325 330 335 Ile Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala
Ile Ala Gly Phe 340 345 350 Ile Glu Gly Gly Trp Thr Gly Met Val Asp
Gly Trp Tyr Gly Tyr His 355 360 365 His Gln Asn Glu Gln Gly Ser Gly
Tyr Ala Ala Asp Gln Lys Ser Thr 370 375 380 Gln Asn Ala Ile Asn Gly
Ile Thr Asn Lys Val Asn Ser Val Ile Glu 385 390 395 400 Lys Met Asn
Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu 405 410 415 Glu
Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly Phe Ile 420 425
430 Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu
435 440 445 Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr
Glu Lys 450 455 460 Val Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile
Gly Asn Gly Cys 465 470 475 480 Phe Glu Phe Tyr His Lys Cys Asn Asp
Glu Cys Met Glu Ser Val Lys 485 490 495 Asn Gly Thr Tyr Asp Tyr Pro
Lys Tyr Ser Glu Glu Ser Lys Leu Asn 500 505 510 Arg Glu Lys Ile Asp
515 361551DNAInfluenza virus 36atcgatcttc tcccggttca gcttgctttc
ctcgctgtac ttggggtagt cgtaggtgcc 60gttcttcacg ctttccatgc actcgtcgtt
gcacttgtgg tagaactcga agcagccgtt 120gccgatctct ttggcgttgt
tcttcagctg gctcttcact ttctcgtaca ggttcttcac 180gttgctgtcg
tggaagtcca gggtcctctc attttccagg aggaccagga gttcggcgtt
240gtaggtccag atgtcgatga agccgtcgtc caccttcttg ttcaggtttt
ccatccgccg 300ttccagcttg ttgaactctt tgcccacggc ggtgaactgg
gtgttcatct tctcgatcac 360gctgttcact ttgttggtga tgccgttgat
ggcgttctgg gtgctcttct gatcggcggc 420atagccagat ccctgctcat
tctggtggtg gtagccgtac cacccatcca ccatgcctgt 480ccatccgccc
tcgataaagc cggcaatggc gccaaacagg cctctgctct ggatgctggg
540gatattcctc aggccggtca ccattctcag cttggcgctc cgcacgtatt
taggacactc 600gccgatggtc acagggtgca cattctggaa gggcaggctg
ctattgatag ctccctgtgg 660tgtctgacac ttggcgtcac acttgtccat
gggggcgttg ctgttgatga tgccgctgcc 720aaagcctctg ctcagggcaa
aggcgtatct aggggcgatc agattgccgt tggcctcgaa 780gatgatggtg
tcgccaggtt ccagcagggt ccagtagtag ttgatcctgc cttcctggtc
840ccgcactttg ggtcttttgg cgatctcggg ggtgaacttt ctgctgtagt
ggctggacac 900cacggacacg taggcattct cggtgtggta cagggccttc
tggatgccga tgttaggagg 960gtggtgcact ccccagagga ccagcacttc
tttttctttg ttgttggcgt agctcttgct 1020caggttgggg tacaggccgt
tcttgccggt cagccacagc aggtttctgt agaagctgct 1080ctcgccattg
tggctacaag aggcgctcac gccggtcact gtatgattgg gccagctgct
1140ctctttgggg aagatctcga atctctcgaa gctggacacg ctggacagct
gttctctcag 1200ttcctcgtaa tcggcgaagt ggccagggta gcaggtgcca
ttctcagggt tgggcttctc 1260cacgatgtag gaccaggact ctttggaaat
cagcagctcg cactcaggat tgcccagaat 1320ccagccggcc acgctacaat
ttcccagctg cagaggggca atgcctttca gcagacacag 1380cttgccgttg
tggctgtttt ccagcaggtt cacgctgtgg gtcacggtca cgttcttttc
1440cagcacggta tccacggtgt cggtgctatt gttggcgtgg tagccgatac
agatggtatc 1500ggcgtaggtg gcggtaaagg tacacagcag caccagcagc
ttcactttca t 1551371605DNAInfluenza virus 37atgaaggcca tcatcgtgct
gctgatggtg gtcacaagca acgccgatag aatctgtacc 60ggcatcacca gcagcaatag
ccctcacgtc gtgaaaacag ctacacaggg cgaagtgaat 120gtgaccggcg
tgatccctct gaccacaaca cctacaaaga gccacttcgc caatctgaag
180ggcacagaga caagaggcaa gctgtgtccc aagtgcctga attgcacaga
tctggatgtg 240gctctgggca gacctaagtg tacaggcaaa atccctagcg
ccagagtgtc cattctgcat 300gaagtgcgac ctgtgaccag cggctgtttt
cctattatgc acgaccggac caagatcaga 360cagctgccta atctgctgag
aggctacgag cacatcagac tgagcaccca caatgtgatc 420aacgccgaaa
atgctcctgg cggcccttat aagatcggca catctggcag ctgccccaac
480attacaaatg gcaatggctt ctttgccacc atggcttggg ccgtgcctaa
gaacgataag 540aacaagaccg ccaccaaccc cctgacaatc gaggtgccat
atatctgtac agagggcgag 600gatcagatca ccgtgtgggg atttcacagc
gacaacgaaa cacagatggc caagctgtac 660ggcgatagca agcctcagaa
gtttaccagc tctgccaatg gcgtgaccac acactatgtg 720tctcagatcg
gcggcttccc taatcagaca gaagatggcg gactgcctca gtctggaaga
780atcgtggtgg attacatggt gcagaagtct ggcaagaccg gcaccatcac
atatcagaga 840ggaatcctgc tgccccagaa agtgtggtgc gcttctggaa
gatccaaagt gatcaagggc 900agcctgcctc tgattggaga agccgattgt
ctgcacgaga aatacggcgg cctgaacaag 960agcaagcctt actatacagg
cgagcacgcc aaggccatcg gcaattgtcc tatttgggtc 1020aagacccctc
tgaagctggc caatggcaca aagtatagac ctccagccaa gctgctgaaa
1080gagagaggct tttttggagc tatcgccggc tttctggaag gcggatggga
gggaatgatt 1140gctggatggc atggctacac atctcatggc gcacatggcg
tggcagtggc tgctgatctg 1200aaatctacac aggaagccat caacaagatc
accaagaacc tgaacagcct gagcgagctg 1260gaagtgaaga atctgcagag
actgtctggc gccatggacg aactgcacaa tgagatcctg 1320gaactggacg
agaaggtgga cgatctgaga gccgatacaa tcagcagcca gattgaactg
1380gctgtgctgc tgtctaacga gggcatcatc aatagcgagg acgaacatct
gctggccctg 1440gaaagaaagc tgaagaagat gctgggacct agcgccgtgg
aaatcggcaa tggatgcttt 1500gagacaaagc acaagtgcaa ccagacctgc
ctggatagaa ttgccgccgg aacatttgat 1560gccggcgagt tttctctgcc
caccttcgat agcctgaata tcaca 160538535PRTInfluenza virus 38Met Lys
Ala Ile Ile Val Leu Leu Met Val Val Thr Ser Asn Ala Asp 1 5 10 15
Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys 20
25 30 Thr Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu
Thr 35 40 45 Thr Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly
Thr Glu Thr 50 55 60 Arg Gly Lys Leu Cys Pro Lys Cys Leu Asn Cys
Thr Asp Leu Asp Val 65 70 75 80 Ala Leu Gly Arg Pro Lys Cys Thr Gly
Lys Ile Pro Ser Ala Arg Val 85 90 95 Ser Ile Leu His Glu Val Arg
Pro Val Thr Ser Gly Cys Phe Pro Ile 100 105 110 Met His Asp Arg Thr
Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly 115 120 125 Tyr Glu His
Ile Arg Leu Ser Thr His Asn Val Ile Asn Ala Glu Asn 130 135 140 Ala
Pro Gly Gly Pro Tyr Lys Ile Gly Thr Ser Gly Ser Cys Pro Asn 145 150
155 160 Ile Thr Asn Gly Asn Gly Phe Phe Ala Thr Met Ala Trp Ala Val
Pro 165 170 175 Lys Asn Asp Lys Asn Lys Thr Ala Thr Asn Pro Leu Thr
Ile Glu Val 180 185 190 Pro Tyr Ile Cys Thr Glu Gly Glu Asp Gln Ile
Thr Val Trp Gly Phe 195 200 205 His Ser Asp Asn Glu Thr Gln Met Ala
Lys Leu Tyr Gly Asp Ser Lys 210 215 220 Pro Gln Lys Phe Thr Ser Ser
Ala Asn Gly Val Thr Thr His Tyr Val 225 230 235 240 Ser Gln Ile Gly
Gly Phe Pro Asn Gln Thr Glu Asp Gly Gly Leu Pro 245 250 255 Gln Ser
Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Ser Gly Lys 260 265 270
Thr Gly Thr Ile Thr Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys Val 275
280 285 Trp Cys Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro
Leu 290 295 300 Ile Gly Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly
Leu Asn Lys 305 310 315 320 Ser Lys Pro Tyr Tyr Thr Gly Glu His Ala
Lys Ala Ile Gly Asn Cys 325 330 335 Pro Ile Trp Val Lys Thr Pro Leu
Lys Leu Ala Asn Gly Thr Lys Tyr 340 345 350 Arg Pro Pro Ala Lys Leu
Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile 355 360 365 Ala Gly Phe Leu
Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His 370 375 380 Gly Tyr
Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu 385 390
395 400 Lys Ser Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn
Ser 405 410 415 Leu Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser
Gly Ala Met 420 425 430 Asp Glu Leu His Asn Glu Ile Leu Glu Leu Asp
Glu Lys Val Asp Asp 435 440 445 Leu Arg Ala Asp Thr Ile Ser Ser Gln
Ile Glu Leu Ala Val Leu Leu 450 455 460 Ser Asn Glu Gly Ile Ile Asn
Ser Glu Asp Glu His Leu Leu Ala Leu 465 470 475 480 Glu Arg Lys Leu
Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly 485 490 495 Asn Gly
Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp 500 505 510
Arg Ile Ala Ala Gly Thr Phe Asp Ala Gly Glu Phe Ser Leu Pro Thr 515
520 525 Phe Asp Ser Leu Asn Ile Thr 530 535 391605DNAInfluenza
virus 39tgtgatattc aggctatcga aggtgggcag agaaaactcg ccggcatcaa
atgttccggc 60ggcaattcta tccaggcagg tctggttgca cttgtgcttt gtctcaaagc
atccattgcc 120gatttccacg gcgctaggtc ccagcatctt cttcagcttt
ctttccaggg ccagcagatg 180ttcgtcctcg ctattgatga tgccctcgtt
agacagcagc acagccagtt caatctggct 240gctgattgta tcggctctca
gatcgtccac cttctcgtcc agttccagga tctcattgtg 300cagttcgtcc
atggcgccag acagtctctg cagattcttc acttccagct cgctcaggct
360gttcaggttc ttggtgatct tgttgatggc ttcctgtgta gatttcagat
cagcagccac 420tgccacgcca tgtgcgccat gagatgtgta gccatgccat
ccagcaatca ttccctccca 480tccgccttcc agaaagccgg cgatagctcc
aaaaaagcct ctctctttca gcagcttggc 540tggaggtcta tactttgtgc
cattggccag cttcagaggg gtcttgaccc aaataggaca 600attgccgatg
gccttggcgt gctcgcctgt atagtaaggc ttgctcttgt tcaggccgcc
660gtatttctcg tgcagacaat cggcttctcc aatcagaggc aggctgccct
tgatcacttt 720ggatcttcca gaagcgcacc acactttctg gggcagcagg
attcctctct gatatgtgat 780ggtgccggtc ttgccagact tctgcaccat
gtaatccacc acgattcttc cagactgagg 840cagtccgcca tcttctgtct
gattagggaa gccgccgatc tgagacacat agtgtgtggt 900cacgccattg
gcagagctgg taaacttctg aggcttgcta tcgccgtaca gcttggccat
960ctgtgtttcg ttgtcgctgt gaaatcccca cacggtgatc tgatcctcgc
cctctgtaca 1020gatatatggc acctcgattg tcagggggtt ggtggcggtc
ttgttcttat cgttcttagg 1080cacggcccaa gccatggtgg caaagaagcc
attgccattt gtaatgttgg ggcagctgcc 1140agatgtgccg atcttataag
ggccgccagg agcattttcg gcgttgatca cattgtgggt 1200gctcagtctg
atgtgctcgt agcctctcag cagattaggc agctgtctga tcttggtccg
1260gtcgtgcata ataggaaaac agccgctggt cacaggtcgc acttcatgca
gaatggacac 1320tctggcgcta gggattttgc ctgtacactt aggtctgccc
agagccacat ccagatctgt 1380gcaattcagg cacttgggac acagcttgcc
tcttgtctct gtgcccttca gattggcgaa 1440gtggctcttt gtaggtgttg
tggtcagagg gatcacgccg gtcacattca cttcgccctg 1500tgtagctgtt
ttcacgacgt gagggctatt gctgctggtg atgccggtac agattctatc
1560ggcgttgctt gtgaccacca tcagcagcac gatgatggcc ttcat
1605402058DNAArtificial SequenceSynthetic 40atgaaggcca aactgctggt
gctgctgtgt acctttaccg ccacctacgc cgacacaatc 60tgtatcggct accacgccaa
caatagcacc gacaccgtgg atacagtgct ggagaagaac 120gtgaccgtga
cccactctgt gaacctgctg gaggacagcc acaatggcaa gctgtgtctg
180ctgaaaggca ttgcccctct gcagctgggc aattgttctg tggccggatg
gattctgggc 240aaccccgagt gtgagctgct gatttctaag gagagctgga
gctacatcgt ggagaccccc 300aatcctgaga atggcacctg ctaccctggc
tacttcgccg attacgagga gctgcgcgag 360cagctgtcta gcgtgtccag
cttcgagaga ttcgagatct tccccaagga gtccagctgg 420cctaatcaca
cagtgacagg cgtgtctgcc agctgtagcc acaacggcaa aagcagcttc
480taccggaacc tgctgtggct gacaggcaag aatggcctgt accccaacct
gagcaagagc 540tacgtgaaca acaaggaaaa ggaagtgctg gtgctgtggg
gagtgcacca ccctcccaac 600atcggaaatc agcgggccct gtaccacaca
gagaacgcct atgtgagcgt ggtgtccagc 660cactacagca gaagattcac
ccccgagatc gccaagagac ccaaagtgag agaccaggag 720ggccggatca
attactactg gaccctgctg gagcctggcg ataccatcat cttcgaggcc
780aacggcaatc tgatcgcccc ttggtatgcc tttgccctga gcagaggctt
tggcagcggc 840atcatcacaa gcaacgcccc catggatgag tgtgatgcca
agtgccagac acctcagggc 900gccatcaata gcagcctgcc cttccagaat
gtgcaccctg tgaccatcgg cgagtgcccc 960aagtatgtga gaagcgccaa
gctgagaatg gtgaccggcc tgagaaacat ccctcagagg 1020gagaccagag
gactgtttgg agccatcgcc ggattcatcg agggaggatg gacaggcatg
1080gtggatggct ggtacggcta ccaccaccag aatgagcagg gctctggata
tgccgccgat 1140cagaagtcta cccagaacgc catcaacggc atcaccaaca
aggtgaacag cgtgatcgag 1200aagatgaaca cccagtttac cgctgtgggc
aaggagttca acaagctgga gcggaggatg 1260gagaacctga acaagaaggt
ggacgacggc tttctggaca tctggaccta caatgccgaa 1320ctcctggtcc
tcctcgagaa tgagaggacc ctggacttcc acgacagcaa cgtgaagaac
1380ctgtatgaga aggtgaagag ccagctgaag aacaacgcca aggagatcgg
caacggctgc 1440ttcgagttct accacaagtg taacaacgag tgtatggaga
gcgtgaagaa cggcacctac 1500gactacccta agtacagcga ggagagcaag
ctgaaccggg agaagatcga ttccggaggc 1560gacatcatca agctgctgaa
cgagcaggtg aacaaggaga tgcagagcag caacctgtac 1620atgagcatga
gcagctggtg ctacacccac agcctggacg gcgccggcct gttcctgttc
1680gaccacgccg ccgaggagta cgagcacgcc aagaagctga tcatcttcct
gaacgagaac 1740aacgtgcccg tgcagctgac cagcatcagc gcccccgagc
acaagttcga gggcctgacc 1800cagatcttcc agaaggccta cgagcacgag
cagcacatca gcgagagcat caacaacatc 1860gtggaccacg ccatcaagag
caaggaccac gccaccttca acttcctgca gtggtacgtg 1920gccgagcagc
acgaggagga ggtgctgttc aaggacatcc tggacaagat cgagctgatc
1980ggcaacgaga accacggcct gtacctggcc gaccagtacg tgaagggcat
cgccaagagc 2040aggaagagcg gatcctag 205841685PRTArtificial
SequenceSynthetic 41Met Lys Ala Lys Leu Leu Val Leu Leu Cys Thr Phe
Thr Ala Thr Tyr 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His Ala
Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn
Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Ser His
Asn Gly Lys Leu Cys Leu Leu Lys Gly Ile 50 55 60 Ala Pro Leu Gln
Leu Gly Asn Cys Ser Val Ala Gly Trp Ile Leu Gly 65 70 75 80 Asn Pro
Glu Cys Glu Leu Leu Ile Ser Lys Glu Ser Trp Ser Tyr Ile 85 90 95
Val Glu Thr Pro Asn Pro Glu Asn Gly Thr Cys Tyr Pro Gly Tyr Phe 100
105 110 Ala Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser
Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro
Asn His Thr 130 135 140 Val Thr Gly Val Ser Ala Ser Cys Ser His Asn
Gly Lys Ser Ser Phe 145 150 155 160 Tyr Arg Asn Leu Leu Trp Leu Thr
Gly Lys Asn Gly Leu Tyr Pro Asn 165 170 175 Leu Ser Lys Ser Tyr Val
Asn Asn Lys Glu Lys Glu Val Leu Val Leu 180 185 190 Trp Gly Val His
His Pro Pro Asn Ile Gly Asn Gln Arg Ala Leu Tyr 195 200 205 His Thr
Glu Asn Ala Tyr Val Ser Val Val Ser Ser His Tyr Ser Arg 210 215 220
Arg Phe Thr Pro Glu Ile Ala Lys Arg Pro Lys Val Arg Asp Gln Glu 225
230 235 240 Gly Arg Ile Asn Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp
Thr Ile 245 250 255 Ile Phe Glu Ala Asn Gly Asn Leu Ile Ala Pro Trp
Tyr Ala Phe Ala 260 265 270 Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile
Thr Ser Asn Ala Pro Met 275 280 285 Asp Glu Cys Asp Ala Lys Cys Gln
Thr Pro Gln Gly Ala Ile Asn Ser 290 295 300 Ser Leu Pro Phe Gln Asn
Val His Pro Val Thr Ile Gly Glu Cys Pro 305 310 315 320 Lys Tyr Val
Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn 325 330 335 Ile
Pro Gln Arg Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe 340 345
350 Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His
355 360 365 His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys
Ser Thr 370 375 380 Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn
Ser Val Ile Glu 385 390 395 400 Lys Met Asn Thr Gln Phe Thr Ala Val
Gly Lys Glu Phe Asn Lys Leu 405 410 415 Glu Arg Arg Met Glu Asn Leu
Asn Lys Lys Val Asp Asp Gly Phe Leu 420 425 430 Asp Ile Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu 435 440 445 Arg Thr Leu
Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys 450 455 460 Val
Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys 465 470
475 480 Phe Glu Phe Tyr His Lys Cys Asn Asn Glu Cys Met Glu Ser Val
Lys 485 490 495 Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser
Lys Leu Asn 500 505 510 Arg Glu Lys Ile Asp Ser Gly Gly Asp Ile Ile
Lys Leu Leu Asn Glu 515 520 525 Gln Val Asn Lys Glu Met Gln Ser Ser
Asn Leu Tyr Met Ser Met Ser 530 535 540 Ser Trp Cys Tyr Thr His Ser
Leu Asp Gly Ala Gly Leu Phe Leu Phe 545 550 555 560 Asp His Ala Ala
Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe 565 570 575 Leu Asn
Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro 580 585 590
Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu 595
600 605 His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His
Ala 610 615 620 Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln
Trp Tyr Val 625 630 635 640 Ala Glu Gln His Glu Glu Glu Val Leu Phe
Lys Asp Ile Leu Asp Lys 645 650 655 Ile Glu Leu Ile Gly Asn Glu Asn
His Gly Leu Tyr Leu Ala Asp Gln 660 665 670 Tyr Val Lys Gly Ile Ala
Lys Ser Arg Lys Ser Gly Ser 675 680 685 422058DNAArtificial
SequenceSynthetic 42ctaggatccg ctcttcctgc tcttggcgat gcccttcacg
tactggtcgg ccaggtacag 60gccgtggttc tcgttgccga tcagctcgat cttgtccagg
atgtccttga acagcacctc 120ctcctcgtgc tgctcggcca cgtaccactg
caggaagttg aaggtggcgt ggtccttgct 180cttgatggcg tggtccacga
tgttgttgat gctctcgctg atgtgctgct cgtgctcgta 240ggccttctgg
aagatctggg tcaggccctc gaacttgtgc tcgggggcgc tgatgctggt
300cagctgcacg ggcacgttgt tctcgttcag gaagatgatc agcttcttgg
cgtgctcgta 360ctcctcggcg gcgtggtcga acaggaacag gccggcgccg
tccaggctgt gggtgtagca 420ccagctgctc atgctcatgt acaggttgct
gctctgcatc tccttgttca cctgctcgtt 480cagcagcttg atgatgtcgc
ctccggaatc gatcttctcc cggttcagct tgctctcctc 540gctgtactta
gggtagtcgt aggtgccgtt cttcacgctc tccatacact cgttgttaca
600cttgtggtag aactcgaagc agccgttgcc gatctccttg gcgttgttct
tcagctggct 660cttcaccttc tcatacaggt tcttcacgtt gctgtcgtgg
aagtccaggg tcctctcatt 720ctcgaggagg accaggagtt cggcattgta
ggtccagatg tccagaaagc cgtcgtccac 780cttcttgttc aggttctcca
tcctccgctc cagcttgttg aactccttgc ccacagcggt 840aaactgggtg
ttcatcttct cgatcacgct gttcaccttg ttggtgatgc cgttgatggc
900gttctgggta gacttctgat cggcggcata tccagagccc tgctcattct
ggtggtggta 960gccgtaccag ccatccacca tgcctgtcca tcctccctcg
atgaatccgg cgatggctcc 1020aaacagtcct ctggtctccc tctgagggat
gtttctcagg ccggtcacca ttctcagctt 1080ggcgcttctc acatacttgg
ggcactcgcc gatggtcaca gggtgcacat tctggaaggg 1140caggctgcta
ttgatggcgc cctgaggtgt ctggcacttg gcatcacact catccatggg
1200ggcgttgctt gtgatgatgc cgctgccaaa gcctctgctc agggcaaagg
cataccaagg 1260ggcgatcaga ttgccgttgg cctcgaagat gatggtatcg
ccaggctcca gcagggtcca 1320gtagtaattg atccggccct cctggtctct
cactttgggt ctcttggcga tctcgggggt 1380gaatcttctg ctgtagtggc
tggacaccac gctcacatag gcgttctctg tgtggtacag 1440ggcccgctga
tttccgatgt tgggagggtg gtgcactccc cacagcacca gcacttcctt
1500ttccttgttg ttcacgtagc tcttgctcag gttggggtac aggccattct
tgcctgtcag 1560ccacagcagg ttccggtaga agctgctttt gccgttgtgg
ctacagctgg cagacacgcc 1620tgtcactgtg tgattaggcc agctggactc
cttggggaag atctcgaatc tctcgaagct 1680ggacacgcta gacagctgct
cgcgcagctc ctcgtaatcg gcgaagtagc cagggtagca 1740ggtgccattc
tcaggattgg gggtctccac gatgtagctc cagctctcct tagaaatcag
1800cagctcacac tcggggttgc ccagaatcca tccggccaca gaacaattgc
ccagctgcag 1860aggggcaatg cctttcagca gacacagctt gccattgtgg
ctgtcctcca gcaggttcac 1920agagtgggtc acggtcacgt tcttctccag
cactgtatcc acggtgtcgg tgctattgtt 1980ggcgtggtag ccgatacaga
ttgtgtcggc gtaggtggcg gtaaaggtac acagcagcac 2040cagcagtttg gccttcat
2058432061DNAArtificial SequenceSynthetic 43atgaaggcca tcctggtggt
gctgctgtac accttcgcca ccgccaacgc cgacaccctg 60tgcatcggct accacgccaa
caacagcacc gacaccgtgg acaccgtgct ggagaagaac 120gtgaccgtga
cccacagcgt gaacctgctg gaggacaagc acaacggcaa gctgtgcaag
180ctgcggggcg tggcccccct gcacctgggc aagtgcaaca tcgccggctg
gattctgggc 240aaccccgagt gcgagagcct gagcaccgcc agcagctgga
gctacatcgt ggagaccccc 300agcagcgaca acggcacctg ctaccccggc
gacttcatcg actacgagga gctgcgggag 360cagctgagca gcgtgagcag
cttcgagcgg ttcgagatct tccccaagac cagcagctgg 420cccaaccacg
acagcaacaa gggcgtgacc gccgcctgcc cccacgccgg cgccaagagc
480ttctacaaga acctgatctg gctggtgaag aagggcaaca gctaccccaa
gctgagcaag 540agctacatca acgacaaggg caaggaggtg ctggtgctgt
ggggcatcca ccaccccagc 600accagcgccg accagcagag cctgtaccag
aacgccgaca cctacgtgtt cgtgggcagc 660agccggtaca gcaagaagtt
caagcccgag atcgccatcc ggcccaaggt gcgggaccag 720gagggccgga
tgaactacta ctggaccctg gtggagcccg gcgacaagat caccttcgag
780gccaccggca acctggtggt gccccggtac gccttcgcca tggagcggaa
cgccggcagc 840ggcatcatca tcagcgacac ccccgtgcac gactgcaaca
ccacctgcca gacccccaag 900ggcgccatca acaccagcct gcccttccag
aacatccacc ccatcaccat cggcaagtgc 960cccaagtacg tgaagagcac
caagctgcgg ctggccaccg gcctgcggaa catccccagc 1020atccagagcc
ggggcctgtt cggcgccatc gccggcttca tcgagggcgg ctggaccggc
1080atggtggacg gctggtacgg ctaccaccac cagaacgagc agggcagcgg
ctacgccgcc 1140gacctgaaga gcacccagaa cgccatcgac gagatcacca
acaaggtgaa cagcgtgatc 1200gagaagatga acacccagtt caccgccgtg
ggcaaggagt tcaaccacct ggagaagcgg 1260atcgagaacc tgaacaagaa
ggtggacgac ggcttcctgg acatctggac ctacaacgcc 1320gagctgctgg
tgctgctgga gaacgagcgg accctggact accacgacag caacgtgaag
1380aacctgtacg agaaggtgcg gagccagctg aagaacaacg ccaaggagat
cggcaacggc 1440tgcttcgagt tctaccacaa gtgcgacaac acctgcatgg
agagcgtgaa gaacggcacc 1500tacgactacc ccaagtacag cgaggaggcc
aagctgaacc gggaggagat cgactccgga 1560ggcgacatca tcaagctgct
gaacgagcag gtgaacaagg agatgcagag cagcaacctg 1620tacatgagca
tgagcagctg gtgctacacc cacagcctgg acggcgccgg cctgttcctg
1680ttcgaccacg ccgccgagga gtacgagcac gccaagaagc tgatcatctt
cctgaacgag 1740aacaacgtgc ccgtgcagct gaccagcatc agcgcccccg
agcacaagtt cgagggcctg 1800acccagatct tccagaaggc ctacgagcac
gagcagcaca tcagcgagag catcaacaac 1860atcgtggacc acgccatcaa
gagcaaggac cacgccacct tcaacttcct gcagtggtac 1920gtggccgagc
agcacgagga ggaggtgctg ttcaaggaca tcctggacaa gatcgagctg
1980atcggcaacg agaaccacgg cctgtacctg gccgaccagt acgtgaaggg
catcgccaag 2040agcaggaaga gcggatccta g 206144686PRTArtificial
SequenceSynthetic 44Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe
Ala Thr Ala Asn 1 5 10 15 Ala Asp Thr Leu Cys Ile Gly Tyr His Ala
Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn
Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Leu Glu Asp Lys His
Asn Gly Lys Leu Cys Lys Leu Arg Gly Val 50 55 60 Ala Pro Leu His
Leu Gly Lys Cys Asn Ile Ala Gly Trp Ile Leu Gly 65 70 75 80 Asn Pro
Glu Cys Glu Ser Leu Ser Thr Ala Ser Ser Trp Ser Tyr Ile 85 90 95
Val Glu Thr Pro Ser Ser Asp Asn Gly Thr Cys Tyr Pro Gly Asp Phe 100
105 110 Ile Asp Tyr Glu Glu Leu Arg Glu Gln Leu Ser Ser Val Ser Ser
Phe 115 120 125 Glu Arg Phe Glu Ile Phe Pro Lys Thr Ser Ser Trp Pro
Asn His Asp 130 135 140 Ser Asn Lys Gly Val Thr Ala Ala Cys Pro His
Ala Gly Ala Lys Ser 145 150 155 160 Phe Tyr Lys Asn Leu Ile Trp Leu
Val Lys Lys Gly Asn Ser Tyr Pro 165 170 175 Lys Leu Ser Lys Ser Tyr
Ile Asn Asp Lys Gly Lys Glu Val Leu Val 180 185 190 Leu Trp Gly Ile
His His Pro Ser Thr Ser Ala Asp Gln Gln Ser Leu 195 200 205 Tyr Gln
Asn Ala Asp Thr Tyr Val Phe Val Gly Ser Ser Arg Tyr Ser 210 215 220
Lys Lys Phe Lys Pro Glu Ile Ala Ile Arg Pro Lys Val Arg Asp Gln 225
230
235 240 Glu Gly Arg Met Asn Tyr Tyr Trp Thr Leu Val Glu Pro Gly Asp
Lys 245 250 255 Ile Thr Phe Glu Ala Thr Gly Asn Leu Val Val Pro Arg
Tyr Ala Phe 260 265 270 Ala Met Glu Arg Asn Ala Gly Ser Gly Ile Ile
Ile Ser Asp Thr Pro 275 280 285 Val His Asp Cys Asn Thr Thr Cys Gln
Thr Pro Lys Gly Ala Ile Asn 290 295 300 Thr Ser Leu Pro Phe Gln Asn
Ile His Pro Ile Thr Ile Gly Lys Cys 305 310 315 320 Pro Lys Tyr Val
Lys Ser Thr Lys Leu Arg Leu Ala Thr Gly Leu Arg 325 330 335 Asn Ile
Pro Ser Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly 340 345 350
Phe Ile Glu Gly Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr 355
360 365 His His Gln Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu Lys
Ser 370 375 380 Thr Gln Asn Ala Ile Asp Glu Ile Thr Asn Lys Val Asn
Ser Val Ile 385 390 395 400 Glu Lys Met Asn Thr Gln Phe Thr Ala Val
Gly Lys Glu Phe Asn His 405 410 415 Leu Glu Lys Arg Ile Glu Asn Leu
Asn Lys Lys Val Asp Asp Gly Phe 420 425 430 Leu Asp Ile Trp Thr Tyr
Asn Ala Glu Leu Leu Val Leu Leu Glu Asn 435 440 445 Glu Arg Thr Leu
Asp Tyr His Asp Ser Asn Val Lys Asn Leu Tyr Glu 450 455 460 Lys Val
Arg Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly 465 470 475
480 Cys Phe Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val
485 490 495 Lys Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala
Lys Leu 500 505 510 Asn Arg Glu Glu Ile Asp Ser Gly Gly Asp Ile Ile
Lys Leu Leu Asn 515 520 525 Glu Gln Val Asn Lys Glu Met Gln Ser Ser
Asn Leu Tyr Met Ser Met 530 535 540 Ser Ser Trp Cys Tyr Thr His Ser
Leu Asp Gly Ala Gly Leu Phe Leu 545 550 555 560 Phe Asp His Ala Ala
Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile 565 570 575 Phe Leu Asn
Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala 580 585 590 Pro
Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr 595 600
605 Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His
610 615 620 Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln
Trp Tyr 625 630 635 640 Val Ala Glu Gln His Glu Glu Glu Val Leu Phe
Lys Asp Ile Leu Asp 645 650 655 Lys Ile Glu Leu Ile Gly Asn Glu Asn
His Gly Leu Tyr Leu Ala Asp 660 665 670 Gln Tyr Val Lys Gly Ile Ala
Lys Ser Arg Lys Ser Gly Ser 675 680 685 452061DNAArtificial
SequenceSynthetic 45ctaggatccg ctcttcctgc tcttggcgat gcccttcacg
tactggtcgg ccaggtacag 60gccgtggttc tcgttgccga tcagctcgat cttgtccagg
atgtccttga acagcacctc 120ctcctcgtgc tgctcggcca cgtaccactg
caggaagttg aaggtggcgt ggtccttgct 180cttgatggcg tggtccacga
tgttgttgat gctctcgctg atgtgctgct cgtgctcgta 240ggccttctgg
aagatctggg tcaggccctc gaacttgtgc tcgggggcgc tgatgctggt
300cagctgcacg ggcacgttgt tctcgttcag gaagatgatc agcttcttgg
cgtgctcgta 360ctcctcggcg gcgtggtcga acaggaacag gccggcgccg
tccaggctgt gggtgtagca 420ccagctgctc atgctcatgt acaggttgct
gctctgcatc tccttgttca cctgctcgtt 480cagcagcttg atgatgtcgc
ctccggagtc gatctcctcc cggttcagct tggcctcctc 540gctgtacttg
gggtagtcgt aggtgccgtt cttcacgctc tccatgcagg tgttgtcgca
600cttgtggtag aactcgaagc agccgttgcc gatctccttg gcgttgttct
tcagctggct 660ccgcaccttc tcgtacaggt tcttcacgtt gctgtcgtgg
tagtccaggg tccgctcgtt 720ctccagcagc accagcagct cggcgttgta
ggtccagatg tccaggaagc cgtcgtccac 780cttcttgttc aggttctcga
tccgcttctc caggtggttg aactccttgc ccacggcggt 840gaactgggtg
ttcatcttct cgatcacgct gttcaccttg ttggtgatct cgtcgatggc
900gttctgggtg ctcttcaggt cggcggcgta gccgctgccc tgctcgttct
ggtggtggta 960gccgtaccag ccgtccacca tgccggtcca gccgccctcg
atgaagccgg cgatggcgcc 1020gaacaggccc cggctctgga tgctggggat
gttccgcagg ccggtggcca gccgcagctt 1080ggtgctcttc acgtacttgg
ggcacttgcc gatggtgatg gggtggatgt tctggaaggg 1140caggctggtg
ttgatggcgc ccttgggggt ctggcaggtg gtgttgcagt cgtgcacggg
1200ggtgtcgctg atgatgatgc cgctgccggc gttccgctcc atggcgaagg
cgtaccgggg 1260caccaccagg ttgccggtgg cctcgaaggt gatcttgtcg
ccgggctcca ccagggtcca 1320gtagtagttc atccggccct cctggtcccg
caccttgggc cggatggcga tctcgggctt 1380gaacttcttg ctgtaccggc
tgctgcccac gaacacgtag gtgtcggcgt tctggtacag 1440gctctgctgg
tcggcgctgg tgctggggtg gtggatgccc cacagcacca gcacctcctt
1500gcccttgtcg ttgatgtagc tcttgctcag cttggggtag ctgttgccct
tcttcaccag 1560ccagatcagg ttcttgtaga agctcttggc gccggcgtgg
gggcaggcgg cggtcacgcc 1620cttgttgctg tcgtggttgg gccagctgct
ggtcttgggg aagatctcga accgctcgaa 1680gctgctcacg ctgctcagct
gctcccgcag ctcctcgtag tcgatgaagt cgccggggta 1740gcaggtgccg
ttgtcgctgc tgggggtctc cacgatgtag ctccagctgc tggcggtgct
1800caggctctcg cactcggggt tgcccagaat ccagccggcg atgttgcact
tgcccaggtg 1860caggggggcc acgccccgca gcttgcacag cttgccgttg
tgcttgtcct ccagcaggtt 1920cacgctgtgg gtcacggtca cgttcttctc
cagcacggtg tccacggtgt cggtgctgtt 1980gttggcgtgg tagccgatgc
acagggtgtc ggcgttggcg gtggcgaagg tgtacagcag 2040caccaccagg
atggccttca t 2061462049DNAArtificial SequenceSynthetic 46atggccatca
tctacctgat cctgctgttt acagctgtgc ggggcgatca gatctgtatc 60ggctaccacg
ccaacaatag caccgagaag gtggacacca tcctggaaag aaatgtgacc
120gtgacccacg ccaaggatat tctggaaaag acccacaacg gcaagctgtg
caagctgaat 180ggcattcctc ctctggaact gggcgattgt tctattgctg
gctggctgct gggaaatcct 240gagtgcgata gactgctgtc tgtgcctgag
tggagctaca tcatggaaaa agagaaccct 300agggacggac tgtgttaccc
cggcagcttc aacgattacg aggaactgaa gcacctgctg 360tccagcgtga
agcacttcga gaaagtgaag atcctgccca aggatagatg gacccagcat
420acaacaacag gcggaagcag agcttgtgct gtgtccggca accccagctt
cttcagaaat 480atggtctggc tgaccaagaa gggctctaat tatcctgtgg
ccaagggcag ctacaataat 540acaagcggcg agcagatgct gattatttgg
ggcgtgcacc accctaatga tgagacagag 600cagagaaccc tgtaccagaa
tgtgggcaca tacgtgtctg tgggcaccag cacactgaat 660aagagaagca
cccccgatat tgccaccaga cccaaagtga atggacaggg cggcagaatg
720gaattttcct ggaccctgct ggatatgtgg gacaccatca actttgagag
caccgggaat 780ctgattgccc ctgagtacgg cttcaagatc agcaagagag
gcagcagcgg catcatgaaa 840acagagggca ccctggaaaa ctgtgaaacc
aagtgtcaga cacctctggg cgccattaat 900accaccctgc ccttccataa
tgtgcaccct ctgacaatcg gcgagtgccc taagtacgtg 960aagtctgaga
aactggtgct ggccacagga ctgagaaatg tgccccagat cgagtcaaga
1020ggcctgtttg gagccattgc cggctttatt gaaggcggat ggcagggaat
ggtggatggg 1080tggtacggct atcaccacag caatgatcag ggatctggct
atgccgccga taaagagagc 1140acccagaagg cctttgacgg catcaccaac
aaagtgaaca gcgtgatcga gaagatgaac 1200acccagtttg aggccgtggg
caaagagttc agcaatctgg aaagacggct ggaaaacctg 1260aacaagaaaa
tggaagatgg cttcctggac gtgtggacat ataatgccga gctgctggtg
1320ctgatggaaa acgagaggac cctggacttt cacgacagca acgtgaagaa
cctgtacgac 1380aaagtgcgga tgcagctgag agacaatgtg aaagagctgg
gcaacggctg ctttgagttc 1440taccacaagt gcgacgacga gtgcatgaat
agcgtgaaga acggcaccta cgactaccct 1500aagtatgagg aagagagcaa
gctgaacaga aacgagatca agtccggagg cgacatcatc 1560aagctgctga
acgagcaggt gaacaaggag atgcagagca gcaacctgta catgagcatg
1620agcagctggt gctacaccca cagcctggac ggcgccggcc tgttcctgtt
cgaccacgcc 1680gccgaggagt acgagcacgc caagaagctg atcatcttcc
tgaacgagaa caacgtgccc 1740gtgcagctga ccagcatcag cgcccccgag
cacaagttcg agggcctgac ccagatcttc 1800cagaaggcct acgagcacga
gcagcacatc agcgagagca tcaacaacat cgtggaccac 1860gccatcaaga
gcaaggacca cgccaccttc aacttcctgc agtggtacgt ggccgagcag
1920cacgaggagg aggtgctgtt caaggacatc ctggacaaga tcgagctgat
cggcaacgag 1980aaccacggcc tgtacctggc cgaccagtac gtgaagggca
tcgccaagag caggaagagc 2040ggatcctag 204947682PRTArtificial
SequenceSynthetic 47Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala
Val Arg Gly Asp 1 5 10 15 Gln Ile Cys Ile Gly Tyr His Ala Asn Asn
Ser Thr Glu Lys Val Asp 20 25 30 Thr Ile Leu Glu Arg Asn Val Thr
Val Thr His Ala Lys Asp Ile Leu 35 40 45 Glu Lys Thr His Asn Gly
Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro 50 55 60 Leu Glu Leu Gly
Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro 65 70 75 80 Glu Cys
Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Met Glu 85 90 95
Lys Glu Asn Pro Arg Asp Gly Leu Cys Tyr Pro Gly Ser Phe Asn Asp 100
105 110 Tyr Glu Glu Leu Lys His Leu Leu Ser Ser Val Lys His Phe Glu
Lys 115 120 125 Val Lys Ile Leu Pro Lys Asp Arg Trp Thr Gln His Thr
Thr Thr Gly 130 135 140 Gly Ser Arg Ala Cys Ala Val Ser Gly Asn Pro
Ser Phe Phe Arg Asn 145 150 155 160 Met Val Trp Leu Thr Lys Lys Gly
Ser Asn Tyr Pro Val Ala Lys Gly 165 170 175 Ser Tyr Asn Asn Thr Ser
Gly Glu Gln Met Leu Ile Ile Trp Gly Val 180 185 190 His His Pro Asn
Asp Glu Thr Glu Gln Arg Thr Leu Tyr Gln Asn Val 195 200 205 Gly Thr
Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Lys Arg Ser Thr 210 215 220
Pro Asp Ile Ala Thr Arg Pro Lys Val Asn Gly Gln Gly Gly Arg Met 225
230 235 240 Glu Phe Ser Trp Thr Leu Leu Asp Met Trp Asp Thr Ile Asn
Phe Glu 245 250 255 Ser Thr Gly Asn Leu Ile Ala Pro Glu Tyr Gly Phe
Lys Ile Ser Lys 260 265 270 Arg Gly Ser Ser Gly Ile Met Lys Thr Glu
Gly Thr Leu Glu Asn Cys 275 280 285 Glu Thr Lys Cys Gln Thr Pro Leu
Gly Ala Ile Asn Thr Thr Leu Pro 290 295 300 Phe His Asn Val His Pro
Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val 305 310 315 320 Lys Ser Glu
Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro Gln 325 330 335 Ile
Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly 340 345
350 Gly Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn
355 360 365 Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln
Lys Ala 370 375 380 Phe Asp Gly Ile Thr Asn Lys Val Asn Ser Val Ile
Glu Lys Met Asn 385 390 395 400 Thr Gln Phe Glu Ala Val Gly Lys Glu
Phe Ser Asn Leu Glu Arg Arg 405 410 415 Leu Glu Asn Leu Asn Lys Lys
Met Glu Asp Gly Phe Leu Asp Val Trp 420 425 430 Thr Tyr Asn Ala Glu
Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu 435 440 445 Asp Phe His
Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met 450 455 460 Gln
Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe 465 470
475 480 Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly
Thr 485 490 495 Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn
Arg Asn Glu 500 505 510 Ile Lys Ser Gly Gly Asp Ile Ile Lys Leu Leu
Asn Glu Gln Val Asn 515 520 525 Lys Glu Met Gln Ser Ser Asn Leu Tyr
Met Ser Met Ser Ser Trp Cys 530 535 540 Tyr Thr His Ser Leu Asp Gly
Ala Gly Leu Phe Leu Phe Asp His Ala 545 550 555 560 Ala Glu Glu Tyr
Glu His Ala Lys Lys Leu Ile Ile Phe Leu Asn Glu 565 570 575 Asn Asn
Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu His Lys 580 585 590
Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His Glu Gln 595
600 605 His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala Ile Lys
Ser 610 615 620 Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val
Ala Glu Gln 625 630 635 640 His Glu Glu Glu Val Leu Phe Lys Asp Ile
Leu Asp Lys Ile Glu Leu 645 650 655 Ile Gly Asn Glu Asn His Gly Leu
Tyr Leu Ala Asp Gln Tyr Val Lys 660 665 670 Gly Ile Ala Lys Ser Arg
Lys Ser Gly Ser 675 680 482049DNAArtificial SequenceSynthetic
48ctaggatccg ctcttcctgc tcttggcgat gcccttcacg tactggtcgg ccaggtacag
60gccgtggttc tcgttgccga tcagctcgat cttgtccagg atgtccttga acagcacctc
120ctcctcgtgc tgctcggcca cgtaccactg caggaagttg aaggtggcgt
ggtccttgct 180cttgatggcg tggtccacga tgttgttgat gctctcgctg
atgtgctgct cgtgctcgta 240ggccttctgg aagatctggg tcaggccctc
gaacttgtgc tcgggggcgc tgatgctggt 300cagctgcacg ggcacgttgt
tctcgttcag gaagatgatc agcttcttgg cgtgctcgta 360ctcctcggcg
gcgtggtcga acaggaacag gccggcgccg tccaggctgt gggtgtagca
420ccagctgctc atgctcatgt acaggttgct gctctgcatc tccttgttca
cctgctcgtt 480cagcagcttg atgatgtcgc ctccggactt gatctcgttt
ctgttcagct tgctctcttc 540ctcatactta gggtagtcgt aggtgccgtt
cttcacgcta ttcatgcact cgtcgtcgca 600cttgtggtag aactcaaagc
agccgttgcc cagctctttc acattgtctc tcagctgcat 660ccgcactttg
tcgtacaggt tcttcacgtt gctgtcgtga aagtccaggg tcctctcgtt
720ttccatcagc accagcagct cggcattata tgtccacacg tccaggaagc
catcttccat 780tttcttgttc aggttttcca gccgtctttc cagattgctg
aactctttgc ccacggcctc 840aaactgggtg ttcatcttct cgatcacgct
gttcactttg ttggtgatgc cgtcaaaggc 900cttctgggtg ctctctttat
cggcggcata gccagatccc tgatcattgc tgtggtgata 960gccgtaccac
ccatccacca ttccctgcca tccgccttca ataaagccgg caatggctcc
1020aaacaggcct cttgactcga tctggggcac atttctcagt cctgtggcca
gcaccagttt 1080ctcagacttc acgtacttag ggcactcgcc gattgtcaga
gggtgcacat tatggaaggg 1140cagggtggta ttaatggcgc ccagaggtgt
ctgacacttg gtttcacagt tttccagggt 1200gccctctgtt ttcatgatgc
cgctgctgcc tctcttgctg atcttgaagc cgtactcagg 1260ggcaatcaga
ttcccggtgc tctcaaagtt gatggtgtcc cacatatcca gcagggtcca
1320ggaaaattcc attctgccgc cctgtccatt cactttgggt ctggtggcaa
tatcgggggt 1380gcttctctta ttcagtgtgc tggtgcccac agacacgtat
gtgcccacat tctggtacag 1440ggttctctgc tctgtctcat cattagggtg
gtgcacgccc caaataatca gcatctgctc 1500gccgcttgta ttattgtagc
tgcccttggc cacaggataa ttagagccct tcttggtcag 1560ccagaccata
tttctgaaga agctggggtt gccggacaca gcacaagctc tgcttccgcc
1620tgttgttgta tgctgggtcc atctatcctt gggcaggatc ttcactttct
cgaagtgctt 1680cacgctggac agcaggtgct tcagttcctc gtaatcgttg
aagctgccgg ggtaacacag 1740tccgtcccta gggttctctt tttccatgat
gtagctccac tcaggcacag acagcagtct 1800atcgcactca ggatttccca
gcagccagcc agcaatagaa caatcgccca gttccagagg 1860aggaatgcca
ttcagcttgc acagcttgcc gttgtgggtc ttttccagaa tatccttggc
1920gtgggtcacg gtcacatttc tttccaggat ggtgtccacc ttctcggtgc
tattgttggc 1980gtggtagccg atacagatct gatcgccccg cacagctgta
aacagcagga tcaggtagat 2040gatggccat 2049492064DNAArtificial
SequenceSynthetic 49atgaaaacca tcattgccct gagctacatc ttttgtctgg
ctctgggcca ggatctgccc 60ggcaatgata atagcaccgc caccctgtgt ctgggacacc
acgccgtgcc taatggcacc 120ctggtgaaaa ccattaccga cgaccagatc
gaagtgacca atgccaccga gctggtgcag 180agcagcagca ccggcaagat
ctgcaacaac ccccacagaa tcctggatgg catcgactgt 240accctgatcg
atgccctgct gggcgatcct cactgcgacg tgttccagaa cgagacatgg
300gacctgttcg tggagagaag caaggccttc agcaactgct acccctacga
tgtgcccgat 360tacgcctctc tgagaagcct ggtggccagc agcggcacac
tggaattcat caccgagggc 420tttacctgga caggcgtgac ccagaatggc
ggcagcaatg cctgtaaaag aggccctggc 480agcggcttct tcagcagact
gaactggctg accaagtccg gcagcaccta ccctgtgctg 540aacgtgacca
tgcccaacaa cgacaacttc gacaagctgt acatctgggg cgtgcaccac
600cctagcacca atcaggaaca gaccagcctg tacgtgcagg ccagcggcag
agtgaccgtg 660tctaccagac ggtcccagca gaccatcatc cccaacatcg
agtcaagacc ttgggtgcgc 720ggcctgagca gcagaatcag catctactgg
accatcgtga aacctggcga cgtgctggtg 780atcaacagca atggcaacct
gatcgccccc agaggctact tcaagatgcg gaccggcaag 840agcagcatca
tgagaagcga cgcccccatc gatacctgta tcagcgagtg catcaccccc
900aacggcagca tccccaacga caagcccttc cagaacgtga acaagatcac
ctacggcgcc 960tgccctaagt acgtgaagca gaacaccctg aagctggcca
ccggcatgag aaatgtgccc 1020gagaagcaga caagaggcct gtttggcgcc
attgccggct ttatcgagaa cggctgggag 1080ggcatgatcg atgggtggta
cggcttcaga caccagaatt ctgagggcac aggacaggcc 1140gccgatctga
agtctacaca ggccgccatc gaccagatca
acggcaagct gaacagagtg 1200atcgagaaaa ccaacgagaa gttccaccag
atcgagaaag aattcagcga ggtggagggc 1260agaatccagg acctggaaaa
atacgtggag gacaccaaga tcgacctgtg gagctacaat 1320gccgaactgc
tggtcgccct ggaaaaccag cacaccatcg acctgaccga cagcgagatg
1380aataagctgt tcgaaaagac cagacggcag ctgagagaaa acgccgagga
catgggcaac 1440ggctgcttca agatctacca caagtgcgac aacgcctgca
tcgagagcat cagaaacggc 1500acctacgacc acgatgtgta cagggacgag
gccctgaaca acagattcca gatcaagtcc 1560ggaggcgaca tcatcaagct
gctgaacgag caggtgaaca aggagatgca gagcagcaac 1620ctgtacatga
gcatgagcag ctggtgctac acccacagcc tggacggcgc cggcctgttc
1680ctgttcgacc acgccgccga ggagtacgag cacgccaaga agctgatcat
cttcctgaac 1740gagaacaacg tgcccgtgca gctgaccagc atcagcgccc
ccgagcacaa gttcgagggc 1800ctgacccaga tcttccagaa ggcctacgag
cacgagcagc acatcagcga gagcatcaac 1860aacatcgtgg accacgccat
caagagcaag gaccacgcca ccttcaactt cctgcagtgg 1920tacgtggccg
agcagcacga ggaggaggtg ctgttcaagg acatcctgga caagatcgag
1980ctgatcggca acgagaacca cggcctgtac ctggccgacc agtacgtgaa
gggcatcgcc 2040aagagcagga agagcggatc ctag 206450687PRTArtificial
SequenceSynthetic 50Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys
Leu Ala Leu Gly 1 5 10 15 Gln Asp Leu Pro Gly Asn Asp Asn Ser Thr
Ala Thr Leu Cys Leu Gly 20 25 30 His His Ala Val Pro Asn Gly Thr
Leu Val Lys Thr Ile Thr Asp Asp 35 40 45 Gln Ile Glu Val Thr Asn
Ala Thr Glu Leu Val Gln Ser Ser Ser Thr 50 55 60 Gly Lys Ile Cys
Asn Asn Pro His Arg Ile Leu Asp Gly Ile Asp Cys 65 70 75 80 Thr Leu
Ile Asp Ala Leu Leu Gly Asp Pro His Cys Asp Val Phe Gln 85 90 95
Asn Glu Thr Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Phe Ser Asn 100
105 110 Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu
Val 115 120 125 Ala Ser Ser Gly Thr Leu Glu Phe Ile Thr Glu Gly Phe
Thr Trp Thr 130 135 140 Gly Val Thr Gln Asn Gly Gly Ser Asn Ala Cys
Lys Arg Gly Pro Gly 145 150 155 160 Ser Gly Phe Phe Ser Arg Leu Asn
Trp Leu Thr Lys Ser Gly Ser Thr 165 170 175 Tyr Pro Val Leu Asn Val
Thr Met Pro Asn Asn Asp Asn Phe Asp Lys 180 185 190 Leu Tyr Ile Trp
Gly Val His His Pro Ser Thr Asn Gln Glu Gln Thr 195 200 205 Ser Leu
Tyr Val Gln Ala Ser Gly Arg Val Thr Val Ser Thr Arg Arg 210 215 220
Ser Gln Gln Thr Ile Ile Pro Asn Ile Glu Ser Arg Pro Trp Val Arg 225
230 235 240 Gly Leu Ser Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys
Pro Gly 245 250 255 Asp Val Leu Val Ile Asn Ser Asn Gly Asn Leu Ile
Ala Pro Arg Gly 260 265 270 Tyr Phe Lys Met Arg Thr Gly Lys Ser Ser
Ile Met Arg Ser Asp Ala 275 280 285 Pro Ile Asp Thr Cys Ile Ser Glu
Cys Ile Thr Pro Asn Gly Ser Ile 290 295 300 Pro Asn Asp Lys Pro Phe
Gln Asn Val Asn Lys Ile Thr Tyr Gly Ala 305 310 315 320 Cys Pro Lys
Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met 325 330 335 Arg
Asn Val Pro Glu Lys Gln Thr Arg Gly Leu Phe Gly Ala Ile Ala 340 345
350 Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Ile Asp Gly Trp Tyr Gly
355 360 365 Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala Ala Asp
Leu Lys 370 375 380 Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys
Leu Asn Arg Val 385 390 395 400 Ile Glu Lys Thr Asn Glu Lys Phe His
Gln Ile Glu Lys Glu Phe Ser 405 410 415 Glu Val Glu Gly Arg Ile Gln
Asp Leu Glu Lys Tyr Val Glu Asp Thr 420 425 430 Lys Ile Asp Leu Trp
Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu 435 440 445 Asn Gln His
Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe 450 455 460 Glu
Lys Thr Arg Arg Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 465 470
475 480 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Glu
Ser 485 490 495 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp
Glu Ala Leu 500 505 510 Asn Asn Arg Phe Gln Ile Lys Ser Gly Gly Asp
Ile Ile Lys Leu Leu 515 520 525 Asn Glu Gln Val Asn Lys Glu Met Gln
Ser Ser Asn Leu Tyr Met Ser 530 535 540 Met Ser Ser Trp Cys Tyr Thr
His Ser Leu Asp Gly Ala Gly Leu Phe 545 550 555 560 Leu Phe Asp His
Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile 565 570 575 Ile Phe
Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser 580 585 590
Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala 595
600 605 Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val
Asp 610 615 620 His Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe
Leu Gln Trp 625 630 635 640 Tyr Val Ala Glu Gln His Glu Glu Glu Val
Leu Phe Lys Asp Ile Leu 645 650 655 Asp Lys Ile Glu Leu Ile Gly Asn
Glu Asn His Gly Leu Tyr Leu Ala 660 665 670 Asp Gln Tyr Val Lys Gly
Ile Ala Lys Ser Arg Lys Ser Gly Ser 675 680 685 512064DNAArtificial
SequenceSynthetic 51ctaggatccg ctcttcctgc tcttggcgat gcccttcacg
tactggtcgg ccaggtacag 60gccgtggttc tcgttgccga tcagctcgat cttgtccagg
atgtccttga acagcacctc 120ctcctcgtgc tgctcggcca cgtaccactg
caggaagttg aaggtggcgt ggtccttgct 180cttgatggcg tggtccacga
tgttgttgat gctctcgctg atgtgctgct cgtgctcgta 240ggccttctgg
aagatctggg tcaggccctc gaacttgtgc tcgggggcgc tgatgctggt
300cagctgcacg ggcacgttgt tctcgttcag gaagatgatc agcttcttgg
cgtgctcgta 360ctcctcggcg gcgtggtcga acaggaacag gccggcgccg
tccaggctgt gggtgtagca 420ccagctgctc atgctcatgt acaggttgct
gctctgcatc tccttgttca cctgctcgtt 480cagcagcttg atgatgtcgc
ctccggactt gatctggaat ctgttgttca gggcctcgtc 540cctgtacaca
tcgtggtcgt aggtgccgtt tctgatgctc tcgatgcagg cgttgtcgca
600cttgtggtag atcttgaagc agccgttgcc catgtcctcg gcgttttctc
tcagctgccg 660tctggtcttt tcgaacagct tattcatctc gctgtcggtc
aggtcgatgg tgtgctggtt 720ttccagggcg accagcagtt cggcattgta
gctccacagg tcgatcttgg tgtcctccac 780gtatttttcc aggtcctgga
ttctgccctc cacctcgctg aattctttct cgatctggtg 840gaacttctcg
ttggttttct cgatcactct gttcagcttg ccgttgatct ggtcgatggc
900ggcctgtgta gacttcagat cggcggcctg tcctgtgccc tcagaattct
ggtgtctgaa 960gccgtaccac ccatcgatca tgccctccca gccgttctcg
ataaagccgg caatggcgcc 1020aaacaggcct cttgtctgct tctcgggcac
atttctcatg ccggtggcca gcttcagggt 1080gttctgcttc acgtacttag
ggcaggcgcc gtaggtgatc ttgttcacgt tctggaaggg 1140cttgtcgttg
gggatgctgc cgttgggggt gatgcactcg ctgatacagg tatcgatggg
1200ggcgtcgctt ctcatgatgc tgctcttgcc ggtccgcatc ttgaagtagc
ctctgggggc 1260gatcaggttg ccattgctgt tgatcaccag cacgtcgcca
ggtttcacga tggtccagta 1320gatgctgatt ctgctgctca ggccgcgcac
ccaaggtctt gactcgatgt tggggatgat 1380ggtctgctgg gaccgtctgg
tagacacggt cactctgccg ctggcctgca cgtacaggct 1440ggtctgttcc
tgattggtgc tagggtggtg cacgccccag atgtacagct tgtcgaagtt
1500gtcgttgttg ggcatggtca cgttcagcac agggtaggtg ctgccggact
tggtcagcca 1560gttcagtctg ctgaagaagc cgctgccagg gcctctttta
caggcattgc tgccgccatt 1620ctgggtcacg cctgtccagg taaagccctc
ggtgatgaat tccagtgtgc cgctgctggc 1680caccaggctt ctcagagagg
cgtaatcggg cacatcgtag gggtagcagt tgctgaaggc 1740cttgcttctc
tccacgaaca ggtcccatgt ctcgttctgg aacacgtcgc agtgaggatc
1800gcccagcagg gcatcgatca gggtacagtc gatgccatcc aggattctgt
gggggttgtt 1860gcagatcttg ccggtgctgc tgctctgcac cagctcggtg
gcattggtca cttcgatctg 1920gtcgtcggta atggttttca ccagggtgcc
attaggcacg gcgtggtgtc ccagacacag 1980ggtggcggtg ctattatcat
tgccgggcag atcctggccc agagccagac aaaagatgta 2040gctcagggca
atgatggttt tcat 2064522064DNAArtificial SequenceSynthetic
52atgaaaacca tcattgccct gagctacatc ctgtgcctgg tgttcacaca gaagctgccc
60ggcaacgata atagcaccgc cacactgtgt ctgggacacc acgccgtgcc taatggcacc
120atcgtgaaaa caatcaccaa cgaccagatc gaagtgacca atgccacaga
gctggtgcag 180agcagcagca caggcgagat ctgtgacagc ccccaccaga
tcctggatgg cgagaactgt 240accctgatcg atgccctgct gggcgatcct
cagtgcgacg gcttccagaa caagaaatgg 300gacctgttcg tggagagaag
caaggcctac agcaactgct acccctacga cgtgcctgat 360tacgccagcc
tgagaagcct ggtggcctct agcggcaccc tggaattcaa caacgagagc
420ttcaactgga ccggcgtgac acagaatggc accagcagcg cctgcatcag
acggtccaac 480aacagcttct tcagtagact gaattggctg acccacctga
agttcaagta ccccgccctg 540aacgtgacca tgcccaacaa tgagaagttc
gacaagctgt acatctgggg agtgcaccac 600cctggcaccg acaacgatca
gatcttccct tacgcccagg ccagcggcag aatcaccgtg 660tccaccaaga
gaagccagca gaccgtgatc cccaatatcg gcagcagacc cagagtgcgg
720aacatcccca gcaggatcag catctactgg acaatcgtga agcctggcga
catcctgctg 780atcaacagca ccggcaacct gatcgcccct cggggctact
ttaagatcag aagcggcaag 840agcagcatca tgagatccga cgcccccatc
ggcaagtgca acagcgagtg catcacccca 900aacggcagca tccccaacga
caagcccttc cagaacgtga acaggatcac ctacggcgcc 960tgccctagat
acgtgaagca gaacaccctg aagctggcca ccggcatgag aaatgtgccc
1020gagaagcaga ccagaggcat ctttggcgcc attgccggct ttatcgagaa
tggctgggag 1080ggaatggtgg atgggtggta cggcttcaga caccagaata
gcgagggaat tggacaggcc 1140gccgatctga aatctaccca ggccgccatc
gaccagatca acggcaagct gaacaggctg 1200atcggcaaga ccaacgagaa
gttccaccag atcgagaaag aattcagcga ggtggagggc 1260agaatccagg
acctggaaaa atacgtggag gacaccaaga tcgacctgtg gagctacaat
1320gccgaactgc tggtcgccct ggaaaaccag cacacaattg atctgacaga
cagtgagatg 1380aataagctgt tcgagaaaac caagaagcag ctgagagaaa
acgccgagga catgggcaac 1440ggctgcttca agatctacca caagtgcgac
aacgcctgca tcggcagcat cagaaacggc 1500acctacgacc acgacgtgta
cagagatgag gccctgaaca accggtttca gatcaagtcc 1560ggaggcgaca
tcatcaagct gctgaacgag caggtgaaca aggagatgca gagcagcaac
1620ctgtacatga gcatgagcag ctggtgctac acccacagcc tggacggcgc
cggcctgttc 1680ctgttcgacc acgccgccga ggagtacgag cacgccaaga
agctgatcat cttcctgaac 1740gagaacaacg tgcccgtgca gctgaccagc
atcagcgccc ccgagcacaa gttcgagggc 1800ctgacccaga tcttccagaa
ggcctacgag cacgagcagc acatcagcga gagcatcaac 1860aacatcgtgg
accacgccat caagagcaag gaccacgcca ccttcaactt cctgcagtgg
1920tacgtggccg agcagcacga ggaggaggtg ctgttcaagg acatcctgga
caagatcgag 1980ctgatcggca acgagaacca cggcctgtac ctggccgacc
agtacgtgaa gggcatcgcc 2040aagagcagga agagcggatc ctag
206453687PRTArtificial SequenceSynthetic 53Met Lys Thr Ile Ile Ala
Leu Ser Tyr Ile Leu Cys Leu Val Phe Thr 1 5 10 15 Gln Lys Leu Pro
Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly 20 25 30 His His
Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile Thr Asn Asp 35 40 45
Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln Ser Ser Ser Thr 50
55 60 Gly Glu Ile Cys Asp Ser Pro His Gln Ile Leu Asp Gly Glu Asn
Cys 65 70 75 80 Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro Gln Cys Asp
Gly Phe Gln 85 90 95 Asn Lys Lys Trp Asp Leu Phe Val Glu Arg Ser
Lys Ala Tyr Ser Asn 100 105 110 Cys Tyr Pro Tyr Asp Val Pro Asp Tyr
Ala Ser Leu Arg Ser Leu Val 115 120 125 Ala Ser Ser Gly Thr Leu Glu
Phe Asn Asn Glu Ser Phe Asn Trp Thr 130 135 140 Gly Val Thr Gln Asn
Gly Thr Ser Ser Ala Cys Ile Arg Arg Ser Asn 145 150 155 160 Asn Ser
Phe Phe Ser Arg Leu Asn Trp Leu Thr His Leu Lys Phe Lys 165 170 175
Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu Lys Phe Asp Lys 180
185 190 Leu Tyr Ile Trp Gly Val His His Pro Gly Thr Asp Asn Asp Gln
Ile 195 200 205 Phe Pro Tyr Ala Gln Ala Ser Gly Arg Ile Thr Val Ser
Thr Lys Arg 210 215 220 Ser Gln Gln Thr Val Ile Pro Asn Ile Gly Ser
Arg Pro Arg Val Arg 225 230 235 240 Asn Ile Pro Ser Arg Ile Ser Ile
Tyr Trp Thr Ile Val Lys Pro Gly 245 250 255 Asp Ile Leu Leu Ile Asn
Ser Thr Gly Asn Leu Ile Ala Pro Arg Gly 260 265 270 Tyr Phe Lys Ile
Arg Ser Gly Lys Ser Ser Ile Met Arg Ser Asp Ala 275 280 285 Pro Ile
Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro Asn Gly Ser Ile 290 295 300
Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala 305
310 315 320 Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr
Gly Met 325 330 335 Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe
Gly Ala Ile Ala 340 345 350 Gly Phe Ile Glu Asn Gly Trp Glu Gly Met
Val Asp Gly Trp Tyr Gly 355 360 365 Phe Arg His Gln Asn Ser Glu Gly
Ile Gly Gln Ala Ala Asp Leu Lys 370 375 380 Ser Thr Gln Ala Ala Ile
Asp Gln Ile Asn Gly Lys Leu Asn Arg Leu 385 390 395 400 Ile Gly Lys
Thr Asn Glu Lys Phe His Gln Ile Glu Lys Glu Phe Ser 405 410 415 Glu
Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr Val Glu Asp Thr 420 425
430 Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu
435 440 445 Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys
Leu Phe 450 455 460 Glu Lys Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu
Asp Met Gly Asn 465 470 475 480 Gly Cys Phe Lys Ile Tyr His Lys Cys
Asp Asn Ala Cys Ile Gly Ser 485 490 495 Ile Arg Asn Gly Thr Tyr Asp
His Asp Val Tyr Arg Asp Glu Ala Leu 500 505 510 Asn Asn Arg Phe Gln
Ile Lys Ser Gly Gly Asp Ile Ile Lys Leu Leu 515 520 525 Asn Glu Gln
Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser 530 535 540 Met
Ser Ser Trp Cys Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe 545 550
555 560 Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu
Ile 565 570 575 Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr
Ser Ile Ser 580 585 590 Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln
Ile Phe Gln Lys Ala 595 600 605 Tyr Glu His Glu Gln His Ile Ser Glu
Ser Ile Asn Asn Ile Val Asp 610 615 620 His Ala Ile Lys Ser Lys Asp
His Ala Thr Phe Asn Phe Leu Gln Trp 625 630 635 640 Tyr Val Ala Glu
Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu 645 650 655 Asp Lys
Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala 660 665 670
Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser Gly Ser 675 680
685 542064DNAArtificial SequenceSynthetic 54ctaggatccg ctcttcctgc
tcttggcgat gcccttcacg tactggtcgg ccaggtacag 60gccgtggttc tcgttgccga
tcagctcgat cttgtccagg atgtccttga acagcacctc 120ctcctcgtgc
tgctcggcca cgtaccactg caggaagttg aaggtggcgt ggtccttgct
180cttgatggcg tggtccacga tgttgttgat gctctcgctg atgtgctgct
cgtgctcgta 240ggccttctgg aagatctggg tcaggccctc gaacttgtgc
tcgggggcgc tgatgctggt 300cagctgcacg ggcacgttgt tctcgttcag
gaagatgatc agcttcttgg cgtgctcgta 360ctcctcggcg gcgtggtcga
acaggaacag gccggcgccg tccaggctgt gggtgtagca 420ccagctgctc
atgctcatgt acaggttgct gctctgcatc tccttgttca cctgctcgtt
480cagcagcttg atgatgtcgc ctccggactt gatctgaaac cggttgttca
gggcctcatc 540tctgtacacg tcgtggtcgt aggtgccgtt tctgatgctg
ccgatgcagg cgttgtcgca 600cttgtggtag atcttgaagc agccgttgcc
catgtcctcg gcgttttctc tcagctgctt 660cttggttttc tcgaacagct
tattcatctc actgtctgtc agatcaattg tgtgctggtt 720ttccagggcg
accagcagtt cggcattgta gctccacagg tcgatcttgg tgtcctccac
780gtatttttcc aggtcctgga ttctgccctc cacctcgctg aattctttct
cgatctggtg 840gaacttctcg ttggtcttgc cgatcagcct gttcagcttg
ccgttgatct ggtcgatggc 900ggcctgggta gatttcagat cggcggcctg
tccaattccc tcgctattct ggtgtctgaa 960gccgtaccac ccatccacca
ttccctccca gccattctcg ataaagccgg caatggcgcc 1020aaagatgcct
ctggtctgct tctcgggcac atttctcatg ccggtggcca gcttcagggt
1080gttctgcttc acgtatctag ggcaggcgcc gtaggtgatc ctgttcacgt
tctggaaggg 1140cttgtcgttg gggatgctgc cgtttggggt gatgcactcg
ctgttgcact tgccgatggg 1200ggcgtcggat ctcatgatgc tgctcttgcc
gcttctgatc ttaaagtagc cccgaggggc 1260gatcaggttg ccggtgctgt
tgatcagcag gatgtcgcca ggcttcacga ttgtccagta 1320gatgctgatc
ctgctgggga tgttccgcac tctgggtctg ctgccgatat tggggatcac
1380ggtctgctgg cttctcttgg tggacacggt gattctgccg ctggcctggg
cgtaagggaa 1440gatctgatcg ttgtcggtgc cagggtggtg cactccccag
atgtacagct tgtcgaactt 1500ctcattgttg ggcatggtca cgttcagggc
ggggtacttg aacttcaggt gggtcagcca 1560attcagtcta ctgaagaagc
tgttgttgga ccgtctgatg caggcgctgc tggtgccatt 1620ctgtgtcacg
ccggtccagt tgaagctctc gttgttgaat tccagggtgc cgctagaggc
1680caccaggctt ctcaggctgg cgtaatcagg cacgtcgtag gggtagcagt
tgctgtaggc 1740cttgcttctc tccacgaaca ggtcccattt cttgttctgg
aagccgtcgc actgaggatc 1800gcccagcagg gcatcgatca gggtacagtt
ctcgccatcc aggatctggt gggggctgtc 1860acagatctcg cctgtgctgc
tgctctgcac cagctctgtg gcattggtca cttcgatctg 1920gtcgttggtg
attgttttca cgatggtgcc attaggcacg gcgtggtgtc ccagacacag
1980tgtggcggtg ctattatcgt tgccgggcag cttctgtgtg aacaccaggc
acaggatgta 2040gctcagggca atgatggttt tcat 2064552067DNAArtificial
SequenceSynthetic 55atggaaaaga tcgtgctgct gctggccatt gtgagcctgg
tgaagagcga ccagatctgc 60attggctacc acgccaacaa tagcacagag caggtggaca
ccatcatgga aaaaaacgtg 120accgtgaccc acgctcagga catcctggaa
aagacccaca acggcaagct gtgtgatctg 180gacggcgtga agcctctgat
cctgagagat tgtagcgtgg ctggatggct gctgggcaac 240cctatgtgcg
acgagttcat caacgtgccc gagtggagct atatcgtgga gaaggccaac
300cccaccaacg atctgtgtta ccccggcagc ttcaacgatt acgaggaact
gaagcacctg 360ctgtcccgga tcaaccactt cgagaagatc cagatcatcc
ccaagtcctc ttggagcgat 420cacgaagcct ctagcggagt gtctagcgcc
tgtccttacc tgggcagccc cagcttcttc 480agaaacgtgg tgtggctgat
caagaagaac agcacctacc ccaccatcaa gaagagctac 540aacaacacca
accaggaaga tctgctggtc ctgtggggaa tccaccaccc taatgatgcc
600gccgagcaga ccagactgta ccagaacccc accacctata tcagcatcgg
caccagcacc 660ctgaatcaga gactggtgcc caagatcgcc accagatcca
aggtgaacgg ccagagcggc 720aggatggaat tcttctggac catcctgaag
cccaacgacg ccatcaactt cgagagcaac 780ggcaacttta tcgcccctga
gtacgcctac aagatcgtga agaagggcga cagcgccatc 840atgaagagcg
agctggaata cggcaactgc aacaccaagt gccagacacc tatgggcgcc
900atcaacagca gcatgccctt ccacaacatc caccctctga ccatcggcga
gtgccctaag 960tacgtgaaga gcaacagact ggtgctggcc acaggcctga
gaaatagccc ccagcgggag 1020agcagaagaa agaagagggg cctgtttgga
gccatcgccg gctttattga aggcggctgg 1080cagggaatgg tggatggctg
gtacggctac caccacagca atgagcaggg ctctggatat 1140gccgccgaca
aagagtctac ccagaaggcc atcgacggcg tcaccaacaa ggtgaacagc
1200atcatcgaca agatgaacac ccagttcgag gctgtgggca gagagttcaa
caacctggaa 1260cggcggatcg agaacctgaa caagaaaatg gaagatggct
tcctggatgt gtggacctac 1320aatgccgaac tgctggtgct gatggaaaac
gagcggaccc tggacttcca cgacagcaac 1380gtgaagaacc tgtacgacaa
agtgcggctg cagctgagag acaacgccaa agagctgggc 1440aacggctgct
tcgagttcta ccacaagtgc gacaacgagt gcatggaaag catcaggaac
1500ggcacctaca actaccctca gtacagcgag gaagccaggc tgaagaggga
agagatcagc 1560tccggaggcg acatcatcaa gctgctgaac gagcaggtga
acaaggagat gcagagcagc 1620aacctgtaca tgagcatgag cagctggtgc
tacacccaca gcctggacgg cgccggcctg 1680ttcctgttcg accacgccgc
cgaggagtac gagcacgcca agaagctgat catcttcctg 1740aacgagaaca
acgtgcccgt gcagctgacc agcatcagcg cccccgagca caagttcgag
1800ggcctgaccc agatcttcca gaaggcctac gagcacgagc agcacatcag
cgagagcatc 1860aacaacatcg tggaccacgc catcaagagc aaggaccacg
ccaccttcaa cttcctgcag 1920tggtacgtgg ccgagcagca cgaggaggag
gtgctgttca aggacatcct ggacaagatc 1980gagctgatcg gcaacgagaa
ccacggcctg tacctggccg accagtacgt gaagggcatc 2040gccaagagca
ggaagagcgg atcctag 206756688PRTArtificial SequenceSynthetic 56Met
Glu Lys Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser 1 5 10
15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val
20 25 30 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln
Asp Ile 35 40 45 Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu
Asp Gly Val Lys 50 55 60 Pro Leu Ile Leu Arg Asp Cys Ser Val Ala
Gly Trp Leu Leu Gly Asn 65 70 75 80 Pro Met Cys Asp Glu Phe Ile Asn
Val Pro Glu Trp Ser Tyr Ile Val 85 90 95 Glu Lys Ala Asn Pro Thr
Asn Asp Leu Cys Tyr Pro Gly Ser Phe Asn 100 105 110 Asp Tyr Glu Glu
Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 115 120 125 Lys Ile
Gln Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser 130 135 140
Ser Gly Val Ser Ser Ala Cys Pro Tyr Leu Gly Ser Pro Ser Phe Phe 145
150 155 160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Ser Thr Tyr Pro
Thr Ile 165 170 175 Lys Lys Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu
Leu Val Leu Trp 180 185 190 Gly Ile His His Pro Asn Asp Ala Ala Glu
Gln Thr Arg Leu Tyr Gln 195 200 205 Asn Pro Thr Thr Tyr Ile Ser Ile
Gly Thr Ser Thr Leu Asn Gln Arg 210 215 220 Leu Val Pro Lys Ile Ala
Thr Arg Ser Lys Val Asn Gly Gln Ser Gly 225 230 235 240 Arg Met Glu
Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 245 250 255 Phe
Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 260 265
270 Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Leu Glu Tyr Gly
275 280 285 Asn Cys Asn Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn
Ser Ser 290 295 300 Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly
Glu Cys Pro Lys 305 310 315 320 Tyr Val Lys Ser Asn Arg Leu Val Leu
Ala Thr Gly Leu Arg Asn Ser 325 330 335 Pro Gln Arg Glu Ser Arg Arg
Lys Lys Arg Gly Leu Phe Gly Ala Ile 340 345 350 Ala Gly Phe Ile Glu
Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355 360 365 Gly Tyr His
His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370 375 380 Glu
Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser 385 390
395 400 Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu
Phe 405 410 415 Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys
Met Glu Asp 420 425 430 Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu
Leu Leu Val Leu Met 435 440 445 Glu Asn Glu Arg Thr Leu Asp Phe His
Asp Ser Asn Val Lys Asn Leu 450 455 460 Tyr Asp Lys Val Arg Leu Gln
Leu Arg Asp Asn Ala Lys Glu Leu Gly 465 470 475 480 Asn Gly Cys Phe
Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu 485 490 495 Ser Ile
Arg Asn Gly Thr Tyr Asn Tyr Pro Gln Tyr Ser Glu Glu Ala 500 505 510
Arg Leu Lys Arg Glu Glu Ile Ser Ser Gly Gly Asp Ile Ile Lys Leu 515
520 525 Leu Asn Glu Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr
Met 530 535 540 Ser Met Ser Ser Trp Cys Tyr Thr His Ser Leu Asp Gly
Ala Gly Leu 545 550 555 560 Phe Leu Phe Asp His Ala Ala Glu Glu Tyr
Glu His Ala Lys Lys Leu 565 570 575 Ile Ile Phe Leu Asn Glu Asn Asn
Val Pro Val Gln Leu Thr Ser Ile 580 585 590 Ser Ala Pro Glu His Lys
Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys 595 600 605 Ala Tyr Glu His
Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val 610 615 620 Asp His
Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln 625 630 635
640 Trp Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile
645 650 655 Leu Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu
Tyr Leu 660 665 670 Ala Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg
Lys Ser Gly Ser 675 680 685 572067DNAArtificial SequenceSynthetic
57ctaggatccg ctcttcctgc tcttggcgat gcccttcacg tactggtcgg ccaggtacag
60gccgtggttc tcgttgccga tcagctcgat cttgtccagg atgtccttga acagcacctc
120ctcctcgtgc tgctcggcca cgtaccactg caggaagttg aaggtggcgt
ggtccttgct 180cttgatggcg tggtccacga tgttgttgat gctctcgctg
atgtgctgct cgtgctcgta 240ggccttctgg aagatctggg tcaggccctc
gaacttgtgc tcgggggcgc tgatgctggt 300cagctgcacg ggcacgttgt
tctcgttcag gaagatgatc agcttcttgg cgtgctcgta 360ctcctcggcg
gcgtggtcga acaggaacag gccggcgccg tccaggctgt gggtgtagca
420ccagctgctc atgctcatgt acaggttgct gctctgcatc tccttgttca
cctgctcgtt 480cagcagcttg atgatgtcgc ctccggagct gatctcttcc
ctcttcagcc tggcttcctc 540gctgtactga gggtagttgt aggtgccgtt
cctgatgctt tccatgcact cgttgtcgca 600cttgtggtag aactcgaagc
agccgttgcc cagctctttg gcgttgtctc tcagctgcag 660ccgcactttg
tcgtacaggt tcttcacgtt gctgtcgtgg aagtccaggg tccgctcgtt
720ttccatcagc accagcagtt cggcattgta ggtccacaca tccaggaagc
catcttccat 780tttcttgttc aggttctcga tccgccgttc caggttgttg
aactctctgc ccacagcctc 840gaactgggtg ttcatcttgt cgatgatgct
gttcaccttg ttggtgacgc cgtcgatggc 900cttctgggta gactctttgt
cggcggcata tccagagccc tgctcattgc tgtggtggta 960gccgtaccag
ccatccacca ttccctgcca gccgccttca ataaagccgg cgatggctcc
1020aaacaggccc ctcttctttc ttctgctctc ccgctggggg ctatttctca
ggcctgtggc 1080cagcaccagt ctgttgctct tcacgtactt agggcactcg
ccgatggtca gagggtggat 1140gttgtggaag ggcatgctgc tgttgatggc
gcccataggt gtctggcact tggtgttgca 1200gttgccgtat tccagctcgc
tcttcatgat ggcgctgtcg cccttcttca cgatcttgta 1260ggcgtactca
ggggcgataa agttgccgtt gctctcgaag ttgatggcgt cgttgggctt
1320caggatggtc cagaagaatt ccatcctgcc gctctggccg ttcaccttgg
atctggtggc 1380gatcttgggc accagtctct gattcagggt gctggtgccg
atgctgatat aggtggtggg 1440gttctggtac agtctggtct gctcggcggc
atcattaggg tggtggattc cccacaggac 1500cagcagatct tcctggttgg
tgttgttgta gctcttcttg atggtggggt aggtgctgtt 1560cttcttgatc
agccacacca cgtttctgaa gaagctgggg ctgcccaggt aaggacaggc
1620gctagacact ccgctagagg cttcgtgatc gctccaagag gacttgggga
tgatctggat 1680cttctcgaag tggttgatcc gggacagcag gtgcttcagt
tcctcgtaat cgttgaagct 1740gccggggtaa cacagatcgt tggtggggtt
ggccttctcc acgatatagc tccactcggg 1800cacgttgatg aactcgtcgc
acatagggtt gcccagcagc catccagcca cgctacaatc 1860tctcaggatc
agaggcttca cgccgtccag atcacacagc ttgccgttgt gggtcttttc
1920caggatgtcc tgagcgtggg tcacggtcac gtttttttcc atgatggtgt
ccacctgctc 1980tgtgctattg ttggcgtggt agccaatgca gatctggtcg
ctcttcacca ggctcacaat 2040ggccagcagc agcacgatct tttccat
2067582109DNAArtificial SequenceSynthetic 58atgaaggcca tcatcgtgct
gctgatggtg gtgaccagca acgccgatag aatctgcacc 60ggcatcacca gcagcaatag
cccccatgtg gtgaaaacag ccacccaggg cgaagtgaat 120gtgacaggcg
tgatccctct gaccaccacc cccaccaaga gctacttcgc caacctgaag
180ggcaccagaa ccagaggcaa gctgtgcccc gattgcctga actgcaccga
tctggatgtg 240gctctgggca gacctatgtg tgtgggcacc acaccatctg
ccaaggccag catcctgcac 300gaagtgaagc ctgtgaccag cggctgcttc
cccatcatgc acgaccggac caagatcaga 360cagctgccca acctgctgag
aggctacgag aacatccggc tgtccaccca gaatgtgatc 420gatgccgaga
aagcccctgg cggaccttat agactgggca ccagcggctc ttgtcccaat
480gccacctcca agagcggctt ttttgccaca atggcctggg ccgtgcctaa
ggacaacaac 540aagaacgcca ccaaccctct gaccgtggag gtgccctaca
tctgtacaga gggcgaggat 600cagatcacag tgtggggctt ccacagcgac
gacaagaccc agatgaagaa cctgtacggc 660gacagcaacc cccagaagtt
taccagcagc gccaatggcg tgaccaccca ctacgtgtcc 720cagatcggca
gctttcccga tcagacagag gatggcggac tgcctcagtc tggcaggatc
780gtggtggact acatgatgca gaagcctggc aagaccggca ccatcgtgta
tcagagaggc 840gtgctgctgc ctcagaaagt gtggtgtgcc agcggcaggt
ctaaagtgat caagggcagc 900ctgcctctga ttggcgaggc cgactgtctg
cacgaaaagt acggcggcct gaacaagagc 960aagccctact acacaggcga
gcacgccaag gccatcggca attgccccat ctgggtgaaa 1020acccccctga
agctggccaa tggcaccaag tacagacctc ccgccaagct gctgaaagag
1080agaggcttct ttggcgccat tgccggattt ctggaaggcg gctgggaggg
aatgattgcc 1140ggctggcacg gctatacatc tcatggggcc catggcgtgg
ctgtggccgc cgatctgaag 1200tctacccagg aagccatcaa caagatcacc
aagaacctga acagcctgag cgagctggaa 1260gtgaagaatc tgcagagact
gagcggcgcc atggatgagc tgcacaacga gatcctggaa 1320ctggacgaga
aagtggatga tctccgcgcc gatacaattt cctcccagat tgaactggcc
1380gtgctgctgt ccaacgaggg catcatcaac agcgaggatg aacacctgct
ggccctggaa 1440cggaagctga agaagatgct gggcccttct gccgtggaga
tcggcaacgg ctgcttcgag 1500acaaagcaca agtgcaacca gacctgcctg
gatagaatcg ccgctggcac cttcaatgcc 1560ggcgagttca gcctgcctac
cttcgacagc ctgaatatca cctccggagg cgacatcatc 1620aagctgctga
acgagcaggt gaacaaggag atgcagagca gcaacctgta catgagcatg
1680agcagctggt gctacaccca cagcctggac ggcgccggcc tgttcctgtt
cgaccacgcc 1740gccgaggagt acgagcacgc caagaagctg atcatcttcc
tgaacgagaa caacgtgccc 1800gtgcagctga ccagcatcag cgcccccgag
cacaagttcg agggcctgac ccagatcttc 1860cagaaggcct acgagcacga
gcagcacatc agcgagagca tcaacaacat cgtggaccac 1920gccatcaaga
gcaaggacca cgccaccttc aacttcctgc agtggtacgt ggccgagcag
1980cacgaggagg aggtgctgtt caaggacatc ctggacaaga tcgagctgat
cggcaacgag 2040aaccacggcc tgtacctggc cgaccagtac gtgaagggca
tcgccaagag caggaagagc 2100ggatcctag 210959702PRTArtificial
SequenceSynthetic 59Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr
Ser Asn Ala Asp 1 5 10 15 Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn
Ser Pro His Val Val Lys 20 25 30 Thr Ala Thr Gln Gly Glu Val Asn
Val Thr Gly Val Ile Pro Leu Thr 35 40 45 Thr Thr Pro Thr Lys Ser
Tyr Phe Ala Asn Leu Lys Gly Thr Arg Thr 50 55 60 Arg Gly Lys Leu
Cys Pro Asp Cys Leu Asn Cys Thr Asp Leu Asp Val 65 70 75 80 Ala Leu
Gly Arg Pro Met Cys Val Gly Thr Thr Pro Ser Ala Lys Ala 85 90 95
Ser Ile Leu His Glu Val Lys Pro Val Thr Ser Gly Cys Phe Pro Ile 100
105 110 Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg
Gly 115 120 125 Tyr Glu Asn Ile Arg Leu Ser Thr Gln Asn Val Ile Asp
Ala Glu Lys 130 135 140 Ala Pro Gly Gly Pro Tyr Arg Leu Gly Thr Ser
Gly Ser Cys Pro Asn 145 150 155 160 Ala Thr Ser Lys Ser Gly Phe Phe
Ala Thr Met Ala Trp Ala Val Pro 165 170 175 Lys Asp Asn Asn Lys Asn
Ala Thr Asn Pro Leu Thr Val Glu Val Pro 180 185 190 Tyr Ile Cys Thr
Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His 195 200 205 Ser Asp
Asp Lys Thr Gln Met Lys Asn Leu Tyr Gly Asp Ser Asn Pro 210 215 220
Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser 225
230 235 240 Gln Ile Gly Ser Phe Pro Asp Gln Thr Glu Asp Gly Gly Leu
Pro Gln 245 250 255 Ser Gly Arg Ile Val Val Asp Tyr Met Met Gln Lys
Pro Gly Lys Thr 260 265 270 Gly Thr Ile Val Tyr Gln Arg Gly Val Leu
Leu Pro Gln Lys Val Trp 275 280 285 Cys Ala Ser Gly Arg Ser Lys Val
Ile Lys Gly Ser Leu Pro Leu Ile 290 295 300 Gly Glu Ala Asp Cys Leu
His Glu Lys Tyr Gly Gly Leu Asn Lys Ser 305 310 315 320 Lys Pro Tyr
Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro 325 330 335 Ile
Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg 340 345
350 Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala
355 360 365 Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp
His Gly 370 375 380 Tyr Thr Ser His Gly Ala His Gly Val Ala Val Ala
Ala Asp Leu Lys 385 390 395 400 Ser Thr Gln Glu Ala Ile Asn Lys Ile
Thr Lys Asn Leu Asn Ser Leu 405 410
415 Ser Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met Asp
420 425 430 Glu Leu His Asn Glu Ile Leu Glu Leu Asp Glu Lys Val Asp
Asp Leu 435 440 445 Arg Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala
Val Leu Leu Ser 450 455 460 Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu
His Leu Leu Ala Leu Glu 465 470 475 480 Arg Lys Leu Lys Lys Met Leu
Gly Pro Ser Ala Val Glu Ile Gly Asn 485 490 495 Gly Cys Phe Glu Thr
Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg 500 505 510 Ile Ala Ala
Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr Phe 515 520 525 Asp
Ser Leu Asn Ile Thr Ser Gly Gly Asp Ile Ile Lys Leu Leu Asn 530 535
540 Glu Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met
545 550 555 560 Ser Ser Trp Cys Tyr Thr His Ser Leu Asp Gly Ala Gly
Leu Phe Leu 565 570 575 Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala
Lys Lys Leu Ile Ile 580 585 590 Phe Leu Asn Glu Asn Asn Val Pro Val
Gln Leu Thr Ser Ile Ser Ala 595 600 605 Pro Glu His Lys Phe Glu Gly
Leu Thr Gln Ile Phe Gln Lys Ala Tyr 610 615 620 Glu His Glu Gln His
Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His 625 630 635 640 Ala Ile
Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr 645 650 655
Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp 660
665 670 Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala
Asp 675 680 685 Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser Gly
Ser 690 695 700 602109DNAArtificial SequenceSynthetic 60ctaggatccg
ctcttcctgc tcttggcgat gcccttcacg tactggtcgg ccaggtacag 60gccgtggttc
tcgttgccga tcagctcgat cttgtccagg atgtccttga acagcacctc
120ctcctcgtgc tgctcggcca cgtaccactg caggaagttg aaggtggcgt
ggtccttgct 180cttgatggcg tggtccacga tgttgttgat gctctcgctg
atgtgctgct cgtgctcgta 240ggccttctgg aagatctggg tcaggccctc
gaacttgtgc tcgggggcgc tgatgctggt 300cagctgcacg ggcacgttgt
tctcgttcag gaagatgatc agcttcttgg cgtgctcgta 360ctcctcggcg
gcgtggtcga acaggaacag gccggcgccg tccaggctgt gggtgtagca
420ccagctgctc atgctcatgt acaggttgct gctctgcatc tccttgttca
cctgctcgtt 480cagcagcttg atgatgtcgc ctccggaggt gatattcagg
ctgtcgaagg taggcaggct 540gaactcgccg gcattgaagg tgccagcggc
gattctatcc aggcaggtct ggttgcactt 600gtgctttgtc tcgaagcagc
cgttgccgat ctccacggca gaagggccca gcatcttctt 660cagcttccgt
tccagggcca gcaggtgttc atcctcgctg ttgatgatgc cctcgttgga
720cagcagcacg gccagttcaa tctgggagga aattgtatcg gcgcggagat
catccacttt 780ctcgtccagt tccaggatct cgttgtgcag ctcatccatg
gcgccgctca gtctctgcag 840attcttcact tccagctcgc tcaggctgtt
caggttcttg gtgatcttgt tgatggcttc 900ctgggtagac ttcagatcgg
cggccacagc cacgccatgg gccccatgag atgtatagcc 960gtgccagccg
gcaatcattc cctcccagcc gccttccaga aatccggcaa tggcgccaaa
1020gaagcctctc tctttcagca gcttggcggg aggtctgtac ttggtgccat
tggccagctt 1080caggggggtt ttcacccaga tggggcaatt gccgatggcc
ttggcgtgct cgcctgtgta 1140gtagggcttg ctcttgttca ggccgccgta
cttttcgtgc agacagtcgg cctcgccaat 1200cagaggcagg ctgcccttga
tcactttaga cctgccgctg gcacaccaca ctttctgagg 1260cagcagcacg
cctctctgat acacgatggt gccggtcttg ccaggcttct gcatcatgta
1320gtccaccacg atcctgccag actgaggcag tccgccatcc tctgtctgat
cgggaaagct 1380gccgatctgg gacacgtagt gggtggtcac gccattggcg
ctgctggtaa acttctgggg 1440gttgctgtcg ccgtacaggt tcttcatctg
ggtcttgtcg tcgctgtgga agccccacac 1500tgtgatctga tcctcgccct
ctgtacagat gtagggcacc tccacggtca gagggttggt 1560ggcgttcttg
ttgttgtcct taggcacggc ccaggccatt gtggcaaaaa agccgctctt
1620ggaggtggca ttgggacaag agccgctggt gcccagtcta taaggtccgc
caggggcttt 1680ctcggcatcg atcacattct gggtggacag ccggatgttc
tcgtagcctc tcagcaggtt 1740gggcagctgt ctgatcttgg tccggtcgtg
catgatgggg aagcagccgc tggtcacagg 1800cttcacttcg tgcaggatgc
tggccttggc agatggtgtg gtgcccacac acataggtct 1860gcccagagcc
acatccagat cggtgcagtt caggcaatcg gggcacagct tgcctctggt
1920tctggtgccc ttcaggttgg cgaagtagct cttggtgggg gtggtggtca
gagggatcac 1980gcctgtcaca ttcacttcgc cctgggtggc tgttttcacc
acatgggggc tattgctgct 2040ggtgatgccg gtgcagattc tatcggcgtt
gctggtcacc accatcagca gcacgatgat 2100ggccttcat
2109612064DNAArtificial SequenceSynthetic 61atgaaaacca taattgcgct
gtcctacata ctgtgtctgg tgtttgccca gaaactgccg 60ggcaatgaca actcaacagc
cacgctctgc ttggggcacc atgccgtccc taacgggacc 120attgtgaaaa
ccattactaa cgatcagata gaggtgacta atgccaccga gctggtgcaa
180agtagctcca caggagagat ctgcgatagt ccccaccaga ttctggacgg
aaagaattgt 240acgctgatcg acgcgctgtt gggcgaccct cagtgtgacg
gatttcagaa taagaagtgg 300gatctgtttg tggaaaggtc aaaggcttat
tcaaattgct acccttacga tgtgcctgat 360tatgccagcc tgcggtccct
cgtcgcgtct agtgggactc tggagttcaa caacgagtca 420tttaactgga
ctggcgttac acagaacggg actagttccg cttgcataag gagaagcaaa
480aatagtttct tcagcagact gaattggctg acacatctga acttcaagta
ccctgcactg 540aatgtaacca tgcccaacaa cgagcagttc gataagcttt
acatttgggg agttcatcat 600cctggcactg acaaggatca gatctttctg
tatgcccagg cttccggcag gattaccgtg 660tctacaaaga gaagccagca
aactgtgtct cccaatatcg gcagtagacc cagagtacgg 720aacatcccta
gtcgcatcag tatttactgg accatcgtga aaccaggcga tattctcctg
780attaacagta ctggcaacct gatcgccccc cggggatact ttaaaatccg
ctctggaaag 840tcctccatta tgagatcaga tgcaccgatc ggaaaatgca
actctgagtg tatcacaccc 900aatgggagca ttcccaatga caaacctttc
cagaacgtta atcgaataac ttatggggcc 960tgtccacggt acgtgaagca
aaataccttg aaactggcga ccggtatgcg caatgtcccc 1020gaaaaacaga
cccgcgggat atttggggct atcgcaggct ttatcgagaa tggctgggaa
1080gggatggtgg atggttggta tggttttaga catcaaaact ccgaaggcag
aggccaggct 1140gccgatctca agagcacgca ggccgctata gatcagatca
atggaaagct caacagactg 1200atcgggaaaa ccaacgaaaa attccatcag
atcgagaaag agttctccga agtcgagggg 1260cgcatacagg acctggagaa
gtatgttgag gatacaaaga ttgatctgtg gtcctacaat 1320gccgagctgc
tggtggctct ggagaatcag cacactattg acctgaccga ttcagagatg
1380aacaaacttt ttgagaagac gaagaagcag cttagagaaa atgcagagga
catggggaac 1440ggatgcttta aaatatatca taagtgtgat aatgcctgca
tcggatcaat tagaaatggt 1500acctatgatc acgatgttta cagggacgaa
gcgctgaata acaggttcca gataaaatcc 1560ggaggcgaca tcatcaagct
gctgaacgag caggtgaaca aggagatgca gagcagcaac 1620ctgtacatga
gcatgagcag ctggtgctac acccacagcc tggacggcgc cggcctgttc
1680ctgttcgacc acgccgccga ggagtacgag cacgccaaga agctgatcat
cttcctgaac 1740gagaacaacg tgcccgtgca gctgaccagc atcagcgccc
ccgagcacaa gttcgagggc 1800ctgacccaga tcttccagaa ggcctacgag
cacgagcagc acatcagcga gagcatcaac 1860aacatcgtgg accacgccat
caagagcaag gaccacgcca ccttcaactt cctgcagtgg 1920tacgtggccg
agcagcacga ggaggaggtg ctgttcaagg acatcctgga caagatcgag
1980ctgatcggca acgagaacca cggcctgtac ctggccgacc agtacgtgaa
gggcatcgcc 2040aagagcagga agagcggatc ctag 206462687PRTArtificial
SequenceSynthetic 62Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys
Leu Val Phe Ala 1 5 10 15 Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr
Ala Thr Leu Cys Leu Gly 20 25 30 His His Ala Val Pro Asn Gly Thr
Ile Val Lys Thr Ile Thr Asn Asp 35 40 45 Gln Ile Glu Val Thr Asn
Ala Thr Glu Leu Val Gln Ser Ser Ser Thr 50 55 60 Gly Glu Ile Cys
Asp Ser Pro His Gln Ile Leu Asp Gly Lys Asn Cys 65 70 75 80 Thr Leu
Ile Asp Ala Leu Leu Gly Asp Pro Gln Cys Asp Gly Phe Gln 85 90 95
Asn Lys Lys Trp Asp Leu Phe Val Glu Arg Ser Lys Ala Tyr Ser Asn 100
105 110 Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Arg Ser Leu
Val 115 120 125 Ala Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu Ser Phe
Asn Trp Thr 130 135 140 Gly Val Thr Gln Asn Gly Thr Ser Ser Ala Cys
Ile Arg Arg Ser Lys 145 150 155 160 Asn Ser Phe Phe Ser Arg Leu Asn
Trp Leu Thr His Leu Asn Phe Lys 165 170 175 Tyr Pro Ala Leu Asn Val
Thr Met Pro Asn Asn Glu Gln Phe Asp Lys 180 185 190 Leu Tyr Ile Trp
Gly Val His His Pro Gly Thr Asp Lys Asp Gln Ile 195 200 205 Phe Leu
Tyr Ala Gln Ala Ser Gly Arg Ile Thr Val Ser Thr Lys Arg 210 215 220
Ser Gln Gln Thr Val Ser Pro Asn Ile Gly Ser Arg Pro Arg Val Arg 225
230 235 240 Asn Ile Pro Ser Arg Ile Ser Ile Tyr Trp Thr Ile Val Lys
Pro Gly 245 250 255 Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile
Ala Pro Arg Gly 260 265 270 Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser
Ile Met Arg Ser Asp Ala 275 280 285 Pro Ile Gly Lys Cys Asn Ser Glu
Cys Ile Thr Pro Asn Gly Ser Ile 290 295 300 Pro Asn Asp Lys Pro Phe
Gln Asn Val Asn Arg Ile Thr Tyr Gly Ala 305 310 315 320 Cys Pro Arg
Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Gly Met 325 330 335 Arg
Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe Gly Ala Ile Ala 340 345
350 Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp Gly Trp Tyr Gly
355 360 365 Phe Arg His Gln Asn Ser Glu Gly Arg Gly Gln Ala Ala Asp
Leu Lys 370 375 380 Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys
Leu Asn Arg Leu 385 390 395 400 Ile Gly Lys Thr Asn Glu Lys Phe His
Gln Ile Glu Lys Glu Phe Ser 405 410 415 Glu Val Glu Gly Arg Ile Gln
Asp Leu Glu Lys Tyr Val Glu Asp Thr 420 425 430 Lys Ile Asp Leu Trp
Ser Tyr Asn Ala Glu Leu Leu Val Ala Leu Glu 435 440 445 Asn Gln His
Thr Ile Asp Leu Thr Asp Ser Glu Met Asn Lys Leu Phe 450 455 460 Glu
Lys Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu Asp Met Gly Asn 465 470
475 480 Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala Cys Ile Gly
Ser 485 490 495 Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg Asp
Glu Ala Leu 500 505 510 Asn Asn Arg Phe Gln Ile Lys Ser Gly Gly Asp
Ile Ile Lys Leu Leu 515 520 525 Asn Glu Gln Val Asn Lys Glu Met Gln
Ser Ser Asn Leu Tyr Met Ser 530 535 540 Met Ser Ser Trp Cys Tyr Thr
His Ser Leu Asp Gly Ala Gly Leu Phe 545 550 555 560 Leu Phe Asp His
Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile 565 570 575 Ile Phe
Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser 580 585 590
Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala 595
600 605 Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val
Asp 610 615 620 His Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe
Leu Gln Trp 625 630 635 640 Tyr Val Ala Glu Gln His Glu Glu Glu Val
Leu Phe Lys Asp Ile Leu 645 650 655 Asp Lys Ile Glu Leu Ile Gly Asn
Glu Asn His Gly Leu Tyr Leu Ala 660 665 670 Asp Gln Tyr Val Lys Gly
Ile Ala Lys Ser Arg Lys Ser Gly Ser 675 680 685 632064DNAArtificial
SequenceSynthetic 63ctaggatccg ctcttcctgc tcttggcgat gcccttcacg
tactggtcgg ccaggtacag 60gccgtggttc tcgttgccga tcagctcgat cttgtccagg
atgtccttga acagcacctc 120ctcctcgtgc tgctcggcca cgtaccactg
caggaagttg aaggtggcgt ggtccttgct 180cttgatggcg tggtccacga
tgttgttgat gctctcgctg atgtgctgct cgtgctcgta 240ggccttctgg
aagatctggg tcaggccctc gaacttgtgc tcgggggcgc tgatgctggt
300cagctgcacg ggcacgttgt tctcgttcag gaagatgatc agcttcttgg
cgtgctcgta 360ctcctcggcg gcgtggtcga acaggaacag gccggcgccg
tccaggctgt gggtgtagca 420ccagctgctc atgctcatgt acaggttgct
gctctgcatc tccttgttca cctgctcgtt 480cagcagcttg atgatgtcgc
ctccggattt tatctggaac ctgttattca gcgcttcgtc 540cctgtaaaca
tcgtgatcat aggtaccatt tctaattgat ccgatgcagg cattatcaca
600cttatgatat attttaaagc atccgttccc catgtcctct gcattttctc
taagctgctt 660cttcgtcttc tcaaaaagtt tgttcatctc tgaatcggtc
aggtcaatag tgtgctgatt 720ctccagagcc accagcagct cggcattgta
ggaccacaga tcaatctttg tatcctcaac 780atacttctcc aggtcctgta
tgcgcccctc gacttcggag aactctttct cgatctgatg 840gaatttttcg
ttggttttcc cgatcagtct gttgagcttt ccattgatct gatctatagc
900ggcctgcgtg ctcttgagat cggcagcctg gcctctgcct tcggagtttt
gatgtctaaa 960accataccaa ccatccacca tcccttccca gccattctcg
ataaagcctg cgatagcccc 1020aaatatcccg cgggtctgtt tttcggggac
attgcgcata ccggtcgcca gtttcaaggt 1080attttgcttc acgtaccgtg
gacaggcccc ataagttatt cgattaacgt tctggaaagg 1140tttgtcattg
ggaatgctcc cattgggtgt gatacactca gagttgcatt ttccgatcgg
1200tgcatctgat ctcataatgg aggactttcc agagcggatt ttaaagtatc
cccggggggc 1260gatcaggttg ccagtactgt taatcaggag aatatcgcct
ggtttcacga tggtccagta 1320aatactgatg cgactaggga tgttccgtac
tctgggtcta ctgccgatat tgggagacac 1380agtttgctgg cttctctttg
tagacacggt aatcctgccg gaagcctggg catacagaaa 1440gatctgatcc
ttgtcagtgc caggatgatg aactccccaa atgtaaagct tatcgaactg
1500ctcgttgttg ggcatggtta cattcagtgc agggtacttg aagttcagat
gtgtcagcca 1560attcagtctg ctgaagaaac tatttttgct tctccttatg
caagcggaac tagtcccgtt 1620ctgtgtaacg ccagtccagt taaatgactc
gttgttgaac tccagagtcc cactagacgc 1680gacgagggac cgcaggctgg
cataatcagg cacatcgtaa gggtagcaat ttgaataagc 1740ctttgacctt
tccacaaaca gatcccactt cttattctga aatccgtcac actgagggtc
1800gcccaacagc gcgtcgatca gcgtacaatt ctttccgtcc agaatctggt
ggggactatc 1860gcagatctct cctgtggagc tactttgcac cagctcggtg
gcattagtca cctctatctg 1920atcgttagta atggttttca caatggtccc
gttagggacg gcatggtgcc ccaagcagag 1980cgtggctgtt gagttgtcat
tgcccggcag tttctgggca aacaccagac acagtatgta 2040ggacagcgca
attatggttt tcat 2064642058DNAArtificial SequenceSynthetic
64atgaaagtga agctgctggt gctgctgtgt acctttaccg ccacctacgc cgataccatc
60tgtatcggct accacgccaa caatagcacc gacaccgtgg ataccgtgct ggaaaagaac
120gtgaccgtga cccacagcgt gaacctgctg gaaaacagcc acaacggcaa
gctgtgtctg 180ctgaaaggca ttgcccctct gcagctggga aattgtagcg
tggccggctg gattctgggc 240aatcctgagt gcgagctgct gatttccaaa
gagtcctggt cctacatcgt ggagaagccc 300aaccctgaga atggcacctg
ctaccctggc cacttcgccg attacgagga actgagagaa 360cagctgtcca
gcgtgtccag cttcgagaga ttcgagatct tccccaaaga gagcagctgg
420cccaatcata cagtgaccgg cgtgagcgcc tcttgtagcc acaatggcga
gagcagcttc 480tacagaaacc tgctgtggct gaccggcaag aacggcctgt
accccaacct gagcaagagc 540tacgccaaca acaaagaaaa agaagtgctg
gtcctctggg gagtgcacca ccctcctaac 600atcggcatcc agaaggccct
gtaccacacc gagaatgcct acgtgtccgt ggtgtccagc 660cactacagca
gaaagttcac ccccgagatc gccaaaagac ccaaagtgcg ggaccaggaa
720ggcaggatca actactactg gaccctgctg gaacctggcg acaccatcat
cttcgaggcc 780aacggcaatc tgatcgcccc tagatacgcc tttgccctga
gcagaggctt tggcagcggc 840atcatcaaca gcaacgcccc catggacaag
tgtgacgcca agtgtcagac accacaggga 900gctatcaata gcagcctgcc
cttccagaat gtgcaccctg tgaccatcgg cgagtgtcct 960aaatacgtgc
ggagcgccaa gctgagaatg gtgaccggcc tgaggaatat ccccagcatc
1020cagagcagag gcctgtttgg cgccattgcc ggctttatcg agggcggatg
gacaggcatg 1080gtggatgggt ggtacggcta ccaccaccag aatgagcagg
gatctggcta tgccgccgat 1140cagaagagca cccagaacgc catcaacggc
atcaccaaca aagtgaacag cgtgatcgag 1200aagatgaaca cccagttcac
cgccgtgggc aaagagttca acaagctgga acggcggatg 1260gaaaacctga
acaagaaggt ggacgacggc ttcatcgaca tctggaccta caacgccgaa
1320ctcctggtcc tcctggaaaa tgagaggacc ctggacttcc acgacagcaa
cgtgaagaac 1380ctgtacgaga aagtgaagag ccagctgaag aacaacgcca
aagagatcgg caacggctgc 1440ttcgagttct accacaagtg caacgacgag
tgcatggaaa gcgtgaagaa cggcacctac 1500gactacccca agtacagcga
ggaaagcaag ctgaaccggg agaagatcga ttccggaggc 1560gacatcatca
agctgctgaa cgagcaggtg aacaaggaga tgcagagcag caacctgtac
1620atgagcatga gcagctggtg ctacacccac agcctggacg gcgccggcct
gttcctgttc 1680gaccacgccg ccgaggagta cgagcacgcc aagaagctga
tcatcttcct gaacgagaac 1740aacgtgcccg tgcagctgac cagcatcagc
gcccccgagc acaagttcga gggcctgacc 1800cagatcttcc agaaggccta
cgagcacgag cagcacatca gcgagagcat caacaacatc 1860gtggaccacg
ccatcaagag caaggaccac gccaccttca acttcctgca gtggtacgtg
1920gccgagcagc acgaggagga ggtgctgttc aaggacatcc tggacaagat
cgagctgatc 1980ggcaacgaga accacggcct gtacctggcc gaccagtacg
tgaagggcat cgccaagagc 2040aggaagagcg gatcctag
205865685PRTArtificial SequenceSynthetic 65Met Lys
Val Lys Leu Leu Val Leu Leu Cys Thr Phe Thr Ala Thr Tyr 1 5 10 15
Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20
25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val
Asn 35 40 45 Leu Leu Glu Asn Ser His Asn Gly Lys Leu Cys Leu Leu
Lys Gly Ile 50 55 60 Ala Pro Leu Gln Leu Gly Asn Cys Ser Val Ala
Gly Trp Ile Leu Gly 65 70 75 80 Asn Pro Glu Cys Glu Leu Leu Ile Ser
Lys Glu Ser Trp Ser Tyr Ile 85 90 95 Val Glu Lys Pro Asn Pro Glu
Asn Gly Thr Cys Tyr Pro Gly His Phe 100 105 110 Ala Asp Tyr Glu Glu
Leu Arg Glu Gln Leu Ser Ser Val Ser Ser Phe 115 120 125 Glu Arg Phe
Glu Ile Phe Pro Lys Glu Ser Ser Trp Pro Asn His Thr 130 135 140 Val
Thr Gly Val Ser Ala Ser Cys Ser His Asn Gly Glu Ser Ser Phe 145 150
155 160 Tyr Arg Asn Leu Leu Trp Leu Thr Gly Lys Asn Gly Leu Tyr Pro
Asn 165 170 175 Leu Ser Lys Ser Tyr Ala Asn Asn Lys Glu Lys Glu Val
Leu Val Leu 180 185 190 Trp Gly Val His His Pro Pro Asn Ile Gly Ile
Gln Lys Ala Leu Tyr 195 200 205 His Thr Glu Asn Ala Tyr Val Ser Val
Val Ser Ser His Tyr Ser Arg 210 215 220 Lys Phe Thr Pro Glu Ile Ala
Lys Arg Pro Lys Val Arg Asp Gln Glu 225 230 235 240 Gly Arg Ile Asn
Tyr Tyr Trp Thr Leu Leu Glu Pro Gly Asp Thr Ile 245 250 255 Ile Phe
Glu Ala Asn Gly Asn Leu Ile Ala Pro Arg Tyr Ala Phe Ala 260 265 270
Leu Ser Arg Gly Phe Gly Ser Gly Ile Ile Asn Ser Asn Ala Pro Met 275
280 285 Asp Lys Cys Asp Ala Lys Cys Gln Thr Pro Gln Gly Ala Ile Asn
Ser 290 295 300 Ser Leu Pro Phe Gln Asn Val His Pro Val Thr Ile Gly
Glu Cys Pro 305 310 315 320 Lys Tyr Val Arg Ser Ala Lys Leu Arg Met
Val Thr Gly Leu Arg Asn 325 330 335 Ile Pro Ser Ile Gln Ser Arg Gly
Leu Phe Gly Ala Ile Ala Gly Phe 340 345 350 Ile Glu Gly Gly Trp Thr
Gly Met Val Asp Gly Trp Tyr Gly Tyr His 355 360 365 His Gln Asn Glu
Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr 370 375 380 Gln Asn
Ala Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu 385 390 395
400 Lys Met Asn Thr Gln Phe Thr Ala Val Gly Lys Glu Phe Asn Lys Leu
405 410 415 Glu Arg Arg Met Glu Asn Leu Asn Lys Lys Val Asp Asp Gly
Phe Ile 420 425 430 Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
Leu Glu Asn Glu 435 440 445 Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu Tyr Glu Lys 450 455 460 Val Lys Ser Gln Leu Lys Asn Asn
Ala Lys Glu Ile Gly Asn Gly Cys 465 470 475 480 Phe Glu Phe Tyr His
Lys Cys Asn Asp Glu Cys Met Glu Ser Val Lys 485 490 495 Asn Gly Thr
Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn 500 505 510 Arg
Glu Lys Ile Asp Ser Gly Gly Asp Ile Ile Lys Leu Leu Asn Glu 515 520
525 Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser
530 535 540 Ser Trp Cys Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe
Leu Phe 545 550 555 560 Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys
Lys Leu Ile Ile Phe 565 570 575 Leu Asn Glu Asn Asn Val Pro Val Gln
Leu Thr Ser Ile Ser Ala Pro 580 585 590 Glu His Lys Phe Glu Gly Leu
Thr Gln Ile Phe Gln Lys Ala Tyr Glu 595 600 605 His Glu Gln His Ile
Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala 610 615 620 Ile Lys Ser
Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val 625 630 635 640
Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys 645
650 655 Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp
Gln 660 665 670 Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser Gly Ser
675 680 685 662058DNAArtificial SequenceSynthetic 66ctaggatccg
ctcttcctgc tcttggcgat gcccttcacg tactggtcgg ccaggtacag 60gccgtggttc
tcgttgccga tcagctcgat cttgtccagg atgtccttga acagcacctc
120ctcctcgtgc tgctcggcca cgtaccactg caggaagttg aaggtggcgt
ggtccttgct 180cttgatggcg tggtccacga tgttgttgat gctctcgctg
atgtgctgct cgtgctcgta 240ggccttctgg aagatctggg tcaggccctc
gaacttgtgc tcgggggcgc tgatgctggt 300cagctgcacg ggcacgttgt
tctcgttcag gaagatgatc agcttcttgg cgtgctcgta 360ctcctcggcg
gcgtggtcga acaggaacag gccggcgccg tccaggctgt gggtgtagca
420ccagctgctc atgctcatgt acaggttgct gctctgcatc tccttgttca
cctgctcgtt 480cagcagcttg atgatgtcgc ctccggaatc gatcttctcc
cggttcagct tgctttcctc 540gctgtacttg gggtagtcgt aggtgccgtt
cttcacgctt tccatgcact cgtcgttgca 600cttgtggtag aactcgaagc
agccgttgcc gatctctttg gcgttgttct tcagctggct 660cttcactttc
tcgtacaggt tcttcacgtt gctgtcgtgg aagtccaggg tcctctcatt
720ttccaggagg accaggagtt cggcgttgta ggtccagatg tcgatgaagc
cgtcgtccac 780cttcttgttc aggttttcca tccgccgttc cagcttgttg
aactctttgc ccacggcggt 840gaactgggtg ttcatcttct cgatcacgct
gttcactttg ttggtgatgc cgttgatggc 900gttctgggtg ctcttctgat
cggcggcata gccagatccc tgctcattct ggtggtggta 960gccgtaccac
ccatccacca tgcctgtcca tccgccctcg ataaagccgg caatggcgcc
1020aaacaggcct ctgctctgga tgctggggat attcctcagg ccggtcacca
ttctcagctt 1080ggcgctccgc acgtatttag gacactcgcc gatggtcaca
gggtgcacat tctggaaggg 1140caggctgcta ttgatagctc cctgtggtgt
ctgacacttg gcgtcacact tgtccatggg 1200ggcgttgctg ttgatgatgc
cgctgccaaa gcctctgctc agggcaaagg cgtatctagg 1260ggcgatcaga
ttgccgttgg cctcgaagat gatggtgtcg ccaggttcca gcagggtcca
1320gtagtagttg atcctgcctt cctggtcccg cactttgggt cttttggcga
tctcgggggt 1380gaactttctg ctgtagtggc tggacaccac ggacacgtag
gcattctcgg tgtggtacag 1440ggccttctgg atgccgatgt taggagggtg
gtgcactccc cagaggacca gcacttcttt 1500ttctttgttg ttggcgtagc
tcttgctcag gttggggtac aggccgttct tgccggtcag 1560ccacagcagg
tttctgtaga agctgctctc gccattgtgg ctacaagagg cgctcacgcc
1620ggtcactgta tgattgggcc agctgctctc tttggggaag atctcgaatc
tctcgaagct 1680ggacacgctg gacagctgtt ctctcagttc ctcgtaatcg
gcgaagtggc cagggtagca 1740ggtgccattc tcagggttgg gcttctccac
gatgtaggac caggactctt tggaaatcag 1800cagctcgcac tcaggattgc
ccagaatcca gccggccacg ctacaatttc ccagctgcag 1860aggggcaatg
cctttcagca gacacagctt gccgttgtgg ctgttttcca gcaggttcac
1920gctgtgggtc acggtcacgt tcttttccag cacggtatcc acggtgtcgg
tgctattgtt 1980ggcgtggtag ccgatacaga tggtatcggc gtaggtggcg
gtaaaggtac acagcagcac 2040cagcagcttc actttcat
2058672112DNAArtificial SequenceSynthetic 67atgaaggcca tcatcgtgct
gctgatggtg gtcacaagca acgccgatag aatctgtacc 60ggcatcacca gcagcaatag
ccctcacgtc gtgaaaacag ctacacaggg cgaagtgaat 120gtgaccggcg
tgatccctct gaccacaaca cctacaaaga gccacttcgc caatctgaag
180ggcacagaga caagaggcaa gctgtgtccc aagtgcctga attgcacaga
tctggatgtg 240gctctgggca gacctaagtg tacaggcaaa atccctagcg
ccagagtgtc cattctgcat 300gaagtgcgac ctgtgaccag cggctgtttt
cctattatgc acgaccggac caagatcaga 360cagctgccta atctgctgag
aggctacgag cacatcagac tgagcaccca caatgtgatc 420aacgccgaaa
atgctcctgg cggcccttat aagatcggca catctggcag ctgccccaac
480attacaaatg gcaatggctt ctttgccacc atggcttggg ccgtgcctaa
gaacgataag 540aacaagaccg ccaccaaccc cctgacaatc gaggtgccat
atatctgtac agagggcgag 600gatcagatca ccgtgtgggg atttcacagc
gacaacgaaa cacagatggc caagctgtac 660ggcgatagca agcctcagaa
gtttaccagc tctgccaatg gcgtgaccac acactatgtg 720tctcagatcg
gcggcttccc taatcagaca gaagatggcg gactgcctca gtctggaaga
780atcgtggtgg attacatggt gcagaagtct ggcaagaccg gcaccatcac
atatcagaga 840ggaatcctgc tgccccagaa agtgtggtgc gcttctggaa
gatccaaagt gatcaagggc 900agcctgcctc tgattggaga agccgattgt
ctgcacgaga aatacggcgg cctgaacaag 960agcaagcctt actatacagg
cgagcacgcc aaggccatcg gcaattgtcc tatttgggtc 1020aagacccctc
tgaagctggc caatggcaca aagtatagac ctccagccaa gctgctgaaa
1080gagagaggct tttttggagc tatcgccggc tttctggaag gcggatggga
gggaatgatt 1140gctggatggc atggctacac atctcatggc gcacatggcg
tggcagtggc tgctgatctg 1200aaatctacac aggaagccat caacaagatc
accaagaacc tgaacagcct gagcgagctg 1260gaagtgaaga atctgcagag
actgtctggc gccatggacg aactgcacaa tgagatcctg 1320gaactggacg
agaaggtgga cgatctgaga gccgatacaa tcagcagcca gattgaactg
1380gctgtgctgc tgtctaacga gggcatcatc aatagcgagg acgaacatct
gctggccctg 1440gaaagaaagc tgaagaagat gctgggacct agcgccgtgg
aaatcggcaa tggatgcttt 1500gagacaaagc acaagtgcaa ccagacctgc
ctggatagaa ttgccgccgg aacatttgat 1560gccggcgagt tttctctgcc
caccttcgat agcctgaata tcacatccgg aggcgacatc 1620atcaagctgc
tgaacgagca ggtgaacaag gagatgcaga gcagcaacct gtacatgagc
1680atgagcagct ggtgctacac ccacagcctg gacggcgccg gcctgttcct
gttcgaccac 1740gccgccgagg agtacgagca cgccaagaag ctgatcatct
tcctgaacga gaacaacgtg 1800cccgtgcagc tgaccagcat cagcgccccc
gagcacaagt tcgagggcct gacccagatc 1860ttccagaagg cctacgagca
cgagcagcac atcagcgaga gcatcaacaa catcgtggac 1920cacgccatca
agagcaagga ccacgccacc ttcaacttcc tgcagtggta cgtggccgag
1980cagcacgagg aggaggtgct gttcaaggac atcctggaca agatcgagct
gatcggcaac 2040gagaaccacg gcctgtacct ggccgaccag tacgtgaagg
gcatcgccaa gagcaggaag 2100agcggatcct ag 211268703PRTArtificial
SequenceSynthetic 68Met Lys Ala Ile Ile Val Leu Leu Met Val Val Thr
Ser Asn Ala Asp 1 5 10 15 Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn
Ser Pro His Val Val Lys 20 25 30 Thr Ala Thr Gln Gly Glu Val Asn
Val Thr Gly Val Ile Pro Leu Thr 35 40 45 Thr Thr Pro Thr Lys Ser
His Phe Ala Asn Leu Lys Gly Thr Glu Thr 50 55 60 Arg Gly Lys Leu
Cys Pro Lys Cys Leu Asn Cys Thr Asp Leu Asp Val 65 70 75 80 Ala Leu
Gly Arg Pro Lys Cys Thr Gly Lys Ile Pro Ser Ala Arg Val 85 90 95
Ser Ile Leu His Glu Val Arg Pro Val Thr Ser Gly Cys Phe Pro Ile 100
105 110 Met His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg
Gly 115 120 125 Tyr Glu His Ile Arg Leu Ser Thr His Asn Val Ile Asn
Ala Glu Asn 130 135 140 Ala Pro Gly Gly Pro Tyr Lys Ile Gly Thr Ser
Gly Ser Cys Pro Asn 145 150 155 160 Ile Thr Asn Gly Asn Gly Phe Phe
Ala Thr Met Ala Trp Ala Val Pro 165 170 175 Lys Asn Asp Lys Asn Lys
Thr Ala Thr Asn Pro Leu Thr Ile Glu Val 180 185 190 Pro Tyr Ile Cys
Thr Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe 195 200 205 His Ser
Asp Asn Glu Thr Gln Met Ala Lys Leu Tyr Gly Asp Ser Lys 210 215 220
Pro Gln Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val 225
230 235 240 Ser Gln Ile Gly Gly Phe Pro Asn Gln Thr Glu Asp Gly Gly
Leu Pro 245 250 255 Gln Ser Gly Arg Ile Val Val Asp Tyr Met Val Gln
Lys Ser Gly Lys 260 265 270 Thr Gly Thr Ile Thr Tyr Gln Arg Gly Ile
Leu Leu Pro Gln Lys Val 275 280 285 Trp Cys Ala Ser Gly Arg Ser Lys
Val Ile Lys Gly Ser Leu Pro Leu 290 295 300 Ile Gly Glu Ala Asp Cys
Leu His Glu Lys Tyr Gly Gly Leu Asn Lys 305 310 315 320 Ser Lys Pro
Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys 325 330 335 Pro
Ile Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr 340 345
350 Arg Pro Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile
355 360 365 Ala Gly Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly
Trp His 370 375 380 Gly Tyr Thr Ser His Gly Ala His Gly Val Ala Val
Ala Ala Asp Leu 385 390 395 400 Lys Ser Thr Gln Glu Ala Ile Asn Lys
Ile Thr Lys Asn Leu Asn Ser 405 410 415 Leu Ser Glu Leu Glu Val Lys
Asn Leu Gln Arg Leu Ser Gly Ala Met 420 425 430 Asp Glu Leu His Asn
Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp 435 440 445 Leu Arg Ala
Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu 450 455 460 Ser
Asn Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu 465 470
475 480 Glu Arg Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile
Gly 485 490 495 Asn Gly Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr
Cys Leu Asp 500 505 510 Arg Ile Ala Ala Gly Thr Phe Asp Ala Gly Glu
Phe Ser Leu Pro Thr 515 520 525 Phe Asp Ser Leu Asn Ile Thr Ser Gly
Gly Asp Ile Ile Lys Leu Leu 530 535 540 Asn Glu Gln Val Asn Lys Glu
Met Gln Ser Ser Asn Leu Tyr Met Ser 545 550 555 560 Met Ser Ser Trp
Cys Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe 565 570 575 Leu Phe
Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile 580 585 590
Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser 595
600 605 Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys
Ala 610 615 620 Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn
Ile Val Asp 625 630 635 640 His Ala Ile Lys Ser Lys Asp His Ala Thr
Phe Asn Phe Leu Gln Trp 645 650 655 Tyr Val Ala Glu Gln His Glu Glu
Glu Val Leu Phe Lys Asp Ile Leu 660 665 670 Asp Lys Ile Glu Leu Ile
Gly Asn Glu Asn His Gly Leu Tyr Leu Ala 675 680 685 Asp Gln Tyr Val
Lys Gly Ile Ala Lys Ser Arg Lys Ser Gly Ser 690 695 700
692112DNAArtificial SequenceSynthetic 69ctaggatccg ctcttcctgc
tcttggcgat gcccttcacg tactggtcgg ccaggtacag 60gccgtggttc tcgttgccga
tcagctcgat cttgtccagg atgtccttga acagcacctc 120ctcctcgtgc
tgctcggcca cgtaccactg caggaagttg aaggtggcgt ggtccttgct
180cttgatggcg tggtccacga tgttgttgat gctctcgctg atgtgctgct
cgtgctcgta 240ggccttctgg aagatctggg tcaggccctc gaacttgtgc
tcgggggcgc tgatgctggt 300cagctgcacg ggcacgttgt tctcgttcag
gaagatgatc agcttcttgg cgtgctcgta 360ctcctcggcg gcgtggtcga
acaggaacag gccggcgccg tccaggctgt gggtgtagca 420ccagctgctc
atgctcatgt acaggttgct gctctgcatc tccttgttca cctgctcgtt
480cagcagcttg atgatgtcgc ctccggatgt gatattcagg ctatcgaagg
tgggcagaga 540aaactcgccg gcatcaaatg ttccggcggc aattctatcc
aggcaggtct ggttgcactt 600gtgctttgtc tcaaagcatc cattgccgat
ttccacggcg ctaggtccca gcatcttctt 660cagctttctt tccagggcca
gcagatgttc gtcctcgcta ttgatgatgc cctcgttaga 720cagcagcaca
gccagttcaa tctggctgct gattgtatcg gctctcagat cgtccacctt
780ctcgtccagt tccaggatct cattgtgcag ttcgtccatg gcgccagaca
gtctctgcag 840attcttcact tccagctcgc tcaggctgtt caggttcttg
gtgatcttgt tgatggcttc 900ctgtgtagat ttcagatcag cagccactgc
cacgccatgt gcgccatgag atgtgtagcc 960atgccatcca gcaatcattc
cctcccatcc gccttccaga aagccggcga tagctccaaa 1020aaagcctctc
tctttcagca gcttggctgg aggtctatac tttgtgccat tggccagctt
1080cagaggggtc ttgacccaaa taggacaatt gccgatggcc ttggcgtgct
cgcctgtata 1140gtaaggcttg ctcttgttca ggccgccgta tttctcgtgc
agacaatcgg cttctccaat 1200cagaggcagg ctgcccttga tcactttgga
tcttccagaa gcgcaccaca ctttctgggg 1260cagcaggatt cctctctgat
atgtgatggt gccggtcttg ccagacttct gcaccatgta 1320atccaccacg
attcttccag actgaggcag tccgccatct tctgtctgat tagggaagcc
1380gccgatctga gacacatagt gtgtggtcac gccattggca gagctggtaa
acttctgagg 1440cttgctatcg ccgtacagct tggccatctg tgtttcgttg
tcgctgtgaa atccccacac 1500ggtgatctga tcctcgccct ctgtacagat
atatggcacc tcgattgtca gggggttggt 1560ggcggtcttg ttcttatcgt
tcttaggcac ggcccaagcc
atggtggcaa agaagccatt 1620gccatttgta atgttggggc agctgccaga
tgtgccgatc ttataagggc cgccaggagc 1680attttcggcg ttgatcacat
tgtgggtgct cagtctgatg tgctcgtagc ctctcagcag 1740attaggcagc
tgtctgatct tggtccggtc gtgcataata ggaaaacagc cgctggtcac
1800aggtcgcact tcatgcagaa tggacactct ggcgctaggg attttgcctg
tacacttagg 1860tctgcccaga gccacatcca gatctgtgca attcaggcac
ttgggacaca gcttgcctct 1920tgtctctgtg cccttcagat tggcgaagtg
gctctttgta ggtgttgtgg tcagagggat 1980cacgccggtc acattcactt
cgccctgtgt agctgttttc acgacgtgag ggctattgct 2040gctggtgatg
ccggtacaga ttctatcggc gttgcttgtg accaccatca gcagcacgat
2100gatggccttc at 211270837DNAInfluenza virus 70atgaaggcca
agctgctggt gctgctgtgc acctttaccg ccacctacgc cgacaccatc 60tgcattggct
accacgccaa caacagcacc gacaccgtgg ataccgtgct ggaaaagaac
120gtgaccgtga cccacagcgt gaacctggga tccggactga gaatggtcac
cggcctgaga 180aacatcccca gcatccagag cagaggcctg tttggagcca
ttgccggctt tattgagggc 240ggatggaccg gaatggtgga tgggtggtac
ggctaccacc accagaatga gcagggctct 300ggctatgccg ccgatcagaa
gtctacccag aacgccatca acggcatcac caacaaagtg 360aacagcgtga
tcgagaagat gggcggcgat cctgaatggg acagagagat caacaactac
420accagcatca tctacagcct gatcgaggaa agccagaacc agcaggaaaa
cggcacaggc 480ggcggatctg gaattgtgca gcagcagaac aacctgctga
gagccattga ggcccagcag 540catctgctgc agctgacagt gtggggcatc
aagcagctgc agacctacaa tgccgagctg 600ctggtcctcc tggaaaacga
gagaaccctg gacttccacg acagcaacgt gaagaacctg 660tacgagaaag
tgaagtccca gctgaagaac aacgccaaag agatcggcaa cggctgcttc
720gagttctacc acaagtgcaa caacgagtgc atggaaagcg tgaagaacgg
cacctacgac 780taccccaagt acagcgagga aagcaagctg aacagagaga
agatcgactc cggaggc 83771279PRTInfluenza virus 71Met Lys Ala Lys Leu
Leu Val Leu Leu Cys Thr Phe Thr Ala Thr Tyr 1 5 10 15 Ala Asp Thr
Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val
Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40
45 Leu Gly Ser Gly Leu Arg Met Val Thr Gly Leu Arg Asn Ile Pro Ser
50 55 60 Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile
Glu Gly 65 70 75 80 Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr
His His Gln Asn 85 90 95 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln
Lys Ser Thr Gln Asn Ala 100 105 110 Ile Asn Gly Ile Thr Asn Lys Val
Asn Ser Val Ile Glu Lys Met Gly 115 120 125 Gly Asp Pro Glu Trp Asp
Arg Glu Ile Asn Asn Tyr Thr Ser Ile Ile 130 135 140 Tyr Ser Leu Ile
Glu Glu Ser Gln Asn Gln Gln Glu Asn Gly Thr Gly 145 150 155 160 Gly
Gly Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile 165 170
175 Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln
180 185 190 Leu Gln Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn
Glu Arg 195 200 205 Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
Tyr Glu Lys Val 210 215 220 Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu
Ile Gly Asn Gly Cys Phe 225 230 235 240 Glu Phe Tyr His Lys Cys Asn
Asn Glu Cys Met Glu Ser Val Lys Asn 245 250 255 Gly Thr Tyr Asp Tyr
Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg 260 265 270 Glu Lys Ile
Asp Ser Gly Gly 275 72837DNAInfluenza virus 72gcctccggag tcgatcttct
ctctgttcag cttgctttcc tcgctgtact tggggtagtc 60gtaggtgccg ttcttcacgc
tttccatgca ctcgttgttg cacttgtggt agaactcgaa 120gcagccgttg
ccgatctctt tggcgttgtt cttcagctgg gacttcactt tctcgtacag
180gttcttcacg ttgctgtcgt ggaagtccag ggttctctcg ttttccagga
ggaccagcag 240ctcggcattg taggtctgca gctgcttgat gccccacact
gtcagctgca gcagatgctg 300ctgggcctca atggctctca gcaggttgtt
ctgctgctgc acaattccag atccgccgcc 360tgtgccgttt tcctgctggt
tctggctttc ctcgatcagg ctgtagatga tgctggtgta 420gttgttgatc
tctctgtccc attcaggatc gccgcccatc ttctcgatca cgctgttcac
480tttgttggtg atgccgttga tggcgttctg ggtagacttc tgatcggcgg
catagccaga 540gccctgctca ttctggtggt ggtagccgta ccacccatcc
accattccgg tccatccgcc 600ctcaataaag ccggcaatgg ctccaaacag
gcctctgctc tggatgctgg ggatgtttct 660caggccggtg accattctca
gtccggatcc caggttcacg ctgtgggtca cggtcacgtt 720cttttccagc
acggtatcca cggtgtcggt gctgttgttg gcgtggtagc caatgcagat
780ggtgtcggcg taggtggcgg taaaggtgca cagcagcacc agcagcttgg ccttcat
83773828DNAInfluenza virus 73atgaaggcta tcctggtggt gctgctgtac
acctttgcca ccgccaatgc cgacaccctg 60tgtattggct accacgccaa caacagcacc
gacaccgtgg ataccgtgct ggaaaagaac 120gtgaccgtga cccacagcgt
gaacctgggc tccggcctga gactggccac cggcctgaga 180aacatcccca
gcattcagag cagaggcctg tttggagcca ttgccggctt tattgagggc
240ggatggaccg gaatggtgga tgggtggtac ggctaccacc accagaatga
gcagggctct 300ggctatgccg ccgacctgaa gtctacccag aacgccatcg
acgagatcac caacaaagtg 360aacagcgtga tcgagaagat gggcggctgg
gacccatggg acagagagat caacaactac 420accagcatca tctacagcct
gatcgaggaa agccagaacc agcaggaaaa cggcacaggc 480ggcggatctg
gaattgtgca gcagcagaac aacctgctga gagccattga ggcccagcag
540catctgctgc agctgacagt gtggggcatc aagcagctgc agacctacaa
cgccgagctg 600ctggtgctgc tcgagaatga gagaaccctg gactaccacg
acagcaacgt gaagaacctg 660tacgagaaag tgcggagcca gctgaagaac
aacgccaaag agatcggcaa cggctgcttc 720gagttctacc acaagtgcga
caatacctgc atggaaagcg tgaagaacgg cacctacgac 780taccccaagt
acagcgagga agccaagctg aaccgggaag agatcgat 82874276PRTInfluenza
virus 74Met Lys Ala Ile Leu Val Val Leu Leu Tyr Thr Phe Ala Thr Ala
Asn 1 5 10 15 Ala Asp Thr Leu Cys Ile Gly Tyr His Ala Asn Asn Ser
Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val
Thr His Ser Val Asn 35 40 45 Leu Gly Ser Gly Leu Arg Leu Ala Thr
Gly Leu Arg Asn Ile Pro Ser 50 55 60 Ile Gln Ser Arg Gly Leu Phe
Gly Ala Ile Ala Gly Phe Ile Glu Gly 65 70 75 80 Gly Trp Thr Gly Met
Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn 85 90 95 Glu Gln Gly
Ser Gly Tyr Ala Ala Asp Leu Lys Ser Thr Gln Asn Ala 100 105 110 Ile
Asp Glu Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Gly 115 120
125 Gly Trp Asp Pro Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Ile Ile
130 135 140 Tyr Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Asn Gly
Thr Gly 145 150 155 160 Gly Gly Ser Gly Ile Val Gln Gln Gln Asn Asn
Leu Leu Arg Ala Ile 165 170 175 Glu Ala Gln Gln His Leu Leu Gln Leu
Thr Val Trp Gly Ile Lys Gln 180 185 190 Leu Gln Thr Tyr Asn Ala Glu
Leu Leu Val Leu Leu Glu Asn Glu Arg 195 200 205 Thr Leu Asp Tyr His
Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val 210 215 220 Arg Ser Gln
Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe 225 230 235 240
Glu Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys Asn 245
250 255 Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn
Arg 260 265 270 Glu Glu Ile Asp 275 75828DNAInfluenza virus
75atcgatctct tcccggttca gcttggcttc ctcgctgtac ttggggtagt cgtaggtgcc
60gttcttcacg ctttccatgc aggtattgtc gcacttgtgg tagaactcga agcagccgtt
120gccgatctct ttggcgttgt tcttcagctg gctccgcact ttctcgtaca
ggttcttcac 180gttgctgtcg tggtagtcca gggttctctc attctcgagc
agcaccagca gctcggcgtt 240gtaggtctgc agctgcttga tgccccacac
tgtcagctgc agcagatgct gctgggcctc 300aatggctctc agcaggttgt
tctgctgctg cacaattcca gatccgccgc ctgtgccgtt 360ttcctgctgg
ttctggcttt cctcgatcag gctgtagatg atgctggtgt agttgttgat
420ctctctgtcc catgggtccc agccgcccat cttctcgatc acgctgttca
ctttgttggt 480gatctcgtcg atggcgttct gggtagactt caggtcggcg
gcatagccag agccctgctc 540attctggtgg tggtagccgt accacccatc
caccattccg gtccatccgc cctcaataaa 600gccggcaatg gctccaaaca
ggcctctgct ctgaatgctg gggatgtttc tcaggccggt 660ggccagtctc
aggccggagc ccaggttcac gctgtgggtc acggtcacgt tcttttccag
720cacggtatcc acggtgtcgg tgctgttgtt ggcgtggtag ccaatacaca
gggtgtcggc 780attggcggtg gcaaaggtgt acagcagcac caccaggata gccttcat
82876822DNAInfluenza virus 76atggccatca tctacctgat tctgctgttt
acagccgtca gaggcgatca gatctgtatt 60ggctaccacg ccaacaatag caccgagaaa
gtggatacca tcctggaaag aaatgtgaca 120gtgacacacg ccaaggatat
tggatcagga ctggtgctgg ctacaggact gagaaatgtg 180cctcagattg
agagcagagg cctgtttgga gccattgctg gctttattga aggcggatgg
240cagggaatga ttgatgggtg gtacggctac caccactcta atgatcaggg
atctggatat 300gccgccgaca aagaatctac acagaaagcc ttcgacggca
tcaccaacaa agtgaatagc 360gtgatcgaga agatgggcgg agatcccgaa
tgggacagag agatcaacaa ctacaccagc 420atcatctaca gcctgatcga
ggaaagccag aatcagcagg aaaatggaac aggcggagga 480tctggaattg
tgcagcagca gaacaatctg ctgagagcta ttgaagctca gcagcatctg
540ctgaatctga cagtgtgggg aatcaaacag ctgcagacat acaatgctga
gctgctggtg 600ctgatggaaa atgagagaac cctggacttc cacgacagca
atgtgaagaa cctgtacgac 660aaagtgcgga tgcagctgag agacaatgtg
aaagaactgg gcaatggctg cttcgagttc 720taccacaagt gcgacgatga
gtgtatgaac agcgtgaaga acggcaccta cgactaccct 780aagtacgagg
aagagagcaa gctgaacaga aatgagatca ag 82277274PRTInfluenza virus
77Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr Ala Val Arg Gly Asp 1
5 10 15 Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val
Asp 20 25 30 Thr Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys
Asp Ile Gly 35 40 45 Ser Gly Leu Val Leu Ala Thr Gly Leu Arg Asn
Val Pro Gln Ile Glu 50 55 60 Ser Arg Gly Leu Phe Gly Ala Ile Ala
Gly Phe Ile Glu Gly Gly Trp 65 70 75 80 Gln Gly Met Ile Asp Gly Trp
Tyr Gly Tyr His His Ser Asn Asp Gln 85 90 95 Gly Ser Gly Tyr Ala
Ala Asp Lys Glu Ser Thr Gln Lys Ala Phe Asp 100 105 110 Gly Ile Thr
Asn Lys Val Asn Ser Val Ile Glu Lys Met Gly Gly Asp 115 120 125 Pro
Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Ile Ile Tyr Ser 130 135
140 Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Asn Gly Thr Gly Gly Gly
145 150 155 160 Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala
Ile Glu Ala 165 170 175 Gln Gln His Leu Leu Asn Leu Thr Val Trp Gly
Ile Lys Gln Leu Gln 180 185 190 Thr Tyr Asn Ala Glu Leu Leu Val Leu
Met Glu Asn Glu Arg Thr Leu 195 200 205 Asp Phe His Asp Ser Asn Val
Lys Asn Leu Tyr Asp Lys Val Arg Met 210 215 220 Gln Leu Arg Asp Asn
Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe 225 230 235 240 Tyr His
Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys Asn Gly Thr 245 250 255
Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu 260
265 270 Ile Lys 78822DNAInfluenza virus 78cttgatctca tttctgttca
gcttgctctc ttcctcgtac ttagggtagt cgtaggtgcc 60gttcttcacg ctgttcatac
actcatcgtc gcacttgtgg tagaactcga agcagccatt 120gcccagttct
ttcacattgt ctctcagctg catccgcact ttgtcgtaca ggttcttcac
180attgctgtcg tggaagtcca gggttctctc attttccatc agcaccagca
gctcagcatt 240gtatgtctgc agctgtttga ttccccacac tgtcagattc
agcagatgct gctgagcttc 300aatagctctc agcagattgt tctgctgctg
cacaattcca gatcctccgc ctgttccatt 360ttcctgctga ttctggcttt
cctcgatcag gctgtagatg atgctggtgt agttgttgat 420ctctctgtcc
cattcgggat ctccgcccat cttctcgatc acgctattca ctttgttggt
480gatgccgtcg aaggctttct gtgtagattc tttgtcggcg gcatatccag
atccctgatc 540attagagtgg tggtagccgt accacccatc aatcattccc
tgccatccgc cttcaataaa 600gccagcaatg gctccaaaca ggcctctgct
ctcaatctga ggcacatttc tcagtcctgt 660agccagcacc agtcctgatc
caatatcctt ggcgtgtgtc actgtcacat ttctttccag 720gatggtatcc
actttctcgg tgctattgtt ggcgtggtag ccaatacaga tctgatcgcc
780tctgacggct gtaaacagca gaatcaggta gatgatggcc at
82279858DNAInfluenza virus 79atgaagacca tcatcgccct gagctacatc
ttctgcctgg ccctgggcca ggacctgccc 60ggcaacgaca acagcaccgc caccctgtgc
ctgggccacc acgccgtgcc caacggcacc 120ctggtgaaga ccatcaccga
cgaccagatc gaggtgacca acgccaccga gctgggctcc 180ggcctgaagc
tggccaccgg catgcggaac gtgcccgaga agcagacccg gggcctgttc
240ggcgccatcg ccggcttcat cgagaacggc tgggagggca tgatcgacgg
ctggtacggc 300ttccggcacc agaacagcga gggcaccggc caggccgccg
acctgaagag cacccaggcc 360gccatcgacc agatcaacgg caagctgaac
cgggtgatcg agaagaccgg cggcgatccc 420gagtgggacc gggagatcaa
caactacacc agcatcatct acagcctgat cgaggagagc 480cagaaccagc
aggagaacgg caccggcggc ggcagcggca tcgtgcagca gcagaacaac
540ctgctgcggg ccatcgaggc ccagcagcac ctgctgcagc tgaccgtgtg
gggcatcaag 600cagctgcaga gctacaacgc cgagctgctg gtggccctgg
agaaccagca caccatcgac 660ctgaccgaca gcgagatgaa caagctgttc
gagaagaccc ggcggcagct gcgggagaac 720gccgaggaca tgggcaacgg
ctgcttcaag atctaccaca agtgcgacaa cgcctgcatc 780gagagcatcc
ggaacggcac ctacgaccac gacgtgtacc gggacgaggc cctgaacaac
840cggttccaga tcaagggc 85880286PRTInfluenza virus 80Met Lys Thr Ile
Ile Ala Leu Ser Tyr Ile Phe Cys Leu Ala Leu Gly 1 5 10 15 Gln Asp
Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly 20 25 30
His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asp Asp 35
40 45 Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Gly Ser Gly Leu Lys
Leu 50 55 60 Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg
Gly Leu Phe 65 70 75 80 Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp
Glu Gly Met Ile Asp 85 90 95 Gly Trp Tyr Gly Phe Arg His Gln Asn
Ser Glu Gly Thr Gly Gln Ala 100 105 110 Ala Asp Leu Lys Ser Thr Gln
Ala Ala Ile Asp Gln Ile Asn Gly Lys 115 120 125 Leu Asn Arg Val Ile
Glu Lys Thr Gly Gly Asp Pro Glu Trp Asp Arg 130 135 140 Glu Ile Asn
Asn Tyr Thr Ser Ile Ile Tyr Ser Leu Ile Glu Glu Ser 145 150 155 160
Gln Asn Gln Gln Glu Asn Gly Thr Gly Gly Gly Ser Gly Ile Val Gln 165
170 175 Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu
Leu 180 185 190 Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ser Tyr
Asn Ala Glu 195 200 205 Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile
Asp Leu Thr Asp Ser 210 215 220 Glu Met Asn Lys Leu Phe Glu Lys Thr
Arg Arg Gln Leu Arg Glu Asn 225 230 235 240 Ala Glu Asp Met Gly Asn
Gly Cys Phe Lys Ile Tyr His Lys Cys Asp 245 250 255 Asn Ala Cys Ile
Glu Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val 260 265 270 Tyr Arg
Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly 275 280 285
81858DNAInfluenza virus 81gcccttgatc tggaaccggt tgttcagggc
ctcgtcccgg tacacgtcgt ggtcgtaggt 60gccgttccgg atgctctcga tgcaggcgtt
gtcgcacttg tggtagatct tgaagcagcc 120gttgcccatg tcctcggcgt
tctcccgcag ctgccgccgg gtcttctcga acagcttgtt 180catctcgctg
tcggtcaggt cgatggtgtg ctggttctcc agggccacca gcagctcggc
240gttgtagctc tgcagctgct tgatgcccca cacggtcagc tgcagcaggt
gctgctgggc 300ctcgatggcc cgcagcaggt tgttctgctg ctgcacgatg
ccgctgccgc cgccggtgcc 360gttctcctgc tggttctggc tctcctcgat
caggctgtag atgatgctgg tgtagttgtt 420gatctcccgg tcccactcgg
gatcgccgcc ggtcttctcg atcacccggt tcagcttgcc 480gttgatctgg
tcgatggcgg cctgggtgct cttcaggtcg gcggcctggc cggtgccctc
540gctgttctgg tgccggaagc cgtaccagcc gtcgatcatg ccctcccagc
cgttctcgat 600gaagccggcg atggcgccga acaggccccg ggtctgcttc
tcgggcacgt tccgcatgcc 660ggtggccagc ttcaggccgg agcccagctc
ggtggcgttg gtcacctcga tctggtcgtc 720ggtgatggtc ttcaccaggg
tgccgttggg cacggcgtgg tggcccaggc acagggtggc 780ggtgctgttg
tcgttgccgg gcaggtcctg gcccagggcc aggcagaaga tgtagctcag
840ggcgatgatg gtcttcat 85882867DNAInfluenza virus 82atgaaaacca
tcattgccct gagctacatc ctgtgcctgg tgttcacaca gaagctgccc 60ggcaacgata
atagcaccgc cacactgtgt ctgggacacc
acgccgtgcc taatggcacc 120atcgtgaaaa caatcaccaa cgaccagatc
gaagtgacca atgccacaga gctgggctcc 180ggcctgaagc tggccaccgg
catgagaaat gtgcccgaga agcagaccag aggcatcttt 240ggcgccattg
ccggctttat cgagaatggc tgggagggaa tggtggatgg gtggtacggc
300ttcagacacc agaatagcga gggaattgga caggccgccg atctgaaatc
tacccaggcc 360gccatcgacc agatcaacgg caagctgaac aggctgatcg
gcaagaccgg cggcgatccc 420gagtgggacc gggagatcaa caactacacc
agcatcatct acagcctgat cgaggagagc 480cagaaccagc aggagaacgg
caccggcggc ggcagcggca tcgtgcagca gcagaacaac 540ctgctgcggg
ccatcgaggc ccagcagcac ctgctgcagc tgaccgtgtg gggcatcaag
600cagctgcaga gctacaatgc cgaactgctg gtcgccctgg aaaaccagca
cacaattgat 660ctgacagaca gtgagatgaa taagctgttc gagaaaacca
agaagcagct gagagaaaac 720gccgaggaca tgggcaacgg ctgcttcaag
atctaccaca agtgcgacaa cgcctgcatc 780ggcagcatca gaaacggcac
ctacgaccac gacgtgtaca gagatgaggc cctgaacaac 840cggtttcaga
tcaagggctc cggaggc 86783289PRTInfluenza virus 83Met Lys Thr Ile Ile
Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Thr 1 5 10 15 Gln Lys Leu
Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly 20 25 30 His
His Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile Thr Asn Asp 35 40
45 Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Gly Ser Gly Leu Lys Leu
50 55 60 Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg Gly
Ile Phe 65 70 75 80 Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu
Gly Met Val Asp 85 90 95 Gly Trp Tyr Gly Phe Arg His Gln Asn Ser
Glu Gly Ile Gly Gln Ala 100 105 110 Ala Asp Leu Lys Ser Thr Gln Ala
Ala Ile Asp Gln Ile Asn Gly Lys 115 120 125 Leu Asn Arg Leu Ile Gly
Lys Thr Gly Gly Asp Pro Glu Trp Asp Arg 130 135 140 Glu Ile Asn Asn
Tyr Thr Ser Ile Ile Tyr Ser Leu Ile Glu Glu Ser 145 150 155 160 Gln
Asn Gln Gln Glu Asn Gly Thr Gly Gly Gly Ser Gly Ile Val Gln 165 170
175 Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu
180 185 190 Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln Ser Tyr Asn
Ala Glu 195 200 205 Leu Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp
Leu Thr Asp Ser 210 215 220 Glu Met Asn Lys Leu Phe Glu Lys Thr Lys
Lys Gln Leu Arg Glu Asn 225 230 235 240 Ala Glu Asp Met Gly Asn Gly
Cys Phe Lys Ile Tyr His Lys Cys Asp 245 250 255 Asn Ala Cys Ile Gly
Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val 260 265 270 Tyr Arg Asp
Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Ser Gly 275 280 285 Gly
84867DNAInfluenza virus 84gcctccggag cccttgatct gaaaccggtt
gttcagggcc tcatctctgt acacgtcgtg 60gtcgtaggtg ccgtttctga tgctgccgat
gcaggcgttg tcgcacttgt ggtagatctt 120gaagcagccg ttgcccatgt
cctcggcgtt ttctctcagc tgcttcttgg ttttctcgaa 180cagcttattc
atctcactgt ctgtcagatc aattgtgtgc tggttttcca gggcgaccag
240cagttcggca ttgtagctct gcagctgctt gatgccccac acggtcagct
gcagcaggtg 300ctgctgggcc tcgatggccc gcagcaggtt gttctgctgc
tgcacgatgc cgctgccgcc 360gccggtgccg ttctcctgct ggttctggct
ctcctcgatc aggctgtaga tgatgctggt 420gtagttgttg atctcccggt
cccactcggg atcgccgccg gtcttgccga tcagcctgtt 480cagcttgccg
ttgatctggt cgatggcggc ctgggtagat ttcagatcgg cggcctgtcc
540aattccctcg ctattctggt gtctgaagcc gtaccaccca tccaccattc
cctcccagcc 600attctcgata aagccggcaa tggcgccaaa gatgcctctg
gtctgcttct cgggcacatt 660tctcatgccg gtggccagct tcaggccgga
gcccagctct gtggcattgg tcacttcgat 720ctggtcgttg gtgattgttt
tcacgatggt gccattaggc acggcgtggt gtcccagaca 780cagtgtggcg
gtgctattat cgttgccggg cagcttctgt gtgaacacca ggcacaggat
840gtagctcagg gcaatgatgg ttttcat 86785837DNAInfluenza virus
85atggagaaga tcgtgctgct gctggccatc gtgagcctgg tgaagagcga ccagatctgc
60atcggctacc acgccaacaa cagcaccgag caggtggaca ccatcatgga gaagaacgtg
120accgtgaccc acgcccagga catcggctcc ggcctggtgc tggccaccgg
cctgcggaac 180agcccccagc gggagagccg gcggaagaag cggggcctgt
tcggcgccat cgccggcttc 240atcgagggcg gctggcaggg catggtggac
ggctggtacg gctaccacca cagcaacgag 300cagggcagcg gctacgccgc
cgacaaggag agcacccaga aggccatcga cggcgtgacc 360aacaaggtga
acagcatcat cgacaagatg ggcggcgatc ccgagtggga ccgggagatc
420aacaactaca ccagcatcat ctacagcctg atcgaggaga gccagaacca
gcaggagaac 480ggcaccggcg gcggcagcgg catcgtgcag cagcagaaca
acctgctgcg ggccatcgag 540gcccagcagc acctgctgca gctgaccgtg
tggggcatca agcagctgca gacctacaac 600gccgagctgc tggtgctgat
ggagaacgag cggaccctgg acttccacga cagcaacgtg 660aagaacctgt
acgacaaggt gcggctgcag ctgcgggaca acgccaagga gctgggcaac
720ggctgcttcg agttctacca caagtgcgac aacgagtgca tggagagcat
ccggaacggc 780acctacaact acccccagta cagcgaggag gcccggctga
agcgggagga gatcagc 83786279PRTInfluenza virus 86Met Glu Lys Ile Val
Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser 1 5 10 15 Asp Gln Ile
Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30 Asp
Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40
45 Gly Ser Gly Leu Val Leu Ala Thr Gly Leu Arg Asn Ser Pro Gln Arg
50 55 60 Glu Ser Arg Arg Lys Lys Arg Gly Leu Phe Gly Ala Ile Ala
Gly Phe 65 70 75 80 Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp
Tyr Gly Tyr His 85 90 95 His Ser Asn Glu Gln Gly Ser Gly Tyr Ala
Ala Asp Lys Glu Ser Thr 100 105 110 Gln Lys Ala Ile Asp Gly Val Thr
Asn Lys Val Asn Ser Ile Ile Asp 115 120 125 Lys Met Gly Gly Asp Pro
Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr 130 135 140 Ser Ile Ile Tyr
Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Asn 145 150 155 160 Gly
Thr Gly Gly Gly Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu 165 170
175 Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly
180 185 190 Ile Lys Gln Leu Gln Thr Tyr Asn Ala Glu Leu Leu Val Leu
Met Glu 195 200 205 Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu Tyr 210 215 220 Asp Lys Val Arg Leu Gln Leu Arg Asp Asn
Ala Lys Glu Leu Gly Asn 225 230 235 240 Gly Cys Phe Glu Phe Tyr His
Lys Cys Asp Asn Glu Cys Met Glu Ser 245 250 255 Ile Arg Asn Gly Thr
Tyr Asn Tyr Pro Gln Tyr Ser Glu Glu Ala Arg 260 265 270 Leu Lys Arg
Glu Glu Ile Ser 275 87837DNAInfluenza virus 87gctgatctcc tcccgcttca
gccgggcctc ctcgctgtac tgggggtagt tgtaggtgcc 60gttccggatg ctctccatgc
actcgttgtc gcacttgtgg tagaactcga agcagccgtt 120gcccagctcc
ttggcgttgt cccgcagctg cagccgcacc ttgtcgtaca ggttcttcac
180gttgctgtcg tggaagtcca gggtccgctc gttctccatc agcaccagca
gctcggcgtt 240gtaggtctgc agctgcttga tgccccacac ggtcagctgc
agcaggtgct gctgggcctc 300gatggcccgc agcaggttgt tctgctgctg
cacgatgccg ctgccgccgc cggtgccgtt 360ctcctgctgg ttctggctct
cctcgatcag gctgtagatg atgctggtgt agttgttgat 420ctcccggtcc
cactcgggat cgccgcccat cttgtcgatg atgctgttca ccttgttggt
480cacgccgtcg atggccttct gggtgctctc cttgtcggcg gcgtagccgc
tgccctgctc 540gttgctgtgg tggtagccgt accagccgtc caccatgccc
tgccagccgc cctcgatgaa 600gccggcgatg gcgccgaaca ggccccgctt
cttccgccgg ctctcccgct gggggctgtt 660ccgcaggccg gtggccagca
ccaggccgga gccgatgtcc tgggcgtggg tcacggtcac 720gttcttctcc
atgatggtgt ccacctgctc ggtgctgttg ttggcgtggt agccgatgca
780gatctggtcg ctcttcacca ggctcacgat ggccagcagc agcacgatct tctccat
83788837DNAInfluenza virus 88atgaaggcca tcatcgtgct gctgatggtg
gtgaccagca acgccgatag aatctgcacc 60ggcatcacca gcagcaatag cccccatgtg
gtgaaaacag ccacccaggg cgaagtgaat 120gtgacaggcg tgatccctct
gggatcagga ctgaagctgg ccaatggcac caagtacaga 180cctcccgcca
agctgctgaa agagagaggc ttctttggcg ccattgccgg atttctggaa
240ggcggctggg agggaatgat tgccggctgg cacggctata catctcatgg
ggcccatggc 300gtggctgtgg ccgccgatct gaagtctacc caggaagcca
tcaacaagat caccaagaac 360ctgaacagcc tgagcgagct ggaaggaggc
gaccccgagt gggatcgcga aatcaacaac 420tacacatcta tcatctacag
tctgattgag gaaagccaga accagcagga gaatgggact 480gggggaggct
ccggaatcgt gcagcagcag aacaatctgc tgcgagccat tgaagctcag
540cagcacctgc tgcagctgac agtgtggggc atcaagcagc tgcaggggtc
ccagattgaa 600ctggccgtgc tgctgtccaa cgagggcatc atcaacagcg
aggatgaaca cctgctggcc 660ctggaacgga agctgaagaa gatgctgggc
ccttctgccg tggagatcgg caacggctgc 720ttcgagacaa agcacaagtg
caaccagacc tgcctggata gaatcgccgc tggcaccttc 780aatgccggcg
agttcagcct gcctaccttc gacagcctga atatcacctc cggaggc
83789279PRTInfluenza virus 89Met Lys Ala Ile Ile Val Leu Leu Met
Val Val Thr Ser Asn Ala Asp 1 5 10 15 Arg Ile Cys Thr Gly Ile Thr
Ser Ser Asn Ser Pro His Val Val Lys 20 25 30 Thr Ala Thr Gln Gly
Glu Val Asn Val Thr Gly Val Ile Pro Leu Gly 35 40 45 Ser Gly Leu
Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys 50 55 60 Leu
Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu 65 70
75 80 Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser
His 85 90 95 Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser
Thr Gln Glu 100 105 110 Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser
Leu Ser Glu Leu Glu 115 120 125 Gly Gly Asp Pro Glu Trp Asp Arg Glu
Ile Asn Asn Tyr Thr Ser Ile 130 135 140 Ile Tyr Ser Leu Ile Glu Glu
Ser Gln Asn Gln Gln Glu Asn Gly Thr 145 150 155 160 Gly Gly Gly Ser
Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala 165 170 175 Ile Glu
Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys 180 185 190
Gln Leu Gln Gly Ser Gln Ile Glu Leu Ala Val Leu Leu Ser Asn Glu 195
200 205 Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg
Lys 210 215 220 Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly
Asn Gly Cys 225 230 235 240 Phe Glu Thr Lys His Lys Cys Asn Gln Thr
Cys Leu Asp Arg Ile Ala 245 250 255 Ala Gly Thr Phe Asn Ala Gly Glu
Phe Ser Leu Pro Thr Phe Asp Ser 260 265 270 Leu Asn Ile Thr Ser Gly
Gly 275 90837DNAInfluenza virus 90gcctccggag gtgatattca ggctgtcgaa
ggtaggcagg ctgaactcgc cggcattgaa 60ggtgccagcg gcgattctat ccaggcaggt
ctggttgcac ttgtgctttg tctcgaagca 120gccgttgccg atctccacgg
cagaagggcc cagcatcttc ttcagcttcc gttccagggc 180cagcaggtgt
tcatcctcgc tgttgatgat gccctcgttg gacagcagca cggccagttc
240aatctgggac ccctgcagct gcttgatgcc ccacactgtc agctgcagca
ggtgctgctg 300agcttcaatg gctcgcagca gattgttctg ctgctgcacg
attccggagc ctcccccagt 360cccattctcc tgctggttct ggctttcctc
aatcagactg tagatgatag atgtgtagtt 420gttgatttcg cgatcccact
cggggtcgcc tccttccagc tcgctcaggc tgttcaggtt 480cttggtgatc
ttgttgatgg cttcctgggt agacttcaga tcggcggcca cagccacgcc
540atgggcccca tgagatgtat agccgtgcca gccggcaatc attccctccc
agccgccttc 600cagaaatccg gcaatggcgc caaagaagcc tctctctttc
agcagcttgg cgggaggtct 660gtacttggtg ccattggcca gcttcagtcc
tgatcccaga gggatcacgc ctgtcacatt 720cacttcgccc tgggtggctg
ttttcaccac atgggggcta ttgctgctgg tgatgccggt 780gcagattcta
tcggcgttgc tggtcaccac catcagcagc acgatgatgg ccttcat
83791867DNAInfluenza virus 91atgaaaacca taattgcgct gtcctacata
ctgtgtctgg tgtttgccca gaaactgccg 60ggcaatgaca actcaacagc cacgctctgc
ttggggcacc atgccgtccc taacgggacc 120attgtgaaaa ccattactaa
cgatcagata gaggtgacta atgccaccga gctgggctcc 180ggcttgaaac
tggcgaccgg tatgcgcaat gtccccgaaa aacagacccg cgggatattt
240ggggctatcg caggctttat cgagaatggc tgggaaggga tggtggatgg
ttggtatggt 300tttagacatc aaaactccga aggcagaggc caggctgccg
atctcaagag cacgcaggcc 360gctatagatc agatcaatgg aaagctcaac
agactgatcg ggaaaaccgg cggcgatccc 420gagtgggacc gggagatcaa
caactacacc agcatcatct acagcctgat cgaggagagc 480cagaaccagc
aggagaacgg caccggcggc ggcagcggca tcgtgcagca gcagaacaac
540ctgctgcggg ccatcgaggc ccagcagcac ctgctgcagc tgaccgtgtg
gggcatcaag 600cagctgcagt cctacaatgc cgagctgctg gtggctctgg
agaatcagca cactattgac 660ctgaccgatt cagagatgaa caaacttttt
gagaagacga agaagcagct tagagaaaat 720gcagaggaca tggggaacgg
atgctttaaa atatatcata agtgtgataa tgcctgcatc 780ggatcaatta
gaaatggtac ctatgatcac gatgtttaca gggacgaagc gctgaataac
840aggttccaga taaaaggctc cggaggc 86792289PRTInfluenza virus 92Met
Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Ala 1 5 10
15 Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly
20 25 30 His His Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile Thr
Asn Asp 35 40 45 Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Gly Ser
Gly Leu Lys Leu 50 55 60 Ala Thr Gly Met Arg Asn Val Pro Glu Lys
Gln Thr Arg Gly Ile Phe 65 70 75 80 Gly Ala Ile Ala Gly Phe Ile Glu
Asn Gly Trp Glu Gly Met Val Asp 85 90 95 Gly Trp Tyr Gly Phe Arg
His Gln Asn Ser Glu Gly Arg Gly Gln Ala 100 105 110 Ala Asp Leu Lys
Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys 115 120 125 Leu Asn
Arg Leu Ile Gly Lys Thr Gly Gly Asp Pro Glu Trp Asp Arg 130 135 140
Glu Ile Asn Asn Tyr Thr Ser Ile Ile Tyr Ser Leu Ile Glu Glu Ser 145
150 155 160 Gln Asn Gln Gln Glu Asn Gly Thr Gly Gly Gly Ser Gly Ile
Val Gln 165 170 175 Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln
Gln His Leu Leu 180 185 190 Gln Leu Thr Val Trp Gly Ile Lys Gln Leu
Gln Ser Tyr Asn Ala Glu 195 200 205 Leu Leu Val Ala Leu Glu Asn Gln
His Thr Ile Asp Leu Thr Asp Ser 210 215 220 Glu Met Asn Lys Leu Phe
Glu Lys Thr Lys Lys Gln Leu Arg Glu Asn 225 230 235 240 Ala Glu Asp
Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp 245 250 255 Asn
Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val 260 265
270 Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Ser Gly
275 280 285 Gly 93867DNAInfluenza virus 93gcctccggag ccttttatct
ggaacctgtt attcagcgct tcgtccctgt aaacatcgtg 60atcataggta ccatttctaa
ttgatccgat gcaggcatta tcacacttat gatatatttt 120aaagcatccg
ttccccatgt cctctgcatt ttctctaagc tgcttcttcg tcttctcaaa
180aagtttgttc atctctgaat cggtcaggtc aatagtgtgc tgattctcca
gagccaccag 240cagctcggca ttgtaggact gcagctgctt gatgccccac
acggtcagct gcagcaggtg 300ctgctgggcc tcgatggccc gcagcaggtt
gttctgctgc tgcacgatgc cgctgccgcc 360gccggtgccg ttctcctgct
ggttctggct ctcctcgatc aggctgtaga tgatgctggt 420gtagttgttg
atctcccggt cccactcggg atcgccgccg gttttcccga tcagtctgtt
480gagctttcca ttgatctgat ctatagcggc ctgcgtgctc ttgagatcgg
cagcctggcc 540tctgccttcg gagttttgat gtctaaaacc ataccaacca
tccaccatcc cttcccagcc 600attctcgata aagcctgcga tagccccaaa
tatcccgcgg gtctgttttt cggggacatt 660gcgcataccg gtcgccagtt
tcaagccgga gcccagctcg gtggcattag tcacctctat 720ctgatcgtta
gtaatggttt tcacaatggt cccgttaggg acggcatggt gccccaagca
780gagcgtggct gttgagttgt cattgcccgg cagtttctgg gcaaacacca
gacacagtat 840gtaggacagc gcaattatgg ttttcat 86794837DNAInfluenza
virus 94atgaaagtga agctgctggt gctgctgtgt acctttaccg ccacctacgc
cgataccatc 60tgtatcggct accacgccaa caatagcacc gacaccgtgg ataccgtgct
ggaaaagaac 120gtgaccgtga cccacagcgt gaacctggga tcaggactga
gaatggtgac cggcctgagg 180aatatcccca gcatccagag cagaggcctg
tttggcgcca ttgccggctt tatcgagggc 240ggatggacag gcatggtgga
tgggtggtac ggctaccacc accagaatga gcagggatct 300ggctatgccg
ccgatcagaa gagcacccag aacgccatca acggcatcac caacaaagtg
360aacagcgtga tcgagaagat gggcggcgat cctgaatggg acagagagat
caacaactac 420accagcatca tctacagcct gatcgaggaa agccagaacc
agcaggaaaa cggcacaggc 480ggcggatctg gaattgtgca gcagcagaac
aacctgctga gagccattga ggcccagcag 540catctgctgc agctgacagt
gtggggcatc aagcagctgc agacctacaa cgccgaactc 600ctggtcctcc
tggaaaatga gaggaccctg gacttccacg acagcaacgt gaagaacctg
660tacgagaaag tgaagagcca gctgaagaac aacgccaaag agatcggcaa
cggctgcttc 720gagttctacc acaagtgcaa cgacgagtgc atggaaagcg
tgaagaacgg cacctacgac 780taccccaagt acagcgagga aagcaagctg
aaccgggaga agatcgattc cggaggc 83795276PRTInfluenza virus 95Met Lys
Val Lys Leu Leu Val Leu Leu Cys Thr Phe Thr Ala Thr Tyr 1 5 10 15
Ala Asp Thr Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20
25 30 Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val
Asn 35 40 45 Leu Gly Ser Gly Leu Arg Met Val Thr Gly Leu Arg Asn
Ile Pro Ser 50 55 60 Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala
Gly Phe Ile Glu Gly 65 70 75 80 Gly Trp Thr Gly Met Val Asp Gly Trp
Tyr Gly Tyr His His Gln Asn 85 90 95 Glu Gln Gly Ser Gly Tyr Ala
Ala Asp Gln Lys Ser Thr Gln Asn Ala 100 105 110 Ile Asn Gly Ile Thr
Asn Lys Val Asn Ser Val Ile Glu Lys Met Gly 115 120 125 Gly Asp Pro
Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser Ile Ile 130 135 140 Tyr
Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Asn Gly Thr Gly 145 150
155 160 Gly Gly Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala
Ile 165 170 175 Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly
Ile Lys Gln 180 185 190 Leu Gln Thr Tyr Asn Ala Glu Leu Leu Val Leu
Leu Glu Asn Glu Arg 195 200 205 Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu Tyr Glu Lys Val 210 215 220 Lys Ser Gln Leu Lys Asn Asn
Ala Lys Glu Ile Gly Asn Gly Cys Phe 225 230 235 240 Glu Phe Tyr His
Lys Cys Asn Asp Glu Cys Met Glu Ser Val Lys Asn 245 250 255 Gly Thr
Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg 260 265 270
Glu Lys Ile Asp 275 96837DNAInfluenza virus 96gcctccggaa tcgatcttct
cccggttcag cttgctttcc tcgctgtact tggggtagtc 60gtaggtgccg ttcttcacgc
tttccatgca ctcgtcgttg cacttgtggt agaactcgaa 120gcagccgttg
ccgatctctt tggcgttgtt cttcagctgg ctcttcactt tctcgtacag
180gttcttcacg ttgctgtcgt ggaagtccag ggtcctctca ttttccagga
ggaccaggag 240ttcggcgttg taggtctgca gctgcttgat gccccacact
gtcagctgca gcagatgctg 300ctgggcctca atggctctca gcaggttgtt
ctgctgctgc acaattccag atccgccgcc 360tgtgccgttt tcctgctggt
tctggctttc ctcgatcagg ctgtagatga tgctggtgta 420gttgttgatc
tctctgtccc attcaggatc gccgcccatc ttctcgatca cgctgttcac
480tttgttggtg atgccgttga tggcgttctg ggtgctcttc tgatcggcgg
catagccaga 540tccctgctca ttctggtggt ggtagccgta ccacccatcc
accatgcctg tccatccgcc 600ctcgataaag ccggcaatgg cgccaaacag
gcctctgctc tggatgctgg ggatattcct 660caggccggtc accattctca
gtcctgatcc caggttcacg ctgtgggtca cggtcacgtt 720cttttccagc
acggtatcca cggtgtcggt gctattgttg gcgtggtagc cgatacagat
780ggtatcggcg taggtggcgg taaaggtaca cagcagcacc agcagcttca ctttcat
83797837DNAInfluenza virus 97atgaaggcca tcatcgtgct gctgatggtg
gtcacaagca acgccgatag aatctgtacc 60ggcatcacca gcagcaatag ccctcacgtc
gtgaaaacag ctacacaggg cgaagtgaat 120gtgaccggcg tgatccctct
gggatcagga ctgaagctgg ccaatggcac aaagtataga 180cctccagcca
agctgctgaa agagagaggc ttttttggag ctatcgccgg ctttctggaa
240ggcggatggg agggaatgat tgctggatgg catggctaca catctcatgg
cgcacatggc 300gtggcagtgg ctgctgatct gaaatctaca caggaagcca
tcaacaagat caccaagaac 360ctgaacagcc tgagcgagct ggaaggaggc
gaccccgagt gggatcgcga aatcaacaac 420tacacatcta tcatctacag
tctgattgag gaaagccaga accagcagga gaatgggact 480gggggaggct
ccggaatcgt gcagcagcag aacaatctgc tgcgagccat tgaagctcag
540cagcacctgc tgcagctgac agtgtggggc atcaagcagc tgcaggggag
ccagattgaa 600ctggctgtgc tgctgtctaa cgagggcatc atcaatagcg
aggacgaaca tctgctggcc 660ctggaaagaa agctgaagaa gatgctggga
cctagcgccg tggaaatcgg caatggatgc 720tttgagacaa agcacaagtg
caaccagacc tgcctggata gaattgccgc cggaacattt 780gatgccggcg
agttttctct gcccaccttc gatagcctga atatcacatc cggaggc
83798279PRTInfluenza virus 98Met Lys Ala Ile Ile Val Leu Leu Met
Val Val Thr Ser Asn Ala Asp 1 5 10 15 Arg Ile Cys Thr Gly Ile Thr
Ser Ser Asn Ser Pro His Val Val Lys 20 25 30 Thr Ala Thr Gln Gly
Glu Val Asn Val Thr Gly Val Ile Pro Leu Gly 35 40 45 Ser Gly Leu
Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys 50 55 60 Leu
Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu 65 70
75 80 Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser
His 85 90 95 Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser
Thr Gln Glu 100 105 110 Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser
Leu Ser Glu Leu Glu 115 120 125 Gly Gly Asp Pro Glu Trp Asp Arg Glu
Ile Asn Asn Tyr Thr Ser Ile 130 135 140 Ile Tyr Ser Leu Ile Glu Glu
Ser Gln Asn Gln Gln Glu Asn Gly Thr 145 150 155 160 Gly Gly Gly Ser
Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala 165 170 175 Ile Glu
Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys 180 185 190
Gln Leu Gln Gly Ser Gln Ile Glu Leu Ala Val Leu Leu Ser Asn Glu 195
200 205 Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg
Lys 210 215 220 Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly
Asn Gly Cys 225 230 235 240 Phe Glu Thr Lys His Lys Cys Asn Gln Thr
Cys Leu Asp Arg Ile Ala 245 250 255 Ala Gly Thr Phe Asp Ala Gly Glu
Phe Ser Leu Pro Thr Phe Asp Ser 260 265 270 Leu Asn Ile Thr Ser Gly
Gly 275 99837DNAInfluenza virus 99gcctccggat gtgatattca ggctatcgaa
ggtgggcaga gaaaactcgc cggcatcaaa 60tgttccggcg gcaattctat ccaggcaggt
ctggttgcac ttgtgctttg tctcaaagca 120tccattgccg atttccacgg
cgctaggtcc cagcatcttc ttcagctttc tttccagggc 180cagcagatgt
tcgtcctcgc tattgatgat gccctcgtta gacagcagca cagccagttc
240aatctggctc ccctgcagct gcttgatgcc ccacactgtc agctgcagca
ggtgctgctg 300agcttcaatg gctcgcagca gattgttctg ctgctgcacg
attccggagc ctcccccagt 360cccattctcc tgctggttct ggctttcctc
aatcagactg tagatgatag atgtgtagtt 420gttgatttcg cgatcccact
cggggtcgcc tccttccagc tcgctcaggc tgttcaggtt 480cttggtgatc
ttgttgatgg cttcctgtgt agatttcaga tcagcagcca ctgccacgcc
540atgtgcgcca tgagatgtgt agccatgcca tccagcaatc attccctccc
atccgccttc 600cagaaagccg gcgatagctc caaaaaagcc tctctctttc
agcagcttgg ctggaggtct 660atactttgtg ccattggcca gcttcagtcc
tgatcccaga gggatcacgc cggtcacatt 720cacttcgccc tgtgtagctg
ttttcacgac gtgagggcta ttgctgctgg tgatgccggt 780acagattcta
tcggcgttgc ttgtgaccac catcagcagc acgatgatgg ccttcat
8371001332DNAArtificial SequenceSynthetic 100atgaaggcca agctgctggt
gctgctgtgc acctttaccg ccacctacgc cgacaccatc 60tgcattggct accacgccaa
caacagcacc gacaccgtgg ataccgtgct ggaaaagaac 120gtgaccgtga
cccacagcgt gaacctggga tccggactga gaatggtcac cggcctgaga
180aacatcccca gcatccagag cagaggcctg tttggagcca ttgccggctt
tattgagggc 240ggatggaccg gaatggtgga tgggtggtac ggctaccacc
accagaatga gcagggctct 300ggctatgccg ccgatcagaa gtctacccag
aacgccatca acggcatcac caacaaagtg 360aacagcgtga tcgagaagat
gggcggcgat cctgaatggg acagagagat caacaactac 420accagcatca
tctacagcct gatcgaggaa agccagaacc agcaggaaaa cggcacaggc
480ggcggatctg gaattgtgca gcagcagaac aacctgctga gagccattga
ggcccagcag 540catctgctgc agctgacagt gtggggcatc aagcagctgc
agacctacaa tgccgagctg 600ctggtcctcc tggaaaacga gagaaccctg
gacttccacg acagcaacgt gaagaacctg 660tacgagaaag tgaagtccca
gctgaagaac aacgccaaag agatcggcaa cggctgcttc 720gagttctacc
acaagtgcaa caacgagtgc atggaaagcg tgaagaacgg cacctacgac
780taccccaagt acagcgagga aagcaagctg aacagagaga agatcgactc
cggaggcgac 840atcatcaagc tgctgaacga gcaggtgaac aaggagatgc
agagcagcaa cctgtacatg 900agcatgagca gctggtgcta cacccacagc
ctggacggcg ccggcctgtt cctgttcgac 960cacgccgccg aggagtacga
gcacgccaag aagctgatca tcttcctgaa cgagaacaac 1020gtgcccgtgc
agctgaccag catcagcgcc cccgagcaca agttcgaggg cctgacccag
1080atcttccaga aggcctacga gcacgagcag cacatcagcg agagcatcaa
caacatcgtg 1140gaccacgcca tcaagagcaa ggaccacgcc accttcaact
tcctgcagtg gtacgtggcc 1200gagcagcacg aggaggaggt gctgttcaag
gacatcctgg acaagatcga gctgatcggc 1260aacgagaacc acggcctgta
cctggccgac cagtacgtga agggcatcgc caagagcagg 1320aagagcggat cc
1332101444PRTArtificial SequenceSynthetic 101Met Lys Ala Lys Leu
Leu Val Leu Leu Cys Thr Phe Thr Ala Thr Tyr 1 5 10 15 Ala Asp Thr
Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val
Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40
45 Leu Gly Ser Gly Leu Arg Met Val Thr Gly Leu Arg Asn Ile Pro Ser
50 55 60 Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile
Glu Gly 65 70 75 80 Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr
His His Gln Asn 85 90 95 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln
Lys Ser Thr Gln Asn Ala 100 105 110 Ile Asn Gly Ile Thr Asn Lys Val
Asn Ser Val Ile Glu Lys Met Gly 115 120 125 Gly Asp Pro Glu Trp Asp
Arg Glu Ile Asn Asn Tyr Thr Ser Ile Ile 130 135 140 Tyr Ser Leu Ile
Glu Glu Ser Gln Asn Gln Gln Glu Asn Gly Thr Gly 145 150 155 160 Gly
Gly Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile 165 170
175 Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln
180 185 190 Leu Gln Thr Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn
Glu Arg 195 200 205 Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu
Tyr Glu Lys Val 210 215 220 Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu
Ile Gly Asn Gly Cys Phe 225 230 235 240 Glu Phe Tyr His Lys Cys Asn
Asn Glu Cys Met Glu Ser Val Lys Asn 245 250 255 Gly Thr Tyr Asp Tyr
Pro Lys Tyr Ser Glu Glu Ser Lys Leu Asn Arg 260 265 270 Glu Lys Ile
Asp Ser Gly Gly Asp Ile Ile Lys Leu Leu Asn Glu Gln 275 280 285 Val
Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser Ser 290 295
300 Trp Cys Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp
305 310 315 320 His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile
Ile Phe Leu 325 330 335 Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser
Ile Ser Ala Pro Glu 340 345 350 His Lys Phe Glu Gly Leu Thr Gln Ile
Phe Gln Lys Ala Tyr Glu His 355 360 365 Glu Gln His Ile Ser Glu Ser
Ile Asn Asn Ile Val Asp His Ala Ile 370 375 380 Lys Ser Lys Asp His
Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala 385 390 395 400 Glu Gln
His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys Ile 405 410 415
Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln Tyr 420
425 430 Val Lys Gly Ile Ala Lys Ser Arg Lys Ser Gly Ser 435 440
1021332DNAArtificial SequenceSynthetic 102ggatccgctc ttcctgctct
tggcgatgcc cttcacgtac tggtcggcca ggtacaggcc 60gtggttctcg ttgccgatca
gctcgatctt gtccaggatg tccttgaaca gcacctcctc 120ctcgtgctgc
tcggccacgt accactgcag gaagttgaag gtggcgtggt ccttgctctt
180gatggcgtgg tccacgatgt tgttgatgct ctcgctgatg tgctgctcgt
gctcgtaggc 240cttctggaag atctgggtca ggccctcgaa cttgtgctcg
ggggcgctga tgctggtcag 300ctgcacgggc acgttgttct cgttcaggaa
gatgatcagc ttcttggcgt gctcgtactc 360ctcggcggcg tggtcgaaca
ggaacaggcc ggcgccgtcc aggctgtggg tgtagcacca 420gctgctcatg
ctcatgtaca ggttgctgct ctgcatctcc ttgttcacct gctcgttcag
480cagcttgatg atgtcgcctc cggagtcgat cttctctctg ttcagcttgc
tttcctcgct 540gtacttgggg tagtcgtagg tgccgttctt cacgctttcc
atgcactcgt tgttgcactt 600gtggtagaac tcgaagcagc cgttgccgat
ctctttggcg ttgttcttca gctgggactt 660cactttctcg tacaggttct
tcacgttgct gtcgtggaag tccagggttc tctcgttttc 720caggaggacc
agcagctcgg cattgtaggt ctgcagctgc ttgatgcccc acactgtcag
780ctgcagcaga tgctgctggg cctcaatggc tctcagcagg ttgttctgct
gctgcacaat 840tccagatccg ccgcctgtgc cgttttcctg ctggttctgg
ctttcctcga tcaggctgta 900gatgatgctg gtgtagttgt tgatctctct
gtcccattca ggatcgccgc ccatcttctc 960gatcacgctg ttcactttgt
tggtgatgcc gttgatggcg ttctgggtag acttctgatc 1020ggcggcatag
ccagagccct gctcattctg gtggtggtag ccgtaccacc catccaccat
1080tccggtccat ccgccctcaa taaagccggc aatggctcca aacaggcctc
tgctctggat 1140gctggggatg tttctcaggc cggtgaccat tctcagtccg
gatcccaggt tcacgctgtg 1200ggtcacggtc acgttctttt ccagcacggt
atccacggtg tcggtgctgt tgttggcgtg 1260gtagccaatg cagatggtgt
cggcgtaggt ggcggtaaag gtgcacagca gcaccagcag 1320cttggccttc at
13321031332DNAArtificial SequenceSynthetic 103atgaaggcta tcctggtggt
gctgctgtac acctttgcca ccgccaatgc cgacaccctg 60tgtattggct accacgccaa
caacagcacc gacaccgtgg ataccgtgct ggaaaagaac 120gtgaccgtga
cccacagcgt gaacctgggc tccggcctga gactggccac cggcctgaga
180aacatcccca gcattcagag cagaggcctg tttggagcca ttgccggctt
tattgagggc 240ggatggaccg gaatggtgga tgggtggtac ggctaccacc
accagaatga gcagggctct 300ggctatgccg ccgacctgaa gtctacccag
aacgccatcg acgagatcac caacaaagtg 360aacagcgtga tcgagaagat
gggcggctgg gacccatggg acagagagat caacaactac 420accagcatca
tctacagcct gatcgaggaa agccagaacc agcaggaaaa cggcacaggc
480ggcggatctg gaattgtgca gcagcagaac aacctgctga gagccattga
ggcccagcag 540catctgctgc agctgacagt gtggggcatc aagcagctgc
agacctacaa cgccgagctg 600ctggtgctgc tcgagaatga gagaaccctg
gactaccacg acagcaacgt gaagaacctg 660tacgagaaag tgcggagcca
gctgaagaac aacgccaaag agatcggcaa cggctgcttc 720gagttctacc
acaagtgcga caatacctgc atggaaagcg tgaagaacgg cacctacgac
780taccccaagt acagcgagga agccaagctg aaccgggaag agatcgattc
cggaggcgac 840atcatcaagc tgctgaacga gcaggtgaac aaggagatgc
agagcagcaa cctgtacatg 900agcatgagca gctggtgcta cacccacagc
ctggacggcg ccggcctgtt cctgttcgac 960cacgccgccg aggagtacga
gcacgccaag aagctgatca tcttcctgaa cgagaacaac 1020gtgcccgtgc
agctgaccag catcagcgcc cccgagcaca agttcgaggg cctgacccag
1080atcttccaga aggcctacga gcacgagcag cacatcagcg agagcatcaa
caacatcgtg 1140gaccacgcca tcaagagcaa ggaccacgcc accttcaact
tcctgcagtg gtacgtggcc 1200gagcagcacg aggaggaggt gctgttcaag
gacatcctgg acaagatcga gctgatcggc 1260aacgagaacc acggcctgta
cctggccgac cagtacgtga agggcatcgc caagagcagg 1320aagagcggat cc
1332104444PRTArtificial SequenceSynthetic 104Met Lys Ala Ile Leu
Val Val Leu Leu Tyr Thr Phe Ala Thr Ala Asn 1 5 10 15 Ala Asp Thr
Leu Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val
Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser Val Asn 35 40
45 Leu Gly Ser Gly Leu Arg Leu Ala Thr Gly Leu Arg Asn Ile Pro Ser
50 55 60 Ile Gln Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile
Glu Gly 65 70 75 80 Gly Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr
His His Gln Asn 85 90 95 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Leu
Lys Ser Thr Gln Asn Ala 100 105 110 Ile Asp Glu Ile Thr Asn Lys Val
Asn Ser Val Ile Glu Lys Met Gly 115 120 125 Gly Trp Asp Pro Trp Asp
Arg Glu Ile Asn Asn Tyr Thr Ser Ile Ile 130 135 140 Tyr Ser Leu Ile
Glu Glu Ser Gln Asn Gln Gln Glu Asn Gly Thr Gly 145 150 155 160 Gly
Gly Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala Ile 165 170
175 Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln
180 185 190 Leu Gln Thr Tyr Asn Ala Glu Leu
Leu Val Leu Leu Glu Asn Glu Arg 195 200 205 Thr Leu Asp Tyr His Asp
Ser Asn Val Lys Asn Leu Tyr Glu Lys Val 210 215 220 Arg Ser Gln Leu
Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe 225 230 235 240 Glu
Phe Tyr His Lys Cys Asp Asn Thr Cys Met Glu Ser Val Lys Asn 245 250
255 Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Lys Leu Asn Arg
260 265 270 Glu Glu Ile Asp Ser Gly Gly Asp Ile Ile Lys Leu Leu Asn
Glu Gln 275 280 285 Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met
Ser Met Ser Ser 290 295 300 Trp Cys Tyr Thr His Ser Leu Asp Gly Ala
Gly Leu Phe Leu Phe Asp 305 310 315 320 His Ala Ala Glu Glu Tyr Glu
His Ala Lys Lys Leu Ile Ile Phe Leu 325 330 335 Asn Glu Asn Asn Val
Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu 340 345 350 His Lys Phe
Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His 355 360 365 Glu
Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala Ile 370 375
380 Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala
385 390 395 400 Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu
Asp Lys Ile 405 410 415 Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr
Leu Ala Asp Gln Tyr 420 425 430 Val Lys Gly Ile Ala Lys Ser Arg Lys
Ser Gly Ser 435 440 1051332DNAArtificial SequenceSynthetic
105ggatccgctc ttcctgctct tggcgatgcc cttcacgtac tggtcggcca
ggtacaggcc 60gtggttctcg ttgccgatca gctcgatctt gtccaggatg tccttgaaca
gcacctcctc 120ctcgtgctgc tcggccacgt accactgcag gaagttgaag
gtggcgtggt ccttgctctt 180gatggcgtgg tccacgatgt tgttgatgct
ctcgctgatg tgctgctcgt gctcgtaggc 240cttctggaag atctgggtca
ggccctcgaa cttgtgctcg ggggcgctga tgctggtcag 300ctgcacgggc
acgttgttct cgttcaggaa gatgatcagc ttcttggcgt gctcgtactc
360ctcggcggcg tggtcgaaca ggaacaggcc ggcgccgtcc aggctgtggg
tgtagcacca 420gctgctcatg ctcatgtaca ggttgctgct ctgcatctcc
ttgttcacct gctcgttcag 480cagcttgatg atgtcgcctc cggaatcgat
ctcttcccgg ttcagcttgg cttcctcgct 540gtacttgggg tagtcgtagg
tgccgttctt cacgctttcc atgcaggtat tgtcgcactt 600gtggtagaac
tcgaagcagc cgttgccgat ctctttggcg ttgttcttca gctggctccg
660cactttctcg tacaggttct tcacgttgct gtcgtggtag tccagggttc
tctcattctc 720gagcagcacc agcagctcgg cgttgtaggt ctgcagctgc
ttgatgcccc acactgtcag 780ctgcagcaga tgctgctggg cctcaatggc
tctcagcagg ttgttctgct gctgcacaat 840tccagatccg ccgcctgtgc
cgttttcctg ctggttctgg ctttcctcga tcaggctgta 900gatgatgctg
gtgtagttgt tgatctctct gtcccatggg tcccagccgc ccatcttctc
960gatcacgctg ttcactttgt tggtgatctc gtcgatggcg ttctgggtag
acttcaggtc 1020ggcggcatag ccagagccct gctcattctg gtggtggtag
ccgtaccacc catccaccat 1080tccggtccat ccgccctcaa taaagccggc
aatggctcca aacaggcctc tgctctgaat 1140gctggggatg tttctcaggc
cggtggccag tctcaggccg gagcccaggt tcacgctgtg 1200ggtcacggtc
acgttctttt ccagcacggt atccacggtg tcggtgctgt tgttggcgtg
1260gtagccaata cacagggtgt cggcattggc ggtggcaaag gtgtacagca
gcaccaccag 1320gatagccttc at 13321061326DNAArtificial
SequenceSynthetic 106atggccatca tctacctgat tctgctgttt acagccgtca
gaggcgatca gatctgtatt 60ggctaccacg ccaacaatag caccgagaaa gtggatacca
tcctggaaag aaatgtgaca 120gtgacacacg ccaaggatat tggatcagga
ctggtgctgg ctacaggact gagaaatgtg 180cctcagattg agagcagagg
cctgtttgga gccattgctg gctttattga aggcggatgg 240cagggaatga
ttgatgggtg gtacggctac caccactcta atgatcaggg atctggatat
300gccgccgaca aagaatctac acagaaagcc ttcgacggca tcaccaacaa
agtgaatagc 360gtgatcgaga agatgggcgg agatcccgaa tgggacagag
agatcaacaa ctacaccagc 420atcatctaca gcctgatcga ggaaagccag
aatcagcagg aaaatggaac aggcggagga 480tctggaattg tgcagcagca
gaacaatctg ctgagagcta ttgaagctca gcagcatctg 540ctgaatctga
cagtgtgggg aatcaaacag ctgcagacat acaatgctga gctgctggtg
600ctgatggaaa atgagagaac cctggacttc cacgacagca atgtgaagaa
cctgtacgac 660aaagtgcgga tgcagctgag agacaatgtg aaagaactgg
gcaatggctg cttcgagttc 720taccacaagt gcgacgatga gtgtatgaac
agcgtgaaga acggcaccta cgactaccct 780aagtacgagg aagagagcaa
gctgaacaga aatgagatca agtccggagg cgacatcatc 840aagctgctga
acgagcaggt gaacaaggag atgcagagca gcaacctgta catgagcatg
900agcagctggt gctacaccca cagcctggac ggcgccggcc tgttcctgtt
cgaccacgcc 960gccgaggagt acgagcacgc caagaagctg atcatcttcc
tgaacgagaa caacgtgccc 1020gtgcagctga ccagcatcag cgcccccgag
cacaagttcg agggcctgac ccagatcttc 1080cagaaggcct acgagcacga
gcagcacatc agcgagagca tcaacaacat cgtggaccac 1140gccatcaaga
gcaaggacca cgccaccttc aacttcctgc agtggtacgt ggccgagcag
1200cacgaggagg aggtgctgtt caaggacatc ctggacaaga tcgagctgat
cggcaacgag 1260aaccacggcc tgtacctggc cgaccagtac gtgaagggca
tcgccaagag caggaagagc 1320ggatcc 1326107442PRTArtificial
SequenceSynthetic 107Met Ala Ile Ile Tyr Leu Ile Leu Leu Phe Thr
Ala Val Arg Gly Asp 1 5 10 15 Gln Ile Cys Ile Gly Tyr His Ala Asn
Asn Ser Thr Glu Lys Val Asp 20 25 30 Thr Ile Leu Glu Arg Asn Val
Thr Val Thr His Ala Lys Asp Ile Gly 35 40 45 Ser Gly Leu Val Leu
Ala Thr Gly Leu Arg Asn Val Pro Gln Ile Glu 50 55 60 Ser Arg Gly
Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp 65 70 75 80 Gln
Gly Met Ile Asp Gly Trp Tyr Gly Tyr His His Ser Asn Asp Gln 85 90
95 Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Phe Asp
100 105 110 Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Gly
Gly Asp 115 120 125 Pro Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr Ser
Ile Ile Tyr Ser 130 135 140 Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu
Asn Gly Thr Gly Gly Gly 145 150 155 160 Ser Gly Ile Val Gln Gln Gln
Asn Asn Leu Leu Arg Ala Ile Glu Ala 165 170 175 Gln Gln His Leu Leu
Asn Leu Thr Val Trp Gly Ile Lys Gln Leu Gln 180 185 190 Thr Tyr Asn
Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu 195 200 205 Asp
Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met 210 215
220 Gln Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe
225 230 235 240 Tyr His Lys Cys Asp Asp Glu Cys Met Asn Ser Val Lys
Asn Gly Thr 245 250 255 Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys
Leu Asn Arg Asn Glu 260 265 270 Ile Lys Ser Gly Gly Asp Ile Ile Lys
Leu Leu Asn Glu Gln Val Asn 275 280 285 Lys Glu Met Gln Ser Ser Asn
Leu Tyr Met Ser Met Ser Ser Trp Cys 290 295 300 Tyr Thr His Ser Leu
Asp Gly Ala Gly Leu Phe Leu Phe Asp His Ala 305 310 315 320 Ala Glu
Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu Asn Glu 325 330 335
Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu His Lys 340
345 350 Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His Glu
Gln 355 360 365 His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala
Ile Lys Ser 370 375 380 Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp
Tyr Val Ala Glu Gln 385 390 395 400 His Glu Glu Glu Val Leu Phe Lys
Asp Ile Leu Asp Lys Ile Glu Leu 405 410 415 Ile Gly Asn Glu Asn His
Gly Leu Tyr Leu Ala Asp Gln Tyr Val Lys 420 425 430 Gly Ile Ala Lys
Ser Arg Lys Ser Gly Ser 435 440 1081326DNAArtificial
SequenceSynthetic 108ggatccgctc ttcctgctct tggcgatgcc cttcacgtac
tggtcggcca ggtacaggcc 60gtggttctcg ttgccgatca gctcgatctt gtccaggatg
tccttgaaca gcacctcctc 120ctcgtgctgc tcggccacgt accactgcag
gaagttgaag gtggcgtggt ccttgctctt 180gatggcgtgg tccacgatgt
tgttgatgct ctcgctgatg tgctgctcgt gctcgtaggc 240cttctggaag
atctgggtca ggccctcgaa cttgtgctcg ggggcgctga tgctggtcag
300ctgcacgggc acgttgttct cgttcaggaa gatgatcagc ttcttggcgt
gctcgtactc 360ctcggcggcg tggtcgaaca ggaacaggcc ggcgccgtcc
aggctgtggg tgtagcacca 420gctgctcatg ctcatgtaca ggttgctgct
ctgcatctcc ttgttcacct gctcgttcag 480cagcttgatg atgtcgcctc
cggacttgat ctcatttctg ttcagcttgc tctcttcctc 540gtacttaggg
tagtcgtagg tgccgttctt cacgctgttc atacactcat cgtcgcactt
600gtggtagaac tcgaagcagc cattgcccag ttctttcaca ttgtctctca
gctgcatccg 660cactttgtcg tacaggttct tcacattgct gtcgtggaag
tccagggttc tctcattttc 720catcagcacc agcagctcag cattgtatgt
ctgcagctgt ttgattcccc acactgtcag 780attcagcaga tgctgctgag
cttcaatagc tctcagcaga ttgttctgct gctgcacaat 840tccagatcct
ccgcctgttc cattttcctg ctgattctgg ctttcctcga tcaggctgta
900gatgatgctg gtgtagttgt tgatctctct gtcccattcg ggatctccgc
ccatcttctc 960gatcacgcta ttcactttgt tggtgatgcc gtcgaaggct
ttctgtgtag attctttgtc 1020ggcggcatat ccagatccct gatcattaga
gtggtggtag ccgtaccacc catcaatcat 1080tccctgccat ccgccttcaa
taaagccagc aatggctcca aacaggcctc tgctctcaat 1140ctgaggcaca
tttctcagtc ctgtagccag caccagtcct gatccaatat ccttggcgtg
1200tgtcactgtc acatttcttt ccaggatggt atccactttc tcggtgctat
tgttggcgtg 1260gtagccaata cagatctgat cgcctctgac ggctgtaaac
agcagaatca ggtagatgat 1320ggccat 13261091362DNAArtificial
SequenceSynthetic 109atgaagacca tcatcgccct gagctacatc ttctgcctgg
ccctgggcca ggacctgccc 60ggcaacgaca acagcaccgc caccctgtgc ctgggccacc
acgccgtgcc caacggcacc 120ctggtgaaga ccatcaccga cgaccagatc
gaggtgacca acgccaccga gctgggctcc 180ggcctgaagc tggccaccgg
catgcggaac gtgcccgaga agcagacccg gggcctgttc 240ggcgccatcg
ccggcttcat cgagaacggc tgggagggca tgatcgacgg ctggtacggc
300ttccggcacc agaacagcga gggcaccggc caggccgccg acctgaagag
cacccaggcc 360gccatcgacc agatcaacgg caagctgaac cgggtgatcg
agaagaccgg cggcgatccc 420gagtgggacc gggagatcaa caactacacc
agcatcatct acagcctgat cgaggagagc 480cagaaccagc aggagaacgg
caccggcggc ggcagcggca tcgtgcagca gcagaacaac 540ctgctgcggg
ccatcgaggc ccagcagcac ctgctgcagc tgaccgtgtg gggcatcaag
600cagctgcaga gctacaacgc cgagctgctg gtggccctgg agaaccagca
caccatcgac 660ctgaccgaca gcgagatgaa caagctgttc gagaagaccc
ggcggcagct gcgggagaac 720gccgaggaca tgggcaacgg ctgcttcaag
atctaccaca agtgcgacaa cgcctgcatc 780gagagcatcc ggaacggcac
ctacgaccac gacgtgtacc gggacgaggc cctgaacaac 840cggttccaga
tcaagggctc cggaggcgac atcatcaagc tgctgaacga gcaggtgaac
900aaggagatgc agagcagcaa cctgtacatg agcatgagca gctggtgcta
cacccacagc 960ctggacggcg ccggcctgtt cctgttcgac cacgccgccg
aggagtacga gcacgccaag 1020aagctgatca tcttcctgaa cgagaacaac
gtgcccgtgc agctgaccag catcagcgcc 1080cccgagcaca agttcgaggg
cctgacccag atcttccaga aggcctacga gcacgagcag 1140cacatcagcg
agagcatcaa caacatcgtg gaccacgcca tcaagagcaa ggaccacgcc
1200accttcaact tcctgcagtg gtacgtggcc gagcagcacg aggaggaggt
gctgttcaag 1260gacatcctgg acaagatcga gctgatcggc aacgagaacc
acggcctgta cctggccgac 1320cagtacgtga agggcatcgc caagagcagg
aagagcggat cc 1362110454PRTArtificial SequenceSynthetic 110Met Lys
Thr Ile Ile Ala Leu Ser Tyr Ile Phe Cys Leu Ala Leu Gly 1 5 10 15
Gln Asp Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly 20
25 30 His His Ala Val Pro Asn Gly Thr Leu Val Lys Thr Ile Thr Asp
Asp 35 40 45 Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Gly Ser Gly
Leu Lys Leu 50 55 60 Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln
Thr Arg Gly Leu Phe 65 70 75 80 Gly Ala Ile Ala Gly Phe Ile Glu Asn
Gly Trp Glu Gly Met Ile Asp 85 90 95 Gly Trp Tyr Gly Phe Arg His
Gln Asn Ser Glu Gly Thr Gly Gln Ala 100 105 110 Ala Asp Leu Lys Ser
Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys 115 120 125 Leu Asn Arg
Val Ile Glu Lys Thr Gly Gly Asp Pro Glu Trp Asp Arg 130 135 140 Glu
Ile Asn Asn Tyr Thr Ser Ile Ile Tyr Ser Leu Ile Glu Glu Ser 145 150
155 160 Gln Asn Gln Gln Glu Asn Gly Thr Gly Gly Gly Ser Gly Ile Val
Gln 165 170 175 Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln
His Leu Leu 180 185 190 Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln
Ser Tyr Asn Ala Glu 195 200 205 Leu Leu Val Ala Leu Glu Asn Gln His
Thr Ile Asp Leu Thr Asp Ser 210 215 220 Glu Met Asn Lys Leu Phe Glu
Lys Thr Arg Arg Gln Leu Arg Glu Asn 225 230 235 240 Ala Glu Asp Met
Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp 245 250 255 Asn Ala
Cys Ile Glu Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val 260 265 270
Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Ser Gly 275
280 285 Gly Asp Ile Ile Lys Leu Leu Asn Glu Gln Val Asn Lys Glu Met
Gln 290 295 300 Ser Ser Asn Leu Tyr Met Ser Met Ser Ser Trp Cys Tyr
Thr His Ser 305 310 315 320 Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp
His Ala Ala Glu Glu Tyr 325 330 335 Glu His Ala Lys Lys Leu Ile Ile
Phe Leu Asn Glu Asn Asn Val Pro 340 345 350 Val Gln Leu Thr Ser Ile
Ser Ala Pro Glu His Lys Phe Glu Gly Leu 355 360 365 Thr Gln Ile Phe
Gln Lys Ala Tyr Glu His Glu Gln His Ile Ser Glu 370 375 380 Ser Ile
Asn Asn Ile Val Asp His Ala Ile Lys Ser Lys Asp His Ala 385 390 395
400 Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala Glu Gln His Glu Glu Glu
405 410 415 Val Leu Phe Lys Asp Ile Leu Asp Lys Ile Glu Leu Ile Gly
Asn Glu 420 425 430 Asn His Gly Leu Tyr Leu Ala Asp Gln Tyr Val Lys
Gly Ile Ala Lys 435 440 445 Ser Arg Lys Ser Gly Ser 450
1111362DNAArtificial SequenceSynthetic 111ggatccgctc ttcctgctct
tggcgatgcc cttcacgtac tggtcggcca ggtacaggcc 60gtggttctcg ttgccgatca
gctcgatctt gtccaggatg tccttgaaca gcacctcctc 120ctcgtgctgc
tcggccacgt accactgcag gaagttgaag gtggcgtggt ccttgctctt
180gatggcgtgg tccacgatgt tgttgatgct ctcgctgatg tgctgctcgt
gctcgtaggc 240cttctggaag atctgggtca ggccctcgaa cttgtgctcg
ggggcgctga tgctggtcag 300ctgcacgggc acgttgttct cgttcaggaa
gatgatcagc ttcttggcgt gctcgtactc 360ctcggcggcg tggtcgaaca
ggaacaggcc ggcgccgtcc aggctgtggg tgtagcacca 420gctgctcatg
ctcatgtaca ggttgctgct ctgcatctcc ttgttcacct gctcgttcag
480cagcttgatg atgtcgcctc cggagccctt gatctggaac cggttgttca
gggcctcgtc 540ccggtacacg tcgtggtcgt aggtgccgtt ccggatgctc
tcgatgcagg cgttgtcgca 600cttgtggtag atcttgaagc agccgttgcc
catgtcctcg gcgttctccc gcagctgccg 660ccgggtcttc tcgaacagct
tgttcatctc gctgtcggtc aggtcgatgg tgtgctggtt 720ctccagggcc
accagcagct cggcgttgta gctctgcagc tgcttgatgc cccacacggt
780cagctgcagc aggtgctgct gggcctcgat ggcccgcagc aggttgttct
gctgctgcac 840gatgccgctg ccgccgccgg tgccgttctc ctgctggttc
tggctctcct cgatcaggct 900gtagatgatg ctggtgtagt tgttgatctc
ccggtcccac tcgggatcgc cgccggtctt 960ctcgatcacc cggttcagct
tgccgttgat ctggtcgatg gcggcctggg tgctcttcag 1020gtcggcggcc
tggccggtgc cctcgctgtt ctggtgccgg aagccgtacc agccgtcgat
1080catgccctcc cagccgttct cgatgaagcc ggcgatggcg ccgaacaggc
cccgggtctg 1140cttctcgggc acgttccgca tgccggtggc cagcttcagg
ccggagccca gctcggtggc 1200gttggtcacc tcgatctggt cgtcggtgat
ggtcttcacc agggtgccgt tgggcacggc 1260gtggtggccc aggcacaggg
tggcggtgct gttgtcgttg ccgggcaggt cctggcccag 1320ggccaggcag
aagatgtagc tcagggcgat gatggtcttc at 13621121341DNAArtificial
SequenceSynthetic 112atggagaaga tcgtgctgct gctggccatc gtgagcctgg
tgaagagcga ccagatctgc 60atcggctacc acgccaacaa cagcaccgag caggtggaca
ccatcatgga gaagaacgtg 120accgtgaccc acgcccagga catcggctcc
ggcctggtgc tggccaccgg cctgcggaac 180agcccccagc gggagagccg
gcggaagaag cggggcctgt tcggcgccat cgccggcttc 240atcgagggcg
gctggcaggg catggtggac ggctggtacg gctaccacca cagcaacgag
300cagggcagcg gctacgccgc cgacaaggag agcacccaga aggccatcga
cggcgtgacc 360aacaaggtga acagcatcat
cgacaagatg ggcggcgatc ccgagtggga ccgggagatc 420aacaactaca
ccagcatcat ctacagcctg atcgaggaga gccagaacca gcaggagaac
480ggcaccggcg gcggcagcgg catcgtgcag cagcagaaca acctgctgcg
ggccatcgag 540gcccagcagc acctgctgca gctgaccgtg tggggcatca
agcagctgca gacctacaac 600gccgagctgc tggtgctgat ggagaacgag
cggaccctgg acttccacga cagcaacgtg 660aagaacctgt acgacaaggt
gcggctgcag ctgcgggaca acgccaagga gctgggcaac 720ggctgcttcg
agttctacca caagtgcgac aacgagtgca tggagagcat ccggaacggc
780acctacaact acccccagta cagcgaggag gcccggctga agcgggagga
gatcagctcc 840ggaggcgaca tcatcaagct gctgaacgag caggtgaaca
aggagatgca gagcagcaac 900ctgtacatga gcatgagcag ctggtgctac
acccacagcc tggacggcgc cggcctgttc 960ctgttcgacc acgccgccga
ggagtacgag cacgccaaga agctgatcat cttcctgaac 1020gagaacaacg
tgcccgtgca gctgaccagc atcagcgccc ccgagcacaa gttcgagggc
1080ctgacccaga tcttccagaa ggcctacgag cacgagcagc acatcagcga
gagcatcaac 1140aacatcgtgg accacgccat caagagcaag gaccacgcca
ccttcaactt cctgcagtgg 1200tacgtggccg agcagcacga ggaggaggtg
ctgttcaagg acatcctgga caagatcgag 1260ctgatcggca acgagaacca
cggcctgtac ctggccgacc agtacgtgaa gggcatcgcc 1320aagagcagga
agagcggatc c 1341113447PRTArtificial SequenceSynthetic 113Met Glu
Lys Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser 1 5 10 15
Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 20
25 30 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp
Ile 35 40 45 Gly Ser Gly Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
Pro Gln Arg 50 55 60 Glu Ser Arg Arg Lys Lys Arg Gly Leu Phe Gly
Ala Ile Ala Gly Phe 65 70 75 80 Ile Glu Gly Gly Trp Gln Gly Met Val
Asp Gly Trp Tyr Gly Tyr His 85 90 95 His Ser Asn Glu Gln Gly Ser
Gly Tyr Ala Ala Asp Lys Glu Ser Thr 100 105 110 Gln Lys Ala Ile Asp
Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp 115 120 125 Lys Met Gly
Gly Asp Pro Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr 130 135 140 Ser
Ile Ile Tyr Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln Glu Asn 145 150
155 160 Gly Thr Gly Gly Gly Ser Gly Ile Val Gln Gln Gln Asn Asn Leu
Leu 165 170 175 Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr
Val Trp Gly 180 185 190 Ile Lys Gln Leu Gln Thr Tyr Asn Ala Glu Leu
Leu Val Leu Met Glu 195 200 205 Asn Glu Arg Thr Leu Asp Phe His Asp
Ser Asn Val Lys Asn Leu Tyr 210 215 220 Asp Lys Val Arg Leu Gln Leu
Arg Asp Asn Ala Lys Glu Leu Gly Asn 225 230 235 240 Gly Cys Phe Glu
Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu Ser 245 250 255 Ile Arg
Asn Gly Thr Tyr Asn Tyr Pro Gln Tyr Ser Glu Glu Ala Arg 260 265 270
Leu Lys Arg Glu Glu Ile Ser Ser Gly Gly Asp Ile Ile Lys Leu Leu 275
280 285 Asn Glu Gln Val Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met
Ser 290 295 300 Met Ser Ser Trp Cys Tyr Thr His Ser Leu Asp Gly Ala
Gly Leu Phe 305 310 315 320 Leu Phe Asp His Ala Ala Glu Glu Tyr Glu
His Ala Lys Lys Leu Ile 325 330 335 Ile Phe Leu Asn Glu Asn Asn Val
Pro Val Gln Leu Thr Ser Ile Ser 340 345 350 Ala Pro Glu His Lys Phe
Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala 355 360 365 Tyr Glu His Glu
Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp 370 375 380 His Ala
Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp 385 390 395
400 Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu
405 410 415 Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr
Leu Ala 420 425 430 Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys
Ser Gly Ser 435 440 445 1141341DNAArtificial SequenceSynthetic
114ggatccgctc ttcctgctct tggcgatgcc cttcacgtac tggtcggcca
ggtacaggcc 60gtggttctcg ttgccgatca gctcgatctt gtccaggatg tccttgaaca
gcacctcctc 120ctcgtgctgc tcggccacgt accactgcag gaagttgaag
gtggcgtggt ccttgctctt 180gatggcgtgg tccacgatgt tgttgatgct
ctcgctgatg tgctgctcgt gctcgtaggc 240cttctggaag atctgggtca
ggccctcgaa cttgtgctcg ggggcgctga tgctggtcag 300ctgcacgggc
acgttgttct cgttcaggaa gatgatcagc ttcttggcgt gctcgtactc
360ctcggcggcg tggtcgaaca ggaacaggcc ggcgccgtcc aggctgtggg
tgtagcacca 420gctgctcatg ctcatgtaca ggttgctgct ctgcatctcc
ttgttcacct gctcgttcag 480cagcttgatg atgtcgcctc cggagctgat
ctcctcccgc ttcagccggg cctcctcgct 540gtactggggg tagttgtagg
tgccgttccg gatgctctcc atgcactcgt tgtcgcactt 600gtggtagaac
tcgaagcagc cgttgcccag ctccttggcg ttgtcccgca gctgcagccg
660caccttgtcg tacaggttct tcacgttgct gtcgtggaag tccagggtcc
gctcgttctc 720catcagcacc agcagctcgg cgttgtaggt ctgcagctgc
ttgatgcccc acacggtcag 780ctgcagcagg tgctgctggg cctcgatggc
ccgcagcagg ttgttctgct gctgcacgat 840gccgctgccg ccgccggtgc
cgttctcctg ctggttctgg ctctcctcga tcaggctgta 900gatgatgctg
gtgtagttgt tgatctcccg gtcccactcg ggatcgccgc ccatcttgtc
960gatgatgctg ttcaccttgt tggtcacgcc gtcgatggcc ttctgggtgc
tctccttgtc 1020ggcggcgtag ccgctgccct gctcgttgct gtggtggtag
ccgtaccagc cgtccaccat 1080gccctgccag ccgccctcga tgaagccggc
gatggcgccg aacaggcccc gcttcttccg 1140ccggctctcc cgctgggggc
tgttccgcag gccggtggcc agcaccaggc cggagccgat 1200gtcctgggcg
tgggtcacgg tcacgttctt ctccatgatg gtgtccacct gctcggtgct
1260gttgttggcg tggtagccga tgcagatctg gtcgctcttc accaggctca
cgatggccag 1320cagcagcacg atcttctcca t 13411151332DNAArtificial
SequenceSynthetic 115atgaaagtga agctgctggt gctgctgtgt acctttaccg
ccacctacgc cgataccatc 60tgtatcggct accacgccaa caatagcacc gacaccgtgg
ataccgtgct ggaaaagaac 120gtgaccgtga cccacagcgt gaacctggga
tcaggactga gaatggtgac cggcctgagg 180aatatcccca gcatccagag
cagaggcctg tttggcgcca ttgccggctt tatcgagggc 240ggatggacag
gcatggtgga tgggtggtac ggctaccacc accagaatga gcagggatct
300ggctatgccg ccgatcagaa gagcacccag aacgccatca acggcatcac
caacaaagtg 360aacagcgtga tcgagaagat gggcggcgat cctgaatggg
acagagagat caacaactac 420accagcatca tctacagcct gatcgaggaa
agccagaacc agcaggaaaa cggcacaggc 480ggcggatctg gaattgtgca
gcagcagaac aacctgctga gagccattga ggcccagcag 540catctgctgc
agctgacagt gtggggcatc aagcagctgc agacctacaa cgccgaactc
600ctggtcctcc tggaaaatga gaggaccctg gacttccacg acagcaacgt
gaagaacctg 660tacgagaaag tgaagagcca gctgaagaac aacgccaaag
agatcggcaa cggctgcttc 720gagttctacc acaagtgcaa cgacgagtgc
atggaaagcg tgaagaacgg cacctacgac 780taccccaagt acagcgagga
aagcaagctg aaccgggaga agatcgattc cggaggcgac 840atcatcaagc
tgctgaacga gcaggtgaac aaggagatgc agagcagcaa cctgtacatg
900agcatgagca gctggtgcta cacccacagc ctggacggcg ccggcctgtt
cctgttcgac 960cacgccgccg aggagtacga gcacgccaag aagctgatca
tcttcctgaa cgagaacaac 1020gtgcccgtgc agctgaccag catcagcgcc
cccgagcaca agttcgaggg cctgacccag 1080atcttccaga aggcctacga
gcacgagcag cacatcagcg agagcatcaa caacatcgtg 1140gaccacgcca
tcaagagcaa ggaccacgcc accttcaact tcctgcagtg gtacgtggcc
1200gagcagcacg aggaggaggt gctgttcaag gacatcctgg acaagatcga
gctgatcggc 1260aacgagaacc acggcctgta cctggccgac cagtacgtga
agggcatcgc caagagcagg 1320aagagcggat cc 1332116444PRTArtificial
SequenceSynthetic 116Met Lys Val Lys Leu Leu Val Leu Leu Cys Thr
Phe Thr Ala Thr Tyr 1 5 10 15 Ala Asp Thr Ile Cys Ile Gly Tyr His
Ala Asn Asn Ser Thr Asp Thr 20 25 30 Val Asp Thr Val Leu Glu Lys
Asn Val Thr Val Thr His Ser Val Asn 35 40 45 Leu Gly Ser Gly Leu
Arg Met Val Thr Gly Leu Arg Asn Ile Pro Ser 50 55 60 Ile Gln Ser
Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly 65 70 75 80 Gly
Trp Thr Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Gln Asn 85 90
95 Glu Gln Gly Ser Gly Tyr Ala Ala Asp Gln Lys Ser Thr Gln Asn Ala
100 105 110 Ile Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys
Met Gly 115 120 125 Gly Asp Pro Glu Trp Asp Arg Glu Ile Asn Asn Tyr
Thr Ser Ile Ile 130 135 140 Tyr Ser Leu Ile Glu Glu Ser Gln Asn Gln
Gln Glu Asn Gly Thr Gly 145 150 155 160 Gly Gly Ser Gly Ile Val Gln
Gln Gln Asn Asn Leu Leu Arg Ala Ile 165 170 175 Glu Ala Gln Gln His
Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln 180 185 190 Leu Gln Thr
Tyr Asn Ala Glu Leu Leu Val Leu Leu Glu Asn Glu Arg 195 200 205 Thr
Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Glu Lys Val 210 215
220 Lys Ser Gln Leu Lys Asn Asn Ala Lys Glu Ile Gly Asn Gly Cys Phe
225 230 235 240 Glu Phe Tyr His Lys Cys Asn Asp Glu Cys Met Glu Ser
Val Lys Asn 245 250 255 Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu
Ser Lys Leu Asn Arg 260 265 270 Glu Lys Ile Asp Ser Gly Gly Asp Ile
Ile Lys Leu Leu Asn Glu Gln 275 280 285 Val Asn Lys Glu Met Gln Ser
Ser Asn Leu Tyr Met Ser Met Ser Ser 290 295 300 Trp Cys Tyr Thr His
Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp 305 310 315 320 His Ala
Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu 325 330 335
Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu 340
345 350 His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu
His 355 360 365 Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp
His Ala Ile 370 375 380 Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu
Gln Trp Tyr Val Ala 385 390 395 400 Glu Gln His Glu Glu Glu Val Leu
Phe Lys Asp Ile Leu Asp Lys Ile 405 410 415 Glu Leu Ile Gly Asn Glu
Asn His Gly Leu Tyr Leu Ala Asp Gln Tyr 420 425 430 Val Lys Gly Ile
Ala Lys Ser Arg Lys Ser Gly Ser 435 440 1171332DNAArtificial
SequenceSynthetic 117ggatccgctc ttcctgctct tggcgatgcc cttcacgtac
tggtcggcca ggtacaggcc 60gtggttctcg ttgccgatca gctcgatctt gtccaggatg
tccttgaaca gcacctcctc 120ctcgtgctgc tcggccacgt accactgcag
gaagttgaag gtggcgtggt ccttgctctt 180gatggcgtgg tccacgatgt
tgttgatgct ctcgctgatg tgctgctcgt gctcgtaggc 240cttctggaag
atctgggtca ggccctcgaa cttgtgctcg ggggcgctga tgctggtcag
300ctgcacgggc acgttgttct cgttcaggaa gatgatcagc ttcttggcgt
gctcgtactc 360ctcggcggcg tggtcgaaca ggaacaggcc ggcgccgtcc
aggctgtggg tgtagcacca 420gctgctcatg ctcatgtaca ggttgctgct
ctgcatctcc ttgttcacct gctcgttcag 480cagcttgatg atgtcgcctc
cggaatcgat cttctcccgg ttcagcttgc tttcctcgct 540gtacttgggg
tagtcgtagg tgccgttctt cacgctttcc atgcactcgt cgttgcactt
600gtggtagaac tcgaagcagc cgttgccgat ctctttggcg ttgttcttca
gctggctctt 660cactttctcg tacaggttct tcacgttgct gtcgtggaag
tccagggtcc tctcattttc 720caggaggacc aggagttcgg cgttgtaggt
ctgcagctgc ttgatgcccc acactgtcag 780ctgcagcaga tgctgctggg
cctcaatggc tctcagcagg ttgttctgct gctgcacaat 840tccagatccg
ccgcctgtgc cgttttcctg ctggttctgg ctttcctcga tcaggctgta
900gatgatgctg gtgtagttgt tgatctctct gtcccattca ggatcgccgc
ccatcttctc 960gatcacgctg ttcactttgt tggtgatgcc gttgatggcg
ttctgggtgc tcttctgatc 1020ggcggcatag ccagatccct gctcattctg
gtggtggtag ccgtaccacc catccaccat 1080gcctgtccat ccgccctcga
taaagccggc aatggcgcca aacaggcctc tgctctggat 1140gctggggata
ttcctcaggc cggtcaccat tctcagtcct gatcccaggt tcacgctgtg
1200ggtcacggtc acgttctttt ccagcacggt atccacggtg tcggtgctat
tgttggcgtg 1260gtagccgata cagatggtat cggcgtaggt ggcggtaaag
gtacacagca gcaccagcag 1320cttcactttc at 13321181362DNAArtificial
SequenceSynthetic 118atgaaaacca tcattgccct gagctacatc ctgtgcctgg
tgttcacaca gaagctgccc 60ggcaacgata atagcaccgc cacactgtgt ctgggacacc
acgccgtgcc taatggcacc 120atcgtgaaaa caatcaccaa cgaccagatc
gaagtgacca atgccacaga gctgggctcc 180ggcctgaagc tggccaccgg
catgagaaat gtgcccgaga agcagaccag aggcatcttt 240ggcgccattg
ccggctttat cgagaatggc tgggagggaa tggtggatgg gtggtacggc
300ttcagacacc agaatagcga gggaattgga caggccgccg atctgaaatc
tacccaggcc 360gccatcgacc agatcaacgg caagctgaac aggctgatcg
gcaagaccgg cggcgatccc 420gagtgggacc gggagatcaa caactacacc
agcatcatct acagcctgat cgaggagagc 480cagaaccagc aggagaacgg
caccggcggc ggcagcggca tcgtgcagca gcagaacaac 540ctgctgcggg
ccatcgaggc ccagcagcac ctgctgcagc tgaccgtgtg gggcatcaag
600cagctgcaga gctacaatgc cgaactgctg gtcgccctgg aaaaccagca
cacaattgat 660ctgacagaca gtgagatgaa taagctgttc gagaaaacca
agaagcagct gagagaaaac 720gccgaggaca tgggcaacgg ctgcttcaag
atctaccaca agtgcgacaa cgcctgcatc 780ggcagcatca gaaacggcac
ctacgaccac gacgtgtaca gagatgaggc cctgaacaac 840cggtttcaga
tcaagggctc cggaggcgac atcatcaagc tgctgaacga gcaggtgaac
900aaggagatgc agagcagcaa cctgtacatg agcatgagca gctggtgcta
cacccacagc 960ctggacggcg ccggcctgtt cctgttcgac cacgccgccg
aggagtacga gcacgccaag 1020aagctgatca tcttcctgaa cgagaacaac
gtgcccgtgc agctgaccag catcagcgcc 1080cccgagcaca agttcgaggg
cctgacccag atcttccaga aggcctacga gcacgagcag 1140cacatcagcg
agagcatcaa caacatcgtg gaccacgcca tcaagagcaa ggaccacgcc
1200accttcaact tcctgcagtg gtacgtggcc gagcagcacg aggaggaggt
gctgttcaag 1260gacatcctgg acaagatcga gctgatcggc aacgagaacc
acggcctgta cctggccgac 1320cagtacgtga agggcatcgc caagagcagg
aagagcggat cc 1362119454PRTArtificial SequenceSynthetic 119Met Lys
Thr Ile Ile Ala Leu Ser Tyr Ile Leu Cys Leu Val Phe Thr 1 5 10 15
Gln Lys Leu Pro Gly Asn Asp Asn Ser Thr Ala Thr Leu Cys Leu Gly 20
25 30 His His Ala Val Pro Asn Gly Thr Ile Val Lys Thr Ile Thr Asn
Asp 35 40 45 Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Gly Ser Gly
Leu Lys Leu 50 55 60 Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln
Thr Arg Gly Ile Phe 65 70 75 80 Gly Ala Ile Ala Gly Phe Ile Glu Asn
Gly Trp Glu Gly Met Val Asp 85 90 95 Gly Trp Tyr Gly Phe Arg His
Gln Asn Ser Glu Gly Ile Gly Gln Ala 100 105 110 Ala Asp Leu Lys Ser
Thr Gln Ala Ala Ile Asp Gln Ile Asn Gly Lys 115 120 125 Leu Asn Arg
Leu Ile Gly Lys Thr Gly Gly Asp Pro Glu Trp Asp Arg 130 135 140 Glu
Ile Asn Asn Tyr Thr Ser Ile Ile Tyr Ser Leu Ile Glu Glu Ser 145 150
155 160 Gln Asn Gln Gln Glu Asn Gly Thr Gly Gly Gly Ser Gly Ile Val
Gln 165 170 175 Gln Gln Asn Asn Leu Leu Arg Ala Ile Glu Ala Gln Gln
His Leu Leu 180 185 190 Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln
Ser Tyr Asn Ala Glu 195 200 205 Leu Leu Val Ala Leu Glu Asn Gln His
Thr Ile Asp Leu Thr Asp Ser 210 215 220 Glu Met Asn Lys Leu Phe Glu
Lys Thr Lys Lys Gln Leu Arg Glu Asn 225 230 235 240 Ala Glu Asp Met
Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp 245 250 255 Asn Ala
Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val 260 265 270
Tyr Arg Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Ser Gly 275
280 285 Gly Asp Ile Ile Lys Leu Leu Asn Glu Gln Val Asn Lys Glu Met
Gln 290 295 300 Ser Ser Asn Leu Tyr Met Ser Met Ser Ser Trp Cys Tyr
Thr His Ser 305 310 315 320 Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp
His Ala Ala Glu Glu Tyr 325 330 335 Glu His Ala Lys Lys Leu Ile Ile
Phe Leu Asn Glu Asn Asn Val Pro 340 345 350 Val Gln Leu Thr Ser Ile
Ser Ala Pro Glu His Lys Phe Glu Gly Leu 355 360 365 Thr Gln Ile Phe
Gln Lys Ala Tyr Glu His Glu Gln His
Ile Ser Glu 370 375 380 Ser Ile Asn Asn Ile Val Asp His Ala Ile Lys
Ser Lys Asp His Ala 385 390 395 400 Thr Phe Asn Phe Leu Gln Trp Tyr
Val Ala Glu Gln His Glu Glu Glu 405 410 415 Val Leu Phe Lys Asp Ile
Leu Asp Lys Ile Glu Leu Ile Gly Asn Glu 420 425 430 Asn His Gly Leu
Tyr Leu Ala Asp Gln Tyr Val Lys Gly Ile Ala Lys 435 440 445 Ser Arg
Lys Ser Gly Ser 450 1201362DNAArtificial SequenceSynthetic
120ggatccgctc ttcctgctct tggcgatgcc cttcacgtac tggtcggcca
ggtacaggcc 60gtggttctcg ttgccgatca gctcgatctt gtccaggatg tccttgaaca
gcacctcctc 120ctcgtgctgc tcggccacgt accactgcag gaagttgaag
gtggcgtggt ccttgctctt 180gatggcgtgg tccacgatgt tgttgatgct
ctcgctgatg tgctgctcgt gctcgtaggc 240cttctggaag atctgggtca
ggccctcgaa cttgtgctcg ggggcgctga tgctggtcag 300ctgcacgggc
acgttgttct cgttcaggaa gatgatcagc ttcttggcgt gctcgtactc
360ctcggcggcg tggtcgaaca ggaacaggcc ggcgccgtcc aggctgtggg
tgtagcacca 420gctgctcatg ctcatgtaca ggttgctgct ctgcatctcc
ttgttcacct gctcgttcag 480cagcttgatg atgtcgcctc cggagccctt
gatctgaaac cggttgttca gggcctcatc 540tctgtacacg tcgtggtcgt
aggtgccgtt tctgatgctg ccgatgcagg cgttgtcgca 600cttgtggtag
atcttgaagc agccgttgcc catgtcctcg gcgttttctc tcagctgctt
660cttggttttc tcgaacagct tattcatctc actgtctgtc agatcaattg
tgtgctggtt 720ttccagggcg accagcagtt cggcattgta gctctgcagc
tgcttgatgc cccacacggt 780cagctgcagc aggtgctgct gggcctcgat
ggcccgcagc aggttgttct gctgctgcac 840gatgccgctg ccgccgccgg
tgccgttctc ctgctggttc tggctctcct cgatcaggct 900gtagatgatg
ctggtgtagt tgttgatctc ccggtcccac tcgggatcgc cgccggtctt
960gccgatcagc ctgttcagct tgccgttgat ctggtcgatg gcggcctggg
tagatttcag 1020atcggcggcc tgtccaattc cctcgctatt ctggtgtctg
aagccgtacc acccatccac 1080cattccctcc cagccattct cgataaagcc
ggcaatggcg ccaaagatgc ctctggtctg 1140cttctcgggc acatttctca
tgccggtggc cagcttcagg ccggagccca gctctgtggc 1200attggtcact
tcgatctggt cgttggtgat tgttttcacg atggtgccat taggcacggc
1260gtggtgtccc agacacagtg tggcggtgct attatcgttg ccgggcagct
tctgtgtgaa 1320caccaggcac aggatgtagc tcagggcaat gatggttttc at
13621211362DNAArtificial SequenceSynthetic 121atgaaaacca taattgcgct
gtcctacata ctgtgtctgg tgtttgccca gaaactgccg 60ggcaatgaca actcaacagc
cacgctctgc ttggggcacc atgccgtccc taacgggacc 120attgtgaaaa
ccattactaa cgatcagata gaggtgacta atgccaccga gctgggctcc
180ggcttgaaac tggcgaccgg tatgcgcaat gtccccgaaa aacagacccg
cgggatattt 240ggggctatcg caggctttat cgagaatggc tgggaaggga
tggtggatgg ttggtatggt 300tttagacatc aaaactccga aggcagaggc
caggctgccg atctcaagag cacgcaggcc 360gctatagatc agatcaatgg
aaagctcaac agactgatcg ggaaaaccgg cggcgatccc 420gagtgggacc
gggagatcaa caactacacc agcatcatct acagcctgat cgaggagagc
480cagaaccagc aggagaacgg caccggcggc ggcagcggca tcgtgcagca
gcagaacaac 540ctgctgcggg ccatcgaggc ccagcagcac ctgctgcagc
tgaccgtgtg gggcatcaag 600cagctgcagt cctacaatgc cgagctgctg
gtggctctgg agaatcagca cactattgac 660ctgaccgatt cagagatgaa
caaacttttt gagaagacga agaagcagct tagagaaaat 720gcagaggaca
tggggaacgg atgctttaaa atatatcata agtgtgataa tgcctgcatc
780ggatcaatta gaaatggtac ctatgatcac gatgtttaca gggacgaagc
gctgaataac 840aggttccaga taaaaggctc cggaggcgac atcatcaagc
tgctgaacga gcaggtgaac 900aaggagatgc agagcagcaa cctgtacatg
agcatgagca gctggtgcta cacccacagc 960ctggacggcg ccggcctgtt
cctgttcgac cacgccgccg aggagtacga gcacgccaag 1020aagctgatca
tcttcctgaa cgagaacaac gtgcccgtgc agctgaccag catcagcgcc
1080cccgagcaca agttcgaggg cctgacccag atcttccaga aggcctacga
gcacgagcag 1140cacatcagcg agagcatcaa caacatcgtg gaccacgcca
tcaagagcaa ggaccacgcc 1200accttcaact tcctgcagtg gtacgtggcc
gagcagcacg aggaggaggt gctgttcaag 1260gacatcctgg acaagatcga
gctgatcggc aacgagaacc acggcctgta cctggccgac 1320cagtacgtga
agggcatcgc caagagcagg aagagcggat cc 1362122454PRTArtificial
SequenceSynthetic 122Met Lys Thr Ile Ile Ala Leu Ser Tyr Ile Leu
Cys Leu Val Phe Ala 1 5 10 15 Gln Lys Leu Pro Gly Asn Asp Asn Ser
Thr Ala Thr Leu Cys Leu Gly 20 25 30 His His Ala Val Pro Asn Gly
Thr Ile Val Lys Thr Ile Thr Asn Asp 35 40 45 Gln Ile Glu Val Thr
Asn Ala Thr Glu Leu Gly Ser Gly Leu Lys Leu 50 55 60 Ala Thr Gly
Met Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe 65 70 75 80 Gly
Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp 85 90
95 Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Arg Gly Gln Ala
100 105 110 Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asp Gln Ile Asn
Gly Lys 115 120 125 Leu Asn Arg Leu Ile Gly Lys Thr Gly Gly Asp Pro
Glu Trp Asp Arg 130 135 140 Glu Ile Asn Asn Tyr Thr Ser Ile Ile Tyr
Ser Leu Ile Glu Glu Ser 145 150 155 160 Gln Asn Gln Gln Glu Asn Gly
Thr Gly Gly Gly Ser Gly Ile Val Gln 165 170 175 Gln Gln Asn Asn Leu
Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu 180 185 190 Gln Leu Thr
Val Trp Gly Ile Lys Gln Leu Gln Ser Tyr Asn Ala Glu 195 200 205 Leu
Leu Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser 210 215
220 Glu Met Asn Lys Leu Phe Glu Lys Thr Lys Lys Gln Leu Arg Glu Asn
225 230 235 240 Ala Glu Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His
Lys Cys Asp 245 250 255 Asn Ala Cys Ile Gly Ser Ile Arg Asn Gly Thr
Tyr Asp His Asp Val 260 265 270 Tyr Arg Asp Glu Ala Leu Asn Asn Arg
Phe Gln Ile Lys Gly Ser Gly 275 280 285 Gly Asp Ile Ile Lys Leu Leu
Asn Glu Gln Val Asn Lys Glu Met Gln 290 295 300 Ser Ser Asn Leu Tyr
Met Ser Met Ser Ser Trp Cys Tyr Thr His Ser 305 310 315 320 Leu Asp
Gly Ala Gly Leu Phe Leu Phe Asp His Ala Ala Glu Glu Tyr 325 330 335
Glu His Ala Lys Lys Leu Ile Ile Phe Leu Asn Glu Asn Asn Val Pro 340
345 350 Val Gln Leu Thr Ser Ile Ser Ala Pro Glu His Lys Phe Glu Gly
Leu 355 360 365 Thr Gln Ile Phe Gln Lys Ala Tyr Glu His Glu Gln His
Ile Ser Glu 370 375 380 Ser Ile Asn Asn Ile Val Asp His Ala Ile Lys
Ser Lys Asp His Ala 385 390 395 400 Thr Phe Asn Phe Leu Gln Trp Tyr
Val Ala Glu Gln His Glu Glu Glu 405 410 415 Val Leu Phe Lys Asp Ile
Leu Asp Lys Ile Glu Leu Ile Gly Asn Glu 420 425 430 Asn His Gly Leu
Tyr Leu Ala Asp Gln Tyr Val Lys Gly Ile Ala Lys 435 440 445 Ser Arg
Lys Ser Gly Ser 450 1231362DNAArtificial SequenceSynthetic
123ggatccgctc ttcctgctct tggcgatgcc cttcacgtac tggtcggcca
ggtacaggcc 60gtggttctcg ttgccgatca gctcgatctt gtccaggatg tccttgaaca
gcacctcctc 120ctcgtgctgc tcggccacgt accactgcag gaagttgaag
gtggcgtggt ccttgctctt 180gatggcgtgg tccacgatgt tgttgatgct
ctcgctgatg tgctgctcgt gctcgtaggc 240cttctggaag atctgggtca
ggccctcgaa cttgtgctcg ggggcgctga tgctggtcag 300ctgcacgggc
acgttgttct cgttcaggaa gatgatcagc ttcttggcgt gctcgtactc
360ctcggcggcg tggtcgaaca ggaacaggcc ggcgccgtcc aggctgtggg
tgtagcacca 420gctgctcatg ctcatgtaca ggttgctgct ctgcatctcc
ttgttcacct gctcgttcag 480cagcttgatg atgtcgcctc cggagccttt
tatctggaac ctgttattca gcgcttcgtc 540cctgtaaaca tcgtgatcat
aggtaccatt tctaattgat ccgatgcagg cattatcaca 600cttatgatat
attttaaagc atccgttccc catgtcctct gcattttctc taagctgctt
660cttcgtcttc tcaaaaagtt tgttcatctc tgaatcggtc aggtcaatag
tgtgctgatt 720ctccagagcc accagcagct cggcattgta ggactgcagc
tgcttgatgc cccacacggt 780cagctgcagc aggtgctgct gggcctcgat
ggcccgcagc aggttgttct gctgctgcac 840gatgccgctg ccgccgccgg
tgccgttctc ctgctggttc tggctctcct cgatcaggct 900gtagatgatg
ctggtgtagt tgttgatctc ccggtcccac tcgggatcgc cgccggtttt
960cccgatcagt ctgttgagct ttccattgat ctgatctata gcggcctgcg
tgctcttgag 1020atcggcagcc tggcctctgc cttcggagtt ttgatgtcta
aaaccatacc aaccatccac 1080catcccttcc cagccattct cgataaagcc
tgcgatagcc ccaaatatcc cgcgggtctg 1140tttttcgggg acattgcgca
taccggtcgc cagtttcaag ccggagccca gctcggtggc 1200attagtcacc
tctatctgat cgttagtaat ggttttcaca atggtcccgt tagggacggc
1260atggtgcccc aagcagagcg tggctgttga gttgtcattg cccggcagtt
tctgggcaaa 1320caccagacac agtatgtagg acagcgcaat tatggttttc at
13621241332DNAArtificial SequenceSynthetic 124atgaaggcca tcatcgtgct
gctgatggtg gtcacaagca acgccgatag aatctgtacc 60ggcatcacca gcagcaatag
ccctcacgtc gtgaaaacag ctacacaggg cgaagtgaat 120gtgaccggcg
tgatccctct gggatcagga ctgaagctgg ccaatggcac aaagtataga
180cctccagcca agctgctgaa agagagaggc ttttttggag ctatcgccgg
ctttctggaa 240ggcggatggg agggaatgat tgctggatgg catggctaca
catctcatgg cgcacatggc 300gtggcagtgg ctgctgatct gaaatctaca
caggaagcca tcaacaagat caccaagaac 360ctgaacagcc tgagcgagct
ggaaggaggc gaccccgagt gggatcgcga aatcaacaac 420tacacatcta
tcatctacag tctgattgag gaaagccaga accagcagga gaatgggact
480gggggaggct ccggaatcgt gcagcagcag aacaatctgc tgcgagccat
tgaagctcag 540cagcacctgc tgcagctgac agtgtggggc atcaagcagc
tgcaggggag ccagattgaa 600ctggctgtgc tgctgtctaa cgagggcatc
atcaatagcg aggacgaaca tctgctggcc 660ctggaaagaa agctgaagaa
gatgctggga cctagcgccg tggaaatcgg caatggatgc 720tttgagacaa
agcacaagtg caaccagacc tgcctggata gaattgccgc cggaacattt
780gatgccggcg agttttctct gcccaccttc gatagcctga atatcacatc
cggaggcgac 840atcatcaagc tgctgaacga gcaggtgaac aaggagatgc
agagcagcaa cctgtacatg 900agcatgagca gctggtgcta cacccacagc
ctggacggcg ccggcctgtt cctgttcgac 960cacgccgccg aggagtacga
gcacgccaag aagctgatca tcttcctgaa cgagaacaac 1020gtgcccgtgc
agctgaccag catcagcgcc cccgagcaca agttcgaggg cctgacccag
1080atcttccaga aggcctacga gcacgagcag cacatcagcg agagcatcaa
caacatcgtg 1140gaccacgcca tcaagagcaa ggaccacgcc accttcaact
tcctgcagtg gtacgtggcc 1200gagcagcacg aggaggaggt gctgttcaag
gacatcctgg acaagatcga gctgatcggc 1260aacgagaacc acggcctgta
cctggccgac cagtacgtga agggcatcgc caagagcagg 1320aagagcggat cc
1332125444PRTArtificial SequenceSynthetic 125Met Lys Ala Ile Ile
Val Leu Leu Met Val Val Thr Ser Asn Ala Asp 1 5 10 15 Arg Ile Cys
Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys 20 25 30 Thr
Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Gly 35 40
45 Ser Gly Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro Pro Ala Lys
50 55 60 Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly Phe
Leu Glu 65 70 75 80 Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly
Tyr Thr Ser His 85 90 95 Gly Ala His Gly Val Ala Val Ala Ala Asp
Leu Lys Ser Thr Gln Glu 100 105 110 Ala Ile Asn Lys Ile Thr Lys Asn
Leu Asn Ser Leu Ser Glu Leu Glu 115 120 125 Gly Gly Asp Pro Glu Trp
Asp Arg Glu Ile Asn Asn Tyr Thr Ser Ile 130 135 140 Ile Tyr Ser Leu
Ile Glu Glu Ser Gln Asn Gln Gln Glu Asn Gly Thr 145 150 155 160 Gly
Gly Gly Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Arg Ala 165 170
175 Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys
180 185 190 Gln Leu Gln Gly Ser Gln Ile Glu Leu Ala Val Leu Leu Ser
Asn Glu 195 200 205 Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala
Leu Glu Arg Lys 210 215 220 Leu Lys Lys Met Leu Gly Pro Ser Ala Val
Glu Ile Gly Asn Gly Cys 225 230 235 240 Phe Glu Thr Lys His Lys Cys
Asn Gln Thr Cys Leu Asp Arg Ile Ala 245 250 255 Ala Gly Thr Phe Asp
Ala Gly Glu Phe Ser Leu Pro Thr Phe Asp Ser 260 265 270 Leu Asn Ile
Thr Ser Gly Gly Asp Ile Ile Lys Leu Leu Asn Glu Gln 275 280 285 Val
Asn Lys Glu Met Gln Ser Ser Asn Leu Tyr Met Ser Met Ser Ser 290 295
300 Trp Cys Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp
305 310 315 320 His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile
Ile Phe Leu 325 330 335 Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser
Ile Ser Ala Pro Glu 340 345 350 His Lys Phe Glu Gly Leu Thr Gln Ile
Phe Gln Lys Ala Tyr Glu His 355 360 365 Glu Gln His Ile Ser Glu Ser
Ile Asn Asn Ile Val Asp His Ala Ile 370 375 380 Lys Ser Lys Asp His
Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala 385 390 395 400 Glu Gln
His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys Ile 405 410 415
Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln Tyr 420
425 430 Val Lys Gly Ile Ala Lys Ser Arg Lys Ser Gly Ser 435 440
1261332DNAArtificial SequenceSynthetic 126ggatccgctc ttcctgctct
tggcgatgcc cttcacgtac tggtcggcca ggtacaggcc 60gtggttctcg ttgccgatca
gctcgatctt gtccaggatg tccttgaaca gcacctcctc 120ctcgtgctgc
tcggccacgt accactgcag gaagttgaag gtggcgtggt ccttgctctt
180gatggcgtgg tccacgatgt tgttgatgct ctcgctgatg tgctgctcgt
gctcgtaggc 240cttctggaag atctgggtca ggccctcgaa cttgtgctcg
ggggcgctga tgctggtcag 300ctgcacgggc acgttgttct cgttcaggaa
gatgatcagc ttcttggcgt gctcgtactc 360ctcggcggcg tggtcgaaca
ggaacaggcc ggcgccgtcc aggctgtggg tgtagcacca 420gctgctcatg
ctcatgtaca ggttgctgct ctgcatctcc ttgttcacct gctcgttcag
480cagcttgatg atgtcgcctc cggatgtgat attcaggcta tcgaaggtgg
gcagagaaaa 540ctcgccggca tcaaatgttc cggcggcaat tctatccagg
caggtctggt tgcacttgtg 600ctttgtctca aagcatccat tgccgatttc
cacggcgcta ggtcccagca tcttcttcag 660ctttctttcc agggccagca
gatgttcgtc ctcgctattg atgatgccct cgttagacag 720cagcacagcc
agttcaatct ggctcccctg cagctgcttg atgccccaca ctgtcagctg
780cagcaggtgc tgctgagctt caatggctcg cagcagattg ttctgctgct
gcacgattcc 840ggagcctccc ccagtcccat tctcctgctg gttctggctt
tcctcaatca gactgtagat 900gatagatgtg tagttgttga tttcgcgatc
ccactcgggg tcgcctcctt ccagctcgct 960caggctgttc aggttcttgg
tgatcttgtt gatggcttcc tgtgtagatt tcagatcagc 1020agccactgcc
acgccatgtg cgccatgaga tgtgtagcca tgccatccag caatcattcc
1080ctcccatccg ccttccagaa agccggcgat agctccaaaa aagcctctct
ctttcagcag 1140cttggctgga ggtctatact ttgtgccatt ggccagcttc
agtcctgatc ccagagggat 1200cacgccggtc acattcactt cgccctgtgt
agctgttttc acgacgtgag ggctattgct 1260gctggtgatg ccggtacaga
ttctatcggc gttgcttgtg accaccatca gcagcacgat 1320gatggccttc at
13321271332DNAArtificial SequenceSynthetic 127atgaaggcca tcatcgtgct
gctgatggtg gtgaccagca acgccgatag aatctgcacc 60ggcatcacca gcagcaatag
cccccatgtg gtgaaaacag ccacccaggg cgaagtgaat 120gtgacaggcg
tgatccctct gggatcagga ctgaagctgg ccaatggcac caagtacaga
180cctcccgcca agctgctgaa agagagaggc ttctttggcg ccattgccgg
atttctggaa 240ggcggctggg agggaatgat tgccggctgg cacggctata
catctcatgg ggcccatggc 300gtggctgtgg ccgccgatct gaagtctacc
caggaagcca tcaacaagat caccaagaac 360ctgaacagcc tgagcgagct
ggaaggaggc gaccccgagt gggatcgcga aatcaacaac 420tacacatcta
tcatctacag tctgattgag gaaagccaga accagcagga gaatgggact
480gggggaggct ccggaatcgt gcagcagcag aacaatctgc tgcgagccat
tgaagctcag 540cagcacctgc tgcagctgac agtgtggggc atcaagcagc
tgcaggggtc ccagattgaa 600ctggccgtgc tgctgtccaa cgagggcatc
atcaacagcg aggatgaaca cctgctggcc 660ctggaacgga agctgaagaa
gatgctgggc ccttctgccg tggagatcgg caacggctgc 720ttcgagacaa
agcacaagtg caaccagacc tgcctggata gaatcgccgc tggcaccttc
780aatgccggcg agttcagcct gcctaccttc gacagcctga atatcacctc
cggaggcgac 840atcatcaagc tgctgaacga gcaggtgaac aaggagatgc
agagcagcaa cctgtacatg 900agcatgagca gctggtgcta cacccacagc
ctggacggcg ccggcctgtt cctgttcgac 960cacgccgccg aggagtacga
gcacgccaag aagctgatca tcttcctgaa cgagaacaac 1020gtgcccgtgc
agctgaccag catcagcgcc cccgagcaca agttcgaggg cctgacccag
1080atcttccaga aggcctacga gcacgagcag cacatcagcg agagcatcaa
caacatcgtg 1140gaccacgcca tcaagagcaa ggaccacgcc accttcaact
tcctgcagtg gtacgtggcc 1200gagcagcacg aggaggaggt gctgttcaag
gacatcctgg acaagatcga gctgatcggc 1260aacgagaacc acggcctgta
cctggccgac cagtacgtga agggcatcgc caagagcagg 1320aagagcggat cc
1332128444PRTArtificial SequenceSynthetic 128Met Lys Ala Ile Ile
Val Leu Leu Met Val Val Thr Ser
Asn Ala Asp 1 5 10 15 Arg Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser
Pro His Val Val Lys 20 25 30 Thr Ala Thr Gln Gly Glu Val Asn Val
Thr Gly Val Ile Pro Leu Gly 35 40 45 Ser Gly Leu Lys Leu Ala Asn
Gly Thr Lys Tyr Arg Pro Pro Ala Lys 50 55 60 Leu Leu Lys Glu Arg
Gly Phe Phe Gly Ala Ile Ala Gly Phe Leu Glu 65 70 75 80 Gly Gly Trp
Glu Gly Met Ile Ala Gly Trp His Gly Tyr Thr Ser His 85 90 95 Gly
Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser Thr Gln Glu 100 105
110 Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Ser Leu Ser Glu Leu Glu
115 120 125 Gly Gly Asp Pro Glu Trp Asp Arg Glu Ile Asn Asn Tyr Thr
Ser Ile 130 135 140 Ile Tyr Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln
Glu Asn Gly Thr 145 150 155 160 Gly Gly Gly Ser Gly Ile Val Gln Gln
Gln Asn Asn Leu Leu Arg Ala 165 170 175 Ile Glu Ala Gln Gln His Leu
Leu Gln Leu Thr Val Trp Gly Ile Lys 180 185 190 Gln Leu Gln Gly Ser
Gln Ile Glu Leu Ala Val Leu Leu Ser Asn Glu 195 200 205 Gly Ile Ile
Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg Lys 210 215 220 Leu
Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly Asn Gly Cys 225 230
235 240 Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg Ile
Ala 245 250 255 Ala Gly Thr Phe Asn Ala Gly Glu Phe Ser Leu Pro Thr
Phe Asp Ser 260 265 270 Leu Asn Ile Thr Ser Gly Gly Asp Ile Ile Lys
Leu Leu Asn Glu Gln 275 280 285 Val Asn Lys Glu Met Gln Ser Ser Asn
Leu Tyr Met Ser Met Ser Ser 290 295 300 Trp Cys Tyr Thr His Ser Leu
Asp Gly Ala Gly Leu Phe Leu Phe Asp 305 310 315 320 His Ala Ala Glu
Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu 325 330 335 Asn Glu
Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu 340 345 350
His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His 355
360 365 Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala
Ile 370 375 380 Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp
Tyr Val Ala 385 390 395 400 Glu Gln His Glu Glu Glu Val Leu Phe Lys
Asp Ile Leu Asp Lys Ile 405 410 415 Glu Leu Ile Gly Asn Glu Asn His
Gly Leu Tyr Leu Ala Asp Gln Tyr 420 425 430 Val Lys Gly Ile Ala Lys
Ser Arg Lys Ser Gly Ser 435 440 1291332DNAArtificial
SequenceSynthetic 129ggatccgctc ttcctgctct tggcgatgcc cttcacgtac
tggtcggcca ggtacaggcc 60gtggttctcg ttgccgatca gctcgatctt gtccaggatg
tccttgaaca gcacctcctc 120ctcgtgctgc tcggccacgt accactgcag
gaagttgaag gtggcgtggt ccttgctctt 180gatggcgtgg tccacgatgt
tgttgatgct ctcgctgatg tgctgctcgt gctcgtaggc 240cttctggaag
atctgggtca ggccctcgaa cttgtgctcg ggggcgctga tgctggtcag
300ctgcacgggc acgttgttct cgttcaggaa gatgatcagc ttcttggcgt
gctcgtactc 360ctcggcggcg tggtcgaaca ggaacaggcc ggcgccgtcc
aggctgtggg tgtagcacca 420gctgctcatg ctcatgtaca ggttgctgct
ctgcatctcc ttgttcacct gctcgttcag 480cagcttgatg atgtcgcctc
cggaggtgat attcaggctg tcgaaggtag gcaggctgaa 540ctcgccggca
ttgaaggtgc cagcggcgat tctatccagg caggtctggt tgcacttgtg
600ctttgtctcg aagcagccgt tgccgatctc cacggcagaa gggcccagca
tcttcttcag 660cttccgttcc agggccagca ggtgttcatc ctcgctgttg
atgatgccct cgttggacag 720cagcacggcc agttcaatct gggacccctg
cagctgcttg atgccccaca ctgtcagctg 780cagcaggtgc tgctgagctt
caatggctcg cagcagattg ttctgctgct gcacgattcc 840ggagcctccc
ccagtcccat tctcctgctg gttctggctt tcctcaatca gactgtagat
900gatagatgtg tagttgttga tttcgcgatc ccactcgggg tcgcctcctt
ccagctcgct 960caggctgttc aggttcttgg tgatcttgtt gatggcttcc
tgggtagact tcagatcggc 1020ggccacagcc acgccatggg ccccatgaga
tgtatagccg tgccagccgg caatcattcc 1080ctcccagccg ccttccagaa
atccggcaat ggcgccaaag aagcctctct ctttcagcag 1140cttggcggga
ggtctgtact tggtgccatt ggccagcttc agtcctgatc ccagagggat
1200cacgcctgtc acattcactt cgccctgggt ggctgttttc accacatggg
ggctattgct 1260gctggtgatg ccggtgcaga ttctatcggc gttgctggtc
accaccatca gcagcacgat 1320gatggccttc at 1332
* * * * *
References