U.S. patent application number 15/289106 was filed with the patent office on 2017-05-04 for recombinant viral vectors and methods for inducing a heterosubtypic immune response to influenza a viruses.
The applicant listed for this patent is NANOTHERAPEUTICS, INC.. Invention is credited to P. Noel BARRETT, Hartmut EHRLICH, Falko-Gunter FALKNER, Annett HESSEL, Thomas R. KREIL, Birgit SCHAFER.
Application Number | 20170121735 15/289106 |
Document ID | / |
Family ID | 45561167 |
Filed Date | 2017-05-04 |
United States Patent
Application |
20170121735 |
Kind Code |
A1 |
FALKNER; Falko-Gunter ; et
al. |
May 4, 2017 |
RECOMBINANT VIRAL VECTORS AND METHODS FOR INDUCING A HETEROSUBTYPIC
IMMUNE RESPONSE TO INFLUENZA A VIRUSES
Abstract
The present invention relates to recombinant viral vectors and
methods of using the recombinant viral vectors to induce an immune
response to influenza A viruses. The invention provides recombinant
viral vectors based, for example, on the non-replicating modified
vaccinia virus Ankara. When administered according to methods of
the invention, the recombinant viral vectors are designed to be
cross-protective and induce heterosubtypic immunity to influenza A
viruses.
Inventors: |
FALKNER; Falko-Gunter;
(Orth/Donau, AT) ; SCHAFER; Birgit; (Wien, AT)
; BARRETT; P. Noel; (Klosterneuburg/Weidling, AT)
; KREIL; Thomas R.; (Klosterneuburg, AT) ;
EHRLICH; Hartmut; (Wien, AT) ; HESSEL; Annett;
(Orth/Donau, AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NANOTHERAPEUTICS, INC. |
Alachua |
FL |
US |
|
|
Family ID: |
45561167 |
Appl. No.: |
15/289106 |
Filed: |
October 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13982524 |
Nov 5, 2013 |
9463237 |
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PCT/US2012/023085 |
Jan 30, 2012 |
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15289106 |
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61438024 |
Jan 31, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/16 20180101;
C12N 2710/24123 20130101; C12N 2710/24143 20130101; C07K 2319/00
20130101; C12N 15/86 20130101; C12N 7/00 20130101; A61K 2039/5256
20130101; A61K 2039/70 20130101; C12N 2710/24134 20130101; A61K
39/145 20130101; A61K 39/12 20130101; A61K 2039/5258 20130101; C12N
2760/16134 20130101; C12N 15/62 20130101; A61K 39/285 20130101;
C12N 2710/24122 20130101 |
International
Class: |
C12N 15/86 20060101
C12N015/86; C12N 15/62 20060101 C12N015/62; C12N 7/00 20060101
C12N007/00; A61K 39/145 20060101 A61K039/145; A61K 39/285 20060101
A61K039/285 |
Claims
1. A recombinant vaccinia virus comprising a gene cassette encoding
a fusion protein comprising at least one influenza A M2
extracellular domain (M2e) polypeptide inserted in an influenza A
headless hemagglutinin (h1HA) polypeptide.
2. The recombinant vaccinia virus of claim 1 wherein the fusion
protein comprises one selected from the group consisting of i) the
h1HA/M2e fusion protein amino acid sequence set out in SEQ ID NO:
2, ii) the VN/1203 HA amino acid sequence set out in SEQ ID NO: 3,
and iii) HA1 amino acids 17-58 of SEQ ID NO: 3, a peptide linker,
at least one M2e polypeptide, a peptide linker, HA1 amino acids
290-343 of SEQ ID NO: 3 and HA2 amino acids 344-568 of SEQ ID NO:
3.
3-4. (canceled)
5. The recombinant vaccinia virus of claim 2, wherein the peptide
linkers linking the HA amino acids and M2e amino acids comprise the
amino acids GGG set out in SEQ ID NO: 4.
6. The recombinant vaccinia virus of claim 1, wherein the M2e
polypeptide comprises one selected from the group consisting of i)
the H5N1 M2e amino acid sequence set out in SEQ ID NO: 5, ii) the
H1N1 M2e amino acid sequence set out in SEQ ID NO: 6, iii) the H9N2
M2e amino acid sequence set out in SEQ ID NO: 7, and iv) the H7N2
M2e amino acid sequence set out in SEQ ID NO: 8.
7-9. (canceled)
10. The recombinant vaccinia virus of claim 1, wherein the fusion
protein comprises more than one M2e polypeptide and the M2e
polypeptides are linked by a peptide linker.
11. The recombinant vaccinia virus of claim 10, wherein the peptide
linker linking the M2e polypeptides comprises the amino acids
GSAGSA set out in SEQ ID NO: 9.
12. The recombinant vaccinia virus of claim 1, wherein expression
of the h1HA/M2e fusion protein from the gene cassette is under the
control of an mH5 promoter or a selP promoter.
13. (canceled)
14. A recombinant vaccinia virus comprising the gene cassette set
out in SEQ ID NO: 1.
15. The recombinant vaccinia virus of claim 1, further comprising a
gene cassette encoding an influenza A matrix protein 1 (M1) and a
gene cassette encoding an influenza A nucleoprotein (NP).
16-23. (canceled)
24. A pharmaceutical composition comprising the recombinant
vaccinia virus of claim 1.
25-26. (canceled)
27. A method of inducing a heterosubtypic immune response to
influenza A virus in an individual comprising administering a
pharmaceutical composition comprising the recombinant vaccinia
virus of claim 1 to the individual.
28-31. (canceled)
32. A recombinant vaccinia virus comprising a first gene cassette
encoding a fusion protein comprising at least one influenza A M2
extracellular domain (M2e) polypeptide inserted in an influenza A
headless hemagglutinin (h1HA) polypeptide and a second gene
cassette encoding influenza A nucleoprotein (NP).
33. The recombinant vaccinia virus of claim 32, wherein the fusion
protein comprises one selected from the group consisting of i) the
h1HA/M2e fusion protein amino acid sequence set out in SEQ ID NO:
2, ii) the VN/1203 HA amino acid sequence set out in SEQ ID NO: 3,
and iii) HA1 amino acids 17-58 of SEQ ID NO: 3, a peptide linker,
at least one M2e polypeptide, a peptide linker, HA1 amino acids
290-343 of SEQ ID NO: 3 and HA2 amino acids 344-568 of SEQ ID NO:
3.
34-35. (canceled)
36. The recombinant vaccinia virus of claim 33, wherein the peptide
linkers linking the HA amino acids and M2e amino acids comprise the
amino acids GGG set out in SEQ ID NO: 4.
37. The recombinant vaccinia virus of claim 32, wherein the M2e
polypeptide comprises one selected from the group consisting of i)
the H5N1 M2e amino acid sequence set out in SEQ ID NO: 5, ii) the
H1N1 M2e amino acid sequence set out in SEQ ID NO: 6, iii) the H9N2
M2e amino acid sequence set out in SEQ ID NO: 7, and iv) the H7N2
M2e amino acid sequence set out in SEQ ID NO: 8.
38-43. (canceled)
44. The recombinant vaccinia virus of claim 32, wherein expression
of the h1HA/M2e fusion protein from the first gene cassette is
under the control of an mH5 promoter or a selP promoter.
45-46. (canceled)
47. The recombinant vaccinia virus of claim 32, wherein expression
of NP from the second gene cassette is under the control of an mH5
promoter or selP promoter.
48-59. (canceled)
60. A pharmaceutical composition comprising the recombinant
vaccinia virus of claim 1.
61-62. (canceled)
63. A method of inducing a heterosubtypic immune response to
influenza A viruses in an individual comprising administering a
pharmaceutical composition comprising the recombinant vaccinia
virus of claim 32 to the individual.
64. A pharmaceutical composition for use in inducing a
heterosubtypic immune response to influenza A viruses in an
individual, the method comprising the step of administering a
pharmaceutical composition comprising the recombinant vaccinia
virus of claim 32 to the individual.
65-101. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to recombinant viral vectors
and methods of using the recombinant viral vectors to induce an
immune response to influenza A viruses. The invention provides
recombinant viral vectors based, for example, on the
non-replicating modified vaccinia virus Ankara. When administered
according to methods of the invention, the recombinant viral
vectors are designed to be cross-protective and induce
heterosubtypic immunity to influenza A viruses.
BACKGROUND OF THE INVENTION
[0002] Human influenza or "the flu" is a respiratory disease that
is caused by influenza A and B viruses. Epidemics of influenza
cause significant illness and death worldwide each year, and
vaccination is the most straightforward strategy to prevent
infection and disease. Traditional influenza vaccines expose the
recipient to influenza virus proteins causing the recipient to
mount an immune response to the proteins. Proteins (or
polypeptides) used in vaccines are commonly called "antigens." The
commonly used seasonal influenza vaccines are based on the major
antigen of the viruses, the hemagglutinin (HA). There are numerous
influenza A subtypes having different HA antigens. Influenza A
subtypes are divided and classified based on the HA and
neuraminidase (NA) proteins that are expressed by the viruses. The
influenza A subtype nomenclature is based on the HA subtype (of the
sixteen different HA genes known in the art) and the NA subtype (of
the nine different NA genes known in the art). Exemplary subtypes,
include, but are not limited to, H5N1, H1N1 and H3N2. There are
also variants of the influenza A subtypes which are referred to as
"strains." For example, the virus A/VietNam/1203/2004 is an
influenza A virus, subtype H5N1, with a strain name
A/VietNam/1203/2004.
[0003] Protection from the seasonal vaccines based on the HA is
strain-specific and new strains emerge constantly, so the classical
influenza vaccines have to be re-formulated each year in an attempt
to match the currently circulating strains. See, Lambert and Fauci
2010. It is therefore highly desirable for next generation vaccines
to be cross-protective and induce heterosubtypic immunity, i.e.,
vaccines against one subtype that protect or partially protect
against challenge infection with influenza A of different
subtypes.
[0004] The current `universal vaccines` (i.e., vaccines designed to
elicit heterosubtypic immunity) that are under development are
mainly based on the more conserved internal influenza virus genes
including the influenza matrix proteins (M1 and M2) (Schotsaert et
al. 2009), the nucleoprotein (NP) and conserved parts of the HA
(Bommakanti et al. 2010; Steel et al. 2010). The polymerase
proteins PA, PB1 and PB2 also induce substantial T cell responses
and may be also relevant targets (Assarsson et al. 2008; Greenbaum
et al. 2009; Lee et al. 2008).
[0005] Next generation influenza vaccines currently under
development include recombinant proteins, synthetic peptides,
virus-like particles (VLPs), DNA-based vaccines and viral vector
vaccines (Lambert and Fauci, supra). The advantage of using live
viral vectors is their known property to induce high levels of
cellular immunity, in particular CD8 T cells. Among the most
promising viral vectors are vaccinia virus-based live vaccines
(Rimmelzwaan and Sutter 2009) and adenovirus-based vectors
(Hoelscher et al. 2006; Hoelscher et al. 2007; Price et al. 2010;
Zhou et al. 2010). Single-dose mucosal immunization using an
adenovirus construct expressing NP and M2, for instance, provided
protection from virulent H5N1, H3N2 and H1N1 viruses (Price et al,
supra). In a further study (Price et al. 2009), DNA vaccination
with nucleoprotein (NP) and matrix 2 (M2) plasmids followed by
boosting with antigen-matched recombinant adenovirus (rAd) provided
robust protection against virulent H1N1 and H5N1 challenges in mice
and ferrets.
[0006] Recombinant vaccines based on modified vaccinia virus Ankara
(MVA) have been used in many non-clinical and clinical studies. MVA
has proven to be exceptionally safe. No significant side effects
have been obtained when MVA was administered to more than 120,000
human patients in the context of the smallpox eradication. Due to a
block in virion morphogenesis the highly attenuated vaccinia virus
strain fails to productively replicate in human and most other
mammalian cells. Nevertheless, the ability to express viral and
foreign genes in the early and late stage is retained. These
characteristics make MVA a promising live vaccine vector that
induces humoral and cellular immune responses and that exhibits a
high safety profile.
[0007] U.S. Pat. Nos. 6,998,252; 7,015,024; 7,045,136 and 7,045,313
relate to recombinant poxviruses, such as vaccinia.
[0008] MVA-based vaccines have been used in clinical studies, for
instance, against HIV, tuberculosis, malaria and cancer. In all of
these studies, at least two doses were used. The human dose of an
MVA-based vaccine was 5.times.10.sup.7 to 5.times.10.sup.8 PFU as
applied in clinical trials (Brookes et al. 2008; Cebere et al.
2006; Tykodi and Thompson 2008;).
[0009] MVA has been used recently as a vector in pandemic H5N1
(Kreijtz et al. 2008; Kreijtz et al. PLoS One 2009; Kreijtz et al.
Vaccine 2009; Kreijtz et al. J. Infect. Dis. 2009; Kreijtz et al.
2007; Mayrhofer et al., 2009; Poon et al. 2009) and H1N1 (Hessel et
al. 2010; Kreijtz et al., J. Infect. Dis. 2009) influenza research.
An MVA-based vaccine expressing NP and M1 is currently being tested
in an ongoing clinical trial (Berthoud et al. 2011).
[0010] Thus, there remains a need in the art for a more broadly
protective influenza vaccine.
DETAILED DESCRIPTION
[0011] The present invention provides recombinant viruses (also
referred to as recombinant viral vectors herein) useful for
generating a heterosubtypic immune response to influenza A viruses.
The recombinant viruses are recombinant vaccinia viruses, such as
recombinant MVA or other non-replicating or replicating vaccinia
virus known in the art. Non-replicating vaccinia viruses include,
but are not limited to, defective vaccinia Lister (dVV), MVA-575
(ECACC V00120707), MVA-BN (ECACC V00083008), MVA-F6 and MVA-M4
(Antoine et al. 1998). In some embodiments, the recombinant viruses
encode a fusion protein (h1HA/M2e) comprising an influenza A
hemagglutinin deletion mutant "headless HA" (h1HA) with at least
one influenza A M2 external domain (M2e) insert; an h1HA/M2e fusion
protein and an influenza A nucleoprotein (NP); or an h1HA and NP.
The recombinant viruses of the invention may further encode an
influenza A matrix protein 1 (M1) and/or an influenza A polymerase
PB1. When administered according to methods of the invention, the
recombinant viruses are cross-protective and induce heterosubtypic
humoral and cellular immune responses (including CD8 and CD4 T cell
responses). The recombinant viruses are therefore contemplated to
be useful as universal influenza A vaccines in humans.
[0012] In some embodiments, the h1HA amino acid sequence encoded by
an open reading frame in recombinant viruses of the invention may
be, for example, the h1HA amino acid sequence set out in SEQ ID NO:
15 (based on A/VietNam/1203/2004 H5N1 HA NCBI Genbank AAW80717
which is SEQ ID NO: 3). The h1HA of SEQ ID NO: 15 comprises a
signal sequence, the HA1 residues 17-58 of SEQ ID NO: 3, a linker
peptide of four glycines, the HA1 residues 290-343 of SEQ ID NO: 3
and the HA2 stalk region residues 344-568 of SEQ ID NO: 3.
[0013] In some embodiments, the h1HA/M2e fusion protein amino acid
sequence encoded by an open reading frame in recombinant viruses of
the invention may be, for example, the h1HA/M2e fusion protein
amino acid sequence set out in SEQ ID NO: 2. The fusion protein of
SEQ ID NO: 2 comprises a signal sequence, the HA1 residues 17-58 of
SEQ ID NO: 3, a linker peptide of three glycines (SEQ ID NO: 4),
the M2e of H5N1 (SEQ ID NO: 5 based on A/VietNam/1203/2004 H5N1
NCBI Genbank ABP35634), a six-amino acid linker GSAGSA (SEQ ID NO:
9), the M2e of H1N1 (equivalent to H2N2 and H3N2) (SEQ ID NO: 6
based on A/New York/3315/2009 H1N1 NCBI Genbank ACZ05592), a
six-amino acid linker GSAGSA (SEQ ID NO: 9), the M2e of H9N2 (SEQ
ID NO: 7 based on A/chicken/Korea/SH0913/2009 H9N2 NCBI Genbank
ADQ43641), a six-amino acid linker GSAGSA (SEQ ID NO: 9), the M2e
of H7N2 (SEQ ID NO: 8 based on A/New York/107/2003 H7N2 NCBI
Genbank ACC55276), a linker peptide of three glycines (SEQ ID NO:
4), the HA1 residues 290-343 of SEQ ID NO: 3 and the HA2 region
residues 344-568 of SEQ ID NO: 3.
[0014] In some embodiments, the h1HA/M2e fusion protein may
comprise one, two, three or four of the M2e polypeptides of SEQ ID
NOs: 5, 6, 7 and 8. The h1HA/M2e fusion protein may comprise an
influenza A M2e polypeptide other than an M2e polypeptide of SEQ ID
NOs: 5, 6, 7, and 8.
[0015] In some embodiments, the NP amino acid sequence encoded by
an open reading frame in recombinant viruses of the invention may
be, for example, the NP amino acid sequence set out in SEQ ID NO:
13 (based on A/VietNam/1203/2004 H5N1 NP NCBI Genbank AAW80720). In
some embodiments, the M1 amino acid sequence encoded by an open
reading frame in recombinant viruses of the invention may be, for
example, the M1 amino acid sequence set out in SEQ ID NO: 11 (based
on A/VietNam/1203/2004 H5N1 M1 Genbank AAW80726). In some
embodiments, the PB1 amino acid sequence encoded by an open reading
frame in recombinant viruses of the invention may be, for example,
the PB1 amino acid sequence set out in SEQ ID NO: 17 (based on
A/VietNam/1203/2004 H5N1 PB1 Genbank AAW80711).
[0016] The invention contemplates that polypeptides encoded by an
open reading frame in a recombinant virus may vary in sequence from
SEQ ID NO: 2, 5, 6, 7, 8, 11, 13, 15 and/or 17 if the polypeptides
retain the ability to induce a protective immune response when the
recombinant virus is administered to an individual. In these
embodiments, the polypeptide may be about 80%, about 85%, about
90%, about 91%, about 92%, about 93%, about 94%, about 95%, about
95%, about 97%, about 98% or about 99% identical to SEQ ID NO: 2,
5, 6, 7, 8, 11, 13, 15 and/or 17.
[0017] In other embodiments, h1HA/M2e fusion proteins, h1HA
polypeptides and NP polypeptides encoded by recombinant viruses of
the invention may be based on the same or different influenza A
subtypes including, but not limited to, any combination of H1 to
H16 and N1 to N9 (including H1N1, H2N1, H3N1, H4N1, H5N1, H6N1,
H7N1, H8N1, H9N1, H10N1, H11N1, H12N1, H13N1, H14N1, H15N1, H16N1;
H1N2, H2N2, H3N2, H4N2, H5N2, H6N2, H7N2, H8N2, H9N2, H10N2, H11N2,
H12N2, H13N2, H14N2, H15N2, H16N2; H1N3, H2N3, H3N3, H4N3, H5N3,
H6N3, H7N3, H8N3, H9N3, H10N3, H11N3, H12N3, H13N3, H14N3, H15N3,
H16N3; H1N4, H2N4, H3N4, H4N4, H5N4, H6N4, H7N4, H8N4, H9N4, H10N4,
H11N4, H12N4, H13N4, H14N4, H15N4, H16N4; H1N5, H2N5, H3N5, H4N5,
H5N5, H6N5, H7N5, H8N5, H9N5, H10N5, H11N5, H12N5, H13N5, H14N5,
H15N5, H16N5; H1N6, H2N6, H3N6, H4N6, H5N6, H6N6, H7N6, H8N6, H9N6,
H10N6, H11N6, H12N6, H13N6, H14N6, H15N6, H16N6; H1N7, H2N7, H3N7,
H4N7, H5N7, H6N7, H7N7, H8N7, H9N7, H10N7, H11N7, H12N7, H13N7,
H14N7, H15N7, H16N7; H1N8, H2N8, H3N8, H4N8, H5N8, H6N8, H7N8,
H8N8, H9N8, H10N8, H11N8, H12N8, H13N8, H14N8, H15N8, H16N8; H1N9,
H2N9, H3N9, H4N9, H5N9, H6N9, H7N9, H8N9, H9N9, H10N9, H11N9,
H12N9, H13N9, H14N9, H15N9, and H16N9). In some embodiments the
influenza A subtype is a pandemic influenza A. Exemplary pandemic
influenza subtypes include, but are not limited to, H1N1, H2N2,
H3N2 and H5N1.
[0018] A list of identified Influenza A strains, including
influenza A H1N1 strains, is available from the World Health
Organization (WHO) and United States Centers for Disease Control
(CDC) databases of Influenza A subtypes. The National Center for
Biotechnology Information (NCBI) database maintained by the United
States National Library of Medicine also maintains an updated
database describing the length and sequence of HA, M2, NP, M1 and
PB1 genes of viruses of influenza A species. Strains listed by
these organizations and strains described in other commercial and
academic databases, or in literature publications and known in the
art, are contemplated for use in the invention. It is also
contemplated that additional influenza A strains hereafter
identified and isolated are also useful in the invention as sources
of influenza A protein sequences. Accordingly, any strain
specifically exemplified in the specification and those known or
after discovered in the art are amenable to the recombinant
vaccinia virus, pharmaceutical compositions, and methods of the
invention. Exemplary strains include, but are not limited to, the
strains in Table 1 below. The table also lists exemplary genes and
associated database accession numbers of those strains.
TABLE-US-00001 TABLE 1 Inserted Virus Influenza NCBI gene NCBI
amino acid Subtype gene Virus Strain acc no. acc no. H5N1 HA A/Viet
Nam/1203/2004 AY818135 AAW80717 H5N1 NP A/Viet Nam/1203/2004
AY818138 AAW80720 H5N1 M1 A/Viet Nam/1203/2004 AY818144 AAW80726
H5N1 PB1 A/Viet Nam/1203/2004 AY818129 AAW80711 H5N1 M2 A/Viet
Nam/1203/2004 EF541453 ABP35634 H1N1 sw M2 A/California/07/09
FJ969537 ACP44185 H1N1 M2 A/New York/3315/2009 CY050765 ACZ05592
H2N2 M2 A/Korea/426/68 NC_007377 YP_308853 H3N2 M2 A/New
York/392/2004 NC_007367 YP_308840 H9N2 M2
A/chicken/Korea/SH0913/2009 HQ221654 ADQ43641 H7N2 M2 A/New
York/107/2003 EU587373 ACC55276 H7N3 M2
A/chicken/Pakistan/34668/1995 CY035834 ACJ03948
[0019] In recombinant viruses of the invention, open reading frames
encoding h1HA/M2e, h1HA, NP, M1 and/or PB1 may be codon-optimized
for expression in human cells. In these embodiments, one or more
(or all) of the naturally occurring codons in an open reading frame
have been replaced in the codon-optimized open reading frame with
codons frequently used in genes in human cells (sometimes referred
to as preferred codons). Codons may be optimized to avoid repeat
sequences to stabilize an open reading frame in the rMVA and/or to
avoid unwanted transcription stop signals. Codon-optimization, in
general, has been used in the field of recombinant gene expression
to enhance expression of polypeptides in cells.
[0020] Gene cassettes encoding h1HA/M2e, h1HA, NP, M1 and PB1 in
recombinant viruses of the invention include an open reading frame
under the control of (i.e., operatively linked to) a promoter that
functions (i.e., directs transcription of the open reading frame)
in the recombinant vaccinia viruses. In exemplary embodiments,
expression from gene cassettes is under the control of the strong
early/late vaccinia virus mH5 promoter (SEQ ID NO: 18) or the
synthetic early/late selP promoter (SEQ ID NO: 19) (Chakrabarti et
al. 1997). In the gene cassettes of the invention the open reading
frame is also operatively linked to a transcription stop signal
such as a vaccinia virus early transcription stop signal.
[0021] In one aspect, the invention provides recombinant vaccinia
virus comprising a gene cassette encoding an influenza A h1HA/M2e
fusion protein. In some embodiments, the recombinant vaccinia virus
is a recombinant MVA comprising a gene cassette expressing the
h1HA/M2e fusion protein set out in SEQ ID NO: 2. In some
embodiments, the recombinant vaccinia virus further comprises a
gene cassette expressing the M1 protein (for example, the M1 set
out in SEQ ID NO: 11) and/or a gene cassette expressing the PB1
protein (for example, the PB1 protein set out in SEQ ID NO:
17).
[0022] In another aspect, the invention provides recombinant
vaccinia virus comprising a first gene cassette encoding an
influenza A h1HA/M2e fusion protein, and a second gene cassette
encoding an influenza NP. In some embodiments, the recombinant
vaccinia virus is a recombinant MVA comprising a first gene
cassette expressing the h1HA/M2e fusion protein set out in SEQ ID
NO: 2 and a second gene cassette expressing the NP set out in SEQ
ID NO: 13. In some embodiments, the recombinant vaccinia virus
further comprises a gene cassette expressing the M1 protein (for
example, the M1 set out in SEQ ID NO: 11) and/or a gene cassette
expressing the PB1 protein (for example, the PB1 protein set out in
SEQ ID NO: 17).
[0023] In yet another aspect, the invention provides recombinant
vaccinia virus comprising a first gene cassette encoding an
influenza A h1HA and a second gene cassette encoding an influenza
NP. In some embodiments, the recombinant vaccinia virus is a
recombinant MVA comprising a first gene cassette expressing the
h1HA set out in SEQ ID NO: 15 and a second gene cassette expressing
the NP set out in SEQ ID NO: 13. In some embodiments, the
recombinant vaccinia virus further comprises a gene cassette
expressing the M1 protein (for example, the M1 set out in SEQ ID
NO: 11) and/or a gene cassette expressing the PB1 protein (for
example, the PB1 protein set out in SEQ ID NO: 17).
[0024] In recombinant vaccinia viruses of the invention, the gene
cassettes may be inserted in non-essential regions of the vaccinia
virus genome, such as the deletion I region, the deletion II
region, the deletion III region, the deletion IV region, the
thymidine kinase locus, the D4R/5R intergenic region, or the HA
locus. In exemplified embodiments of recombinant MVA, the insertion
of the h1HA/M2e and h1HA gene cassettes is in the D4R/5R intergenic
region and the insertion of the NP gene cassette is in the deletion
III region. The recombinant MVA is derived from an MVA free of
bovine spongiform encephalopathy (BSE) such as MVA74 LVD6 obtained
from the National Institutes of Health.
[0025] The recombinant viruses of the invention may be formulated
as pharmaceutical compositions according to methods known in the
art. In some embodiments, the recombinant viruses are formulated as
described in International Publication No. WO 2010/056991.
[0026] The invention provides methods of inducing a heterosubtypic
influenza A immune response in an individual comprising
administering compositions of recombinant vaccinia virus of the
invention to the individual. In the methods, the composition may be
administered as a single dose, a double dose or multiple doses. The
administration route in humans may be inhalation, intranasally,
orally, and parenterally. Examples of parenteral routes of
administration include intradermal, intramuscular, intravenous,
intraperitoneal and subcutaneous administration. The range of the
human immunization dose may be about 10.sup.6 to about 10.sup.9
PFU. The methods of the invention induce humoral and cellular
immune responses in the individual. Moreover, in embodiments of the
invention the methods induce a protective immune response in the
individual. The protective immune response may be where the
individual exhibits no symptoms of infection, a reduction in
symptoms, a reduction in virus titer in tissues or nasal
secretions, and/or complete protection against infection by
influenza virus.
[0027] The invention also provides kits for administering
recombinant vaccinia virus of the invention packaged in a manner
which facilitates their use to practice methods of the invention.
In one embodiment, such a kit includes a recombinant virus or
composition described herein, packaged in a container such as a
sealed bottle or vessel, with a label affixed to the container or
included in the package that describes use of the compound or
composition in practicing the method. Preferably, the recombinant
virus or composition is packaged in a unit dosage form. The kit may
further include a device suitable for administration according to a
specific route of administration or for practicing a screening
assay. Preferably, the kit contains a label that describes use of
the recombinant vaccinia virus. In some embodiments, the kit
comprises instructions for administration to a human subject.
[0028] Also provided are methods of producing a recombinant
vaccinia virus expressing a gene cassette of the invention. As
illustrated with MVA, the methods comprise the steps of: a)
infecting primary chicken embryo cells or a suitable permanent cell
line (e.g., avian) with MVA, b) transfecting the infected cells
with a plasmid comprising the gene cassette and comprising DNA
flanking the gene cassette that is homologous to a non-essential
region of the MVA genome, c) growing the cells to allow the plasmid
to recombine with the MVA genome during replication of the MVA in
chicken cells thereby inserting the gene cassette into the MVA
genome in the non-essential region, and d) obtaining the
recombinant MVA produced. Exemplary chicken embryo cells are
described in U.S. Pat. No. 5,391,491. (Slavik et al. 1983) Other
avian cells (e.g., DF-1) are also contemplated. In the methods, the
non-essential MVA region is the deletion I region, the deletion II
region (Meyer et al. 1991), the deletion III region (Antoine et al.
1996), the deletion IV region (Meyer et al., supra; Antoine et al.
1998) the thymidine kinase locus (Mackett et al. 1982), the D4R/5R
intergenic region (Holzer et al. 1998), or the HA locus (Antoine et
al. supra). In one exemplified embodiment, the insertion is in the
deletion III region. In another exemplified embodiment, the
insertion is in the D4R/5R intergenic region. If two gene cassettes
are to be inserted, the two are inserted in different non-essential
regions. Gene cassettes may additionally be inserted into any other
suitable genomic region or intergenomic regions.
[0029] Other vertebrate cell lines are useful for culture and
growth of vaccinia virus of the invention. Exemplary vertebrate
cells useful to culture vaccinia virus of the invention include,
but are not limited to, MRC-5, MRC-9, CV-1 (African Green monkey),
HEK (human embryonic kidney), PerC6 (human retinoblast), BHK-21
cells (baby hamster kidney), BSC (monkey kidney cell), LLC-MK2
(monkey kidney) and permanent avian cell lines such as DF-1.
[0030] Vero cells are an accepted cell line for production of viral
vaccines according to the World Health Organization. In some
embodiments, recombinant replicating vaccinia virus of the
invention are produced in Vero cells.
[0031] Additional aspects and details of the invention will be
apparent from the following examples, which are intended to be
illustrative rather than limiting.
FIGURES
[0032] FIG. 1 shows the amino acid sequence (SEQ ID NO: 15) of the
headless HA protein encoded by recombinant MVA (rMVA) of the
invention. The protein contains a signal sequence (grey), HA1
residues (red), a linker peptide of four glycines (black), HA1
residues (red), and the HA2 stalk region (black). Cysteines 58 and
63 and the polybasic cleavage site (amino acids 112-119) are
underlined. FIG. 2 shows the nucleotide sequence (SEQ ID NO: 14) of
the headless HA protein encoded by rMVA of the invention.
[0033] FIG. 3 shows the amino acid sequence (SEQ ID NO: 2) of
headless HA/M2e fusion protein. The designed protein contains a
signal sequence (grey), HA1 residues (red), a linker peptide of
three glycines (black), the M2e of H5N1 (blue), the six amino acid
linker GSAGSA (black), the M2e of H1N1 (equivalent to H2N2, H3N2;
green), the six amino acid linker GSAGSA, the M2e of H9N2 (orange),
the six amino acid linker GSAGSA, the M2e of H7N2 (pink), a linker
peptide of three glycines (black), HA1 residues (red) and the HA2
stalk region (black). The polybasic cleavage site (amino acids
224-231) is underlined. FIG. 4 shows the nucleotide sequence (SEQ
ID NO: 1) of the headless HA/M2e fusion protein encoded by rMVA of
the invention.
[0034] FIG. 5 shows single-insert rMVAs containing influenza genes.
A) indicates the h1HA, h1HA/M2e, M2, PB1, or M1 gene cassettes that
are located in the recombinant MVA D4R/D5R intergenic locus, at the
position corresponding to nucleotide 87,281 of wild type MVA
(Antoine et al, supra). B) indicates the NP gene cassette is
located in the del 111 locus at the position corresponding to
nucleotide 142,992 of wild type MVA.
[0035] FIG. 6 shows a Western Blot of chicken cell lysates tested
for influenza virus antigens. A) Expression of headless HA and the
headless HA/M2e fusion protein using a detection antibody directed
against HA. Lane 1, protein ladder, size in kDa; lane 2, MVA-h1HA;
lane 3, MVA-h1HA/M2e; lane 4, MVA wt (negative control); and lane
5, MVA-HA-VN (positive control). B) Expression of the headless
HA/M2e fusion protein using a detection antibody directed against
M2. Lane 1, protein ladder, size in kDa; lane 2, MVA-M2-VN; lane 3,
MVA-h1HA/M2e; and lane 4, MVA wt (negative control). The
recombinant MVA-M2-VN expresses the M2 protein (weak band below 15
kDa). The anti-M2-antibody binds a peptide at the N-terminus of the
M2 protein; thus the expression of the h1HA/M2e fusion protein is
also detectable at around 70 kDa (lane 3).
[0036] FIG. 7 shows double-insert rMVAs containing influenza genes.
The h1HA or h1HA/M2e gene cassette is located in the D4R/D5R
intergenic locus, at the position corresponding to nucleotide
87,281 of wild type MVA. The NP gene cassette is located in the del
111 locus at the position corresponding to nucleotide 142,992 of
the wild type MVA.
[0037] FIG. 8 shows a Western Blot of chicken cell lysates tested
for influenza virus antigens. A) Expression of headless HA and the
headless HA/M2e fusion protein using a detection antibody directed
against HA. Lanes 1 and 7, protein ladder, size in kDa; lane 2,
MVA-HA-VN (positive control); lane 3, MVA-h1HA; lane 4, MVA wt
(negative control); lane 5, MVA-h1HA/M2e-NP; and lane 6,
MVA-h1HA-NP. The h1HA/M2e fusion protein expressed by MVA-h1HA/M2e
is visible at around 70 kDa (lane 5). The lower bands at around 40
kDa represent the h1HA expressed by MVA-h1HA-NP and MVA-h1HA. The
control construct (MVA-HA-VN), expressing the full length HA
protein express the HA0 (band around 80 kDa), the HA1 (band around
55 kDa, and the HA2 (band around 25 kDa). The expression of the HA2
protein is also visible in lanes 3, 5 and 6 as the h1HA and
h1HA/M2e proteins also contain the polybasic cleavage site. The
specific HA bands are absent in the negative control (lane 4). B)
NP expression detected with an NP-specific antibody. Lane 1,
protein ladder, size in kDa; lane 2, MVA-D3-NP-VN; lane 3,
MVA-h1HA-NP; lane 4, MVA-h1HA/M2e-NP; and lane 5, MVA wt (negative
control).
[0038] FIG. 9 shows monitoring of weight (A, B), clinical symptoms
(C, D) and survival (E, F) after vaccination with recombinant MVAs
and challenge with H5N1. As controls, mice were vaccinated with
MVA-HA-VN, expressing the full-length HA of A/Vietnam/1203/2004, wt
MVA or were treated with PBS (panels A, C, E). Mice were vaccinated
with the single recombinant MVA-h1HA, MVA-h1HA/M2e, MVA-NP-VN or
the double recombinants MVA-h1HA-NP and MVA-h1HA/M2e-NP (panels B,
D, F). After challenge with wild-type H5N1, mice were monitored for
14 days.
[0039] FIG. 10 shows monitoring of weight (A, B), clinical symptoms
(C, D) and survival (E, F) after vaccination with recombinant MVAs
and challenge with H9N2 virus. As controls, mice were vaccinated
with the whole virus preparation of H9N2, wt MVA or were treated
with PBS (panels A, C, E). Mice were vaccinated with the single
recombinant MVA-h1HA, MVA-h1HA/M2e, MVA-NP-VN or double recombinant
MVA-h1HA-NP and MVA-h1HA/M2e-NP (panels B, D, F). After challenge
with virulent mouse-adapted H9N2 influenza virus, mice were
monitored for 14 days.
[0040] FIG. 11 shows triple-insert rMVAs containing influenza
genes. The h1HA or h1HA/M2e and M1gene cassettes will be located in
the D4R/D5R intergenic locus, at the position 87,281 nt of the wt
MVA sequence. The NP gene cassette will be located in the del 111
locus at the position 142,992 nt of the wt MVA sequence.
EXAMPLES
[0041] The present invention is illustrated by the following
examples wherein Example 1 describes the choice and design of
influenza A antigens in exemplary recombinant MVA of the invention,
Example 2 details the production of single-insert recombinant MVAs,
Example 3 describes animal experiments with the single-insert MVAs,
Example 4 details the production of double-insert recombinant MVAs,
Example 5 describes animal experiments with the double-insert MVAs,
Example 6 details the production of triple-insert recombinant MVAs
and Example 7 describes animal experiments with the triple-insert
MVAs.
Example 1
Choice and Design of Influenza a Antigens
[0042] Influenza headless HA, a headless HA/M2e fusion protein, NP,
M1, M2 and PB1 were the influenza A antigens chosen to be encoded
by exemplary recombinant MVA of the invention.
[0043] Monoclonal antibodies against the HA stalk domain, the HA2
region, are broadly cross-reactive and neutralize several subtypes
of viruses (Ekiert et al. 2009; Kashyap et al. 2008; Okuno et al.
1993; Sanchez-Fauquier et al. 1987; Sui et al. 2009; Throsby et al.
2008). The antibodies target the HA2 region of the molecule and
presumably act by preventing the conformational change of HA at low
pH, thus presumably blocking fusion of viral and host membranes
during influenza infection. However, the production of soluble,
native (neutral pH-like) HA2 immunogen has proven to be difficult,
owing to the metastable nature of HA (Chen et al. 1995). To induce
an immune response against the neutral pH conformation, a headless
HA was chosen as an antigen. The headless HA consists of two HA1
regions that interact with an HA2 subunit, stabilizing the neutral
pH conformation (Bommakanti et al., supra; Steel et al.,
supra).
[0044] The extracellular domain of the M2 protein (M2e, 23AS) is
highly conserved across influenza A virus subtypes. In animals, M2e
specific antibodies reduce the severity of infection with a wide
range of influenza A virus strains (Fan et al. 2004; Neirynck et
al. 1999). Many groups have reported M2e-based vaccine candidates
in different forms (De Filette et al. 2008; Denis et al. 2008;
Eliasson et al. 2008; Fan et al., supra; Neirynck et al., supra).
Recently, Zhao et al. reported that a tetra-branched multiple
antigenic peptide vaccine based on H5N1 M2e induced strong immune
responses and cross protection against different H5N1 clades and
even heterosubtypic protection from 2009 H1N1 (Zhao et al. 2010b;
Zhao et al. 2010a).
[0045] Vaccination using vectors expressing the conserved influenza
NP, or a combination of NP and matrix protein has been studied in
animal models and various degrees of protection against both
homologous and heterologous viruses have been demonstrated (Price
et al., supra; Ulmer et al. 1993). NP elicit a robust CD8.sup.+ T
cell response in mice and in humans (McMichael et al., 1986;
Yewdell et al., 1985) that, as epidemiological studies suggest, may
contribute to resistance against severe disease following influenza
A virus infection (Epstein 2006).
[0046] The headless HA included in rMVA of the invention is a new
headless HA (h1HA) based on the VN/1203 influenza strain. The h1HA
contains a polybasic cleavage site which is cleaved during
expression from the rMVA exposing the fusion peptide for the immune
system. The amino acid sequence of the h1HA is set out in FIG. 1
and in SEQ ID NO: 15. The nucleotide sequence of the MVA insert is
set out FIG. 2 and SEQ ID NO: 14.
[0047] The amino acid sequence of the headless HA/M2e fusion
protein included in rMVA of the invention is set out in FIG. 3
below and in SEQ ID NO: 2. The nucleotide sequence of the fusion
protein is set out in FIG. 4 below and in SEQ ID NO: 1. In the
fusion protein, the M2e domains of H5N1, H9N2, H7N2 and H1N1
(equivalent to H2N2, H3N2) form an M2e "head" on the h1HA. The four
particular M2e domains were chosen to represent the M2e from
seasonal and pandemic strains.
Example 2
Construction and Characterization of Single-Insert MVA Vectors
[0048] The following single-insert, recombinant MVA (rMVA) are
utilized in the experiments described herein.
TABLE-US-00002 TABLE 2 rMVA Inserted influenza gene NCBI gene acc
no. 1. MVA-h1HA headless HA based on AY818135 2. MVA-h1HA/M2e
headless HA/M2e fusion based on AY818135 3. MVA-M1-VN Matrix
protein 1 AY818144 4. MVA-M2-VN Matrix protein 2 EF541453 5.
MVA-PB1-VN Polymerase subunit PB1 AY818129 6. MVA-mNP Nucleoprotein
AY818138 7. Control MVA-HA-VN Hemagglutinin AY818135 8. Control
MVA-wt No insert -- 9. Control PBS No insert --
[0049] For construction of single-insert rMVA vectors expressing
h1HA, the h1HA/M2e fusion protein or PB1, the h1HA, h1HA/M2e and
PB1 genes were chemically synthesized (Geneart, Inc., Regensburg,
Germany). The synthetic genes are driven by the strong vaccinia
early/late promoter mH5 (Wyatt et al. 1996) and terminated with a
vaccinia virus specific stop signal downstream of the coding region
that is absent internally. The gene cassettes were cloned in the
plasmid pDM-D4R (Ricci et al., 2011) resulting in plasmids
pDM-h1HA, pDM-h1HA/M2e and pDM-PB1-VN, respectively. The
introduction of the foreign genes into the D4R/D5R intergenic
region of MVA was done as described elsewhere (Ricci et al. 2011)
resulting in viruses MVA-h1HA, MVA-h1HA/M2e, MVA-PB1-VN.
[0050] For the construction of the rMVA expressing M1, the M1
sequence (accession number AY818144) was placed downstream of the
strong vaccinia early/late promoter selP (Chakrabarti et al. 1997)
and cloned in pDM-D4R, resulting in pDM-M1-VN. The expression
cassette of pDD4-M2-VN--including the M2 sequence (accession number
EF541453) under the control of the mH5 promoter--was cloned in
pDM-D4R resulting in pDM-M2-VN. The plasmids were then used for
recombination with MVA according to Holzer et al, supra resulting
in the viruses MVA-M1-VN and MVA-M2-VN, respectively as shown in
FIG. 5A.
[0051] For the construction of single-insert MVAs expressing the NP
protein, the NP expression cassette of pDD4-mH5-mNP-VN (Mayrhofer
et al., supra) was cloned in plasmid pd3-lacZ-gpt, resulting in
pd3-lacZ-mH5-NP-VN. Plasmid pd3-lacZ-gpt contains a lacZ/gpt
selection marker cassette and a multiple cloning site (MCS) for
insertion of genes of interest. The sequences are framed by genomic
MVA sequences of the del III region. The marker cassette is
destabilized by a tandem repeat of MVA del III flank, thus the
final recombinant is free of any auxiliary sequences. The insertion
plasmid directs the gene cassettes into the MVA deletion III (del
III) region. After infection of primary chicken embryo cells with
MOI 1, cells were transfected with pd3-lacZ-mH5-NP-VN according to
the calcium phosphate technique (Graham and van der Eb 1973),
resulting MVA-NP-VN shown in FIG. 5B. The MVA strain (MVA 1974/NIH
clone 1) was kindly provided by B. Moss (National Institutes of
Health). Recombinant virus is selected using the transient marker
stabilization method (Scheiflinger et al, 1998).
[0052] The single-insert MVA vectors expressing the NP, PB1, M1,
M2, h1HA, and h1HA/M2e were characterized by PCR and Western blot
as described in Hessel et al, supra. Recombinant viruses were grown
in CEC or DF-1 cells and purified by centrifugation through a
sucrose cushion. Primary CEC were produced in-house and cultivated
in Med199 (Gibco.RTM.) supplemented with 5% fetal calf serum (FCS).
The DF-1 (CRL-12203) cell line was obtained from the ATCC (American
Type Culture Collection) and cultivated in DMEM (Biochrom, Inc.)
supplemented with 5% FCS.
[0053] The correct expression of the influenza proteins by the
rMVAs was confirmed by Western blotting. For this purpose CEC or
the permanent chicken cell line DF-1 were infected with a MOI of
0.1 and cell lysates were prepared 48-72 hrs post infections. The
recombinant MVAs that express the h1HA (MVA-h1HA and MVA-h1HA/M2e)
were analyzed in a Western blot using an anti-influenza
A/Vietnam/1194/04 (H5N1) polyclonal serum (NIBSC 04/214) for
detection. Donkey-anti-sheep alkaline phosphatase-conjugated IgG
(Sigma Inc.) was used as a secondary antibody. The recombinant MVAs
that express the M2 and M2e (MVA-M2-VN and MVA-h1HA/M2e) were
analyzed in Western Blots using an anti-avian influenza M2 antibody
binding a peptide present at the amino terminus of the H5N1 M2
(ProSci, Cat#4333). Goat-anti-rabbit alkaline
phosphatase-conjugated IgG (Sigma Inc.) antibody was used as a
secondary antibody. As shown in FIG. 6A, the recombinant MVAs
expressing the h1HA (MVA-h1HA and MVA-h1HA/M2e) gene inserts
induced expression of the HA containing antigens in avian DF-1
cells. The bands around 40 kDa in lane 2 represent the h1HA. The
larger band at around 70 kDa in lane 3 represents the h1HA/M2e. The
large band at around 80 kDa in lane 5 represents the HA0
hemagglutinin-precursor, which is cleaved into the HA1 and HA2
subunits represent by the bands at approximately 55 and 25 kDa. The
specific h1HA, h1HA/M2e or HA bands are absent in the wild-type MVA
control (lane 4).
[0054] FIG. 6B shows the M2 expression by MVA-M2-VN (lane 2) or
MVA-h1HA/M2e (lane 3). The weak but specific band around 10 kDa in
lane 2 represents the wild-type M2 protein whereas the larger band
around 70 kDa represents the h1HA/M2e protein. Both bands are
absent in the wild-type MVA control (lane 4).
[0055] The expression of the M1, NP and PB1 protein is detected
with polyclonal guinea-pig anti-influenza H5N1 serum produced in
house, a polyclonal goat antibody detecting the PB1 of Influenza A
virus (Santa Cruz, Cat#: vC-19), and a monoclonal
mouse-anti-NP-antibody (BioXcell, Cat# BE0159), respectively. The
MVA-M1-VN and MVA-NP-VN induce expression of the M1 protein (around
27 kDa) and the NP protein (around 60 kDa) (not shown).
Example 3
Animal Experiments with the Single-Insert Vaccines
Protection Experiment
[0056] A standard protection experiment consists of two arms
(primed with about 1.times.10.sup.3-1.times.10.sup.5 TCID.sub.50
H1N1v CA/07 and unprimed) of nine groups of mice each (respectively
vaccinated i.m. with 1.times.10.sup.6 pfu of the nine vaccines and
controls shown in Table 2), a group consisting of six animals
resulting in 108 animals, defines one set. The animals of one set
are challenged with one of the six challenge viruses shown in Table
3 below.
TABLE-US-00003 TABLE 3 Pre-treatment Challenge strain Subtype
Abbreviation H1N1v/unprimed A/California/07/2009 H1N1 CA/07
H1N1v/unprimed A/Vietnam/1203/2004 H5N1 VN/1203 H1N1v/unprimed
A/HongKong/G9/ H9N2 HK/G9 H1N1v/unprimed A/Victoria/210/2009 H3N2
VF09 H1N1v/unprimed A/FPV/Rostock/34 H7N1 RO/34 H1N1v/unprimed
A/PR8/1934 H1N1 PR8
[0057] Female Balb/c mice are 8-10 weeks old at the pre-treatment
time point and 14-16 weeks old at the time point of immunization
with the vaccines and controls shown in Table 2. Mice were
immunized intramuscularly twice (days 42 and 63) with 10.sup.6 pfu
of the vaccines or wild type MVA, 3.75 .mu.g whole virus
preparation H9N2 A/HongKong/G9/1997 or with buffer (PBS). At day
84, mice were challenged intranasally with 10.sup.3 TCID.sub.50
H5N1 A/Vietnam/1203/2004 (H5N1, CDC #2004706280), with
2.5.times.10.sup.4 TCID.sub.50 mouse adapted H9N2
A/HongKong/G9/1997 or with 1.66.times.10.sup.4 TCID.sub.50 H7N1
A/FPV/Rostock/34. The challenge doses correspond to approx. 30 LD50
for the H5N1 challenge and 32 LD50 for the H9N2 challenge per
animal. Sera are collected at days 41, 62 and 85 and analyzed for
HA-specific IgG concentration by HI titer or microneutralization
assay.
[0058] The primary outcome of the animal experiments is protection
as measured by lethal endpoint, weight loss, or lung titer. Further
the ELISA titers of pooled pre-challenge sera measured against
inactivated whole virus H5N1 strain A/Vietnam/1203/2004 are
determined.
T Cell Experiments
[0059] Frequencies of influenza-specific CD4 and CD8 T cells are
determined in immunized mice by flow cytometry. In a standard
experiment, groups of 5 female BALB/c mice are immunized twice with
the vaccines or controls listed in Table 2. Splenocytes are
re-stimulated in-vitro using inactivated whole virus antigens of
different influenza strains for CD4 T-cells and, when available,
peptides representing the CD8 T-cell epitopes of the vaccine insert
constructs and IFN-.gamma. production are measured. All experiments
are performed twice, using a total of 140 animals.
Other Experiments
[0060] An evaluation of the cell-mediated immunity after a single
immunization, demonstration of functional activity of cytotoxic
T-cells in a VITAL assay and assessment of recruitment of
influenza-specific T-cells into the lungs of challenged animals are
also carried out. The induction/expansion of vaccine-specific
T-cells is also monitored in the primed mouse model by immunizing
mice which resolved a influenza virus infection once with these
vaccines.
Example 4
Construction and Characterization of Double-Insert rMVA Vectors
[0061] The following double-insert, rMVA and controls are utilized
in the experiments described herein.
TABLE-US-00004 TABLE 4 rMVA Inserted influenza gene Comment 1.
MVA-h1HA-NP headless HA + NP Double insert construct 2.
MVA-h1HA/M2e-NP headless HA/m2e fusion protein + NP Double insert
construct 3. MVA-NP-VN nucleoprotein Control 4. MVA-HA-VN
hemagglutinin Control 5. MVA-wt Empty vector Neg. control 6. PBS --
Neg. control
[0062] For the construction of the double insert rMVA vector
co-expressing either the h1HA or h1HA/M2e gene cassette in
combination with the NP protein gene cassette, the single insert
MVA recombinants of Example 2 containing the h1HA or h1HA/M2e gene
cassette are used. CEC cells were infected with MVA-h1HA or
MVA-h1HA/Me2 and afterwards transfected with pd3-lacZ-mH5-NP-VN
(see Example 2). Homologous recombination and propagation of the
recombinant MVA vectors are performed as described in Example 2.
The resulting double insert MVA vectors, named MVA-h1HA-NP or
MVA-h1HA/M2e-NP, contain the h1HA or h1HA/M2e expression cassette
in the D4R/D5R locus and the NP expression cassette in the del III
locus. See FIG. 7.
[0063] The recombinant MVAs were characterized by Western Blot as
described in Example 2. FIG. 8A shows the expression of the h1HA
and h1HA/M2e after infection of CEC with MVA-h1HA-NP (lane 6) or
MVA-h1HA/M2e-NP (lane 5). The bands around 40 kDa in lanes 3 and 6
represent the h1HA of the MVA-h1HA and MVA-h1HA-NP constructs. The
band around 70 kDa in lane 5 represents the h1HA/M2e fusion
protein. The HA bands are absent in the wild-type control in lane
4. The same samples were used for detection of NP protein
expression in Western Blots (as described in Example 2). As shown
in FIG. 8B, the recombinant MVAs MVA-h1HA-NP and MVA-h1HA/M2e-NP
also induced expression of the NP protein in avian CEC cells. The
bands around 60 kDa in lanes 2 to 4 represent the NP.
Example 5
Animal Experiments with the Double-Insert Vaccines or Vector
Combinations
Protection Experiment
[0064] A standard experiment included eight groups of mice
(vaccinated with the six vaccines and controls shown in Table 5)
each group consisting of six animals. The protection experiments
were carried out as described in Example 3. After challenge mice
were monitored over a time period of 14 days and weight loss or
symptoms including ruffled fur (score of 1), curved posture (score
of 2), apathy (score of 3), and death (score of 4) were recorded.
For ethical reasons, mice were euthanized after weight loss of
.gtoreq.25%. Protection results are compiled in Table 5 and
displayed in FIGS. 9 and 10.
TABLE-US-00005 TABLE 5 Protection of mice from death after double
dose vaccinations with recombinant MVAs and homologous or
heterologous challenge. VN1203.sup.(1) challenge HK/G9.sup.(2)
After H5N1 Clinical Protection After H9N2 Clinical challenge n/nt
Gr Vaccine score at day 14 n/nt.sup.(3) (%) score at day 14 (%) 1
MVA-h1HA-NP 2.83 2/6 (33) 0 6/6 (100) 2 MVA-h1HA/M2e-NP 1 5/6 (83)
0 6/6 (100) 3 MVA-h1HA 2.67 2/6 (33) 3.33 1/6 (17) 4 MVA-h1HA/M2e 4
0/6 (0) 2.67 2/6 (33) 5 MVA-NP-VN 3.33 2/6 (33) 0 6/6 (100) 6
Homologous 0 6/6 (100) 0 6/6 (100) control vaccine.sup.(4) 7
MVA-wt(5) 4 0/6 (0) 2.83 2/6 (33) 8 PBS 2.67 2/6 (33) 4 0/6 (0)
.sup.(1)VN1203, challenge strain A/Vietnam/1203/2004;
.sup.(2)HK/G9, challenge strain A/HongKong/G9/1997; .sup.(3)n/nt,
survival per group, .sup.(4)Homologous control vaccine;
(5)wild-type MVA (NIH74 LVD clone 6).
[0065] As positive control mice were vaccinated with homologous
control constructs. In case of H5N1 challenge mice were vaccinated
with MYA-HA-VN (Hessel et al., 2011) and in case of H9N2 challenge
mice were vaccinated with an inactivated whole virus preparation of
the H9N2 A/HongKong/G9/1997 influenza virus. Both controls induced
full protection (Table 5; FIGS. 9 and 10, panels E). In the
wild-type MVA and buffer groups all mice showed marked weight loss
compared to the positive control groups and nearly all mice died
after challenge. Mice vaccinated with the single recombinant MVAs
(MVA-h1HA, MVA-h1HA/M2e, MVA-D3-NP-VN) showed no significantly
better protection after the strong H5N1 challenge compared to the
negative control groups (FIG. 9 A-F). Also against heterosubtypic
(H9N2) challenge no significant protection was seen in MVA-h1HA and
MVA-h1HA/M2e vaccinated groups (FIG. 10).
[0066] Surprisingly, however, vaccination with the double construct
expressing the fusion protein h1HA/M2e and the NP protein resulted
in nearly full protection (FIG. 9 B, D, F) after the H5N1 challenge
with approx. 30 LD50 per animal. Also after heterosubtypic
challenge (with approx. 32 LD50 H9N2 virus) mice were fully
protected after vaccination with the double recombinant
MVA-h1HA/M2e-NP. Furthermore, the double recombinant MVA-h1HA-NP
and the single recombinant MVA-NP-VN induced full protection
against the heterosubtypic challenge with H9N2 (FIG. 10, B, D, F).
As can be seen in the weight monitoring (FIGS. 9 and 10, panels B)
and in the clinical scores (FIGS. 9 and 10, panels D), the double
construct MVA-h1HA/M2e-NP showed the best results presumably by
combined beneficial effects contributed by the different influenza
antigens.
T Cell Experiments
[0067] Frequencies of influenza-specific CD4 and CD8 T cells are
determined in immunized mice by flow cytometry. In a standard
protocol experiment, groups of 5 female BALB/c mice are immunized
twice with the vaccines or controls listed in Table 4. Splenocytes
are re-stimulated in-vitro using inactivated whole virus antigens
of different influenza strains for CD4 T-cells and, when available,
peptides representing the CD8 T-cell epitopes of the vaccine insert
constructs and IFN-.gamma. production are measured. All experiments
are performed twice.
Other Experiments
[0068] An evaluation of the cell mediated immunity after a single
immunization, demonstration of functional activity of cytotoxic
T-cells in a VITAL assay and assessment of recruitment of
influenza-specific T-cells into the lungs of challenged animals are
also carried out. The induction/expansion of vaccine-specific
T-cells is also monitored in the primed mouse model by immunizing
mice which resolved a influenza virus infection once with these
vaccines.
Example 6
Construction and Characterization of Triple-Insert rMVA Vectors and
Virus-Like Particles
[0069] Influenza virus-like particles (VLPs) induce humoral and
cellular responses and can protect against lethal challenges
(Bright et al. 2007; Pushko et al. 2005; Song et al. 2010). VLPs
chosen for experiments herein comprise either h1HA or h1HA/M2e in
combination with NP and M1. The VLPs are generated from
triple-insert MVA vectors.
[0070] For the construction of the triple-insert MVA vectors
co-expressing either h1HA or h1HA/M2e in combination with the M1
(SEQ ID NO: 11) and the NP protein (SEQ ID NO: 13), the M1 gene
(SEQ ID NO: 10) of pDD4-M1-VN is cloned downstream of the synthetic
early/late promotor selP (Chakrabarti et al. 1997). The resulting
gene cassette is cloned downstream of the h1HA or h1HA/M2e gene
cassette in pDM-h1HA or pDM-h1HA/M2e. The resulting plasmids
harboring a double gene cassette (pDM-h1HA-M1 and pDM-h1HA/M2e-M1)
are used for recombination into defective MVA as described above.
Afterwards, a recombination with an NP gene cassette (SEQ ID NO:
12)-containing plasmid (pD3-lacZ-gpt-NP-VN) is done resulting in a
triple-insert MVA virus. This triple-insert MVA is plaque purified
under transient marker selection.
[0071] The triple-insert MVA vectors, named MVA-h1HA-M1-NP or
MVA-h1HA/M2e-M1-NP contain the h1HA or h1HA/M2e expression cassette
and M1 expression cassette in tandem order in the D4R/D5R locus and
the NP expression cassette in the del III locus (FIG. 7).
[0072] Detection of VLPs is as follows. HeLa or 293 cells are
seeded into T 175 cm.sup.2 flasks and grown in DMEM+10%
FCS+Pen/Strep. To generate VLPs, cells are infected with 1 MOI of
single-insert MVA or triple-insert MVA recombinants, respectively.
Empty MVA vectors or single-insert MVA recombinants without M1 are
used as controls. At 1 h post infection (p.i.), the medium is
replaced by DMEM+Pen/Strep and culture medium is harvested 48 h
p.i. and cellular debris is pelleted by centrifugation at
2.000.times.g for 10 min. The procedure for analyzing VLPs by
sucrose gradient density flotation and sucrose cushion has been
described previously (Chen et al. 2007; Chen et al. 2005;
Gomez-Puertes et al. 2000). The samples are then analyzed by
immunoblotting. Additionally, electron microscopy (EM) analysis
with medium of infected cells is performed.
Example 7
Animal Experiments with the Triple-Insert Vaccines or Vector
Combinations
[0073] A standard experiment includes 6 groups of primed and
unprimed mice (vaccinated with the 6 vaccines and controls shown in
Table 5), each group consisting of 6 animals, resulting in 36
animals (1 set). The animals are challenged with one of the 6
challenge viruses shown in Table 3. In sum, there are 6 sets of 72
animals each requiring 432 mice to assess cross-protection in the
primed and naive models.
TABLE-US-00006 TABLE 5 rMVA Inserted influenza gene(s) comment 1.
MVA-h1HA-M1-NP headless HA + nucleoprotein + 3 inserts matrix 1 2.
MVA-h1HA/M2e-M1-NP headless HA/m2e fusion 3 inserts protein +
nucleoprotein + matrix 1 3. MVA-tbd best construct from previous
control screening 4. MVA-HA-VN hemagglutinin control 5. MVA-wt
empty vector neg. control 6. PBS -- neg. control
[0074] The present invention is illustrated by the foregoing
examples and variations thereof will be apparent to those skilled
in the art. Therefore, no limitations other than those set out in
the following claims should be placed on the invention.
[0075] All documents cited in this application are hereby
incorporated by reference in their entirety for their disclosure
described.
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Sequence CWU 1
1
1911362DNAArtificial SequenceSynthetic nucleotide 1atggagaaaa
tagtgcttct ttttgcaata gtcagtcttg ttaaaagtga tcagatttgc 60attggttacc
atgcaaacaa ctcgacagag caggttgaca caataatgga aaagaacgtt
120actgttacac atgcccaaga catactggaa aagaaacaca acgggaagct
ctgcggagga 180ggaagtcttc taaccgaggt cgaaacgcct accagaaacg
aatgggagtg cagatgcagc 240gattcaagtg atggaagtgc aggatcagcg
agtcttctaa ccgaggtcga aacgcctatc 300agaaacgaat gggggtgcag
atgcaacgat tcaagtgatg gaagtgcagg atcagcgagt 360cttctaaccg
aggtcgaaac gcctaccaga aacggatggg agtgcaaatg cagcgattca
420agtgatggaa gtgcaggatc agcgagtctt ctaaccgagg tcgaaacgcc
tatcagaaaa 480ggatgggagt gcaactgcag cgattcaagt gatggaggag
gatgcaacac caagtgtcaa 540actccaatgg gggcgataaa ctctagcatg
ccattccaca atatacaccc tctcaccatt 600ggggaatgcc ccaaatatgt
gaaatcaaac agattagtcc ttgcgactgg gctcagaaat 660agccctcaaa
gagagagaag aagaaaaaag agaggattat ttggagctat agcaggtttt
720atagagggag gatggcaggg aatggtagat ggttggtatg ggtaccacca
tagcaatgag 780caggggagtg ggtacgctgc agacaaagaa tccactcaaa
aggcaataga tggagtcacc 840aataaggtca actcgatcat tgacaaaatg
aacactcagt ttgaggccgt tggaagggaa 900tttaacaact tagaaaggag
aatagagaat ttaaacaaga agatggaaga cgggttccta 960gatgtctgga
cttataatgc tgaacttctg gttctcatgg aaaatgagag aactctagac
1020tttcatgact caaatgtcaa gaacctttac gacaaggtcc gactacagct
tagggataat 1080gcaaaggagc tgggtaacgg ttgtttcgag ttctatcata
aatgtgataa tgaatgtatg 1140gaaagtgtaa gaaatggaac gtatgactac
ccgcagtatt cagaagaagc gagactaaaa 1200agagaggaaa taagtggagt
aaaattggaa tcaataggaa tttaccaaat actgtcaatt 1260tattctacag
tggcgagttc cctagcactg gcaatcatgg tagctggtct atccttatgg
1320atgtgctcca atggatcgtt acaatgcaga atttgcattt aa
13622453PRTArtificial SequenceSynthetic peptide 2Met Glu Lys Ile
Val Leu Leu Phe 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 Lys His Asn Gly Lys Leu Cys Gly Gly Gly Ser Leu
Leu 50 55 60 Thr Glu Val Glu Thr Pro Thr Arg Asn Glu Trp Glu Cys
Arg Cys Ser 65 70 75 80 Asp Ser Ser Asp Gly Ser Ala Gly Ser Ala Ser
Leu Leu Thr Glu Val 85 90 95 Glu Thr Pro Ile Arg Asn Glu Trp Gly
Cys Arg Cys Asn Asp Ser Ser 100 105 110 Asp Gly Ser Ala Gly Ser Ala
Ser Leu Leu Thr Glu Val Glu Thr Pro 115 120 125 Thr Arg Asn Gly Trp
Glu Cys Lys Cys Ser Asp Ser Ser Asp Gly Ser 130 135 140 Ala Gly Ser
Ala Ser Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Lys 145 150 155 160
Gly Trp Glu Cys Asn Cys Ser Asp Ser Ser Asp Gly Gly Gly Cys Asn 165
170 175 Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser Met Pro
Phe 180 185 190 His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys
Tyr Val Lys 195 200 205 Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg
Asn Ser Pro Gln Arg 210 215 220 Glu Arg Arg Arg Lys Lys Arg Gly Leu
Phe Gly Ala Ile Ala Gly Phe 225 230 235 240 Ile Glu Gly Gly Trp Gln
Gly Met Val Asp Gly Trp Tyr Gly Tyr His 245 250 255 His Ser Asn Glu
Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr 260 265 270 Gln Lys
Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp 275 280 285
Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn Leu 290
295 300 Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe
Leu 305 310 315 320 Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
Met Glu Asn Glu 325 330 335 Arg Thr Leu Asp Phe His Asp Ser Asn Val
Lys Asn Leu Tyr Asp Lys 340 345 350 Val Arg Leu Gln Leu Arg Asp Asn
Ala Lys Glu Leu Gly Asn Gly Cys 355 360 365 Phe Glu Phe Tyr His Lys
Cys Asp Asn Glu Cys Met Glu Ser Val Arg 370 375 380 Asn Gly Thr Tyr
Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys 385 390 395 400 Arg
Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Ile Tyr Gln 405 410
415 Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Ile
420 425 430 Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser
Leu Gln 435 440 445 Cys Arg Ile Cys Ile 450 3568PRTInfluenza A
virusMISC_FEATUREVN/1203 HA sequence 3Met Glu Lys Ile Val Leu Leu
Phe 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 Lys 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 Val Asn Asp Leu Cys Tyr Pro
Gly Asp 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 Ser His Glu Ala Ser 130 135 140 Leu Gly Val Ser Ser Ala
Cys Pro Tyr Gln Gly Lys Ser 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
Arg 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 Lys Leu Tyr Gln
195 200 205 Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn
Gln Arg 210 215 220 Leu Val Pro Arg 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 Thr 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 Arg 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 Val Arg Asn Gly Thr Tyr Asp
Tyr Pro Gln Tyr Ser Glu Glu Ala 500 505 510 Arg Leu Lys Arg Glu Glu
Ile Ser Gly Val Lys Leu Glu Ser Ile Gly 515 520 525 Ile Tyr Gln Ile
Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala 530 535 540 Leu Ala
Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly 545 550 555
560 Ser Leu Gln Cys Arg Ile Cys Ile 565 43PRTArtificial
SequenceSynthetic linker 4Gly Gly Gly 1 523PRTInfluenza A
virusMISC_FEATUREH5N1 M2e sequence 5Ser Leu Leu Thr Glu Val Glu Thr
Pro Thr Arg Asn Glu Trp Glu Cys 1 5 10 15 Arg Cys Ser Asp Ser Ser
Asp 20 623PRTInfluenza A virusMISC_FEATUREH1N1 M2e sequence 6Ser
Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Asn Glu Trp Gly Cys 1 5 10
15 Arg Cys Asn Asp Ser Ser Asp 20 723PRTInfluenza A
virusMISC_FEATUREH9N2 M2e sequence 7Ser Leu Leu Thr Glu Val Glu Thr
Pro Thr Arg Asn Gly Trp Glu Cys 1 5 10 15 Lys Cys Ser Asp Ser Ser
Asp 20 823PRTInfluenza A virusMISC_FEATUREH7N2 M2e sequence 8Ser
Leu Leu Thr Glu Val Glu Thr Pro Ile Arg Lys Gly Trp Glu Cys 1 5 10
15 Asn Cys Ser Asp Ser Ser Asp 20 96PRTArtificial SequenceSynthetic
linker 9Gly Ser Ala Gly Ser Ala 1 5 10986DNAInfluenza A
virusmisc_featureM1 sequence 10atgagtcttc taaccgaggt cgaaacgtac
gttctctcta tcatcccgtc aggccccctc 60aaagccgaga tcgcacagaa acttgaagat
gtctttgcag gaaagaacac cgatctcgag 120gctctcatgg agtggctaaa
gacaagacca atcctgtcac ctctgactaa agggattttg 180ggatttgtat
tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc
240cagaatgccc taaatggaaa tggagatcca aataatatgg atagggcagt
taagctatat 300aagaagctga aaagagaaat aacattccat ggggctaagg
aggtcgcact cagctactca 360accggtgcac ttgccagttg catgggtctc
atatacaaca ggatgggaac ggtgactacg 420gaagtggctt ttggcctagt
gtgtgccact tgtgagcaga ttgcagattc acagcatcgg 480tctcacagac
agatggcaac tatcaccaac ccactaatca gacatgagaa cagaatggtg
540ctggccagca ctacagctaa ggctatggag cagatggcgg gatcaagtga
gcaggcagcg 600gaagccatgg agatcgctaa tcaggctagg cagatggtgc
aggcaatgag gacaattggg 660actcatccta actctagtgc tggtctgaga
gataatcttc ttgaaaattt gcaggcctac 720cagaaacgaa tgggagtgca
gatgcagcga ttcaagtgat cctattgttg ttgccgcaaa 780tatcattggg
atcttgcact tgatattgtg gattcttgat cgtcttttct tcaaatgcat
840ttatcgtcgc cttaaatacg gtttgaaaag agggcctgct acggcagggg
tacctgagtc 900tatgagggaa gagtaccggc aggaacagca gagtgctgtg
gatgttgacg atggtcattt 960tgtcaacata gaattggagt aaaaaa
98611252PRTInfluenza A virusMISC_FEATUREM1 sequence 11Met Ser Leu
Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro 1 5 10 15 Ser
Gly Pro Leu Lys Ala Glu Ile Ala Gln Lys Leu Glu Asp Val Phe 20 25
30 Ala Gly Lys Asn Thr Asp Leu Glu Ala Leu Met Glu Trp Leu Lys Thr
35 40 45 Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe
Val Phe 50 55 60 Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg
Arg Arg Phe Val 65 70 75 80 Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro
Asn Asn Met Asp Arg Ala 85 90 95 Val Lys Leu Tyr Lys Lys Leu Lys
Arg Glu Ile Thr Phe His Gly Ala 100 105 110 Lys Glu Val Ala Leu Ser
Tyr Ser Thr Gly Ala Leu Ala Ser Cys Met 115 120 125 Gly Leu Ile Tyr
Asn Arg Met Gly Thr Val Thr Thr Glu Val Ala Phe 130 135 140 Gly Leu
Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg 145 150 155
160 Ser His Arg Gln Met Ala Thr Ile Thr Asn Pro Leu Ile Arg His Glu
165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu
Gln Met 180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu
Ile Ala Asn Gln 195 200 205 Ala Arg Gln Met Val Gln Ala Met Arg Thr
Ile Gly Thr His Pro Asn 210 215 220 Ser Ser Ala Gly Leu Arg Asp Asn
Leu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met Gly
Val Gln Met Gln Arg Phe Lys 245 250 121507DNAInfluenza A
virusmisc_featureNP sequence 12atggcgtctc aaggcaccaa acgatcttat
gaacagatgg aaactggtgg ggaacgccag 60aatgctactg agatcagggc atctgttgga
agaatggtta gtggcattgg gaggttctac 120atacagatgt gcacagaact
caaactcagt gactatgaag ggaggctgat ccagaacagc 180ataacaatag
agagaatggt actctctgca tttgatgaaa gaaggaacag atacctggaa
240gaacacccca gtgcgggaaa ggacccgaag aagactggag gtccaattta
tcggaggaga 300gacgggaaat gggtgagaga gctaattctg tacgacaaag
aggagatcag gaggatttgg 360cgtcaagcga acaatggaga ggacgcaact
gctggtctta cccacctgat gatatggcat 420tccaatctaa atgatgccac
atatcagaga acgagagctc tcgtgcgtac tggaatggac 480ccaaggatgt
gctctctgat gcaagggtca actctcccga ggagatctgg agctgccggt
540gcagcagtaa agggggtagg gacaatggtg atggagctga ttcggatgat
aaaacgaggg 600atcaacgacc ggaatttctg gagaggcgaa aatggaagaa
gaacaaggat tgcatatgag 660agaatgtgca acatcctcaa agggaaattc
caaacagcag cacaaagagc aatgatggat 720caagtgcgag agagcagaaa
tcctgggaat gctgaaattg aagatctcat ttttctggca 780cggtctgcac
tcatcctgag aggatcagtg gcccataagt cctgcttgcc tgcttgtgtg
840tacggacttg cagtggccag tggatatgac tttgagagag aagggtactc
tctggttgga 900atagatcctt tccgcctgct tcaaaacagc caggtcttta
gtctcattag accaaatgag 960aatccagcac ataagagtca attagtgtgg
atggcatgcc actctgcagc atttgaggac 1020cttagagtct caagtttcat
cagagggaca agagtggtcc caagaggaca gctatccacc 1080agaggggttc
aaattgcttc aaatgagaac atggaggcaa tggactccaa cactcttgaa
1140ctgagaagca gatattgggc tataagaacc agaagcggag gaaacaccaa
ccagcagagg 1200gcatctgcag gacagatcag cgttcagccc actttctcgg
tccagagaaa ccttcccttc 1260gaaagagcga ccattatggc agcatttaca
ggaaatactg agggcagaac gtctgacatg 1320aggactgaaa tcataagaat
gatggaaagt gccagaccag aagatgtgtc attccagggg 1380cggggagtct
tcgagctctc ggacgaaaag gcaacgaacc cgatcgtgcc ttcctttgac
1440atgaataatg aaggatctta tttcttcgga gacaatgcag aggagtatga
caattaaaga 1500aaaatac 150713498PRTInfluenza A virusMISC_FEATURENP
sequence 13Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu
Thr Gly 1 5 10 15 Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser
Val Gly Arg Met 20 25 30 Val Ser Gly Ile Gly Arg Phe Tyr Ile Gln
Met Cys Thr Glu Leu Lys 35 40 45 Leu Ser Asp Tyr Glu Gly Arg Leu
Ile Gln Asn Ser Ile Thr Ile Glu 50 55 60 Arg Met Val Leu Ser Ala
Phe Asp Glu Arg Arg Asn Arg Tyr Leu Glu 65 70 75 80 Glu His Pro Ser
Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile 85 90 95 Tyr Arg
Arg Arg Asp Gly Lys Trp Val Arg Glu Leu Ile Leu Tyr Asp 100 105 110
Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp 115
120 125 Ala Thr Ala Gly Leu Thr His Leu Met Ile Trp His Ser Asn Leu
Asn 130 135 140 Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr
Gly Met Asp 145 150 155 160 Pro Arg Met Cys Ser Leu Met Gln Gly Ser
Thr Leu Pro Arg Arg Ser 165 170 175 Gly Ala Ala Gly Ala Ala Val Lys
Gly Val Gly Thr Met Val Met Glu 180 185 190 Leu Ile Arg Met Ile Lys
Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg 195 200 205 Gly Glu Asn Gly
Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn 210 215 220 Ile Leu
Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Met Asp 225 230
235
240 Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu
245 250 255 Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val
Ala His 260 265 270 Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala
Val Ala Ser Gly 275 280 285 Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Leu
Val Gly Ile Asp Pro Phe 290 295 300 Arg Leu Leu Gln Asn Ser Gln Val
Phe Ser Leu Ile Arg Pro Asn Glu 305 310 315 320 Asn Pro Ala His Lys
Ser Gln Leu Val Trp Met Ala Cys His Ser Ala 325 330 335 Ala Phe Glu
Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Thr Arg Val 340 345 350 Val
Pro Arg Gly Gln Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn 355 360
365 Glu Asn Met Glu Ala Met Asp Ser Asn Thr Leu Glu Leu Arg Ser Arg
370 375 380 Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Asn Thr Asn Gln
Gln Arg 385 390 395 400 Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr
Phe Ser Val Gln Arg 405 410 415 Asn Leu Pro Phe Glu Arg Ala Thr Ile
Met Ala Ala Phe Thr Gly Asn 420 425 430 Thr Glu Gly Arg Thr Ser Asp
Met Arg Thr Glu Ile Ile Arg Met Met 435 440 445 Glu Ser Ala Arg Pro
Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe 450 455 460 Glu Leu Ser
Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp 465 470 475 480
Met Asn Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr 485
490 495 Asp Asn 141026DNAArtificial SequenceSynthetic nucleotide
14atggagaaaa tagtgcttct ttttgcaata gtcagtcttg ttaaaagtga tcagatttgc
60attggttacc atgcaaacaa ctcgacagag caggttgaca caataatgga aaagaacgtt
120actgttacac atgcccaaga catactggaa aagaaacaca acgggaagct
ctgcggagga 180ggaggatgca acaccaagtg tcaaactcca atgggggcga
taaactctag catgccattc 240cacaatatac accctctcac cattggggaa
tgccccaaat atgtgaaatc aaacagatta 300gtccttgcga ctgggctcag
aaatagccct caaagagaga gaagaagaaa aaagagagga 360ttatttggag
ctatagcagg ttttatagag ggaggatggc agggaatggt agatggttgg
420tatgggtacc accatagcaa tgagcagggg agtgggtacg ctgcagacaa
agaatccact 480caaaaggcaa tagatggagt caccaataag gtcaactcga
tcattgacaa aatgaacact 540cagtttgagg ccgttggaag ggaatttaac
aacttagaaa ggagaataga gaatttaaac 600aagaagatgg aagacgggtt
cctagatgtc tggacttata atgctgaact tctggttctc 660atggaaaatg
agagaactct agactttcat gactcaaatg tcaagaacct ttacgacaag
720gtccgactac agcttaggga taatgcaaag gagctgggta acggttgttt
cgagttctat 780cataaatgtg ataatgaatg tatggaaagt gtaagaaatg
gaacgtatga ctacccgcag 840tattcagaag aagcgagact aaaaagagag
gaaataagtg gagtaaaatt ggaatcaata 900ggaatttacc aaatactgtc
aatttattct acagtggcga gttccctagc actggcaatc 960atggtagctg
gtctatcctt atggatgtgc tccaatggat cgttacaatg cagaatttgc 1020atttaa
102615341PRTArtificial SequenceSynthetic peptide 15Met Glu Lys Ile
Val Leu Leu Phe 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 Lys His Asn Gly Lys Leu Cys Gly Gly Gly Gly Cys
Asn 50 55 60 Thr Lys Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser
Met Pro Phe 65 70 75 80 His Asn Ile His Pro Leu Thr Ile Gly Glu Cys
Pro Lys Tyr Val Lys 85 90 95 Ser Asn Arg Leu Val Leu Ala Thr Gly
Leu Arg Asn Ser Pro Gln Arg 100 105 110 Glu Arg Arg Arg Lys Lys Arg
Gly Leu Phe Gly Ala Ile Ala Gly Phe 115 120 125 Ile Glu Gly Gly Trp
Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His 130 135 140 His Ser Asn
Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr 145 150 155 160
Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile Ile Asp 165
170 175 Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn Asn
Leu 180 185 190 Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp
Gly Phe Leu 195 200 205 Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val
Leu Met Glu Asn Glu 210 215 220 Arg Thr Leu Asp Phe His Asp Ser Asn
Val Lys Asn Leu Tyr Asp Lys 225 230 235 240 Val Arg Leu Gln Leu Arg
Asp Asn Ala Lys Glu Leu Gly Asn Gly Cys 245 250 255 Phe Glu Phe Tyr
His Lys Cys Asp Asn Glu Cys Met Glu Ser Val Arg 260 265 270 Asn Gly
Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala Arg Leu Lys 275 280 285
Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Ile Tyr Gln 290
295 300 Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu Ala
Ile 305 310 315 320 Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn
Gly Ser Leu Gln 325 330 335 Cys Arg Ile Cys Ile 340
162274DNAInfluenza A virusmisc_featurePB1 sequence 16atggatgtca
atccgacttt acttttcttg aaagtaccag tgcaaaatgc tataagtacc 60accttccctt
atactggaga ccctccatac agccatggaa cagggacagg atacaccatg
120gacacagtca acagaacaca ccaatattca gaaaagggga agtggacaac
aaacacagag 180actggagcac cccaactcaa cccgattgat ggaccactac
ctgaggataa tgagcccagt 240gggtacgcac aaacagattg tgtattggaa
gcaatggctt tccttgaaga atcccaccca 300gggatctttg aaaactcgtg
tcttgaaacg atggaaattg ttcaacaaac aagagtggat 360aaactgaccc
aaggtcgcca gacctatgac tggacattga atagaaacca accggctgca
420actgctttgg ccaacactat agaaatcttc agatcgaacg gtctaacagc
caatgaatcg 480ggacggctaa tagatttcct caaggatgtg atggagtcaa
tggataagga agaaatggag 540ataacaacac atttccagag aaagagaagg
gtgagggaca acatgaccaa gaaaatggtc 600acacaaagaa caatagggaa
gaaaaaacaa aggctgaaca aaaagagcta cctgataaga 660gcactgacac
tgaacacaat gacaaaagat gcagaaagag gcaaattgaa gaggcgagcg
720attgcaacac ccggaatgca aatcagagga ttcgtgtact ttgttgaaac
actagcgagg 780agtatctgtg agaaacttga gcaatctgga ctcccagtcg
gagggaatga gaagaaggct 840aaattggcaa acgtcgtgag gaagatgatg
actaactcac aagatactga actctccttt 900acaattactg gagacaatac
caaatggaat gagaatcaga atcctaggat gtttctggca 960atgataacgt
acatcacaag gaaccagcca gaatggtttc ggaatgtctt aagcatagct
1020cctataatgt tctcaaacaa aatggcgaga ctaggaaaag gatacatgtt
cgaaagtaag 1080agcatgaagt tacgaacaca aataccagca gaaatgcttg
caaacattga tcttaaatac 1140ttcaatgaat taacgaaaaa gaaaattgag
aaaataaggc ctctattaat agatggtaca 1200gcctcattga gccctggaat
gatgatgggc atgttcaaca tgctgagtac agtcctagga 1260gtttcaatcc
tgaatcttgg acagaaaagg tacaccaaaa ccacatattg gtgggacgga
1320ctccaatcct ctgatgattt cgctctcatc gtaaatgcac cgaatcatga
gggaatacaa 1380gcaggagtgg ataggtttta taggacttgt aaactagttg
gaatcaatat gagcaagaag 1440aagtcttaca taaatcggac agggacattt
gaattcacga gctttttcta ccgctatgga 1500tttgtagcca atttcagtat
ggagctgccc agttttggag tgtctggaat taatgaatcg 1560gccgacatga
gcattggtgt tacagtgata aaaaacaata tgataaacaa cgaccttggg
1620ccagcaacag ctcagatggc tcttcagtta ttcatcaagg actacagata
cacataccga 1680tgccacagag gggatacgca aatccaaaca aggagatcat
tcgagctgaa gaagctgtgg 1740gagcaaaccc gttcaaaggc aggactgttg
gtttcagatg gaggaccaaa tctatacaat 1800atccgaaacc tccatattcc
tgaagtctgc ttaaaatggg aattgatgga tgaagattac 1860cagggcagac
tgtgtaatcc tctgaatcca ttcgtcagcc ataaggaaat tgaatctgtc
1920aacaatgctg tagtaatgcc agctcatggc ccggccaaga gtatggaata
tgatgccgtt 1980gcaactacac attcatggat tcctaaaagg aaccgttcca
ttctcaatac gagtcaaagg 2040ggaattcttg aggatgaaca gatgtaccag
aagtgctgca atctattcga gaaattcttc 2100cccagcagtt catatcggag
gccagttgga atttccagca tggtggaggc catggtgtct 2160agggcccgaa
ttgacgcacg aatcgatttc gagtctggaa ggattaagaa agaagagttt
2220gccgagatca tgaagatctg ttccaccatt gaagaactca gacggcaaaa atag
227417757PRTInfluenza A virusMISC_FEATUREPB1 sequence 17Met Asp Val
Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Val Gln Asn 1 5 10 15 Ala
Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His 20 25
30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln
35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Thr Gly
Ala Pro 50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp
Asn Glu Pro Ser 65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu
Ala Met Ala Phe Leu Glu 85 90 95 Glu Ser His Pro Gly Ile Phe Glu
Asn Ser Cys Leu Glu Thr Met Glu 100 105 110 Ile Val Gln Gln Thr Arg
Val Asp Lys Leu Thr Gln Gly Arg Gln Thr 115 120 125 Tyr Asp Trp Thr
Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala 130 135 140 Asn Thr
Ile Glu Ile Phe Arg Ser Asn Gly Leu Thr Ala Asn Glu Ser 145 150 155
160 Gly Arg Leu Ile Asp Phe Leu Lys Asp Val Met Glu Ser Met Asp Lys
165 170 175 Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg
Val Arg 180 185 190 Asp Asn Met Thr Lys Lys Met Val Thr Gln Arg Thr
Ile Gly Lys Lys 195 200 205 Lys Gln Arg Leu Asn Lys Lys Ser Tyr Leu
Ile Arg Ala Leu Thr Leu 210 215 220 Asn Thr Met Thr Lys Asp Ala Glu
Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 Ile Ala Thr Pro Gly
Met Gln Ile Arg Gly Phe Val Tyr Phe Val Glu 245 250 255 Thr Leu Ala
Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro 260 265 270 Val
Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 275 280
285 Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly
290 295 300 Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe
Leu Ala 305 310 315 320 Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu
Trp Phe Arg Asn Val 325 330 335 Leu Ser Ile Ala Pro Ile Met Phe Ser
Asn Lys Met Ala Arg Leu Gly 340 345 350 Lys Gly Tyr Met Phe Glu Ser
Lys Ser Met Lys Leu Arg Thr Gln Ile 355 360 365 Pro Ala Glu Met Leu
Ala Asn Ile Asp Leu Lys Tyr Phe Asn Glu Leu 370 375 380 Thr Lys Lys
Lys Ile Glu Lys Ile Arg Pro Leu Leu Ile Asp Gly Thr 385 390 395 400
Ala Ser Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser 405
410 415 Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Lys Arg Tyr
Thr 420 425 430 Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp
Asp Phe Ala 435 440 445 Leu Ile Val Asn Ala Pro Asn His Glu Gly Ile
Gln Ala Gly Val Asp 450 455 460 Arg Phe Tyr Arg Thr Cys Lys Leu Val
Gly Ile Asn Met Ser Lys Lys 465 470 475 480 Lys Ser Tyr Ile Asn Arg
Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe 485 490 495 Tyr Arg Tyr Gly
Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe 500 505 510 Gly Val
Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr 515 520 525
Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala 530
535 540 Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr
Arg 545 550 555 560 Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg
Ser Phe Glu Leu 565 570 575 Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys
Ala Gly Leu Leu Val Ser 580 585 590 Asp Gly Gly Pro Asn Leu Tyr Asn
Ile Arg Asn Leu His Ile Pro Glu 595 600 605 Val Cys Leu Lys Trp Glu
Leu Met Asp Glu Asp Tyr Gln Gly Arg Leu 610 615 620 Cys Asn Pro Leu
Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val 625 630 635 640 Asn
Asn Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu 645 650
655 Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg
660 665 670 Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu
Gln Met 675 680 685 Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe
Pro Ser Ser Ser 690 695 700 Tyr Arg Arg Pro Val Gly Ile Ser Ser Met
Val Glu Ala Met Val Ser 705 710 715 720 Arg Ala Arg Ile Asp Ala Arg
Ile Asp Phe Glu Ser Gly Arg Ile Lys 725 730 735 Lys Glu Glu Phe Ala
Glu Ile Met Lys Ile Cys Ser Thr Ile Glu Glu 740 745 750 Leu Arg Arg
Gln Lys 755 1899DNAArtificial SequencePromoter 18aaaaattgaa
aataaataca aaggttcttg agggttgtgt taaattgaaa gcgagaaata 60atcataaata
atttcattat cgcgatatcc gttaagttt 991939DNAArtificial
SequenceSynthetic promoter 19aaaaattgaa attttatttt ttttttttgg
aatataaat 39
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