U.S. patent application number 12/666246 was filed with the patent office on 2010-06-03 for replication deficient influenza virus for the expression of heterologous sequences.
This patent application is currently assigned to AVIR Green Hills Biotechnology Research Development Trade AG. Invention is credited to Michael Bergmann, Andrej Egorov, Christian Kittel, Thomas Muster, Markus Wolschek.
Application Number | 20100136052 12/666246 |
Document ID | / |
Family ID | 39034019 |
Filed Date | 2010-06-03 |
United States Patent
Application |
20100136052 |
Kind Code |
A1 |
Wolschek; Markus ; et
al. |
June 3, 2010 |
REPLICATION DEFICIENT INFLUENZA VIRUS FOR THE EXPRESSION OF
HETEROLOGOUS SEQUENCES
Abstract
The present invention covers a novel replication deficient
influenza virus comprising a modified NS1 segment coding for a NS1
protein lacking a functional RNA binding domain and functional
effector domain and a heterologous sequence inserted between the
splice donor site and the splice acceptor site of the NS gene
segment. Further the use of the virus as vector for expression of
various proteins like chemokines, cytokines or antigenic structures
is covered, methods for producing virus particles using said virus
vector as well as its use for production of vaccines. Also a fusion
peptide comprising part of the N-terminus of an NS1 protein and a
signal sequence fused to the C-terminus of said NS1 peptide is
covered.
Inventors: |
Wolschek; Markus; (Vienna,
AT) ; Egorov; Andrej; (Vienna, AT) ; Bergmann;
Michael; (Klosterneuburg, AT) ; Muster; Thomas;
(Vienna, AT) ; Kittel; Christian; (Vienna,
AT) |
Correspondence
Address: |
SHELDON MAK ROSE & ANDERSON PC
100 Corson Street, Third Floor
PASADENA
CA
91103-3842
US
|
Assignee: |
AVIR Green Hills Biotechnology
Research Development Trade AG
Wien
AT
|
Family ID: |
39034019 |
Appl. No.: |
12/666246 |
Filed: |
June 26, 2008 |
PCT Filed: |
June 26, 2008 |
PCT NO: |
PCT/EP08/58154 |
371 Date: |
December 22, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60946644 |
Jun 27, 2007 |
|
|
|
Current U.S.
Class: |
424/206.1 ;
435/235.1; 435/320.1; 435/454; 530/350; 530/387.3 |
Current CPC
Class: |
C12N 2760/16132
20130101; C12N 2760/16243 20130101; A61K 2039/5256 20130101; C07K
14/005 20130101; C12N 7/00 20130101; C12N 15/86 20130101; C12N
2760/16143 20130101; A61K 2039/525 20130101; A61K 39/00
20130101 |
Class at
Publication: |
424/206.1 ;
435/454; 435/235.1; 435/320.1; 530/350; 530/387.3 |
International
Class: |
A61K 39/145 20060101
A61K039/145; C12N 15/00 20060101 C12N015/00; C12N 7/01 20060101
C12N007/01; C12N 15/63 20060101 C12N015/63; C07K 14/00 20060101
C07K014/00; C07K 16/00 20060101 C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 5, 2007 |
EP |
07450176.8 |
Claims
1. Replication deficient influenza virus characterized in that it
comprises a) a modified NS segment coding for a NS1 protein
comprising at least one amino acid modification within positions 1
to 73 resulting in complete lack of its functional RNA binding and
at least one amino acid modification between position 74 and the
carboxy-terminal amino acid residue resulting in complete lack of
its effector function and b) a heterologous sequence between a
functional splice donor site and functional splice acceptor site
inserted in the NS gene segment
2. Replication deficient influenza virus according to claim 1
characterized in that it comprises at least 10 amino acids and
preferably up to 14, preferably up to 30 amino acids of the
N-terminus of the NS1 protein.
3. Replication deficient influenza virus according to any one of
claim 1 or 2 characterized in that amino acids 134 to 161 are
deleted.
4. Replication deficient influenza virus according to any one of
claim 1 or 2 characterized in that amino acids 117 to 161 are
deleted.
5. Replication deficient influenza virus according to any one of
claims 2 to 4 comprising a signal peptide or part thereof fused to
the C-terminus of NS1 protein.
6. Replication deficient influenza virus according to any one of
claims 1 to 5 characterized in that the heterologous sequence is
expressed from the NS1 open reading frame.
7. Replication deficient influenza virus according to any one of
claims 1 to 5 characterized in that the heterologous sequence is
expressed from a separate open reading frame.
8. Replication deficient influenza virus according to any one of
claims 1 to 7 characterized in that the heterologous sequence is
selected from the group consisting of biologically active proteins,
antigens or derivatives or fragments thereof.
9. Replication deficient influenza virus according to any one of
claims 1 to 8 characterized in that the heterologous sequence is a
chemokine or cytokine or derivative or fragment thereof.
10. Replication deficient influenza virus according to any one of
claims 1 to 9 characterized in that the heterologous sequence is
derived from mycobacterium tuberculosis.
11. Replication deficient influenza virus according to any one of
claims 1 to 10 characterized in that the heterologous sequence
comprises a signal peptide.
12. Replication deficient influenza virus according to claim 11
characterized in that the signal peptide is derived from an
antibody light chain, preferably from an Ig kappa chain, more
preferably from mouse Ig kappa chain.
13. Replication deficient influenza virus according to claim 12
characterized in that the Ig kappa signal peptide comprises at
least 10 amino acids, more preferred at least 12 amino acids.
14. Replication deficient influenza virus according to any one of
claim 12 or 13 characterized in that the Ig kappa signal peptide
comprises the sequence METDTLLLWVLLLWVPGSTGD (SEQ ID No. 11) or
METDTLLLWVLLLWVPRSHG (SEQ ID No. 82) or part or derivatives
thereof.
15. Replication deficient influenza virus according to any one of
claims 1 to 14 characterized in that the heterologous sequence
comprises a fusion protein of a biologically active protein and an
antigen.
16. Replication deficient influenza virus according to any one of
claims 1 to 15 characterized in that the heterologous sequence is
selected from the group consisting of IL2, GM-CSF, IL15, MIP 1
alpha and MIP 3 alpha, ESAT-6 or a derivative or fragment
thereof.
17. Replication deficient influenza virus according to any one of
claims 1 to 16 characterized in that the translation of said NS1
protein is terminated by at least one STOP codon and expression of
said heterologous sequence is reinitiated by a START codon.
18. Replication deficient influenza virus according to any one of
claims 1 to 17 characterized in that the heterologous open reading
frame is at least partially overlapping with the NS1 open reading
frame.
19. Replication deficient influenza virus according to any one of
claims 1 to 18 characterized in that translation of the
heterologous open reading frame is initiated from an overlapping
STOP/START codon sequence.
20. Replication deficient influenza virus according to claim 19
characterized in that the overlapping START/STOP codon is TAATG
(SEQ ID. No. 81) or UAAUG (SEQ ID No. 53).
21. Replication deficient influenza virus according to any one of
claims 1 to 20 characterized in that translation of the
heterologous open reading frame is initiated from an optimized
translation initiation sequence, preferably a Kozak consensus
sequence.
22. Replication deficient influenza virus according to any one of
claims 1 to 21 containing an altered sequence downstream of the
splice donor site and/or upstream of the splice acceptor site in
the NS segment.
23. Replication deficient influenza virus according to any one of
claims 1 to 22 characterized in that the heterologous sequence is
secreted from the infected cell.
24. Replication deficient influenza virus according to any one of
claims 1 to 23 with a sequence as shown in SEQ ID Nos 1 to 10, 67,
68 or with at least 98% homology therewith.
25. Combination of at least two replication deficient influenza
viruses according to any one of claims 1 to 24 comprising at least
one biologically active molecule or derivative or fragment thereof
and at least one antigenic structure.
26. Vaccine comprising a replication deficient influenza virus
according to any one of claims 1 to 24.
27. Use of a replication deficient influenza virus according to any
one of claims 1 to 24 for the preparation of a medicament for
therapeutic treatment in patients.
28. Use of a replication deficient influenza virus according to any
one of claims 1 to 24 for the preparation of a medicament for the
tumor treatment in patients.
29. Vector comprising a nucleotide sequence coding for a
replication deficient influenza virus according to any one of
claims 1 to 24.
30. Method for producing a replication deficient influenza virus
according to any one of claims 1 to 24, comprising the steps of:
transfecting cells, preferably Vero cells, with at least one vector
according to claim 29, incubating the transfected cells to allow
for the development of viral progeny containing the heterologous
protein.
31. Method for producing a replication deficient influenza virus
according to any one of claims 1 to 24, comprising the steps of:
transforming a cell, preferably a Vero cell, with a vector
according to claim 29 preferably together with a purified
preparation of influenza virus RNP complex, infecting the selected
cells with an influenza helper virus, incubating the infected cells
to allow for the development of viral progeny and selecting
transformed cells that express the modified NS gene and the
heterologous sequence,
32. Method for the creation of a replication deficient influenza
virus according to any one of claims 1 to 24, comprising the steps
of: transfecting a cell line, preferably a Vero cell line, with a
DNA vector comprising a modified NS gene free of functional RNA
binding domain and a heterologous sequence inserted between a
functional splice donor site and the splice acceptor site of the NS
gene, selecting transfected cells that express the modified NS gene
and the heterologous sequence, infecting the selected cells with a
desired influenza virus, incubating the infected cells to allow for
the development of viral progeny containing the heterologous
protein, selecting and harvesting said viral progeny containing the
heterologous protein.
33. The method according to claim 29, characterized in that the DNA
vector is a transcription system for minus sense influenza RNA.
34. The method according to claim 29 or 30, characterized in that
said viral progeny is further combined with a pharmaceutically
acceptable carrier for use as a vaccine.
35. Fusion protein comprising between 10 and 30 amino acids of the
N-terminus of an NS1 protein, a heterologous sequence and a signal
peptide fused to the C-terminus of said NS1 peptide.
36. Fusion protein according to claim 32 characterized in that the
signal peptide consists of 8-50 amino acids.
37. Fusion protein according to any one of claim 32 or 33
characterized in that the signal peptide is derived from an
antibody light chain, preferably from an Ig kappa chain, more
preferably from mouse Ig kappa chain or a derivative thereof.
38. Fusion protein according to any one of claims 35 to 37
characterized in that the Ig kappa signal peptide comprises at
least 10 amino acids, more preferred at least 12 amino acids.
39. Fusion protein according to any one of claims 35 to 38
characterized in that the Ig kappa signal peptide comprises the
sequence METDTLLLWVLLLWVPGSTGD (SEQ ID No. 11) or
METDTLLLWVLLLWVPRSHG (SEQ ID No. 82) or part or derivatives
thereof.
Description
[0001] The present invention covers a replication deficient
influenza virus comprising a modified NS1 segment coding for an NS1
protein lacking a functional RNA binding domain and functional
effector domain and a heterologous sequence inserted between the
splice donor site and the splice acceptor site of the NS segment.
Said heterologous sequence can be expressed either from the NS1
open reading frame or an open reading frame different from the NS1
open reading frame.
[0002] Further therapeutic preparations containing said replication
deficient influenza virus and their use are covered as well as the
process for manufacturing said virus.
[0003] The influenza virions consist of an internal
ribonucleoprotein core (a helical nucleocapsid) containing the
single-stranded RNA genome, and an outer lipoprotein envelope lined
inside by a matrix protein (M1). The segmented genome of influenza
A and B virus consists of eight molecules (seven for influenza C)
of linear, negative polarity, single-stranded RNAs which encodes
eleven (some influenza A strains ten) polypeptides, including: the
RNA-dependent RNA polymerase proteins (PB2, PB1 and PA) and
nucleoprotein (NP) which form the nucleocapsid; the matrix membrane
proteins (M1, M2 or BM2 for influenza B, respectively); two surface
glycoproteins which project from the lipid containing envelope:
hemagglutinin (HA) and neuraminidase (NA); the nonstructural
protein (NS1) and the nuclear export protein (NEP). Influenza B
viruses encode also NB, a membrane protein which might have ion
channel activity and most influenza A strains also encode an
eleventh protein (PB1-F2) believed to have proapoptotic
properties.
[0004] Transcription and replication of the genome takes place in
the nucleus and assembly occurs via budding on the plasma membrane.
The viruses can reassort genes during mixed infections. Influenza
virus adsorbs via HA to sialyloligosaccharides in cell membrane
glycoproteins and glycolipids. Following endocytosis of the virion,
a conformational change in the HA molecule occurs within the
cellular endosome which facilitates membrane fusion, thus
triggering uncoating. The nucleocapsid migrates to the nucleus
where viral mRNA is transcribed. Viral mRNA is transcribed and
processed by a unique mechanism in which viral endonuclease cleaves
the capped 5'-terminus from cellular heterologous mRNAs which then
serve as primers for transcription from viral RNA templates by the
viral transcriptase. Transcripts terminate at sites 15 to 22 bases
from the ends of their templates, where oligo(U) sequences act as
signals for the addition of poly(A) tracts. Of the eight viral RNA
molecules of influenza A virus so produced, six are monocistronic
messages that are translated directly into the proteins
representing HA, NA, NP and the viral polymerase proteins, PB2, PB1
and PA. The other two transcripts undergo splicing, each yielding
two mRNAs which are translated in different reading frames to
produce M1, M2, NS1 and NEP. In most of influenza A viruses,
segment 2 also encodes for a second protein (PB1-F2), expressed
from an overlapping reading frame. In other words, the eight viral
RNA segments code for eleven proteins: nine structural and 2
nonstructural (NS1 and the recently identified PB1-F2)
proteins.
[0005] The application of viral vectors for delivery of foreign
proteins and biologically active molecules is an attractive
approach for gene therapy, treatment of cancer and prevention of
infectious diseases. Influenza viruses are especially considered as
potential vaccine vectors. In contrast to other vectors such as
adenoviruses or retroviruses, influenza does not contain a DNA
intermediate and is therefore not able to integrate into the host's
chromosomes. There are several options to manipulate the influenza
genome depending on the desired aims and possibilities to produce
recombinant viruses. These strategies include the insertion of
foreign proteins into the surface glycoproteins NA and HA (Muster
T. et al., 1994, J. Virol., 68, 4031-4034; Percy N. et al., 1994,
J. Virol., 68, 4486-4492), the creation of additional genomic
fragments (Flick R and Hobom G., 1999, Virology, 262, 93-103;
Watanabe T. et al., 2003, J. Virol., 77, 10575-10583) and the
manipulation of the non-structural NS1 protein (Ferko B. et al.,
2001, J. Virol., 8899-8908; Takasuka N. et al., 2002, Vaccine, 20,
1579-1585). The influenza NS1 protein has several advantages as a
target for engineering since it does not presumably interfere with
the structure of the virions, but is synthesized in large
quantities in infected cells and tolerates long insertions up to
several hundred nucleotides.
[0006] As NS1 is only expressed intracellulary and less exposed to
the humoral arm of the immune system, the development of the immune
response to the NS1 protein or to the proteins fused to NS1 is
limited mainly to the induction of CD8.sup.+ T cell immunity.
Obviously, for the induction of B-cell response or for the
expression of biologically active molecules, efficient delivery of
the recombinant protein to the cell surface is required.
[0007] Vaccination is presently seen as the best way to protect
humans against influenza. Annual human influenza epidemics (caused
by influenza type A or type B viruses) are manifested as highly
infectious acute respiratory disease with high morbidity and
significant mortality. Vaccination is accomplished with
commercially available, chemically inactivated (killed) or live
attenuated influenza virus vaccines. The concept of the current
live attenuated vaccine is based on the generation of a temperature
sensitive attenuated "master strain" adapted to grow at 25.degree.
C. (cold adaptation). Live cold adapted (ca) and inactivated virus
vaccine stimulate the immune system differently, yet in both cases
lack of sufficient immunogenicity especially in elderly persons is
one of the most important drawbacks in influenza vaccination.
Although ca live influenza virus vaccines are considered as
sufficiently safe, the exact genetic and molecular mechanisms of
attenuation are not completely understood. It is claimed that the
nature of the safety of ca influenza vaccines is based on a large
number of point mutations distributed across the internal gene
segments. However, only a small number of mapped mutations
localized in the polymerase genes are responsible for the
attenuation of ca virus strains that are unable to replicate at
normal body temperature (Herlocher, M. L., A. C. Clavo, and H. F.
Maassab. 1996, Virus Res. 42:11-25; Herlocher, M. L., H. F. et al.,
1993, Proc Natl Acad Sci USA. 90:6032-6036). In fact, the genetic
stability of live vaccine strains are often questioned since
viruses re-isolated from vaccinated hosts reveal additional point
mutations which might eventually function as "suppressor" mutations
causing enhanced replication properties and a possible loss of the
temperature sensitive phenotype of the revertant virus (Herlocher,
M. L., H. F. et al., 1993, Proc. Natl. Acad. Sci. 90:6032-6036,
Treanor, J., M. et al., 1994 J Virol. 68:7684-7688.)
[0008] Reflecting the potential risks of the ca live attenuated
influenza virus vaccines and in view of the low stability often
combined with low expression rate of foreign proteins in influenza
virus vectors, there is still a high demand to create a completely
attenuated influenza virus vector inducing cellular and/or humoral
immunogenicity and stably expressing high amounts of foreign
proteins.
[0009] It has been surprisingly shown by the inventors that an
influenza virus vector as developed according to the invention does
fulfill these unmet demands, i.e. providing an influenza virus
vector that is of high safety due to complete attenuation and which
shows stable expression of foreign genes inserted into the virus
vector. Preferably, the foreign genes show high expression rates
when inserted into the inventive virus vector.
[0010] Although various attempts have been made to overcome the
issues of low genetic stability and low expression rate of proteins
or peptides in attenuated virus vectors, none of these constructs
have been efficiently successful yet.
[0011] Kittel et al. (Virology, 2004, 324, 67-73) described an
influenza A virus consisting of an NS1 protein of 125 aa length
(approx. one half of the wt NS1 protein) and expressing green
fluorescence protein (GFP) from the NS1 reading frame, which was
replicating in PKR knock out mice. In interferon competent cells
the virus was not stably expressing GFP but the virus was loosing
its fluorescent activity due to the appearance of various deletions
within the GFP sequence.
[0012] A bicistronic expression strategy based on the insertion of
an overlapping stop-start codon cassette into the NS gene for
expressing GFP was disclosed by Kittel et al. (2005, J. Virol., 79,
10672-10677). Although being genetically stable, the expression
level of the GFP from this reading frame was significantly lower
than that obtained from an influenza virus vector expressing GFP
from the NS1 ORF (Kittel et al., 2004, see above).
[0013] Ferko et al. did not describe a replication deficient virus
but a ca influenza virus expressing human interleukin 2 (J. Virol.,
2006, 11621-11627). Yet, the genetic stability and safety of a cold
adapted virus has to be questioned in view of the genetic structure
leading to temperature sensitivity (Herlocher M. et al., Proc.
Natl. Acad. Sci, 1993, 90, 6032-6036). Additionally, the IL-2
expression levels were low.
[0014] The present invention relates to the development of a
replication deficient influenza virus comprising a modified NS
segment coding for an NS1 protein lacking a functional RNA binding
domain and functional effector domain and a heterologous sequence
inserted between the splice donor site and the splice acceptor site
of the NS1 gene segment. According to the invention the
heterologous sequence can be expressed from the NS1 reading frame
or from a separate open reading frame.
[0015] Although WO 07/016715 describes that influenza virus wherein
the NS gene (sometimes referred to also as NS1 gene) comprises
deletions and wherein the virus can be used to express an
immunostimulatory cytokine, there is no disclosure on the specific
influenza vector which could successfully express foreign
proteins.
[0016] In contrast, the inventors have surprisingly shown that the
heterologous sequences, which can be even larger than the natural
intron, can be stably expressed at high levels from the NS segment
if inserted between a functional splice donor site and functional
splice acceptor site, provided NS splicing efficiency is adjusted
according to insert size.
[0017] This was neither shown nor indicated in WO 06/088481 and WO
01/64680.
[0018] According to a preferred embodiment of the invention, the
functional splice donor site and the splice acceptor site of the NS
gene segment is the natural splice site.
[0019] According to the invention the heterologous sequences can be
selected from any biologically active proteins or peptides or
antigenic structures.
[0020] Antigenic peptides or proteins are characterized by
comprising epitopes which can lead to immunomodulatory activities,
like binding of antibodies or antibody like structures or induction
of cellular immune responses.
[0021] Preferably, proteins or peptides are selected from the group
consisting of antigens, preferably bacterial antigens like ESAT6,
growth factors, cytokines like interleukins, lymphokines and
chemokines and fragments or derivatives thereof, more preferred
from Mycobacterium tuberculosis, GM-CSF, CCL-3, CCL-20, interleukin
2, interleukin 15 or a fragment or derivative thereof.
[0022] The present invention further relates to therapeutic
preparations, preferably vaccine preparations containing said
replication deficient influenza viruses. Exemplarily these
preparations can be used for the prevention and treatment of
infectious diseases or cancer.
[0023] Further, methods for producing the inventive influenza
viruses by transfecting cell lines (e.g. Vero cells, MDCK cells
etc.) and expressing viral particles are disclosed.
FIGURES
[0024] FIG. 1 (a-j): Nucleic acid sequence of various vector
constructs.
[0025] a: Sequence of the deINS1-IL-2-10 segment (SEQ ID No. 1)
[0026] b: Sequence of the deINS1-IL-2-11 segment (SEQ ID No. 2)
[0027] c: Sequence of the deINS1-IL-2-14 segment (SEQ ID No. 3)
[0028] d: Sequence of deINS1-IL2-13 segment (SEQ ID No. 4)
[0029] e: Sequence of deINS1-IL-2-21 segment (SEQ ID No. 5)
[0030] f: Sequence of deINS1-IL-2-17 segment (SEQ ID No. 6),
[0031] g: Sequence of deINS1-IL-15-21 segment (SEQ ID No. 7)
[0032] h: Sequence of deINS1-GM-CSF-21 segment (SEQ ID No. 8)
[0033] i: Sequence of deINS1-CCL-3-21 segment (SEQ ID No. 9)
[0034] j: Sequence of deINS1-CCL20-21 segment (SEQ ID No. 10)
[0035] k: Sequence of deINS1-ESAT-6s-21 segment (SEQ ID No. 67)
[0036] l: Sequence of deINS1-ESAT-6i-21 segment (SEQ ID No. 68)
[0037] m: Sequence of deINS1-IL2-23 segment (SEQ ID No. 78)
[0038] n: Sequence of deINS1-IL2-24 segment (SEQ ID No. 79)
[0039] FIG. 2: Schematic representation of the influenza A
wild-type NS segment and the three chimeric IL-2 NS segments
deINS1-IL-2-10 and deINS1-IL-2-11 and deINS1-IL-2-14.
[0040] FIG. 3: Human IL-2 levels in supernatants from Vero cells
infected with GHB-IL-2-10, GHB-IL-2-11 or GHB01.
[0041] FIG. 4: RT-PCR analysis of the NS segment after five
passages on Vero cells
[0042] FIG. 5: Human IL-2 levels in supernatants from Vero cells
infected with GHB-IL-2-11, GHB-IL-2-13, GHB-IL2-14 and
GHB-IL2-21.
[0043] FIG. 6: Amino acid sequence of wt influenza virus PR8
NS1
[0044] FIG. 7: deINS1-IL-2 mRNA splicing can be altered by either
modifying the sequence surrounding the splice donor site or the
sequences 5' to the splice acceptor site.
[0045] FIG. 8: Schematic IL-2 expression construct. The ORF of the
truncated NS1 consists of nucleotides 45-158; the human IL-2 ORF
consists of nucleotides 161-619; the 5' intron boundary is between
nucleotides 77 and 78; the 3' intron boundary is between
nucleotides 657 and 658.
[0046] FIG. 9: Nucleotide sequence of .DELTA.NS1-38IL2 (SEQ ID No.
77).
[0047] The invention provides replication-deficient influenza
viruses comprising a modified NS segment coding for a NS1 protein
comprising at least one amino acid modification within positions 1
to 73 resulting in complete lack of its functional RNA binding and
at least one amino acid between position 74 and the
carboxy-terminal amino acid residue, specifically until amino acid
position 167, resulting in complete lack of its effector function
and a heterologous sequence between a functional splice donor site
and functional splice acceptor site inserted in the NS gene
segment.
[0048] Preferably the influenza virus is derived from influenza A
virus, influenza B virus or influenza C virus. Vectors based on or
derived from Influenza A or influenza B virus sequences are
preferred.
[0049] The replication deficient influenza virus according to the
invention can be used as viral vector for immunization against any
pathogens or antigenic structures to induce an immune response
against the heterologous structures expressed by said viral vector.
The immune response can comprise a cellular immune response and/or
a humoral immune response. By using heterologous sequences
expressing immunomodulating proteins or peptides, the immune
response towards the influenza virus can be further boosted,
resulting in an improved influenza vaccine formulation. This is
especially relevant for vaccination of elderly or immunosuppressed
individuals.
[0050] The virus selected for use in the invention comprises a
modified NS gene leading to an influenza virus that is attenuated,
i.e. it is infectious and can replicate in vivo in interferon
deficient cells or cell systems but does not replicate in
interferon competent cells. According to the invention the term
"replication deficient" is defined as replication rate in
interferon competent host cells that is at least less than 5%,
preferably less than 1%, preferably less than 0.1% than wild type
influenza virus as determined by hemagglutination assay, TCID50
assay or plaque assay as well known in the art.
[0051] The NS gene segment according to the invention must contain
functional splice donor and splice acceptor sites.
[0052] According to a specific embodiment of the invention, the
influenza gene segments can be derived from different influenza
strains, either pandemic or interpandemic ones. This can result in
reassorted influenza viruses which combine the genes for the
surface glycoproteins hemagglutinin (HA) and/or neuraminidase (NA)
of actual interpandemic viruses with five or six or seven RNA
segments coding for other proteins from the attenuated master
strain (6/2 combination) or 7/1 reassortants or 5/3 reassortants
containing HA, NA and M segments of a circulating strain
respectively.
[0053] The inventors have used a reverse genetics system on Vero
cells for developing reassortants and/or expression of modified
influenza virus strains. The technology is already well known in
the art (Pleschka S. et al., 1996, J. Virol., 70(6), 4188-4192,
Neumann and Kawaoka, 1999, Adv. Virus Res., 53, 265-300, Hoffmann
et al. 2000, Proc Natl Acad Sci USA. 97:6108-13). Alternatively,
the technology based on RNPs as described by Enami and Enami (J.
Virol, 2000, 74,12, pp. 5556-5561) can be used for developing
reassortants.
[0054] The NS1 protein of influenza A virus is a multifunctional
protein that consists of approximately 230 amino acids and is early
and abundantly synthesized in infection. It counters cellular
antiviral activities and is a virulence factor. By the activity of
its carboxy terminal region, the NS1 protein is able to inhibit the
host mRNA's processing mechanisms. Second, it facilitates the
preferential translation of viral mRNA by direct interaction with
the cellular translation initiation factor. Third, by binding to
dsRNA and interaction with putative cellular kinase(s), the NS1
protein is able to prevent the activation of interferon (IFN-)
inducible dsRNA-activated kinase (PKR), 2'5'-oligoadenylate
synthetase system and cytokine transcription factors. Fourth, the N
terminal part of NS1 binds to RIG-I and inhibits downstream
activation of IRF-3, preventing the transcriptional induction of
IFN-.beta.. Therefore the NS1 protein inhibits the expression of
IFN-.alpha. or IFN-.beta. genes, delays the development of
apoptosis in the infected cells, and prevents the formation of the
antiviral state in neighbouring cells. Influenza viruses containing
modifications within the NS1 protein are known in the art. For
example, WO 99/64571 describes the complete knock out of the NS
gene segment, WO 99/64068 discloses various NS gene segments that
have been partially deleted, yet none of the described
modifications disclose an influenza virus vector according to the
present invention.
[0055] According to the present invention the modification within
the NS1 protein can be a deletion, an insertion or substitution of
at least one amino acid resulting in a replication deficient
influenza virus.
[0056] Preferably the modified NS1 protein comprises a deletion of
at least 50% of the NS1 amino acids, preferably of at least 70%,
more preferably of at least 90%.
[0057] Alternatively, the functionality of the NS1 protein can be
completely diminished.
[0058] The NS1 protein of the influenza virus vector according to
the invention lacks the functional RNA binding domain. The primary
function of this domain located at the amino end of the NS1 protein
(amino acids 1-73, the wild type amino acid sequence is attached as
SEQ ID No. 80) is binding dsRNA and inhibiting the 2''5''oligo (A)
synthetase/RNase L pathway (Min J. et al., Proc. Natl. Acad. Sci,
2006, 103, 7100-7105, Chien et al., Biochemistry. 2004 Feb. 24;
43(7)1950-62) as well as the activation of a cytoplasmic RNA
helicase, RIG-I, retinoic acid-inducible protein I (Yoneyama M. et
al., Nat. Immunol., 2004, 5, 730-737).
[0059] Lack of a functional RNA binding domain is defined according
to the present invention as complete lack of dsRNA binding ability
leading to an influenza virus that does not replicate in interferon
competent cells.
[0060] According to the invention the effector domain of the NS1
protein of influenza virus vector is not functional. The effector
domain interacts with cellular proteins to inhibit mRNA nuclear
export. The effector domain is located at the C-terminal part of
the NS1 protein. According to Schultz et al. the effector domain is
specifically located between amino acid residues 117 and 161, other
literature locates the effector domain between 134 and 161. The NS1
effector domain can be completely or partially deleted as well as
amino acids can be substituted or inserted and the remaining
effector domain can be tested for functionality as described in the
art (Schultz-Cherry S. et al., J. Virol., 2001, 7875-7881).
[0061] According to the invention the C-terminal amino acids
relevant for effector binding activity are modified to inhibit
effector function. Specifically amino acids at positions 74 to 230,
more specifically amino acids at positions 116 to 161, more
specifically at positions 134 to 161 are modified. According to a
preferred embodiment, the modification is a deletion of said amino
acids.
[0062] The heterologous sequence according to the present invention
can be any biologically active protein or peptide or antigenic
structure.
[0063] For example, antigenic structures can be proteins or
carbohydrate structures which can be recognized by the immune
system, e.g. antibodies or antibody-like structures can bind to
these structures. These epitope structures can contain signal
peptides or can be directly linked to the modified NS1 protein. For
example, foreign epitope structures can be derived from other
pathogens, from tumor associated antigens or retroviral epitopes
expressed on the surface of tumour cells. Carbohydrate antigens are
often of particularly weak immunogenicity. Their immunogenicity can
be improved by conjugating the carbohydrate to a protein carrier.
Proteins or peptides can also be linked to transmembrane domain
sequences preferably containing stretches of hydrophobic amino
acids or other leader sequences known to be needed for transporting
the protein/peptide through the cellular membrane barriers.
Transmembrane domain usually denotes a single transmembrane alpha
helix of a transmembrane protein. An alpha-helix in a membrane can
be folded independently from the rest of the protein, similar to
domains of water-soluble proteins. A transmembrane domain can be
any three-dimensional protein structure which is thermodynamically
stable in a membrane. This may be a single alpha helix, a stable
complex of several transmembrane alpha helices, a transmembrane
beta barrel, a beta-helix of gramicidin A, or any other
structure.
[0064] Transmembrane helices are usually about 20 amino acids in
length, although they may be much longer or shorter.
[0065] For example these could be HA transmembrane sequences or any
other known viral transmembrane domains.
[0066] The biologically active protein used according to the
invention can comprise a signal peptide. The signal peptide can be
any signal sequence being either a naturally occurring signal
sequence or a synthetic one. For example it can be the naturally
existing signal sequence of the heterologous sequence.
Alternatively, it can also be derived from an antibody, preferably
from an Ig kappa chain, more preferably from Ig kappa signal
peptide. Preferably, the Ig kappa chain is derived from mouse Ig
kappa chain.
[0067] According to a preferred embodiment of the invention the
heterologous sequence expresses cytokines or chemokines or
fragments or derivatives thereof.
[0068] Cytokines are small secreted proteins which mediate and
regulate immunity, inflammation and hematopoiesis. The largest
group of cytokines are those which promote proliferation and
differentiation of immune cells. Included within this group are
interleukins, which are cytokines produced by leukocytes, and
interferons, which may be produced by a variety of cell types.
[0069] Interferons (IFN) are a family of naturally occurring
glycoproteins produced by cells of the immune system of
vertebrates, including mammals, birds, reptiles and fish, in
response to challenge by agents such as bacteria, viruses,
parasites and tumour cells. In humans there are three major classes
of interferons. The type I interferons include 14 IFN-alpha
subtypes and single IFN-beta, omega, kappa and epsilon isoforms.
Type II interferons consist of IFN-gamma and a recently discovered
third class consists of IFN-lambda with three different
isoforms.
[0070] Th1 cells secrete mainly IL-2, IFN-.gamma., and TNF-.beta.,
whereas Th2 cells which are relevant in humoral immune responses
secrete cytokines such as IL-4, IL-5, and IL-10. Th2-type cytokines
mediate delayed type hypersensitivity responses against
intracellular pathogens and inhibit the Th1 responses.
[0071] Chemokines, originally derived from chemoattractant
cytokines, actually comprise more than 50 members and represent a
family of small, inducible, and secreted proteins of low molecular
weight (6-12 kDa in their monomeric form) that play a decisive role
during immunosurveillance and inflammatory processes. Depending on
their function in immunity and inflammation, they can be
distinguished into two classes. Inflammatory chemokines are
produced by many different tissue cells as well as by immigrating
leukocytes in response to bacterial toxins and inflammatory
cytokines like IL-1, TNF and interferons. Their main function is to
recruit leukocytes for host defence and in the process of
inflammation. Homing chemokines, on the other hand, are expressed
constitutively in defined areas of the lymphoid tissues. They
direct the traffic and homing of lymphocytes and dendritic cells
within the immune system. These chemokines, as illustrated by
BCA-I, SDF-1 or SLC, control the relocation and recirculation of
lymphocytes in the context of maturation, differentiation,
activation and ensure their correct homing within secondary
lymphoid organs.
[0072] According to the present invention it has been shown that
biologically active cytokines or chemokines or derivatives or
fragments thereof can be stably and efficiently expressed using an
open reading frame different from the ORF expressing the NS1
protein. Alternatively additional leader sequences other than the
natural signal peptides can be fused to the cytokines or chemokines
which may further support efficient secretion of the protein and
show a highly efficient induction of immune response in vivo.
[0073] Surprisingly, chemokines and cytokines can also be
efficiently expressed when the amino acid sequence corresponding to
the mature cytokine/chemokine is fused to a part of the NS1 protein
via an amino acid sequence acting as a signal peptide , For
example, this can be a part of the mouse IgKappa signal
peptide.
[0074] According to the present invention the heterologous sequence
preferably codes for interleukin 2 (IL-2) or a fragment or
derivative thereof. IL-2 comprises secretory signal sequences and
is an immunomodulatory, T-cell derived molecule required for the
clonal expansion of antigen-activated T-cells. The secretion of
IL-2 by CD4+ T lymphocytes has multiple biological effects, such as
the induction of proliferation of T-helper and T-killer cells and
the stimulation of T-cells to produce other cytokines. Furthermore,
IL-2 can also activate B-cells, NK cells and macrophages. When IL-2
is expressed from recombinant viruses infecting non-lymphoid cells,
its secretion could significantly decrease the pathogenesis of
viral infection and modify the immune response. It is also known
that IL-2 acts as immune adjuvant.
[0075] According to the present invention any fragment or
derivative of the cytokines and chemokines is included that is
still biologically active, i.e. shows immunomodulatory
activities.
[0076] Alternatively, the cytokines/chemokines can also be selected
from the group consisting of IL-15, GM-CSF, CCL3 or CCL20 or
derivatives or fragments thereof.
[0077] Alternatively, it can be also any epitope or
immunomodulatory region derived from Mycobacterium tuberculosis,
for example ESAT-6.
[0078] Alternatively the heterologous sequences can also comprise
chimeric proteins being cytokines or chemokines or fragments or
derivatives thereof fused to antigenic proteins or antigenic
peptides. Fusion can be either directly or via peptide linker
sequences having a length of at least 4 amino acids, preferably at
least 5 amino acids. For example, the linker sequences according to
the invention are GGGS or GGGGS.
[0079] Examples for IL-2 chimeric proteins are known in the art.
Exemplarily, this could be IL-2-PE40 (wherein PE is Pseudomonas
exotoxin A), DAB389-IL-2 (where DAB is diphtheria toxin) or IL-2
Bax (wherein Bax is a proapoptotic protein of human origin)
(Aqeilan R. et al., Biochem. J., 2003, 129-140).
[0080] According to the present invention the nucleotide sequences
of the heterologous sequences which are introduced into the
replication deficient influenza vector show at least 80% identity
with their native sequences, preferably at least 85% identity, more
preferred at least 90% identity. Any optimization of the nucleotide
sequence in view of codon usage is included thereby.
[0081] Alternatively, the heterologous sequence can comprise B-cell
or T-cell-epitopes, for example a B cell epitope from influenza
hemagglutinin (HATB), for example the A loop epitope from the
influenza virus hemagglutinin (HA) or parts thereof, or peptides
representing one of the immunodominant epitopes of HA corresponding
to amino acid sequence 150 to 159 (Caton et al., 1982, Cell,
417-427).
[0082] The epitope can also be derived from melanoma-associated
endogenous retrovirus (MERV) as described in WO06/119527. It can be
an epitope derived from the gag, pol or env protein of the virus,
preferably from env. Especially, it can be one or more of the
following peptides: EMQRKAPPRRRRHRNRA (SEQ ID. No 12);
RMKLPSTKKAEPPTWAQ (SEQ ID. No 13); TKKAEPPTWAQLKKLTQ (SEQ ID. No
14); MPAGAAAANYTYWAYVP (SEQ ID. No 15); PIDDRCPAKPEEEGMMI (SEQ ID.
No 16); YPPICLGRAPGCLMPAV (SEQ ID. No 17); YQRSLKFRPKGKPCPKE (SEQ
ID. No 18); FRPKGKPCPKEIPKESK (SEQ ID. No 19); GKPCPKEIPKESKNTEV
(SEQ ID. No 20); GTIIDWAPRGQFYHNCS (SEQ ID. No 21);
RGQFYHNCSGQTQSCPS (SEQ ID. No 22); DLTESLDKHKHKKLQSF (SEQ ID. No
23); PWGWGEKGISTPRPKIV (SEQ ID. No 24); PKIVSPVSGPEHPELWR (SEQ ID.
No 25); PRVNYLQDFSQRSLKF (SEQ ID. No 26); RVNYLQDFSYQRSLKFR (SEQID.
No 27); VNYLQDFSYQRSLKFRP (SEQ ID. No 28); VNYLQDFSYQRSLKFRSP (SEQ
ID. No 29); NYLQDFSYQRSLKFRPK (SEQ ID. No 30); YLQDFSYQRSLKFRPKG
(SEQ ID. No 31); LQDFSYQRSLKFRPKGK (SEQ ID. No 32);
QDFSYQRSLKFRPKGKP (SEQ ID. No 33); DFSYQRSLKFRPKGKPC (SEQ ID. No
34); FSYQRSLKFRPKGKPCP (SEQ ID. No 35); SYQRSLKFRPKGKPCPK (SEQ ID.
No 36); YQRSLKFRPKGKPCPKE (SEQ ID. No 37); QRSLKFRPKGKPCPKEI (SEQ
ID. No 38); RSLKFRPKGKPCPKEIP (SEQ ID. No 39); SLKFRPKGKPCPKEIPK
(SEQ ID. No 40); LKFRPKGKPCPKEIPKE (SEQ ID. No 41);
KFRPKGKPCPKEIPKES (SEQ ID. No 42); FRPKGKPCPKEIPKESK (SEQ ID. No
43); RPKGKPCPKEIPKESKN (SEQ ID. No 44); PKGKPCPKEIPKESKNT (SEQ ID.
No 45); KGKPCPKEIPKESKNTE (SEQ ID. No 46); GKPCPKEIPKESKNTEV (SEQ
ID. No 47); KPCPKEIPKESKNTEVL (SEQ ID. No 48); PCPKEIPKESKNTEVLV
(SEQ ID. No 49); CPKEIPKESKNTEVLVW (SEQ ID. No 50);
PKEIPKESKNTEVLVWE (SEQ ID. No 51); SYQRSLKFRPKGKPCPKEIP (SEQ ID. No
52).
[0083] According to an alternative embodiment of the invention the
heterologous sequence is expressed from an open reading frame (ORF)
different from the NS1 ORF. Another method for generating a second
ORF can be achieved by incorporation of an internal ribosome entry
site element (Garcia-Sastre A., et al., 1999, J. Virol., 75,
9029-9036) or doubling of influenza virus promoter sequences
(Machado A. et al., 2003, Virology, 313, 235-249).
[0084] According to the present invention it has been surprisingly
shown that even if the first approx. 12 amino acids of the NS1
protein are still present, secretion of the heterologous sequence
is not prohibited
[0085] Therefore, according to the present invention, the virus
vector can contain at least 10 amino acids, preferably up to 30,
preferably up to 20, preferred up to 14 amino acids of the
N-terminus of the NS1 protein and a signal peptide or part thereof
fused to the NS1 C-terminus. The C-terminal signal sequence is
preferably present in case the NS1 protein contains not more than
30 amino acids of the N-terminus.
[0086] By using this specific construct, i.e. the fusion of a
signal peptide or part thereof with said N-terminal amino acids of
the NS1 protein, the so derived NS1 protein can be functionally
modified to act as a signal peptide. Expression of heterologous
sequences by said fusion peptides can increase the secretory
characteristics of said heterologous sequences.
[0087] According to a preferred embodiment of the invention the
translation of the NS1 protein is terminated by at least one stop
codon and expression of said heterologous sequence is reinitiated
by a start codon. For example, a stop-start cassette having the
sequence UAAUG (SEQ ID. No 53) can be inserted into the influenza A
virus NS gene coding sequence followed by the insertion of the
heterologous sequence. In view of the short Stop-Start codon
sequence and the limited capacity of the virus to express long
sequence inserts when fused directly to or posttranslationally
cleaved from NS1, the stop-start system can be highly advantageous
compared to the incorporation of long sequences, i.e. of an
internal ribosome entry site element. The stop-start codon can be
inserted at any position within the NS gene between the splice
donor and the splice acceptor site without modifying the nucleotide
sequences of the functional splice sites.
[0088] In an alternative embodiment the stop-start codon is
inserted at a position wherein at least 4 nucleotides, more
preferred at least 6 nucleotides, (more preferred at least 8
nucleotides downstream) of the 5'' splice donor site of the NS gene
are expressed. The NS 5' and 3' intron boundaries are defined as
the cleavage site between the first exon and the intron and the
cleavage site between the intron and the second exon. In case of
influenza A, the insertion of the start-stop codon is placed at any
position within the NS gene, although at least 10 N-terminal amino
acids of the NS1 protein, alternatively at least 12 N-terminal
amino acids of the NS1 protein are expressed. Alternatively, the
heterologous open reading frame can also be at least partially
overlapping with the NS1 open reading frame.
[0089] In an embodiment of the invention the translation of the
heterologous open reading frame is initiated from an optimized
translation initiation sequence, preferably the translation
initiation sequence is a Kozak consensus sequence (Kozak M.,
Nucleic Acids Research, 1984, 12, 857-872). This consensus sequence
can comprise at least part of the sequence CCRGCCAUGG, wherein R
can be A or G (SEQ ID NO. 54). Positions -3 (i.e., 3 nucleotides
upstream from the ATG codon) and +4 have the strongest influence on
translation (Kozak M., Nucleic Acids Research, 1987, 15,
8125-8148). Thus, the consensus sequence can also be RXXAUGG,
XXAUGG or RXXAUG.
[0090] Furthermore according to the invention the NS gene segment
contains a functional splice donor and/or acceptor splice site.
According to the invention the splice donor and acceptor sites of
the NS gene are consisting of the two nucleotides 3' to the 5'
intron boundary and the two nucleotides 5' to the 3' intron
boundary. Homology to U1 snRNA or pyrimidine stretch can also be
tested and developed to improve functional splice sites.
[0091] According to a specific embodiment, the NS gene segment
contains a functional natural splice donor and acceptor splice
site, i.e. the splice donor and acceptor sites are kept as natural
sites, i.e. the nucleotides are not modified by artificial
techniques.
[0092] Any nucleotide modifications at the splice sites occurring
naturally due to modifications of influenza viruses based on
environmental adaptations or natural strain developments are
natural modifications and do not fall under the term synthetic or
artificial modifications.
[0093] Alternatively, the sequences surrounding the splice donor
and/or upstream of the acceptor site can be altered, Preferably,
alteration or modification can be performed within 3 nucleotides 5'
to the and/or 8 nucleotides 3' to the 5' border of the NS intron,
as well as 100 nucleotides 5' to the and/or 2 nucleotides 3' to the
3' border of the NS intron. This is preferably by introducing
synthetic sequences in order to modify splicing activity.
[0094] If e.g. insertion of a heterologous sequence increases NS
intron size it may be preferable to modify the sequences
surrounding the splice donor and/or acceptor site in order to
increase splicing efficacy and thus genetic stability of the
recombinant NS segment.
[0095] For example, it can be modified in that either the sequence
surrounding the splice donor site is altered to increase the
homology to the 5' end of the human U1 snRNA and/or the sequence
upstream of the splice acceptor site containing the branch point
(Plotch et al. 1986, Proc Natl Acad Sci USA. 83:5444-8; Nemeroff et
al. 1992, Mol Cell Biol. 12:962-70) and the pyrimidine stretch is
replaced by a sequence that enhances splicing of the NS
segment.
[0096] For example, the sequence surrounding the 5' splice site can
be changed from (as found in the PR8 NS segment, (SEQ ID. No 55) to
(nucleotides complementary to the 5' end of the human U1 snRNA are
shown in bold italic letters, the splice donor site is indicated by
"/", (SEQ ID. No 56).
[0097] In order to optimize splicing, the a preferred sequence
introduced 5' of the splice acceptor site comprises a lariat
consensus sequence and a pyrimidine stretch. For example, the
sequence upstream of the synthetic splice acceptor site can be as
follows:
TABLE-US-00001 TACTAAC GACAG/ (SEQ ID. No 57)
[0098] The lariat consensus sequence is underlined, the pyrimidine
stretch is bold, the 3' intron boundary is indicated by "/".
[0099] In view of stability of the virus vector and the expression
rate of the heterologous sequence it can be important to introduce
the synthetic/modified sequence containing a lariat consensus
sequence and a pyrimidine stretch at a specific position within the
NS gene, e.g. directly upstream of the slice acceptor site.
[0100] Furthermore, it may be necessary to vary the distance
between the lariat consensus sequence and the pyrimidine stretch to
modify the splicing rate of the NS segment (Plotch S. and Krug R.,
1986, Proc. Natl. Acad. Sci., 83, 5444-5448; Nemeroff M. et al.,
1992, Mol. Cell. Biol., 962-970).
[0101] In a preferred embodiment the replication deficient
influenza virus according to the invention comprises a nucleotide
sequence as shown in FIG. 1 (a-j) or is at least 96% homologous,
alternatively at least 98% homologous.
[0102] In an additional embodiment, also a combination of at least
two replication deficient influenza viruses according to the
invention comprising at least one biologically active molecule or
derivative or fragment thereof and at least one antigenic structure
is claimed. Such combination comprising different heterologous
sequences might be advantageous in view of further increasing
humoral as well as cellular immunogenicity. For example, one of the
vectors can contain a cytokine or fragment or derivative thereof
like IL2 and a second virus vector can comprise an antigenic
peptide or polypeptide.
[0103] Alternatively the heterologous sequences can also comprise
fusion proteins wherein cytokines or chemokines or fragments or
derivatives thereof are fused to antigenic proteins or antigenic
peptides or linked directly or via a linker peptide to the NS1
protein derivative.
[0104] The present invention covers also a signal peptide
comprising part of the N-terminal amino acids of an NS1 protein,
for example 10-12 amino acids of the N-terminus of the NS1 protein,
and a signal peptide or part thereof fused to the C-terminus of
said NS1 peptide. Said signal peptide can consist of 8 to 30,
preferably up to 50 amino acids.
[0105] The signal sequence can be derived from an antibody light
chain, preferably from an Ig kappa chain, more preferably from
mouse Ig kappa chain. According to an alternative embodiment, the
Ig Kappa chain can comprise at least 10 amino acids, more preferred
at least 12 amino acids, for example comprising the sequence
METDTLLLWVLLLWVPGSTGD (SEQ ID. No. 11) or METDTLLLWVLLLWVPRSHG (SEQ
ID No. 82) or part thereof.
[0106] A vaccine formulation comprising the replication deficient
influenza virus vector according to the invention is also
covered.
[0107] According to the invention the replication deficient
influenza virus can be used for the preparation of a medicament for
therapeutic treatment in patients, for example for the treatment of
infectious diseases or cancer.
[0108] Methods of introduction include but are not limited to
intradermal, intramuscular, intraperitoneal, intravenous,
intranasal, epidural or oral routes. Introduction by intranasal
routes is preferred.
[0109] In a preferred embodiment it may be desirable to introduce
the medicament into the lungs by any suitable route. Pulmonary
administration can also be employed, using e.g. an inhaler or
nebulizer or formulate it with an aerosolizing agent.
[0110] The pharmaceutical preparation can also be delivered by a
controlled release system, like a pump.
[0111] The medicament according to the invention can comprise a
therapeutically effective amount of the replication deficient virus
and a pharmaceutically acceptable carrier. "Pharmaceutically
acceptable" means approved by regulatory authorities like FDA or
EMEA. The term "carrier" refers to a diluent, adjuvant, excipient
or vehicle with which the preparation is administered. Saline
solutions, dextrose and glycerol solutions as liquid carriers or
excipients like glucose, lactose, sucrose or any other excipients
as known in the art to be useful for pharmaceutical preparations
can be used. Additionally, also stabilizing agents can be included
to increase shelf live of the medicament.
[0112] Preferably, a ready-to-use infusion solution is provided.
Alternatively, the preparation can be formulated as powder which is
solved in appropriate aqueous solutions immediately before
application.
[0113] The amount of the pharmaceutical composition of the
invention which will be effective in the treatment of a particular
disorder or condition will depend on the nature of the disorder or
condition, and can be determined by standard clinical techniques.
In addition, in vitro assays may optionally be employed to help
identify optimal dosage ranges. The precise dose to be employed in
the formulation will also depend on the route of administration,
and the seriousness of the disease or disorder, and should be
decided according to the judgment of the practitioner and each
patient's circumstances. However, suitable dosage ranges for
administration are generally about 10.sup.4-5.times.10.sup.7 pfu
and can be administered once, or multiple times with intervals as
often as needed. Pharmaceutical compositions of the present
invention comprising 10.sup.4-5.times.10.sup.7 pfu of mutant
replication deficient viruses can be administered intranasally,
intratracheally, intramuscularly or subcutaneously Effective doses
may be extrapolated from dose-response curves derived from in vitro
or animal model test systems.
[0114] Furthermore, a vector comprising a nucleotide sequence
coding for a replication deficient influenza virus according to the
invention is covered.
[0115] If a DNA vector is used, said vector is a transcription
system for minus sense influenza RNA. For example it can be a
vector as used by Hoffmann et al., 2000, Proc Natl Acad Sci USA.
97:6108-13. Alternatively, also an RNA comprising the sequence
coding for the inventive replication deficient virus can be
used.
[0116] Method for producing the inventive replication deficient
influenza virus comprising the steps of: transfecting cells,
preferably Vero cells, with at least one vector comprising the
sequence for the inventive virus, incubating the transfected cells
to allow for the development of viral progeny containing the
heterologous protein is of course also covered by the
invention.
[0117] Alternatively, a method for producing a replication
deficient influenza virus is also provided, comprising the steps
of: transforming a cell, preferably a Vero cell, with a vector
comprising a nucleotide sequence coding for a replication deficient
influenza virus according to the invention preferably together with
a purified preparation of influenza virus RNP complex, infecting
the selected cells with an influenza helper virus, incubating the
infected cells to allow for the development of viral progeny and
selecting transformed cells that express the modified NS gene and
the heterologous sequence,
[0118] The foregoing description will be more fully understood with
reference to the following examples. Such examples are, however,
merely representative of methods of practicing one or more
embodiments of the present invention and should not be read as
limiting the scope of invention.
EXAMPLES
Example 1
Expression of Human Interleukin-2 From a Separate Open Reading
Frame
[0119] A cDNA coding for human IL-2 was inserted into a modified NS
segment of the influenza A strain Puerto Rico/8/34 that does not
code for a functional NS1 protein. The NS1 protein was terminated
after amino acid 21 by means of an artificially introduced Stop
codon and thus does neither contain the RNA binding domain nor the
effector domain.
[0120] To allow IL-2 translation the artificially introduced NS1
stop codon overlaps with the Start codon of IL-2 to give the
sequence TAATG (SEQ ID No. 81). Two constructs were generated (see
FIG. 2).
[0121] In both constructs (deINS1-IL-2-10 and deINS-IL-2-11) the
IL-2 cDNA including the overlapping Stop/start codon replaces
nucleotides 90-345 of the wild-type NS segment corresponding to
amino acids 22-106 of the NS1 protein.
[0122] Construct deINS-IL-2-10 thus comprises the natural splice
acceptor site, the natural branch point 20 nucleotides upstream of
the splice acceptor site (Plotch et al. 1986, Proc Natl Acad Sci
USA. 83:5444-8; Nemeroff et al. 1992, Mol Cell Biol. 12:962-70) as
well as the natural 11-nucleotide pyrimidine stretch of the
wild-type NS segment. A lariat consensus sequence (CTRAY or
YNYYRAY) that is found 72 nucleotides upstream of the 3' splicing
site in the wild-type NS segment is also present in the
deINS-IL-2-10 segment.
[0123] In addition, in the deINS1-IL-2-11 segment a synthetic
sequence of 29 nucleotides comprising a lariat consensus sequence
followed by a 20-base pyrimidine stretch segment replaces
nucleotides 361-525 of the wild-type NS segment corresponding to
amino acids 112-166 of the NS1 protein. Thus also the natural
branch point, the pyrimidine stretch as well as the lariat
consensus sequence found 72 bases upstream of the 3' splicing site
in the NS segment were replaced.
[0124] Furthermore, in both chimeric IL-2 NS segments the sequence
downstream of the 5' intron boundary was changed to achieve 100%
complementarity to the 5' end of the human U1 snRNA (i.e. /GTAGATTG
as found in the wild type NS segment was changed to GTAAGTAT). In
addition a methionine found in alternative reading frame at
position 76 of the wild-type NS segment was changed to a
valine.
[0125] Thus the amino acid sequence of the truncated NS1 protein is
MDPNTVSSFQVSIFLWRVRKR (letters shown underlined in bold denote
changes from the wild-type NS sequence, (SEQ ID No. 59).
[0126] Description of the deINS1-IL-2-10 segment as shown in FIG.
1a: the ORF is consisting of the truncated NS1, i.e. the
nucleotides 27-92; the human IL-2 ORF consists of nucleotides
92-553; The 5' intron boundary is located between nucleotides 56
and 57; the 3' intron boundary is between nucleotides 739 and 740
(SEQ ID No 1).
[0127] Description of the deINS1-IL-2-11 segment as shown in FIG.
1b: the ORF of the truncated NS1 consists of nucleotides 27-92; the
human IL-2 ORF consists of nucleotides 92-553; the splice donor
site is between nucleotides 56 and 57; the splice acceptor site is
between nucleotides 603 and 604 (SEQ ID No 2);
[0128] Plasmid Constructions
[0129] As a backbone for construction of chimeric human
Interleukin-2 NS segments the plasmid pKW2000 was used. pKW2000 was
obtained by deleting the CMV promoter in pHW2000 (Hoffmann et al.
2000, Proc Natl Acad Sci USA. 97:6108-13). Thus upon transfection
only vRNA is transcribed from pKW2000 derivatives.
[0130] DeINS1-IL-2-10 and deINS1-IL-2-11 segments were constructed
by PCR standard methods and cloned into pKW2000 to yield the
plasmids pKW-deINS-IL2-10 and pKW-deINS-IL-2-11, respectively.
Analogously, a pKW2000 derivative containing the PR8 deINS segment
(Garcia-Sastre et al. 1998, Virology. 252:324-30) was constructed
(pKW-deINS1).
[0131] PA, PB1, PB2, HA, NA, M and NP segments derived from a
Vero-cell adapted influenza A H1N1 virus strain (GHB01) were cloned
into pHW2000.
[0132] All plasmids were sequenced to ensure the absence of
unwanted mutations.
[0133] Generation of Viruses
[0134] Vero cells were maintained in DMEM/F12 medium containing 10%
foetal calf serum and 1% Glutamax-I supplement at 37.degree. C.
[0135] For virus generation seven pHW2000 derivatives containing
the segments PA, PB1, PB2, HA, NA, M and NP derived from GHBO1 as
well as two protein expression plasmids coding for Influenza A PR8
NS1 (pCAGGS-NS1(SAM); (Salvatore et al. 2002, J Virol. 76:1206-12))
and NEP (pcDNA-NEP) were used together with either
pKW-deINS-IL-2-10, pKW-deINS-IL-2-11 or pKWdeINS1 for
cotransfection of Vero cells. Following transfection, to support
virus replication Vero cells were cultured in serum-free medium
(Opti-Pro; Invitrogen) in the presence of 5 .mu.g/ml trypsin. Three
days after transfection 50-100% CPE was observed and rescued
viruses were frozen or further amplified on Vero cells. In addition
chimeric IL-2 expressing viruses were plaque purified once. After
amplification on Vero cells several plaques were frozen for further
analysis.
[0136] The generated viruses are designated GHB-IL-2-10,
GHB-IL-2-11 and GHB01.
[0137] Analysis of Interleukin-2 Expression
[0138] Vero cells were infected at a multiplicity of infection of
0.1 with GHB-deINS1, GHB-IL-2-10 or GHB-IL-2-11 and incubated for
16 h at 37.degree. C. in serum-free medium in the presence of 1
.mu.g/ml trypsin. Subsequently, foetal calf serum (final
concentration 10%) as well as soy bean trypsin inhibitor (final
concentration 100 .mu.g/ml) was added and incubation at 37.degree.
C. was continued for another 24 h.
[0139] Supernatants were analysed for secreted IL-2 by ELISA.
[0140] IL-2 expression was found to be about 5-fold higher for the
GHB-IL-2-10 virus compared to the GHB-IL-2-11 virus (see FIG. 3).
As expected, no IL-2 was detected in supernatants infected with
GHBO1 virus lacking the IL-2 cDNA.
[0141] The human-IL2 expression level in Vero cells was approx.
2600 pg/ml in GHB-IL-2-10 and approx. 500 pg/ml GHB-IL-2-11. In
contrast, the expression level according to the state of the art
was between 250-350 pg/ml (Kittel et al., 2005, s. above).
[0142] Analysis of Virus Stability
[0143] Chimeric IL-2 influenza viruses obtained either directly
after transfection or after one round of plaque purification were
serially passaged five times on Vero cells. RNA was extracted using
a ViralAmp kit (Qiagen) and reverse transcribed. Whole NS segments
were PCR amplified and subjected to agarose gel electrophoresis to
evaluate the presence of deletions.
[0144] As shown in FIG. 4, deletion bands were found for all
GHB-IL-2-10 virus samples regardless of plaque purification. In
contrast, PCR products obtained for the GHB-IL-2-11 virus samples
migrated at the expected size (see FIG. 4).
Example 2
Expression of Human Interleukin-2 From the NS1 Open Reading
Frame
[0145] A cDNA coding for human IL-2 was inserted into a modified NS
segment of the influenza A strain Puerto Rico/8/34 that does not
code for a functional NS1 protein. In contrast to example 1, the
IL-2 cDNA was directly fused to a truncated (12 amino acid) NS1
protein. Thus, IL-2 is expressed from the NS1 open reading frame
(see FIG. 1c, deINS1-IL-2-14)
[0146] To allow IL-2 secretion, a cDNA coding for the mature IL-2
was fused to the first 12 aa of the NS1 protein via a modified Ig
kappa signal peptide resulting in the following amino acid
sequence:
TABLE-US-00002 (SEQ ID No. 60) MDPNTVSSFQVS- -
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA
TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE
TTFMCEYADETATIVEFLNRWITFCQSIISTLT.
[0147] The first 12 amino acids of the above sequence correspond to
the truncated NS1 protein, the amino acids corresponding to the
modified mouse Ig kappa signal peptide are depicted in italic bold
letter, and the remaining amino acid sequence corresponds to the
mature human IL-2.
[0148] Description of the deINS1-IL-2-14 segment: ORF of the
NS1-IgKappa-IL-2 fusion: nucleotides 27-509; Splice donor site
between nucleotides 56 and 57; Splice acceptor site between
nucleotides 559 and 560 (FIG. 1c)
[0149] Virus generation and analysis of IL-2 expression was done as
described in example 1. The generated viruses was designated
GHB-IL-2-14.
[0150] IL-2 expression levels were found to be about 17-times
higher than for GHB-IL-2-11 (see FIG. 5). Thus, high level IL-2
expression from the truncated NS1 open reading frame is
feasible.
Example 3
Influence of the Sequence Surrounding the Splice Donor Site on IL-2
Expression
[0151] To analyse the influence of the sequence surrounding the
splice donor site on IL-2 expression, deINS1-IL-2-11 and
deINS1-IL-2-14 were further modified.
[0152] DeINS1-IL-2-13 was constructed from deINS1-IL-2-11 by
changing the 8 nucleotides downstream of the 5' intron boundary
from to as found in the wild type PR8 NS segment (nucleotides
complementary to the 5' end of the human U1 snRNA are shown in bold
italic letters, the 5' intron boundary is indicated by "/"). The
deINS1-IL2-13 sequence is shown in FIG. 1d.
[0153] Similarly, deINS1-IL-2-21 was constructed from
deINS1-IL-2-14 by changing the sequence to (nucleotides
complementary to the 5' end of the human U1 snRNA are shown in bold
italic letters, the 5' intron boundary is indicated by "/").
[0154] The deINS1-IL2-21 sequence is shown in FIG. 1e.
[0155] Thus, in both constructs homology to the 5' end of the U1
snRNA was decreased when compared to their progenitor
constructs.
[0156] For deINS1-IL-2-13 the amino acid sequence for the truncated
NS1 protein is: MDPNTVSSFQVDCFLWRVRKR (SEQ ID NO. 61)
[0157] For deINS1-IL-2-21 the amino acid sequence for the NS1-IgK
signal peptide-IL-2 fusion protein is:
MDPNTVSSFQV-FAAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQC
LEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITFCQSIISTLT (SEQ ID NO. 62)
[0158] The first 11 amino acids of the above sequence correspond to
the truncated NS1 protein, the amino acids corresponding to the
modified mouse Ig kappa signal peptide are depicted in italic bold
letter, and the remaining amino acid sequence corresponds to the
mature human IL-2.
[0159] Viruses were generated and analysed for IL-2 expression as
described in example 1. The generated viruses were designated
GHB-IL-2-13and GHB-IL-2-21. Genetic stability of the deINS1-IL-2-13
and deINS1-11-2-21 segment was analysed after 5 consecutive
passages on Vero cells as described in example 1.
[0160] IL-2 expression levels were found to be higher for the
respective constructs that have a lower homology to the U1 sRNA
around their splice donor site (see FIG. 5).
[0161] Levels for GHB-IL-2-13 were found to be about 13-times
higher than for the corresponding virus that exhibits a high
homology to the U1 snRNA (GHB-IL-2-13; 9,4 ng/ml versus 0,7 ng/ml;
FIG. 5). Similarly, IL-2 levels for GHB-IL-2-21 were found to be
roughly 2,6-times higher than for GHB-IL-2-14 (31,1 ng/ml versus
12,1 ng/ml; FIG. 5).
[0162] Thus, by modifying the sequence around the NS splice donor
site IL-2 expression levels can be tuned.
[0163] For both viruses, deINS1-IL-2-13 and deINS1-Il-2-21 no
deletion bands were found after 5 consecutive passages indicating
genetic stability.
Example 4
Expression of IL-2 From a Separate Open Reading Frame: Translation
Initiation via a Kozak Consensus Sequence
[0164] The stop/start codon sequence in deINS1-IL-2-11 was replaced
by a Kozak consensus sequence (i.e. the TAATG was replaced with
TAAGCCGCCACCATG; the stop and start codon are indicated in bold
underlined letters, SEQ ID No. 63) to yield the segment
deINS1-IL-2-17.
[0165] The deINS1-IL-2-17 nucleotide sequence is shown in FIG.
1f.
[0166] Virus generation and analysis of IL-2 expression for
GHB-IL-2-17 was performed as described in example 1. IL-2
expression levels were found to be about twice as high as for
GHB-IL-2-11 (data not shown).
Example 5
Expression of Human IL-15 From the NS1 Open Reading Frame
[0167] A cDNA coding for human IL-15 is inserted into a modified NS
segment of the influenza A strain Puerto Rico/8/34 that does not
code for a functional NS1 protein. To allow secretion, the a cDNA
encoding mature IL-15 is fused to a truncated (11 amino acid) NS1
ORF via a modified mouse Ig kappa signal peptide resulting in the
following amino acid sequence:
MDPNTVSSFQV-FANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQV-
ISLESGDASI HDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
(SEQ ID No. 69)
[0168] The first 11 amino acids of the above sequence correspond to
the truncated NS1 protein, the amino acids corresponding to the
modified mouse Ig kappa signal peptide are depicted in italic bold
letter, and the remaining amino acid sequence corresponds to the
mature human IL-15.
[0169] The resulting chimeric IL-15 NS segment is referred to as
deINS1-IL-15-21. The deINS1-IL-15-21 nucleotide sequence is shown
in FIG. 1g
[0170] Virus generation is performed as described in example 1.
[0171] IL-15 expression levels in the supernatants of infected Vero
cells were assessed by ELISA and were found to be in the range of
1-2 ng/ml.
Example 6
Expression of Human GM-CSF From the NS1 Open Reading Frame
[0172] A cDNA coding for human GM-CSF is inserted into a modified
NS segment of the influenza A strain Puerto Rico/8/34 that does not
code for a functional NS1 protein. To allow secretion, the mature
GM-CSF cDNA is fused to a truncated (11 amino acid) NS1 protein via
a modified mouse Ig kappa signal peptide resulting in the following
amino acid sequence:
MDPNTVSSFQV-FAAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTAAEMNETVEVISEMFDLQEPTCLQ
TRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDF
LLVIPFDCWEPVQE (SEQ ID NO. 64)
[0173] The first 11 amino acids of the above sequence correspond to
the truncated NS1 protein, the amino acids corresponding to the
modified mouse Ig kappa signal peptide are depicted in italic bold
letter, and the remaining amino acid sequence corresponds to the
mature human GM-CSF.
[0174] The resulting chimeric GM-CSF NS segment is referred to as
deINS1-GM-CSF-21. The deINS1-GM-CSF-21 nucleotide sequence is shown
in FIG. 1h
[0175] Virus generation is performed as described in example 1.
Example 7
Expression of Human CCL-3 From the NS1 Open Reading Frame
[0176] A cDNA coding for human CCL-3 (MIP-1alpha) is inserted into
a modified NS segment of the influenza A strain Puerto Rico/8/34
that does not code for a functional NS1 protein.
[0177] To allow secretion, the mature CCL-3 cDNA is fused to a
truncated (11 amino acid) NS1 protein via a modified mouse Ig kappa
signal peptide resulting in the following amino acid sequence:
MDPNTVSSFQV-FAAPLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPSVIFLTKRGRQVCADPSEE
WVQKYVSDLELSA (SEQ ID NO. 65)
[0178] The first 11 amino acids of the above sequence correspond to
the truncated NS1 protein, the amino acids corresponding to the
modified mouse Ig kappa signal peptide are depicted in italic bold
letter, and the remaining amino acid sequence corresponds to the
mature human CCL-3.
[0179] The resulting chimeric CCL-3 NS segment is referred to as
deINS1-CCL-3-21. The deINS1-CCL-3-21 nucleotide sequence is shown
in FIG. 1i
[0180] Virus generation is performed as described in example 1.
Example 8
Expression of Human CCL-20 From the NS1 Open Reading Frame
[0181] A cDNA coding for human CCL-20 (MIP-3alpha) was inserted
into a modified NS segment of the influenza A strain Puerto
Rico/8/34 that does not code for a functional NS1 protein.
[0182] To allow secretion, the mature CCL-20 cDNA was fused to a
truncated (11 amino acid) NS1 protein via a modified mouse Ig kappa
signal peptide resulting in the following amino acid sequence:
MDPNTVSSFQV-FAASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVCANPKQTWVKYI
VRLLSKKVKNM (SEQ ID NO. 66)
[0183] The first 11 amino acids of the above sequence correspond to
the truncated NS1 protein, the amino acids corresponding to the
modified mouse Ig kappa signal peptide are depicted in italic bold
letter, and the remaining amino acid sequence corresponds to the
mature human CCL-20.
[0184] The resulting chimeric CCL-20 NS segment is referred to as
deINS1-CCL-20-21. The deINS1-CCL-20-21 nucleotide sequence is shown
in FIG. 1j
[0185] Virus generation is performed as described in example 1.
[0186] CCL-20 expression levels in the supernatants of infected
Vero cells were assessed by ELISA was found to be in the range of
25 ng/ml.
Example 9
Expression of Secreted Mycobacterium tuberculosis ESAT-6 From the
NS1 Open Reading Frame
[0187] A cDNA coding for mycobacterium tuberculosis ESAT-6 was
inserted into a modified NS segment of the influenza A strain
Puerto Rico/8/34 that does not code for a functional NS1
protein.
[0188] To allow secretion, an ESAT-6 cDNA was fused to a truncated
(11 amino acid) NS1 protein via a modified mouse Ig kappa signal
peptide resulting in the following amino acid sequence:
MDPNTVSSFQV-FAMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQ
QKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA (SEQ ID NO. 70)
[0189] The first 11 amino acids of the above sequence correspond to
the truncated NS1 protein, the amino acids corresponding to the
modified mouse Ig kappa signal peptide are depicted in italic bold
letter, and the remaining amino acid sequence corresponds to
ESAT-6.
[0190] The resulting chimeric ESAT-6 NS segment is referred to as
deINS1-ESAT-6s-21. The deINS1-ESAT-6s-21 nucleotide sequence is
shown in FIG. 1k
[0191] Virus generation was performed as described in example
1.
Example 10
Intracellular Expression of Mycobacterium tuberculosis ESAT-6 From
the NS1 Open Reading Frame
[0192] A cDNA coding for mycobacterium tuberculosis ESAT-6 was
inserted into a modified NS segment of the influenza A strain
Puerto Rico/8/34 that does not code for a functional NS1
protein.
[0193] In contrast to example 9 an ESAT-6 cDNA was directly fused
(i.e. without an amino acid sequence acting as a signal peptide) to
a truncated (11 amino acid) NS1 protein resulting in the following
amino acid sequence:
TABLE-US-00003 (SEQ ID NO. 71) MDPNTVSSFQVFA
[0194] The first 11 amino acids of the above sequence correspond to
the truncated NS1 protein, while the amino acid sequence shown in
italic bold letters corresponds to ESAT-6.
[0195] The resulting chimeric ESAT-6 NS segment is referred to as
deINS1-ESAT-6i-21. The deINS1-ESAT-6i-21 nucleotide sequence is
shown in FIG. 1l.
[0196] Virus generation was performed as described in example
1.
Example 11
Expression of IL-2 From the NS1 Open Reading Frame Using
Alternative Signal Peptide Sequences
[0197] The deINS1-IL2-21 segment (example 3) was modified by
replacing the partial mouse IgK signal peptide sequence with other
sequences.
[0198] For deINS1-IL2-23 the amino acid sequence LLWVLLLWVPGSTG
(SEQ ID No. 58) in deINS1-IL2-21 was replaced by the sequence
WVLFILLLFLFLPRSHG (SEQ ID No. 72) resulting in the amino acid
sequence
TABLE-US-00004 (SEQ ID No. 73) MDPNTVSSFQVFAWVLFILLLFLFLPRSHG-
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA
TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE
TTFMCEYADETATIVEFLNRWITFCQSIISTLT.
[0199] The deINS1-IL2-23 nucleotide sequence is shown in FIG.
1m.
[0200] For deINS1-IL2-24 the amino acid sequence LLWVLLLWVPGSTG
(SEQ ID No. 58) in deINS1-IL2-21 was replaced by the sequence
AGAALLALLAALLPASRA (SEQ ID No. 74) which is derived from the human
epidermal growth factor (hEGF) signal peptide
(MRPSGTAGAALLALLAALCPASRA, (SEQ ID No. 75)) resulting in the amino
acid sequence
TABLE-US-00005 (SEQ ID No. 76)
MDPNTVSSFQVFAAGAALLALLAALLPASRAAPTSSSTKKTQLQLEHLLL
DLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEV
LNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLN
RWITFCQSIISTLT.
[0201] The deINS1-IL2-24 nucleotide sequence is shown in FIG.
1n.
[0202] Virus generation was performed as described in example
1.
[0203] IL-2 expression levels in the supernatants of infected Vero
cells were assessed by ELISA.
[0204] Thus, the partial mouse IgK signal peptide can be replaced
by other sequences acting as a signal peptide.
Example 12
Modification of Sequences Surrounding the Splice Donor and Acceptor
Site Affects NS Splicing Efficiency
[0205] To analyse the influence of the sequences surrounding the
intron boundaries on splicing efficiency deINS1-IL-2-10 (see
example 1) was further modified. DeINS1-IL-2-12 was constructed
from deINS1-IL-2-10 by changing the 8 nucleotides downstream of the
5' intron boundary from to as found in the wild type PR8 NS segment
(nucleotides complementary to the 5' end of the human U1 snRNA are
shown in bold italic letters, the splice donor site is indicated by
"/"). Otherwise the deINS1-IL-2-12 nucleotide sequence is identical
to deINS1-IL-2-10.
[0206] Virus generation was done as described in example 1.
[0207] Genetic stability of the deINS1-IL-2-12 segment was analysed
after 5 consecutive passages as described in example 1. Clear
deletion bands were found (data not shown).
[0208] To analyse splicing efficacy, Vero cells were cotransfected
with four plasmids expressing PB1, PB2, PA and NP proteins along
with a plasmid expressing vRNA of deINS1-IL-2-10, deINS1-IL-2-11
(see example 1), deINS1-IL-2-12 or deINS1-IL-2-13 (see example
3).
[0209] 24 hours later mRNA was extracted from transfected cells and
analysed for spliced and unspliced deINS1-IL-2 mRNA species by Real
Time PCR.
[0210] The following table summarises the sequence modifications
performed either 3' to the splice donor site or 5' to the splice
acceptor site as well as genetic stability and IL-2 expression
levels (IL-2 expression for deINS1-IL-2-10 and deLNS1-IL-2-12 are
not given since both segments appeared genetically unstable).
TABLE-US-00006 segment delNS1- delNS1- delNS1- delNS1- IL-2-12
IL-2-10 IL-2-13 IL-2-11 Sequence 3' to wild-type modified wild-type
modified splice donor site Sequence 5' to 3' wild-type wild-type
modified modified splice acceptor site Genetic stability negative
negative positive positive IL-2 expression na na 8 ng 700 pg
[0211] As shown in FIG. 7, deINS1-IL-2 mRNA splicing can be altered
by either modifying the sequence surrounding the splice donor site
or the sequences 5' to the splice acceptor site.
[0212] It is also apparent, that increasing splicing efficiency
above a certain threshold necessary to achieve genetic stability
reduces IL-2 expression (deINS1-IL-2-13 versus deINS1-IL-2-11).
Example 13
Expression of Human Interleukin-2 From a Separate Open Reading
Frame of Influenza B
[0213] A cDNA coding for human IL-2 was inserted into a modified NS
segment of the influenza B strain B/Vienna/33/06. The NS1 protein
was terminated after amino acid 38 by means of an artificially
introduced Stop codon and thus does neither contain the RNA binding
domain nor C-terminal domain of NS1.
[0214] To allow IL-2 translation the artificially introduced NS1
stop codon overlaps with the Start codon of IL-2 to give the
sequence TAATG. A schematic expression scheme is given in FIG. 8.
In this construct (.DELTA.NS1-38IL2), the IL-2 cDNA including the
overlapping Stop/start codon replaces nucleotides 159-728 of the
wild-type NS segment corresponding to amino acids 38-228 of the NS1
protein.
[0215] In this construct, a synthetic sequence of 29 nucleotides
comprising a lariat consensus sequence followed by a 20-base
pyrimidine stretch segment replaces the natural splice acceptor
site plus the natural pyrimidine stretch analogous to the influenza
A construct deINS1-IL-2-11.
[0216] Description of the .DELTA.NS1-38IL2 segment as shown in FIG.
8: the ORF of the truncated NS1 consists of nucleotides 45-158; the
human IL-2 ORF consists of nucleotides 161-619; the 5' intron
boundary is between nucleotides 77 and 78; the 3' intron boundary
is between nucleotides 657 and 658.
[0217] Generation of Plasmids and Viruses
[0218] Plasmids for influenza B Viruses were generated analogous to
the influenza A plasmids using standard cloning techniques. HA and
NA derived from a Vero-cell adapted influenza B/Thuringen/2/06
strain and PA, PB1, PB2, M and NP segments derived from a Vero-cell
adapted influenza B/Vienna/33/06 virus strain and were cloned into
pHW2006. All plasmids were sequenced to ensure the absence of
unwanted mutations.
[0219] The IL2 expressing influenza virus was generated as
described for influenza A and designated .DELTA.NS1-38IL2.
[0220] Analysis of Virus Stability
[0221] Chimeric IL-2 influenza viruses obtained directly after
transfection were serially passaged four times on Vero cells. RNA
was extracted using a ViralAmp kit (Qiagen) and reverse
transcribed. Whole NS segments were PCR amplified and subjected to
agarose gel electrophoresis to evaluate the presence of deletions.
PCR products obtained for the .DELTA.NS1-38IL2 virus samples after
1 and 4 passages migrated at the expected size, indicating that the
IL2 expressing vector is stable.
[0222] Immunogenicity in Mice
[0223] To investigate the immunogenic potential, mice were
immunized with 1*10.sup.5TCID.sub.50/mouse with wt influenza B
Virus, .DELTA.NS1-38IL2, .DELTA.NS1-38 (a control Virus which was
constructed similar to .DELTA.NS1-38IL2 but without the insertion
of IL2) or PBS as a control. Four weeks post immunization, mice
were challenged with 2*10.sup.5TCID.sub.50/mouse of homologous
influenza B wt virus. Three days post infection, mice were
sacrificed and viral replication was investigated in lungs and
nasal turbinates were. Mice which were immunized with the wt
influenza Virus were protected in lungs and noses whereas in the
control mice immunized with PBS, viral titres of approximately 3
logs in both, nasal and lung tissues. At the dose of
1*10.sup.5TCID50/mouse none of the mice immunized with
.DELTA.NS1-38 was protected from wt influenza challenge manifesting
nasal and lung tissues comparable to the naive animals. In
contrast, no virus could be isolated from any mouse immunized with
virus .DELTA.NS1-38IL2 at the same dose, indicating that all mice
were protected.
Sequence CWU 1
1
8211101DNAhuman 1agcaaaagca gggtgacaaa aacataatgg atccaaacac
tgtgtcaagc tttcaggtaa 60gtatctttct ttggcgtgtc cgcaaacgat aatgtacagg
atgcaactcc tgtcttgcat 120tgcactaagt cttgcacttg tcacaaacag
tgcacctact tcttcgtcga caaagaaaac 180acagctacaa ctggagcatt
tactgctgga tttacagatg attttgaatg gaattaataa 240ttacaagaat
cccaaactca ccaggatgct cacatttaag ttttacatgc ccaagaaggc
300cacagaactg aaacatcttc agtgtctaga agaagaactc aaacctctgg
aggaagtgct 360aaatttagct caaagcaaaa actttcactt aagacccagg
gacttaatca gcaatatcaa 420cgtaatagtt ctggaactaa agggatctga
aacaacattc atgtgtgaat atgctgatga 480gacagcaacc attgtagaat
ttctgaacag atggattacc ttttgtcaaa gcatcatctc 540aacactaact
tgataaccaa gcagaaagtg gcaggccctc tttgtatcag aatggaccag
600gcgatcatgg ataagaacat catactgaaa gcgaacttca gtgtgatttt
tgaccggctg 660gagactctaa tattgctaag ggctttcacc gaagagggag
caattgttgg cgaaatttca 720ccattgcctt ctcttccagg acatactgct
gaggatgtca aaaatgcagt tggagtcctc 780atcgggggac ttgaatggaa
tgataacaca gttcgagtct ctgaaactct acagagattc 840gcttggagaa
gcagtaatga gaatgggaga cctccactca ctccaaaaca gaaacgagaa
900atggcgggaa caattaggtc agaagtttga agaaataaga tggttgattg
aagaagtgag 960acacaaactg aagataacag agaatagttt tgagcaaata
acatttatgc aagccttaca 1020tctattgctt gaagtggagc aagagataag
aactttctcg tttcagctta tttaataata 1080aaaaacaccc ttgtttctac t
11012965DNAhuman 2agcaaaagca gggtgacaaa aacataatgg atccaaacac
tgtgtcaagc tttcaggtaa 60gtatctttct ttggcgtgtc cgcaaacgat aatgtacagg
atgcaactcc tgtcttgcat 120tgcactaagt cttgcacttg tcacaaacag
tgcacctact tcttcgtcga caaagaaaac 180acagctacaa ctggagcatt
tactgctgga tttacagatg attttgaatg gaattaataa 240ttacaagaat
cccaaactca ccaggatgct cacatttaag ttttacatgc ccaagaaggc
300cacagaactg aaacatcttc agtgtctaga agaagaactc aaacctctgg
aggaagtgct 360aaatttagct caaagcaaaa actttcactt aagacccagg
gacttaatca gcaatatcaa 420cgtaatagtt ctggaactaa agggatctga
aacaacattc atgtgtgaat atgctgatga 480gacagcaacc attgtagaat
ttctgaacag atggattacc ttttgtcaaa gcatcatctc 540aacactaact
tgataaccaa gcagaaagtg gtactaacct tcttctcttt cttctcctga
600caggacatac tgctgaggat gtcaaaaatg cagttggagt cctcatcggg
ggacttgaat 660ggaatgataa cacagttcga gtctctgaaa ctctacagag
attcgcttgg agaagcagta 720atgagaatgg gagacctcca ctcactccaa
aacagaaacg agaaatggcg ggaacaatta 780ggtcagaagt ttgaagaaat
aagatggttg attgaagaag tgagacacaa actgaagata 840acagagaata
gttttgagca aataacattt atgcaagcct tacatctatt gcttgaagtg
900gagcaagaga taagaacttt ctcgtttcag cttatttaat aataaaaaac
acccttgttt 960ctact 9653921DNAhuman 3agcaaaagca gggtgacaaa
aacataatgg atccaaacac tgtgtcaagc tttcaggtaa 60gtctcctgct ttgggtactg
ctgctctggg ttccaggttc cactggtgca cctacttctt 120cgtcgacaaa
gaaaacacag ctacaactgg agcatttact gctggattta cagatgattt
180tgaatggaat taataattac aagaatccca aactcaccag gatgctcaca
tttaagtttt 240acatgcccaa gaaggccaca gaactgaaac atcttcagtg
tctagaagaa gaactcaaac 300ctctggagga agtgctaaat ttagctcaaa
gcaaaaactt tcacttaaga cccagggact 360taatcagcaa tatcaacgta
atagttctgg aactaaaggg atctgaaaca acattcatgt 420gtgaatatgc
tgatgagaca gcaaccattg tagaatttct gaacagatgg attacctttt
480gtcaaagcat catctcaaca ctaacttgat aaccaagcag aaagtggtac
taaccttctt 540ctctttcttc tcctgacagg acatactgct gaggatgtca
aaaatgcagt tggagtcctc 600atcgggggac ttgaatggaa tgataacaca
gttcgagtct ctgaaactct acagagattc 660gcttggagaa gcagtaatga
gaatgggaga cctccactca ctccaaaaca gaaacgagaa 720atggcgggaa
caattaggtc agaagtttga agaaataaga tggttgattg aagaagtgag
780acacaaactg aagataacag agaatagttt tgagcaaata acatttatgc
aagccttaca 840tctattgctt gaagtggagc aagagataag aactttctcg
tttcagctta tttaataata 900aaaaacaccc ttgtttctac t 9214965DNAhuman
4agcaaaagca gggtgacaaa aacataatgg atccaaacac tgtgtcaagc tttcaggtag
60attgctttct ttggcgtgtc cgcaaacgat aatgtacagg atgcaactcc tgtcttgcat
120tgcactaagt cttgcacttg tcacaaacag tgcacctact tcttcgtcga
caaagaaaac 180acagctacaa ctggagcatt tactgctgga tttacagatg
attttgaatg gaattaataa 240ttacaagaat cccaaactca ccaggatgct
cacatttaag ttttacatgc ccaagaaggc 300cacagaactg aaacatcttc
agtgtctaga agaagaactc aaacctctgg aggaagtgct 360aaatttagct
caaagcaaaa actttcactt aagacccagg gacttaatca gcaatatcaa
420cgtaatagtt ctggaactaa agggatctga aacaacattc atgtgtgaat
atgctgatga 480gacagcaacc attgtagaat ttctgaacag atggattacc
ttttgtcaaa gcatcatctc 540aacactaact tgataaccaa gcagaaagtg
gtactaacct tcttctcttt cttctcctga 600caggacatac tgctgaggat
gtcaaaaatg cagttggagt cctcatcggg ggacttgaat 660ggaatgataa
cacagttcga gtctctgaaa ctctacagag attcgcttgg agaagcagta
720atgagaatgg gagacctcca ctcactccaa aacagaaacg agaaatggcg
ggaacaatta 780ggtcagaagt ttgaagaaat aagatggttg attgaagaag
tgagacacaa actgaagata 840acagagaata gttttgagca aataacattt
atgcaagcct tacatctatt gcttgaagtg 900gagcaagaga taagaacttt
ctcgtttcag cttatttagt actaaaaaac acccttgttt 960ctact
9655921DNAhuman 5agcaaaagca gggtgacaaa aacataatgg atccaaacac
tgtgtcaagc tttcaggtat 60ttgccctgct ttgggtactg ctgctctggg ttccaggttc
cactggtgca cctacttctt 120cgtcgacaaa gaaaacacag ctacaactgg
agcatttact gctggattta cagatgattt 180tgaatggaat taataattac
aagaatccca aactcaccag gatgctcaca tttaagtttt 240acatgcccaa
gaaggccaca gaactgaaac atcttcagtg tctagaagaa gaactcaaac
300ctctggagga agtgctaaat ttagctcaaa gcaaaaactt tcacttaaga
cccagggact 360taatcagcaa tatcaacgta atagttctgg aactaaaggg
atctgaaaca acattcatgt 420gtgaatatgc tgatgagaca gcaaccattg
tagaatttct gaacagatgg attacctttt 480gtcaaagcat catctcaaca
ctaacttgat aaccaagcag aaagtggtac taaccttctt 540ctctttcttc
tcctgacagg acatactgct gaggatgtca aaaatgcagt tggagtcctc
600atcgggggac ttgaatggaa tgataacaca gttcgagtct ctgaaactct
acagagattc 660gcttggagaa gcagtaatga gaatgggaga cctccactca
ctccaaaaca gaaacgagaa 720atggcgggaa caattaggtc agaagtttga
agaaataaga tggttgattg aagaagtgag 780acacaaactg aagataacag
agaatagttt tgagcaaata acatttatgc aagccttaca 840tctattgctt
gaagtggagc aagagataag aactttctcg tttcagctta tttaataata
900aaaaacaccc ttgtttctac t 9216975DNAhuman 6agcaaaagca gggtgacaaa
aacataatgg atccaaacac tgtgtcaagc tttcaggtaa 60gtatctttct ttggcgtgtc
cgcaaacgat aagccgccac catgtacagg atgcaactcc 120tgtcttgcat
tgcactaagt cttgcacttg tcacaaacag tgcacctact tcttcgtcga
180caaagaaaac acagctacaa ctggagcatt tactgctgga tttacagatg
attttgaatg 240gaattaataa ttacaagaat cccaaactca ccaggatgct
cacatttaag ttttacatgc 300ccaagaaggc cacagaactg aaacatcttc
agtgtctaga agaagaactc aaacctctgg 360aggaagtgct aaatttagct
caaagcaaaa actttcactt aagacccagg gacttaatca 420gcaatatcaa
cgtaatagtt ctggaactaa agggatctga aacaacattc atgtgtgaat
480atgctgatga gacagcaacc attgtagaat ttctgaacag atggattacc
ttttgtcaaa 540gcatcatctc aacactaact tgataaccaa gcagaaagtg
gtactaacct tcttctcttt 600cttctcctga caggacatac tgctgaggat
gtcaaaaatg cagttggagt cctcatcgga 660ggacttgaat ggaatgataa
cacagttcga gtctctgaaa ctctacagag attcgcttgg 720agaagcagta
atgagaatgg gagacctcca ctcactccaa aacagaaacg agaaatggcg
780ggaacaatta ggtcagaagt ttgaagaaat aagatggttg attgaagaag
tgagacacaa 840actgaagata acagagaata gttttgagca aataacattt
atgcaagcct tacatctatt 900gcttgaagtg gagcaagaga taagaacttt
ctcgtttcag cttatttagt actaaaaaac 960acccttgttt ctact
9757864DNAhuman 7agcaaaagca gggtgacaaa gacataatgg atccaaacac
tgtgtcaagc tttcaggtat 60ttgccctcct gtgggtgctg ctgctgtggg tgccccgcag
ccacggcaac tgggtgaacg 120tgatcagcga cctgaagaag atcgaggacc
tgatccagag catgcacatc gacgccaccc 180tgtacaccga gagcgacgtg
caccccagct gcaaggtgac cgccatgaag tgctttctgc 240tggaactgca
ggtgatcagc ctggaaagcg gcgacgccag catccacgac accgtggaga
300acctgatcat cctggccaac aacagcctga gcagcaacgg caacgtgacc
gagagcggct 360gcaaagagtg cgaggaactg gaagagaaga acatcaaaga
gtttctgcag agcttcgtgc 420acatcgtgca gatgttcatc aacaccagct
gatgaccaag cagaaagtgg tactaacctt 480cttctctttc ttctcctgac
aggacatact gctgaggatg tcaaaaatgc agttggagtc 540ctcatcgggg
gacttgaatg gaatgataac acagttcgag tctctgaaac tctacagaga
600ttcgcttgga gaagcagtaa tgagaatggg agacctccac tcactccaaa
acagaaacga 660gaaatggcgg gaacaattag gtcagaagtt tgaagaaata
agatggttga ttgaagaagt 720gagacacaaa ctgaagataa cagagaatag
ttttgagcaa ataacattta tgcaagcctt 780acatctattg cttgaagtgg
agcaagagat aagaactttc tcgtttcagc ttatttaata 840ataaaaaaca
cccttgtttc tact 8648903DNAhuman 8agcaaaagca gggtgacaaa gacataatgg
atccaaacac tgtgtcaagc tttcaggtat 60ttgccctgct gtgggtgctg ctcctctggg
tgcccagaag ccacggagcc cctgccagaa 120gccccagccc ctccacccag
ccctgggagc acgtgaacgc catccaggaa gccaggcggc 180tgctgaacct
gagccgggac acagccgccg agatgaacga gaccgtggag gtgatcagcg
240agatgttcga cctccaggaa cccacctgcc tgcagacccg gctggaactg
tacaagcagg 300gcctgcgggg cagcctgacc aagctgaagg gccccctgac
catgatggcc agccactaca 360agcagcactg cccccccacc cccgagacca
gctgcgccac ccagatcatc accttcgaga 420gcttcaaaga gaacctgaag
gacttcctgc tggtgatccc cttcgactgc tgggagcccg 480tgcaggaatg
atgaccaagc agaaagtggt actaaccttc ttctctttct tctcctgaca
540ggacatactg ctgaggatgt caaaaatgca gttggagtcc tcatcggggg
acttgaatgg 600aatgataaca cagttcgagt ctctgaaact ctacagagat
tcgcttggag aagcagtaat 660gagaatggga gacctccact cactccaaaa
cagaaacgag aaatggcggg aacaattagg 720tcagaagttt gaagaaataa
gatggttgat tgaagaagtg agacacaaac tgaagataac 780agagaatagt
tttgagcaaa taacatttat gcaagcctta catctattgc ttgaagtgga
840gcaagagata agaactttct cgtttcagct tatttaataa taaaaaacac
ccttgtttct 900act 9039732DNAhuman 9agcaaaagca gggtgacaaa gacataatgg
atccaaacac tgtgtcaagc tttcaggtat 60ttgccctgct gtgggtgctg ctcctctggg
tgcccagaag ccacggagcc cccctggccg 120ccgatacccc caccgcctgc
tgcttcagct acaccagccg gcagatcccc cagaacttca 180tcgccgacta
cttcgagacc agcagccagt gcagcaagcc cagcgtgatc ttcctgacca
240agcggggcag gcaggtctgc gccgacccca gcgaggaatg ggtgcagaaa
tacgtgagcg 300acctggaact gagcgcctga tgaccaagca gaaagtggta
ctaaccttct tctctttctt 360ctcctgacag gacatactgc tgaggatgtc
aaaaatgcag ttggagtcct catcggggga 420cttgaatgga atgataacac
agttcgagtc tctgaaactc tacagagatt cgcttggaga 480agcagtaatg
agaatgggag acctccactc actccaaaac agaaacgaga aatggcggga
540acaattaggt cagaagtttg aagaaataag atggttgatt gaagaagtga
gacacaaact 600gaagataaca gagaatagtt ttgagcaaat aacatttatg
caagccttac atctattgct 660tgaagtggag caagagataa gaactttctc
gtttcagctt atttaataat aaaaaacacc 720cttgtttcta ct 73210732DNAhuman
10agcaaaagca gggtgacaaa gacataatgg atccaaacac tgtgtcaagc tttcaggtat
60ttgccctgct gtgggtgctg ctcctctggg tccccagaag ccacggcgcc agcaacttcg
120actgctgcct gggctacacc gaccggatcc tgcaccctaa gttcatcgtg
ggcttcacca 180ggcagctggc caacgagggc tgcgacatca acgccatcat
cttccacacc aagaaaaagc 240tgtccgtgtg cgccaacccc aagcagacct
gggtgaagta catcgtgcgg ctgctgtcca 300agaaagtgaa gaacatgtga
tgaccaagca gaaagtggta ctaaccttct tctctttctt 360ctcctgacag
gacatactgc tgaggatgtc aaaaatgcag ttggagtcct catcggggga
420cttgaatgga atgataacac agttcgagtc tctgaaactc tacagagatt
cgcttggaga 480agcagtaatg agaatgggag acctccactc actccaaaac
agaaacgaga aatggcggga 540acaattaggt cagaagtttg aagaaataag
atggttgatt gaagaagtga gacacaaact 600gaagataaca gagaatagtt
ttgagcaaat aacatttatg caagccttac atctattgct 660tgaagtggag
caagagataa gaactttctc gtttcagctt atttaataat aaaaaacacc
720cttgtttcta ct 7321121PRTmouse 11Met Glu Thr Asp Thr Leu Leu Leu
Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Gly Ser Thr Gly Asp
201217PRTHuman endogenous retrovirus 12Glu Met Gln Arg Lys Ala Pro
Pro Arg Arg Arg Arg His Arg Asn Arg1 5 10 15Ala1317PRTHuman
endogenous retrovirus 13Arg Met Lys Leu Pro Ser Thr Lys Lys Ala Glu
Pro Pro Thr Trp Ala1 5 10 15Gln1417PRTHuman endogenous retrovirus
14Thr Lys Lys Ala Glu Pro Pro Thr Trp Ala Gln Leu Lys Lys Leu Thr1
5 10 15Gln1517PRTHuman endogenous retrovirus 15Met Pro Ala Gly Ala
Ala Ala Ala Asn Tyr Thr Tyr Trp Ala Tyr Val1 5 10 15Pro1617PRTHuman
endogenous retrovirus 16Pro Ile Asp Asp Arg Cys Pro Ala Lys Pro Glu
Glu Glu Gly Met Met1 5 10 15Ile1717PRTHuman endogenous retrovirus
17Tyr Pro Pro Ile Cys Leu Gly Arg Ala Pro Gly Cys Leu Met Pro Ala1
5 10 15Val1817PRTHuman endogenous retrovirus 18Tyr Gln Arg Ser Leu
Lys Phe Arg Pro Lys Gly Lys Pro Cys Pro Lys1 5 10 15Glu1917PRTHuman
endogenous retrovirus 19Phe Arg Pro Lys Gly Lys Pro Cys Pro Lys Glu
Ile Pro Lys Glu Ser1 5 10 15Lys2017PRTHuman endogenous retrovirus
20Gly Lys Pro Cys Pro Lys Glu Ile Pro Lys Glu Ser Lys Asn Thr Glu1
5 10 15Val2117PRTHuman endogenous retrovirus 21Gly Thr Ile Ile Asp
Trp Ala Pro Arg Gly Gln Phe Tyr His Asn Cys1 5 10 15Ser2217PRTHuman
endogenous retrovirus 22Arg Gly Gln Phe Tyr His Asn Cys Ser Gly Gln
Thr Gln Ser Cys Pro1 5 10 15Ser2317PRTHuman endogenous retrovirus
23Asp Leu Thr Glu Ser Leu Asp Lys His Lys His Lys Lys Leu Gln Ser1
5 10 15Phe2417PRTHuman endogenous retrovirus 24Pro Trp Gly Trp Gly
Glu Lys Gly Ile Ser Thr Pro Arg Pro Lys Ile1 5 10 15Val2517PRTHuman
endogenous retrovirus 25Pro Lys Ile Val Ser Pro Val Ser Gly Pro Glu
His Pro Glu Leu Trp1 5 10 15Arg2616PRTHuman endogenous retrovirus
26Pro Arg Val Asn Tyr Leu Gln Asp Phe Ser Gln Arg Ser Leu Lys Phe1
5 10 152717PRTHuman endogenous retrovirus 27Arg Val Asn Tyr Leu Gln
Asp Phe Ser Tyr Gln Arg Ser Leu Lys Phe1 5 10 15Arg2817PRTHuman
endogenous retrovirus 28Val Asn Tyr Leu Gln Asp Phe Ser Tyr Gln Arg
Ser Leu Lys Phe Arg1 5 10 15Pro2918PRTHuman endogenous retrovirus
29Val Asn Tyr Leu Gln Asp Phe Ser Tyr Gln Arg Ser Leu Lys Phe Arg1
5 10 15Ser Pro3017PRTHuman endogenous retrovirus 30Asn Tyr Leu Gln
Asp Phe Ser Tyr Gln Arg Ser Leu Lys Phe Arg Pro1 5 10
15Lys3118PRTHuman endogenous retrovirus 31Tyr Leu Gln Asp Phe Ser
Tyr Gln Arg Ser Leu Lys Phe Arg Pro Lys1 5 10 15Gly Lys3217PRTHuman
endogenous retrovirus 32Leu Gln Asp Phe Ser Tyr Gln Arg Ser Leu Lys
Phe Arg Pro Lys Gly1 5 10 15Lys3317PRTHuman endogenous retrovirus
33Gln Asp Phe Ser Tyr Gln Arg Ser Leu Lys Phe Arg Pro Lys Gly Lys1
5 10 15Pro3417PRTHuman endogenous retrovirus 34Asp Phe Ser Tyr Gln
Arg Ser Leu Lys Phe Arg Pro Lys Gly Lys Pro1 5 10 15Cys3517PRTHuman
endogenous retrovirus 35Phe Ser Tyr Gln Arg Ser Leu Lys Phe Arg Pro
Lys Gly Lys Pro Cys1 5 10 15Pro3617PRTHuman endogenous retrovirus
36Ser Tyr Gln Arg Ser Leu Lys Phe Arg Pro Lys Gly Lys Pro Cys Pro1
5 10 15Lys3717PRTHuman endogenous retrovirus 37Tyr Gln Arg Ser Leu
Lys Phe Arg Pro Lys Gly Lys Pro Cys Pro Lys1 5 10 15Glu3817PRTHuman
endogenous retrovirus 38Gln Arg Ser Leu Lys Phe Arg Pro Lys Gly Lys
Pro Cys Pro Lys Glu1 5 10 15Ile3917PRTHuman endogenous retrovirus
39Arg Ser Leu Lys Phe Arg Pro Lys Gly Lys Pro Cys Pro Lys Glu Ile1
5 10 15Pro4017PRTHuman endogenous retrovirus 40Ser Leu Lys Phe Arg
Pro Lys Gly Lys Pro Cys Pro Lys Glu Ile Pro1 5 10 15Lys4117PRTHuman
endogenous retrovirus 41Leu Lys Phe Arg Pro Lys Gly Lys Pro Cys Pro
Lys Glu Ile Pro Lys1 5 10 15Glu4217PRTHuman endogenous retrovirus
42Lys Phe Arg Pro Lys Gly Lys Pro Cys Pro Lys Glu Ile Pro Lys Glu1
5 10 15Ser4317PRTHuman endogenous retrovirus 43Phe Arg Pro Lys Gly
Lys Pro Cys Pro Lys Glu Ile Pro Lys Glu Ser1 5 10 15Lys4417PRTHuman
endogenous retrovirus 44Arg Pro Lys Gly Lys Pro Cys Pro Lys Glu Ile
Pro Lys Glu Ser Lys1 5 10 15Asn4517PRTHuman endogenous retrovirus
45Pro Lys Gly Lys Pro Cys Pro Lys Glu Ile Pro Lys Glu Ser Lys Asn1
5 10 15Thr4617PRTHuman endogenous retrovirus 46Lys Gly Lys Pro Cys
Pro Lys Glu Ile Pro Lys Glu Ser Lys Asn Thr1 5 10 15Glu4717PRTHuman
endogenous retrovirus 47Gly Lys Pro Cys Pro Lys Glu Ile Pro Lys Glu
Ser Lys Asn Thr Glu1 5 10 15Val4817PRTHuman endogenous retrovirus
48Lys Pro Cys Pro Lys Glu Ile Pro Lys Glu Ser Lys Asn Thr Glu Val1
5 10 15Leu4917PRTHuman endogenous retrovirus 49Pro Cys Pro Lys Glu
Ile Pro Lys Glu Ser Lys Asn Thr Glu Val Leu1 5 10 15Val5017PRTHuman
endogenous retrovirus 50Cys Pro Lys Glu Ile Pro Lys Glu Ser Lys Asn
Thr Glu Val Leu Val1 5 10 15Trp5117PRTHuman
endogenous retrovirus 51Pro Lys Glu Ile Pro Lys Glu Ser Lys Asn Thr
Glu Val Leu Val Trp1 5 10 15Glu5220PRTHuman endogenous retrovirus
52Ser Tyr Gln Arg Ser Leu Lys Phe Arg Pro Lys Gly Lys Pro Cys Pro1
5 10 15Lys Glu Ile Pro 20535RNAartificial sequenceStop-start
cassette 53uaaug 55410RNAartificial sequenceKozak Sequence
54ccrgccaugg 105511DNAInfluenza A virus 55caggtagatt g
115611DNAartificial sequenceUS snRNA complementary strand
56caggtaagta t 115732DNAartificial sequencelariat consensus
sequence 57tactaacctt cttctctttc ttctcctgac ag 325814PRTmouse 58Leu
Leu Trp Val Leu Leu Leu Trp Val Pro Gly Ser Thr Gly1 5
105921PRTInfluenza A virus 59Met Asp Pro Asn Thr Val Ser Ser Phe
Gln Val Ser Ile Phe Leu Trp1 5 10 15Arg Val Arg Lys Arg
2060160PRThuman 60Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Ser
Leu Leu Leu Trp1 5 10 15Val Leu Leu Leu Trp Val Pro Gly Ser Thr Gly
Ala Pro Thr Ser Ser 20 25 30Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu
His Leu Leu Leu Asp Leu 35 40 45Gln Met Ile Leu Asn Gly Ile Asn Asn
Tyr Lys Asn Pro Lys Leu Thr 50 55 60Arg Met Leu Thr Phe Lys Phe Tyr
Met Pro Lys Lys Ala Thr Glu Leu65 70 75 80Lys His Leu Gln Cys Leu
Glu Glu Glu Leu Lys Pro Leu Glu Glu Val 85 90 95Leu Asn Leu Ala Gln
Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu 100 105 110Ile Ser Asn
Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr 115 120 125Thr
Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe 130 135
140Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu
Thr145 150 155 1606121PRTInfluenza A virus 61Met Asp Pro Asn Thr
Val Ser Ser Phe Gln Val Asp Cys Phe Leu Trp1 5 10 15Arg Val Arg Lys
Arg 2062160PRTartificial sequenceNS1-IgKappa-IL2 construct 62Met
Asp Pro Asn Thr Val Ser Ser Phe Gln Val Phe Ala Leu Leu Trp1 5 10
15Val Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Ala Pro Thr Ser Ser
20 25 30Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp
Leu 35 40 45Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys
Leu Thr 50 55 60Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala
Thr Glu Leu65 70 75 80Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
Pro Leu Glu Glu Val 85 90 95Leu Asn Leu Ala Gln Ser Lys Asn Phe His
Leu Arg Pro Arg Asp Leu 100 105 110Ile Ser Asn Ile Asn Val Ile Val
Leu Glu Leu Lys Gly Ser Glu Thr 115 120 125Thr Phe Met Cys Glu Tyr
Ala Asp Glu Thr Ala Thr Ile Val Glu Phe 130 135 140Leu Asn Arg Trp
Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr145 150 155
1606315DNAartificial sequenceKozak Sequence 63taagccgcca ccatg
1564154PRTartificial sequenceNS1-GMCSF-IgKappa construct 64Met Asp
Pro Asn Thr Val Ser Ser Phe Gln Val Phe Ala Leu Leu Trp1 5 10 15Val
Leu Leu Leu Trp Val Pro Arg Ser His Gly Ala Pro Ala Arg Ser 20 25
30Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val Asn Ala Ile Gln Glu
35 40 45Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp Thr Ala Ala Glu Met
Asn 50 55 60Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp Leu Gln Glu
Pro Thr65 70 75 80Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln Gly
Leu Arg Gly Ser 85 90 95Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met
Ala Ser His Tyr Lys 100 105 110Gln His Cys Pro Pro Thr Pro Glu Thr
Ser Cys Ala Thr Gln Ile Ile 115 120 125Thr Phe Glu Ser Phe Lys Glu
Asn Leu Lys Asp Phe Leu Leu Val Ile 130 135 140Pro Phe Asp Cys Trp
Glu Pro Val Gln Glu145 1506597PRTartificial sequenceCCL-3
NS1-IgKappa construct 65Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val
Phe Ala Leu Leu Trp1 5 10 15Val Leu Leu Leu Trp Val Pro Arg Ser His
Gly Ala Pro Leu Ala Ala 20 25 30Asp Thr Pro Thr Ala Cys Cys Phe Ser
Tyr Thr Ser Arg Gln Ile Pro 35 40 45Gln Asn Phe Ile Ala Asp Tyr Phe
Glu Thr Ser Ser Gln Cys Ser Lys 50 55 60Pro Ser Val Ile Phe Leu Thr
Lys Arg Gly Arg Gln Val Cys Ala Asp65 70 75 80Pro Ser Glu Glu Trp
Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser 85 90
95Ala6697PRTartificial sequenceCCL-20-NS1IgKappa construct 66Met
Asp Pro Asn Thr Val Ser Ser Phe Gln Val Phe Ala Leu Leu Trp1 5 10
15Val Leu Leu Leu Trp Val Pro Arg Ser His Gly Ala Ser Asn Phe Asp
20 25 30Cys Cys Leu Gly Tyr Thr Asp Arg Ile Leu His Pro Lys Phe Ile
Val 35 40 45Gly Phe Thr Arg Gln Leu Ala Asn Glu Gly Cys Asp Ile Asn
Ala Ile 50 55 60Ile Phe His Thr Lys Lys Lys Leu Ser Val Cys Ala Asn
Pro Lys Gln65 70 75 80Thr Trp Val Lys Tyr Ile Val Arg Leu Leu Ser
Lys Lys Val Lys Asn 85 90 95Met67807DNAMycobacterium tuberculosis
67agcaaaagca gggtgacaaa gacataatgg atccaaacac tgtgtcaagc tttcaggtat
60ttgccctgct ctgggtgctg ctgctgtggg tgccccggtc ccacggcatg accgagcagc
120agtggaactt cgccggcatc gaggccgccg ctagcgccat ccagggcaac
gtgaccagca 180tccacagcct gctggacgag ggcaagcaga gcctgaccaa
gctggcagct gcctggggcg 240gctctggcag cgaggcctac cagggcgtgc
agcagaagtg ggacgccacc gccaccgagc 300tgaacaacgc cctgcagaac
ctggcccgga ccatcagcga ggccggacag gccatggcca 360gcaccgaggg
caatgtgaca ggcatgttcg cctgatgacc aagcagaaag tggtactaac
420cttcttctct ttcttctcct gacaggacat actgctgagg atgtcaaaaa
tgcagttgga 480gtcctcatcg ggggacttga atggaatgat aacacagttc
gagtctctga aactctacag 540agattcgctt ggagaagcag taatgagaat
gggagacctc cactcactcc aaaacagaaa 600cgagaaatgg cgggaacaat
taggtcagaa gtttgaagaa ataagatggt tgattgaaga 660agtgagacac
aaactgaaga taacagagaa tagttttgag caaataacat ttatgcaagc
720cttacatcta ttgcttgaag tggagcaaga gataagaact ttctcgtttc
agcttattta 780ataataaaaa acacccttgt ttctact
80768765DNAMycobacterium tuberculosis 68agcaaaagca gggtgacaaa
gacataatgg atccaaacac tgtgtcaagc tttcaggtat 60ttgccatgac cgagcagcag
tggaacttcg ccggcatcga ggccgcagcc agcgccatcc 120agggcaacgt
gaccagcatc cacagcctgc tggacgaggg caagcagagc ctgaccaagc
180tggccgcagc ctggggcggc tctggcagcg aggcctacca gggcgtgcag
cagaagtggg 240acgccaccgc caccgagctg aacaacgccc tgcagaacct
ggcccggacc atcagcgagg 300ccggacaggc catggccagc accgagggca
atgtgacagg catgttcgcc tgatgaccaa 360gcagaaagtg gtactaacct
tcttctcttt cttctcctga caggacatac tgctgaggat 420gtcaaaaatg
cagttggagt cctcatcggg ggacttgaat ggaatgataa cacagttcga
480gtctctgaaa ctctacagag attcgcttgg agaagcagta atgagaatgg
gagacctcca 540ctcactccaa aacagaaacg agaaatggcg ggaacaatta
ggtcagaagt ttgaagaaat 600aagatggttg attgaagaag tgagacacaa
actgaagata acagagaata gttttgagca 660aataacattt atgcaagcct
tacatctatt gcttgaagtg gagcaagaga taagaacttt 720ctcgtttcag
cttatttaat aataaaaaac acccttgttt ctact 76569141PRTartificial
sequenceIL-15 NS1 IgKappa construct 69Met Asp Pro Asn Thr Val Ser
Ser Phe Gln Val Phe Ala Leu Leu Trp1 5 10 15Val Leu Leu Leu Trp Val
Pro Arg Ser His Gly Asn Trp Val Asn Val 20 25 30Ile Ser Asp Leu Lys
Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile 35 40 45Asp Ala Thr Leu
Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val 50 55 60Thr Ala Met
Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu65 70 75 80Ser
Gly Asp Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu 85 90
95Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys
100 105 110Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe
Leu Gln 115 120 125Ser Phe Val His Ile Val Gln Met Phe Ile Asn Thr
Ser 130 135 14070122PRTMycobacterium tuberculosis 70Met Asp Pro Asn
Thr Val Ser Ser Phe Gln Val Phe Ala Leu Leu Trp1 5 10 15Val Leu Leu
Leu Trp Val Pro Arg Ser His Gly Met Thr Glu Gln Gln 20 25 30Trp Asn
Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala Ile Gln Gly Asn 35 40 45Val
Thr Ser Ile His Ser Leu Leu Asp Glu Gly Lys Gln Ser Leu Thr 50 55
60Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly Ser Glu Ala Tyr Gln Gly65
70 75 80Val Gln Gln Lys Trp Asp Ala Thr Ala Thr Glu Leu Asn Asn Ala
Leu 85 90 95Gln Asn Leu Ala Arg Thr Ile Ser Glu Ala Gly Gln Ala Met
Ala Ser 100 105 110Thr Glu Gly Asn Val Thr Gly Met Phe Ala 115
12071108PRTartificialFusion protein NS1 Mycobacterium tuberculosis
ESAT6 71Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Phe Ala Met Thr
Glu1 5 10 15Gln Gln Trp Asn Phe Ala Gly Ile Glu Ala Ala Ala Ser Ala
Ile Gln 20 25 30Gly Asn Val Thr Ser Ile His Ser Leu Leu Asp Glu Gly
Lys Gln Ser 35 40 45Leu Thr Lys Leu Ala Ala Ala Trp Gly Gly Ser Gly
Ser Glu Ala Tyr 50 55 60Gln Gly Val Gln Gln Lys Trp Asp Ala Thr Ala
Thr Glu Leu Asn Asn65 70 75 80Ala Leu Gln Asn Leu Ala Arg Thr Ile
Ser Glu Ala Gly Gln Ala Met 85 90 95Ala Ser Thr Glu Gly Asn Val Thr
Gly Met Phe Ala 100 1057217PRTartificialsynthetic signal peptide
72Trp Val Leu Phe Ile Leu Leu Leu Phe Leu Phe Leu Pro Arg Ser His1
5 10 15Gly73163PRTartificialdelNS1-IL2-21 segment 73Met Asp Pro Asn
Thr Val Ser Ser Phe Gln Val Phe Ala Trp Val Leu1 5 10 15Phe Ile Leu
Leu Leu Phe Leu Phe Leu Pro Arg Ser His Gly Ala Pro 20 25 30Thr Ser
Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu 35 40 45Leu
Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro 50 55
60Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala65
70 75 80Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro
Leu 85 90 95Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu
Arg Pro 100 105 110Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu
Glu Leu Lys Gly 115 120 125Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala
Asp Glu Thr Ala Thr Ile 130 135 140Val Glu Phe Leu Asn Arg Trp Ile
Thr Phe Cys Gln Ser Ile Ile Ser145 150 155 160Thr Leu
Thr7418PRThuman 74Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala Ala Leu
Leu Pro Ala Ser1 5 10 15Arg Ala7524PRThuman 75Met Arg Pro Ser Gly
Thr Ala Gly Ala Ala Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro
Ala Ser Arg Ala 2076164PRTartificialdelNS1-IL2-24 76Met Asp Pro Asn
Thr Val Ser Ser Phe Gln Val Phe Ala Ala Gly Ala1 5 10 15Ala Leu Leu
Ala Leu Leu Ala Ala Leu Leu Pro Ala Ser Arg Ala Ala 20 25 30Pro Thr
Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu 35 40 45Leu
Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn 50 55
60Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys65
70 75 80Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys
Pro 85 90 95Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His
Leu Arg 100 105 110Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val
Leu Glu Leu Lys 115 120 125Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr
Ala Asp Glu Thr Ala Thr 130 135 140Ile Val Glu Phe Leu Asn Arg Trp
Ile Thr Phe Cys Gln Ser Ile Ile145 150 155 160Ser Thr Leu
Thr771023DNAartificialdelta NS influenza B IL2 construct
77agcagaagca gaggatttgt ttagtcactg gcaaacagga aaaaatggcg aacaacatga
60ccacaacaca aattgaggtg ggtccgggag caaccaatgc caccataaac tttgaagcag
120gaattctgga gtgctatgaa aggctttcat ggcaaagata atgtacagga
tgcaactcct 180gtcttgcatt gcactaagtc ttgcacttgt cacaaacagt
gcacctactt cttcgtcgac 240aaagaaaaca cagctacaac tggagcattt
actgctggat ttacagatga ttttgaatgg 300aattaataat tacaagaatc
ccaaactcac caggatgctc acatttaagt tttacatgcc 360caagaaggcc
acagaactga aacatcttca gtgtctagaa gaagaactca aacctctgga
420ggaagtgcta aatttagctc aaagcaaaaa ctttcactta agacccaggg
acttaatcag 480caatatcaac gtaatagttc tggaactaaa gggatctgaa
acaacattca tgtgtgaata 540tgctgatgag acagcaacca ttgtagaatt
tctgaacaga tggattacct tttgtcaaag 600catcatctca acactaactt
gataatacta accttcttct ctttcttctc ctgacagtgg 660aggatgaaga
agatggccat cggatcctca actcactctt cgagcgtctt aatgaaggac
720attcaaagcc aattcgagca gctgaaactg cggtgggagt cttatcccaa
tttggtcaag 780agcaccgatt atcaccagaa gagggagaca attagactgg
tcacggaaga actttatctt 840ttaagtaaaa gaattgatga taacatatta
ttccacaaaa cagtaatagc taacagctcc 900ataatagctg acatggttgt
atcattatca ttattagaaa cattgtatga aatgaaggat 960gtggttgaag
tgtacagcag gcagtgcttg tgaatttaaa ataaaaatcc tcttgttact 1020act
102378930DNAartificialdelNS1-IL2-23 sequence 78agcaaaagca
gggtgacaaa aacataatgg atccaaacac tgtgtcaagc tttcaggtat 60ttgcctgggt
gcttttcata cttctgcttt tcctgttcct tccaagatca catggtgcac
120ctacttcttc gtcgacaaag aaaacacagc tacaactgga gcatttactg
ctggatttac 180agatgatttt gaatggaatt aataattaca agaatcccaa
actcaccagg atgctcacat 240ttaagtttta catgcccaag aaggccacag
aactgaaaca tcttcagtgt ctagaagaag 300aactcaaacc tctggaggaa
gtgctaaatt tagctcaaag caaaaacttt cacttaagac 360ccagggactt
aatcagcaat atcaacgtaa tagttctgga actaaaggga tctgaaacaa
420cattcatgtg tgaatatgct gatgagacag caaccattgt agaatttctg
aacagatgga 480ttaccttttg tcaaagcatc atctcaacac taacttgata
accaagcaga aagtggtact 540aaccttcttc tctttcttct cctgacagga
catactgctg aggatgtcaa aaatgcagtt 600ggagtcctca tcgggggact
tgaatggaat gataacacag ttcgagtctc tgaaactcta 660cagagattcg
cttggagaag cagtaatgag aatgggagac ctccactcac tccaaaacag
720aaacgagaaa tggcgggaac aattaggtca gaagtttgaa gaaataagat
ggttgattga 780agaagtgaga cacaaactga agataacaga gaatagtttt
gagcaaataa catttatgca 840agccttacat ctattgcttg aagtggagca
agagataaga actttctcgt ttcagcttat 900ttaataataa aaaacaccct
tgtttctact 93079933DNAartificialdelNS1-IL2-24 sequence 79agcaaaagca
gggtgacaaa aacataatgg atccaaacac tgtgtcaagc tttcaggtat 60ttgccgcagg
agctgcactt ttggcacttc ttgctgcact tcttcctgct tcaagagctg
120cacctacttc ttcgtcgaca aagaaaacac agctacaact ggagcattta
ctgctggatt 180tacagatgat tttgaatgga attaataatt acaagaatcc
caaactcacc aggatgctca 240catttaagtt ttacatgccc aagaaggcca
cagaactgaa acatcttcag tgtctagaag 300aagaactcaa acctctggag
gaagtgctaa atttagctca aagcaaaaac tttcacttaa 360gacccaggga
cttaatcagc aatatcaacg taatagttct ggaactaaag ggatctgaaa
420caacattcat gtgtgaatat gctgatgaga cagcaaccat tgtagaattt
ctgaacagat 480ggattacctt ttgtcaaagc atcatctcaa cactaacttg
ataaccaagc agaaagtggt 540actaaccttc ttctctttct tctcctgaca
ggacatactg ctgaggatgt caaaaatgca 600gttggagtcc tcatcggggg
acttgaatgg aatgataaca cagttcgagt ctctgaaact 660ctacagagat
tcgcttggag aagcagtaat gagaatggga gacctccact cactccaaaa
720cagaaacgag aaatggcggg aacaattagg tcagaagttt gaagaaataa
gatggttgat 780tgaagaagtg agacacaaac
tgaagataac agagaatagt tttgagcaaa taacatttat 840gcaagcctta
catctattgc ttgaagtgga gcaagagata agaactttct cgtttcagct
900tatttaataa taaaaaacac ccttgtttct act 93380230PRTInfluenza A
virus 80Met Asp Pro Asn Thr Val Ser Ser Phe Gln Val Asp Cys Phe Leu
Trp1 5 10 15His Val Arg Lys Arg Val Ala Asp Gln Glu Leu Gly Asp Ala
Pro Phe 20 25 30Leu Asp Arg Leu Arg Arg Asp Gln Lys Ser Leu Arg Gly
Arg Gly Ser 35 40 45Thr Leu Gly Leu Asp Ile Glu Thr Ala Thr Arg Ala
Gly Lys Gln Ile 50 55 60Val Glu Arg Ile Leu Lys Glu Glu Ser Asp Glu
Ala Leu Lys Met Thr65 70 75 80Met Ala Ser Val Pro Ala Ser Arg Tyr
Leu Thr Asp Met Thr Leu Glu 85 90 95Glu Met Ser Arg Asp Trp Ser Met
Leu Ile Pro Lys Gln Lys Val Ala 100 105 110Gly Pro Leu Cys Ile Arg
Met Asp Gln Ala Ile Met Asp Lys Asn Ile 115 120 125Ile Leu Lys Ala
Asn Phe Ser Val Ile Phe Asp Arg Leu Glu Thr Leu 130 135 140Ile Leu
Leu Arg Ala Phe Thr Glu Glu Gly Ala Ile Val Gly Glu Ile145 150 155
160Ser Pro Leu Pro Ser Leu Pro Gly His Thr Ala Glu Asp Val Lys Asn
165 170 175Ala Val Gly Val Leu Ile Gly Gly Leu Glu Trp Asn Asp Asn
Thr Val 180 185 190Arg Val Ser Glu Thr Leu Gln Arg Phe Ala Trp Arg
Ser Ser Asn Glu 195 200 205Asn Gly Arg Pro Pro Leu Thr Pro Lys Gln
Lys Arg Glu Met Ala Gly 210 215 220Thr Ile Arg Ser Glu Val225
230815DNAartificialstop/start codon 81taatg
58220PRTartificialmodified Ig Kappa signal sequence 82Met Glu Thr
Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro1 5 10 15Arg Ser
His Gly 20
* * * * *