REPLICATION DEFICIENT INFLUENZA VIRUS FOR THE EXPRESSION OF HETEROLOGOUS SEQUENCES

Abstract

ers 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.

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° 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 ΔNS1-38IL2 (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-β. Therefore the NS1 protein inhibits the expression of IFN-α or IFN-β 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-γ, and TNF-β, 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 10⁴-5×10⁷ pfu and can be administered once, or multiple times with intervals as often as needed. Pharmaceutical compositions of the present invention comprising 10⁴-5×10⁷ 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° 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 μg/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° C. in serum-free medium in the presence of 1 μg/ml trypsin. Subsequently, foetal calf serum (final concentration 10%) as well as soy bean trypsin inhibitor (final concentration 100 μg/ml) was added and incubation at 37° 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 (ΔNS1-38IL2), 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 ΔNS1-38IL2 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 ΔNS1-38IL2.

[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 ΔNS1-38IL2 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*10⁵TCID₅₀/mouse with wt influenza B Virus, ΔNS1-38IL2, ΔNS1-38 (a control Virus which was constructed similar to ΔNS1-38IL2 but without the insertion of IL2) or PBS as a control. Four weeks post immunization, mice were challenged with 2*10⁵TCID₅₀/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*10⁵TCID50/mouse none of the mice immunized with ΔNS1-38 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 ΔNS1-38IL2 at the same dose, indicating that all mice were protected.

Sequence CWU 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

Claims

1. Replication deficient influenza virus characterized in that it comprisesa) 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 andb) 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.

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