INFLUENZA VIRUS-LIKE PARTICLES (VLPS)

Abstract

Disclosed are influenza virus-like particles (VLPs), wherein the VLPs include hemagglutinin (HA) protein and neuraminidase (NA) protein on the surface of the VLPs, a nucleoprotein (NP) ribonucleoprotein complex, and wherein the VLPs do not contain a ribonucleoprotein complex of at least one of PB1, PB2, and NS.

Claims

1.-17. (canceled)

18. A vaccine comprising RNA expressing Influenza virus-like particles (RNA-VLPs), and a pharmaceutically acceptable excipient, wherein the RNA-VLPs comprise: hemagglutinin (HA) protein and neuraminidase (NA) protein on the surface of the VLPs, a nucleoprotein (NP) ribonucleoprotein complex, wherein the VLPs do not contain a ribonucleoprotein complex of PB1, PB2, and NS2; wherein the RNA-VLPs further comprise a matrix protein (M) ribonucleoprotein complex; and wherein genetic information for RNA dependent RNA polymerases are missing.

19. The vaccine according to claim 18, wherein the HA protein is selected from the group of seasonal, non-pandemic HA proteins.

20. The vaccine according to claim 18, wherein the NA protein is selected from the group of seasonal, non-pandemic NA proteins.

21. The vaccine according to claim 18, wherein the RNA-VLPs further comprise a genetically modified M ribonucleoprotein complex.

22. The vaccine according to claim 18, wherein the M ribonucleoprotein complex contains viral RNA encoding an antigen of a non-influenza virus pathogen, an oncogene or a malignant tissue protein, especially wherein the M ribonucleoprotein complex contains viral RNA encoding a fusion protein containing an M protein and a protein or antigenic protein fragment of a non-influenza virus pathogen.

23. The vaccine according to claim 22, wherein the non-influenza virus pathogen is selected from a non-influenza virus, a gram-positive or gram-negative bacterium, a fungus, a protozoan, or a prion, preferably a non-influenza RNA virus, a DNA virus, or a retrovirus, especially wherein the virus is SARS-COV-1, SARS-COV-2, or Dengue virus.

24. The vaccine according to claim 18, wherein the RNA-VLPs comprise: HA protein and NA protein on the surface of the RNA-VLPs, a NP ribonucleoprotein complex, and an M ribonucleoprotein complex, wherein the RNA-VLPs do not contain a ribonucleoprotein complex of PB1, PB2, NS1 and NS2.

25. The vaccine according to claim 18, triggering inflammatory cell death (necroptosis) upon entry of the RNA-VLPs into a cell.

26. The vaccine according to claim 18, wherein the vaccine is an intranasal vaccine, preferably an aerosol or nasal spray.

27. An influenza virus-like particles (RNA-VLPs), wherein the RNA-VLPs comprise: hemagglutinin (HA) protein and neuraminidase (NA) protein on the surface of the RNA-VLPs, a nucleoprotein (NP) ribonucleoprotein complex, a matrix protein (M) ribonucleoprotein complex, wherein the RNA-VLPs do not contain a ribonucleoprotein complex of at least PB1, PB2, and NS; and wherein genetic information for RNA dependent RNA polymerases are missing.

28. The RNA-VLPs according to claim 27, further comprising a genetically modified M ribonucleoprotein complex, preferably wherein the M ribonucleoprotein complex contains viral RNA encoding an antigen of a non-influenza virus pathogen, an oncogene or a malignant tissue protein, especially wherein the M ribonucleoprotein complex contains viral RNA encoding a fusion protein containing an M protein and a protein or antigenic protein fragment of a non-influenza virus pathogen.

29. The RNA-VLPs according to claim 28, wherein the non-influenza virus pathogen is selected from a non-influenza virus, a gram-positive or gram-negative bacterium, a fungus, a protozoan, or a prion, preferably a non-influenza RNA virus, a DNA virus, or a retrovirus, especially wherein the virus is SARS-COV-1, SARS-COV-2, or Dengue virus.

30. A pharmaceutical composition comprising a vaccine according to claim 18.

31. A pharmaceutical composition comprising the RNA-VLPs according to claim 27.

32. A method for producing a vaccine according to claim 18, comprising the steps of providing unidirectional vectors for HA, NA, PA, PB1 and PB2; providing a bidirectional vector for NP; providing a bidirectional vector for M; and expressing the vectors in a recombinant cell system so as to obtain RNA-VLPs.

33. The method according to claim 32, further comprising the steps of filling the RNA-VLPs into a final container and finishing the RNA-VLPs to a pharmaceutical composition ready for use for administration to human individuals.

34. The method according to claim 32, wherein the vectors are expressed in Madin-Darby Canine Kidney (MDCK) cells, African green monkey kidney cells (Vero) cells, 293 cells, 293T cells, porcine kidney (PK) cells, owl monkey kidney (OMK) cells, Madin-Darby bovine kidney (MDBK) cells, chicken embryo kidney (CEK) cells, chicken embryo fibroblasts, primary chick kidney cells, or cells isolated from the chorioallantoic membrane of embryonated chicken eggs, preferably in MDCK cells or Vero cells, especially in MDCK cells.

35. The RNA-VLPs as defined in claim 27, for medical use, preferably for preventing pathogen-caused diseases, especially for preventing influenza.

Description

[0073] The invention is further illustrated by the following examples and the figures, yet without being limited thereto.

[0074] FIG. 1 shows the principle of triggering cellular immunity by viral RNA. Infection of dendritic cells by influenza or expression of viral RNA triggers necroptosis. The process of necroptosis involves the perforation of cell membranes, which in turn leads to the release of inner-cell constituents, which are perceived as a danger signal by other cells. In addition, the perforation also leaves viral nucleic acids and proteins in the intercellular space and can thus be better processed by the innate immune cells, which were activated by those danger associated molecular patterns (DAMPs), and then more effectively presented to cells of the adaptive immune system such as T cells;

[0075] FIG. 2 shows the structure of the influenza (IAV) virion. a) Electron microscope image of an influenza virus particle. b) Graphic representation of the IAV virion: In the lipid bilayer, which originates from a host cell, there are HA and NA spikes and M2 ion channels. The strength of the virion is supported by an envelope made of M1 matrix protein. The genetic information of the virus is divided into 8 segments, which are located inside the virion as a ribonucleoprotein complex. c) Schematic representation of a ribonucleoprotein complex (RNP): The genetic information is wound as a negative single-stranded RNA around a strand of nucleoproteins (NP). Furthermore, the RNPs have their own polymerase complex (PB1, PB2 and PA), which begins to replicate the viral RNA independently after the virion has successfully penetrated the host cell;

[0076] FIG. 3 shows the schematic representation of the RNA-VLP and the plasmids necessary for its production: The surface of the VLP is identical to that of an IAV virion (see FIG. 2). Inside the RNA-VLP are two ribonucleoprotein complexes that express viral RNA for NP and M after inoculation de novo. When the RNA-VLP is formed in cell culture, HA, NA, and the polymerases PA, PB1, PB2 are required only as proteins, so the expression of these proteins is accomplished by a plasmid that only produces messenger RNA and the proteins that is translated by the host's cell machinery. Since NP and M are required both as protein and as viral copy RNA (vRNA), a bidirectional plasmid is required which expresses messenger RNA as well as vRNA;

[0077] FIG. 4 shows cell death triggered by RNA-VLPs. Human dendritic cells were infected with various constructs of VLPs and, after an eight-hour incubation, analysed for cell death using imaging flow cytometry. An empty VLP (HA/NA (Prot.)) and vehicle (cell culture medium) were used as negative controls. Infection with an influenza virus and a VLP with all three polymerases as additional RNPs served as a positive control;

[0078] FIG. 5 shows the experimental set-up to measure the induction of cellular immunity by RNA-VLPs. Human DCs were infected with RNA-VLPs or controls. After a four-hour incubation, DCs from the same donor were added and after another four hours, T cells from another donor were added. The cell death in the infected DCs activates the added DCs, which in turn cause the T cells to proliferate (see FIG. 1);

[0079] FIG. 6 shows the cellular immunity triggered by RNA-VLPs. Human dendritic cells were infected with various constructs of VLPs and cultured with DCs and foreign T cells (see FIG. 5). T cell proliferation was measured using flow cytometry. An empty VLP (HA/NA (Prot.)) and vehicle infection (cell culture medium) were used as negative controls. Infection with an influenza virus and a VLP containing all three polymerase RNPs served as a positive control;

[0080] FIG. 7 shows the sample plan IAV challenge study;

[0081] FIG. 8 shows the sample plan for the challenge study involving a chimeric vaccine against IAV and PRCv;

[0082] FIG. 9 illustrates the production of the chimeric bidirectional plasmid containing genetic information of the IAV M segment as well as genetic information from the SARS-CoV-2 using Gibson assembly. In short: The phW2000 plasmid containing the IAV M Segment as well as a specific region of the SARS-COV-2 are amplified by using specific primers creating a 20 base pair overlap between the two amplicons. During the assembly process a nuclease chews back DNA from the 5″end allowing the two amplicons to anneal. A DNA polymerase and ligase fill any gaps and join the DNA removing any nicks in the DNA. The product of this process is a bidirectional plasmid containing the chimeric sequence of IAV M and a SARS-CoV-2 protein;

[0083] FIG. 10 shows a scheme of chimeric IAV SARS-CoV-2 RNA-VLP production. a) HA, NA, PB1, PB2 and PA segments are rescued in unidirectional (pUNI) plasmids by reverse genetics. NP and M are rescued in bidirectional plasmids (pBI) b) A specific region of the SARS-CoV-2 is reverse transcribed and amplified by PCR. c) A chimeric bidirectional plasmid containing the genetic information of the IAV M segment and a part of SARS-CoV-2 is produced by Gibson assembly (see FIG. 9). d) The HA, NA, PB1, PB2, PA, NP and chimeric M plasmid is transformed into a MDCK cell line culture. e) The chimeric IAV-SARS-CoV-2 RNA-VLP arises through self-assembly in the cell line and is harvested in the cell culture supernatant. f) The chimeric IAV-SARS-CoV-2 RNA-VLP in detail;

[0084] FIG. 11 shows the plasmid map of pCAGGS;

[0085] FIG. 12 shows the plasmid map of pHWX

EXAMPLES

I. In Vitro Proof of Concept for the Present Invention

[0086] To show the functionality of the present invention, the following VLPs with HA and NA surface proteins and different configurations of RNPs were constructed: [0087] 1. An empty VLP that has only HA and NA proteins on the surface [0088] 2. VLPs with an NP ribonucleoprotein complex [0089] 3. VLPs with an NP and M ribonucleoprotein complex. It should be mentioned that due to the bidirectional nature of the pHWX plasmid M, both RNP and surface protein are present in addition to HA and NA [0090] 4. VLPs with NP and all polymerases as a ribonucleoprotein complex. This construct was designed as a positive control and is not suitable as a vaccine due to biological safety concerns.

[0091] For the prototype vaccine, HA and NA from virus strain A/New Caledonia/20/1999 and M, polymerases and NP from virus strain A/California/4/2009 were used. The individual IAV segments were isolated according to Hoffmann et al. (Arch. Virol. 146 (2001), 2275-2289, 10.1007/s007050170002). First, the vRNA was extracted from virus isolates. The vRNA was rewritten into cDNA using the UNI12 primer and a reverse transcriptase. Segment-specific sequences were then amplified by PCR using segment specific primers and purified by gel electrophoresis (Hoffmann et al., 2001). The individual segments were cloned into their intended plasmids using restriction enzymes and ligases, transformed into E. coli bacteria and plated out on agar plates. Individual colonies were selected to check the cloned insert for completeness and correctness using Sanger sequencing. The plasmids were then transformed together into MDCK cells by lipofectamine. After the transformation, the RNA-VLPs were created by “self-assembly” in the MDCK cells (Nayaket et al., Virus Res. 106 (2004), 147-165; 10.1016/j.virusres.2004.08.012). Next, the RNA-VLPs were harvested in the cell culture medium. In the future, it is planned to use simpler and more effective methods for creating the plasmids and RNA-VLPs, e.g. one pot cloning (Choi et al., Sci. Rep. 9, (2019) 8318; 10.1038/s41598-019-44813-z).

[0092] The cell death induction of the VLPs was tested in human dendritic cells, as the ability to trigger necroptosis in immune cells is crucial for vaccine effectiveness (Hartmann et al., 2017). For this purpose, dendritic cells from monocytes, which were obtained from anonymous blood donations, were grown by adding the cytokines GMCSF and IL-4. These dendritic cells were then infected with the various VLP constructs or with the influenza strain NC/99 as a positive control. Eight hours after infection, cell death induction was measured using flow cytometry. While the inclusion of the NP RNP alone into the VLP did not result in a significant increase in cell death rate compared to the uninfected negative control, there was a significant increase in the inclusion of both NP and M RNPs, which was surprisingly higher than the VLPs, where also all polymerases were included as RNPs (FIG. 4).

[0093] The ability of the VLPs to elicit a cellular immune response was checked by a T-cell dendritic-cell co-cultivation method. For this purpose, human dendritic cells were infected with the VLP constructs or the controls (virus or vehicle). After 4 hours, dendritic cells from the same blood donor were added so that they could be activated by the danger signals from the dead infected cells. This cell mixture was again cultured after 4 hours with T cells from another donor for 2.5 days. These foreign T cells were activated on the one hand by the graft-versus-host reaction and on the other hand by the status of the dendritic cells and thereby driven to proliferation. The proliferation was then measured by flow cytometry using the dilution of a dye, which is associated with each cell division (proliferation) (FIG. 5).

[0094] Similar to the cell death experiments, the empty VLPs could not trigger an increased T cell proliferation compared to the negative control. This is an indication that vaccination with surface proteins primarily elicits antibody-mediated immunity. The VLPs with M and NP RNPs, on the other hand, were able to produce at least a similarly high T cell proliferation as an infection with a real influenza virus (positive control). Interestingly, the M/NP RNP combination was even more effective than the combination of NP and all three polymerases. The VLP construct containing only the NP RNP was able to cause a slight increase in T cell proliferation. These data show that a VLP consisting of a surface including HA, NA and M proteins filled with NP and M RNPs is a good trigger of cellular anti-viral immunity (FIG. 6).

[0095] In summary, the present invention consists of combining the technology of the VLPs and complexes expressing viral RNA and the resulting vaccine looking like an infectious particle for the immune system. After being absorbed into an organism, these RNA-VLPs behave like an infectious IAV virion by penetrating the cell nucleus and beginning to express viral RNA through the polymerases they have brought with them. This newly synthesized RNA then triggers an inflammatory signal cascade that boosts cellular immunity. Exactly this step is not used in current vaccination strategies. We have already proven that this innovation creates the cellular immunity necessary for long-lasting protection in the cell culture system.

II. In Vivo Proof of Concept for the Present Invention

[0096] In vivo proof of concept is provided by two experiments. The first experiment focuses on testing the effect of RNA-VLP as a sole vaccination against porcine influenza. In a second experiment, an RNA-VLP vaccine is constructed with broader activity against influenza and a porcine corona virus, which causes pathologies in pigs comparable to those observed in humans when infected with the current SARS-CoV-2.

II.1. Proof of Concept of Vaccination Against IAV with RNA-VLPs in Pigs.

[0097] An RNA-VLP with segments from a recently isolated porcine H1N1 IAV isolate is constructed as described in the sections above. The efficacy of the RNA-VLP as a vaccine against influenza is tested in a challenge experiment where the usefulness of the RNA-VLP is compared with a conventional veterinary anti influenza vaccine. 30 animals are grouped in three groups of 10: (i) animals getting the RNA-VLPs plus vehicle, (ii) animals taking the conventional vaccine, and vehicle and (iii) vehicle group. After vaccination 5 of the ten animals are challenged with an IAV infection (FIG. 7). Clinical signs, laboratory measurements (such as viral load, and cytokine levels), and cell immunological measurements serve as readouts for vaccine efficacy.

II.2 Proof of Concept of Combinatorial Vaccination Against IAV and Coronavirus

[0098] For this experiment a RNA-VLP is constructed where a part of the nucleoprotein sequence of the porcine respiratory corona virus (PRCv) is added to the M segment RNP. PRCv has a similar structure to SARS-CoV-2 and serves as a model as there is currently no animal model to test SARS-CoV-2 infections. 40 animals are grouped in two groups of 20: (i) animals getting the RNA-VLPs plus vehicle, and (ii) vehicle group. After vaccination, groups of five of the twenty animals are challenged with an IAV, PRCv, a combination of IAV and PRCv or vehicle (FIG. 8). Clinical signs, laboratory measurements (such as viral load, and cytokine levels), and cell immunological measurements serve as readouts for vaccine efficacy.

II.3 Proof of Concept of a Combinatorial Vaccination Against IAV and SARS-CoV-2

[0099] For this experiment, a RNA-VLP with a chimeric IAV M RNP will be constructed. The chimeric M RNP will contain genetic information of the IAV M segment as well as genetic information fully or partially describing one of the proteins of the SARS-CoV-2. VLP will be constructed as described above. Those VLPs will be used to vaccinate pigs. After vaccination and a possible booster vaccination blood will be drawn from the animals, which will be used in serological and in vitro cell immunological tests. As soon as a valid and useable animal model for SARS-CoV-2 is identified the anti SARS-CoV-2 RNA-VLP will be tested in a in vivo challenge study (FIGS. 9 and 10).

Abbreviations

[0100] DAI DNA-dependent activator of IFN-regulatory factors [0101] DAMPS danger associated molecular patterns [0102] DC dendritic cells [0103] HA hemagglutinin [0104] IAV Influenza A Virus [0105] LAIV attenuated live virus vaccine [0106] NA neuraminidase [0107] NP nucleoprotein [0108] PAMPs pathogen associated molecular patterns [0109] PRCv porcine respiratory corona virus [0110] RNA Ribonucleic acid [0111] RNP ribonucleoprotein complexes [0112] ZBP1 Z-DNA-binding protein 1

TABLE-US-00001 Sequences: SEQ ID NO: 1 (pCAGGS): GTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATAT GGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTG ACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACT ATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGT CAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAG TACATCTACGTATTAGTCATCGCTATTACCATGGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCC ATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGG CGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAG GTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCG GCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGTTGCCTTCGCCCCGTGCCCCGCTCCGC GCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCC CTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTCGTTTCTTTTCTGTGGCTGCGTGAAAG CCTTAAAGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTG CGTGGGGAGCGCCGCGTGCGGCCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTT GTGCGCTCCGCGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGG GGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGGCGGTCGGGCT GTAACCCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTGC GGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGG CCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCGGAGCGCCGGCGGCTGTCGAGGCGC GGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATC TGGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGCGAAGCGGTGCGGCGCC GGCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCATCTCCAGC CTCGGGGCTGCCGCAGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGT GTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCA ACGTGCTGGTTGTTGTGCTGTCTCATCATTTTGGCAAAGAATTCCTCGAGGAATTCACTCCTCAGGTGCA GGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTT CCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAAT TTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAA TCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGA ACAAAGGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGA AAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAA AATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCC TCTTCTCTTATGAAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGT GTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGT GCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGT CGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCC CTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCG AGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAA AAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATA AAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGAT CCGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCT CGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAAT ACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGG AACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATC GACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTC CCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGC GTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCC GGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCG CTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGG TAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTG ATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTAT CAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGA GTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGT TCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCA GTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGG AAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAA GCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGT CACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCC CATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTG TTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTG TGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGC GTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCG GGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACT GATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAA AAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATT TATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTC CGCGCACATTTCCCCGAAAAGTGCCACCTG SEQ ID NO: 2 (pHWX): CAGCTGGTTCTTTCCGCCTCAGAAGCCATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCC AATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCAGACAATGCGA TGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGG GAGGGGCAAACAACAGATGGCTGGCAACTAGAAGGCACAGTCGAGGCTGATCAGCGAGCTCTAGCATTTA GGTGACCGCCGGGAGGGCGTCCCCGGCCCGGCGCTGCTCCCGCGTGTGTCCTGGGGTTGACCAGAGGGCC CCGGGCGCTCCGTGTGTGGCTGCGATGGTGGCGTTTTTGGGGACAGGTGTCCGTGTCGCGCGTCGCCTGG GCCGGCGGCGTGGTCGGTGACGCGACCTCCCGGCCCCGGGGGAGGTATATCTTTCGCTCCGAGTCGGCAT TTTGGGCCGCCGGGTTATTGGAGACGGGGTCGACCTGCAGACTGGCTGTGTATAAGGGAGCCTGACATTT ATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTC TTCCCCGATAACGGAAACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCATCCCCGATA TGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCA TCCGTCGCCCGGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTT ATAGGTGTAAACCTTAAACTGCATTTCACCAGCCCCTGTTCTCGTCAGCAAAAGAGCCGTTCATTTCAAT AAACCGGGCGACCTCAGCCATCCCTTCCTGATTTTCCGCTTTCCAGCGTTCGGCACGCAGACGACGGGCT TCATTCTGCATGGTTGTGCTTACCAGACCGGAGATATTGACATCATATATGCCTTGAGCAACTGATAGCT GTCGCTGTCAACTGTCACTGTAATACGCTGCTTCATAGCATACCTCTTTTTGACATACTTCGGGTATACA TATCAGTATATATTCTTATACCGCAAAAATCAGCGCGCAAATACGCATACTGTTATCTGGCTTTTAGTAA GCCGGATCCACGCGTCGTCTCCTCCCCCCCAACTTCGGAGGTCGACCAGTACTCCGGTTAACTGCTAGAC CCTATAGTGAGTCGTATTAATTTCGATAAGCCAGTAAGCAGTGGGTTCTCTAGTTAGCCAGAGAGCTCTG CTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATT TTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAA TCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCG ATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGG CGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATCGACGTCAGGTGGCACTTTTCG GGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGA CAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCG CCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAA AGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTT GAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTAT TATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGA GTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATA ACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTT TTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACC AAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAA CTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTC TGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGTTCTCGCGG TATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAG GCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGT CAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGT GAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGAC CCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAAC TGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATA AGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGG GGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTA TGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCT CTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG GCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATT CTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAG CGAGTCAGTGAGCGAGGAAGCGGAAGagcgcccaatacgcaaaccgcctctccccgcgcgttggccgatt cattaatg SEQ ID NO: 3 (Influenza A virus (A/California/04/2009(H1N1)) seg- ment 1 polymerase PB2 (PB2) gene; FJ969516.1 Influenza A virus (A/California/04/2009(H1N1)) segment 1 polymerase PB2 (PB2) gene, complete cds) ATGGAGAGAATAAAAGAACTGAGAGATCTAATGTCGCAGTCCCGCACTCGCGAGATACTCACTAAGACCA CTGTGGACCATATGGCCATAATCAAAAAGTACACATCAGGAAGGCAAGAGAAGAACCCCGCACTCAGAAT GAAGTGGATGATGGCAATGAGATACCCAATTACAGCAGACAAGAGAATAATGGACATGATTCCAGAGAGG AATGAACAAGGACAAACCCTCTGGAGCAAAACAAACGATGCTGGATCAGACCGAGTGATGGTATCACCTC TGGCCGTAACATGGTGGAATAGGAATGGCCCAACAACAAGTACAGTTCATTACCCTAAGGTATATAAAAC TTATTTCGAAAAGGTCGAAAGGTTGAAACATGGTACCTTCGGCCCTGTCCACTTCAGAAATCAAGTTAAA ATAAGGAGGAGAGTTGATACAAACCCTGGCCATGCAGATCTCAGTGCCAAGGAGGCACAGGATGTGATTA TGGAAGTTGTTTTCCCAAATGAAGTGGGGGCAAGAATACTGACATCAGAGTCACAGCTGGCAATAACAAA AGAGAAGAAAGAAGAGCTCCAGGATTGTAAAATTGCTCCCTTGATGGTGGCGTACATGCTAGAAAGAGAA TTGGTCCGTAAAACAAGGTTTCTCCCAGTAGCCGGCGGAACAGGCAGTGTTTATATTGAAGTGTTGCACT TAACCCAAGGGACGTGCTGGGAGCAGATGTACACTCCAGGAGGAGAAGTGAGAAATGATGATGTTGACCA AAGTTTGATTATCGCTGCTAGAAACATAGTAAGAAGAGCAGCAGTGTCAGCAGACCCATTAGCATCTCTC TTGGAAATGTGCCACAGCACACAGATTGGAGGAGTAAGGATGGTGGACATCCTTAGACAGAATCCAACTG AGGAACAAGCCGTAGACATATGCAAGGCAGCAATAGGGTTGAGGATTAGCTCATCTTTCAGTTTTGGTGG GTTCACTTTCAAAAGGACAAGCGGATCATCAGTCAAGAAAGAAGAAGAAGTGCTAACGGGCAACCTCCAA ACACTGAAAATAAGAGTACATGAAGGGTATGAAGAATTCACAATGGTTGGGAGAAGAGCAACAGCTATTC TCAGAAAGGCAACCAGGAGATTGATCCAGTTGATAGTAAGCGGGAGAGACGAGCAGTCAATTGCTGAGGC AATAATTGTGGCCATGGTATTCTCACAGGAGGATTGCATGATCAAGGCAGTTAGGGGCGATCTGAACTTT GTCAATAGGGCAAACCAGCGACTGAACCCCATGCACCAACTCTTGAGGCATTTCCAAAAAGATGCAAAAG TGCTTTTCCAGAACTGGGGAATTGAATCCATCGACAATGTGATGGGAATGATCGGAATACTGCCCGACAT GACCCCAAGCACGGAGATGTCGCTGAGAGGGATAAGAGTCAGCAAAATGGGAGTAGATGAATACTCCAGC ACGGAGAGAGTGGTAGTGAGTATTGACCGATTTTTAAGGGTTAGAGATCAAAGAGGGAACGTACTATTGT CTCCCGAAGAAGTCAGTGAAACGCAAGGAACTGAGAAGTTGACAATAACTTATTCGTCATCAATGATGTG GGAGATCAATGGCCCTGAGTCAGTGCTAGTCAACACTTATCAATGGATAATCAGGAACTGGGAAATTGTG AAAATTCAATGGTCACAAGATCCCACAATGTTATACAACAAAATGGAATTTGAACCATTTCAGTCTCTTG TCCCTAAGGCAACCAGAAGCCGGTACAGTGGATTCGTAAGGACACTGTTCCAGCAAATGCGGGATGTGCT TGGGACATTTGACACTGTCCAAATAATAAAACTTCTCCCCTTTGCTGCTGCCCCACCAGAACAGAGTAGG ATGCAATTTTCCTCATTGACTGTGAATGTGAGAGGATCAGGGTTGAGGATACTGGTAAGAGGCAATTCTC CAGTATTCAATTACAACAAGGCAACCAAACGACTTACAGTTCTTGGAAAGGATGCAGGTGCATTGACTGA AGATCCAGATGAAGGCACATCTGGGGTGGAGTCTGCTGTCCTGAGAGGATTTCTCATTTTGGGCAAAGAA GACAAGAGATATGGCCCAGCATTAAGCATCAATGAACTGAGCAATCTTGCAAAAGGAGAGAAGGCTAATG TGCTAATTGGGCAAGGGGACGTAGTGTTGGTAATGAAACGAAAACGGGACTCTAGCATACTTACTGACAG CCAGACAGCGACCAAAAGAATTCGGATGGCCATCAATTAG SEQ ID NO: 4 (Influenza A virus (A/California/04/2009(H1N1)) seg- ment 2 polymerase PB1 (PB1) gene; MK159411.1 Influenza A virus (A/California/04/2009(H1N1)) segment 2 polymerase PB1 (PB1) gene, complete cds; and nonfunctional PB1-F2 protein (PB1-F2) gene, com- plete sequence) AGCAAAAGCAGGCAAACCATTTGAATGGATGTCAATCCGACTCTACTTTTCCTAAAAATTCCAGCGCAAA ATGCCATAAGCACCACATTCCCTTATACTGGAGATCCTCCATACAGCCATGGAACAGGAACAGGATACAC CATGGACACAGTAAACAGAACACACCAATACTCAGAAAAGGGAAAGTGGACGACAAACACAGAGACTGGT GCACCCCAGCTCAACCCGATTGATGGACCACTACCTGAGGATAATGAACCAAGTGGGTATGCACAAACAG ACTGTGTTCTAGAGGCTATGGCTTTCCTTGAAGAATCCCACCCAGGAATATTTGAGAATTCATGCCTTGA AACAATGGAAGTTGTTCAACAAACAAGGGTAGATAAACTAACTCAAGGTCGCCAGACTTATGATTGGACA TTAAACAGAAATCAACCGGCAGCAACTGCATTGGCCAACACCATAGAAGTCTTTAGATCGAATGGCCTAA CAGCTAATGAGTCAGGAAGGCTAATAGATTTCTTAAAGGATGTAATGGAATCAATGAACAAAGAGGAAAT AGAGATAACAACCCACTTTCAAAGAAAAAGGAGAGTAAGAGACAACATGACCAAGAAGATGGTCACGCAA AGAACAATAGGGAAGAAAAAACAAAGACTGAATAAGAGAGGCTATCTAATAAGAGCACTGACATTAAATA CGATGACCAAAGATGCAGAGAGAGGCAAGTTAAAAAGAAGGGCTATCGCAACACCTGGGATGCAGATTAG AGGTTTCGTATACTTTGTTGAAACTTTAGCTAGGAGCATTTGCGAAAAGCTTGAACAGTCTGGGCTCCCA GTAGGGGGCAATGAAAAGAAGGCCAAACTGGCAAATGTTGTGAGAAAGATGATGACTAATTCACAAGACA CAGAGATTTCTTTCACAATCACTGGGGACAACACTAAGTGGAATGAAAATCAAAATCCTCGAATGTTCCT GGCGATGATTACATATATCACCAGAAATCAACCCGAGTGGTTCAGAAACATCCTGAGCATGGCACCCATA ATGTTCTCAAACAAAATGGCAAGACTAGGGAAAGGGTACATGTTCGAGAGTAAAAGAATGAAGATTCGAA CACAAATACCAGCAGAAATGCTAGCAAGCATTGACCTGAAGTACTTCAATGAATCAACAAAGAAGAAAAT TGAGAAAATAAGGCCTCTTCTAATAGATGGCACAGCATCACTGAGTCCTGGGATGATGATGGGCATGTTC AACATGCTAAGTACGGTCTTGGGAGTCTCGATACTGAATCTTGGACAAAAGAAATACACCAAGACAATAT ACTGGTGGGATGGGCTCCAATCATCCGACGATTTTGCTCTCATAGTGAATGCACCAAACCATGAGGGAAT ACAAGCAGGAGTGGACAGATTCTACAGGACCTGCAAGTTAGTGGGAATCAACATGAGCAAAAAGAAGTCC TATATAAATAAGACAGGGACATTTGAATTCACAAGCTTTTTTTATCGCTATGGATTTGTGGCTAATTTTA GCATGGAGCTACCCAGCTTTGGAGTGTCTGGAGTAAATGAATCAGCTGACATGAGTATTGGAGTAACAGT GATAAAGAACAACATGATAAACAATGACCTTGGACCTGCAACGGCCCAGATGGCTCTTCAATTGTTCATC AAAGACTACAGATACACATATAGGTGCCATAGGGGAGACACACAAATTCAGACGAGAAGATCATTTGAGT TAAAGAAGCTGTGGGATCAAACCCAATCAAAAGTAGGGCTATTAGTATCAGATGGAGGACCAAACTTATA CAATATACGGAATCTTCACATTCCTGAAGTCTGCTTAAAATGGGAGCTAATGGATGATGATTATCGGGGA AGACTTTGTAATCCCCTGAATCCCTTTGTCAGTCATAAAGAGATTGATTCTGTAAACAATGCTGTGGTAA TGCCAGCCCATGGTCCAGCCAAAAGCATGGAATATGATGCCGTTGCAACTACACATTCCTGGATTCCCAA GAGGAATCGTTCTATTCTCAACACAAGCCAAAGGGGAATTCTTGAGGATGAACAGATGTACCAGAAGTGC TGCAATCTATTCGAGAAATTTTTCCCTAGCAGTTCATATAGGAGACCGGTTGGAATTTCTAGCATGGTGG AGGCCATGGTGTCTAGGGCCCGGATTGATGCCAGGGTCGACTTCGAGTCTGGACGGATCAAGAAAGAAGA GTTCTCTGAGATCATGAAGATCTGTTCCACCATTGAAGAACTCAGACGGCAAAAATAATGAATTTAACTT GTCCTTCATGAAAAAATGCCTTGTTTCTACT SEQ ID NO: 5 (Influenza A virus (A/California/04/2009(H1N1)) seg- ment 3 polymerase PA (PA) gene; FJ966081.1 Influenza A virus (A/California/04/2009(H1N1)) segment 3 polymerase PA (PA) gene, complete cds) ATGGAAGACTTTGTGCGACAATGCTTCAATCCAATGATCGTCGAGCTTGCGGAAAAGGCAATGAAAGAAT ATGGGGAAGATCCGAAAATCGAAACTAACAAGTTTGCTGCAATATGCACACATTTGGAAGTTTGTTTCAT GTATTCGGATTTCCATTTCATCGACGAACGGGGTGAATCAATAATTGTAGAATCTGGTGACCCGAATGCA CTATTGAAGCACCGATTTGAGATAATTGAAGGAAGAGACCGAATCATGGCCTGGACAGTGGTGAACAGTA TATGTAACACAACAGGGGTAGAGAAGCCTAAATTTCTTCCTGATTTGTATGATTACAAAGAGAACCGGTT CATTGAAATTGGAGTAACACGGAGGGAAGTCCACATATATTACCTAGAGAAAGCCAACAAAATAAAATCT GAGAAGACACACATTCACATCTTTTCATTCACTGGAGAGGAGATGGCCACCAAAGCGGACTACACCCTTG ACGAAGAGAGCAGGGCAAGAATCAAAACTAGGCTTTTCACTATAAGACAAGAAATGGCCAGTAGGAGTCT ATGGGATTCCTTTCGTCAGTCCGAAAGAGGCGAAGAGACAATTGAAGAAAAATTTGAGATTACAGGAACT ATGCGCAAGCTTGCCGACCAAAGTCTCCCACCGAACTTCCCCAGCCTTGAAAACTTTAGAGCCTATGTAG ATGGATTCGAGCCGAACGGCTGCATTGAGGGCAAGCTTTCCCAAATGTCAAAAGAAGTGAACGCCAAAAT TGAACCATTCTTGAGGACGACACCACGCCCCCTCAGATTGCCTGATGGGCCTCTTTGCCATCAGCGGTCA AAGTTCCTGCTGATGGATGCTCTGAAATTAAGTATTGAAGACCCGAGTCACGAGGGGGAGGGAATACCAC TATATGATGCAATCAAATGCATGAAGACATTCTTTGGCTGGAAAGAGCCTAACATAGTCAAACCACATGA GAAAGGCATAAATCCCAATTACCTCATGGCTTGGAAGCAGGTGCTAGCAGAGCTACAGGACATTGAAAAT GAAGAGAAGATCCCAAGGACAAAGAACATGAAGAGAACAAGCCAATTGAAGTGGGCACTCGGTGAAAATA TGGCACCAGAAAAAGTAGACTTTGATGACTGCAAAGATGTTGGAGACCTTAAACAGTATGACAGTGATGA GCCAGAGCCCAGATCTCTAGCAAGCTGGGTCCAAAATGAATTCAATAAGGCATGTGAATTGACTGATTCA AGCTGGATAGAACTTGATGAAATAGGAGAAGATGTTGCCCCGATTGAACATATCGCAAGCATGAGGAGGA ACTATTTTACAGCAGAAGTGTCCCACTGCAGGGCTACTGAATACATAATGAAGGGAGTGTACATAAATAC GGCCTTGCTCAATGCATCCTGTGCAGCCATGGATGACTTTCAGCTGATCCCAATGATAAGCAAATGTAGG ACCAAAGAAGGAAGACGGAAAACAAACCTGTATGGGTTCATTATAAAAGGAAGGTCTCATTTGAGAAATG ATACTGATGTGGTGAACTTTGTAAGTATGGAGTTCTCACTCACTGACCCGAGACTGGAGCCACACAAATG GGAAAAATACTGTGTTCTTGAAATAGGAGACATGCTCTTGAGGACTGCGATAGGCCAAGTGTCGAGGCCC ATGTTCCTATATGTGAGAACCAATGGAACCTCCAAGATCAAGATGAAATGGGGCATGGAAATGAGGCGCT GCCTTCTTCAGTCTCTTCAGCAGATTGAGAGCATGATTGAGGCCGAGTCTTCTGTCAAAGAGAAAGACAT GACCAAGGAATTCTTTGAAAACAAATCGGAAACATGGCCAATCGGAGAGTCACCCAGGGGAGTGGAGGAA GGCTCTATTGGGAAAGTGTGCAGGACCTTACTGGCAAAATCTGTATTCAACAGTCTATATGCGTCTCCAC AACTTGAGGGGTTTTCGGCTGAATCTAGAAAATTGCTTCTCATTGTTCAGGCACTTAGGGACAACCTGGA ACCTGGAACCTTCGATCTTGGGGGGCTATATGAAGCAATCGAGGAGTGCCTGATTAATGATCCCTGGGTT TTGCTTAATGCATCTTGGTTCAACTCCTTCCTCACACATGCACTGAAGTAG SEQ ID NO: 6 (Influenza A virus (A/California/04/2009(H1N1)) seg- ment 5 nucleocapsid protein (NP) gene; FJ966083.1 Influenza A virus (A/California/04/2009(H1N1)) segment 5 nucleocapsid protein (NP) gene, complete cds) ATGGCGTCTCAAGGCACCAAACGATCATATGAACAAATGGAGACTGGTGGGGAGCGCCAGGATGCCACAG AAATCAGAGCATCTGTCGGAAGAATGATTGGTGGAATCGGGAGATTCTACATCCAAATGTGCACTGAACT CAAACTCAGTGATTATGATGGACGACTAATCCAGAATAGCATAACAATAGAGAGGATGGTGCTTTCTGCT TTTGATGAGAGAAGAAATAAATACCTAGAAGAGCATCCCAGTGCTGGGAAGGACCCTAAGAAAACAGGAG GACCCATATATAGAAGAGTAGACGGAAAGTGGATGAGAGAACTCATCCTTTATGACAAAGAAGAAATAAG GAGAGTTTGGCGCCAAGCAAACAATGGCGAAGATGCAACAGCAGGTCTTACTCATATCATGATTTGGCAT TCCAACCTGAATGATGCCACATATCAGAGAACAAGAGCGCTTGTTCGCACCGGAATGGATCCCAGAATGT GCTCTCTAATGCAAGGTTCAACACTTCCCAGAAGGTCTGGTGCCGCAGGTGCTGCGGTGAAAGGAGTTGG AACAATAGCAATGGAGTTAATCAGAATGATCAAACGTGGAATCAATGACCGAAATTTCTGGAGGGGTGAA AATGGACGAAGGACAAGGGTTGCTTATGAAAGAATGTGCAATATCCTCAAAGGAAAATTTCAAACAGCTG CCCAGAGGGCAATGATGGATCAAGTAAGAGAAAGTCGAAACCCAGGAAACGCTGAGATTGAAGACCTCAT TTTCCTGGCACGGTCAGCACTCATTCTGAGGGGATCAGTTGCACATAAATCCTGCCTGCCTGCTTGTGTG TATGGGCTTGCAGTAGCAAGTGGGCATGACTTTGAAAGGGAAGGGTACTCACTGGTCGGGATAGACCCAT TCAAATTACTCCAAAACAGCCAAGTGGTCAGCCTGATGAGACCAAATGAAAACCCAGCTCACAAGAGTCA ATTGGTGTGGATGGCATGCCACTCTGCTGCATTTGAAGATTTAAGAGTATCAAGTTTCATAAGAGGAAAG AAAGTGATTCCAAGAGGAAAGCTTTCCACAAGAGGGGTCCAGATTGCTTCAAATGAGAATGTGGAAACCA TGGACTCCAATACCCTGGAACTGAGAAGCAGATACTGGGCCATAAGGACCAGGAGTGGAGGAAATACCAA TCAACAAAAGGCATCCGCAGGCCAGATCAGTGTGCAGCCTACATTCTCAGTGCAGCGGAATCTCCCTTTT GAAAGAGCAACCGTTATGGCAGCATTCAGCGGGAACAATGAAGGACGGACATCCGACATGCGAACAGAAG TTATAAGAATGATGGAAAGTGCAAAGCCAGAAGATTTGTCCTTCCAGGGGCGGGGAGTCTTCGAGCTCTC GGACGAAAAGGCAACGAACCCGATCGTGCCTTCCTTTGACATGAGTAATGAAGGGTCTTATTTCTTCGGA GACAATGCAGAGGAGTATGAGAGTTGA SEQ ID NO: 7 (Influenza A virus (A/California/04/2009(H1N1)) seg- ment 7 matrix protein 2(M2) and matrix protein 1 (Ml) genes; FJ969513.1 Influenza A virus (A/California/04/2009(H1N1)) segment 7 matrix protein 2 (M2) and matrix protein 1 (Ml) genes, complete cds) ATGAGTCTTCTAACCGAGGTCGAAACGTACGTTCTTTCTATCATCCCGTCAGGCCCCCTCAAAGCCGAGA TCGCGCAGAGACTGGAAAGTGTCTTTGCAGGAAAGAACACAGATCTTGAGGCTCTCATGGAATGGCTAAA GACAAGACCAATCTTGTCACCTCTGACTAAGGGAATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGT GAGCGAGGACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTAAATGGGAATGGGGACCCGAACAACATGG ATAGAGCAGTTAAACTATACAAGAAGCTCAAAAGAGAAATAACGTTCCATGGGGCCAAGGAGGTGTCACT AAGCTATTCAACTGGTGCACTTGCCAGTTGCATGGGCCTCATATACAACAGGATGGGAACAGTGACCACA GAAGCTGCTTTTGGTCTAGTGTGTGCCACTTGTGAACAGATTGCTGATTCACAGCATCGGTCTCACAGAC AGATGGCTACTACCACCAATCCACTAATCAGGCATGAAAACAGAATGGTGCTGGCTAGCACTACGGCAAA GGCTATGGAACAGATGGCTGGATCGAGTGAACAGGCAGCGGAGGCCATGGAGGTTGCTAATCAGACTAGG CAGATGGTACATGCAATGAGAACTATTGGGACTCATCCTAGCTCCAGTGCTGGTGTGAAAGATGACCTTC TTGAAAATTTGCAGGCCTACCAGAAGCGAATGGGAGTGCAGATGCAGCGATTCAAGTGATCCTCTCGTCA TTGCAGCAAATATCATTGGGATCTTGCACCTGATATTGTGGATTACTGATCGTCTTTTTTTCAAATGTAT TTATCGTCGCTTTAAATACGGTTTGAAAAGAGGGCCTTCTACGGAAGGAGTGCCTGAGTCCATGAGGGAA GAATATCAACAGGAACAGCAGAGTGCTGTGGATGTTGACGATGGTCATTTTGTCAACATAGAGCTAGAGT AA SEQ ID NO: 8 (Influenza A virus (A/New Caledonia/20/1999(H1N1)) segment 4 HA gene; CY031336.1 Influenza A virus (A/New Caledo- nia/20/1999 (H1N1)) segment 4 sequence) AGCAAAAGCAGGGGAAAATAAAAACAACCAAAATGAAAGCAAAACTACTGGTCCTGTTATGTACATTTAC AGCTACATATGCAGACACAATATGTATAGGCTACCATGCCAACAACTCAACCGACACTGTTGACACAGTA CTTGAGAAGAATGTGACAGTGACACACTCTGTCAACCTACTTGAGGACAGTCACAATGGAAAACTATGTC TACTAAAAGGAATAGCCCCACTACAATTGGGTAATTGCAGCGTTGCCGGATGGATCTTAGGAAACCCAGA ATGCGAATTACTGATTTCCAAGGAATCATGGTCCTACATTGTAGAAACACCAAATCCTGAGAATGGAACA TGTTACCCAGGGTATTTCGCCGACTATGAGGAACTGAGGGAGCAATTGAGTTCAGTATCTTCATTTGAGA GATTCGAAATATTCCCCAAAGAAAGCTCATGGCCCACCCACACCGTAACCGGAGTATCAGCATCATGCTC CCATAATGGGAAAAGCAGTTTTTACAGAAATTTGCTATGGCTGACGGGGAAGAATGGTTTGTACCCAAAC CTGAGCAAGTCCTATGTAAACAACAAAGAGAAAGAAGTCCTTGTACTATGGGGTGTTCATCACCCGCCTA ACATAGGGGACCAAAGGGCCCTCTATCATACAGAAAATGCTTATGTCTCTGTAGTGTCTTCACATTATAG CAGAAGATTCACCCCAGAAATAGCCAAAAGACCCAAAGTAAGAGATCAGGAAGGAAGAATCAACTACTAC TGGACTCTGCTGGAACCTGGGGATACAATAATATTTGAGGCAAATGGAAATCTAATAGCGCCATGGTATG CTTTTGCACTGAGTAGAGGCTTTGGATCAGGAATCATCACCTCAAATGCACCAATGGATGAATGTGATGC GAAGTGTCAAACACCTCAGGGAGCTATAAACAGCAGTCTTCCTTTCCAGAATGTACACCCAGTCACAATA GGAGAGTGTCCAAAGTATGTCAGGAGTGCAAAATTAAGGATGGTTACAGGACTAAGGAACATCCCATCCA TTCAATCCAGAGGTTTGTTTGGAGCCATTGCCGGTTTCATTGAAGGGGGGTGGACTGGAATGGTAGATGG GTGGTATGGTTATCATCATCAGAATGAGCAAGGATCTGGCTATGCTGCAGATCAAAAAAGTACACAAAAT GCCATTAACGGGATTACAAACAAGGTGAATTCTGTAATTGAGAAAATGAACACTCAATTCACAGCTGTGG GCAAAGAATTCAACAAATTGGAAAGAAGGATGGAAAACTTAAATAAAAAAGTTGATGATGGGTTTCTAGA CATTTGGACATATAATGCAGAATTGTTGGTTCTACTGGAAAATGAAAGGACTTTGGATTTCCATGACTCC AATGTGAAGAATCTGTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATAGGAAACGGGT GTTTTGAATTCTATCACAAGTGTAACAATGAATGCATGGAGAGTGTGAAAAATGGAACTTATGACTATCC AAAATATTCCGAAGAATCAAAGTTAAACAGGGAGAAAATTGATGGAGTGAAATTGGAATCAATGGGAGTC TATCAGATTCTGGCGATCTACTCAACTGTCGCCAGTTCCCTGGTTCTTTTGGTCTCCCTGGGGGCAATCA GCTTCTGGATGTGTTCCAATGGGTCTTTGCAGTGTAGAATATGCATCTGAGACCAGAATTTCAGAAATAT AAGAAAAAACACCCTTGTTTCTACT SEQ ID NO: 9 (Influenza A virus (A/New Caledonia/20/1999(H1N1)) segment 6 NA gene; CY033624.1 Influenza A virus (A/New Caledo- nia/20/1999 (H1N1)) segment 6, complete sequence) AATGAATCCAAATCAAAAAATAATAACCATTGGATCAATCAGTATAGCAATCGGAATAATTAGTCTAATG TTGCAAATAGGAAATATTATTTCAATATGGGCTAGTCACTCAATCCAAACTGGAAGTCAAAACCACACTG GAGTATGCAACCAAAGAATCATCACATATGAAAACAGCACCTGGGTGAATCACACATATGTTAATATTAA CAACACTAATGTTGTTGCTGGAAAGGACAAAACTTCAGTGACATTGGCCGGCAATTCATCTCTTTGTTCT ATCAGTGGATGGGCTATATACACAAAAGACAACAGCATAAGAATTGGCTCCAAAGGAGATGTTTTTGTCA TAAGAGAACCTTTCATATCATGTTCTCACTTGGAATGCAGAACCTTTTTTCTGACCCAAGGTGCTCTATT AAATGACAAACATTCAAATGGGACCGTTAAGGACAGAAGTCCTTATAGGGCCTTAATGAGCTGTCCTCTA GGTGAAGCTCCGTCCCCATACAATTCAAAGTTTGAATCAGTTGCATGGTCAGCAAGCGCATGCCATGATG GCATGGGCTGGTTAACAATCGGAATTTCTGGTCCAGACAATGGAGCTGTGGCTGTACTAAAATACAACGG CATAATAACTGAAACCATAAAAAGTTGGAAAAAGCGAATATTAAGAACACAAGAGTCTGAATGTGTCTGT GTGAACGGGTCATGTTTCACCATAATGACCGATGGCCCGAGTAATGGGGCCGCCTCGTACAAAATCTTCA AGATCGAAAAGGGGAAGGTTACTAAATCAATAGAGTTGAATGCACCCAATTTTCATTATGAGGAATGTTC CTGTTACCCAGACACTGGCACAGTGATGTGTGTATGCAGGGACAACTGGCATGGTTCAAATCGACCTTGG GTGTCTTTTAATCAAAACCTGGATTATCAAATAGGATACATCTGCAGTGGGGTGTTCGGTGACAATCCGC GTCCCAAAGATGGAGAGGGCAGCTGTAATCCAGTGACTGTTGATGGAGCAGACGGAGTAAAGGGGTTTTC ATACAAATATGGTAATGGTGTTTGGATAGGAAGGACTAAAAGTAACAGACTTAGAAAGGGGTTTGAGATG ATTTGGGATCCTAATGGATGGACAGATACCGACAGTGATTTCTCAGTGAAACAGGATGTTGTGGCAATAA CTGATTGGTCAGGGTACAGCGGAAGTTTCGTTCAACATCCTGAGTTAACAGGATTGGACTGTATAAGACC TTGCTTCTGGGTTGAGTTAGTCAGAGGACTGCCTAGAGAAAATACAACAATCTGGACTAGTGGGAGCAGC ATTTCTTTTTGTGGCGTAAATAGTGATACTGCAAACTGGTCTTGGCCAGACGGTGCTGAGTTGCCGTTCA CCATTGACAAGTAG SEQ ID Nos: 3 to 9 are all available in the UNIPROT database.

[0113] The present invention discloses the following preferred embodiments:

1. Influenza virus-like particles (VLPs), wherein the VLPs comprise:

[0114] hemagglutinin (HA) protein and neuraminidase (NA) protein on the surface of the VLPs,

[0115] a nucleoprotein (NP) ribonucleoprotein complex,

wherein the VLPs do not contain a ribonucleoprotein complex of at least one of PB1, PB2, and NS2.
2. VLPs according to embodiment 1, further comprising a matrix protein (M) ribonucleoprotein complex.
3. VLPs according to embodiment 1 or 2, wherein the HA protein is selected from the group of seasonal, non-pandemic HA proteins.
4. VLPs according to any one of embodiments 1 to 3, wherein the NA protein is selected from the group of seasonal, non-pandemic NA proteins.
5. VLPs according to any one of embodiments 1 to 4, further comprising a genetically modified M ribonucleoprotein complex, preferably wherein the M ribonucleoprotein complex contains viral RNA encoding an antigen of a non-influenza virus pathogen, an oncogene or a malignant tissue protein, especially wherein the M ribonucleoprotein complex contains viral RNA encoding a fusion protein containing an M protein and a protein or antigenic protein fragment of a non-influenza virus pathogen.
6. VLPs according to embodiment 5, wherein especially the non-influenza virus pathogen is selected from a non-influenza virus, a gram-positive or gram-negative bacterium, a fungus, a protozoan, or a prion, preferably a non-influenza RNA virus, a DNA virus, or a retrovirus, especially wherein the virus is SARS-COV-1, SARSCOV-2, or Dengue virus.
7. VLPs according to any one of embodiments 1 to 6, wherein the VLPs comprise: [0116] HA protein and NA protein on the surface of the VLPs, [0117] a NP ribonucleoprotein complex, and, preferably an M ribonucleoprotein complex,
wherein the VLPs do not contain a ribonucleoprotein complex of at least two of PB1, PB2, NS1 and NS2; preferably wherein the VLPs do not contain a ribonucleoprotein complex of at least three of PB1, PB2, NS1 and NS2; even more preferred wherein the VLPs do not contain a ribonucleoprotein complex of NS1; especially wherein the VLPs do not contain a ribonucleoprotein complex of PB1, PB2, NS1 and NS2.
8. VLPs according to any one of embodiments 1 to 7, triggering inflammatory cell death (necroptosis) upon entry into a cell.
9. Vaccine comprising the VLPs according to any one of embodiments 1 to 8, and a pharmaceutically acceptable excipient.
10. Vaccine according to embodiment 9, wherein the vaccine is an intranasal vaccine, preferably an aerosol or nasal spray.
11. Pharmaceutical composition comprising the VLPs according to any one of embodiments 1 to 8.
12. Method for producing VLPs according to any one of embodiments 1 to 8, comprising the steps of [0118] providing unidirectional vectors for HA, NA, PA, PB1 and PB2; [0119] providing a bidirectional vector for NP, [0120] providing either a unidirectional or bidirectional vector for M, [0121] expressing the vectors in a recombinant cell system to obtain VLPs according to any one of embodiments 1 to 8.
13. Method according to embodiment 12, further comprising the steps of filling the VLPs into a final container and finishing the VLPs to a pharmaceutical composition ready for use for administration to human individuals.
14. Method according to embodiment 12 or 13, wherein the vectors are expressed in Madin-Darby Canine Kidney (MDCK) cells, African green monkey kidney cells (Vero) cells, 293 cells, 293T cells, porcine kidney (PK) cells, owl monkey kidney (OMK) cells, Madin-Darby bovine kidney (MDBK) cells, chicken embryo kidney (CEK) cells, chicken embryo fibroblasts, primary chick kidney cells, or cells isolated from the chorioallantoic membrane of embryonated chicken eggs, preferably in MDCK cells or Vero cells, especially in MDCK cells.
15. VLPs according to any one of embodiments 1 to 8, for medical use, preferably for preventing pathogen-caused diseases, especially for preventing influenza.