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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K2039/525—Virus
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  • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
  • A61K2039/541—Mucosal route
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• • A—HUMAN NECESSITIES
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  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
  • A61K2039/552—Veterinary vaccine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • A61K2039/70—Multivalent vaccine
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  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16123—Virus like particles [VLP]
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/16011—Orthomyxoviridae
  • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
  • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• VLPs Influenza virus-like particles
• the present invention discloses influenza virus-like parti- cles (VLPs) and vaccines comprising such VLPs.
• VLPs influenza virus-like parti- cles
• a viral infection or vaccination is first recognized by the host's innate immune system. Innate immune cells identify the virus or vaccine as foreign, process it in order to present it to the adaptive immune system and thereby activate it. Depending on the nature of the vaccine, the protection initiated by the adaptive immune system is based more on antibodies (humoral immune response) or on cells that kill newly infected cells quickly and efficiently, making it impossible for the virus to spread (cellular immune response).
• IAV influenza A virus
• LAIV attenuated live viruses
• vaccines based on attenuated viruses have a viral RNA expressing polymerase that is not effective at body temperature, vaccination in the host does not express the necessary new viral RNA, which triggers the microinflamination required for cellular immunity. For these reasons, the current vaccine strategies have no lasting immune protection against IAV.
• Influenza virus causes 250,000-500,000 deaths worldwide an- nually; a global pandemic could kill millions. Vaccination is the most cost-effective public health measure to prevent disease and mortality caused by influenza virus infection.
• the influenza virion is an enveloped virus whose lipid bilayer comes from the plasma membrane of a host cell.
• Two different types of glycoprotein spikes are embedded in the shell. Approximately 80 percent of the spikes are hemagglutinin (HA), a trimeric protein that acts when the virus attaches to a host cell. The remaining approximately 20 percent of the glycoprotein spikes consist of neuraminidase (NA), which is believed to primarily facilitate the release of newly produced virus particles from the host cell.
• HA hemagglutinin
• NA neuraminidase
• RNA is packaged with nucleoprotein (NP) forming a helical ribonucleoprotein complex, containing three polymerase peptides for each RNA segment.
• NP nucleoprotein
• Most segments encode a single protein, but some describe several, such as the M segment, which encodes the matrix protein M1 and the ion channel protein M2 (Fig. 2).
• Influenza VLPs are potential candidates for inclusion in im- munogenic compositions. VLPs closely resemble mature virions, but they do not contain viral genomic material (see e.g. most recently: WO 2020/00101 A1 ([0017], [00254]); WO 2020/014656 A1 (p. 23)). In fact, VLPs are - per definition in the prior art - empty virus envelopes. Therefore, VLPs are non-replicative in nature, which makes them safe for administration as a vaccine. In addition, VLPs can be engineered to express viral glycoproteins on the surface of the VLP, which is their most native physiological configuration. Moreover, since VLPs resemble intact virions and are multivalent particulate structures, VLPs may be more effective in inducing neutralizing antibodies to the glycoprotein than soluble envelope protein antigens.
• US 2017/058265 A1 discloses the generation of influenza vi- ruses from virus-like particles by using sets of isolated host cells with virus segments.
• Watanabe et al. J. Virol. 76(2002): 767-773 report immunogenicity and protective efficacy of repli- cation-incompetent influenza VLPs.
• US 9,150,620 B2 describes a vaccine against multitypes of avian influenza viruses with spe- cifically mutated influenza HA protein.
• Liu et al. Antiviral. Res. 133 (2016): 110-118) report on multi-subtype influenza VLPs incorporated with flagellin and GM-CSF for vaccine design.
• a universal influenza vaccine based on heter- ologous multiple matrix protein 2 extracellular domain (M2e) pro- teins.
• M2e multiple matrix protein 2 extracellular domain
• RNA-VLPs RNA expressing VLPs
• the present invention provides influenza virus- like particles (VLPs), wherein the 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 at least one of PB1, PB2, NS1 and NS2.
• 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 at least one of PB1, PB2, NS1 and NS2.
• VLPs comprise: hemagglutinin (HA) protein and neuraminidase (NA) protein on the surface of the VLPs, a nucleoprotein (NP) ribon
• the present invention further provides a vaccine comprising influenza virus-like particles (VLPs), and a pharmaceutically ac- ceptable excipient, wherein the 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 at least one of PB1, PB2, and NS2.
• 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 at least one of PB1, PB2, and NS2.
• VLPs comprise: hemagglutinin (HA) protein and neuraminidase (NA) protein on the surface of the
• the present invention provides RNA-VLPs which are used in a vaccine that appears like a real viral infection to the immune system but cannot multiply.
• virus-like particles VLPs
• VLPs virus-like particles
• NP preferably two (i.e. NP and M)
• RNP IAV-ribonucleoprotein complexes
• RNA-VLPs due to their similarity, enter the cell on the same route as a functional IAV virion and discharge their two RNPs into the cell nucleus.
• Necroptosis causes the host cell to burst, which has several advantages for the immune system:
• the internal ingredients such as nuclear DNA and proteins are a danger signal that attracts and activates uninvolved cells of the innate immune system; viral proteins and RNA are rendered non-functional by the cell death of infected cells; those non- functional viral parts now serve as pathogen-associated molecular patterns (PAMPs), which are taken up by the now activated cells of the innate immune system and, after processing, are presented to the cells of the adaptive immune system.
• PAMPs pathogen-associated molecular patterns
• RNA-VLPs are biologically safer than pseudo- typed vaccines, which are in short IAV viruses with a non-func- tional NA protein (Powell et al., J. Virol. 86 (2012), 13397- 13406), because they do not replicate segments expressing viral RNA polymerases.
• the VLPs according to the present invention trig- ger inflammatory cell death (necroptosis) upon entry into a cell but do not trigger apoptosis of the cell.
• necroptosis results in the release of danger- associated molecular patterns (DAMPs), and thus is associated with inflammation and immune cell activation.
• DAMPs danger- associated molecular patterns
• RNA-VLPs are an im- provement of the existing VLP technology (disclosed e.g. Kang et al., PloS one 14 (2019), e0216871; 10.1371/journal.pone.0216871; Haynes, Exp. Rev. Vacc. 8 (2009), 435-445; Schotsaert et al., Sci. Rep.
• RNA-VLPs for the immune system simulate infection with a living virus through RNA expression .
• RNA-VLPs can be pro- symbolized using "reverse genetics” technology (Perez et al., Meth. Mol. Biol. 1602 (2017), 251-273; 10.1007/978-1-4939-6964-7_16).
• the individual influenza segments are isolated from a field virus, transcribed from RNA into DNA and cloned into DNA plasmids.
• the plasmids containing the DNA of each IAV segment are then transfected into a cell line.
• the plasmids produce either viral copy RNA, and/or viral messenger RNA for the production of viral proteins.
• the placement of pro- moters on the plasmids determines whether viral RNA, viral proteins or both are expressed after transfection of the plasmids into a cell culture. This technology has long been used to produce chi- meric viruses.
• VLPs are known in the present field of technology as particles made up of one or more viral structural proteins, but lacking the viral genome i.a. encoding these viral structural pro- teins of the VLP. Because VLPs lack a viral genome, they are not able to multiply in cells to generate infectious particles and yield safer and potentially more-economical vaccines and vaccine products. In addition, VLPs can often be produced by heterologous expression and can be easily purified. Most VLPs disclosed in the prior art comprise at least a viral core protein that drives bud- ding and release of particles from a host cell. Influenza VLPs can be produced by transfection of host cells with plasmids encoding the HA, NA and M proteins.
• VLPs can be isolated from cell culture supernatants.
• a protocol for purifying or isolating influenza VLPs from cell supernatants involves low speed centrifugation (to remove cell debris), vacuum filtration and ultracentrifugation of the VLPs through 20% glycerol.
• a virus- like particle may also include a subviral particle (SVP), which is typically smaller in size than a virus and constitutes a particle without a virus capsid or genome.
• SVP subviral particle
• the present VLPs - although containing certain viral nucleic acids - are clearly different from rescued viruses known in the art (e.g. Fodor et al., J. Virol. 73 (1999), 9679-9682; Hoffman et al., Arch. Virol. 146 (2001), 2275-2289; WO 01/04333 A1; WO 2007/016598 A2) due to the lack of the ability to be reproduced in a cell to obtain infectious viral particles.
• US 2017/058265 A1 uses various VLP constructs and cells, yet without suggesting them to be used for vaccines; this document aims at replication compe- tent fully infectious viruses to be used in various vaccine tech- nologies listed (yet without referring to use these VLPs in a vaccination context).
• VLPs according to the present invention do not contain a ribonucleoprotein complex of NS1.
• Watanabe et al., 2002 describe the delta NS1 recombinant influenza virus (which is not a VLP) which can propagate in IFN deficient cells (cells that have a diminished intrinsic immune response). It may be remarked that the VLPs according to the present invention - in contrast to all these constructs - cannot propagate in any cell line as the genetic information for the (for virus replication absolutely necessary) RNA dependent RNA polymerases are missing.
• the minimum requirement for the VLPs in the vac- cine according to the present invention are: only one or two RNPs inside; in any case PA, PB1, PB2 or NS1 could never be used as the template for the RNA in the RNP; this ensures that the VLP is unable to replicate (PA, PB1, PB2) or exert any immune antagonistic function.
• the VLPs according to the present invention are free of a nucleic acid set-up which would enable the creation of infectious particles in a host cell. This is why - by definition - the VLPs according to the present invention lack at least one of the ribo- nucleoprotein complexes which are indispensable for viral repli- cation, i.e.
• the VLPs according to the present invention lack even more than just one ribonucleoprotein complex of PB1, PB2, and NS2, e.g.
• the VLPs according to the present invention lack an NS1 ribonucleoprotein complex.
• the VLPs according to the present invention only contain an NP ribonucleoprotein complex, preferably with an M ribonucleoprotein complex, as the only ribonucleoprotein complex.
• the VLPs according to the present invention are - due to the mandatory lack of indispensable genetic material for reproducibility within a host cell (i.e. PB1, PB2, and/or NS2) - non-infectious, since they are only able to bind to and enter a cell but not able to be propagated by any host cell to produce infectious particles which then are able to enter further cells.
• a host cell i.e. PB1, PB2, and/or NS2
• the VLPs according to the present invention contain viral genetic material (a NP ribo- nucleoprotein complex; preferably also an M ribonucleoprotein com- plex) and may therefore also be referred to as "RNA-VLPs" but are - as the prior art VLPs - non-replicating, non-infectious viral capsid structures of influenza. Accordingly, the VLPs according to the present invention are able to enter a cell but no reproduction of the virus/VLP in the cell occurs. Accordingly, it is clear that as used herein e.g.
• a nucleoprotein (NP) ribonucleopro- tein complex means the ribonucleoprotein complex (RNP) encoding NP (i.e. an RNP with a viral RNA (vRNA) encoding NP; see also e.g. Fig. 11).
• the VLPs used in the vaccines of the present invention are therefore VLPs expressing HA and NA at their surface and have integrated an RNP with a vRNA encoding NP but no RNP encoding at least one of PB1, PB2 and NS2 (and preferably also no RNP encoding NS1), especially no RNP encoding all of PB1, PB2, NS1 and NS2.
• RNA-VLPs As a minimum the IAV surface proteins hemagglutinin (HA) and neuraminidase (NA) are required, for the construction of the VLPs envelope.
• the M protein is added, for greater stability and also to make the VLP externally identical to a true virus.
• the RNP of the NP segment will serve as a source for the viral RNA.
• NP has the advantages that it is expressed in relatively high numbers early after infection and it also has no known immune antagonistic function.
• the addition of the M segment as an RNP enables RNA-VLPs to be used as vectors in vaccination against pathogens other than influenza, since foreign sequences of other pathogens can also be added to the M segment without severely restricting its function- ality (Fig. 3).
• the VLPs according to the present invention pref- erably exhibit hemagglutinin activity and/or neuraminidase activ- ity.
• hemagglutinin activity refers to the ability of HA-containing VLPs, or portions thereof to bind and agglutinate red blood cells (erythrocytes).
• neuraminidase activity refers to the enzymatic activity of NA-containing VLPs, or portions thereof to cleave sialic acid res- idues from substrates including proteins such as fetuin.
• Influenza virus hemagglutinin HA is a viral surface glycopro- tein that generally comprises approximately 560 amino acids (e.g., 566 amino acids) and represents 25% of the total virus protein.
• HA is a protein antigen that is highly useful as an immunogen and is responsible for adhesion of the viral particle to, and its pene- tration into, a host cell, particularly, in the respiratory epi- thelium, in the early stages of infection (or entry into a cell (for the VLPs according to the present invention)).
• Eighteen dif- ferent HA subtypes have been identified in influenza viruses (H1- H18).
• Influenza virus neuraminidase (NA) is a second membrane gly- coprotein of influenza viruses.
• NA is 413 amino acid in length, and is encoded by a gene of 1413 nucleotides.
• NI terminal neuraminic acid
• N6 N7, N8 and N9 are cleaving terminal neuraminic acid (also called sialic acid) resi- dues from carbohydrate moieties on the surfaces of infected cells.
• NA also cleaves sialic acid residues from viral proteins, prevent- ing aggregation of viruses. Using this mechanism, it is hypothe- sized that NA facilitates the release of viral progeny by prevent- ing newly formed viral particles from accumulating along the cell membrane, as well as by promoting transportation of the virus through the mucus present on the mucosal surface.
• NA is an im- portant antigenic determinant that is subject to antigenic varia- tion.
• Sequences of HA and NA proteins to be used according to the present invention are e.g. available from the Influenza Research Database (IRD).
• the IRD contains avian and non-human mammalian influenza surveillance data, human clinical data associated with virus extracts, phenotypic characteristics of viruses isolated from extracts and all genomic and proteomic data available in public repositories for influenza viruses.
• IRD links host surveil- lance and clinical data to sequence and phenotypic data for all well characterized influenza virus strains as well as data obtained from public data sources for well-characterized virus strains will be supplemented with IRD generated data.
• the IRD contains 95,032 entries for HA proteins and 82,794 entries for NA proteins.
• the HA protein in the VLPs according to the present invention is selected from the group of HA proteins disclosed in the IRD, preferably a seasonal, non- pandemic HA. In the IRD, this can be selected by e.g. excluding 2009 pHINl sequences from the "Clade Classification" in a search for HA proteins.
• the NA protein in the VLPs according to the pre- sent invention is selected from the group of NA proteins disclosed in the IRD, preferably a seasonal, non-pandemic NA.
• Influenza A virus RNA segment 5 encodes NP (a polypeptide of 498 amino acids in length), which is rich in arginine, glycine and serine residues and has a net positive charge at neutral pH. The majority of the polypeptide has a preponderance of basic amino acids and an overall predicted pi of 9.3, but the C-terminal 30 residues of NP are, with a pi of 3.7, markedly acidic. In influenza B and C viruses, the length of the homologous NP polypeptide is 560 and 565 residues, respectively. Alignment of the predicted amino acid sequences of the NP genes of the three influenza virus types reveals significant similarity among the three proteins, with the type A and B NPs showing the highest degree of conserva- tion. Phylogenetic analysis of virus strains isolated from dif- ferent hosts reveals that the NP gene is relatively well conserved.
• the seventh RNA segment of influenza viruses encodes both, the M1 and the M2 protein.
• M1 is a matrix protein that lies just beneath the viral envelope in the form of dimers and interacts with viral ribonucleoprotein (vRNP) complex, forming a bridge between the inner core components and the membrane pro- teins.
• vRNPs harbour the determinants for host range.
• M1 contacts with both viral RNA and NP, promoting the formation of RNP com- plexes and causing the dissociation of RNP from the nuclear matrix.
• M1 plays a vital role in assembly by recruiting the viral compo- nents to the site of assembly and essential role in the budding process including formation of viral particles.
• the M1 protein forms a layer under the patches of host cell membrane that are rich with the viral hemagglutinin, neuraminidase and M2 transmem- brane proteins, and facilitates budding of the mature viruses.
• M1 consists of two domains connected by a linker sequence.
• the N- terminal domain has a multi-helical structure that can be divided into two subdomains.
• the C-terminal domain also contains alpha- helical structure.
• M2 is a membrane protein, which is inserted into the viral envelope and projects from the surface of the virus as tetramers.
• the extracellular domain of M2 is recognized by hosts' immune system.
• Transmembrane domain of M2 has ion channel activity, which is involved in the uncoating process of the virus in the cell.
• the M2 protein is a proton-selective viroporin, in- tegral in the viral envelope of the influenza A virus.
• the channel itself is a homotetramer (consists of four identical M2 units), where the units are helices stabilized by two disulfide bonds, and is activated by low pH. Proton conductance by the M2 protein in influenza A is essential for viral replication.
• the M gene there- fore encodes both matrix and membrane proteins, and has multiple functions (see e.g. Furuse et al., Virol. J. 6 (2009), 67; doi:10.1186/1743-422X-6-67) .
• a preferred embodiment of the present VLPs further comprises a matrix protein (M) ribonucleoprotein complex.
• M matrix protein
• VLPs according to the present invention comprise:
• HA protein and NA protein on the surface of the VLPs a NP ribonucleoprotein complex, and, preferably an M ribonu- cleoprotein complex
• 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.
• the M ribonucleoprotein complex in the VLPs ac- cording to the present invention contains nucleic acids selected from the group of M genes disclosed in the IRD.
• the M protein encoding nucleic acid sequences in the VLPs according to the present invention may be provided as encoding a modified pro- tein, for example a fusion protein comprising or consisting of (an) influenza M protein (s) and an antigen from a non-influenza virus pathogen.
• the VLPs according to the present invention further comprise a genetically modified M ribonucleoprotein complex, preferably wherein the M ribonucleoprotein complex contains viral RNA encoding an antigen of a non-influenza virus pathogen, especially wherein the M ribo- nucleoprotein 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.
• M pro- tein or "M ribonucleoprotein complex”
• M pro- tein or "M ribonucleoprotein complex”
• the non-influenza virus pathogen can be 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.
• the M ribonucleoprotein complex comprises an RNA encoding a fusion protein comprising the (influenza) M protein and a pathogen antigen or an immunogenic portion thereof.
• the pathogen is a viral, bacterial, fungal or protozoan pathogen.
• Suitable examples of such antigens are disclosed e.g. in WO 2015/149944 A2.
• the VLP technology according to the present invention can also a be used to elicit an immune response against oncogenes or malignant tissue proteins (for ex- ample, tumour antigens located at the surface of tumour cells and being specific for the tumour cells (i.e. not present on benign cells)).
• the present invention also re- fers to a vaccine comprising the VLPs according to the present invention and a pharmaceutically acceptable excipient.
• the VLPs according to the present invention are provided as a pharmaceutical preparation suitable to be applied to human individuals. Therefore, the VLPs according to the present invention may be combined with pharmaceutically acceptable excip- ients which are the conventional carriers (vehicles) and excipi- ents that are physiologically and pharmaceutically acceptable for use in human subjects.
• pharmaceutically acceptable vehicles are known to the skilled practitioner in the pertinent art and can be readily found, e.g. in Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., in its updated/cur- rent editions, which describes compositions and formulations suit- able for pharmaceutical delivery of one or more therapeutic com- positions, such as one or more influenza vaccines, and additional pharmaceutical agents.
• parenteral formulations usually comprise injectable fluids/liquids that include pharma- ceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
• physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
• solid compositions for example, powder, pill, tablet, or capsule forms
• conventional non-toxic solid carriers may include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate, which typically stabilize and/or increase the half-life of a composition or drug.
• compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
• auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
• Suitable pharmaceutical compositions of the VLPs according to the present invention for parenteral administration include, with- out limitation, sterile aqueous or non-aqueous solutions, suspen- sions, and emulsions.
• non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and canola oil, and injectable organic esters, such as ethyl oleate.
• Aqueous carriers include water, alco- holic/aqueous solutions, emulsions or suspensions, including sa- line and buffered media.
• Parenteral vehicles include, for example, sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
• Intravenous vehicles include, for example, fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
• Preservatives and other additives may also be present in such compositions and preparations, such as, for example, antimi- crobials, antioxidants, chelating agents, colorants, stabilizers, inert gases and the like.
• compositions may potentially be administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids, such as hydrochloric acid, hy- drobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, ma- leic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl and aryl amines and substituted ethanolamines.
• inorganic acids such as hydrochloric acid, hy- drobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
• compositions which include a therapeutically effective amount of the VLPs according to the present invention, alone, or in combination with a pharmaceuti- cally acceptable carrier.
• a pharmaceutical formulation suitable for the present purposes e.g. there are many appropriate examples and suggestions for the pharmaceutical formulations of influenza vaccines, which are also applicable for the VLPs according to the present inven- tion.
• Pharmaceutically acceptable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
• the carrier and composition can be sterile, and the formulation should suit the mode of admin- istration.
• the composition can also contain minor amounts of wet- ting or emulsifying agents, or pH buffering agents.
• the composition is preferably a liquid or aqueous solution, suspension, emulsion, or dispersion.
• a liquid or aqueous composition can be lyophilized and reconstituted with a solution or buffer prior to use.
• the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Any of the commonly known pharmaceutical carriers, such as sterile saline solution or sesame oil, can be used.
• the medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceu- tically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like.
• the vaccine according to the present invention is designed as an intranasal delivered vaccine, i.e. a vaccine which can be administered to an individual intranasally.
• this intranasal vaccine formulation is preferably designed as aer- osol or nasal spray.
• the RNA-VLP according to the present in- vention causes the necessary micro inflammation itself, no addi- tional adjuvant is necessary. Accordingly, this vaccine may be provided even free of any further adjuvant. This is specifically preferred, if a fast, easy and cheap production method is needed.
• the present invention relates to a pharmaceutical composition comprising the VLPs according to the present invention.
• a specifically preferred aspect of the present invention is drawn to influenza virus-like particles (VLPs), wherein the VLPs comprise: hemagglutinin (HA) protein and neuraminidase (NA) protein on the surface of the VLPs, a nucleoprotein (NP) ribonucleoprotein complex, a matrix protein (M) ribonucleoprotein complex, wherein the VLPs do not contain a ribonucleoprotein complex of at least PB1, PB2, and NS.
• VLPs comprise: hemagglutinin (HA) protein and neuraminidase (NA) protein on the surface of the VLPs, a nucleoprotein (NP) ribonucleoprotein complex, a matrix protein (M) ribonucleoprotein complex, wherein the VLPs do not contain a ribonucleoprotein complex of at least PB1, PB2, and NS.
• these VLPs according to the present invention further comprise a genetically modified M ribonucleoprotein com- plex, 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.
• this non-influenza virus pathogen is selected from a non-influenza virus, a gram-positive or gram-negative bac- terium, a fungus, a protozoan, or a prion, preferably a non-in- fluenza RNA virus, a DNA virus, or a retrovirus, especially wherein the virus is SARS-COV-1, SARS-COV-2, or Dengue virus.
• Another aspect of the present invention is a method for pro- ducing VLPs according to the present invention, comprising the steps of providing unidirectional vectors for HA, NA, PA, PB1 and PB2; providing a bidirectional vector for NP, providing either a unidirectional or bidirectional vector for
• VLPs according to the present invention.
• bidirectional vectors may be used as well (as long this set-up does not lead to a reproductive viral particle, i.e. a particle which is infectious after produc- tion in the host cell to which the VLP has been delivered); how- ever, preferably only one (for NP), even more preferred only two (for NP and M), bidirectional vectors are used in the production of the VLPs according to the present invention.
• the present method is preferably intended to provide compositions for use in human medicine
• the present method further comprises 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.
• VPLs according to the present invention is any cell that supports replication of influenza virus and meets acceptable standards for regulatory approval.
• the VLPs according to the present invention are therefore preferably produced in cells/cell lines which are ap- proved by the regulatory authorities.
• MDCK Madin-Darby Canine Kidney
• African green monkey kidney cells Vero
• PK porcine kidney
• OMK owl monkey kidney
• MDCK Madin-Darby bovine kid- ney
• CEK chicken embryo kidney
• Cockayne syndrome e.g., IL-12
• MDCK cell culture is specifically preferred, since MDCK cells were already approved by the FDA and EMA as cell lines for vaccine production (e.g. Flucelvax and Optaflu).
• MDCK cells are a model mammalian cell line used in biomedical research. MDCK cells are used for a wide variety of cell biology studies including cell polarity, cell-cell adhesions, collective cell motility, as well as responses to growth factors. It is one of few cell culture models that is suited for 3D cell culture and multicellular rear- rangements known as branching morphogenesis.
• the present invention also relates to the use of the present VLPs for medical use, preferably for preventing pathogen-caused diseases and for the treatment and prevention of tumour diseases (i.e. in immune oncology), preferably for the prevention or treat- ment of emerging viral diseases, especially for preventing influ- enza.
• tumour diseases i.e. in immune oncology
• Preferred virus-caused emerging diseases to be prevented and treated with the vaccines according to the present invention are those identified in Graham et al. (Graham et al., Nat. Immunol.
• Paramyxoviridaea especially Measles virus, mumps virus, Nipah virus; Togaviridaea(-alphaviridae), especially Rubella virus, Chikungunya virus, Western equine encephalitis vi- rus, Eastern equine encephalitis virus, Venezuelan equine enceph- alitis virus, Mayaro virus, Ross River virus, Barmah Forest virus, O'nyong'nyong virus, Semliki Forest virus, Getah virus, Sindbis virus; Reoviridaea, especially Rotavirus, New rotaviruses, Banna virus, Nelson Bay orthoreoviruses; Orthomyxoviridaea, especially Influenza virus A and B, multiple subtypes of influenza A virus, Dhori virus, Thogoto virus, Bourbon virus; Adenoviridaea, espe- cially Adenovirus 4 and 7, Adenovirus 14 or 81 or other serotype
• viral antigens of these viruses can be intro-ucked in the M gene to elicit immune responses against this re- combinant antigen upon administration of the VLPs according to the present invention (as vaccines) to a (human) individual.
• Methods of treating or preventing a disease or infection, or symptoms thereof, caused by influenza virus are provided.
• the methods comprise administering a thera- Chamberically effective amount of a pharmaceutical composition com- prising the VLPs according to the present invention (e.g., a VLP vaccine) to a subject (e.g., a mammal, in particular, a human subject).
• a pharmaceutical composition com- prising the VLPs according to the present invention
• a subject e.g., a mammal, in particular, a human subject.
• One embodiment involves a method of treating a subject suffering from, or at risk of or susceptible to disease or infec- tion, or a symptom thereof, caused by the pathogen, especially the influenza virus.
• the method includes administering to the subject (e.g., a mammalian subject), an amount or a therapeutic amount of a vaccine comprising VLPs, sufficient to treat the disease, in- fection, or symptoms thereof, caused by the pathogen, especially the influenza virus, under conditions in which the disease, in- fection, and/or the symptoms thereof are treated.
• the subject e.g., a mammalian subject
• an amount or a therapeutic amount of a vaccine comprising VLPs sufficient to treat the disease, in- fection, or symptoms thereof, caused by the pathogen, especially the influenza virus, under conditions in which the disease, in- fection, and/or the symptoms thereof are treated.
• the methods herein include administering to the subject (including a human subject identified as in need of such treatment) an effective amount of a vaccine according to the present invention to produce such effect.
• the treatment methods are suitably administered to subjects, particularly humans, suf- fering from, having, susceptible to, or at risk of having a dis- ease, disorder, infection, or symptom thereof, namely, caused by the pathogen, especially a flu or influenza. Identifying a subject in need of such treatment can be based on the judgment of the subject or of a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diag- nostic method).
• prophylactic methods of preventing or protecting against a disease or infection, or symptoms thereof, caused by the pathogen, especially the influenza virus are provided.
• Such meth- ods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising an immunogenic composition according to the present invention (e.g., a VLP vaccine) to a subject (e.g., a mammal such as a human), in particular, prior to infection of the subject or prior to onset of the disease, such as influenza.
• RNA-VLP the surface molecules HA, NA and the polymer- ases PA, PB1, PB2 expressed as proteins and the segments NP and M expressed as proteins and RNA are used.
• HA, NA PA, PB1 and PB2 are used in the construction of the RNA-VLPs in a unidirectional plas- mid, which only produces viral protein.
• PCAGGS Fig. 11
• this plasmid After transfection into a cell, this plasmid only expresses messenger RNAs, which are then translated into viral proteins by the cell's own machinery.
• RNA 12 can be used for the expression NP and M, into both mRNA as well as viral copy RNA (vRNA).
• vRNA viral copy RNA
• the expression of the negative sense genome RNA, (vRNA) is fostered by a human RNA polymerase I promoter and a terminator sequence, e.g. the mouse terminator sequence on pHWXccdB.
• a polymerase II transcription cassette e.g. the chicken b-actin promoter and polyA
• all plasmids are transformed together into a suitable cell line, e.g. the MDCK target line.
• RNA-VLPS Self-assembly unites the proteins and RNAs into viral shells with the surface proteins as well as the two RNPs, which contain viral M and NP RNA.
• the finished RNA-VLPS are then released into the cell culture medium and har- vested there.
• 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 in- volves 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.
• 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 adap- tive immune system such as T cells;
• DAMPs danger associated molecular patterns
• Fig. 2 shows the structure of the influenza (IAV) virion
• 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
• 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;
• 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.
• 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.
• 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;
• 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;
• 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 ac- tivates the added DCs, which in turn cause the T cells to prolif- erate (see Fig. 1);
• 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;
• Fig. 7 shows the sample plan IAV challenge study
• Fig. 8 shows the sample plan for the challenge study involving a chimeric vaccine against IAV and PRCv;
• Fig. 9 illustrates the production of the chimeric bidirec- tional plasmid containing genetic information of the IAV M segment as well as genetic information from the SARS-CoV-2 using Gibson assembly.
• the phW2000 plasmid containing the IAV M Seg- ment 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.
• 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 bidirec- tional plasmid containing the chimeric sequence of IAV M and a SARS-CoV-2 protein;
• 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)
• pBI bidirectional plasmids
• a specific region of the SARS-CoV-2 is reverse transcribed and amplified by PCR.
• 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).
• 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;
• Fig. 11 shows the plasmid map of pCAGGS
• Fig. 12 shows the plasmid map of pHWX
• the fol- lowing VLPs with HA and NA surface proteins and different config- urations of RNPs were constructed:
• VLPs with an NP ribonucleoprotein complex VLPs with an NP ribonucleoprotein complex
• VLPs with an NP and M ribonucleoprotein complex Due to the bidirectional nature of the pHWX plasmid M, both RNP and surface protein are present in addi- tion to HA and NA
• 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.
• 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 iso- lated according to Hoffmann et al. (Arch. Virol. 146 (2001), 2275- 2289, 10.1007/s007050170002).
• 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 in- dividual 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.vi- rusres.2004.08 .012). Next, the RNA-VLPs were harvested in the cell culture medium.
• 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).
• dendritic cells from monocytes which were ob- tained 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.
• the ability of the VLPs to elicit a cellular immune response was checked by a T-cell dendritic-cell co-cultivation method.
• 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).
• VLPs with M and NP RNPs were able to produce at least a similarly high T cell proliferation as an infection with a real influenza virus (positive control).
• 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 prolif- eration.
• 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 im- mune system.
• these RNA-VLPs behave like an infectious IAV virion by penetrating the cell nu- cleus and beginning to express viral RNA through the polymerases they have brought with them.
• This newly synthesized RNA then trig- gers an inflammatory signal cascade that boosts cellular immunity. Exactly this step is not used in current vaccination strategies.
• RNA-VLP vaccine is constructed with broader activity against influenza and a porcine corona virus, which causes pathol- ogies in pigs comparable to those observed in humans when infected with the current SARS-CoV-2.
• 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 vac- cine.
• 30 animals are grouped in three groups of 10: (i) animals getting the RNA-VLPs plus vehicle, (ii) animals taking the con- ventional vaccine, and vehicle and (iii) vehicle group. After vac- cination 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.
• 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 porcine respiratory corona virus
• 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
• 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 vaccina- tion 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).
• 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)
• 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)
• 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)
• 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)
• SEQ ID NO: 7 Influenza A virus (A/California/04/2009(H1N1)) seg- ment 7 matrix protein 2 (M2) and matrix protein 1 (M1) genes; FJ969513.1 Influenza A virus (A/California/04/2009(H1N1)) segment 7 matrix protein 2 (M2) and matrix protein 1 (M1) genes, complete cds)
• 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)
• 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)
• SEQ ID Nos: 3 to 9 are all available in the UNIPROT database.
• the present invention discloses the following preferred em- bodiments:
• VLPs Influenza virus-like particles
• the VLPs com- prise: 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 at least one of PB1, PB2, and NS2.
• M matrix protein
• VLPs according to embodiment 1 or 2 wherein the HA protein is selected from the group of seasonal, non-pandemic HA proteins.
• 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.
• a non-influenza virus 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.
• HA protein and NA protein on the surface of the VLPs a NP ribonucleoprotein complex, and, preferably an M ribonu- cleoprotein complex
• 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.
• VLPs according to any one of embodiments 1 to 7, triggering inflammatory cell death (necroptosis) upon entry into a cell.
• Vaccine comprising the VLPs according to any one of embodi- ments 1 to 8, and a pharmaceutically acceptable excipient.
• Vaccine according to embodiment 9, wherein the vaccine is an intranasal vaccine, preferably an aerosol or nasal spray.
• composition comprising the VLPs according to any one of embodiments 1 to 8.
• Method for producing VLPs comprising the steps of providing unidirectional vectors for HA, NA, PA, PB1 and PB2; providing a bidirectional vector for NP, providing either a unidirectional or bidirectional vector for
• MDCK Madin-Darby Canine Kidney
• Vero African green monkey kidney cells
• PK porcine kidney
• OMK owl monkey kidney
• MDBK Madin- Darby bovine kidney
• CEK chicken embryo kidney
• primary chick kidney cells or cells

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