AU2015202195B2 - New influenza virus immunizing epitope - Google Patents

Abstract

Abstract A method for synthesizing influenza virus-like particles (VLPs) within a plant or a portion of a plant is provided. The method involves expression of a novel influenza HA protein in plants and its purification The invention is also directed towards a VLP comprising influenza HA protein and plants lipids. The invention is also directed to a nucleic acid encoding improved influenza HA as well as vectors. The VLPs may be used to formulate influenza vaccines, or may be used to enrich existing vaccines.

Description

NEW INFLUENZA VIRUS IMMUNIZING EPITOPE FIELD OF INVENTION

[0001] The present application is a divisional application of Australian Application No. 2009270404, which is incorporated in its entirety herein by reference.

[0001a] The present invention relates to the production of virus-like particles. More specifically, the present invention is directed to the production of virus-like particles comprising influenza antigens, most particularly modified influenza antigens that have broad cross-reactivity with other influenza strains.

BACKGROUND OF THE INVENTION

[0001b] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0002] Influenza is the leading cause of death in humans due to a respiratory virus. Common symptoms include fever, sore throat, shortness of breath, and muscle soreness, among others. During flu season, influenza viruses infect 10-20% of the population worldwide, leading to 250-500,000 deaths annually [0003] Influenza viruses are enveloped viruses that bud from the plasma membrane of infected mammalian cells. They are classified into types A, B, or C, based on the nucleoproteins and matrix protein antigens present. Influenza type A viruses may be further divided into subtypes according to the combination of hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins presented. HA governs the ability of the virus to bind to and penetrate the host cell. NA removes terminal sialic acid residues from glycan chains on host cell and viral surface proteins, which prevents viral aggregation and facilitates virus mobility. Currently, 16 HA (HI-HI6) and 9 ΝΑ (N1-N9) subtypes are recognized. Each type A influenza virus presents one type of HA and one type of NA glycoprotein. Generally, each subtype exhibits species specificity; for example, all HA and NA subtypes are known to infect birds, while only subtypes HI, H2, H3, H5, H7, H9, H10, Nl, N2, N3 and N7 have been shown to infect humans (Horimoto 2006; Suzuki 2005). Influenza viruses comprising H5, H7 and H9 are considered the most highly pathogenic forms of influenza A viruses, and are most likely to cause future pandemics.

[0004] Influenza pandemics are usually caused by highly transmissible and virulent influenza viruses, and can lead to elevated levels of illness and death globally. The emergence of mw infiuenxa A subtypes resulted pandemics ii*3h$'20fh century. Tile Spam sh tin, caused by an HI Ml virus, in 1918-1919 led to the deaths of over 50 million people worldwide between 1917 and 1920* Presently, the risk of the emergence of a new subtype, or of the transmission ίο humans of a subtype endemic in animals, is always present. Qf particular concern is aMgily virulent form of avian influenza (also called “bird ila”), outbreaks of which have been reported in several countries around the world, in many cases, this bird fin can result in mortality rates approaching 100% within 48 hours. The spread of the avian influenza virus (H5NI), first identiSed in Hong Kong in 1997, to other Asian countries and Europe has been pdsmiated tn be linked to the migratory patterns of wild birds. 10005]: The current method of combating influenza in humans is by annual vaccination. The vaccine is usually a combinati on of several strains that are predicted to he the :*tflM^easoiniL,-*i:: The prediction is coordinated by the World Health Organisation, Generally, the number of vaccine doses produced each year is not sUffiei'Cnt·to "vaccinate;:the world’s population* For examples Canada and the United-States obtain enough vaccines doses to Immunize about one third of their jiopulation, while only 17% of the population of the European Unioneanhe vaccinated, fl is evident that current worldwide production of inilueamvaecane would be msnfiicieat in the face of a: wotidwidb; Bu pandemic* Even if the necessai^ mmuai pmdumipn could somehow be met In a given year, the dominant strains changethus stockpiling at low-need times in the year is nbtpraetical. Economical, large scale production of an effective influenza vaccine isof sijpificant interest^ government and private industry alike, [0O06] Currently, the most important source viral stocks Jm use in vaccines are produced in fertilised eggs. The virus particles are harvested, and far an inactivated viral vaccine, disrupted by detergent to inactivate it. Live attennaied vaccines am made of influenza viruses that were adapted tor growth atlow temperature which nieans that at normal body temperature, the vaccine is attenuated. Such a vaccine is licensed in USA for use in individuals horn 5 to 49 years of age, InaeiivaW whole virus vaccines are rendered harmless by inactivation with chemical agents and they have been produced in embryonic eggs or mammalian cell culture. All these types of vaccine show some specific advantages and disadvantages, One advantage of vaccines deriv#-froni;'^iiejie vimses: is the type of imnumify induced fey such vaccines, in general, split vaccines induce a strong antibody response while vaccines made of whole vi ruses induce both an :cMtiilar response. Even though a functional antibody response is a criterion for licensure that correlates with protection induced by a vaccine, there is increasing evidence that a T~ce!l response is also important in influenza immunity ----- this may also provide better protection; in the dderly, [0907] in order to induce a cellular immune response, vaccines made of whole viruses were developed. Due to the high pathogenicity of the influenza strain Cd-g, M5N1), these vaccines are produced in Bli-*· facility, For highly pathogenic influenza strains such as MSN 1, some manuiactnrers have modified the hemagglutinin gene sequence in order to reduce the pathogenicity of the influenza strain and to make it avirulent and more easily produced in embryonic eggs or mammalian eel! culture. Others also use reassortant influenza strains in which the genetic sequences for the hemagglutinin and neuraminidase proteins are cloned in a high-yielding low pathogenic influenza donor strain (A/FR/8/34; Quad F-S et al, 200?)v While these methods may produce useful vaccines, they do not provide a solution to the need for Mgh-voiume, low cost and fast production of vaccines in the scale necessary to meet the ^ohal need in a normal year, and would almost certainly he insufficient in the face of a panderaic, [0008] Mstag this reverse geneticmchnology, onemight also need to mutate the genetic sequence of the MA protein to make it avirelent. For highly pathogenic influenza strains, the production of whole virus vaccines either requires confinement procedures or the resulting vaccines do not exactly match the genetic sequenceof the circulating vires. In theease of live-attenuated vaccines, there is still a risk that the administered vaccine can recombine with an influenza virus hpm the host, leading to a new influenza virus.

[0009] While this method maintains the antigenic epitope and gost-translational modifications, there are a number of drawbacks including the risk of amt animation due to the use of Whole virus and variable yields depending on virus strain. Sub-optimal levels of protection may result fioni genetic heterogeneity In the virus doe to its mtmdoelion into eggs, .0&er'!®sa(lv®i^|6S::&dbde:exlM^ve'^i^m»g-:%^fet^wng eggs, contamination risks due to and long production times. Also, persons be eligible candidates fdr receiving the vaccine, [0010] In the ease of a pandemic, split vaccine production is slowed by the need to adapt thestrain lor growth in eggs and the variable pmdnmibh yields achieved; Although this technology has been used tor years for the production of seasonal vaccines, it ean hardly respond in a reasonable timeframe to a paMemlc since worldwide maaufaetnring capacity is limited. 10011] The recentoutbreak in Mexico of Influenza type A MINI also highlights the ergentmMieal need to develop rapid methodology for vaccine production of newly emerging strains. 10012] To avoid the use of eggs, infiuenza viruses have also been produced in mammalian cell culture,: lor example in MDCK or PERC.6 cells, or the like. Another approach is reverse genetics, in which viruses are produced by cell rianslbrmation with viral genes. These methods, however, also require the use of whole vims as: well as elaborate methods and specific culture environments. {0013] Several recombinant products have been developed as recombinant influenza vaccine eandidat^ylhe^.gppmadK«s have focused on the expression, production, and purification of inSuenea type A HA and N A proteins, including expression of these proteins usingbaduipvirtriAniected insect cells (Crawford ei al, 199¾ Johansson, 1999), viral vectors, and IJHA vaccine eonstmcts fOlsen n al ,1997).

[0014] infection are well known. Briefly, the infectious cycle is initiated by the attaehment of the virion surface H A protein to a sialic acid* containing cellular receptor (glyepproteins and glycolipids). The NA protein mediates processing: of the sialic acid receptor, and 'Virus penetration into the cell depends on: HA-dependent receptor-mediated endocytosis. M the aeidie confines of internalized ettdosonaes containing an influenza virion, the HA protein undergoes conformational changes that lead to fusion of viral and cell menihranos and virus uncoadng and M2-mediated release of MI proteins fkn« nucleocapsid-associated nboftudeoproteins (RNPs), which migrate into the cell nucleus for viral ERA 'Synthesis, Antibodies to HA proteins prevent vims infection by neutralizing virus infecuvity, Whereas antibodies to NA proteins ntediate their effect on the early steps of viral replication.

[00!$] Crawford -et&i. (1999) disclose expression of influenza HA in baeulovrrus·· infected insect ceils. The expressed proteins are described as being capable of preventing lethal influenza disease eadsed by avian MS and H7 influenza subtypes. Johansson e! of (1999) teach that baenlovims-expressed iniuenxa MA and Dl A proteins induce immune responses in animal superior to those induced by a conventional vaOeine,

Immunogenicity and efficacy of baeulovims^expressed hmnagglatinm of equine influenza vims was compared to a homologous,©NA vaccine candidate (Olsen & αί, 1997% Collectively, these data demonstrate that a high degree of protection against influenza vims challenge can be induced with recombinant HA or NA proteins, using various experimental approaches and in different animal models, [0016] Since previous research has shown that the surface influenza glycoproteins, HA and NA, are the primary targets for eliciting protective immunity against influenza virus and that MI provides a conserved target for cellular immunity to influenza* a new vaccine candidate:msy include these viral antigens as a protein macromolecular particle, such as virus-like particles (VLFs), As vaccine products, VLPs offer the advanfage of being more immunogenie iban subunit or recombinant antigens and are able to stimulate both humoral and cellular immunc response (Grgacic and Anderson, 2096). Further, the particle with these influenza antigens may display conformational epitopes that elicit neutralizing antibodies to multiple strains of influenza viruses.

[0017] Production of a non-infections influenza virus strain for vaccine purposes is one way to avoid inadvertent infection. Alternatively, virus-like particles (VLPs) as substitutes for the cultured vims have been investigated. VLPs mimic the structure of the viral capsid, but lack a genome, and thus cahaoi replicate or provide a means for a secondary1 infection.

[0018] Several studies have demoosfrated that recombinantinfluenza proteins self-assemble into VIPs in cell culture using mammalian expression plasmids or baculovirus vectors (Gomex-Puertas et «1,1999; Hewr«^;^:d4?;'2.^®i,titthian and Galarza, 2001). Gomez-Pucrtas et ai. (1999) discloses that efficient formation of influenza YIP depends on the expression levels of several viral pmteins. Neomaini er of (2000) establi shed a :.i»^mali^^3pfepsioii plasmid” based system for generating infections inffoenza viras-ike particles entirely from, cloned eDMAs, Latham and Galarza (2001) reported the formation of-inSuima'IiCk.Fs in insect cells infected with recombinant haeolovirns eo-expressing BA, NA, Ml f and M2 genes. These studies demonstrated that influenza virion proteins may selfrasseufole upon co-expression In eukaryotic cells.

[0019] Gomez-Fnertas::# «/· (2000) teach that, in addition to the hemagglutinin (HA), the matrix protein (Ml) of the influenza vires is essential for VLP budding from insect cells. However, Chen e? al (2007) teach that Ml might not bemtrured for VLP formation, and observed that efficient release Of Ml and VIPs required the presence of HA and sialidsse activity provided by IM A, The IMA cleaves the sialic acids of foe glycoproteins at the surface of foe cells producing the YLPs, apd releasing the VIPs in the medium, (00.203 Qua» et al (2007) teach that a VLP vaccine pfodneed in a haeulovirp expression system (insect· ceil) induces a protective immunity apdnst some strains of inflnenza virus (A/PR.8/34 (HIM!)). The VIPs studied by Quan were observed to bud from foe plasma membrane, and were considered to be of the correct size and^ nmrphoiogy, similar fo those obtained in a mammalian system (MOCK cells), [00211 Enveloped viruses may obtain their lipid envelope when 'budding5 out of the infected cell and obtain the membrane from the plasma membrane, or from that of an .internal' organelle, Influenza virus particles and VIPs bud from the plasma membrane of the host cell. In mammalian or baeuiovirus cell systems, for example, influenza buds from the plasma membrane (Quan et al 2007).

[00223 Only a fow enveloped viruses are known to infect plants (for example, members of the Topoviruses and Ehabdo viruses). Of the known plant enveloped viruses, they are characterized by budding from internal membrarfos of foe host Cell, and not from the plasma membrane. Although a small number of recombinant VLPs have been produced in plant hosts, none were derived from the plasma membrane. Current influenza VLP production technologies rely on the co-expression of multiple viral proteins, and this dependence represents a drawback of these technologies since in case of a pandemic and of yearly epidemics, response time is crucial for vaccination. A simpler VLP production system, relying on the expression of only one viral protein is desirable to accelerate the development of vaccine.

[0023] The production of influenza HA VLP in plant based system has been described in WO 2009/009876 that essentially showed that the influenza HA is able to self-assemble in plant host cells and bud from plasma membranes in virus-like particles.

[0024] In order to protect the world population from influenza and to stave off future pandemics, vaccine manufacturers will need to develop effective, rapid methods producing vaccine doses. The current use of fertilized eggs to produce vaccines is insufficient and involves a lengthy process. HA proteins used are specific for each strain and do not cross-react with other strains to provide broader spectrum vaccines thus necessitating constant production or short reaction time once a new strain is identified.

[0025] Certain modifications and/or mutations may be brought to the HA native protein used for producing VLP, such modifications bringing about a hemagglutinin protein that has broader spectrum to induce antibody neutralizing to more than one, or several strains of flu, even after only a single administration.

SUMMARY OF THE INVENTION

[0025a] According to a first aspect, the present invention provides a nucleic acid comprising a nucleotide sequence encoding an influenza virus hemagglutinin (HA) comprising a HA1 domain, wherein said HA1 domain is modified to be free of N-linked glycosylation sites at positions 154, 165, and 286, by a. modifying an amino acid at position 154 to be a non-asparagine and/or modifying an amino acid at position 156 to be a non-serine or non-threonine; b. modifying an amino acid at position 165 to be a non-asparagine and/or modifying an amino acid position 167 to be a non-serine or non-threonine; and c. modifying an amino acid at position 286 to be a non-asparagine and/or modifying an amino acid at position 288 to be a non-serine or non-threonine, wherein the numbering is in accordance with strain A/Vietnam/1194/04 and wherein the nucleotide sequence is operatively linked to a regulatory region active in a plant.

[0025b] According to a second aspect, the present invention provides a method of producing influenza virus like particles (VLPs) in a plant, a portion thereof, or a plant cell, the method comprising: (a) introducing the nucleic acid according to the first aspect into the plant, portion thereof, or the plant cell and (b) incubating the plant, portion thereof, or the plant cell under conditions that permit expression of the nucleotide sequence, thereby producing the VLPs.

[0025c] According to a third aspect, the present invention provides a virus like particle (VLP) produced by the method according to the second aspect.

[0025d] According to a fourth aspect, the present invention provides a virus like particle (VLP) comprising an influenza virus HA molecule encoded by the nucleotide sequence as described in the first aspect.

[0025e] According to a fifth aspect, the present invention provides use of the VLP according to the third aspect or the fourth aspect for the manufacture of a medicament for the prevention or treatment of an influenza infection in a subject.

[0025f] According to a sixth aspect, the present invention provides a composition comprising an effective dose of the VLP according to the third aspect or the fourth aspect, in admixture with a pharmaceutically acceptable carrier.

[0025g] According to a seventh aspect, the present invention provides a method of inducing immunity to an influenza infection in a subject, comprising administering the composition according to the sixth aspect.

[0025h] According to an eighth aspect, the present application provides a transgenic plant or a transgenic plant cell transiently or stably transformed with the nucleic acid according to the first aspect.

[0025i] Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

[0026] It is a further aspect of the invention to provide an improved influenza vaccine.

[0027] It is a further aspect of the invention to provide novel influenza virus-like particles.

[0028] It is a further aspect of the invention to provide an hemagglutinin protein which has been modified to provide a broader spectrum antibody reaction.

[0029] contemplates a polypeptide having an amino acid residue sequence substantially identical to that of a viral envelope Kdloked glycoprotein but. that is partially or totally free Of H-linfced carbohydrates (he, has one Of more glycdsylatiou site that is abolished when compared to an original native HA sequence), as well as methods of producing and utilizing the polypeptide. £0030] It is a further ppect of the invention to provide a HA protein wherein one or more of the Ndiuked glycosylaiion sites femvHAi domain has been m&uerria VLPs fbffhe preparation of a broad spectrum iaflueMa vaccine.

[0031] Particularly, the HA1 domain comprises ammo addslocated at positions 1 to 331 as numbered in accordance with strdn A/^etnam/1194/04; S1Q II> N0,34)*. More particularly,lheHA1 domain comprises the globular head portion and the domain of the protein, corresponding to. 331 of the protein as mimbered in accordance 194/04; SECj I0iNC>J4}. Particularly, the giycosylation site that is abolished is originally present on the globular head portioh of the protein, particularly corresponding to amino acids located between positions 39 to 273 of SEQ ID No.34. More particularly, the abolished glycosyiatkm site is originally located in the F2 domain of the protein, particularly corresponding to amino acids located between positions 274-331 of SEQ Π) NO.34, [0032] The present invention provides for amina acid substitutions In the hemagglutinin (NA) molecule of I ntluenza A that can alter the antigenicity and immunogenicity of the HA. These substitutions may alter antigenic sites by altering receptor specificity and/or antibodyvsntigen binding. In variety of embodiments, the from the substitution may be useful for the production of vaccines with broader cross» reactivity for influenza, PartlcUlaflyi the amino add substitution results th molecules with fee immenogeoadty diaracteristics of the amino acid substitution of nomasparagme residue of the HA protein at the location corresponding to the receptor binding-site and particulariy eorrespondmgto location 154 and/or 165 and/or 286 (whemin ntmlhering is maeeordanee with strain A/¥t^am/ii94/04; SBQ IB NO*34). In particular embodiments, the amino add substitution [0033] The influenza virus increased arttigeaidiyMA moletadtemay itsehtde o&eprmore nomglyeosylated amino add dnTespunding to positions 154 and/or 165 and/or 286 in H5 HA* where removal of any one of these glyeosylation sites results in an increase reactivity with antisera derived fro&fan animal exposed to ^ infinenza vims with a wildtype HA molecule, [0034] In order to destroy a glyeosylation site, the triad signal N-X-S/T (where N is a Asn, X can be any amino acid except Fro, and S/T can be both Ser or Thr) may be modified by protein engineering. The first appmaeh used can be to replace the Asn by another:amino acid. The second approach is to replace the S/T ammo add at position nr2 relative to the asparagine to be glycosylated, by any other amino acid residue, An appropriate amino add used to replace the asparagine, sedhe pr direonine is alanine, but other amino acid can also be used. For example, Asn can fee replaced by Leu, He, Val,

Thr, Ser or Ala, Also, Ser or Thr can be replaced by Ala,3¾ fie or Leu, [00331 Fertiddlafly, fhelnfinensia virus increased antigenicity HA molecule may include a non-asparagine amino add at positions 154 and/or 165 and/or 286 in H5 HA, [0036] The influenza virus increased antigenicity HA molecule may include MA protein wherein head portion is devoid of H-linfced glycosyiahon sites i,e. all three glyeosylation sites have been abolished.

[01)37] The induerma vims increased amigodcity I1A molecule may include one or more than one glyeosylation site that is removed, selected from the group consisting of: N-154, N-165 and N-286 (wherein the numbering is in aeeordanee with strain A/Viethaor/l 194/04), (00381 The present invention provides a modified: hemagglutinin (HA) horn different influenza strains, (0039] The present invention also provides a method of producing lofiuenaa virus like particles (VLPs) ip a npn-siaiytaing: host organism comprising: a) introducing a nucleic add encoding a» influenza hemagglutinin (HA) antigen as defiii^iaJ^V'^-.op^lidvely linked ίο a regulatory region active in a nomsialylating host organism or a portion thereof, and

Is) incubating the host or a portion thereof tinder conditions that permit the sttpression of the nucleic acid, thereby producing the Y'LPs.

[0040] The present invendon Inelndes the above method wherein, in the step of introducing (step a), the nuehrie acid may be either hansiently expressed in the host, or stably expressed in the host Furthermore, the YLPs t^y^j^^edmsin^for example, size exclusion eiuonMtography, [0041J Addihonaliy the present invention relates to a. non-sialylating host organism used for the production of vims like particle (YLP) comprising influenza vims HA protein. Particularly. suitable host capable of producing a YLP, is fer example, a plant or a portion thereof a plant cell, an insect or a portion thereof or an insect cell, or a yeast or portion thereof or nyeast cell. |004^].^«kiOiding.fb inyention there is provided a nncleic add comprising, a nucleotide seqnenee encoding a modiied influenza HA as defined above operatively linked to a regulatory region active in a non-sialylating host organism. The antigen may he an inflyenza hemaggintmm (HA) devoid of one or more the M-linked glycosyiation sites from the head portion of the molecule (antigenic sites that are normally present in the -native sequence).

[0043] The present invention also provides a virus like particle (YLP) comprising an mduenxa virus HA protein as defined herein and one or more than ope host lipid. If the ^stisdnseck^m^the/vimlite particle (YLP) may comprise an influenza virus If A protein and ohe or mofethan one insect lipid, or if the host is a yeast, then the virus like particle (YLP) may eotnprise an influenza virus HA protein and one or more than one yeast lipid, if the host is a plant, then.: theYirtiS like particle (YLP) may comprise ah influenza virus HA protein and one or more than one plant lipid. £0044! Tie invention limber provides VLPs that are produced in a plant thereby containing one or more than one lipid of plant origin (generally referred to as lipidk*}- [0045] The invention further provides 'VLPs produced in insect cells comprising lipids imm the plasma membrane of insect cells (generally referred to as ‘Insect lipids”); [0046] The invention .further provides VLPs produced in yeast comprising lipids from the plasma membrane of yeast ceils (geoeraliy referred': to as “yeast lipids5'}, [0047] Also included in the present invention is a .imposition, comprising an effective dose of a VLP comprising an influenza virus BA protein, one or more than ope lipid derived from a non~riaiyiatmg host production ceil, in admixture with a pharmaeentieaiiy acceptable carrier. The pharmaceutically acceptable carrier may be suitable for oral, intradermal, intranasal, intramuscular, inlraperitoneal,.intravenous* or subcutaneous administration, [0048] further included in the present invention is a vaccine composition comprising an immumologieally effective dose of u VLP M deiued heroin In admixture with a pharmaceutically aeOepiable carrier with or without the presence of an adjutant The vaccine may be admlmstered orally, intradermally, intranasally, immmuseaiariy, intraperitonealiy, intravenously, or sofxmaneousiy, Particularly, the vaccine is administered without the use of an adju vant.

[0049} The present invention also provides for a method Of inducing Ismmnity to an influenza virus infection in a subject, the method comprising administering to the subject the vims like particles comprising an influenza virus HA protein, one or more than one host lipid, and a phammoeufieally acceptable currier, The virus-like particle may he administered to a subject orally, mfetdermally, iotranasally. intramuscularly, intxaperiioneally, inriavemmsly, of subcutaneously.

[0050] The present invention pertains to a method for inducing immunity to influenza virus infection in a subject comprising administering to the subject an effective dose of a vaccine comprising one or more than one VLP as defined herein.

[0051] The sirtpet being treated by the methods as defined above may be selected km die group comprising humans, primaSos, horses, pigs, birds fa%d^)/water foiv1s migratory birds, quail, duck, geese, poultry, clueless, camel, canine, dogs, feline,cats, tiger, leopard, civet, mink, stone marten, ferrets, bouse pets, livestock, mice, fats, seal, whales and the like, Particiiiarly, the^ss^eta niay be a boman patient or birds in genera! (including water fowl, migratory birds, poultry such m gua.il, dock, geese, turkey, chicken}, particularly migratory birds or poultry for human consumptiou (guail, duck, geese, turkey, chicken).

[0052] The present ihventidn also provides for a container such as a syringe as well as kits comprising such a container, all of which comprising the vaccine composition as defined herein.

[0053] This summary of tlte i nvention does not necessarily describe all aspects of the invention.

UEfAILE© BESCIRirriCp OF THE INVENTION BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Ihese and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

Figure 1A represeitts the localisation of glycosyiation sites on the infiueifoa virus HA US acids identity, position, and location are indicated by analog)·' on the structure of the A/Vietnam/i 194/04: SEQ IB NO. 34 (PDB foe: 2IBX). The triple mutant has been made by the destruction of the glyeosylation sites N154,

Ml65, and 14236 Ideated on the globular head. The study from Bright et αί (2003) has been used to locate the potential antigenic sites, Glyeosylation type has been determined based on what is written In the literature about HAs HI , H3 and H7 (Abe ¥, et cd. (2004); Virprust DJ et ol. (2007); and. ICuroda et αί (1990);

Figure IB Is an illustration of the subdomains of the B A monomer: The F 1 (1-38 as numbered according to A/Vietnam/1104/04; SEQ1R NO04), F’2 (274-331) andF subdomains are represented. The receptor binding site and esterase sub-domains that together form the globular head (39-273). The fusion peptide is represented as a white box, lire TmD and cytotail cannot he seen on any HA strocte^s shiee o»ly the soluble bromelain products of HAs have been crystallized. and structure elucidated;

Figure 2 represents the sfoueiuim of a moisotuer of HA from different A subtypes. The lipid Mayer, with: its alphstie counterpart and its polar headis presented as well .·&ό^Ε4·. Ha et ai (Ha A, Stevens EH, Sltehel 12» Wiley DC 0002) H$ ayian;

Figure d shows a sequence of an alfalfa plastoeyanm-hased expression cassette used for the expression of HI in accordance with an embodiment of the present invention (SEQ ID NO:S). signal peptide is underlined, Bglll (ACATCT) and SacI (¢3 A13GTC) restriction sites used fer cloning are shown in hold;

Figure 4 shows a. representation of plasmid 660 assembled tor the expression of'w&fi type H A subtype H S Imm Afindonesia/S/OS;

Figure S shows a represeniation of plasmid 680 assembled for the expression of Unglycosylaied mutated HA subtype MS from A/lndonesia/S/05;:

Figure 6 shows antibody titers against Whole Inactivated ¥iruses (WIY) first and second dose; The reactivity of sera from rats imniimized wifo elfoef the wt VTF or the triple mutant AHT^oon-glyeosylated) was messed alter the first 04 days) or the second immunization (35 days), Ihnnunoreaetivlty xvas assessed against several HSMi viruses;

Figure 7 represents hemagginiinatteofinkihitiOU (HI) ardibody titers after first and second dose, HI titers from rats immunized with the wt or the triple mutant VLP (non-glycosylated) were assessed 14 days alter the first (Day14) orthe second (Day 35) immunization, hnmuooreaetiviiy was assessed agaiust several H5H1 viruses add one Hi Ml virus;

Figure t represents the sequence listinglor an luiuenxa HAO;

Figure 9 represents the sequence listing for anlniluenza HA protein subtype H2;

Figure if represents foe seqneuce listlhg for an Influenza HA protein subtype H3;

Figure 11 represents foe sequence listing for an Influenza HA protein subtype H4;

Figure 12 represents the sequence Msiing for an MfluenmHA protein subtype H5;

Figure 13 represents the sequence listing ter an infinenna MA protein subtype H6; 14 represents the sequence listing for an Influenza HA protein subtype H7; l^gare IS r^resents the sequence Esting for an Influenza HA protein subtype HS;

Figure 10 represents the sequence listing for an Influenza HA protein subtype H9;

Figure 17 represents the sequence listing for an influenza HA protein subtype H10;

Figure 18 represents foe sequence listing for an Influenza HA protein subtype HI I ;

Figure .1.9 represents the sequence listing for an Inifoenza HA protein suht)^ H12;

Figure 30 represents the sequence listing for an Influenza HA protein subtype HI 3;

Figure 31 represents the sequence listing for an Influenza HA protein subtype H14;

Figure 22 represents the sequence listing for sat Influenza HA protein stfoiype HIS;

Figure 23 represents the sequotce Mstiug for an foSueuza HA protein Subtype 1116;

Figure 24 represents the sequence listing for the 660 pCAMBIA expression vector containing the complete wild type HS sequence;

Figure 25 A~3 represent the sequence listings of primers Used for PGR amplification;

Figure 26 represents the sequence listing for the fragment produced, containing foe complete HS coding region including the native signal peptide flanked by a Hindi!! site immediately upstreanxoithe initial ATG, and a Sac! site immediately downstream of foe Stop (ΤΛΑ) codon;

Figure 27 represents fee sequence li sting for foe Segment produced^ containing the complete H5 eoding repon modified to remove all three glyoosylaflon sites, including the native signal peptide flanked hv a HindM site immediately upstream of foe initial ATG, and a Sad site imm^mtely downstream of the stop (TAA) codon;

Figure 28 AH> represent foe sequence listings for primers for FOR amplification.

Figure 29 represent amino acid sequence of mature H5 from strain ΑΛΊ einam/i 194/()4; and

Figwe 30 AriB txposenf the nudeie add and amino acid sequences respeedwly of mature H A from strain B / FlondaM/2CM)6.

DETAILED DESCRIPTION Of' FABTICULAR EMBODIMENTS

[0055] The present invention relates to-thei'jprddiacticm' of vmis-like particles CVLP),

More particularly: the present Invention Is directed to the prod uction of vi rus-like partides comprising influenza antigens.

[0056] Hie following description is of a particular embodiment. l·- HA protein [0057] As used herein, a "protein" refers generally to a string of amino acids connected by a peptide bond, which may be folded into secondary, tertiary or quaternary stmcture to achieve a particular ntojphology. Alternatively, the teams polypeptide, peptide or peptide fragments may be used in a similar context [0058] The term “htanaigfuimin domain” refers to a peptide comprismg dther tire HAO precursor polypeptide, or the HA1 and HA2 domains. The hemagglutinin domain does not include the signal peptide, transraemhrane domain, or the cytoplasmic tail found in the naturally occurring protein, [0059] With:reference to influenza vims, the term ^henui^hiiinin" or “HA” as used here# refers to a glycoprotein found on die outside of mduenxa viral particles. HA is a homotrimerie membrane type I glyeoprotein, generally comprising a si gnal peptide, an ΗΑΪ domain, and an HA2 domain comprising a membrane-spanning anchor site at the GL terminus and a small cytoplasmic tail (Figure IB);. Nucleotide sequences encoding HA are well known and are available “ set;, for example, the BioDefencePublic Health base (Influmrza Virus; see URL: hiohealtldiase.org) or National Center for Biotechnology information(see URL: ncblmlm-hih,gdv), both Of -which are incoiporated herein By reference.

Stmeiurai influenza HAs [OOiOJ The HA monomer can he subdivided in 2 -distant functional domains, fee globular head domain and the stem domain. The correspondence of these domains between the primary sequence and the structure of HA is illustrated at Figures IB and I, The stem of fee vims via fee extraordinary conformational change it can perforns at acidic pH, It is further described as 4 subdomains, fee fusion peptide (hydrophobic stretch of 2b amino acids responsible for fosion wife fee host membrane in fee fow«pH conformational stale); the stem domain, itself' {that can accommodate 2 extremely diifofept conformations}, fee transmembrane domain (Tmf>) (dmermine fee afSnity of HA for lipid rafts) fee cytoplasmic taii( Ctaft) (is involved in secretion of HA). bmdi»g'i^5;d0maift:and:fee''yestigial esterase domain (E). The esterase subdomain is rather buried #om fee surface of the protein and therefore the mfeority of antibodies raised against HA binds to the receptor binding domain (represented by the uppermost part of the head in Figure 2).

[00§i] The term Htomotrinieff or “bomohimeric^ indieates that an oligomer is formed by feree HA pretdn molecules. HA protein is spdhesis'ed as a 75 iePa monomeric precursor protein (ΗΑ0), which assembles at fee surface info an elongated trimeric protein. For highly pathogenic avian strains, fee precursor protein is cleaved intraeeMariy at a conserved activation cleavage site (also referred to as i^ion peptide) info chains, HAI (328 amino acids) and HA2 (221 ammo acids; edmprising fee transmembrane region), linked by a disulfide bond before trirnerixation oecnrs. Although this step is central fofvirdsl^ essential for the trirnerfoatfon of fee protein, For mammalian and apathogenle avian influenza virus stmins, the precursor ΗΑ0 is cleaved exiracdlulariy by proteases secreted by cells of fee msplrafory tract of fee host, or by eoTnfecting bacteria or mycoplasma. Insertion of HA within fee endoplasmic reticulum (HR) membrane of the host cell, signal peptide cleavage and protein giycosylafton are coAranslationa! events, Correct of fee protein and formation of 6 intra-chain disnlftde bonds. The HA trimer assembles within fee pis- and trans-Clolgl complex, fee transmembrane domain playing a role ip fee trimerization process. The crystal structures of bromelain-treated HA proteins, which laek the transmembrane domain, have shown a. highly conserved structure amongst influenza strains. It has also been established that HA undergoes tnejor conformational changes during the infection process, which requires the precursor HAD to he cleaved into the 2 polypeptide chains jMl and HA2. The HA protein may he processed (i.c,, comprise HAi and HA2 domains), or may he unprocessed (ie. comprise the HAG domain):, [0062] The present invention pertains to the use of an HA prof ein comprising the trsnsmenibrane domain and includes HAI and HA2 domains, for example the HA protein may he HAD, or processed HA comprising HAI and MA2.

[0063] The HA of the presem Invention may be obtained from any subtype. For example, the HA mayH3, H4, H5, H6, H7, 1i8,H9,H10, Hlj, HI2, Η13,ΗΙ4;Η15, or Hi6. )0064] The present invention includes ATJP’s comprising HA having modified N~glyean$, The recombinant HA of the present invention may also comprise an amino acid sequence based on the sequence any hemagglutinin known in the art- see, for example^ the BioOefencc Public Health base (IntluenmMrus; see Mohealfebase-org) or National Center for Biotechnology Information (see HRl; ndii.nlininih.gov) wherein the native N-linked glyeosylation sites have been removedAnutatedAleleted/modifted to rempve/the sugar residues that mask, the peptidtc antigenic sites.

[0065] Furthermore, the HA may be based on the seguence of a hemagglutinin that is isolated from one or more emerging or newlyridsntiBed influenza viruses,

[0066] Furthermore, YLPs 'may he produced that comprise a combination of HA subtypes, For example^AfUPs may comprise one of more than one HA from the: subtype HI, H2,B3S H4, HS, H6, H7,B8, H9,.1410, HI 1, H12, HI3, HI4, H15, H16,or a combination thereof. Selection. of the combination of HAs may be determined by the intended use of the vaccine prepared from the V LP. For example, a vaccine for use in inoouiatlng birds may: comprise any combination of H A subtypes, while YLPs useful for inoculating humans may comprise subtypes lorn one or more than one of subtypes H I, 142, M3 or M5, combinations may be prepared depending

Upon the use of feeVLP, In order to produce VLPs comprising combinations of HA sdbt^p^v^rdi^«d:MA^nb^pe may fee ee-expressed within the same cell, for example a plant ceil.

[OOfe?] PariiaiWyi VLPs produced m desenhed herein do sot comprise neuraminidase (NA)· However; NA may be eo-expressed wife HA should VLPs edmprising HA and NA fee desired, 2- Fiu subtypes [0068]The Invention Includes all types of human influenza vims, including for example, but not limited to fee very prevalent A safe-types, and the less common B type, and € type, and to MAs ©fetained feom other influenza subtypes.

[096^1 The pmsent inv^tion else ineMIes VLPs feat comprise HAs obtained from one or more than one influenza subtype. For example VLPs may comprise one or more than, one HA feom the subtype HI (encoded fey SEQTP MO:1), H2 (encoded fey SEQ ID N0:2), H3 (encoded by SEQ IS N0:3), H4 (encoded by SEQ IS NG:4>, H5 (encoded fey SEQ ID MO;5), H6 (encoded by SEQ ID NO;6), H7 (encoded: by SEQ ID NO:?), H8 (encoded by SEQ ID NO'S), H9 (encoded fey SEQ ID N0:9), HI 0 (eneoded fey SEQ ID HOtltfe HI ! (encoded by SEQ ID NO: 11), HI2 (eneoded fey SEQ ID MO: I2), HI3 (encoded fey SEQ ID NO: 13), HI4 (encoded by SEQ IS NO: 14), HI5 (encoded by SEQ ID NO::1:5),, Η16 (encoded by SEQ IP NO: 16), or a combination thereof One of more that one HA from .the one or more fend one influenza subtypes may be co-expressed within; a plant or i nsect eel), Id ehsure that the swthesis of fee one of more fearrohe HA results in the formation of VLPs comprising a combination of HAs obtained irom one: or more than one influenza snhtype. Selection of fee eomfeinatlon of HAs may fee determined fey fee Mended use of the vaccine prepared from fee VLP, For example a vaccine for Use in moculating humans may Mmprise any comfemation of HA sifelypes, particularly, one or mom than one of subtypes HI , H2> BQ, H5, H7, H9, H10, Nl , N2, N3 and N7, Particularly, Hi, H2, H3, H5, [0070] However, other HA subtype combinations may be prepared depending upon the use of the isoeulup- 3 - Method of production [0071] a method of producing virus like particles (¥LPs) iu a host, themiibre, the invention provides for VIPs, and a method for preduemg viral VLBs ina host expression system, from the expression of a stogie envelope ptoteim 3lie method in vol ves imrod«ci3% a nnefeiie aeid encoding an antigen Opet^vefy linked to a regulatory region active in the host or a portion thereof] and inenfeafrng the host or a portion of the host under condi tions that permit the expression of die nucleic acid, thereby producing the VLPs. 10072] The regulatoiY elements of the present invention may also be combined with coding region of inter^t lor expression Within a range of host organisms that ate amenable to transformation, or transient expression, such as particularly...plant, insect or yeast, [0073] Pardeuiatiy, such organism are plants, froth monocots and dleots, for example hut not limited to com, cereal plants, wheat, barley, 'Swmice spp> soybean, hears, pea, alfalfa, potato, tomato, ginseng, and Arabldopsis.

[0074] lytefhods for stable trafrsfoimaiiors, and regeneration of these organisms are established in the art aid known to one of sMl in the art. The methods of obtaining transformed arid regenerated plants are also well known in the art, [0075] By ^^sformatfonT it is meant the stable interspecific transfer of genetic ihfbnnation (nucleotide sequence) that .is manifested ^notyplealiy, phenotypiealiy or bofrr The interspecific transfer of genetic information from a chimeric construct to a host may he beri table and the transfer of genetic information considered stable, or the transfer may be transient and the; transfer of genetic information is not inheritable.

[0070] Transient expression methods may Be used to express the cons tructs of the preseat Invention (see Liu and Lomonossoff, 1002, journalof Vlrological Methods, 10S;343bj4k; which is Incorporated herein bymforenceh Alternatively, a vacuomfoased ffoosient expression method, as described by Kapila et al 199? (incorporated herein by reforcuee) may be used. These methods may include,, for example, but are not limited to, a method of Agro-inoculation or Agrodnftliration, however, other transient methods may also be used as noted above, With either Agio-inoculation or Agro-infiltration, a mixture of A|robacteria comprising foe desired sueleic add enter the intercellular spaces of a ti ssue, for example the leaves, aerial portion of the plant fmcl udiag std»,; leaves and flo wer) , other portion of foe plant (stem, root, floworj, or foe whole plant. After crossing the epidermis, the Agrobaeteriuin infeed and transfer i-DN A copies into the cells. The t-DNA is episomatiy transcribed and the mRBa translated, leading to the production of the protein of interest in infected cells, however, the passage of ΐ-DN A inside the nucleus is transient, 4- Host organ!sin [0077] ITre VLPs of foe present mvemioixmay be produced ip a host cell that is characterized by laehing foe ability to sialylate proteins, for example lacking siahdase, such as a plant cell, an inseet ceil, fungi, and other organisms including sponge, eoelenterara, ahnelida, arthmppoda, motlusca, nemathelinintfeea, troebehnintes, plafoelminthes, ehaetognatha, tentaculate, chlamydia, spinoehetes, gram-positive bacteria, ^i^obacteria, as identified in glycofonim (see, for example, the fJMc gij^fonmt.grjp/sdene^^rd/evolutionfES-AOBE.btml}.

[0078] The VLPs produced as described heroin do not typically comprise neuraminidase (NA). However, jSLA may be c»<-expressed with HA should VLPs comprising HA and NA be desired.

[0079] Particularly, the ¥LPs of the present invention may be· produced: m plant cells, a whole plant or portions thereof such as leaf, seeds, or any Other plant matter.

[0080] By foe term “plant matter\ it is meant any material derived from a plant. Plant matter may comprise an enfrre plant, tissue, cells, or any fraction thereof Further, plant matter may comprise Intracellular plant components, extracellular plant components, liquid or solid extracts of plants, or a coirfoination thereof, further, plant matter may comprise plasts, plant iceiis^ ^ssoe, aliquid extract, or a combination thereof pom plant leaves, stems, dowers, frod, roots or a comMmtion tlmreof. Plant isatter rsay comprise a plant or portico thereof which has not Peers subjected to any processing steps. However, it is also may be subjected to minimal processing steps as defined below, dr more rigorous processing, including partial or substantial pjptein pbrificafion using techniques commonly; known wi&amp;jh'lbe art limited to ehramatography, electrophoresis and the like.

[0081 ] By the term ''minimal processing’* it is meant plant matter, for example, a plant or portion thereof comprising aprotein of interest which is pinfially purified to yield a plant extract, homogenate, traction of plant homogenate or the like (ie, minimally processed!. Partial purification may comprise, but is not limited to disrupting plant eeilnlar structures thereby creating a composition comprising sohfole insoluble plant components which may be separated for example, but not iindted^ .by:t^tri&amp;^3hiDiti: filtration or a combination thereof In this regard, proteins secreted within the extracellular space of leaf or other tissues could be readily: obtained using vacuum or centrifugal extraction, dr tissues could he extracted under pressure by passage through rollers or grinding or the like to squeeze or liberate the protein free from within Jte extracellular space. Minimal proeesslog could also involve preparation of crude extracts of soluble proteins, since these preparations would have negligible contamination from Secondary plant products,Further, minima! processing may invdlye aqueous extraction of soluble protein from leaves, followed by precipitation with any suitable salt. Other methods may 'include·'isprge scale maceration and: juice extraction in order to permit the direct use of the extract.

[0082] The plaint matter, in the form of plant material or tissue may be orally deli vered to a subject. The plant matter may be administered as part of a dietary supplemerit, along with other foods, or encapsulated, The plant matter or tissue may also he concentrated to improve Of increase palatabdity, or provided along with other materials, ingredients, or pharmaceutical excipients, as required, [0083] Also consideredpari of this mveaioii are tmnsgeme plants, plant cells or seeds containing present invention. Methods of regenerating whole plants tom plant cells are also known in the art. In general, transformed plant cells are cultured which may contain selectiveagents such-as antibiotics, where selectable markers ate used to facilitate identifeation of transformed plant cells. Once eallbs forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and die shoots transferred to footing medium for regeneration of plants. The plants may then be used to establish repetitive generations, either from seeds er asing vegetative propagation teehnkjues. Transgenic plants can also he generated without using tissue cultures.

[0084] Also considered part of this invention are fransgenie plants, trees, yeast, bacteria, fungi, insect and animal cells eomaiPing the elntnmie gene construct comprising a nucleic acid encoding recombinant HA® for VLP production, in accordance with the present indention.

[ObS S] It i s contemplated that a plant comprising the grotem of interest, or expressing the VLP comprising the protein of interest may be administered to a subject or target organism, in a variety of ways depMdmg upon the need and the situation. Fen examples the protein of interest obtained fern the plant may be extracted prior to its use m either a crude, partially pimfied, or punried form. If the protein is to be purified, then it may be produced in either edible or non-edible plants, furthermore, if the prmein is orally administered, the pl ant tissue may be harvested and directly feed to the subject,- or the harvested tissue may be dried prior to feeding, or an animal may be permitted to graxe on the plant wi th no prior hafoesf taking place, It is also considered vrithin the scope of this invention for the harvested plant tissues to be provided as a food supplement within animal iced. If the plant tissue is being feed to an animat with little or not further processing S is preferred that tire plant tissue heiug administered is edible, [008b] Post-inmseriptienal -gene siltmeing (PTOS) may fee invol ved in limiting expression of tratisgenes in plants, and cu-expressioh of a suppressor of silencing from the potato virus Y (HePro) may be used to counteract the specific degradation of transgeoe mRNAs (Brigneti et&amp;l, 1998), Alternate suppressors of silencing are well known in the art and may he used as described herein (Chiba ei at., 2006,. Virology 346:7-14; which is incorporated herein by mierence| &amp; example hot not limited to, TBV -plAiC-Pro (Tobacco etch vjrtis*|jl/BC-Pro), EYY ~p2I, pl9 of Tomato bushy StimiVirps (TBS V pi 9), capsid protein of Tomato crinkle vires (TOY ~CF)S 2b of Cucumber mosaic : virus; CMV-2h), p25 of Potato: virus X iPVX~p25), pi 1 of Potato virus M (PVM-pl 1), pi I of Potato vims S (FYS-pll), p!6 of Blueberry scorch virus, (BScY -~pl6), p23 of Citrus tristexa virus (CW~p23), p24 of Ompevineleaffeil-assoeiaied virus-2, {GLRaY-2: p24), p! 0 of Grapevine virus A* (GVA-plO), p:14 of'Grapevine vims BfGVB~pi4)s plO of Hemcleum latent viras;(BLV-'piO),: or pi 6 of Garlic common latent virus (GCLV-pl6), Therefore, a suppressor of ^silencing, tor example, but not limiter! to, HePro, THY ~ pVSCLPro, BYY~p21, TESY $19, TCV-CP, CMV~2b, PYX-p2S, PVM-pi 1, PVS-pU, BSbY~pl6, €TV~p23, GLRaV-2 p24, GBV~pI4> HLV-pIO, GCLY-plb or GVA-plO* may he eo-eapressed along with the nucleic -acid'sequence encoding the protein .of-interest to further ensure high levels of protein production within a plant, [008¾ VLPs of enveloped viruses generally acquire their envelope from die membrane they hud through. Plant plasma membranes have a phytosierol complement that may have knmtumsdmulatory effects» To Investigate this possibility, plant-made HS VLPs mem admimstered to animals in the presence of an adjuvant,, amd the HAI (hemagglutinatton inhibition antibody response) determined '(Figure ?}.

[0088] The production of VLPs in plants presents several advantages o ver the produetibn of these particles in insect cell culture. Plant lipids can stimulate specific immune cells and enhance tbp immune response induced. Plant membranes are made of lipids, phosphatidylcholine (PC) and phosphatidylcthanolamine (PE), and also contain giycosphmgolipids that are unique to plants and some bacteria and protozoa, Sphingoliplds are unusudl in that they are not esters of glycerol like PC or PE hut rather consist of a long chain amino alcohol that forms an amide linkage to a fatty acid chain containing more than I S carbons. PC and PE as well as gSycesphingolipids can bind to GDI molecules expressed by mammalian immune cells such as antigen-presenting cells (APCs) like denmtic cells and macrophages and other cells including E and "Γ lymphocytes in the thymus and liver (Tsuii M,, :2006).: Furthermore, in addition to the potential adjuvant effect of the presence of plant lipids, the ability of plant N-glycans to facilitate the capture of glycoprotein antigens fey antigen presenting cells (SainfLtore-Degas, 2007), may fee advantageous of the pRxluetieh ofVLFs.;m,pia«ts. f00S9| Without wishing to feebound fey theory, it will mince &amp; stronger immune maetion than VLPs made in other femduetioh/mamdmihmng systems and that the immune reactioninduced fey these plant* made YLPs will fee stronger when compared to the hmmme reaction induced fey live or attenuated whole virus vaccines, [0090] Contrary m vaeeines tnade of whole viruses, YLPs provide die advantage as they arenon-infeoiious, thus restrictive biological containment is not as significant an issue as it would fee working with a whole, infectious vims, and is not required tor production, Plant-made YLPs provide a further advantage again fey allowing the expression system to fee grown in a greenhouse or field, thus feeing significantly mere economical and suitable for scale-up. (0091] Additionally, plants do not comprise the enzymes involved in synthesizing and adding sialic acid residues to proteins, YLPs may be produced in the absence of neuptninidase (NA),::and there is feu used to co-express KA, or to treat the producing cells or extract with siaiidase (neuraminidase), to ensure YLP production in plants.

[1)092] Particularly the YLPs produced m accordance with the present invention do not comprise Ml protein which is known to hind RNA, RNA is a contaminant of the YLP preparation aid is midesired when obtaining regulatory approval for the VLP product for use as a human vaccine. 5- nucleic adds [0093] The present invention provides a nucleic acid comprising a nucleotide sequence (HA) antigen, operatively linked to a regulatory region active in a non-siai>iating host organism.

[0094] The present invention describes, but is not limited to, the cloning of a nucleic acid encoding HA, for example but not limited to, a human influenza A virus HA into a host the podaetipp,ofinfiuenza'VLF$^m':3iebo$t> suitable tor vaccine production. The VLPs may also be used to produce reagents composed of :teeomBn^Ti«itt^K8a. structural proteins that self-assemble into functional and itomunogenie homotypic maeromoieeular protein structures, including sub vi ral influenza p^eies and inSuenea ¥tP, in transformed hosts ceils, tor example plant cells or insect cells,· [0095] The present invention also includes nucleotide sequences HI (eneoded by SEQ10 MO:I), H2 (encoded by SEQ ID NO:2), H3 (encodedby SEQ ID N0;3), H4 (encoded by SEQ IDN©:4), H5 (encoded by SEQ 113 NO:5), H6 (encoded by SEQ ID NO:6), M7 (encoded by SEQ ID $0:7), H8 (encoded by SEQ ID $0:8:), H9 (encoded by SEQ ID NO:9), H10 (encoded by SEQ ID NO: 10), Ml 1 (encoded by SEQ ID NO: 11), HI2 (encoded by SEQ ID NO; 12), HI3 (encoded by SEQ ID NO: 13), HI 4 (encoded by SEQ ID NO: 14), Μ15 (encoded by SEQ ID NOT5), and Hid (encoded by SEQ ID NO:!6).

[009b] Partionlarly, the present invention loclndes nucleotide sequences SEQ ID $0:1; SEQ ID NO: 5; SEQ ID NO:7 encoding HA from HI, MS or H7 fespeetiveiy; a micfeotide sequeuce SEQ ID NO: 1; SEQ ID MO;5; SEQ ID $0:7, that hybridizes under stringent hybridisation conditious to a uueleic acid that encodes the HA from HI, H5 or H7, respecbvaly; or a nbcleotide seqnence SEQ ID NOT; SEQ ID NOrS; SEQ ID NO:7, that hybridizes under stringent -hybridization conditions to a complement of a nucleic acid encoding the MA^irem HI , H5 or H7 respectively; wherein the nucleotide sequence encodes a hemagglutinin protein that when expressed torms a 17TP, and that the VLP induces the production of an antibody. For exaniple, expression of the nucleotide sequence within a host cell tonus a VLP, and the VLP may he used to produce an antibody that is capable of binding HA, including mature HA, HAO, HA1, or I1A2. The VLP, when administered to a subject. Induces an hnmune response.

[ 0097] Hybridization under stnngent hybridization conditions are known in the art (sec tor example Carrent Protocols in Molecular Biology, Ansuhel &amp; alf eds, 3995 and '•supplements; MmMis et of, in Molecular Cloning (A Laboratory Manual), Colei Spring Harbor Ufooratory, 1982; Ssmbrook and. Russell, in Molecular Cloning; A Lahqi$tq:ry Manual, 3rd edition 2001; each of whsch is incorporated herein by rderence). An example of one such may he about 16-20 hours hybridization in 4 A SSC at 6$°C, followed by washing in 0,1 X SSC at 65¾ for an hour; or 2 washes in OH X SSC at 65°C each for 20dr30 mlmttes, Alternati vely, an exemplary stringent hybridization condition could be overnight (16-20 hoursfin 50% forinamide, 4 X SSC at 42X1, followed by washing in 0,1 X SBC at 65¾ for an hour, or 2 wMhes in 0,1 X SSC at 65¾ each for 20 or 30 minutes, or overnight (16-20 hours), or hybridization in Church aqueous phosphate butler (7% SDS; 10 mM EITFA) at 65X2, with 2 washes either at 50¾ in Q J X SSC,-0,-1%. SDS tor 20 or 30 minutes each, or 2 washes at 65¾ in 2 X SSC, 0,1% SDS tor 20 or 30 minutes each.

[0098] Additionally; the present invention includes nucleotide sequences that are characterised as having about 70,75,80, 85, 87,90,91,92, 93 94,95,96,97,98,99, 100% or any amount therebetween, sequence identity, or sequence similarity, with the nucleotide sequence encoding HA horn HI (SBQ IDMO’I), HS (SEQ119 MO: 5) or E? (SEC) ID MD: 7), wherein the nucleotide sequence encodes a hemagglutinin protein that when expressed forins a VLP, and that the VLP induces the production of an antibody. For example, expression of the micleotide sequence within a plant cell forms a VLP, and Sue Vl.P may be used to produce an antibody that is capable of binding HA, including mature HA, If A.0, HA!, or HA2* The VLP, when administered to a subject, induces an immune response.

[0099] Sequence identity or sequence similarity may be determined usi ng a nucleotide sequence comparison program, such as that provided within DMAS1S (for example, using, but not limdud to, the following parameters; OAF penalty 5, fof top diagonals 5, fixed OAF penalty 10, k-fople 2, floating gap 10, and window size 5). However, other methods of aligumeht of sequences: for comparison are well-known in trie art for example foe algorithms of Smith &amp; Waterman (1981, Adv, Appi Math. 2:482), Xeedleraan &amp; Wunseh (J, Mol, Biol. 48:443,1970), Pearson &amp; Lipman (1988, Proc. Natl. Acad, Set, USA 85:2444), and by computerized implementations of these algorithms (e.g, GAP, BESTP1T, PASTA, and BLAST)., or by manual alignment and visual: inspection, [00100] Therefore, the present invention further includes a suitable vector comprising the cMmeric construct suitable for use with either stable or transient expression systems. The genetic information may be also provided within one or more than one construct.

For example, a mml cofide sequence encoding a protein of interest may be introduced in one construct, and a second nucleotide sequence encoding a protein that modi ties glycosylation of the protein of interest may be introduced using a separate construct These nucleotide sequences muy then be eo-expressed within a host. However, a construct comprising a nucleotide sequence encoding both the protein of interest and the protein that modifies glycosyi atfon profile of the protein of interest may^also he used, In tills ease the nucleotide sequence would comprise a first sequence comprising a first nucleic acid sequence encoding the protein of interest operatively linked to a promoter or regulatory region, and a second sequence comprising a second nucleic acid sequence encoding th e protein that modifies th e glycosy fatten profile of the protein of interest, the second sequence operafi vely liithed to a promoter of regulatory region.

[00101] By “eo-expressed” it is meant thattwo, dr more than two, nucleotide sequences are expressed at about the apd within the sente tissue of the host. However, the expressed at exactly the same time,

Rather, the two or more nucleotide sequences are expressed in a manfier such that the encoded products have a chance to interact. For example, the protein that modifies glyeosylafion of the protein of interest may be expressed either before or during the period when the protein of interest is expressed so that modification of the glyeosylafion of the protein of interest takes place. The two or more than two nucleotide sequences cap he co~expressed using a translent expression system, where the two or more· sequences are introduced within the host at about the same time under conditions that both sequences are expressed . Alternatively, a platform host comprising one of the nUcleofide sequences, for example the sequence encoding the protein that modifies the ^ycosylaiion profile of: the protein of interest, may he: trausfanned, either transiently or in a stable manner, with an additional sequence encoding the protein of impest, in thi s case, the sequence: eroding the proton that modifies the glyposylation profile of the protein of interest may he expressed within a desired tissue, during a desired stage of development, or Its expression may fee mdeeed^ and the additional sequence encoding the protein of inierest may be expressed under similar conditions and in the same tissue, to ^«^-β^ίί,^'ΐιβοΐβ^δο sequences am eo-expressed, 100102] The eonstmots of the present invention can be introduced into plant cells using 11 plashiids, Ri plasmids, plant vims vectors, direct DNA. transformation, micro-injection, electroporation, etc. For reviews of such techniques see f or example Weisshaeh and Weisshaeh, Methods for Plant Molecular Biology, Academy Press» New York ¥111» pp. 421-463--(1 OSS); Geierson and Corey, Plant Molecular Biology, 2d Ed. (1088); and Mlki and Iyer, Fundamentals of Gene Transfer in Plants, la Plant Metaholisuy 2d Ed, IYF, Dennis, OH Turpin, DD Lefehwe. DB Eaj^ell (eds), Addison Wesly, hangmans' Ltd. Eondon, pp. 561-579 (1997). Other methods include direct DNA 'uptake, the use of liposomes, electroporation, tor example using protoplasts, micro-injeetion, miofoprojeetfles or whiskers, and vacuum mdimatton. See, for example,

Silang, ei al (Gene 1.00:247-250 (1991), Scheid ei αί, (Mol, Gen. Genet. 228:104-112, 1991), Guerehe ef # (Plant Science 52: 111-116» 1987), Neuhause ei αί (Theor. Appl Genet. 75: 30-36, 1987), Klein cf a/., Nature327::70-73 (1987)»Howell eial. (Science; 208: 1265, 1980), Horschetm. (Science 227: 1229-1231.1985), DeBlock e: a/., .Plant Physiology 91: 694-701,1989), Methods for Plant Molecular Biology (Weisshaeh and Weisshaeh, edsTS Academic Press;Ino,, 1988), Methods in Plant Molecular Biology (Schuler and Eielinski, eds., Academic Press Me,» 1989), Liu and Lomonossoif (3. Virol Meth, 105:343448,2002,), US. Pat. Nos. 4,945,050):5,036,0065000 5,100,792» US. patient application Ser, Nos. 08/438,666) filed May 10,1995, and 07/951,715, filed Sep. 25, 1902, (all of which are hereby incorporated by reference), (00103] By ‘hegdlafory region” ^regulatory element” or ''promoter” it is meant a portion of nucleic acid typically», hot not always, upstream of dmprotein coding region of a gene, which may he comprised of either f>N A or RNA, or both DNA and RNA. When a regulatory region is acti ve, arm in opemtive assoeiation, or operatively linked, with a gene of interest, this may result in expression of the gene of interest. A foipilafoiy element may be capable of mediating organ ^ecifiipityv:qfr:^»frt)lHiig developmental or temporal gene activation. A Regulatory region” meludes promoter elements, com promoter elements exhibiting a basal promoter actmtv, elements that are inducible in response to an external stiordns, elements thatmediafe pronmier aePvsty SPelr as negative regulatory elements or transcriptional enhancers, ‘'Regulatory region \ as used herein, also includes elements that are active following transcription, for example, regulatory elements that nipdulate: gene expression such as translational and transcriptional enhancers, translational and transcriptional repressors, upstream activadng sequences, and mR.NA instability determinants. Several the coding region.

[pQf 04} In the context of this disclosure, the term “regulatory element" or ‘Regulatory regio# typically refers to a sequence of phi A, usually, hat not always, upstream (5?) to th e coding sequence Of a structural pn% which controls the expression of the coding region by providing the recognition slur RH A polymerase and/wA^^te'^td-r^liot' transcription to start at a particular site; However, it is to he understood that other nucleotide sequences, located within introns, or the regulation of expression of a coding region of interest, -As example of a regulatory element that provides for the recognition tor RNA polymerase or other transcriptional factors to ensure initiation at a particular site is a pmnmter element. Most, hut not all, eukaryotic promoter elements contain a TATA box, a conserved nucleic acid sequence comprised of adenosine and thymidine nucleotide base pairs usually situated approxi m ately 2§ base pairs upstream of a transcriptional start site. A promoter element comprises a basal promoter element, responsible for the initiation of transcription, as well as other regulatory elements (as listed above) that modify gene expression, !®3 Qd] There are several types of regoiatory regions, including those that: are development ally regulated, inducible or constitutive, A regulatory region that is developmentally regulated, or controls the differential expression of a gene under its control, is acti vated within certain organs or tissues Of an organ at speci fic times during the development of that organ or tissue, However, some regulatory regions that arc developmentally regulated may preferentially he active within certain organs or tissues at specific ^ piay also be active I» a devefepmentally reflated maimer, or at a basal level is other organs or tissues within the plant as well, Examples of tissue-specific regulatory regions, for example see-specific a regulatory region, include fheuapin promoter, and the cruet ferin promoter (Rask et of, 1998,1. Plant Physiol. 152: 595-599; Bilodeau #oi, 1994, Plant Cell 14; 125-130). An example of a leaf-specific promoter includes the (Figure 3; 0S 7,125,978, which is incorporated herein by refereaee}.

[00106] An inducible regulatory region is one that is enable of directly or indirectly activating transcription of one or more to an inducer- in the absence of an inducer the Obi A sequences or genes will not he transcribed. Typically tee protein factor that binds specifically to an inducible regulatory region to activate transeripion may be present in an inactive form, which is then directly or indirectly converted to the active form by the inducer, Howeyer, the protein factor may also be absent. The inducer can he a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenol to compound or a |teysipiegieai stress imposed directly by heat, cold, salt, or toxic elements or indirectly through the aefion of a pathogen or disease agent such as a vims. A plant cell containing an inducible regulatory region may be. exposed to an inducer by externally applying the inducer to the eelf or plain such as by spraying, watering, heating or similar methods. Inducible regulatory elements may be derived irons either plant or non-plant genes (e.g. Gate, €, and Leak, 1998, Trends Plant Sci 3,352-358; which is incorporated by reference). Example, of potential inducible promoters include, btente limited to, ietraeyeltne-indiioiMe promoter (Gate, C.,1997, Ann, Rev. plate Physiol. Plate Mol Biol, 48, 89-108; which is incorporated by relerencte, steroid indueible pmmoter (Aoyama, T, and Choa, H.H., 1997, Plant i. 2, 397-404; which is incorporated by reference) and ethanol-inducible promoter (Salter, MG., et al, 1998, Plate Journal 16,127-132; Caddiek; MX, et ai,1998; Nature Biotech, 16,177-180, which ape inco^orated by reference) sytokinin indueible 1B6 and CKO genes (Brandstatter, I. andKieber, 1,1.,1998, Plate Cell 10,1009-1019; Kakimoto, T.., 1996, Science 274,982-985; which are incorporated by reference) and the auxin inducible element, DR5 (yibiasov, Plant Cell 9, 1963-197!; which is incorporated fey reference), [OOIOTIJ A constitutive regulatory region directs the expression of a gene throughout the various parts of a plant and eQ)ft^uo«dy':thmw^0u|,pli»t development. Examples of known:· .constitutive regulator)'' elements include promoters associated with the GaMV 35S transcript, [Oddi et of, 1985, Nature, 313: 810-812), the rice actiii 1 fZhang#aT 1991, Plant Cell, 3:11554165), aetm 2 (An et«/., 1996, Plant I, 10:107-121¾ or tms 2 (U.S. 5,428,147, which is incorporated herein by reference), and triosephosphate isqmerase 1 (Xu et, at, 1994, Plant Physiol, 106:: 459d67) genes, the maize obiquitm 1 gene (Coraejo et al, 1993, Plant Mol. Biol, 29: 637-646), the Ai^idopsisnbl^uitin 1 and 6 genes (Boltorf et al, 1995, Plant Mol, Biol. 29: 637-646), and the tobacco tmuslatioosl Initiation factor 4A gene (Mandel et al, 1995 Plant Mol, Biol, 29:9954904), The term "constitutive·* as used herein does not necessarily indicate that a gene under control of the constitutive regulatory region is expmssed af the same level in all cell t ypes, hut that the gene is expressed in a wide range of cell types even thoiqih variation in abundance is often observed, [00108] By "operarively linked" it is meant that the particular sequences, lor example a regulatory element and a coding region of interest, interact either directly or indirectly to carry out an intended function, such as mediation or mpdniation of gene expression. The interaction of operatively linked sequences may, for example, be mediated by proteins that interact with the operatively linked sequences, [00109] The oue or more than one nucleotide sequence of the present invention may be expressed in any suitable plant host that is hansftmnted by the nucleotide sequ^tee, or constructs, or vectors of the present invention. Examples of suitable hosts include, but are not 1 imbed to, agricultural crops including alfalfa, canola, Brassier* spp.; maize. Nicotians spp., alfalfa, potato, ginseng, pea, oat, rice, soybean, wheat, barley, sunflower, cotton and the like.

[991 10] The one or more ebimerie gfiaietic constructs of the present invention can further comprise a 3 s untranslated region. A y untranslated region refers to that portion of a gene comprising a DNA sega^.iiiat contains a poiyadenylation signal and any other regulatory signals capable of effserfing mEMA processing or gene expression. The poiyadenylation signal is osoaliy characterised by effecting the addition of poly adenylic acid irachs to the 3' end of the mRNA precursor, Poiyadenylation signals are ammoniy reeogiibed hy the presence of hpinolo^· to tire canonical form 5;ΐ AATAAA4T although variations arc no t uncommon. One or more of the chimeric genetic constructs of the present invention can also include further enhancers, either translation or transcriptinh enhancers, as may he repaired. These enhancer regions are well known to persons skilled in the art, and can include the ATG initiation codon and adjacent sequences. The initiation codon must he in phase wi th fee reading frame of the coding sequence to ensure translation of the entire sequence, [001111 Nonriipiting examples of suitable 3* regions are the 3* transcribed horn translated regions containing a poiyadenylation signed of Agrobacterium tumor inducing (Ti) plasmid genes, sueb au the nopaline synthase (Nos gene) and plant genes such as fee soybean storage protein genes, the small subunit of fee rihulose^T Sfeisphosphate caffibxyiase (ssROBISCO; GS 4,962,028; which is incorporated herein by reference^ gene, the promoter' used in regulating plastocyanin expression (Pwee and Gray 1993; which is incorporated herein by rdference), An exainpiepf a piastocyanin promoter is described in US 7425^8.{wM^mim»tport»t^he®s^. fry nefemoe); [001 12j As described herein, promoters comprising enhancer sequences wife demonstrated efficiency in leaf expression, have been found to he ef^tive in transient expression. Without washing to he hound by theory, aitac&amp;nehi of upstream regulatory elements of a photosyntbetie gene by attachment to fee nuclear matrix may mediate strong expression. For example up to -784 from the translation start sits of the pea plasiocyaain gene may he used mediate stmugfeporter gene expression, [00113 j The use Uf a regulatory region horn a photosyntheti c gene, tor example hut not limited to&amp;pl&amp;stoeyanm regulatoryregion (US 7,125,978; which is ineorporated herein hy'.befeheiebh}#diegulafp^if^piii.bbtalhbd.fromRibulose 1,5-bispbosphate cafboxylaseroxygenase (RuBisGG; US 4^962,028; which is incorporated herein by tefefcei&amp;f),; chlomj&amp;ylla?b binding protein (GAB; I9rofyhler#a; 1986; which is -incorporated· herein by reference), ST-ES1 .{associated with the oxygm-e valving complex of photosystem II, Stockhaus mM 1989; wfci eh is incorporated herein by reference) may be used in accordance with the present invention.

[(H) 11 #} To aid in identi fieation of transformed plant ceils, the constructs of this InventtOB tnay be further manipulated to include plant selectable markers. Useful selectable markers include enzymes that provide for resistance to chemicals such as an antibiotic lot example, gentamyem, hygromyeln, kanamycin, or herbicides such as phosphinothtycin, glyphosate, chloroMlfuron, and tire like. Similarly» enzymes providing for production of a compound identifiable by colour change such as GUS (beta-ghmuromdas% or luminescence, such as luciferase or OFF, may be used.

[0011§j The resulti ng cDM A copies of these genes m ay be cloned in a suitable expression vector as required by the host expression syiamn. Examples Of apfuepriaie expression vectors for plants are described below, alternatively, baculovirus expression vector, for ex ample, pFastBael (InVhtogeo), resulting in pFastBael-based plasmids» using known methods, add information provided by the mahufoetmers instructions nay be used, [00! 16] The present invention is foriher directed to a gene construct comprising a nuefoic acid''#p6j|l»g;ht^:#desmbfed above, operatively linked to a rcplaforyfoiement that is operative in a plant. Examples of regulatory elements operati ve in. a plant cell and that may be used in accordance with the present invention include but arc dot limited to a plasfocyanhi regulatory region (US 7,125,978; which is Incorporated herein by reference), or a regulatory region obtained from Rihulosc 1,5-bisphosphate carboxyl asefoxygenase (RuBisCO; US 4,962,028; which is ineopporatedherein by reference), chlorophyll a/b binding protein (CAB; Leuiwilsr et al; 1986; which is irmmqtorated herein by reference), ST-LS X (associated with the oxygen-evolving complex of photosystem R, Stoekhaus # at 1989: whiehis incorporated herein by reference). if the constract is expressed in an insect cell, examples of mediatory elements operative In an insect cell include but are not limited to the polyhedron promoter, the gp64 promoter and the like. |0Οί 0] The present invmtlpii further provides the eloping .of a nucleic acid encoding an HA, for example but .not limited to, human. influenza A/3hdonesia/5/0$ virus HA (H5M1) into a plant, yeast or insect expression vector (e.g. haeulovirus expression vector) aod production of influenza vaccine candidates or reagents comprised of recombinant influenza strodtoral proteins that seif-assemhlc into .functional amt imnnmogCHic homoiypic macfomoiecular protein structures, incloding stdiviral inSuenaa particles and iniuenm VLP, in transformed plant cells or transformedinsect cells.

[00118] The nucleic acid encoding the HA, fjbr example hut not limited to, a human ihSuenza A/New Caledoma/20/99 (HI Hi) virus HA, or the human influenza A/lndouesia^S/OS virus HA gene may he expressed, lot example, using a Baculovirus Expression Systes) in an appropriate cell line, fer example, Spodopters ifuglperda ceils fe,g. SM cell line; ATCC;FTA~4047}, Other insect cell lines may also fee used.

[00119] The nucleic acid encoding the HA may, alternately, fee expressed in a plant cell, or in a plant. The nnclek acid encoding HA may he synthesized by reverse transcription and polymerase chain reaction (PCR) using HA RNA, ,M.m example, the.'RMA may be Isolated from human influenza A/New Caledoniaf2tp§ (HI Ml) virus or human influenza A/Indonesia/5/05 (HSN'i) virus, or from ceils infected with an influenza virus, For reverse franscriptioh and PCR, oligonucleotide primers specific for H A EKA, for example but not limited to, human influenza A/New Caledonia/20/99 (HlN l) virus: HA genes or human influenza A/lndonesi;a/5:/05 (H5M I ) Virus H AO genes cap fee Med. Additionally, the nucleic acid encoding HA may be chemically synthesized using methods as would known to ope of slaiU&amp;#eart 6-Proteins [001201 The present invenrion also ineind.es one or mote than one HA protein encoded by nucleotide sequences SEQID MO:l; SEQ lp:NO:5; SEQ ID NO;? (encoding HA from HI, H5 or M7, mspeclively), a nudeotide sepuenee SEQ ID MO:l; SEQ ID MO:S; SEQ ID MO'7, that hybridizes conditions to a nucleic aeid

that ·Ρηδί>&amp;&amp; H5 or HT, respectively, or anudeodde sequence SEQ ID NO: I ; S EQ IB NO: 5; SEQ ID NO;7, that feybd&amp;es under stringent hybridisation conditions to a GOmpbment ffa nucleic acid encoding the LIA, from HI, RS or HI, respectively, wherein »:bem^lutinm protein that when expressed forms a VLP, and that the VLP induces the production of an antibody, [0012.1 ] Similarly, ihwprcsent invention includes MAS ossbeiated with the following subtypes HI: (encoded by SEQ ID NO; 1), H2 (encoded by SEQ ID NO:2), H3 (encoded by SEQ ID NO:3),114 (encoded by SEQ ID NO:4)r H5 (encoded hy SEQ ID NO:5), H6 (encoded by SEQ ID 110:6),117 (encoded by SEQ ID NO :7), MS (encoded by SEQ ID HO;8), H9 (encoded by SEQ:ID NON). II10 (encoded by SEQ ID NO: 10), Mil (encoded by SEQ ID NO: 1 I), HI 2 (encoded by SEQ ID NO:! 2¾ Hi 3 (encoded by SEQ ID NO: 13), H14 (encoded by SEQ ID NON 4), MIS (encoded by SEQ ID NO: 15), Mid (encoded % SEQ ID NO:1 d); andwcIeotidb;s^ae®e^';lhat:ane characterized as having from about 60 io 100% or any amount Ibsrebetween se«|oence identity, partietdarly fc>m about 70 to 100% of homology or any amount therebetween, 80 to 100% or any amount there between, 90-1:00% or any amount therebetween, or 95-100% or any amount 'the^b^we«h:^umee''i4isxtit3f· witk-Hl (SEQ ID HO:I), H2 (SEQ ID NO: 2), 113 (SEQ ID NON), H4 (SEQ ID NO:4);, H5 (SEQ ID NO:5), H6 (SEQ ID NON), H? (SEQ ID NOB); H8 (SEQ ID NON), 119 (SEQ ID NO:9), B IO (SEQ ID NO: 10), 1111 (SEQ ID NO: 11), H12 (SEQ ID NO: i 2), MB (SEQID NO: 13), HI4 {SEQ ID NO; 14), Mi S (SEQ ID NO: 15), II16 (S EQ ID NO: 16), wherein the nudeotide sequence encodes a hema|gl«iinin protein that when expressed forms a VLB, and that the VLP induces the production of an antibody, For example^ expression of the nucleotide sequence within a pDnl cell forms a YEP, and the VLP may be need to produce i&amp;n antibody that is capable of binding HA, including mature HA, MAC, HAL or HA2, Hie VLP, when administered to a subject, induees an immune response.

7- VLP

[00122] Therefore, the present invention Is directed to a VLB comprising one or more than one H A type or subtype.

[OOISIJ The tens ‘Virus like partide*vpiJR), or "vims-like particles” or !,VLPs" refers to stmetores that selfiassemhle and comprise stmetural proteins such minfieenmBA protein, ¥LPs are generally morphologically and antlgenically similar to virions podueed in an infection, bat lack genetic ioihppation sufficient to replicate and .thus are nonrinfecfious, In some examples, BLPs may comprise a single protein species, or more tkan One protein species, For YLPs comprising more than one protein species, the protein species may fee from the same species of virus:, or may comprise » protein from a different species, genus, subfamily or tknuly of vires (as designated by the ICTV nomenclature}. In other exarnples, one or more of the protein species comprising a YEP may be modified from the naturallyoccurring seqncnce, VLFs may be produced in suitable host cells including plant and insect host cells. Following extraction from the host cell and upon isolation and further purification tinder suitable conditions, YLPs may be purified as intact structures.

[00134] The invention also Includes, but is not limited to, obtain a lipid envelope from the plasma membrane of the cell in which the VIP proteins are expressed. For example, if the VLF is expressed in a plant-based system, the VLP may obtain a lipid epyelope fix>m the plasma membrane of the cell [00125] Generally, the term ‘lipid’* refrrs to a fat-soluble (lipoplnlld^^teally* occurring molecules. The term is also used more specifically to refer to fatty-acids and their derivatives (mcloding fti-s di-, and mono^ycerides iand phospholipids), as well as other Iat~soiuble sterol-containing rnetabolites or sterols. Phospholipids are a .major component of all biological rhembrancS, along with giyeolipids, sterols and proteins. Examples of phospholipids include pho^hatidyletbanoiamine, phosphatidylcholine, phosphatidylinoSitol, phosphatldylserme, and the like, Examples of sterols include zoosicrols (e.g., cholesterol) and phytosterols. Over 200 phytosierols have been identified in various plant species, the most common being eampesferol, stigmasterol, ergosteroi, brassicastcrol, dehs-7-stigmasterOl, deIta-7-ayenasterol, daunosterol, sitosteml, 24-methyIebOlestero!, cholesterol or beta-sitos terol, As one of skill in the art would understand, the lipid composition of the plasma membmne of a cell may vary with the culture or growth ccaiditions pithe ceil or organism from which the ceil is obtained.

[()0125] Gell membranes generally comprise lipid hilayers, as well as proteins for various functions, Localized concentrations of particular lipids may be found in the lipid bilayer, referred fo as ‘lipid rafts'. Without wishing to be bound by theory, lipid rafts may have significant roles in undo and exocyiosts, entry nr egress of viruses or other infections agents, inteweeli signal transduction, interaction with other structural components of the ceil or organism, such as irbraceiiular and extracellular matrices, [00127] The ¥LFf produced from influenza derived proteins, in accordance with the present inve^ftion do not comprise Ml protein. The Ml protein is known to bind feNA (Wakefield and Brownlee, 1989) which is a contaminant Of tie VLP preparation. The presence of 1NA is undesired when obtaining regulatory approval for the VLP product, therefore a MLP preparation lacking RMA may be advantageous.

[00128] A: VLP produced in a plant according to some aspects of the invention may be competed with planftderived lipids. The VLP may comprise an HAD, HA l or HA2 peptide or combinations thereof. The pl anriderived lipids may ho in the form of a lipid bilayer, and may further comprise an envelope surrounding the VLP. The plant derived lipids may comprise lipid components of the plasma membrane o f the plant where the VLP Is producddi including, and one or more than one plant derived lipid, for example but not limited to phosphatidylcholine (PG), phosplmtidyletltanolamme (PE), glyeospfoh^lipids, phytosterols or a combination thereof A plaoLderived lipid may alternately be referred to as a ‘plant lipid5.

[00129] In plants, influenza VLPs hud foom the plasma membrane, therefore the lipid eomposidon of foe VIPs reflects their origin. Use VLPs produced according to the present invention comprise M A, eomplexed with plant derived lipids. Plant lipids can stimulate specific immune cells and enhance foe immune response induced. Plant membranes are made of lipids, phosphatidylcholine (PC) and phpsphatidyfeihanolamine (Pi), and also contain giycosphingchpids, saponins, and phytosterols, Additionally, lipid rafts are also found in plant plasma membranes - these microdomalns are enriched in spfoh^lipids and sterols. M plants, a variety of phytosterols are known to occur. and cholesterol (Montand ..eie'L, f$>130} FC and FE as well as glyeosphingolipids can bind to GDI moleciu1.es expressed by mammalian Immune cells sdeh as ardigen-pmsmting cells (APGs) like dendritic cells and macrophages and other .cells including B and T lymphocytes in the thymus and liver pisix}? M,:. 2006), GDI molecules are structurally similarM major histoeompatihdity complex (ΜΜΟ) molecules of class I and their role is to present glyeolipid antigens to MKT cells (Matural.Killer T cells). Upon activation, MKT cells activate innate Immune cells sncb as MK cells and dendritic cells and also like the antibody-producing B cells and T~cells.

[00131] A variety of phytosterols may be f ound in a plasma membrane - the specific Complement may vary depending on the species, growth conditions, nutrient resources or pathogen state, to name: a Jew factors, Generally, heta^sltosterol is the most abundant phytosterol [00132] The phytosterols present In an ί hfluenxa YLP eomplexed with a lipid bilayer, such as an plasma-membrane derived envelope may provide lor an advantageous vaccine composition. Without vrishing to be bound by theory^ plant-made VLPs eomplexed Mith a lipid bilayer, stseb as a plasma-membrane derived envelope, may induce a stronger immune reaction than VLPs made in other expression systems, and may be similar to the immune reaction induced by live or attenuated whole virus vaccines, [00133] Therefore, in some embodiments, the invention provides tor a VLP eomplexed with a plant-derived lipid bilayer. In some embodiments dm plant-derived lipid Mlsyer may comprise the envelope of the VLP.

[00134} Therefore, the present invention provides a composition comprising an effective dose of a VLP comprising am InfiMenza virus H A protein, one or more than one plant lipid, and a pharmaceutically acceptable carrier. The influenza Virus HA protein may be HS Indonesia, Also provided is a method of Inducing inpnnnity to an influenza virus infection in a subject. The nicfed administering the viass like particle comprising as initoenea vims HA protein, one or mom than one plant lipid, and a plmanac^iticaily acceptable carrier, The virus like particle may he administered to a subject ©raii&amp; iotradennally, intranasally, intrtoPuscutarly, innnperiioneally, intravenously, or subcutaneously. 9- Method of treatment [00135} The present invention provides tor a method of inducing immunity or '‘provoking an frmnuae response” to an inOoesza virus infection in a subject, the method comprising administering the composition as defined herein, [0013¾] An “immune response51 generally refers to a response of the adaptive immune system. The adaptive immune system generally comprises a humoral response, and a cell-mediated response. The humoral response is the aspect of immunity that is mediated by secretod ahtihodies, produced in the cells of the B lymphocyte lineage (B cell). Secreted antibodies bind to antigens on the surfaces of invading micaohes (such as viruses or bacteria), which flags them tor destruction. Humoral immunity is used generally to refer to antibody production and the processes that accompany it, as well as the effector fimetions of antibodies, inclndii^TitS eeii activation and cytokine produetlony meinory cell generation, opsonin promotion of phagocytosis, pathogen elimination and the like.

[00137] A eelbmediated response is ah immune response that does not involve antibodies but rather invol ves the aetiyation of macrophages, natural killer eelk (NK), antigen-specifie cytOtosic T-lymphocytes, and the release of various c>1okines in response to an antigen, Cell-mediated immunity is used generally to refer to some Th ceil acdvatipj^'T0^^H.:at^yi|riphiMd T-cell mediated responses, Cell mediated immunity is of parti cular importance In responding to viral infections, [00138] Tleifecomhinant HA VLPs of the present invention can he used in cenjhnefion with existing ittfiuehM vaccines, to supplement the vaccines* fender them more efficacious, and to reduce the administration dosages necessary. As would 'be known to a person Of skill in the art, the vaccine may he directed agains t one or more than one mfkserjzs virus. Examples of suifoble vaccines include, but arc not limited to those eommemiaiiy available from Sanofi-Pasteur, ID Biomedical Mortal Sinovac, €&amp;on, Roche, Medlmmnne, GlaxoSmithKIme, Novartis. SanoB-Aventis, -Serono, Shire Fhatmaceshcats apd the like, [00139: J If desired> the ¥LPs of the present invention may be admixed with » suitable adjuvant as would he known to one of skill in the art. Furthermore, the ¥LP may be used id an effective dose ofthe VLB for the treatment of a target organism, as defined above. Fnrfoermore, the VhP produced according to the present in vention may be combined with VfTs obtained using difeent m0uenza proteins, for example, neuraminidase (NA), [00140] Therefore, tile present invention provides: arneihod for inducing immuni ty to influenza virus infection in an animal or target organism comprising administering an effective dose of a vaccine comprising one or more than one ¥LP, The vaccine may he admimstered orally, infradennaiiy, intranasaily, inOmmscularly, intraperitonealty, intravenously* or subcutaneously.

[00141 ] As shown in Figures 6 and 7 m vkm assays showing emss-reaetiyity of antibodies raised against the mutated A%vdonesia/5/05 H5 ¥LFs and other influenza ^.:A/yietha«5/i^03/04; A/ Anhui/i/05 and A/Turkey/582/06 (all B5N1. strains^ whereas if showed less hemagglutination reactivity against the only H1N1 tested f ^Ι|^ΐϊ|Γί5 1? jf [00142] §i'jpsigoanf|^;lhd^tibo<iies produced after a single dose of mutated HSN1 [unglycosylated H5 protein) induced a greater response against all MS strains msted after 14 days than antibodies produced againstthe wild-type MS, indicating that this unglyeosylated inanunogen may provide more rapid response that the rvild-iype one, [00143] These data, therefore,: demonstrate that plant-made influenza.: VLPs comprising foe hmiated H5 hemagglutinin viral protein devoid of N-linked carbohydrates induce an immune response specific for pathogenic inBuenza strains, and that this response is cross-reactive and may be rapid after one single dose, Ρδί44| Examples of a select or target organism that the ¥LPs of the present invention may he administered to include, hot are not limited to, humans, primates, Birds, water fowl, migratory birds, quails, duck, geesd»qpd«|iry» Ιο^«^,.φϊόΐ6!^.·^'ίηο, sheep, equine, horse, samel, canine, dogs, feline, cats, tiger, leopard, civets' mink, stone marten, ferrets, house pets, livestock, rabbits, mice, rats, guinea pigs another rodents» seal, whale and the like. Such target organisms are exemplary, and are not to he considered limiting to the applicati ons and uses of the present ipvemiorn [00145] The present invention also plains to influenza viruses which infect other mammals or host animals, for exampie humans, primates, horses, pip» birds, avian water fowl, migratory 'birds, quail, duck» geese, poultry, turkey, chicken, camel, canine, dogs, feline, cats, tiger, leopard» civet, mink, stone marten, ferrets, house pets, livestock, mice, rats, seal, whale and the like.

[00146] Particularly, the subject being treated by the mefhod as defined above may he selected from the group comprising humans, primates, horses, pigs, birds (avian) water fewlmigramry birds, quail, duck, g^^^cken*-id0^,<^»,^s55!fe, livestock and the like, Fariiedariy; dte si^i)eef may he a human patient or birds in general (including water fowl, migratory birds, poultry such as quail, duck, geese, turkey, chicken), parrieularly migratory birds or poultry for human consumption (quail, duck, geese, turkey, chicken). More particularly, the subject is human. 11 - Containers, syringes» and kits etc;.

[00147} The present invention also provides tor &amp; containercomprising the composition as defined herein. Particularly» the container contains single unit dose or in multiple dosage; form with a preservative agent. More particularly* the container-is a syringe “ready-fonuse” pra-ftited with the composition or the vaccine as defined herein, [0(1148] More particularly, the invention:also provides for a kit comprising a eontainer comprising the vaccine or composition as defined herein, and instoctioas on how to use/administer said eomposition/vaecine. |00149] The invention will now be describee! in detail by way of reference only to the following non-lord ting examples.

Example 1

Material and methods 1>.'Mutation of wild-type MS from A/lrdonesiaO/OS (SEQ 1I> NO, 17) to obtain mutated unglyeosylated H5, [0()150] The triple mutant has been made by removing the glycos^mimt sites N154, MT65, and N286 located 0« the globular head of wild type HAs, more Spedieatly by replacing the Tbr or Set enclosed in the glycosylntion serinenee pattern ΝΟΟ'Γ/S by an Ala residim. Therefore, the triple mutant contained the following Three amino add replacements: ΤΙ 56AS Tld7A and S28SA (numbered according to the starting SBQ ID NO, 17), The three amino add replacement were perforated fey PCR-based ligation method presented in Darveau et &amp;L (1995]·using the wild type HA expression voder (660 construct. Fi gure 4} as the template. (00151 j Briefly, three PCR amplification were performed in parallel on OdO pOAMBI A expression vector as the template With 3 difremnt pairs of primers: 1) Platm443e(SBQ ID NO: IS) and HAS-TI56A>r (SEQ ID NO: 19); 2) HA5-T167A.e (SEQ IDN0:2O) and HA5-S2g8A,r (SEQ ID NO: 21); and 3| MA5-S288A.C (SEQ ID NQ:22) and HA(Ind)~$acLr (SEQ ID NO: 23). (Q0152] The amplification produets obtained from the three reactions were mixed together and the mixture served as templatefor a βίΐΐί isaetion (assembling reaction) using FlatO“443e (SEQ ID NO: 1.8) and HA(lfid)»Sao.r (SEQ ID NO: 23) as primers. The resulting tiupneat was digested with Ban® (located in the plasiocyauin promoter) and Saef (at the 3%nd of the fragment) and cloned into pCAMBIAFlaste previously digested with foe sameenxymes, The^suiting plasmid, named is presented in Figure s (SEQ ID »29).

[00153} 413 manipulations were earned out using tie general molecular biology protocols of Sambtpok and .Russell (2001 [which is incorporated herein by reference).

Tire first cloning step consisted is assembling a receptor plasmid containing upstream and downstream regulatory elements of the alfalfa plastoeyanin gene. The plastocyanin promoter and 5 iliXE sequences were amplified horn alfalfa genomic DN A using oligonueleoiide primers XmaI~pFlas.c (SEQ ID NO: 24) and SaelAAfG~pPia$,r (SEQ ID MCk 25).. lire resulting amplification product was digested with Xmal and Sad and ligated ifo0pCAMBIA23QQ (Gambia, Canberra. Australia), previously digested with the same enzymes* to create pCAMBIAprorao Plasto, Similarly, the 3 TJTR sequences and terminator of the plastoeyanin gene was amplified from alfalfa genomic DHA using the following primers: SaeEP!asTer,e (SEQ ID HO: 2d) and EcoRI-PIasTenr (SEQ ID NO: 27), and the product was digested with Sad and EcoRI before being inserted into the same sites of pGANiBIAptomoFlasto to create pCAHIBiAPlast©, 3. Assemble of HS expression cassette [00154] A fragment encoding hemagglutinin from influenza stern A/indonesla75/OS (B5N1; Ace. No, LANE ISDN 125873) was synfoesized fry Epoch Biolabs (Sugar Land, TX, ilSA), The fragment produceds eontainmg the complete HI coding regioft (SBQ IJ> HO,17) Including the native signal peptide flanked by a BindHl site immediately upstream of the initial ATG, and a SacI site immediately dnwnsteam of the stop (TAA) codon, is presented in SEQ ID HO: 28 (and SEQ ID NO.29 in the case of the mutant H5). The Hi coding region was cloned into a plastocyani η-based expression cassette hy the

Darveau et aL (1095). Briefly, a first FOR amplification was obtained using primers P!ah>443c (SEQ ID NO: 30) and SpHAilnd)·· Piaster (SEQ ID NO:31) and pCAMBIA nromoPlasto as template, In parallel, a second amplification was performed with primers Ftasto~SpHA(Ind},e (SEQ ID HO: 6) and HA{ind)~Sac,r (SEQ ID NO:32) With HS coding fragment as template. The amplification obtained fmm both reactions were mixed together and the mixture served as template for a third machoa ^assembling reaction) using Plato-443c (SEQ I D NO: 4} and HA(lnd)~ Sac.r (SEQ ID NO: 33} as primers. Hie resuiiing iragrrjeat was digested With BamHI (in the plastocwanin promoter) and Saul (at the Tend of the fmgmeni) and cloned Into pDAMBlAftlasto previously digesmdM the same enaymes. The resetting plasmid, named 660, is presented in Figure § whereas the plasmid resulting from the ‘homated” H5 protein was named 680. |QQ155] An BcPro construct pSHePro) was prepared as described in Hamilton et ui (2002). All clones were sequenced to confirm the integrity of the constructs. The plasmids were used to transform A^pba^1«pE';tiiraap§ieieos':;(AGLl; ATCG, Manassas, ¥A 20108, USA) by electroporation (MattanoMeh etM, 1980). The integrity of all A, tnmefaeiens strains were confirmed by restriction mapping. 4, Preparation of plant biomass. Inoculum, agromfiltmtiom and batwesting [00156) Meotiana beaithamlana plants were grown from seeds in fiats filled with a .commercial peat moss substrate. The plants were allowed to gaw in the greenhouse under a 16/8 photoperiod and a temperature regime of 25*0 day/2(fC night. Three weeks after seeding, individual plantlets were picked out, transplanted in pots and left to grow in the greenhouse for three additional weeks under the same environmental conditions. Prior to tmnsfonnafion, apical and axillary buds were removed at various timesics indicated below* either by pinching the buds from the plant, or by chemically treating the plant [0015?] Agtbhacferia transfected with plasmids 660 or 680 were grown in a IfEB medium supplemented with 10 mM 2"[Nmt0^holmo)elhan.esulibnic acid (MIES), 20 gM: aeetosyringone, 50 pg/ml kanamycin and 25 pg/ml of carbemcillin pfi5.6 until they reached an 00600 between 0.6 and 1.6. Agrobactedum suspensions were cenififuged before use arid resuspended in infiltration medium (10 mM MgC12 and 10 mM MBS pH 5,6), SvrmgeAufiliration was performed as described by Liu and Lomonosseff :(2002:, Journal of Virologieal Methods, 105:343-34¾}. For vacunm-infiltration, A. tumefaciens suspensions were eenififuged, resuspended ip fbp infiltration medium and stored Overni^t at 4-U. On the day of infiltration* culture batches were diluted in 2.5 culture volumes and allowed to warm before use. Whole plants of Nicotlana benthamiana were placed upside down in the bacterial suspension in an air-tight stainless steel task under a vacuum of20-40 Tort for 2-mis, Following syringe or vacutmt isfilhstion, plauts were returned to the ^reeshouse a 4-5 day incubation period until harvest, 5» Leaf sampling and total protein extraction 100158] Following iscuhation, tie aerial part of plants was harvested, §oges at -SO^C, crushed into pieces. Total soluble proteins were extracted by homogenizing (Folytion) each sample of frozen-crushed plant material in 3 volumes of cold 50 mM Tiis pH 7,4, 0.15 M MaCl, and 1 .n^:;|^<5®ybn0thaii«sal'foayi' fluoride, After homogenization, the Slurries: were centrifuged at 20,000 g for :20 mis at 4*€ and these clarified crude extracts (supernatant] kept for analyses.. The total protein content of clarified crude extracts was determined by tire Bradford assay (BtOrlJad, Hercules, CA) using bovine serum ..albumin as the reference standard, [00159] Protein concentrations were determined by the BCA protein assay (Pierce Biochemical, Rnckport IT], Proteins were separated by SDS-PAGE under reduemg conditions and stained with Ceomassie Blue. Stained gels were scanned and densitometry^^alpls perftmned using MageJ Software (K1H).

[§0160] Proteins from eluhon fiction horn SBC were precipitated with acetone (Bollag el a L, 1996), resuspended in 1/5 volume in equilibrafiosr/elution buffer and separated by SDS-FAGE under reducing conditions and eleehotrasafeired onto polyvinylene difluoride (FVDF) membranes (Roche BkgnosfiesC^ for immuoodeteetiom Prior to imnmnobiotting, the membrvmes xvej^ bloekM with 55¾ skim milk and 0J% TWeen-20 in Ttis-bofeed^jap'CFSS-T):.^ id»i8h at 4*C, (00161] Immunoblotting was performed by incubation with the following antibodies: for the detection: of H i , a htouse anti-influeuza A monoclonal antibody (Fitzgerald Industries hneraational. Concord, MA, USA, Cat, No. 10-150)(2 p.g/ml in 2% skim milk in TBS-Tween 20 0.1%), and for the detection of llS, a rabbit anti~HS (Vietnam) antibody (Immune Technology, Woodside, NY, USA, Cat No. ΓΤ-ΟΟ3-005Υ) diluted 1/4000 in 2% skim milk mTBSGTween 20 0.1¾. A peroxidase-eotgugated goat anti-mouse IgG (H-f L): anfibpdy. (Jayson temnnoreseaKih kabomtoriesy West Grove, PA, USA, Gat. No. 115-03S-140) (diluted 1/12 000 in 2% skim milk in TBS-Tween 20 0,1%) was used as secondary antibody. Imm.unoreaclive complexes were detected by phemiluminescenee using lomtnOl as the substrate (Roche Diagnostics Corporation), Horseradish peroxidase-enzyme; conjugation of human IgG antibody was carried oot by using the EE-Oiik Plus# Activated Peroxidase cphjp^^aMt'CPii^be, Eoekfoid,. IL). £00162] Hemagputinaiion assay for H$ was based on a method described by Nayak, and Reiehl (2004). Briefly, serial douhie dilutions of the test samples (100 pL·) were made in V-bottomed 96~well microtiter plates eontaining 100 pk PBS, leaving 100 p L of diluted sample per well. One hundred microliters of a 0<25%'^me:b^;Md#''p$ns suspension (Bio Link Inc., Syracuse, MY) were added to each well, and plates were incubated for/¾ at room tenrperature. The reciprocal of the highest dilution showing complete hemagglutination was recorded as HA activity. In parallel, a recombinant HA standard (A/Viemam/1203/2004 H5M1) (Protein Selene® Corporation., Meriden, CT) was diluted in PBS and tun as a control on each plate, 1 HS ¥LP purification (00163] Frozen 660- of 680-infilfiated leaves of N. benthamiana were homogenized In 13 volumes of SO mM TrispH 8, NaCl 50 mM and 0.04%; sodium meta»Ms«lfite using a commercial blender, The resulting extract was supplemented with 1 rnM PMSF and adjusted to pH 6 with 1 MI acetic acid before being heated at 42°C for 5 min, Diaiomaceous earth (DE) was added to the heat-treated extract to adsorb the contaminants· precipitated by the pH shift and heat treatment, and the slurry was filtered through a Whatman paper filter. The .resulting clarified (extract was cehtri&amp;ged at 10,000 x g for 10 minutes at RT to remove residual DE, passed through 0,8/0,2 μηι Acropack 20 tilt»» and loaded onfo a fomifoagarose affinity column (Sigma* Aldrich, St-Louis, MO, USA), Following a wash step in 400 mM MaCl, 25 mM ’Pris pH 6, bound proteins were eluted with 1,5 M NaGI, 50 mM MBS pH fir Eluted VLP were supplemented with Tween-SO to a final eeneentrafion of 02)005% (v/v), ¥I,P were concentrated on a 100 kDa MWC0 Anrieon membrane, centrifuged at 10,000 x g for 30 minutes at 4oC ansi i^o^endedin FjBS pH 7,4 with 0.01% Tween-80 and 0.01% ihimerosaf Suspended YLFs were Ste-steriMeed before use. 8, Animal studies

[00164] Studies on the immune response to influenza VLF adndnistratinn were performed with 6-8 week old female Wistar rats (Charles River.Labofutdries); Thirteen rats were randomly divided info three groups ranging from three for the control group to five animals for both the plant-made V3t#-I55 wild type vaeeine (660) and the mutant (680) vaccine groups. Eight groups were used for imTamusetdar immunization and six groups were used to test intranasal route oh administration. All groups were immunized in a two-dose regiment, the boost immnainaticn being: done 14 days following the fust imrmmizatiorL

[00165] For intramuscular admiaistration in Mud legs, nnanaesthetixed rata were immunized with either the piMt-made ¥Lf H5 vaccine (I S gg), the plant-made VLP HS mutant form of the vaccine or FES.

[00166] All antigen preparations were mixed with Alhydrogel to a final cpneentration of ! % (alum: Accurate Chemical and Sfoentiilc Corporation, Wesbury, KY, US) in a U1 volume ratio prior to immunizations.

Blood collection and spleen collection [00! 67] Jugular vein blood collection was performed fourteen days after the first, immunizanon and fourteen days after second immunization on anaesthetized animal. Serum was collected: by centrifuging at :8000 g for 10 min, [00168] Three weeks after second inminniSation, rats were anaesthetized with. CQ2 gas and immediately upon termination, cardiac puncture was used to collect blood, [00169] Spleen collection was performed on rats Collected spleens were placed in RFM1 supplemented with gentamycin and mashed in a SO ml conical tube wi th plunger Ifom a 10 ml syringe. Madfteft spleens were rinsed 2 times and centrifuged at 2000 rpm for S min and resuspended m AC&amp; lysing buffer for 5 min at room temperature, fte splcuocytes were washed in PBS^ntamycin, resuspended in 5% EPMI and counted. Splenocytes were used for proliferation assay.

Antibody diets:

Amemarn/i2Q3m®4 0$mk A/Animi/I/m (mNl): Adiurhy/m-key/J/05 0SMJ); Λ/New Caiedornamm (ΗΊΝ2 [CMH 70] AniMnllumza antibody titers of sera were measured at 14 days after the first itnntnnizafioo as well as 21 days after the second immunisation (at sacrifice)· The titers were detennined by «^jase-lioked imntunosorbeitt assay (ELISA) using: tie inactivated virus A®do«esia(5/05 as the coating antigen. The end-point (titers were expressed as the reciprocal valne of the highest dilution that reached an QD val ue of at least 0.1 higher than that of negative control samples.

[00171] For antibody class determinati on (fgGl, IgG2a> IgG2h, IgCB, IgM), Ore titers were evaluated on final bleeding by ELISA as previously described.

Hemagglutination itikibMm 01) Mitts-

[00172] Hemagglutination inhibiMon (HI) titers of sera were measured at days 14 and 35 afterthe seepnd imnruiusation as pbvi0nsiy deserihed (WHO 2OO2; Kendal 1082). Inactivated virus preparations from strains A/Indonesia/5/0St^Anhnifl/d5 (HSbll)j A/t^ey/Tprfeeyi' 1 /05 (Ή5Η1) or A/VIetnam/t2O3/2O04 were used to test rat serum samples for Hl aetivity. Serawerepre4reatedwiih receptor-destroying enzyme If (RDB II) (Denka Seiken Co., Tokyo, Japan) prepared tmm Vibrio cholerae (Kendal 1982). Ill assays were perfnmred with 0,S% horse red blood cells. HI antibody hires were defined as the reciprocal of the highest dilution causing complete inhibition of agglutination.

Results [001731 The reactivity of the sera fern rats irnmuniaed with either the wi VLP or the mutant VLP was assessed 14 days after the Erst (Day 14) or the second immnrnnaEon (Day 35), Allrais were immunized with 15 pg of the antigen formulated with alum. Itnntunoreaetiviiy was assessed against MSN1 viruses of elade 1 (A'Yiotnam/1203/04), clad© 2,1 (A/fedonesia/S/OS), clade 2 j (A/turkeyf&amp;!i%/l/05) and ©lade 2.3 (A/Anhui/l /05), After the first <3ρ§^ tl$e mutast Vfift induced·» higher antibody reaction than the wt Ibr all H5N1 strains tested (Figure 6). The imnmnereaefivity against the afoan strain statistically Hgnifteant fo<0.05) after the first

dose, im.mucoreaciivity was also assessed against H IN I viruses ( A/New GaledoraaG0/99) shoeing immunorsacttviiy after boost ifoection, GMT: geometric mean liter, Values are the GJdT (In) of reei|n^cal eiid-point titers of Sve rats per group. Bars represent mean deviation, * p< 0.05 compared to the wt VLF

[00174] HI titers fern rats immunized with .'the wt or the momnt VLP were assessed 14 days after the first (Day 14) or the second (Day 35) irmrmnkatiom HI antibody responses were measured using inactivated whole H5N1 viruses. After the first immunization,, the mutant VLF induces a higher HI antibody response than the wt VHP against all H5Ht viruses tested (Figaro 7),. Statistical significance was reached tor A/Indenesifod/Qo and A/turfceyG'mkey/1/05 influenza strains, GMTv geometftc meatt titer. Values 3¾¾ the GMT titers of ft ve tats per group. Bars represent mean deviation, * p< 0,05 and compared to wt VHP, [00175] These data strongly surest that the “mutated” tmglycoaylaied H5 proiein represents a very interesting alternative to the nati ve 155 protein for the production of VLPs as broad-spectrum and fast-active flu vaccine, [00175] Ah citations are'hereby incorporated by reference, [00177] The present invention has been described with regard toone or mom particular embodiments. However if will be apparent to persons skilled in the art that a number of variatiops and modifications can he made without departing from foe scope of the invention as defined in the claims, * Abe Y. et al ^urml t^vir&amp;iogy, (2004) 7$ : 9605-9011. * Boliag, D.M,, ftozycki, MJP., and Edelstein, S.J, (1996) Protein methods (2m edition)-Wilcy-Liss, New York, USA. * Bligh} Ι,β,ϊ &amp; Dyer, W,J, Can. J. Med Sci 37, 911-917 (1959), * Bright\€laiMrohgy. (2003) 308 ; 270-278, * Cffom BJ), Ees^ GJ% Morita, E.s -apd Lamb SLA, (2007) Muenza virus bemaggludnp and nenranbriidase, but not the matrix protein, are required for assembly arid budding of pigsteid^erived virus-like particles, ,1. Virol, SIt 7111-7123. « Crawford, 1., Wilkinson, B>, Voznesensky, A*? Smith, G,, Garcia* M. , Stone, M.,.. add Perdue, M. L, (1990), Baeufovtrtis-derivsdfoemagghdinin vaccines protect against lethal inBuenza infections by avian IIS ami H7 subtypes. Vaccine f 7,2265-2274. * Darveau, A->/Pelletier, A. Sc Perreault, #<·. PCR-mediated synthesis of chimeric moL·mM&amp;Meihoφ:f¢mmsa 26,77--85 (1.903), * Grgacie EVL, Anderson DA, Virus-like particles: passport to immune recognition. Methods 2006; 40; 60-65, * Gillim-Ross, L-, and Suhbsrao, K, (2006) Emerging respiratory viruses; challenges and

Rev, 19,614-636. * Gomeri-PdeRas, P,yMeha, L, Castillo, M,s Vivo, A,, Perez--Pastrana, E, and Portela, A, (1999) Effident fommtioo of influenza virus-likeparticles; dependenceon-the-expression level of viral proteins, J. Gen. Virol. SO, 1635-1645. * Gomez-Puertas, P., Albo, €., Perez-Pastrana, E-, Vivo, A.. and Portela, A. (2000) Influenza Vims protein is the major driving force in virus budding, J Virol, 74, 11..518-I 1:547'. '« H amilton, A. ? Vpinnet, O., Chappell, L, &amp; Baulcombe, D, Two classes of short interfering BN A in RNA silencing. EMBOX 21, 4671-4679 (2002), «. Ηρ%ρη, R, <&amp; Wilhnitzer, L, Storage of competent cells for Agrobaeterpni transformation- Nmteic Acid Res, 16,9877 (1988). « Harbury PB, Zhang T, Kim PS, Alber T, (1993) A switch between two--, three-, and fopr-strarsded coiled coils In GGhi4 leudne zipper mutants. Science; 262: 1401-3407) » Horimotb T-, Kawaoha Y. Strategies for developing vaccines against hSNl influenza a viruses- Trends In Mol Med, 2006; 12(11):506-314. » HuangZ> Elkin G, Maloney B,l, Beuhner N, Arntcen. €-1. Than&amp;vala Y, Mason MS. Viru$~Mke particle expreasfen and assembly in plants: hepatitis B and Norwalk viruses. Vaccine. 2005 Mar 7;23{lS):18Sl-8, * Johansson, B. widvinSnenza A ^^s b^aggiafem aiKl neuramiiiidlas^ pfodneed inrecombinant ba^ balanced and broadened immune responsesageriQr to eonyentioMl vaccine. Vaccine 17,2073-2680. * EapSa, X, De Eycke, 8.., Van Montagu, M, Si Angenon, G. An AgrobacttTiam-mediatcd transient gene expression system for intact leaves. Flam Set. .12¼ 101-108 * Eufoda at $990) fifoi-agy. 174 : 418-429, * Latham, T,, and Galarza, J, M. (2001). Formation of wild-type and chimeric influenza virus-like particles IpHowing sinruhaneous expression of only four shueforal proteins. X Virol 75,6154- 6165, « Ldfebvre». B. Pftps&amp;l 144,402-418 (2007). * Liu, L &amp; LoutonoasoiXGP. Agroinfoetioh as a rapid method for propaiating Cowpea mosaic virus-based constructs. J. Viral Methods 105,543-348 (2002). . MaoakvLJ., Vp,RJL &amp; Ando, Sailed ReseM, 1243-1250 (1983) « bf attanovich, D,, Ruler, F,, da Camara Machado, A,, Laimer, M., Regner, F., Sternkellner, H,, Himmler, 0,, and Ratinger, IL (1989) Elhcimt transformation of .Agmhactemm spp. By electroporation, Nuel Ac. Res. 17,6747, « Mena, I,, Vivo, A,, Perez, E.. and Portela, A. (1996) Rescue of synthetic chloramphenicol acetyltransferase ENA into influenza virus-like particles obtained from recombinant plasmids, X Virol 70, 5016-5024. * Mongrand Ss Morel h Claverol S, Garde JP„ Hartmann MA et al. Lipid rafts in higher plant ceils, The. Journal of BiologicalGhemistry 2004; 279(35): 36277-3:6286, * Neumann, G., WMasabe, f,, and Kawaoka, Y, (2000) Plasmid-driven formation of virus-like particles. X Virol 74, 547-551, * Nayak DP, Reich! U. (2004) Neuraminidase aciiv5ty assays for monitoring MOCK cell culture derived influenza vitas. J Virol Methods 122(1):9-15, * Olsen, €. W., McGregor, M. VV., Dybdahl-Sissoko, N., Schram, B. R., Nelson, K. M, . Limn, D,, Maddin, M, D,, and Swam, W. F, (1997), Immonogemefty and efficacy of baculovirus- expressed and DNA-based equine influenza vims hemagglutinin vaccines in mice. Vaccine I S, 11494.156, » Quan FS, Huang €? Gompans RW, Kang SM. Vlrus-fiMe particle vaccine induces prcfeetive imiBumty against homologous and heterologous strains of kftuenza vims. Journal of Virology 2007; 81(7): 3514-3524. » Sainfijort^Dupas C> Faye L, Goniord V. (2007) From plants to pharma with glycosyiati&amp;n. l a the toolbox, Treads an biotech, 25(7) 317-323 . » Saavbiook 1, and Russell DW. Molecular cloning: a laboratory manual. Gold Spring Harbor, K.Y. Cold Spring Harbor Laboratory Press, 2001. » Suzuki, Y, (2005) Siaiobiology of influenza. Molecular mechanism of host range variation of iniuentta viruses, Sioi, Pharm. Soii28, 399-408. * Tsaji M,, Cell. Mol Life Sea, 63 (2Q06); 18894:898, « Vigerust DJ et at joiiritiil-qf.virology* (2007) Si : 8593-8600, * Wakefield L,, G.G, Brownlee Nue Acid Res, 17 (1989); 8569-8580. * Kendal, AF, f eretta MS, Skeltel J. Concepts and procedures -for laboratory-based intluenza surveillance; Atlanta:CDC; 1982, p.B17-B35, * %H0, Manual on animal iuflneara diagnosis and sorvedttanee. Department of ; eonuannicable disease surveillance and response. World Health Organisstiou Global Infinerika Program. 2002,

Claims (20)

1. CLAIMS:-

   1. A nucleic acid comprising a nucleotide sequence encoding an influenza virus hemagglutinin (HA) comprising a HA1 domain, wherein said HA1 domain is modified to be free of N-linked glycosylation sites at positions 154, 165, and 286, by a. modifying an amino acid at position 154 to be a non-asparagine and/or modifying an amino acid at position 156 to be a non-serine or non-threonine; b. modifying an amino acid at position 165 to be a non-asparagine and/or modifying an amino acid position 167 to be a non-serine or non-threonine; and c. modifying an amino acid at position 286 to be a non-asparagine and/or modifying an amino acid at position 288 to be a non-serine or non-threonine, wherein the numbering is in accordance with strain A/Vietnam/1194/04 and wherein the nucleotide sequence is operatively linked to a regulatory region active in a plant.
2. 2. The nucleic acid according to claim 1, wherein the nucleotide sequence has at least 90% identity to SEQ ID NO. 17, wherein residues encoding amino acids at position 154, 165 or 286 encode a non-asparagine and/or wherein residues encoding amino acids at positions 165, 167 or 288 encode a non-serine, non-threonine or an alanine.
3. 3. The nucleic acid according to claim 1, wherein the nucleotide sequence encodes an influenza virus hemagglutinin (HA) comprising the sequence of SEQ ID NO. 34, and wherein amino acids at positions 154, 165 and 286 encode a non-asparagine and/or wherein the amino acids at position 156, 167 and 288 encode a non-serine, and a non-threonine or an alanine.
4. 4. The nucleic acid according to claim 1, wherein the nucleotide sequence is as defined according to SEQ ID NO. 29.
5. 5. The nucleic acid according to claim 1, wherein amino acids at positions 154, 165 and 286 are leucine, isoleucine, valine, threonine, serine, alanine, or a combination thereof.
6. 6. The nucleic acid according to claim 1, wherein the amino acids at positions 156, 167 and 288 are leucine, isoleucine, valine, alanine, or a combination thereof.
7. 7. The nucleic acid according to claim 1, wherein each amino acid at positions 154, 165 and 286 is modified to be an alanine, and/or wherein each amino acid at positions 156, 167 and 288 is modified to be an alanine.
8. 8. The nucleic acid according to claim 1, wherein the regulatory region is selected from the group consisting of: a plastocyanin regulatory region; a napin promoter, a cruciferin promoter, a Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) regulatory region, a chlorophyll a/b binding protein promoter, an ST-LS1 promoter, a polyhedron promoter, and a gp64 promoter.
9. 9. A method of producing influenza virus like particles (VLPs) in a plant, a portion thereof, or a plant cell, the method comprising: (a) introducing the nucleic acid according to any one of claims 1 to 8 into the plant, portion thereof, or the plant cell and (b) incubating the plant, portion thereof, or the plant cell under conditions that permit expression of the nucleotide sequence, thereby producing the VLPs.
10. 10. The method according to claim 9, wherein, in the step of introducing (step a), the nucleic acid is either transiently expressed in the plant, portion thereof, or the plant cell, or stably expressed in the plant, the portion thereof, or the plant cell.
11. 11. The method according to claim 9, further comprising a step of: (c) harvesting the plant, portion thereof, or the plant cell and purifying the VLPs.
12. 12. A virus like particle (VLP) produced by the method according to any one of claims 9 to 11.
13. 13. A virus like particle (VLP) comprising an influenza virus HA molecule encoded by the nucleotide sequence as defined in any one of claims 1 to 8.
14. 14. The VLP according to claim 12 or claim 13, wherein the influenza virus HA comprises plant-specific N-glycans, modified N-glycans, one or more than one plant lipid, or combinations thereof.
15. 15. The VLP according to any one of claims 12 to 14, wherein the influenza virus HA is of type A or type B, or wherein the HA is from one or more than one A subtype selected from the group consisting of: H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15and H16.
16. 16. Use of the VLP according to any one of claims 12 to 15 for the manufacture of a medicament for the prevention or treatment of an influenza infection in a subject.
17. 17. A composition comprising an effective dose of the VLP according to any one of claims 12 to 15, in admixture with a pharmaceutically acceptable carrier.
18. 18. A method of inducing immunity to an influenza infection in a subject, comprising administering the composition according to claim 17.
19. 19. The method according to claim 18, wherein the composition is administered to a subject orally, intradermally, intranasally, intramuscularly, intraperitoneally, intravenously, or subcutaneously.
20. 20. A transgenic plant or a transgenic plant cell transiently or stably transformed with the nucleic acid according to any one of claims 1 to 8.